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Sample records for human neutral ceramidase

  1. Discovery and evaluation of inhibitors of human ceramidase.

    PubMed

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

    2011-11-01

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

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

    PubMed

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

    2011-07-01

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

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

    PubMed Central

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

    2003-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    1999-12-01

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

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

    PubMed

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

    2011-08-12

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

  7. Acid Ceramidase in Melanoma

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed Central

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

    2008-01-01

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

  10. Acid Ceramidase Promotes Nuclear Export of PTEN through Sphingosine 1-Phosphate Mediated Akt Signaling

    PubMed Central

    Beckham, Thomas H.; Cheng, Joseph C.; Lu, Ping; Marrison, S. Tucker; Norris, James S.; Liu, Xiang

    2013-01-01

    The tumor suppressor PTEN is now understood to regulate cellular processes at the cytoplasmic membrane, where it classically regulates PI3K signaling, as well as in the nucleus where multiple roles in controlling cell cycle and genome stability have been elucidated. Mechanisms that dictate nuclear import and, less extensively, nuclear export of PTEN have been described, however the relevance of these processes in disease states, particularly cancer, remain largely unknown. We investigated the impact of acid ceramidase on the nuclear-cytoplasmic trafficking of PTEN. Immunohistochemical analysis of a human prostate tissue microarray revealed that nuclear PTEN was lost in patients whose tumors had elevated acid ceramidase. We found that acid ceramidase promotes a reduction in nuclear PTEN that is dependent upon sphingosine 1-phosphate-mediated activation of Akt. We were further able to show that sphingosine 1-phosphate promotes formation of a complex between Crm1 and PTEN, and that leptomycin B prevents acid ceramidase and sphingosine 1-phosphate mediated loss of nuclear PTEN, suggesting an active exportin-mediated event. To investigate whether the tumor promoting aspects of acid ceramidase in prostate cancer depend upon its ability to export PTEN from the nucleus, we used enforced nuclear expression of PTEN to study docetaxel-induced apoptosis and cell killing, proliferation, and xenoengraftment. Interestingly, while acid ceramidase was able to protect cells expressing wild type PTEN from docetaxel, promote proliferation and xenoengraftment, acid ceramidase had no impact in cells expressing PTEN-NLS. These findings suggest that acid ceramidase, through sphingosine 1-phosphate, promotes nuclear export of PTEN as a means of promoting tumor formation, cell proliferation, and resistance to therapy. PMID:24098536

  11. Drug targeting of sphingolipid metabolism: sphingomyelinases and ceramidases

    PubMed Central

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

    2011-01-01

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

  12. Ceramidase Regulates Synaptic Vesicle Exocytosis and Trafficking

    PubMed Central

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

    2009-01-01

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

  13. 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.

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

    PubMed Central

    Okino, Nozomu; Ito, Makoto

    2016-01-01

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

  15. 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

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

    PubMed Central

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

    2016-01-01

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

  17. 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.

  18. 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.

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

    PubMed Central

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

    2015-01-01

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

  20. Acid ceramidase is upregulated in AML and represents a novel therapeutic target

    PubMed Central

    Tan, Su-Fern; Liu, Xin; Fox, Todd E.; Barth, Brian M.; Sharma, Arati; Turner, Stephen D.; Awwad, Andy; Dewey, Alden; Doi, Kenichiro; Spitzer, Barbara; Shah, Mithun Vinod; Morad, Samy A.F.; Desai, Dhimant; Amin, Shantu; Zhu, Junjia; Liao, Jason; Yun, Jong; Kester, Mark; Claxton, David F.; Wang, Hong-Gang; Cabot, Myles C.; Schuchman, Edward H.; Levine, Ross L.; Feith, David J.; Loughran, Thomas P.

    2016-01-01

    There is an urgent unmet need for new therapeutics in acute myeloid leukemia (AML) as standard therapy has not changed in the past three decades and outcome remains poor for most patients. Sphingolipid dysregulation through decreased ceramide levels and elevated sphingosine 1-phosphate (S1P) promotes cancer cell growth and survival. Acid ceramidase (AC) catalyzes ceramide breakdown to sphingosine, the precursor for S1P. We report for the first time that AC is required for AML blast survival. Transcriptome analysis and enzymatic assay show that primary AML cells have high levels of AC expression and activity. Treatment of patient samples and cell lines with AC inhibitor LCL204 reduced viability and induced apoptosis. AC overexpression increased the expression of anti-apoptotic Mcl-1, significantly increased S1P and decreased ceramide. Conversely, LCL204 induced ceramide accumulation and decreased Mcl-1 through post-translational mechanisms. LCL204 treatment significantly increased overall survival of C57BL/6 mice engrafted with leukemic C1498 cells and significantly decreased leukemic burden in NSG mice engrafted with primary human AML cells. Collectively, these studies demonstrate that AC plays a critical role in AML survival through regulation of both sphingolipid levels and Mcl-1. We propose that AC warrants further exploration as a novel therapeutic target in AML. PMID:27825124

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

    PubMed Central

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

    2013-01-01

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

  2. Polyomavirus BK Neutralizing Activity in Human Immunoglobulin Preparations

    PubMed Central

    Randhawa, Parmjeet S; Schonder, Kristine; Shapiro, Ron; Farasati, Nousha; Huang, Yuchen

    2011-01-01

    Background Polyomavirus BK (BKV) infection can cause nephropathy in the allograft kidney. No well-established drug treatment is available at this time. Human intravenous immunoglobulins (IVIG) have been used as an empiric therapy without proof of effectiveness. Methods We tested five lots of commercially available IVIG preparations from two different suppliers for polyomavirus neutralizing activity. BKV and mouse polyomavirus were used to infect human and murine host cells, respectively, with or without prior treatment with IVIG. Neutralization activity was measured by quantitation of viral DNA after 7 days in culture. Results Coincubation of BKV but not mouse polyomavirus with clinically relevant concentrations of IVIG derived from healthy and hepatitis B vaccinated subjects caused more than 90% inhibition of viral DNA yield after 7 days in culture. Consistent with a direct neutralizing mechanism, this effect was significantly diminished if viral infection was performed in immunoglobulin pretreated cells or if immunoglobulin treatment was delayed 2 hr after addition of infectious virus. Conclusion Human IVIG preparations contain BKV neutralizing antibodies. Data on neutralizing capacity of these antibodies are presented to aid dose exploration in clinical trials seeking to validate the use of IVIG in patients with BKV infection. PMID:20568674

  3. Human-like antibodies neutralizing Western equine encephalitis virus.

    PubMed

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

    2014-01-01

    This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69-3A2) neutralized 50% of 5x10(4) TCID 50/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69-3A2 antibody is a promising candidate for future passive vaccine development.

  4. Human-like antibodies neutralizing Western equine encephalitis virus

    PubMed Central

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

    2014-01-01

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

  5. 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.

  6. Influenza virus resistance to human neutralizing antibodies.

    PubMed

    Crowe, James E

    2012-01-01

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

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

    PubMed Central

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

    1982-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  9. 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

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

    PubMed

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

    2016-09-12

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

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

    PubMed

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

    2015-04-02

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

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

    PubMed

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

    2001-02-23

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

  13. Protective Efficacy of Broadly Neutralizing Antibodies with Incomplete Neutralization Activity against Simian-Human Immunodeficiency Virus in Rhesus Monkeys.

    PubMed

    Julg, Boris; Sok, Devin; Schmidt, Stephen D; Abbink, Peter; Newman, Ruchi M; Broge, Thomas; Linde, Caitlyn; Nkolola, Joseph; Le, Khoa; Su, David; Torabi, Julia; Pack, Melissa; Pegu, Amarendra; Allen, Todd M; Mascola, John R; Burton, Dennis R; Barouch, Dan H

    2017-10-15

    HIV broadly neutralizing antibodies (bnAbs) have been shown to occasionally display unusual virus neutralization profiles with nonsigmoidal slopes and plateaus at <100% neutralization against a variety of viruses. The significance of incomplete neutralization for the ability of bnAbs to mediate protective effects in vivo, however, is undetermined. In the current study, we selected two bnAbs, PGT121 and 3BNC117, as they incompletely neutralize the clade C simian-human immunodeficiency virus (SHIV) stock (SHIV-327c) at 85% and 70%, respectively, and performed a protection study in rhesus macaques. The animals were intravenously (i.v.) administered PGT121 or 3BNC117 at 10 and 2 mg/kg of body weight before being rectally challenged with a single high dose of SHIV-327c. PGT121 protected 6 out of 7 monkeys, while 6 out of 7 3BNC117-pretreated animals became infected, although with significantly delayed plasma viremia compared to the control animals. These data suggest that complete neutralization is not imperative for bnAbs to prevent infection but that with increasing levels of incomplete neutralization the sterilizing activity diminishes.IMPORTANCE Multiple antibodies have been identified that potently neutralize a broad range of circulating HIV strains. However, not every virus-antibody combination results in complete neutralization of the input virus, suggesting that a fraction of virus particles are resistant to antibody neutralization despite high antibody concentrations. This observation of "incomplete neutralization" is associated with nonsigmoidal neutralization curves plateauing below 100% neutralization, but the significance of the phenomenon for the ability of neutralizing antibodies to mediate protective effects in vivo is undetermined. In this study, we show that the broadly neutralizing antibody PGT121, which neutralized only up to 85% of the SHIV-327c challenge stock in vitro, protected 6 out of 7 rhesus macaques against infection while the antibody 3BNC

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

    PubMed Central

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

    1992-01-01

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

  15. 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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Abraham, Michael H

    2014-07-01

    Analysis of percentage human intestinal absorption (%HIA) for 280 drugs shows that an excellent fit can be obtained using only three descriptors for neutral molecules with a SD of 13.9%. Use of descriptors for individual cations and anions does not lead to any better goodness-of-fit. It is noted that diffusion coefficients in water for ionized molecules are almost identical to those for the corresponding neutral molecules. Comparison of equation coefficients for HIA with those for other processes shows that HIA resembles diffusion in water but does not resemble permeation through biological bilayers. It is shown that compound substituent effects on HIA are near those for diffusion but are far away from substituent effects on permeation through a typical bilayer. Calculations indicate that rates of permeation through an unstirred mucosal layer are of the same order as experimental rates of permeation in HIA. It is concluded that for the 280 compound set, diffusion through the unstirred mucosal layer is the rate determining step. The effect on pK(a) in transfer of acids and bases from water to another solvent, and of diffusion past a negative charge in a phase/bilayer is also considered. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  18. Efficacy of Neutral Electrolyzed Water for Inactivation of Human Norovirus.

    PubMed

    Moorman, Eric; Montazeri, Naim; Jaykus, Lee-Ann

    2017-08-15

    Human norovirus (NoV) is the leading cause of acute gastroenteritis worldwide. Persistence on surfaces and resistance to many conventional disinfectants contribute to widespread transmission of norovirus. We examined the efficacy of neutral electrolyzed water (NEW; pH 7) for inactivation of human NoV GII.4 Sydney in suspension (ASTM method 1052-11) and on stainless steel surfaces (ASTM method 1053-11) with and without an additional soil load. The impact of the disinfectant on viral capsid was assessed using reverse transcriptase quantitative PCR (RT-qPCR; with an RNase pretreatment), SDS-PAGE, transmission electron microscopy, and a histo-blood group antigen (HBGA) receptor-binding assay. These studies were done in parallel with those using Tulane virus (TuV), a cultivable human NoV surrogate. Neutral electrolyzed water at 250 ppm free available chlorine produced a 4.8- and 0.4-log10 reduction in NoV genome copy number after 1 min in suspension and on stainless steel, respectively. Increasing the contact time on surfaces to 5, 10, 15, and 30 min reduced human NoV genomic copies by 0.5, 1.6, 2.4, and 5.0 log10 and TuV infectious titers by 2.4, 3.0, 3.8, and 4.1 log10 PFU, respectively. Increased soil load effectively eliminated antiviral efficacy regardless of testing method and virus. Exposure to NEW induced a near complete loss of receptor binding (5 ppm, 30 s), degradation of VP1 major capsid protein (250 ppm, 5 min), and increased virus particle aggregation (150 ppm, 30 min). Neutral electrolyzed water at 250 ppm shows promise as an antinoroviral disinfectant when used on precleaned stainless steel surfaces.IMPORTANCE Norovirus is the leading cause of acute viral gastroenteritis worldwide. Transmission occurs by fecal-oral or vomitus-oral routes. The persistence of norovirus on contaminated environmental surfaces exacerbates its spread, as does its resistance to many conventional disinfectants. The purpose of this research was to evaluate the antinoroviral

  19. 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

  20. Preclinical development of a humanized neutralizing antibody targeting HGF

    PubMed Central

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

    2017-01-01

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

  1. Acid ceramidase deficiency associated with spinal muscular atrophy with progressive myoclonic epilepsy.

    PubMed

    Gan, Joanna J; Garcia, Virginie; Tian, Jane; Tagliati, Michele; Parisi, Joseph E; Chung, Jeffrey M; Lewis, Richard; Baloh, Robert; Levade, Thierry; Pierson, Tyler Mark

    2015-12-01

    Spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) is an extremely rare disorder related to the lysosomal storage disease, Farber lipogranulomatosis. Both disorders are autosomal recessive conditions caused by mutations in the ASAH1 gene encoding acid ceramidase. Farber disease is associated with joint deformities, lipomatous skin nodules, and often is fatal by 2-3 years of age; while SMA-PME is characterized by childhood-onset motor neuron disease and progressive myoclonic epilepsy. We report a case of SMA-PME with a novel mutation in the ASAH1 gene encoding acid ceramidase. The proband presented with childhood-onset of diffuse muscle atrophy and hypotonia. He also had diffuse weakness with greater proximal than distal involvement. Tongue fasciculations were present and his reflexes were either diminished or absent. He ambulated with an unsteady and hesitant gait. He subsequently developed myoclonic epilepsy along with other associated features including tremor, polymyoclonus, and sensorineural hearing loss. Neurophysiological studies revealed a motor neuron disorder and generalized epilepsy. Exome sequencing analysis identified compound heterozygous variants and biochemical analysis indicated acid ceramidase activity was approximately 12 percent of normal controls. Our proband was phenotypically similar to other cases of SMA-PME, albeit with somewhat lesser severity, slower progression, and greater longevity. As lysosomal disorders are sometimes amendable to early interventions, it is important to make early diagnoses in these cases. The combination of motor neuron disease and progressive myoclonic epilepsy should prompt genetic evaluation of ASAH1. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Neutralizing human monoclonal antibodies prevent Zika virus infection in macaques.

    PubMed

    Magnani, Diogo M; Rogers, Thomas F; Beutler, Nathan; Ricciardi, Michael J; Bailey, Varian K; Gonzalez-Nieto, Lucas; Briney, Bryan; Sok, Devin; Le, Khoa; Strubel, Alexander; Gutman, Martin J; Pedreño-Lopez, Núria; Grubaugh, Nathan D; Silveira, Cassia G T; Maxwell, Helen S; Domingues, Aline; Martins, Mauricio A; Lee, David E; Okwuazi, Erica E; Jean, Sherrie; Strobert, Elizabeth A; Chahroudi, Ann; Silvestri, Guido; Vanderford, Thomas H; Kallas, Esper G; Desrosiers, Ronald C; Bonaldo, Myrna C; Whitehead, Stephen S; Burton, Dennis R; Watkins, David I

    2017-10-04

    Therapies to prevent maternal Zika virus (ZIKV) infection and its subsequent fetal developmental complications are urgently required. We isolated three potent ZIKV-neutralizing monoclonal antibodies (nmAbs) from the plasmablasts of a ZIKV-infected patient-SMZAb1, SMZAb2, and SMZAb5-directed against two different domains of the virus. We engineered these nmAbs with Fc LALA mutations that abrogate Fcγ receptor binding, thus eliminating potential therapy-mediated antibody-dependent enhancement. We administered a cocktail of these three nmAbs to nonhuman primates 1 day before challenge with ZIKV and demonstrated that the nmAbs completely prevented viremia in serum after challenge. Given that numerous antibodies have exceptional safety profiles in humans, the cocktail described here could be rapidly developed to protect uninfected pregnant women and their fetuses. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  3. Detection of human serum antibodies that neutralize infectious human papillomavirus type 11 virions.

    PubMed

    Christensen, N D; Kreider, J W; Shah, K V; Rando, R F

    1992-05-01

    A selection of human sera were tested for the presence of antibodies that neutralized infectious human papillomavirus (HPV) type 11. Neutralizing antibodies were detected by prevention of HPV-11-induced condylomatous transformation of human foreskin chips transplanted subrenally into athymic mice. Test sera were obtained from 21 female patients with genital condylomas and eight patients with laryngeal papillomas. Control patients consisted of 57 adult random blood donors and five asymptomatic children. ELISAs demonstrated that all sera from patients with genital papillomas were strongly reactive to disrupted papillomavirus (PV) antigens of HPV-11, bovine PV type 1 and cottontail rabbit PV, but only two were weakly reactive to intact HPV-11. None of the eight sera from the laryngeal papilloma bearers reacted significantly to disrupted PV antigens, but four of the eight showed strong specific responses to intact HPV-11 only. The majority of the sera that were reactive to intact HPV-11 by ELISA neutralized HPV-11 infectivity in the athymic mouse xenograft system. The data indicated that ELISA reactivity to intact HPV-11 virions was a good predictor for the presence of HPV-11 neutralizing antibodies.

  4. Acid Ceramidase Maintains the Chondrogenic Phenotype of Expanded Primary Chondrocytes and Improves the Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Ge, Yi; He, Xingxuan; DeAngelis, Victor A.; Eliyahu, Efrat; Leong, Daniel J.; Sun, Hui B.; Mason, Jeffrey B.; Haskins, Mark E.; Richardson, Dean W.; Schuchman, Edward H.

    2013-01-01

    Acid ceramidase is required to maintain the metabolic balance of several important bioactive lipids, including ceramide, sphingosine and sphingosine-1-phosphate. Here we show that addition of recombinant acid ceramidase (rAC) to primary chondrocyte culture media maintained low levels of ceramide and led to elevated sphingosine by 48 hours. Surprisingly, after three weeks of expansion the chondrogenic phenotype of these cells also was markedly improved, as assessed by a combination of histochemical staining (Alcian Blue and Safranin-O), western blotting (e.g., Sox9, aggrecan, collagen 2A1), and/or qPCR. The same effects were evident in rat, equine and human cells, and were observed in monolayer and 3-D cultures. rAC also reduced the number of apoptotic cells in some culture conditions, contributing to overall improved cell quality. In addition to these effects on primary chondrocytes, when rAC was added to freshly harvested rat, equine or feline bone marrow cultures an ∼2-fold enrichment of mesenchymal stem cells (MSCs) was observed by one week. rAC also improved the chondrogenic differentiation of MSCs, as revealed by histochemical and immunostaining. These latter effects were synergistic with TGF-beta1. Based on these results we propose that rAC could be used to improve the outcome of cell-based cartilage repair by maintaining the quality of the expanded cells, and also might be useful in vivo to induce endogenous cartilage repair in combination with other techniques. The results also suggest that short-term changes in sphingolipid metabolism may lead to longer-term effects on the chondrogenic phenotype. PMID:23638138

  5. A protective and broadly cross-neutralizing epitope of human papillomavirus L2.

    PubMed

    Gambhira, Ratish; Karanam, Balasubramanyam; Jagu, Subhashini; Roberts, Jeffrey N; Buck, Christopher B; Bossis, Ioannis; Alphs, Hannah; Culp, Timothy; Christensen, Neil D; Roden, Richard B S

    2007-12-01

    We generated a monoclonal antibody, RG-1, that binds to highly conserved L2 residues 17 to 36 and neutralizes human papillomavirus 16 (HPV16) and HPV18. Passive immunotherapy with RG-1 was protective in mice. Antiserum to the HPV16 L2 peptide comprising residues 17 to 36 (peptide 17-36) neutralized pseudoviruses HPV5, HPV6, HPV16, HPV 18, HPV31, HPV 45, HPV 52, HPV 58, bovine papillomavirus 1, and HPV11 native virions. Depletion of HPV16 L2 peptide 17-36-reactive antibodies from cross-neutralizing rabbit and human L2-specific sera abolished cross-neutralization and drastically reduced neutralization of the cognate type. This cross-neutralization of diverse HPVs associated with cervical cancer, genital warts, and epidermodysplasia verruciformis suggests the possibility of a broadly protective, peptide-based vaccine.

  6. 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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2015-11-01

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

  9. 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.

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

    PubMed Central

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

    1992-01-01

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

  11. Human immunodeficiency virus type 1 is trapped by acidic but not by neutralized human cervicovaginal mucus.

    PubMed

    Lai, Samuel K; Hida, Kaoru; Shukair, Shetha; Wang, Ying-Ying; Figueiredo, Anna; Cone, Richard; Hope, Thomas J; Hanes, Justin

    2009-11-01

    To reliably infect a primate model for human immunodeficiency virus (HIV), approximately 10,000-fold more virus must be delivered vaginally than intravenously. However, the vaginal mechanisms that help protect against HIV are poorly understood. Here, we report that human cervicovaginal mucus (CVM), obtained from donors with normal lactobacillus-dominated vaginal flora, efficiently traps HIV, causing it to diffuse more than 1,000-fold more slowly than it does in water. Lactobacilli acidify CVM to pH approximately 4 by continuously producing lactic acid. At this acidic pH, we found that lactic acid, but not HCl, abolished the negative surface charge on HIV without lysing the virus membrane. In contrast, in CVM neutralized to pH 6 to 7, as occurs when semen temporarily neutralizes the vagina, HIV maintained its native surface charge and diffused only 15-fold more slowly than it would in water. Thus, methods that can maintain both a high lactic acid content and acidity for CVM during coitus may contribute to both vaginal and penile protection by trapping HIV before it can reach target cells. Our results reveal that CVM likely plays an important but currently unappreciated role in decreasing the rate of HIV sexual transmission.

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

    PubMed

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

    1996-04-01

    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.

  13. Human Neutralizing Monoclonal Antibody Inhibition of Middle East Respiratory Syndrome Coronavirus Replication in the Common Marmoset.

    PubMed

    Chen, Zhe; Bao, Linlin; Chen, Cong; Zou, Tingting; Xue, Ying; Li, Fengdi; Lv, Qi; Gu, Songzhi; Gao, Xiaopan; Cui, Sheng; Wang, Jianmin; Qin, Chuan; Jin, Qi

    2017-06-15

    Middle East respiratory syndrome coronavirus (MERS-CoV) infection in humans is highly lethal, with a fatality rate of 35%. New prophylactic and therapeutic strategies to combat human infections are urgently needed. We isolated a fully human neutralizing antibody, MCA1, from a human survivor. The antibody recognizes the receptor-binding domain of MERS-CoV S glycoprotein and interferes with the interaction between viral S and the human cellular receptor human dipeptidyl peptidase 4 (DPP4). To our knowledge, this study is the first to report a human neutralizing monoclonal antibody that completely inhibits MERS-CoV replication in common marmosets. Monotherapy with MCA1 represents a potential alternative treatment for human infections with MERS-CoV worthy of evaluation in clinical settings. © Crown copyright 2017.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-05-22

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Cheshire, William P; Jones, Nancy L

    2005-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  1. 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.

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

    PubMed Central

    Ringe, Rajesh

    2013-01-01

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

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    2016-12-15

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

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

    PubMed

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

    2007-06-01

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

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

    PubMed Central

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

    2013-01-01

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

  7. Akt2 and acid ceramidase cooperate to induce cell invasion and resistance to apoptosis.

    PubMed

    Berndt, Norbert; Patel, Ronil; Yang, Hua; Balasis, Maria E; Sebti, Saïd M

    2013-07-01

    Both Akt 2 and acid ceramidase (ASAH1) are found aberrantly overexpressed in cancer cells, but whether these two enzymes cooperate to induce malignant transformation is not known. We found that in immortalized, non-transformed cells, ectopic co-expression of Akt2 and ASAH1 is significantly more effective than expression of each gene alone at inducing cell invasion and at conferring resistance to apoptosis. Consistent with these observations, siRNA-mediated depletion of both Akt2 and ASAH1 is much more potent than depleting each alone at inhibiting cell viability/proliferation and cell invasion. Furthermore, pharmacological inhibitors of Akt (TCN or MK-2206) and ASAH1 (B13) synergize to inhibit cell viability/proliferation, and combinations of these drugs are more effective than single-agent treatments at inhibiting cell invasion. Taken together, the results suggest that these two enzymes cooperate to induce malignant transformation and warrant further preclinical studies to evaluate the potential of combining inhibitors of Akt and ASAH1 to treat cancer.

  8. 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.

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

    PubMed Central

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

    2011-01-01

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

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

    ERIC Educational Resources Information Center

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

    2011-01-01

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

  11. 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

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

    PubMed

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

    2016-09-11

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

  13. Human Immunodeficiency Virus Type 1 Elite Neutralizers: Individuals with Broad and Potent Neutralizing Activity Identified by Using a High-Throughput Neutralization Assay together with an Analytical Selection Algorithm▿ †

    PubMed Central

    Simek, Melissa D.; Rida, Wasima; Priddy, Frances H.; Pung, Pham; Carrow, Emily; Laufer, Dagna S.; Lehrman, Jennifer K.; Boaz, Mark; Tarragona-Fiol, Tony; Miiro, George; Birungi, Josephine; Pozniak, Anton; McPhee, Dale A.; Manigart, Olivier; Karita, Etienne; Inwoley, André; Jaoko, Walter; DeHovitz, Jack; Bekker, Linda-Gail; Pitisuttithum, Punnee; Paris, Robert; Walker, Laura M.; Poignard, Pascal; Wrin, Terri; Fast, Patricia E.; Burton, Dennis R.; Koff, Wayne C.

    2009-01-01

    The development of a rapid and efficient system to identify human immunodeficiency virus type 1 (HIV-1)-infected individuals with broad and potent HIV-1-specific neutralizing antibody responses is an important step toward the discovery of critical neutralization targets for rational AIDS vaccine design. In this study, samples from HIV-1-infected volunteers from diverse epidemiological regions were screened for neutralization responses using pseudovirus panels composed of clades A, B, C, and D and circulating recombinant forms (CRFs). Initially, 463 serum and plasma samples from Australia, Rwanda, Uganda, the United Kingdom, and Zambia were screened to explore neutralization patterns and selection ranking algorithms. Samples were identified that neutralized representative isolates from at least four clade/CRF groups with titers above prespecified thresholds and ranked based on a weighted average of their log-transformed neutralization titers. Linear regression methods selected a five-pseudovirus subset, representing clades A, B, and C and one CRF01_AE, that could identify top-ranking samples with 50% inhibitory concentration (IC50) neutralization titers of ≥100 to multiple isolates within at least four clade groups. This reduced panel was then used to screen 1,234 new samples from the Ivory Coast, Kenya, South Africa, Thailand, and the United States, and 1% were identified as elite neutralizers. Elite activity is defined as the ability to neutralize, on average, more than one pseudovirus at an IC50 titer of 300 within a clade group and across at least four clade groups. These elite neutralizers provide promising starting material for the isolation of broadly neutralizing monoclonal antibodies to assist in HIV-1 vaccine design. PMID:19439467

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

    PubMed Central

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

    1988-01-01

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

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

    PubMed

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

    2012-10-01

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

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

    PubMed

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

    2016-05-10

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

  17. A human inferred germline antibody binds to an immunodominant epitope and neutralizes Zika virus

    PubMed Central

    Ricciardi, Michael J.; Pedreño-Lopez, Núria; Gutman, Martin J.; Bailey, Varian K.; Maxwell, Helen S.; Domingues, Aline; Gonzalez-Nieto, Lucas; Avelino-Silva, Vivian I.; Trindade, Mateus; Nogueira, Juliana; Oliveira, Consuelo S.; Maestri, Alvino; Felix, Alvina Clara; Levi, José Eduardo; Nogueira, Mauricio L.; Martins, Mauricio A.; Martinez-Navio, José M.; Fuchs, Sebastian P.; Whitehead, Stephen S.; Burton, Dennis R.; Desrosiers, Ronald C.; Kallas, Esper G.; Watkins, David I.

    2017-01-01

    The isolation of neutralizing monoclonal antibodies (nmAbs) against the Zika virus (ZIKV) might lead to novel preventative strategies for infections in at-risk individuals, primarily pregnant women. Here we describe the characterization of human mAbs from the plasmablasts of an acutely infected patient. One of the 18 mAbs had the unusual feature of binding to and neutralizing ZIKV despite not appearing to have been diversified by affinity maturation. This mAb neutralized ZIKV (Neut50 ~ 2 μg/ml) but did not react with any of the four dengue virus serotypes. Except for the expected junctional diversity created by the joining of the V-(D)-J genes, there was no deviation from immunoglobulin germline genes. This is a rare example of a human mAb with neutralizing activity in the absence of detectable somatic hypermutation. Importantly, binding of this mAb to ZIKV was specifically inhibited by human plasma from ZIKV-exposed individuals, suggesting that it may be of value in a diagnostic setting. PMID:28604797

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2003-06-01

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

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

    PubMed Central

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

    2003-01-01

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

  1. 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

  2. Acid ceramidase treatment enhances the outcome of autologous chondrocyte implantation in a rat osteochondral defect model.

    PubMed

    Frohbergh, M E; Guevara, J M; Grelsamer, R P; Barbe, M F; He, X; Simonaro, C M; Schuchman, E H

    2016-04-01

    The overall aim of this study was to evaluate how supplementation of chondrocyte media with recombinant acid ceramidase (rhAC) influenced cartilage repair in a rat osteochondral defect model. Primary chondrocytes were grown as monolayers in polystyrene culture dishes with and without rhAC (added once at the time of cell plating) for 7 days, and then seeded onto Bio-Gide® collagen scaffolds and grown for an additional 3 days. The scaffolds were then introduced into osteochondral defects created in Sprague-Dawley rat trochlea by a microdrilling procedure. Analysis was performed 6 weeks post-surgery macroscopically, by micro-CT, histologically, and by immunohistochemistry. Treatment with rhAC led to increased cell numbers and glycosaminoglycan (GAG) production (∼2 and 3-fold, respectively) following 7 days of expansion in vitro. Gene expression of collagen 2, aggrecan and Sox-9 also was significantly elevated. After seeding onto Bio-Gide®, more rhAC treated cells were evident within 4 h. At 6 weeks post-surgery, defects containing rhAC-treated cells exhibited more soft tissue formation at the articular surface, as evidenced by microCT, as well as histological evidence of enhanced cartilage repair. Notably, collagen 2 immunostaining revealed greater surface expression in animals receiving rhAC treated cells as well. Collagen 10 staining was not enhanced. The results further demonstrate the positive effects of rhAC treatment on chondrocyte growth and phenotype in vitro, and reveal for the first time the in vivo effects of the treated cells on cartilage repair. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

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

    PubMed Central

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

    2015-01-01

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

  4. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

    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

    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. 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. 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.

  7. 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

  8. 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

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

    PubMed

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

    2009-11-01

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

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

    PubMed Central

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

    2012-01-01

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

  11. Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice.

    PubMed

    Scallan, Ciaran D; Jiang, Haiyan; Liu, Tongyao; Patarroyo-White, Susannah; Sommer, Jurg M; Zhou, Shangzhen; Couto, Linda B; Pierce, Glenn F

    2006-03-01

    Long-term cures of hemophilia B have been achieved using AAV2 delivering the factor IX gene to the liver of adeno-associated virus (AAV)-naive hemophilic animals. However, the clinical success of this approach requires overcoming pre-existing AAV neutralizing antibodies prevalent in humans. To better define the inhibition of neutralizing antibodies on AAV2-mediated liver transduction, we developed an in vivo passive immunity model. SCID mice were first reconstituted to a defined neutralizing titer with pooled plasma-derived human immunoglobulin. AAV2-FIX vectors then were administered to the liver, and the transduction efficiency was measured by plasma FIX levels. Unexpectedly, AAV2 neutralizing titers lower than 1:10 were sufficient to neutralize 4 to 20 x 10(12) vg/kg of AAV2 vectors in vivo, a capacity that was underestimated by in vitro neutralizing assays. We also evaluated strategies to evade neutralization, including the use of alternative delivery routes, infusion parameters, empty capsids, and alternative AAV serotypes 6 and 8. The results indicate that low AAV2 neutralizing titers can be inhibitory to the tested human and primate AAV vectors delivered into the circulatory system. Therefore, novel nonprimate AAV vectors or compartmentalized delivery may offer more consistent therapeutic effects in the presence of pre-existing AAV neutralizing antibodies.

  12. 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

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

    PubMed

    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

    2011-08-23

    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.

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

    PubMed Central

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

    2015-01-01

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

  15. 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.

  16. 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

  17. 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

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

    PubMed

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

    2015-09-14

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2017-03-01

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

  1. 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.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-12-14

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

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

    PubMed

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

    2003-12-19

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

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

    PubMed

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

    2012-06-01

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

  6. Comparison of Different Cell Substrates on the Measurement of Human Influenza Virus Neutralizing Antibodies

    PubMed Central

    Zhai, Weiguo; Zhang, Dan Ning; Mai, Cecilia; Choy, Justin; Jian, Gary; Sra, Kuldip; Galinski, Mark S

    2012-01-01

    Eight cell lines were systematically compared for their permissivity to primary infection, replication, and spread of seven human influenza viruses. Cell lines were of human origin (Caco-2, A549, HEp-2, and NCI-H292), monkey (Vero, LLC-MK2), mink (Mv1 Lu), and canine (MDCK). The influenza viruses included seasonal types and subtypes and a pandemic virus. The MDCK, Caco-2, and Mv1 Lu cells were subsequently compared for their capacity to report neutralization titers at day one, three and six post-infection. A gradient of sensitivity to primary infection across the eight cell lines was observed. Relative to MDCK cells, Mv1 Lu reported higher titers and the remaining six cell lines reported lower titers. The replication and spread of the seven influenza viruses in the eight cell substrates was determined using hemagglutinin expression, cytopathic effect, and neuraminidase activity. Virus growth was generally concordant with primary infection, with a gradient in virus replication and spread. However, Mv1 Lu cells poorly supported virus growth, despite a higher sensitivity to primary infection. Comparison of MDCK, Caco-2, and Mv1 Lu in neutralization assays using defined animal antiserum confirmed MDCK cells as the preferred cell substrate for influenza virus testing. The results observed for neutralization at one day post-infection showed MDCK cells were similar (<1 log2 lower) or superior (>1 log2 higher) for all seven viruses. Relative to Caco-2 and Mv1 Lu cells, MDCK generally reported the highest titers at three and six days post-infection for the type A viruses and lower titers for the type B viruses and the pandemic H9N2 virus. The reduction in B virus titer was attributed to the complete growth of type B viruses in MDCK cells before day three post-infection, resulting in the systematic underestimation of neutralization titers. This phenomenon was also observed with Caco-2 cells. PMID:23284988

  7. 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

  8. 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

  9. 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.

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

    PubMed Central

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

    2009-01-01

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

  11. Natural Selection Affects Multiple Aspects of Genetic Variation at Putatively Neutral Sites across the Human Genome

    PubMed Central

    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

    2011-01-01

    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. PMID:22022285

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

    PubMed

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

    2013-09-01

    Infection with human cytomegalovirus (CMV) during pregnancy is the most common cause of congenital disorders, and can lead to severe life-long disabilities with associated high cost of care. Since there is no vaccine or effective treatment, current efforts are focused on identifying potent neutralizing antibodies. A panel of CMV monoclonal antibodies identified from patent applications, was synthesized and expressed in order to reproduce data from the literature showing that anti-glycoprotein B antibodies neutralized virus entry into all cell types and that anti-pentameric complex antibodies are highly potent in preventing virus entry into epithelial cells. It had not been established whether antibodies could prevent subsequent rounds of infection that are mediated primarily by direct cell-to-cell transmission. A thorough validation of a plaque reduction assay to monitor cell-to-cell spread led to the conclusion that neutralizing antibodies do not significantly inhibit plaque formation or reduce plaque size when they are added post-infection. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2009-03-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2012-04-17

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

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

    SciTech Connect

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

    1982-12-01

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

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

    SciTech Connect

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

    2016-11-02

    ABSTRACT

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

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

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

    PubMed

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

    2016-12-29

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

  19. 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.

  20. Cross-Neutralizing and Protective Human Antibody Specificities to Poxvirus Infections.

    PubMed

    Gilchuk, Iuliia; Gilchuk, Pavlo; Sapparapu, Gopal; Lampley, Rebecca; Singh, Vidisha; Kose, Nurgun; Blum, David L; Hughes, Laura J; Satheshkumar, Panayampalli S; Townsend, Michael B; Kondas, Ashley V; Reed, Zachary; Weiner, Zachary; Olson, Victoria A; Hammarlund, Erika; Raue, Hans-Peter; Slifka, Mark K; Slaughter, James C; Graham, Barney S; Edwards, Kathryn M; Eisenberg, Roselyn J; Cohen, Gary H; Joyce, Sebastian; Crowe, James E

    2016-10-20

    Monkeypox (MPXV) and cowpox (CPXV) are emerging agents that cause severe human infections on an intermittent basis, and variola virus (VARV) has potential for use as an agent of bioterror. Vaccinia immune globulin (VIG) has been used therapeutically to treat severe orthopoxvirus infections but is in short supply. We generated a large panel of orthopoxvirus-specific human monoclonal antibodies (Abs) from immune subjects to investigate the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV, and VARV and that are determinants of protection in murine challenge models. Optimal protection following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus. This work reveals orthopoxvirus targets for human Abs that mediate cross-protective immunity and identifies new candidate Ab therapeutic mixtures to replace VIG. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Capillary electrophoresis separation of human milk neutral and acidic oligosaccharides derivatized with 2-aminoacridone.

    PubMed

    Galeotti, Fabio; Coppa, Giovanni V; Zampini, Lucia; Maccari, Francesca; Galeazzi, Tiziana; Padella, Lucia; Santoro, Lucia; Gabrielli, Orazio; Volpi, Nicola

    2014-03-01

    Human milk is a unique fluid in glycobiology due to the presence of many free structurally complex oligosaccharides emerging as important dietary factors during early life and having many biological and protective functions. Methods that allow accurate profiling of oligosaccharide mixtures in this complex biological fluid with quantification of the four known genetically determined groups are welcomed. A high-voltage CE separation and detection at 254 nm of 17 neutral and acidic human milk oligosaccharide (HMO) standard along with lactose derivatized with 2-aminoacridone, using a BGE containing 20% methanol as an organic modifier and borate, able to form on-capillary anionic borate-polyol complexes, is reported. This CE approach was able to separate both neutral HMOs and acidic HMOs, with the sialic acid residue, also in the presence of lactose in high content. This method was applied to the four secretory groups individually extracted by a rapid and simple preparative step. LODs were found ranging from ∼50 to 700 fmol. We were able to measure HMO content also in the presence of excess fluorophore, or interference from proteins, peptides, salts, and other impurities normally present in this complex biological fluid. Overall, CE equipped with a UV detector is a common analytical approach and this simple CE separation offers high resolution and sensitivity for the differentiation of human milk samples related to genetic groups and days of lactation by considering that important changes in HMO content are a reflection of the lactation day.

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

    PubMed

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

    2017-01-30

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

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

    PubMed Central

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

    2015-01-01

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

  4. 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

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

    PubMed

    WINSSER, J; SABIN, A B

    1952-11-01

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

  6. 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

  7. 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.

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

    PubMed Central

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

    1997-01-01

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

  9. 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

  10. 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.

  11. Neutral amino acid transport in human synovial cells: substrate specificity of adaptative regulation and transinhibition.

    PubMed

    Aussel, C; Rousseau-Loric, S; Cynober, L; Agneray, J; Ekindjian, O G

    1989-10-01

    Neutral amino acid transport was characterized in human synovial cells. The amino acids tested are transported by all three major neutral amino acid transport systems, that is, A, L, and ASC. The model amino acid 2-aminoisobutyric acid (AIB) was found to be a strong specific substrate for system A in synovial cells. When cells were starved of amino acids, the activity of AIB transport increased, reaching a maximum within 1 h. The stimulation of transport activity was not blocked by cycloheximide and would thus appear to be related to a release from transinhibition. Similarly, the decrease in the activity of AIB transport observed after the addition of alpha-methyl-aminoisobutyric acid (meAIB) appeared to be related to transinhibition. However, using a different approach, that is, amino acid starvation followed by incubation with 10 mM meAIB and transfer to an amino acid-free medium with or without cycloheximide supplementation, a clear increase in AIB uptake, due both to derepression and a release from transinhibition, was observed. Unlike human fibroblasts, the depression of system A in these synovial cells was not serum-dependent. The process of derepression was observed only after preloading with meAIB. Neither AIB nor alanine produced this phenomenon. Moreover, alanine preloading led to a large increase in AIB transport activity due to a release from transinhibition. These observations indicate that the process of derepression and release from transinhibition are specific to the substrates present in the culture medium prior to amino acid starvation.

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

    PubMed Central

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

    2017-01-01

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

  14. 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

  15. 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

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  18. Increased Aggregation Is More Frequently Associated to Human Disease-Associated Mutations Than to Neutral Polymorphisms

    PubMed Central

    De Baets, Greet; Van Doorn, Loic; Rousseau, Frederic; Schymkowitz, Joost

    2015-01-01

    Protein aggregation is a hallmark of over 30 human pathologies. In these diseases, the aggregation of one or a few specific proteins is often toxic, leading to cellular degeneration and/or organ disruption in addition to the loss-of-function resulting from protein misfolding. Although the pathophysiological consequences of these diseases are overt, the molecular dysregulations leading to aggregate toxicity are still unclear and appear to be diverse and multifactorial. The molecular mechanisms of protein aggregation and therefore the biophysical parameters favoring protein aggregation are better understood. Here we perform an in silico survey of the impact of human sequence variation on the aggregation propensity of human proteins. We find that disease-associated variations are statistically significantly enriched in mutations that increase the aggregation potential of human proteins when compared to neutral sequence variations. These findings suggest that protein aggregation might have a broader impact on human disease than generally assumed and that beyond loss-of-function, the aggregation of mutant proteins involved in cancer, immune disorders or inflammation could potentially further contribute to disease by additional burden on cellular protein homeostasis. PMID:26340370

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  1. Human semen cryopreservation: a sperm DNA fragmentation study with alkaline and neutral Comet assay.

    PubMed

    Ribas-Maynou, J; Fernández-Encinas, A; García-Peiró, A; Prada, E; Abad, C; Amengual, M J; Navarro, J; Benet, J

    2014-01-01

    Sperm cryopreservation is widely used for both research and reproduction purposes, but its effect on sperm DNA damage remains controversial. Sperm DNA fragmentation (SDF) has become an important biomarker to assess male infertility. In particular, the differentiation between single- and double-stranded DNA fragmentation (ssSDF and dsSDF) has clinical implications for male infertility where ssSDF is associated with reduced fertility, whereas dsSDF is associated with increased risk of miscarriage. In this study, semen samples from 30 human males have been analysed in both fresh and cryopreserved using the alkaline and neutral Comet assays. Results show an increase of about 10% of ssSDF, assessed by the alkaline Comet assay, regardless of the male fertility status. Neutral Comet analysis of dsSDF does not show any statistical increase when comparing fresh and cryopreserved samples in any of the patient groups. Results support previous reports that oxidative stress is the major effector in DNA damage during sample cryopreservation, as, on one hand, ssSDF has previously been related to oxidative damage and, on the other hand, we have not found any effect on dsSDF. Therefore, there might be a slight risk of decreased fertility after using a freezed sample, but no evidence for increased miscarriage risk from cryopreserved spermatozoa should be expected. © 2013 American Society of Andrology and European Academy of Andrology.

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

    PubMed

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

    2012-05-02

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

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

    PubMed

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

    2012-01-01

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

  4. 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

  5. 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.

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2013-09-03

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

  8. HIV-1 Cross-reactive Primary Virus Neutralizing Antibody Response Elicited by Immunization in Non-human Primate.

    PubMed

    Wang, Yimeng; O'Dell, Sijy; Turner, Hannah L; Chiang, Chi-I; Lei, Lin; Guenaga, Javier; Wilson, Richard; Martinez-Murillo, Paola; Doria-Rose, Nicole; Ward, Andrew B; Mascola, John R; Wyatt, Richard T; Karlsson Hedestam, Gunilla B; Li, Yuxing

    2017-08-23

    Elicitation of broadly neutralizing antibody (bNAb) responses is a major goal for the development of an HIV-1 vaccine. Current HIV-1 envelope glycoprotein (Env) vaccine candidates predominantly elicit tier 1 and/or autologous tier 2 virus neutralizing antibody (NAb) responses, as well as weak and/or sporadic cross-reactive tier 2 virus NAb responses with unknown specificity. To delineate the specificity of vaccine-elicited cross-reactive tier 2 virus NAb responses, we performed single memory B cell sorting from the peripheral blood of a rhesus macaque immunized with YU2gp140-F trimers in adjuvant, using JR-FL SOSIP.664, a native Env trimer mimetic as sorting probe to isolate monoclonal Abs (MAbs). We found striking genetic and functional convergence of the SOSIP-sorted Ig repertoire, with pre-dominant VH4 or VH5 gene family usage and Env V3-specificity. Of these vaccine-elicited V3-specific mAbs, nearly 20% (6/33) displayed cross-reactive tier 2 virus neutralization, which recapitulated the serum neutralization capacity. Substantial similarities in binding specificity, neutralization breadth and potency, and sequence/structural homology were observed between selected macaque cross-reactive V3 NAbs elicited by vaccination and prototypic V3 NAbs derived from natural infections in humans, highlighting the convergence of this subset of primate V3-specific B cell repertories. Our study demonstrated that cross-reactive primary virus neutralizing B cell lineages could be elicited by vaccination as detected using a standardized panel of tier 2 viruses. Whether these lineages could be expanded to acquire increased breadth and potency of neutralization merits further investigation.IMPORTANCE Elicitation of antibody responses capable of neutralizing diverse HIV-1 primary virus isolates (designated as broadly neutralizing antibodies, bNAbs) remains a high priority for the vaccine field. bNAb responses were so far only observed in response to natural infection and only in a

  9. 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

  10. Characterization of neutralizing epitopes within the major capsid protein of human papillomavirus type 33

    PubMed Central

    Roth, Stefanie D; Sapp, Martin; Streeck, Rolf E; Selinka, Hans-Christoph

    2006-01-01

    Background Infections with papillomaviruses induce type-specific immune responses, mainly directed against the major capsid protein, L1. Based on the propensity of the L1 protein to self-assemble into virus-like particles (VLPs), type-specific vaccines have already been developed. In order to generate vaccines that target a broader spectrum of HPV types, extended knowledge of neutralizing epitopes is required. Despite the association of human papillomavirus type 33 (HPV33) with cervical carcinomas, fine mapping of neutralizing conformational epitopes on HPV33 has not been reported yet. By loop swapping between HPV33 and HPV16 capsid proteins, we have identified amino acid sequences critical for the binding of conformation-dependent type-specific neutralizing antibodies to surface-exposed hyper variable loops of HPV33 capsid protein L1. Results Reactivities of monoclonal antibodies (mAbs) H33.B6, H33.E12, H33.J3 and H16.56E with HPV16:33 and HPV33:16 hybrid L1 VLPs revealed the complex structures of their conformational epitopes as well as the major residues contributing to their binding sites. Whereas the epitope of mAb H33.J3 was determined by amino acids (aa) 51–58 in the BC loop of HPV33 L1, sequences of at least two hyper variable loops, DE (aa 132–140) and FGb (aa 282–291), were found to be essential for binding of H33.B6. The epitope of H33.E12 was even more complex, requiring sequences of the FGa loop (aa 260–270), in addition to loops DE and FGb. Conclusion These data demonstrate that neutralizing epitopes in HPV33 L1 are mainly located on the tip of the capsomere and that several hyper variable loops contribute to form these conformational epitopes. Knowledge of the antigenic structure of HPV is crucial for designing hybrid particles as a basis for intertypic HPV vaccines. PMID:17014700

  11. Characterization of a Major Neutralizing Epitope on Human Papillomavirus Type 16 L1

    PubMed Central

    White, Wendy I.; Wilson, Susan D.; Palmer-Hill, Frances J.; Woods, Robert M.; Ghim, Shin-je; Hewitt, Lisa A.; Goldman, Daniel M.; Burke, Steven J.; Jenson, A. Bennett; Koenig, Scott; Suzich, JoAnn A.

    1999-01-01

    Persistent infection with human papillomavirus type 16 (HPV-16) is strongly associated with the development of cervical cancer. Neutralizing epitopes present on the major coat protein, L1, have not been well characterized, although three neutralizing monoclonal antibodies (MAbs) had been identified by using HPV-16 pseudovirions (R. B. Roden et al., J. Virol. 71:6247–6252, 1997). Here, two of these MAbs (H16.V5 and H16.E70) were demonstrated to neutralize authentic HPV-16 in vitro, while the third (H16.U4) did not. Binding studies were conducted with the three MAbs and virus-like particles (VLPs) composed of the reference L1 sequence (114K) and three variant L1 sequences: Rochester-1k (derived from viral stock DNA), GU-1 (derived from cervical biopsy DNA), and GU-2 (derived from biopsy DNA, but containing some sequence changes likely to be artifactual). While all three MAbs bound to 114K and Rochester-1k VLPs, GU-1 VLPs were not recognized by H16.E70, and both H16.E70 and H16.V5 failed to bind to GU-2 VLPs. Site-directed mutagenesis was used to replace disparate amino acids in the GU-2 L1 with those found in the 114K L1. Alteration of the amino acid at position 50, from L to F, completely restored H16.V5 binding and partially restored H16.E70 binding, while complete restoration of H16.E70 binding occurred with GU-2 VLPs containing both L50F and T266A alterations. Immunization of mice with L1 variant VLPs revealed that GU-2 VLPs were poorly immunogenic. The L50F mutant of GU-2 L1, in which the H16.V5 epitope was restored, elicited HPV-16 antibody responses comparable to those obtained with 114K VLPs. These results demonstrate the importance of the H16.V5 epitope in the generation of potent HPV-16 neutralizing antibody responses. PMID:10233949

  12. 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

  13. De novo sequencing and resurrection of a human astrovirus-neutralizing antibody

    DOE PAGES

    Bogdanoff, Walter A.; Morgenstern, David; Bern, Marshall; ...

    2016-03-14

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

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

    PubMed Central

    2016-01-01

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

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

    PubMed

    Bogdanoff, Walter A; Morgenstern, David; Bern, Marshall; Ueberheide, Beatrix M; Sanchez-Fauquier, Alicia; DuBois, Rebecca M

    2016-05-13

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

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

    PubMed

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

    1997-03-01

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

  17. 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.

  18. Structure of IL-17A in complex with a potent, fully human neutralizing antibody.

    PubMed

    Gerhardt, Stefan; Abbott, W Mark; Hargreaves, David; Pauptit, Richard A; Davies, Rick A; Needham, Maurice R C; Langham, Caroline; Barker, Wendy; Aziz, Azad; Snow, Melanie J; Dawson, Sarah; Welsh, Fraser; Wilkinson, Trevor; Vaugan, Tris; Beste, Gerald; Bishop, Sarah; Popovic, Bojana; Rees, Gareth; Sleeman, Matthew; Tuske, Steven J; Coales, Stephen J; Hamuro, Yoshitomo; Russell, Caroline

    2009-12-18

    IL-17A is a pro-inflammatory cytokine produced by the newly identified Th17 subset of T-cells. We have isolated a human monoclonal antibody to IL-17A (CAT-2200) that can potently neutralize the effects of recombinant and native human IL-17A. We determined the crystal structure of IL-17A in complex with the CAT-2200 Fab at 2.6 A resolution in order to provide a definitive characterization of the epitope and paratope regions. Approximately a third of the IL-17A dimer is disordered in this crystal structure. The disorder occurs in both independent copies of the complex in the asymmetric unit and does not appear to be influenced by crystal packing. The complex contains one IL-17A dimer sandwiched between two CAT-2200 Fab fragments. The IL-17A is a disulfide-linked homodimer that is similar in structure to IL-17F, adopting a cystine-knot fold. The structure is not inconsistent with the previous prediction of a receptor binding cavity on IL-17 family members. The epitope recognized by CAT-2200 is shown to involve 12 amino acid residues from the quaternary structure of IL-17A, with each Fab contacting both monomers in the dimer. All complementarity-determining regions (CDRs) in the Fab contribute to a total of 16 amino acid residues in the antibody paratope. In vitro affinity optimization was used to generate CAT-2200 from a parental lead antibody using random mutagenesis of CDR3 loops. This resulted in seven amino acid changes (three in VL-CDR3 and four in VH-CDR3) and gave an approximate 30-fold increase in potency in a cell-based neutralization assay. Two of the seven amino acids form part of the CAT-2200 paratope. The observed interaction site between CAT-2200 and IL-17A is consistent with data from hydrogen/deuterium exchange mass spectrometry and mutagenesis approaches.

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

    PubMed

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

    2017-04-15

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

  20. Evidence for non-V3-specific neutralizing antibodies that interfere with gp120/CD4 binding in human immunodeficiency virus 1-infected humans.

    PubMed Central

    Kang, C Y; Nara, P; Chamat, S; Caralli, V; Ryskamp, T; Haigwood, N; Newman, R; Köhler, H

    1991-01-01

    Total anti-gp120 antibodies (total anti-gp120 Abs) were purified from a pool of four human immunodeficiency virus-positive (HIV+) sera by affinity chromatography on a gp120SF2-Sepharose column and exhibited both type- and group-specific neutralizing activities. To dissect the epitope specificity of the group-specific neutralizing antibodies, CD4 attachment site-specific antibodies (CD4-site Abs) were isolated from total anti-gp120 Abs by using a CD4-blocked gp120SF2-Sepharose column. The CD4-site Abs exhibited group-specific neutralizing activities. Another approach to dissecting type- and group-specific neutralizing activities of total anti-gp120 Abs was to separate the third variable region (V3)-specific antibodies (V3-region Abs) from non-V3-region-specific antibodies (non-V3 Abs). The results indicated that V3-region Abs exhibited type-specific neutralizing activities, whereas non-V3 Abs exhibited group-specific neutralizing activities. By comparing the neutralizing activities of V3-region Abs to those of non-V3 Abs, we concluded that V3-region Abs are more effective than non-V3 Abs in neutralizing a specific HIV isolate. Collectively, this study indicates that group-specific neutralizing anti-gp120 antibodies are specific for the CD4 attachment site. PMID:2068099

  1. Evidence for non-V3-specific neutralizing antibodies that interfere with gp120/CD4 binding in human immunodeficiency virus 1-infected humans.

    PubMed

    Kang, C Y; Nara, P; Chamat, S; Caralli, V; Ryskamp, T; Haigwood, N; Newman, R; Köhler, H

    1991-07-15

    Total anti-gp120 antibodies (total anti-gp120 Abs) were purified from a pool of four human immunodeficiency virus-positive (HIV+) sera by affinity chromatography on a gp120SF2-Sepharose column and exhibited both type- and group-specific neutralizing activities. To dissect the epitope specificity of the group-specific neutralizing antibodies, CD4 attachment site-specific antibodies (CD4-site Abs) were isolated from total anti-gp120 Abs by using a CD4-blocked gp120SF2-Sepharose column. The CD4-site Abs exhibited group-specific neutralizing activities. Another approach to dissecting type- and group-specific neutralizing activities of total anti-gp120 Abs was to separate the third variable region (V3)-specific antibodies (V3-region Abs) from non-V3-region-specific antibodies (non-V3 Abs). The results indicated that V3-region Abs exhibited type-specific neutralizing activities, whereas non-V3 Abs exhibited group-specific neutralizing activities. By comparing the neutralizing activities of V3-region Abs to those of non-V3 Abs, we concluded that V3-region Abs are more effective than non-V3 Abs in neutralizing a specific HIV isolate. Collectively, this study indicates that group-specific neutralizing anti-gp120 antibodies are specific for the CD4 attachment site.

  2. 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.

  3. 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

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

    PubMed

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

    2014-08-01

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

  5. Neutralization of human papillomavirus by specific nanobodies against major capsid protein L1.

    PubMed

    Minaeian, Sara; Rahbarizadeh, Fatemeh; Zarkesh-Esfahani, Sayyed Hamid; Ahmadvand, Davoud; Broom, Oliver Jay

    2012-05-01

    The human papillomavirus (HPV) is the main cause of cervical cancer in developing countries. Rapid diagnosis and initiation of treatment of the HPV infection are critical. Various methods have been employed to reduce the immunogenicity of antibodies targeting HPV serotypes. Nanobodies are the smallest fragments of naturally occurring single-domain antibodies with their antigenbinding site compromised into a single domain. Nanobodies have remarkable properties such as high stability, solubility, and high homology to the human VH3 domain. In this study, a phagemid library was employed to enrich for nanobodies against the L1 protein of the human papilloma virus. Binding reactivity of the selected clones was evaluated using phage enzyme-linked immunosorbent assay (phage-ELISA). Finally, two nanobodies (sm5 and sm8) with the best reactivity against the Gardasil vaccine and the purified HPV-16 L1 protein were expressed and purified using a Ni(+)-NTA column. The accuracy of expression and purification of the nanobodies was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting assays. In vitro studies demonstrated that neutralization was achieved by the selected nanobodies. The ease of generation and unique features of these molecules make nanobodies promising molecules for the new generation of HPV diagnosis and therapy.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-06-25

    HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned. However, recently developed single-cell-based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies to HIV-1 (refs 4, 5). These antibodies can prevent infection and suppress viraemia in humanized mice and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favourable pharmacokinetics. A single 30 mg kg(-1) infusion of 3BNC117 reduced the viral load in HIV-1-infected individuals by 0.8-2.5 log10 and viraemia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that, as a single agent, 3BNC117 is safe and effective in reducing HIV-1 viraemia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy and cure.

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

    PubMed Central

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

    2014-01-01

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

  9. 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

  10. Human Immune Response to Botulinum Pentavalent (ABCDE) Toxoid, Determined by a Neutralization Test and by an ELISA

    DTIC Science & Technology

    1988-05-04

    optimum concentrations of the neurotoxins and the reference positive standard (human botulism immune globulin ) for use in the ELISA were determined...botulism immune globulin as a reference standard, and normal human serum (Fisher Scientific Co., Orangeburg, N.Y.) as a negative control. All test sera were...human botulism immune globulin as the standard in the ELISA, because the W.H.O. Standard Antitoxin, used in the neutralization test, was produced in

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

    PubMed Central

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

    1979-01-01

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

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

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

    1996-01-01

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

  14. A neutralizing human monoclonal antibody protects African Green monkeys from Hendra virus challenge

    PubMed Central

    Bossart, Katharine N.; Geisbert, Thomas W.; Feldmann, Heinz; Zhu, Zhongyu; Feldmann, Friederike; Geisbert, Joan B.; Yan, Lianying; Feng, Yan-Ru; Brining, Doug; Scott, Dana; Wang, Yanping; Dimitrov, Antony S.; Callison, Julie; Chan, Yee-Peng; Hickey, Andrew C.; Dimitrov, Dimiter S.; Broder, Christopher C.; Rockx, Barry

    2012-01-01

    Hendra virus (HeV) is a recently emerged zoonotic paramyxovirus that can cause a severe and often fatal disease in horses and humans. HeV is categorized as a biosafety level 4 agent, which has made the development of animal models and testing of potential therapeutics and vaccines challenging. Infection of African Green monkeys (AGMs) with HeV was recently demonstrated and disease mirrored fatal HeV infection in humans, manifesting as a multisystemic vasculitis with widespread virus replication in vascular tissues and severe pathologic manifestations in the lung, spleen and brain. Here, we demonstrate that m102.4, a potent HeV neutralizing human monoclonal antibody (hmAb), can protect AGMs from disease post infection (p.i.) with HeV. Fourteen AGMs were challenged intratracheally with a lethal dose of HeV and twelve subjects were infused twice with a 100 mg dose of m102.4 beginning at either 10 hr, 24 hr or 72 hr p.i. and again approximately 48 hrs later. The presence of viral RNA, infectious virus and HeV-specific immune responses demonstrated that all subjects were infected following challenge. All twelve AGMs that received m102.4 survived infection; whereas the untreated control subjects succumbed to disease on day 8 p.i.. Animals in the 72 hr treatment group exhibited neurological signs of disease but all animals started to recover by day 16 p.i.. These results represent successful post-exposure in vivo efficacy by an investigational drug against HeV and highlight the potential impact a hmAb can have on human disease. PMID:22013123

  15. 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. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Viscum album neutralizes tumor-induced immunosuppression in a human in vitro cell model

    PubMed Central

    Steinborn, Carmen; Klemd, Amy Marisa; Sauer, Barbara; Garcia-Käufer, Manuel; Urech, Konrad; Follo, Marie; Ücker, Annekathrin; Kienle, Gunver Sophia; Huber, Roman

    2017-01-01

    Tumor cells have the capacity to secrete immunosuppressive substances in order to diminish dendritic cell (DC) activity and thereby escape from immune responses. The impact of mistletoe (Viscum album) extracts (VAE), which are frequently used as an additive anti-cancer therapy to stimulate the immune response, is still unknown. Using a human cellular system, the impact of two different VAE (VAEA + VAEI) on the maturation of human dendritic cells and on T cell function has been investigated using flow cytometry, automated fluorescence microscopy and cytokine bead array assays. Furthermore, we examined whether VAEI was able to counteract tumor-induced immunosuppression within this cellular system using a renal cancer cell model. The role of mistletoe lectin (ML) was analyzed using ML-specific antibodies and ML-depleted VAEI. VAEI and VAEA augmented the maturation of dendritic cells. VAEI abrogated tumor-induced immunosuppression of dendritic cells and both processes were partially mediated by ML since ML-depleted VAEI and ML-specific antibodies almost neutralized the rehabilitative effects of VAEI on DC maturation. Using these settings, co-culture experiments with purified CD4+ T cells had no influence on T cell proliferation and activation but did have an impact on IFN-γ secretion. The study provides a potential mode-of-action of VAE as an additive cancer therapy based on immunomodulatory effects. However, the impact on the in vivo situation has to be evaluated in further studies. PMID:28719632

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

    PubMed Central

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

    2012-01-01

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

  18. 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.

  19. 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.

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

    PubMed

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

    2012-01-01

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

  1. Prokaryotic selectivity and LPS-neutralizing activity of short antimicrobial peptides designed from the human antimicrobial peptide LL-37.

    PubMed

    Nan, Yong Hai; Bang, Jeong-Kyu; Jacob, Binu; Park, Il-Seon; Shin, Song Yub

    2012-06-01

    To develop novel antimicrobial peptides (AMPs) with shorter lengths, improved prokaryotic selectivity and retained lipolysaccharide (LPS)-neutralizing activity compared to human cathelicidin AMP, LL-37, a series of amino acid-substituted analogs based on IG-19 (residues 13-31 of LL-37) were synthesized. Among the IG-19 analogs, the analog a4 showed the highest prokaryotic selectivity, but much lower LPS-neutralizing activity compared to parental LL-37. The analogs, a5, a6, a7 and a8 with higher hydrophobicity displayed LPS-neutralizing activity comparable to that of LL-37, but much lesser prokaryotic selectivity. These results indicate that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of LPS-neutralizing activity and prokaryotic selectivity. Furthermore, to increase LPS-neutralizing activity of the analog a4 without a remarkable decrease in prokaryotic selectivity, we synthesized Trp-substituted analogs (a4-W1 and a4-W2), in which Phe(5) or Phe(15) of a4 is replaced by Trp. Despite their same prokaryotic selectivity, a4-W2 displayed much higher LPS-neutralizing activity compared to a4-W1. When compared with parental LL-37, a4-W2 showed retained LPS-neutralizing activity and 2.8-fold enhanced prokaryotic selectivity. These results suggest that the effective site for Trp-substitution when designing novel AMPs with higher LPS-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. Taken together, the analog a4-W2 can serve as a promising template for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection. Copyright © 2012. Published by Elsevier Inc.

  2. Unusual Features of Vaccinia Virus Extracellular Virion Form Neutralization Resistance Revealed in Human Antibody Responses to the Smallpox Vaccine

    PubMed Central

    Benhnia, Mohammed Rafii-El-Idrissi; Maybeno, Matthew; Blum, David; Aguilar-Sino, Rowena; Matho, Michael; Meng, Xiangzhi; Head, Steven; Felgner, Philip L.; Zajonc, Dirk M.; Koriazova, Lilia; Kato, Shinichiro; Burton, Dennis R.; Xiang, Yan; Crowe, James E.; Peters, Bjoern

    2013-01-01

    The extracellular virion form (EV) of vaccinia virus (VACV) is essential for viral pathogenesis and is difficult to neutralize with antibodies. Why this is the case and how the smallpox vaccine overcomes this challenge remain incompletely understood. We previously showed that high concentrations of anti-B5 antibodies are insufficient to directly neutralize EV (M. R. Benhnia, et al., J. Virol. 83:1201–1215, 2009). This allowed for at least two possible interpretations: covering the EV surface is insufficient for neutralization, or there are insufficient copies of B5 to allow anti-B5 IgG to cover the whole surface of EV and another viral receptor protein remains active. We endeavored to test these possibilities, focusing on the antibody responses elicited by immunization against smallpox. We tested whether human monoclonal antibodies (MAbs) against the three major EV antigens, B5, A33, and A56, could individually or together neutralize EV. While anti-B5 or anti-A33 (but not anti-A56) MAbs of appropriate isotypes were capable of neutralizing EV in the presence of complement, a mixture of anti-B5, anti-A33, and anti-A56 MAbs was incapable of directly neutralizing EV, even at high concentrations. This remained true when neutralizing the IHD-J strain, which lacks a functional version of the fourth and final known EV surface protein, A34. These immunological data are consistent with the possibility that viral proteins may not be the active component of the EV surface for target cell binding and infectivity. We conclude that the protection afforded by the smallpox vaccine anti-EV response is predominantly mediated not by direct neutralization but by isotype-dependent effector functions, such as complement recruitment for antibodies targeting B5 and A33. PMID:23152530

  3. 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. © 2016 UICC.

  4. 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.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-05-01

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

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

    PubMed Central

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

    2015-01-01

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

  8. 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

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

    PubMed

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

    2016-12-01

    Direct-acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection, but this still leaves a large number of treatment failures secondary to the emergence of resistance-associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to use neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies. Therefore, we identified an HMAb that is less likely to elicit RAVs for affinity maturation to increase potency and, more important, breadth of protection. Selected matured antibodies show improved affinity and neutralization against a panel of diverse HCV isolates. Structural and modeling studies reveal that the affinity-matured HMAb mediates virus neutralization, in part, by inducing conformational change to the targeted epitope, and that the maturated light chain is responsible for the improved affinity and breadth of protection. A matured HMAb protected humanized mice when challenged with an infectious HCV human serum inoculum for a prolonged period. However, a single mouse experienced breakthrough infection after 63 days when the serum HMAb concentration dropped by several logs; sequence analysis revealed no viral escape mutation. The findings suggest that a single broadly neutralizing antibody can prevent acute HCV infection without inducing RAVs and may complement DAAs to reduce the emergence of RAVs. (Hepatology 2016;64:1922-1933). © 2016 by the American Association for the Study of Liver Diseases.

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

    PubMed

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

    2013-12-15

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

  11. Structural basis for norovirus neutralization by an HBGA blocking human IgA antibody.

    PubMed

    Shanker, Sreejesh; Czakó, Rita; Sapparapu, Gopal; Alvarado, Gabriela; Viskovska, Maria; Sankaran, Banumathi; Atmar, Robert L; Crowe, James E; Estes, Mary K; Prasad, B V Venkataram

    2016-10-04

    Human noroviruses (HuNoVs) cause sporadic and epidemic gastroenteritis worldwide. They are classified into two major genogroups (GI and GII), with each genogroup further divided into multiple genotypes. Susceptibility to these viruses is influenced by genetically determined histo-blood group antigen (HBGA) expression. HBGAs function as cell attachment factors by binding to a surface-exposed region in the protruding (P) domain of the capsid protein. Sequence variations in this region that result in differential HBGA binding patterns and antigenicity are suggested to form a basis for strain diversification. Recent studies show that serum antibodies that block HBGA binding correlate with protection against illness. Although genogroup-dependent variation in HBGA binding specificity is structurally well characterized, an understanding of how antibodies block HBGA binding and how genotypic variations affect such blockade is lacking. Our crystallographic studies of the GI.1 P domain in complex with the Fab fragment of a human IgA monoclonal antibody (IgA 5I2) with HBGA blocking activity show that the antibody recognizes a conformational epitope formed by two surface-exposed loop clusters in the P domain. The antibody engulfs the HBGA binding site but does not affect its structural integrity. An unusual feature of the antigen recognition by IgA 5I2 is the predominant involvement of the CDR light chain 1 in contrast to the commonly observed CDR heavy chain 3, providing a unique perspective into antibody diversity in antigen recognition. Identification of the antigenic site in the P domain shows how genotypic variations might allow escape from antibody neutralization and exemplifies the interplay between antigenicity and HBGA specificity in HuNoV evolution.

  12. 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.

  13. 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

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

    PubMed

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

    2008-07-01

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

  15. 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.

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

    PubMed

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

    2007-02-01

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

  17. Human Monoclonal Antibodies Generated Following Vaccination with AVA Provide Neutralization by Blocking Furin Cleavage but not by Preventing Oligomerization

    PubMed Central

    Smith, Kenneth; Crowe, Sherry R.; Garman, Lori; Guthridge, Carla; Muther, Jennifer J.; McKee, Emily; Farris, A. Darise; Guthridge, Joel M.; Wilson, Patrick C.; James, Judith A.

    2012-01-01

    In order to identify the combination of antibody-mediated mechanisms of neutralization that result from vaccination with anthrax vaccine adsorbed (AVA), we isolated antibody secreting cells from a single donor seven days after booster vaccination with AVA and generated nine fully human monoclonal antibodies (hmAb) with high specificity for protective antigen (PA). Two of the antibodies were able to neutralize lethal toxin in vitro at low concentrations (IC50: p6C01, 0.12 µg/ml and p6F01, 0.45 µg/ml). Passive transfer of either of these hmAbs to A/J mice prior to challenge with lethal toxin conferred 80–90% protection. We demonstrate that hmAb p6C01 is neutralizing by preventing furin cleavage of PA in a dose-dependent manner, but the mechanism of p6F01 is unclear. Three additional antibodies were found to bind to domain 3 of PA and prevent oligomerization, although they did not confer significant protection in vivo and showed a significant prozone-like effect in vitro. These fully human antibodies provide insight into the neutralizing response to AVA for future subunit vaccine and passive immunotherapeutic cocktail design. PMID:22425791

  18. Cross-Reactive and Cross-Neutralizing Activity of Human Mumps Antibodies Against a Novel Mumps Virus From Bats.

    PubMed

    Beaty, Shannon M; Nachbagauer, Raffael; Hirsh, Ariana; Vigant, Frederic; Duehr, James; Azarm, Kristopher D; Stelfox, Alice J; Bowden, Thomas A; Duprex, W Paul; Krammer, Florian; Lee, Benhur

    2017-01-15

    To evaluate the antigenic relationship between bat mumps virus (BMV) and the JL5 vaccine strain of mumps virus (MuVJL5), we rescued a chimeric virus bearing the F and HN glycoproteins of BMV in the background of a recombinant JL5 genome (rMuVJL5). Cross-reactivity and cross-neutralization between this chimeric recombinant MuV bearing the F and HN glycoproteins of BMV (rMuVJL5-F/HNBMV) virus and rMuVJL5 were demonstrated using hyperimmune mouse serum samples and a curated panel of human serum. All mouse and human serum samples that were able to neutralize rMuVJL5 infection had cross-neutralizing activity against rMuVJL5-F/HNBMV. Our data suggest that persons who have neutralizing antibodies against MuV might be protected from infection by BMV. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

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

    PubMed

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

    2006-06-01

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

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2002-01-01

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

  2. [Modification of Bowie's method of demonstrating specific granules in cells of the human renal juxtaglomerular apparatus fixed in neutral formalin].

    PubMed

    Orduian, S L

    1976-04-01

    A modification of Bowie's method for detection of specific granules of the juxtaglomerular apparatus of the human kidneys, fixed in 10% neutral formalin, is suggested. In order to achieve better staining, sections of material fixed in formalin are additionally treated with Helly's liquid and, following the removal of sublimate deposit, with a 2.5% solution of potassium bichromate. After this the sections are stained by Bowie's method in accordance with Pitcock and Hartroft's prescription.

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

    PubMed

    Bogdanova, L D; Myshkin, A E

    1990-01-01

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

  4. 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

  5. Deleterious Alleles in the Human Genome Are on Average Younger Than Neutral Alleles of the Same Frequency

    PubMed Central

    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. PMID:23468643

  6. 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.

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  9. 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.

  10. Increased Sensitivity to CD4 Binding Site-Directed Neutralization following In Vitro Propagation on Primary Lymphocytes of a Neutralization-Resistant Human Immunodeficiency Virus IIIB Strain Isolated from an Accidentally Infected Laboratory Worker

    PubMed Central

    Beaumont, Tim; Quakkelaar, Esther; van Nuenen, Ad; Pantophlet, Ralph; Schuitemaker, Hanneke

    2004-01-01

    We previously described the adaptation of the neutralization-sensitive human immunodeficiency virus type 1 (HIV-1) strain IIIB to a neutralization-resistant phenotype in an accidentally infected laboratory worker. During long-term propagation of this resistant isolate, designated FF3346, on primary peripheral blood leukocytes in vitro, an HIV-1 variant appeared that had regained sensitivity to neutralization by soluble CD4 (sCD4) and the broadly neutralizing monoclonal antibody b12. When an early passage of FF3346 was subjected to limiting-dilution culture in peripheral blood mononuclear cells, eight virus variants with various degrees of neutralization resistance were isolated. Two of them, the sCD4 neutralization-resistant variant LW_H8res and the sCD4 neutralization-sensitive variant LW_G9sens, were selected for further study. Interestingly, these two viruses were equally resistant to neutralization by agents that recognize domains other than the CD4 binding site. Site-directed mutagenesis revealed that the increased neutralization sensitivity of variant LW_G9sens resulted from only two changes, an Asn-to-Ser substitution at position 164 in the V2 loop and an Ala-to-Glu substitution at position 370 in the C3 domain of gp120. In agreement with this notion, the affinity of b12 for monomeric gp120 containing the N164S and A370E substitutions in the background of the molecular clone LW_H8res was higher than its affinity for the parental gp120. Surprisingly, no correlation was observed between CD4 binding affinity for monomeric gp120 and the level of neutralization resistance, suggesting that differences in sCD4 neutralization sensitivity between these viruses are only manifested in the context of the tertiary or quaternary structure of gp120 on the viral surface. The results obtained here indicate that the neutralization-sensitive strain IIIB can become neutralization resistant in vivo under selective pressure by neutralizing antibodies but that this resistance may

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

    PubMed Central

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

    2012-01-01

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

  12. Human sperm sex chromosome disomy and sperm DNA damage assessed by the neutral comet assay.

    PubMed

    McAuliffe, M E; Williams, P L; Korrick, S A; Dadd, R; Marchetti, F; Martenies, S E; Perry, M J

    2014-10-10

    Is there an association between human sperm sex chromosome disomy and sperm DNA damage? An increase in human sperm XY disomy was associated with higher comet extent; however, there was no other consistent association of sex chromosome disomies with DNA damage. There is limited published research on the association between sex chromosome disomy and sperm DNA damage and the findings are not consistent across studies. We conducted a cross-sectional study of 190 men (25% ever smoker, 75% never smoker) from subfertile couples presenting at the Massachusetts General Hospital Fertility Clinic from January 2000 to May 2003. Multiprobe fluorescence in situ hybridization for chromosomes X, Y and 18 was used to determine XX, YY, XY and total sex chromosome disomy in sperm nuclei using an automated scoring method. The neutral comet assay was used to measure sperm DNA damage, as reflected by comet extent, percentage DNA in the comet tail, and tail distributed moment. Univariate and multiple linear regression models were constructed with sex chromosome disomy (separate models for each of the four disomic conditions) as the independent variable, and DNA damage parameters (separate models for each measure of DNA damage) as the dependent variable. Men with current or past smoking history had significantly greater comet extent (µm: regression coefficients with 95% CI) [XX18: 15.17 (1.98, 28.36); YY18: 14.68 (1.50, 27.86); XY18: 15.41 (2.37, 28.45); Total Sex Chromosome Disomy: 15.23 (2.09, 28.38)], and tail distributed moment [XX18: 3.01 (0.30, 5.72); YY18: 2.95 (0.24, 5.67); XY18: 3.04 (0.36, 5.72); Total Sex Chromosome Disomy: 3.10 (0.31, 5.71)] than men who had never smoked. In regression models adjusted for age and smoking, there was a positive association between XY disomy and comet extent. For an increase in XY disomy from 0.56 to 1.47% (representing the 25th to 75th percentile), there was a mean increase of 5.08 µm in comet extent. No other statistically significant

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

    PubMed

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

    2017-02-01

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

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed

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

    2014-01-01

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

  16. 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

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

    PubMed Central

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

    2015-01-01

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

  18. Human sperm sex chromosome disomy and sperm DNA damage assessed by the neutral comet assay

    PubMed Central

    McAuliffe, M.E.; Williams, P.L.; Korrick, S.A.; Dadd, R.; Marchetti, F.; Martenies, S.E.; Perry, M.J.

    2014-01-01

    STUDY QUESTION Is there an association between human sperm sex chromosome disomy and sperm DNA damage? SUMMARY ANSWER An increase in human sperm XY disomy was associated with higher comet extent; however, there was no other consistent association of sex chromosome disomies with DNA damage. WHAT IS KNOWN ALREADY There is limited published research on the association between sex chromosome disomy and sperm DNA damage and the findings are not consistent across studies. STUDY DESIGN, SIZE, AND DURATION We conducted a cross-sectional study of 190 men (25% ever smoker, 75% never smoker) from subfertile couples presenting at the Massachusetts General Hospital Fertility Clinic from January 2000 to May 2003. PARTICIPANTS/MATERIALS, SETTING, METHODS Multiprobe fluorescence in situ hybridization for chromosomes X, Y and 18 was used to determine XX, YY, XY and total sex chromosome disomy in sperm nuclei using an automated scoring method. The neutral comet assay was used to measure sperm DNA damage, as reflected by comet extent, percentage DNA in the comet tail, and tail distributed moment. Univariate and multiple linear regression models were constructed with sex chromosome disomy (separate models for each of the four disomic conditions) as the independent variable, and DNA damage parameters (separate models for each measure of DNA damage) as the dependent variable. MAIN RESULTS AND THE ROLE OF CHANCE Men with current or past smoking history had significantly greater comet extent (µm: regression coefficients with 95% CI) [XX18: 15.17 (1.98, 28.36); YY18: 14.68 (1.50, 27.86); XY18: 15.41 (2.37, 28.45); Total Sex Chromosome Disomy: 15.23 (2.09, 28.38)], and tail distributed moment [XX18: 3.01 (0.30, 5.72); YY18: 2.95 (0.24, 5.67); XY18: 3.04 (0.36, 5.72); Total Sex Chromosome Disomy: 3.10 (0.31, 5.71)] than men who had never smoked. In regression models adjusted for age and smoking, there was a positive association between XY disomy and comet extent. For an increase in XY

  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.

  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. 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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-12-01

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

  5. Three-dimensional structure of the Fab fragment of a neutralizing antibody to human rhinovirus serotype 2.

    PubMed Central

    Tormo, J.; Stadler, E.; Skern, T.; Auer, H.; Kanzler, O.; Betzel, C.; Blaas, D.; Fita, I.

    1992-01-01

    The crystal structure of the antigen-binding fragment of a monoclonal antibody (8F5) that neutralizes human rhinovirus serotype 2 has been determined by X-ray diffraction studies. Antibody 8F5, obtained by immunization with native HRV2 virions, cross-reacts with peptides of the viral capsid protein VP2, which contribute to the neutralizing immunogenic site B in this serotype. The structure was solved by the molecular replacement method and has been refined to an R-factor of 18.9% at 2.8 A resolution. The elbow angle, relating the variable and constant modules of the molecule is 127 degrees, representing the smallest elbow angle observed so far in an Fab fragment. Furthermore, the charged residues of the epitope can be well accommodated in the antigen-binding site. This is the first crystal structure reported for an antibody directed against an icosahedral virus. PMID:1338980

  6. An Aptamer That Neutralizes R5 Strains of Human Immunodeficiency Virus Type 1 Blocks gp120-CCR5 Interaction

    PubMed Central

    Dey, Antu K.; Khati, Makobetsa; Tang, Min; Wyatt, Richard; Lea, Susan M.; James, William

    2005-01-01

    We recently described the isolation and structural characterization of 2′-fluoropyrimidine-substituted RNA aptamers that bind to gp120 of R5 strains of human immunodeficiency virus type 1 and thereby potently neutralize the infectivity of phylogenetically diverse R5 strains. Here we investigate the physical basis of their antiviral action. We show that both N-linked oligosaccharides and the variable loops V1/V2 and V3 are not required for binding of one aptamer, B40, to gp120. Using surface plasmon resonance binding analyses, we show that the aptamer binds to the CCR5-binding site on gp120 in a relatively CD4-independent manner, providing a mechanistic explanation for its neutralizing potency. PMID:16227301

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

    PubMed Central

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

    1993-01-01

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

  8. 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.

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

    PubMed

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

    2012-04-30

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed Central

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

    1988-01-01

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

  13. 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.

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

    PubMed

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

    2016-03-03

    Increasing studies suggest that ceramides differing in acyl chain length and/or degree of unsaturation have distinct roles in mediating biological responses. However, still much remains unclear about regulation and role of distinct ceramide species in the immune response. Here, we demonstrate that alkaline ceramidase 3 (Acer3) mediates the immune response by regulating the levels of C18:1-ceramide in cells of the innate immune system and that Acer3 deficiency aggravates colitis in a murine model by augmenting the expression of pro-inflammatory cytokines in myeloid and colonic epithelial cells (CECs). According to the NCBI Gene Expression Omnibus (GEO) database, ACER3 is downregulated in immune cells in response to lipopolysaccharides (LPS), a potent inducer of the innate immune response. Consistent with these data, we demonstrated that LPS downregulated both Acer3 mRNA levels and its enzymatic activity while elevating C(18:1)-ceramide, a substrate of Acer3, in murine immune cells or CECs. Knocking out Acer3 enhanced the elevation of C(18:1)-ceramide and the expression of pro-inflammatory cytokines in immune cells and CECs in response to LPS challenge. Similar to Acer3 knockout, treatment with C(18:1)-ceramide, but not C18:0-ceramide, potentiated LPS-induced expression of pro-inflammatory cytokines in immune cells. In the mouse model of dextran sulfate sodium-induced colitis, Acer3 deficiency augmented colitis-associated elevation of colonic C(18:1)-ceramide and pro-inflammatory cytokines. Acer3 deficiency aggravated diarrhea, rectal bleeding, weight loss and mortality. Pathological analyses revealed that Acer3 deficiency augmented colonic shortening, immune cell infiltration, colonic epithelial damage and systemic inflammation. Acer3 deficiency also aggravated colonic dysplasia in a mouse model of colitis-associated colorectal cancer. Taken together, these results suggest that Acer3 has an important anti-inflammatory role by suppressing cellular or tissue C(18

  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. Long-term persistence of Chikungunya virus neutralizing antibodies in human populations of North Eastern Thailand.

    PubMed

    Nitatpattana, Narong; Kanjanopas, Kobkan; Yoksan, Sutee; Satimai, Wichai; Vongba, Narong; Langdatsuwan, Sasiporn; Nakgoi, Khajornpong; Ratchakum, Supot; Wauquier, Nadia; Souris, Marc; Auewarakul, Prasert; Gonzalez, Jean-Paul

    2014-10-21

    Chikungunya virus (CHIKV) outbreak recurrences in Thailand are unpredictable and separated by unexplained and often long silent epidemiological periods that can last for several years. These silent periods could be explained in part by the fact that infection with one CHIKV strain confers lasting natural immunity, even against other CHIKV strains. In this study we evaluated the persistence of CHIKV-specific neutralizing antibodies in the population of Chumpae District, Khon Kaen Province, nineteen years after a CHIKV outbreak occurred in the same area in 1991. Overall 39% (44/111) of 111 former patients had neutralizing antibodies reacting against CHIKV ECSA strain. Consistently high titers of neutralizing antibodies were found in 75% (33/44) of all positively-reacting sera, 70% of which (23/33) were collected from individuals amongst the >60 years old age group. Although the prevalence found in Pong Haeng village (70%) was significantly higher than the prevalence detected in the Nong Thum village (14%), control study villages without known previous Chikungunya epidemics had a high Chikungunya neutralizing antibody prevalence (65%). More than one-third of the pre-exposed population had persisting natural immunity that was more likely boosted by recent and repetitive exposure to the emerging ECSA CHIKV in Thailand. Also, Chikungunya virus appears to largely circulate in the country with a great variability appears between villages or area probably associated with the vector abundance and efficiency. Altogether these results show a potential for a lifelong immunity against CHIKV. Given the rapid spread of the highly pathogenic ECSA strain in Southern Thailand, the development of CHIK vaccine is strongly recommended.

  17. 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. © 2011 Wiley Periodicals, Inc.

  18. 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.

  19. 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

  20. 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

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

    PubMed

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

    1999-05-01

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

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

    PubMed

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

    2012-10-01

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

  3. 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

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

    SciTech Connect

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

    2015-10-26

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

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

    DOE PAGES

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

    2015-10-26

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

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

    PubMed

    Ozaki, Daniel A; Gao, Hongmei; Todd, Christopher A; Greene, Kelli M; Montefiori, David C; Sarzotti-Kelsoe, Marcella

    2012-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Dierickx, Paul J; Scheers, Ellen M

    2002-01-01

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

  9. Evaluation of antiviral drugs and neutralizing antibodies to human immunodeficiency virus by a rapid and sensitive microtiter infection assay.

    PubMed Central

    Montefiori, D C; Robinson, W E; Schuffman, S S; Mitchell, W M

    1988-01-01

    A 96-well microtiter infection assay for the human immunodeficiency virus (HIV) is described. The assay utilizes human T-cell lymphotropic virus type I-immortalized MT-2 cells as targets for infection and requires only 4 to 5 days for completion. Cytolysis was quantitated by vital dye uptake of poly-L-lysine-adhered cells as an endpoint for infection. The assay's efficacy was proven by the sensitive and accurate assessment of several known anti-HIV agents including two inhibitors of reverse transcription (3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine), three biological response modifiers (recombinant interferons alpha and beta and mismatched double-stranded RNA), a direct inactivator of HIV virions (amphotericin B), and neutralizing antibodies from two HIV-positive human subjects. Evaluation of data was facilitated by computer-assisted analysis. This assay provides a means for rapid, sensitive, and inexpensive large-scale in vitro testing of potential anti-HIV therapeutic regimens and quantitation of HIV-neutralizing antibody titers. PMID:3422647

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2016-04-25

    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.

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

    PubMed Central

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

    1988-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2013-11-01

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

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

    PubMed Central

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

    2002-01-01

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

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

    PubMed Central

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

    1994-01-01

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

  18. Development of a 2-Plex Luminex-Based Competitive Immunoassay to Quantify Neutralizing Antibodies Induced by Virus-Like Particles for Human Papillomavirus 16 and 18

    PubMed Central

    Lv, Pin; Zhu, Hongmei; Wang, Hongfang; Wang, George

    2011-01-01

    Human papillomavirus (HPV) L1 virus-like particles (VLPs) were proven an effective vaccine candidate to prevent against HPV-16 and -18 infections. In order to evaluate the potency of our produced HPV-16 and -18 L1 VLPs-based vaccine candidates, also to quantify neutralizing antibodies induced by them, a 2-plex Luminex-based competitive immunoassay was developed. Unlike the published paper, the no-biotin conjugated neutralizing mAbs spiked normal human serum (NHS) was used for standard curve preparation, while phycoerythrin (PE) was not labeled directly to neutralizing mAbs for signaling. After the coupling optimization of VLPs to microspheres and the neutralizing mAbs biotinylation, the 2-plex standard curve was prepared with good fit and high dynamic range. In addition, no cross-reactivity was also confirmed. The 2-plex Luminex-based immunoassay represents good potential not only for vaccine candidate's evaluation but also for its further clinical use. PMID:21808597

  19. 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.

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

    PubMed

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

    1994-05-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2011-03-01

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

  3. Molecular signatures of hemagglutinin stem-directed heterosubtypic human neutralizing antibodies against influenza A viruses.

    PubMed

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

    2014-05-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    1992-01-01

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

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

    PubMed

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

    2007-06-01

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

  7. 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

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

    PubMed

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

    2015-12-01

    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. 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 on a widely used

  9. 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.

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

    PubMed

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

    2009-10-01

    Nipah virus is a broadly tropic and highly pathogenic zoonotic paramyxovirus in the genus Henipavirus whose natural reservoirs are several species of Pteropus fruit bats. Nipah virus has repeatedly caused outbreaks over the past decade associated with a severe and often fatal disease in humans and animals. Here, a new ferret model of Nipah virus pathogenesis is described where both respiratory and neurological disease are present in infected animals. Severe disease occurs with viral doses as low as 500 TCID(50) within 6 to 10 days following infection. The underlying pathology seen in the ferret closely resembles that seen in Nipah virus infected humans, characterized as a widespread multisystemic vasculitis, with virus replicating in highly vascular tissues including lung, spleen and brain, with recoverable virus from a variety of tissues. Using this ferret model a cross-reactive neutralizing human monoclonal antibody, m102.4, targeting the henipavirus G glycoprotein was evaluated in vivo as a potential therapeutic agent. All ferrets that received m102.4 ten hours following a high dose oral-nasal Nipah virus challenge were protected from disease while all controls died. This study is the first successful post-exposure passive antibody therapy for Nipah virus using a human monoclonal antibody.

  11. 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

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

    PubMed Central

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

    1995-01-01

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

  13. 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.

  14. Induction of high-titer neutralizing antibodies, using hybrid human immunodeficiency virus V3-Ty viruslike particles in a clinically relevant adjuvant.

    PubMed Central

    Griffiths, J C; Berrie, E L; Holdsworth, L N; Moore, J P; Harris, S J; Senior, J M; Kingsman, S M; Kingsman, A J; Adams, S E

    1991-01-01

    The localization of neutralization determinants within the envelope glycoproteins of human immunodeficiency virus (HIV) has been largely achieved by immunizing small animals in conjunction with Freund's adjuvant. However, for eventual use in humans, candidate HIV vaccine components must also be efficacious in a nontoxic formulation. We describe here the production of hybrid Ty viruslike particles carrying the major neutralizing domain of HIV and demonstrate the induction of high-titer virus-neutralizing antibodies and an HIV-specific T-cell proliferative response after immunization in conjunction with aluminum hydroxide. As aluminum hydroxide and aluminum phosphate are the only adjuvants currently licensed for use in humans, these observations have implications for the development of an effective vaccine against HIV. Images PMID:1985208

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

  18. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    1998-01-01

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

  1. 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. Copyright

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

    PubMed Central

    Jana, Arundhati; Pahan, Kalipada

    2007-01-01

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

  3. 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.

  4. Determination of the fatty acid profile of neutral lipids, free fatty acids and phospholipids in human plasma.

    PubMed

    Firl, Nina; Kienberger, Hermine; Hauser, Teresa; Rychlik, Michael

    2013-04-01

    Knowledge of the fatty acid composition of lipid classes in human plasma is an important factor in the investigation of human metabolism. Therefore, a method for the analysis of neutral lipid (NL), phospholipid (PL) and free fatty acids (FFA) in human plasma has been developed and validated. Separation of lipid classes was carried out by solid phase extraction of the lipid extract. The fractions were transesterified and the resulting fatty acid methyl esters were determined by GC/FID. For the method to be validated, precision, detection and quantification limits, as well as recovery, were determined for combined lipid extraction, solid phase extraction and GC analysis. The lipid extraction was miniaturized and simplified by application of an ultrasound ‘Sonotrode’. The resolution of lipid classes was optimized with appropriate standards added to a representative plasma sample. In addition, a rapid derivatization procedure using trimethylsulfoniumhydroxide was established. Low determination limits (1.5, 0.2 and 1.3 μg/g plasma for NL, PL and FFA, respectively) indicate that the method’s sensitivity is sufficient to quantify even minor components. Furthermore, recovery for NL and PL fatty acids was found to range from 80% to 110%. The results were similar for FFA apart from more polar free fatty acids due to their higher solubility in water. Repetitive measurements showed very good precision apart from the long chain PUFA for which the coefficients of variation were significantly higher. The present method is applicable to the quantitation of fatty acids in lipid classes of human plasma including several minor components.

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

    PubMed

    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

    2015-07-28

    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.

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

    PubMed

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

    1997-12-10

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

  7. Transintestinal Cholesterol Transport Is Active in Mice and Humans and Controls Ezetimibe-Induced Fecal Neutral Sterol Excretion.

    PubMed

    Jakulj, Lily; van Dijk, Theo H; de Boer, Jan Freark; Kootte, Ruud S; Schonewille, Marleen; Paalvast, Yared; Boer, Theo; Bloks, Vincent W; Boverhof, Renze; Nieuwdorp, Max; Beuers, Ulrich H W; Stroes, Erik S G; Groen, Albert K

    2016-12-13

    Except for conversion to bile salts, there is no major cholesterol degradation pathway in mammals. Efficient excretion from the body is therefore a crucial element in cholesterol homeostasis. Yet, the existence and importance of cholesterol degradation pathways in humans is a matter of debate. We quantified cholesterol fluxes in 15 male volunteers using a cholesterol balance approach. Ten participants repeated the protocol after 4 weeks of treatment with ezetimibe, an inhibitor of intestinal and biliary cholesterol absorption. Under basal conditions, about 65% of daily fecal neutral sterol excretion was bile derived, with the remainder being contributed by direct transintestinal cholesterol excretion (TICE). Surprisingly, ezetimibe induced a 4-fold increase in cholesterol elimination via TICE. Mouse studies revealed that most of ezetimibe-induced TICE flux is mediated by the cholesterol transporter Abcg5/Abcg8. In conclusion, TICE is active in humans and may serve as a novel target to stimulate cholesterol elimination in patients at risk for cardiovascular disease. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2015-01-16

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

  9. 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.

  10. [Detection of neutralizing antibodies and DNA of human papillomavirus 16, 18 in women aged 18-45 years in Funing, Jiangsu province].

    PubMed

    Liu, Wenyu; Wei, Feixue; Tang, Jie; Yang, Shangbo; Gao, Yachun; Wang, Ting; Jiang, Yunfei; Li, Mei; Hong, Ying; Chu, Kai; Chen, Wen; Hu, Yuemei; Zhu, Fengcai

    2016-03-01

    To understand the distributions of DNA and neutralizing antibodies of human papillomavirus (HPV)16, 18 in 18-45 year-old women. Totally, 1494 women were enrolled through multistage random sampling in Funing, Jiangsu province. Cervical exfoliated cells were collected from them for HPV DNA testing, and serum samples were taken from them for the detection of HPV16, 18 neutralizing antibodies by using pseudovirion-based neutralization assay(PBNA). Among the 1494 women, 28(1.9%) and 188(12.6%) were positive for DNA and neutralizing antibody of HPV16 respectively, and 15(1.0%) and 60(4.0%) were positive for DNA and neutralizing antibody of HPV18, respectively. There were no significant differences in the detection rates of DNA and neutralizing antibody of HPV16, 18 among different age groups. About 16.7% of the women were infected with HPV16, 18, or both. In Funing county of Jiangsu province, most women aged 18-45 years has no immunity to HPV16 and 18, indicating that they are appropriate targets for HPV 16/18 vaccination.

  11. Structural Basis for Marburg Virus Neutralization by a Cross-Reactive Human Antibody

    DOE PAGES

    Hashiguchi, Takao; Fusco, Marnie L.; Bornholdt, Zachary A.; ...

    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 theirmore » NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. We find that 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.« less

  12. 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.

  13. 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.

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

    PubMed

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

    2015-08-01

    Broadly neutralizing antibodies (bnAbs) can prevent lentiviral infection in nonhuman primates and may slow the spread of human immunodeficiency virus type 1 (HIV-1). Although protection by passive transfer of human bnAbs has been demonstrated in monkeys, durable expression is essential for its broader use in humans. Gene-based expression of bnAbs provides a potential solution to this problem, although immune responses to the viral vector or to the antibody may limit its durability and efficacy. Here, we delivered an adeno-associated viral vector encoding a simianized form of a CD4bs bnAb, VRC07, and evaluated its immunogenicity and protective efficacy. The expressed antibody circulated in macaques for 16 weeks at levels up to 66 g/ml, although immune suppression with cyclosporine (CsA) was needed to sustain expression. Gene-delivered simian VRC07 protected against simian-human immunodeficiency virus (SHIV) infection in monkeys 5.5 weeks after treatment. Gene transfer of an anti-HIV antibody can therefore protect against infection by viruses that cause AIDS in primates when the host immune responses are controlled.

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

    PubMed Central

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

    2017-01-01

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

  16. Neutralization of Human Interleukin 23 by Multivalent Nanobodies Explained by the Structure of Cytokine-Nanobody Complex.

    PubMed

    Desmyter, Aline; Spinelli, Silvia; Boutton, Carlo; Saunders, Michael; Blachetot, Christophe; de Haard, Hans; Denecker, Geertrui; Van Roy, Maarten; Cambillau, Christian; Rommelaere, Heidi

    2017-01-01

    The heterodimeric cytokine interleukin (IL) 23 comprises the IL12-shared p40 subunit and an IL23-specific subunit, p19. Together with IL12 and IL27, IL23 sits at the apex of the regulatory mechanisms shaping adaptive immune responses. IL23, together with IL17, plays an important role in the development of chronic inflammation and autoimmune inflammatory diseases. In this context, we generated monovalent antihuman IL23 variable heavy chain domain of llama heavy chain antibody (VHH) domains (Nanobodies(®)) with low nanomolar affinity for human interleukin (hIL) 23. The crystal structure of a quaternary complex assembling hIL23 and several nanobodies against p19 and p40 subunits allowed identification of distinct epitopes and enabled rational design of a multivalent IL23-specific blocking nanobody. Taking advantage of the ease of nanobody formatting, multivalent IL23 nanobodies were assembled with properly designed linkers flanking an antihuman serum albumin nanobody, with improved hIL23 neutralization capacity in vitro and in vivo, as compared to the monovalent nanobodies. These constructs with long exposure time are excellent candidates for further developments targeting Crohn's disease, rheumatoid arthritis, and psoriasis.

  17. First administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity.

    PubMed

    Bakker, A B H; Python, C; Kissling, C J; Pandya, P; Marissen, W E; Brink, M F; Lagerwerf, F; Worst, S; van Corven, E; Kostense, S; Hartmann, K; Weverling, G J; Uytdehaag, F; Herzog, C; Briggs, D J; Rupprecht, C E; Grimaldi, R; Goudsmit, J

    2008-11-05

    Immediate passive immune prophylaxis as part of rabies post-exposure prophylaxis (PEP) often cannot be provided due to limited availability of human or equine rabies immunoglobulin (HRIG and ERIG, respectively). We report first clinical data from two phase I studies evaluating a monoclonal antibody cocktail CL184 against rabies. The studies included healthy adult subjects in the USA and India and involved two parts. First, subjects received a single intramuscular dose of CL184 or placebo in a double blind, randomized, dose-escalation trial. Second, open-label CL184 (20IU/kg) was co-administered with rabies vaccine. Safety was the primary objective and rabies virus neutralizing activity (RVNA) was investigated as efficacy parameter. Pain at the CL184 injection site was reported by less than 40% of subjects; no fever or local induration, redness or swelling was observed. RVNA was detectable from day 1 to day 21 after a single dose of CL184 20 or 40IU/kg. All subjects had adequate (>0.5IU/mL) RVNA levels from day 14 onwards when combined with rabies vaccine. CL184 appears promising as an alternative to RIG in PEP.

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

    PubMed

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

    2014-05-01

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

  19. 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

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

    PubMed

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

    2016-04-01

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

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

    PubMed Central

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

    2010-01-01

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

  2. IgA and Neutralizing Antibodies to Influenza A Virus in Human Milk: A Randomized Trial of Antenatal Influenza Immunization

    PubMed Central

    Schlaudecker, Elizabeth P.; Steinhoff, Mark C.; Omer, Saad B.; McNeal, Monica M.; Roy, Eliza; Arifeen, Shams E.; Dodd, Caitlin N.; Raqib, Rubhana; Breiman, Robert F.; Zaman, K.

    2013-01-01

    Background Antenatal immunization of mothers with influenza vaccine increases serum antibodies and reduces the rates of influenza illness in mothers and their infants. We report the effect of antenatal immunization on the levels of specific anti-influenza IgA levels in human breast milk. (ClinicalTrials.gov identifier NCT00142389; http://clinicaltrials.gov/ct2/show/NCT00142389). Methods and Findings The Mother's Gift study was a prospective, blinded, randomized controlled trial that assigned 340 pregnant Bangladeshi mothers to receive either trivalent inactivated influenza vaccine, or 23-valent pneumococcal polysaccharide vaccine during the third trimester. We evaluated breast milk at birth, 6 weeks, 6 months, and 12 months, and serum at 10 weeks and 12 months. Milk and serum specimens from 57 subjects were assayed for specific IgA antibody to influenza A/New Caledonia (H1N1) using an enzyme-linked immunosorbent assay (ELISA) and a virus neutralization assay, and for total IgA using ELISA. Influenza-specific IgA levels in breast milk were significantly higher in influenza vaccinees than in pneumococcal controls for at least 6 months postpartum (p = 0.04). Geometric mean concentrations ranged from 8.0 to 91.1 ELISA units/ml in vaccinees, versus 2.3 to 13.7 ELISA units/mL in controls. Virus neutralization titers in milk were 1.2 to 3 fold greater in vaccinees, and correlated with influenza-specific IgA levels (r = 0.86). Greater exclusivity of breastfeeding in the first 6 months of life significantly decreased the expected number of respiratory illness with fever episodes in infants of influenza-vaccinated mothers (p = 0.0042) but not in infants of pneumococcal-vaccinated mothers (p = 0.4154). Conclusions The sustained high levels of actively produced anti-influenza IgA in breast milk and the decreased infant episodes of respiratory illness with fever suggest that breastfeeding may provide local mucosal protection for the infant for at least 6 months

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

    SciTech Connect

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

    2011-12-31

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

  4. Effects of human anti-spike protein receptor binding domain antibodies on severe acute respiratory syndrome coronavirus neutralization escape and fitness.

    PubMed

    Sui, Jianhua; Deming, Meagan; Rockx, Barry; Liddington, Robert C; Zhu, Quan Karen; Baric, Ralph S; Marasco, Wayne A

    2014-12-01

    The receptor binding domain (RBD) of the spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) is a major target of protective immunity in vivo. Although a large number of neutralizing antibodies (nAbs) have been developed, it remains unclear if a single RBD-targeting nAb or two in combination can prevent neutralization escape and, if not, attenuate viral virulence in vivo. In this study, we used a large panel of human nAbs against an epitope that overlaps the interface between the RBD and its receptor, angiotensin-converting enzyme 2 (ACE2), to assess their cross-neutralization activities against a panel of human and zoonotic SARS-CoVs and neutralization escape mutants. We also investigated the neutralization escape profiles of these nAbs and evaluated their effects on receptor binding and virus fitness in vitro and in mice. We found that some nAbs had great potency and breadth in neutralizing multiple viral strains, including neutralization escape viruses derived from other nAbs; however, no single nAb or combination of two blocked neutralization escape. Interestingly, in mice the neutralization escape mutant viruses showed either attenuation (Urbani background) or increased virulence (GD03 background) consistent with the different binding affinities between their RBDs and the mouse ACE2. We conclude that using either single nAbs or dual nAb combinations to target a SARS-CoV RBD epitope that shows plasticity may have limitations for preventing neutralization escape during in vivo immunotherapy. However, RBD-directed nAbs may be useful for providing broad neutralization and prevention of escape variants when combined with other nAbs that target a second conserved epitope with less plasticity and more structural constraint. The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 has resulted in severe human respiratory disease with high death

  5. Effects of Human Anti-Spike Protein Receptor Binding Domain Antibodies on Severe Acute Respiratory Syndrome Coronavirus Neutralization Escape and Fitness

    PubMed Central

    Sui, Jianhua; Deming, Meagan; Rockx, Barry; Liddington, Robert C.; Zhu, Quan Karen

    2014-01-01

    ABSTRACT The receptor binding domain (RBD) of the spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) is a major target of protective immunity in vivo. Although a large number of neutralizing antibodies (nAbs) have been developed, it remains unclear if a single RBD-targeting nAb or two in combination can prevent neutralization escape and, if not, attenuate viral virulence in vivo. In this study, we used a large panel of human nAbs against an epitope that overlaps the interface between the RBD and its receptor, angiotensin-converting enzyme 2 (ACE2), to assess their cross-neutralization activities against a panel of human and zoonotic SARS-CoVs and neutralization escape mutants. We also investigated the neutralization escape profiles of these nAbs and evaluated their effects on receptor binding and virus fitness in vitro and in mice. We found that some nAbs had great potency and breadth in neutralizing multiple viral strains, including neutralization escape viruses derived from other nAbs; however, no single nAb or combination of two blocked neutralization escape. Interestingly, in mice the neutralization escape mutant viruses showed either attenuation (Urbani background) or increased virulence (GD03 background) consistent with the different binding affinities between their RBDs and the mouse ACE2. We conclude that using either single nAbs or dual nAb combinations to target a SARS-CoV RBD epitope that shows plasticity may have limitations for preventing neutralization escape during in vivo immunotherapy. However, RBD-directed nAbs may be useful for providing broad neutralization and prevention of escape variants when combined with other nAbs that target a second conserved epitope with less plasticity and more structural constraint. IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 has resulted in severe human respiratory

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    1992-01-01

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

  8. 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.

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

    PubMed

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

    2016-11-01

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

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

    PubMed

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

    2017-01-15

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

  11. 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

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

    2016-04-01

    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 SHIVSF162P3Nand 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. 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, but their development

  12. A Human Antibody Recognizing a Conserved Epitope of H5 Hemagglutinin Broadly Neutralizes Highly Pathogenic Avian Influenza H5N1 Viruses

    PubMed Central

    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

    2012-01-01

    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

  13. 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.

  14. Isolation of nanomolar scFvs of non-human primate origin, cross-neutralizing botulinum neurotoxins A1 and A2 by targeting their heavy chain.

    PubMed

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

    2015-09-17

    Botulism is a naturally occurring disease, mainly caused by the ingestion of food contaminated by the botulinum neurotoxins (BoNTs). Botulinum neurotoxins are the most lethal. They are classified among the six major biological warfare agents by the Centers for Disease Control. BoNTs act on the cholinergic motoneurons, where they cleave proteins implicated in acetylcholine vesicle exocytosis. This exocytosis inhibition induces a flaccid paralysis progressively affecting all the muscles and generally engendering a respiratory distress. BoNTs are also utilized in medicine, mainly for the treatment of neuromuscular disorders, preventing large scale vaccination. Botulism specific treatment requires injections of antitoxins, usually of equine origin and thus poorly tolerated. Therefore, development of human or human-like neutralizing antibodies is of a major interest, and it is the subject of the European framework project called "AntiBotABE". In this study, starting from a macaque immunized with the recombinant heavy chain of BoNT/A1 (BoNT/A1-HC), an immune antibody phage-display library was generated and antibody fragments (single chain Fragment variable) with nanomolar affinity were isolated and further characterized. The neutralization capacities of these scFvs were analyzed in the mouse phrenic nerve-hemidiaphragm assay. After a three-round panning, 24 antibody fragments with affinity better than 10 nM were isolated. Three of them neutralized BoNT/A1 efficiently and two cross-neutralized BoNT/A1 and BoNT/A2 subtypes in the mouse phrenic nerve-hemidiaphragm assay. These are the first monoclonal human-like antibodies cross-neutralizing both BoNT/A1 and BoNT/A2. The antibody A1HC38 was selected for further development, and could be clinically developed for the prophylaxis and treatment of botulism.

  15. 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.

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

    PubMed

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

    2014-11-01

    Human monoclonal antibodies (HMAbs) with neutralizing capabilities constitute potential immune-based treatments or prophylaxis against hepatitis C virus (HCV). However, lack of cell culture-derived HCV (HCVcc) harboring authentic envelope proteins (E1/E2) has hindered neutralization investigations across genotypes, subtypes, and isolates. We investigated the breadth of neutralization of 10 HMAbs with therapeutic potential against a panel of 16 JFH1-based HCVcc-expressing patient-derived Core-NS2 from genotypes 1a (strains H77, TN, and DH6), 1b (J4, DH1, and DH5), 2a (J6, JFH1, and T9), 2b (J8, DH8, and DH10), 2c (S83), and 3a (S52, DBN, and DH11). Virus stocks used for in vitro neutralization analysis contained authentic E1/E2, with the exception of full-length JFH1 that acquired the N417S substitution in E2. The 50% inhibition concentration (IC50) for each HMAb against the HCVcc panel was determined by dose-response neutralization assays in Huh7.5 cells with antibody concentrations ranging from 0.0012 to 100 μg/mL. Interestingly, IC50 values against the different HCVcc's exhibited large variations among the HMAbs, and only three HMAbs (HC-1AM, HC84.24, and AR4A) neutralized all 16 HCVcc recombinants. Furthermore, the IC50 values for a given HMAb varied greatly with the HCVcc strain, which supports the use of a diverse virus panel. In cooperation analyses, HMAbs HC84.24, AR3A, and, especially HC84.26, demonstrated synergistic effects towards the majority of the HCVcc's when combined individually with AR4A. Through a neutralization analysis of 10 clinically relevant HMAbs against 16 JFH1-based Core-NS2 recombinants from genotypes 1a, 1b, 2a, 2b, 2c, and 3a, we identified at least three HMAbs with potent and broad neutralization potential. The neutralization synergism obtained when pooling the most potent HMAbs could have significant implications for developing novel strategies to treat and control HCV. © 2014 by the American Association for the Study of Liver

  17. Variant-specific monoclonal and group-specific polyclonal human immunodeficiency virus type 1 neutralizing antibodies raised with synthetic peptides from the gp120 third variable domain.

    PubMed

    Laman, J D; Schellekens, M M; Abacioglu, Y H; Lewis, G K; Tersmette, M; Fouchier, R A; Langedijk, J P; Claassen, E; Boersma, W J

    1992-03-01

    The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external membrane glycoprotein gp120 is of crucial importance in eliciting neutralizing antibodies in infected persons. Polyclonal (PAb) and monoclonal (MAb) antibodies directed against selected epitopes in the V3 domain are valuable tools for analysis of the involvement of such sequences in neutralization and for definition of the relation between amino acid variability and immunological cross-reactions. The aim of this study was to obtain such site-specific antibodies. By using synthetic peptides derived from the V3 domain, a group-specific neutralizing PAb, two high-affinity HIV-1 IIIB neutralizing MAb, and two nonneutralizing MAb were raised. A 15-amino-acid peptide overlapping the tip of the V3 domain of HIV-1 MN was used to produce a rabbit PAb (W0/07). This PAb inhibited syncytium formation induced by HIV-1 IIIB and four field isolates. A similar IIIB-derived peptide was used to generate two murine immunoglobulin G1 (IgG1) MAb (IIIB-V3-13 and IIIB-V3-34). Pepscan analysis mapped the binding site of IIIB-V3-34 to the sequence IRIQRGPGR. The Kds of IIIB-V3-13 and IIIB-V3-34 for gp120 were 6.8 x 10(-11) and 1.6 x 10(-10) M, respectively. These MAb neutralized IIIB but not MN and inhibited syncytium formation induced by IIIB. They are applicable in enzyme-linked immunosorbent assays, immunocytochemistry, and flow cytometry. A peptide covering the left base of the V3 domain was used to generate two murine IgG1 MAb (IIIB-V3-21 and IIIB-V3-26). The binding site of IIIB-V3-21 was mapped to the sequence INCTRPN. These MAb did not neutralize HIV-1 and did not inhibit syncytium formation. This study supports the notion that HIV-1 neutralizing antibodies suitable for multiassay performance can be obtained with synthetic peptides and that high-affinity MAb can be generated. Such site-specific antibodies are useful reagents in the analysis of HIV-1 neutralization. In addition, the cross-neutralization

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

    PubMed Central

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

    2013-01-01

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

  19. 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

  20. Cervical Spine Muscle-Tendon Unit Length Differences Between Neutral and Forward Head Postures: Biomechanical Study Using Human Cadaveric Specimens.

    PubMed

    Khayatzadeh, Saeed; Kalmanson, Olivia A; Schuit, Dale; Havey, Robert M; Voronov, Leonard I; Ghanayem, Alexander J; Patwardhan, Avinash G

    2017-07-01

    Forward head posture (FHP) may be associated with neck pain and poor health-related quality of life. Literature describes only qualitative muscle length changes associated with FHP. The purpose of this study was to quantify how muscle-tendon unit lengths are altered when human cadaveric specimens are placed in alignments representing different severities of FHP. This biomechanical study used 13 fresh-frozen cadaveric cervical spine specimens (Occiput-T1, 54±15 y). Specimens' postural changes simulating increasing FHP severity while maintaining horizontal gaze were assessed. Specimen-specific anatomic models derived from computed tomography-based anatomic data were combined with postural data and specimen-specific anatomy of muscle attachment points to estimate the muscle length changes associated with FHP. Forward head posture was associated with flexion of the mid-lower cervical spine and extension of the upper cervical (sub-occipital) spine. Muscles that insert on the cervical spine and function as flexors (termed "cervical flexors") as well as muscles that insert on the cranium and function as extensors ("occipital extensors") shortened in FHP when compared to neutral posture. In contrast, muscles that insert on the cervical spine and function as extensors ("cervical extensors") as well as muscles that insert on the cranium and function as flexors ("occipital flexors") lengthened. The greatest shortening was seen in the major and minor rectus capitis posterior muscles. These muscles cross the Occiput-C2 segments, which exhibited extension to maintain horizontal gaze. The greatest lengthening was seen in posterior muscles crossing the C4-C6 segments, which exhibited the most flexion. This cadaver study did not incorporate the biomechanical influence of active musculature. This study offers a novel way to quantify postural alignment and muscle length changes associated with FHP. Model predictions are consistent with qualitative descriptions in the literature.

  1. 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.

  2. Molecular basis of in vitro affinity maturation and functional evolution of a neutralizing anti-human GM-CSF antibody

    PubMed Central

    Eylenstein, Roy; Weinfurtner, Daniel; Härtle, Stefan; Strohner, Ralf; Böttcher, Jark; Augustin, Martin; Ostendorp, Ralf; Steidl, Stefan

    2016-01-01

    X-ray structure analysis of 4 antibody Fab fragments, each in complex with human granulocyte macrophage colony stimulating factor (GM-CSF), was performed to investigate the changes at the protein-protein binding interface during the course of in vitro affinity maturation by phage display selection. The parental antibody MOR03929 was compared to its derivatives MOR04252 (CDR-H2 optimized), MOR04302 (CDR-L3 optimized) and MOR04357 (CDR-H2 and CDR-L3 optimized). All antibodies bind to a conformational epitope that can be divided into 3 sub-epitopes. Specifically, MOR04357 binds to a region close to the GM-CSF N-terminus (residues 11–24), a short second sub-epitope (residues 83–89) and a third at the C-terminus (residues 112–123). Modifications introduced during affinity maturation in CDR-H2 and CDR-L3 led to the establishment of additional hydrogen bonds and van der Waals contacts, respectively, providing a rationale for the observed improvement in binding affinity and neutralization potency. Once GM-CSF is complexed to the antibodies, modeling predicts a sterical clash with GM-CSF binding to GM-CSF receptor α and β chain. This predicted mutually exclusive binding was confirmed by a GM-CSF receptor α chain ligand binding inhibition assay. Finally, high throughput sequencing of clones obtained after affinity maturation phage display pannings revealed highly selected consensus sequences for CDR-H2 as well for CDR-L3, which are in accordance with the sequence of the highest affinity antibody MOR04357. The resolved crystal structures highlight the criticality of these strongly selected residues for high affinity interaction with GM-CSF. PMID:26406987

  3. 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.

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

    PubMed

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

    2014-08-01

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

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

    PubMed

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

    2015-02-01

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

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

    PubMed Central

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

    1994-01-01

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

  8. Synergistic neutralization of a chimeric SIV/HIV type 1 virus with combinations of human anti-HIV type 1 envelope monoclonal antibodies or hyperimmune globulins.

    PubMed

    Li, A; Baba, T W; Sodroski, J; Zolla-Pazner, S; Gorny, M K; Robinson, J; Posner, M R; Katinger, H; Barbas, C F; Burton, D R; Chou, T C; Ruprecht, R M

    1997-05-20

    A panel of 14 human IgG monoclonal antibodies (MAbs) specific for envelope antigens of the human immunodeficiency virus type 1 (HIV-1), 2 high-titer human anti-HIV-1 immunoglobulin (HIVIG) preparations, and 15 combinations of MAbs or MAb/HIVIG were tested for their ability to neutralize infection of cultured human T cells (MT-2) with a chimeric simian immunodeficiency virus (SHIV-vpu+), which expressed HIV-1 IIIB envelope antigens. Eleven MAbs and both HIVIGs were neutralizing. When used alone, the anti-CD4-binding site MAb b12, the anti-gp41 MAb 2F5, and the anti-gp120 MAb 2G12 were the most potent. When combination regimens involving two MAbs targeting different epitopes were tested, synergy was seen in all paired MAbs, except for one combination that revealed additive effects. The lowest effective antibody concentration for 50% viral neutralization (EC50) and EC90 were achieved with combinations of MAbs b12, 2F5, 2G12, and the anti-V3 MAb 694/98D. Depending on the combination regimen, the concentration of MAbs required to reach 90% virus neutralization was reduced approximately 2- to 25-fold as compared to the dose requirement of individual MAbs to produce the same effect. Synergy of the combination regimens implies that combinations of antibodies may have a role in passive immunoprophylaxis against HIV-1. The ability of SHIV to replicate in rhesus macaques will allow us to test such approaches in vivo.

  9. Human H5N2 avian influenza infection in Japan and the factors associated with high H5N2-neutralizing antibody titer.

    PubMed

    Ogata, Tsuyoshi; Yamazaki, Yoshinao; Okabe, Nobuhiko; Nakamura, Yosikazu; Tashiro, Masato; Nagata, Noriko; Itamura, Shigeyuki; Yasui, Yoshinori; Nakashima, Kazutoshi; Doi, Mikio; Izumi, Youko; Fujieda, Takashi; Yamato, Shin'ichi; Kawada, Yuichi

    2008-01-01

    H5N2 avian influenza virus infection of humans has not been reported thus far. The first H5N2 avian influenza infection of poultry in Japan occurred in Ibaraki. The subjects were workers at 35 chicken farms in Ibaraki Prefecture, where the H5N2 virus or antibody was isolated from chickens. None of the subjects exhibited influenza symptoms. The H5N2-neutralizing antibody titers of the first and second paired sera samples were compared. To investigate the possible factors for this increase, the H5N2-neutralizing antibody titer (1:40 or more) was calculated for the second samples. A logistic regression analysis was performed to examine the association of these factors with H5N2-neutralizing antibody positivity. We performed Wilcoxon matched-pairs signed-ranked test on data collected from 257 subjects, and determined that the H5N2 antibody titers of the second paired sera samples were significantly higher than those of the first samples (P < 0.001). The H5N2 antibody titers of paired sera of 13 subjects without a history of seasonal influenza vaccination within the previous 12 months increased 4-fold or more. The percentage of antibody positivity was 32% for subjects with a history of seasonal influenza vaccination (28% of all subjects) and 13% for those without a history of the same. The adjusted odds ratio of H5N2-neutralizing antibody positivity was 4.6 (95% confidence interval: 1.6-13.7) for those aged over 40 and 3.1 (95% confidence interval: 1.6-6.1) for those with a history of seasonal influenza vaccination within the previous 12 months. The results suggest that this may have been the first avian influenza H5N2 infection of poultry to affect humans. A history of seasonal influenza vaccination might be associated with H5N2-neutralizing antibody positivity.

  10. 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.

  11. Human Monoclonal Antibodies to a Novel Cluster of Conformational Epitopes on HCV E2 with Resistance to Neutralization Escape in a Genotype 2a Isolate

    PubMed Central

    Keck, Zhen-yong; Xia, Jinming; Wang, Yong; Wang, Wenyan; Krey, Thomas; Prentoe, Jannick; Carlsen, Thomas; Li, Angela Ying-Jian; Patel, Arvind H.; Lemon, Stanley M.; Bukh, Jens; Rey, Felix A.; Foung, Steven K. H.

    2012-01-01

    The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1–6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1–6 HCVcc. Epitope mapping identified a cluster of overlapping epitopes that included nine contact residues in two E2 regions encompassing aa418–446 and aa611–616. Effect on virus entry was measured using H77C HCV retroviral pseudoparticles, HCVpp, bearing an alanine substitution at each of the contact residues. Seven of ten mutant HCVpp showed over 90% reduction compared to wild-type HCVpp and two others showed approximately 80% reduction. Interestingly, four of these antibodies bound to a linear E2 synthetic peptide encompassing aa434–446. This region on E2 has been proposed to elicit non-neutralizing antibodies in humans that interfere with neutralizing antibodies directed at an adjacent E2 region from aa410–425. The isolation of four HC-84 HMAbs binding to the peptide, aa434–446, proves that some antibodies to this region are to highly conserved epitopes mediating broad virus neutralization. Indeed, when HCVcc were passaged in the presence of each of these antibodies, virus escape was not observed. Thus, the cluster of HC-84 epitopes, designated as antigenic domain D, is

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

    PubMed

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

    2001-02-14

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

  13. Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro.

    PubMed

    Parren, P W; Marx, P A; Hessell, A J; Luckay, A; Harouse, J; Cheng-Mayer, C; Moore, J P; Burton, D R

    2001-09-01

    A major unknown in human immunodeficiency virus (HIV-1) vaccine design is the efficacy of antibodies in preventing mucosal transmission of R5 viruses. These viruses, which use CCR5 as a coreceptor, appear to have a selective advantage in transmission of HIV-1 in humans. Hence R5 viruses predominate during primary infection and persist throughout the course of disease in most infected people. Vaginal challenge of macaques with chimeric simian/human immunodeficiency viruses (SHIV) is perhaps one of the best available animal models for human HIV-1 infection. Passive transfer studies are widely used to establish the conditions for antibody protection against viral challenge. Here we show that passive intravenous transfer of the human neutralizing monoclonal antibody b12 provides dose-dependent protection to macaques vaginally challenged with the R5 virus SHIV(162P4). Four of four monkeys given 25 mg of b12 per kg of body weight 6 h prior to challenge showed no evidence of viral infection (sterile protection). Two of four monkeys given 5 mg of b12/kg were similarly protected, whereas the other two showed significantly reduced and delayed plasma viremia compared to control animals. In contrast, all four monkeys treated with a dose of 1 mg/kg became infected with viremia levels close to those for control animals. Antibody b12 serum concentrations at the time of virus challenge corresponded to approximately 400 (25 mg/kg), 80 (5 mg/kg), and 16 (1 mg/kg) times the in vitro (90%) neutralization titers. Therefore, complete protection against mucosal challenge with an R5 SHIV required essentially complete neutralization of the infecting virus. This suggests that a vaccine based on antibody alone would need to sustain serum neutralizing antibody titers (90%) of the order of 1:400 to achieve sterile protection but that lower titers, around 1:100, could provide a significant benefit. The significance of such substerilizing neutralizing antibody titers in the context of a potent

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

    PubMed Central

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

    1991-01-01

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

  15. Neutralizing and ELISA IgG antibodies to human cytomegalovirus glycoprotein complexes may help date the onset of primary infection in pregnancy.

    PubMed

    Lilleri, D; Gerna, G; Furione, M; Zavattoni, M; Spinillo, A

    2016-08-01

    Definition of onset for primary human cytomegalovirus (HCMV) infection during pregnancy is critical for several reasons, including diagnosis of pre-conceptional infections and definition of gestational age at the time of infection. To determine the onset of primary HCMV infection, differential kinetics of antibodies neutralizing infection of epithelial and fibroblast cells, as well as ELISA IgG antibodies to HCMV glycoprotein complexes (gC) gH/gL/pUL128L, gH/gL/gO, and gB were exploited and compared with conventional assays. In a series of 40 pregnant women with primary HCMV infection and ascertained HCMV-related mild clinical symptoms, the kinetics of different types of neutralizing and ELISA IgG antibodies were investigated with the aim of establishing criteria for dating the onset of primary infection in pregnant women without clinical symptoms. IgG antibodies to gB and gH/gL/pUL128L, as well as antibodies neutralizing infection of epithelial cells appeared early after infection onset (within 2-3 weeks) and increased rapidly, whereas antibodies to gH/gL/gO and antibodies neutralizing infection of fibroblasts appeared later (>30 days) and increased slowly. Both the conventional diagnostic assays (IgG, and IgM antibody, and IgG avidity index) and the novel assays for determination of antibody responses directed against HCMV gC allowed the definition of an algorithm indicating the onset of primary HCMV infection in asymptomatic women within a period of 1-2 months. New neutralization and ELISA IgG assays to HCMV gC provide additional tools for dating the onset of primary infection in pregnancy. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Mutations on the FG surface loop of human papillomavirus type 16 major capsid protein affect recognition by both type-specific neutralizing antibodies and cross-reactive antibodies.

    PubMed

    Carpentier, Guillaume S; Fleury, Maxime J J; Touzé, Antoine; Sadeyen, Jean-Rémy; Tourne, Sylvie; Sizaret, Pierre-Yves; Coursaget, Pierre

    2005-12-01

    The aim of this study was to further characterize the conformational neutralizing epitopes present on the surface-exposed FG loop of human papillomavirus (HPV) type 16 L1 major capsid protein. We have generated previously two chimeric L1 proteins by insertion of a foreign peptide encoding an epitope of the hepatitis B core (HBc) antigen within the FG loop. In addition, three other chimeric L1 proteins were obtained by replacing three different FG loop sequences by the HBc motif and three others by point mutations. All these chimeric L1 proteins retained the ability to self-assemble into virus-like particles (VLPs), with the exception of the mutant with substitution of the L1 sequence 274-279 by the HBc motif. The eight chimeric VLPs were then analyzed for differential reactivity with a set of six HPV-16 and HPV-31 monoclonal antibodies that bound to conformational and linear epitopes. The binding patterns of these monoclonal antibodies confirmed that the FG loop contained or contributed to neutralizing conformational epitopes. The results obtained suggested that the H31.F7 antibody, an anti-HPV-31 cross-reacting and neutralizing antibody, recognized a conformational epitope situated before the 266-271 sequence. In addition, H16.E70 neutralizing antibody reactivity was reduced with L1 VLPs with an Asn to Ala point mutation at position 270, suggesting that Asn is a part of the epitope recognized by this antibody. This study contributes to the understanding of the antigenic structure of HPV-16 and -31 L1 proteins by confirming that the FG loop contributes to neutralizing epitopes and suggesting the existence of both type-specific and cross-reactive conformational epitopes within the FG loop. Copyright (c) 2005 Wiley-Liss, inc.

  17. Comprehensive Profiling of Radiosensitive Human Cell Lines with DNA Damage Response Assays Identifies the Neutral Comet Assay as a Potential Surrogate for Clonogenic Survival

    PubMed Central

    Nahas, Shareef A.; Davies, Robert; Fike, Francesca; Nakamura, Kotoka; Du, Liutao; Kayali, Refik; Martin, Nathan T.; Concannon, Patrick; Gatti, Richard A.

    2015-01-01

    In an effort to explore the possible causes of human radiosensitivity and identify more rapid assays for cellular radiosensitivity, we interrogated a set of assays that evaluate cellular functions involved in recognition and repair of DNA double-strand breaks: (1) neutral comet assay, (2) radiation-induced γ-H2AX focus formation, (3) the temporal kinetics of structural maintenance of chromosomes 1 phosphorylation, (4) intra-S-phase checkpoint integrity, and (5) mitochondrial respiration. We characterized a unique panel of 19 “radiosensitive” human lymphoblastoid cell lines from individuals with undiagnosed diseases suggestive of a DNA repair disorder. Radiosensitivity was defined by reduced cellular survival using a clonogenic survival assay. Each assay identified cell lines with defects in DNA damage response functions. The highest concordance rate observed, 89% (17/19), was between an abnormal neutral comet assay and reduced survival by the colony survival assay. Our data also suggested that the neutral comet assay would be a more rapid surrogate for analyzing DNA repair/processing disorders. PMID:21962002

  18. Human Monoclonal Antibodies against Highly Conserved HR1 and HR2 Domains of the SARS-CoV Spike Protein Are More Broadly Neutralizing

    PubMed Central

    Elshabrawy, Hatem A.; Coughlin, Melissa M.; Baker, Susan C.; Prabhakar, Bellur S.

    2012-01-01

    Immune sera from convalescent patients have been shown to be effective in the treatment of patients infected with Severe Acute Respiratory Syndrome Virus (SARS-CoV) making passive immune therapy with human monoclonal antibodies an attractive treatment strategy for SARS. Previously, using Xenomouse (Amgen British Columbia Inc), we produced a panel of neutralizing Human monoclonal antibodies (HmAbs) that could specifically bind to the ectodomain of the SARS-CoV spike (S) glycoprotein. Some of the HmAbs were S1 domain specific, while some were not. In this study, we describe non-S1 binding neutralizing HmAbs that can specifically bind to the conserved S2 domain of the S protein. However, unlike the S1 specific HmAbs, the S2 specific HmAbs can neutralize pseudotyped viruses expressing different S proteins containing receptor binding domain sequences of various clinical isolates. These data indicate that HmAbs which bind to conserved regions of the S protein are more suitable for conferring protection against a wide range of SARS-CoV variants and have implications for generating therapeutic antibodies or subunit vaccines against other enveloped viruses. PMID:23185609

  19. Human monoclonal antibodies against highly conserved HR1 and HR2 domains of the SARS-CoV spike protein are more broadly neutralizing.

    PubMed

    Elshabrawy, Hatem A; Coughlin, Melissa M; Baker, Susan C; Prabhakar, Bellur S

    2012-01-01

    Immune sera from convalescent patients have been shown to be effective in the treatment of patients infected with Severe Acute Respiratory Syndrome Virus (SARS-CoV) making passive immune therapy with human monoclonal antibodies an attractive treatment strategy for SARS. Previously, using Xenomouse (Amgen British Columbia Inc), we produced a panel of neutralizing Human monoclonal antibodies (HmAbs) that could specifically bind to the ectodomain of the SARS-CoV spike (S) glycoprotein. Some of the HmAbs were S1 domain specific, while some were not. In this study, we describe non-S1 binding neutralizing HmAbs that can specifically bind to the conserved S2 domain of the S protein. However, unlike the S1 specific HmAbs, the S2 specific HmAbs can neutralize pseudotyped viruses expressing different S proteins containing receptor binding domain sequences of various clinical isolates. These data indicate that HmAbs which bind to conserved regions of the S protein are more suitable for conferring protection against a wide range of SARS-CoV variants and have implications for generating therapeutic antibodies or subunit vaccines against other enveloped viruses.

  20. Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation.

    PubMed

    Sabbatucci, Michela; Covino, Daniela Angela; Purificato, Cristina; Mallano, Alessandra; Federico, Maurizio; Lu, Jing; Rinaldi, Arturo Ottavio; Pellegrini, Matteo; Bona, Roberta; Michelini, Zuleika; Cara, Andrea; Vella, Stefano; Gessani, Sandra; Andreotti, Mauro; Fantuzzi, Laura

    2015-01-22

    Macrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members. CCL2 neutralization potently reduced the number of p24 Gag+ cells during the course of either productive or single cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viral DNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of the modulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression, to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replication mediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was type I IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expression revealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in the defence response to viruses. Neutralization of endogenous CCL2 determines a profound

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    2008-03-01

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

  3. 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.

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

    PubMed

    Matthews, Luke J; Butler, Paul M

    2011-07-01

    Numerous lines of evidence suggest that Homo sapiens evolved as a distinct species in Africa by 150,000 years before the present (BP) and began major migrations out-of-Africa ∼50,000 BP. By 20,000 BP, our species had effectively colonized the entire Old World, and by 12,000 BP H. sapiens had a global distribution. We propose that this rapid migration into new habitats selected for individuals with low reactivity to novel stressors. Certain dopamine receptor D4 (DRD4) polymorphisms are associated with low neuronal reactivity and increased exploratory behavior, novelty seeking, and risk taking, collectively considered novelty-seeking trait (NS). One previous report (Chen et al.: Evol Hum Behav 20 (1999) 309-324) demonstrated a correlation between migratory distance and the seven-repeat (7R) VNTR DRD4 allele at exon 3 for human populations. This study, however, failed to account for neutral genetic processes (drift and admixture) that might create such a correlation in the absence of natural selection. Furthermore, additional loci surrounding DRD4 are now recognized to influence NS. Herein we account for neutral genetic structure by modeling the nonindependence of neutral allele frequencies between human populations. We retest the DRD4 exon 3 alleles, and also test two other loci near DRD4 that are associated with NS. We conclude there is an association between migratory distance and DRD4 exon 3 2R and 7R alleles that cannot be accounted for by neutral genetic processes alone. Copyright © 2011 Wiley-Liss, Inc.

  5. Cross-reactive antibodies in convalescent SARS patients' sera against the emerging novel human coronavirus EMC (2012) by both immunofluorescent and neutralizing antibody tests.

    PubMed

    Chan, Kwok-Hung; Chan, Jasper Fuk-Woo; Tse, Herman; Chen, Honglin; Lau, Candy Choi-Yi; Cai, Jian-Piao; Tsang, Alan Ka-Lun; Xiao, Xincai; To, Kelvin Kai-Wang; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Zheng, Bo-Jiang; Wang, Ming; Yuen, Kwok-Yung

    2013-08-01

    A severe acute respiratory syndrome (SARS)-like disease due to a novel betacoronavirus, human coronavirus EMC (HCoV-EMC), has emerged recently. HCoV-EMC is phylogenetically closely related to Tylonycteris-bat-coronavirus-HKU4 and Pipistrellus-bat-coronavirus-HKU5 in Hong Kong. We conducted a seroprevalence study on archived sera from 94 game-food animal handlers at a wild life market, 28 SARS patients, and 152 healthy blood donors in Southern China to assess the zoonotic potential and evidence for intrusion of HCoV-EMC and related viruses into humans. Anti-HCoV-EMC and anti-SARS-CoV antibodies were detected using screening indirect immunofluorescence (IF) and confirmatory neutralizing antibody tests. Two (2.1%) animal handlers had IF antibody titer of ≥ 1:20 against both HCoV-EMC and SARS-CoV with neutralizing antibody titer of <1:10. No blood donor had antibody against either virus. Surprisingly, 17/28 (60.7%) of SARS patients had significant IF antibody titers with 7/28 (25%) having anti-HCoV-EMC neutralizing antibodies at low titers which significantly correlated with that of HCoV-OC43. Bioinformatics analysis demonstrated a significant B-cell epitope overlapping the heptad repeat-2 region of Spike protein. Virulence of SARS-CoV over other betacoronaviruses may boost cross-reactive neutralizing antibodies against other betacoronaviruses. Convalescent SARS sera may contain cross-reactive antibodies against other betacoronaviruses and confound seroprevalence study for HCoV-EMC. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  6. 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.

  7. A Virus-Like Particle Vaccine Elicits Broad Neutralizing Antibody Responses in Humans to All Chikungunya Virus Genotypes.

    PubMed

    Goo, Leslie; Dowd, Kimberly A; Lin, Tsai-Yu; Mascola, John R; Graham, Barney S; Ledgerwood, Julie E; Pierson, Theodore C

    2016-11-15

    Chikungunya virus (CHIKV) is an alphavirus that has emerged as a global health burden. There are 3 CHIKV genotypes: Asian, West African, and Eastern/Central/South African. No licensed CHIKV vaccine is available, and whether the antibody response elicited by one genotype can neutralize heterologous genotypes is unclear. We assessed neutralizing antibody (NAb) responses of volunteers in a phase 1 study of a CHIKV vaccine against 9 viral strains representing all 3 genotypes. Minimal differences in vaccine-elicited NAb responses were observed among genotypes, suggesting that vaccination with a single CHIKV strain can elicit cross-protective NAbs against all 3 genotypes. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2010-12-17

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

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

    SciTech Connect

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

    2011-08-09

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

  11. An alphavirus vector overcomes the presence of neutralizing antibodies and elevated numbers of Tregs to induce immune responses in humans with advanced cancer

    PubMed Central

    Morse, Michael A.; Hobeika, Amy C.; Osada, Takuya; Berglund, Peter; Hubby, Bolyn; Negri, Sarah; Niedzwiecki, Donna; Devi, Gayathri R.; Burnett, Bruce K.; Clay, Timothy M.; Smith, Jonathan; Lyerly, H. Kim

    2010-01-01

    Therapeutic anticancer vaccines are designed to boost patients’ immune responses to tumors. One approach is to use a viral vector to deliver antigen to in situ DCs, which then activate tumor-specific T cell and antibody responses. However, vector-specific neutralizing antibodies and suppressive cell populations such as Tregs remain great challenges to the efficacy of this approach. We report here that an alphavirus vector, packaged in virus-like replicon particles (VRP) and capable of efficiently infecting DCs, could be repeatedly administered to patients with metastatic cancer expressing the tumor antigen carcinoembryonic antigen (CEA) and that it overcame high titers of neutralizing antibodies and elevated Treg levels to induce clinically relevant CEA-specific T cell and antibody responses. The CEA-specific antibodies mediated antibody-dependent cellular cytotoxicity against tumor cells from human colorectal cancer metastases. In addition, patients with CEA-specific T cell responses exhibited longer overall survival. These data suggest that VRP-based vectors can overcome the presence of neutralizing antibodies to break tolerance to self antigen and may be clinically useful for immunotherapy in the setting of tumor-induced immunosuppression. PMID:20679728

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

    PubMed

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

    2011-06-10

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

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

    PubMed Central

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

    2011-01-01

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of β-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs. PMID:21489992

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2015-04-17

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

  18. 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

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

    PubMed

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

    2011-11-03

    The majority of cervical cancers are associated with infection by one or more Human Papillomavirus (HPV) types from just two distinct Alpha-Papillomavirus species groups, A7 and A9. The extent to which the current HPV16/18 vaccines will protect against other genetically related HPV types is of interest to inform vaccine implementation, cervical disease surveillance and the development of second generation HPV vaccines. The aim of this study was to determine the frequency and titer of neutralizing antibodies against a range of A7 (18, 39, 45, 59, 68) and A9 (16, 31, 33, 35, 52, 58) HPV types using sera from individuals immunized with the bivalent HPV vaccine within the school-based, UK national HPV immunization programme. Serum samples were collected from 69 girls aged 13-14 years, a median 5.9 months (inter-quartile range, IQR, 5.7-6.0) after their third vaccine dose. Cross-neutralizing antibodies against HPV31, HPV33, HPV35 and HPV45 were common and strongly associated with the titer for the related vaccine-type, but were considerably lower (<1%) than their related vaccine type-specific response. The low prevalence of these HPV types in the population and the ages within the study cohort suggest these responses are due to vaccination. It is unclear whether such low levels of neutralizing antibodies would be sufficient to protect at the site of infection in the absence of other immune effectors but the coincidence with HPV types reported from efficacy studies is intriguing. The utility of neutralizing antibodies as surrogate markers of protection remains to be determined. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    PubMed Central

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

    1995-01-01

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

  1. 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.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-03-15

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

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

    PubMed

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

    2010-10-01

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

  5. Growth characteristics of human parechovirus 1 to 6 on different cell lines and cross- neutralization of human parechovirus antibodies: a comparison of the cytopathic effect and real time PCR.

    PubMed

    Westerhuis, Brenda M; Jonker, Sara C M; Mattao, Sandhia; Benschop, Kimberley S M; Wolthers, Katja C

    2013-05-13

    Human parechoviruses (HPeVs) are among the most frequently detected picornaviruses in humans. HPeVs are usually associated with mild gastrointestinal and respiratory symptoms with the exception of HPeV3 which causes neonatal sepsis and CNS infection. Previous studies showed various results in culturing different HPeV genotypes, inducing only a low cytopathic effect (CPE). In vitro growth characteristics of the different HPeV genotypes in a range of 10 different cell lines are scored with CPE and measured in the supernatant by real time PCR. In the optimal cell line for each genotype a standard neutralization assay with the available HPeV antibodies (Abs) was performed and scored by CPE and measured by real time PCR. All six HPeV types were able to replicate on the RD99, A549, and Vero cell lines. HPeV1 was the only genotype able to replicate on all cell lines. Most efficient growth of HPeV1, 2, 4, 5, and 6 was shown on the HT29 cell line, while HPeV3 was unable to replicate on HT29. In all cases viral replication could be measured by real time PCR before CPE appeared. The polyclonal Abs available against HPeV1, 2, 4 and 5 all showed neutralization of their respective genotype after 7 days with inhibition of >60% in real time PCR and full inhibition of CPE, although cross-neutralization is shown. Replication of HPeV3 could only be inhibited by 12% by the anti-HPeV3 (aHPeV3) Ab and no inhibition of CPE was shown after 7 days. When replication is monitored by PCR, growth of HPeV genotypes 1 to 6 is supported by most of the cell lines tested, where viral replication is measured before appearance of CPE. A combination of HT29 and Vero cells would therefore support replication of all culturable HPeV types, so viral replication could be detected by PCR within 3 days for all genotypes.In addition, we showed efficient neutralization for HPeV1, 2, 4, 5, while cross- neutralization was shown between these types, indicating possible common neutralizing epitopes. For HPeV3 no

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

    SciTech Connect

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

    2006-01-01

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

  7. Primary human cytomegalovirus infections: kinetics of ELISA-IgG and neutralizing antibody in pauci/asymptomatic pregnant women vs symptomatic non-pregnant subjects.

    PubMed

    Fornara, Chiara; Furione, Milena; Lilleri, Daniele; Cane, Ilaria; Revello, M Grazia; Zavattoni, Maurizio; Gerna, Giuseppe

    2015-03-01

    Human cytomegalovirus infections are mostly asymptomatic in infants and young children, while they are often associated with overt clinical symptoms in adults. To verify whether the antibody response to HCMV is more potent in symptomatic non-pregnant adults as compared to asymptomatic/paucisymptomatic pregnant women. Overall, 36 consecutive pregnant women with primary HCMV infection were compared with 10 consecutive symptomatic non-pregnant subjects with primary HCMV infection and overt clinical symptoms. Levels of IgG antibody responses to HCMV-infected cell lysate and the pentamer gH/gL/pUL128L, gH/gL and gB HCMV glycoprotein complexes as well as neutralizing antibodies preventing infection of epithelial cells (ARPE-19) and human embryonic lung fibroblast (HELF) cells were compared at intervals of 1-30, 31-60, 61-90, 91-180 and 181-360 days after onset of infection. In parallel, viral load was quantified by real-time PCR. In symptomatic non-pregnant subjects, the IgG responses to HCMV lysate as well as to gH/gL and ARPE-19 neutralizing antibodies were significantly higher from 31 to 60 through 180 days after infection onset. In the same patients, the IgG antibody responses to the pentamer and HELF-neutralizing antibody were significantly higher starting 90 days post-infection. The presence of overt clinical symptoms is associated with a significantly higher antibody response (concomitantly with a higher viral load) in non-pregnant subjects with symptomatic primary HCMV infection as compared to pregnant women with paucisymptomatic/ asymptomatic primary infection (and lower viral load). Copyright © 2015 Elsevier B.V. All rights reserved.

  8. 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.

  9. Beyond Viral Neutralization.

    PubMed

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

    2017-01-13

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

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

    PubMed

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

    2012-08-01

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

  11. Human BJ Fibroblasts is an Alternative to Mouse BALB/c 3T3 Cells in In Vitro Neutral Red Uptake Assay.

    PubMed

    Mannerström, Marika; Toimela, Tarja; Sarkanen, Jertta-Riina; Heinonen, Tuula

    2017-09-01

    The OECD GD 129 BALB/c 3T3 neutral red uptake (NRU) assay is a standardized test method for estimating starting dose for an acute oral systemic toxicity test in rodents. Mouse BALB/c 3T3 fibroblasts are the most commonly used cells in the NRU assay. We have previously transferred and validated BALB/c 3T3 NRU assay in our GLP laboratory. Subsequently, in order to obtain more human-relevant cytotoxicity data, we performed an intralaboratory validation using human BJ fibroblasts in the NRU assay instead of mouse BALB/c 3T3 fibroblasts. Here, we present comparative cytotoxicity data of 26 different test chemicals (pharmaceuticals, industrial chemicals, pesticides and food additives) produced with both BALB/c 3T3 NRU and BJ NRU assays. © 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

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

    PubMed

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

    2006-02-15

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

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

    PubMed

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

    2014-10-21

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

  14. 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.

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

    PubMed Central

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

    1997-01-01

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

  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. Neutralizing Antibody Responses to Human Herpesviruses 6 and 7 Do Not Cross-React with Each Other, and Maternal Neutralizing Antibodies Contribute to Sequential Infection with These Viruses in Childhood

    PubMed Central

    Yoshida, Mariko; Torigoe, Sadayoshi; Ikeue, Kumiko; Yamada, Masao

    2002-01-01

    Seroprevalence of human herpesvirus 6 (HHV-6) and HHV-7 infections is very high throughout the world, and almost all people are exposed first to HHV-6 and second to HHV-7 in their childhood. However, it is not clear whether the neutralizing (NT) antibody response between each virus is cross-reactive or not. To elucidate the NT antibody response between each virus, 55 serum samples from an adult group (subjects 22 to 88 years old) and 60 serum samples from a young group (subjects 2 to 18 years old) were examined by a dot blot method for detecting viral late antigen. Thirty-nine serum samples obtained from cord bloods and a few serum samples obtained from pediatric patients with exanthem subitum were also examined to assess the maternal transferred NT antibodies against each virus. The NT antibody titers against HHV-7 in the adult group remained high throughout all the individuals, and none were negative. Those against HHV-6 were high values in the young group but low values, including negative values (three samples), in the adult group. These results suggested that the NT antibody response to either HHV-6 or HHV-7 in each individual was specific to each virus and did not cross-react with each other. In the adult group, the NT antibody response to HHV-6 decreased, while that to HHV-7 remained high throughout all the individuals. Maternal transferred NT antibody titers against HHV-7 were higher and remained longer after birth than those of HHV-6, and these findings were in accord with the clinical observation that HHV-6 infection usually occurs earlier than HHV-7 infection. PMID:11874884

  18. Generation of human neutralizing monoclonal antibodies against the 2009 pandemic H1N1 virus from peripheral blood memory B lymphocytes

    PubMed Central

    Wang, Hao; Ma, Chi; Lu, Yanlai; Ji, Xu; Pang, Yongsheng; Hua, Fang; Cui, Lianxian; Ba, Denian; He, Wei

    2013-01-01

    The 2009 H1N1 influenza pandemic demonstrated the significance of a global health threat to human beings. Although pandemic H1N1 vaccines have been rapidly developed, passive serotherapy may offer superior immediate protection against infections in children, the elderly and immune-compromised patients during an influenza pandemic. Here, we applied a novel strategy based on Epstein–Barr virus (EBV)-immortalized peripheral blood memory B cells to screen high viral neutralizing monoclonal antibodies (MAbs) from individuals vaccinated with the 2009 pandemic H1N1 vaccine PANFLU.1. Through a massive screen of 13 090 immortalized memory B-cell clones from three selected vaccinees, seven MAbs were identified with both high viral neutralizing capacities and hemagglutination inhibition (HAI) activities against the 2009 pandemic H1N1 viruses. These MAbs may have important clinical implications for passive serotherapy treatments of infected patients with severe respiratory syndrome, especially children, the elderly and immunodeficient individuals. Our successful strategy for generating high-affinity MAbs from EBV-immortalized peripheral blood memory B cells may also be applicable to other infectious or autoimmune diseases. PMID:23912783

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

    PubMed

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

    2013-10-15

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

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

    PubMed

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

    2013-01-01

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

  1. Influence of elastin-like peptide fusions on the quantity and quality of a tobacco-derived human immunodeficiency virus-neutralizing antibody.

    PubMed

    Floss, Doreen M; Sack, Markus; Arcalis, Elsa; Stadlmann, Johannes; Quendler, Heribert; Rademacher, Thomas; Stoger, Eva; Scheller, Jürgen; Fischer, Rainer; Conrad, Udo

    2009-12-01

    The use of vaginal microbicides containing human immunodeficiency virus (HIV)-neutralizing antibodies (nAbs) is a promising strategy to prevent HIV-1 infection. Although antibodies are predominantly manufactured using mammalian cells, elastin-like peptide (ELP) fusion technology improves the stability of recombinant, plant-produced proteins and facilitates their purification, making plants an alternative platform for antibody production. We generated transgenic tobacco plants accumulating four different formats of the anti-HIV-1 antibody 2G12 in the endoplasmic reticulum (ER), i.e. with ELP on either the light or heavy chain, on both, or on neither. Detailed analysis of affinity-purified antibodies by surface plasmon resonance spectroscopy showed that the kinetic binding parameters of all formats were identical to 2G12 lacking ELP produced in Chinese hamster ovary (CHO) cells. Importantly, protein purification from seeds by inverse transition cycling (ITC) did not affect the binding kinetics. Analysis of heavy chain N-glycans from leaf-derived antibodies showed that retrieval to the ER was efficient for all formats. In seeds, however, N-glycans on the naked antibody were extensively trimmed compared with those on the ELP fusion formats, and were localized to a different subcellular compartment. The in vitro HIV-neutralization properties of the tobacco-derived 2G12 were equivalent to or better than those of the CHO counterpart.

  2. 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

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

    DOE PAGES

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

    2015-08-03

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

  4. 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.

  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; ...

    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.

    PubMed

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

    2015-08-01

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

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

    PubMed Central

    2013-01-01

    Background Differently from HIV-1, HIV-2 disease progression usually takes decades without antiretroviral therapy and the majority of HIV-2 infected individuals survive as elite controllers with normal CD4+ T cell counts and low or undetectable plasma viral load. Neutralizing antibodies (Nabs) are thought to play a central role in HIV-2 evolution and pathogenesis. However, the dynamic of the Nab response and resulting HIV-2 escape during acute infection and their impact in HIV-2 evolution and disease progression remain largely unknown. Our objective was to characterize the Nab response and the molecular and phenotypic evolution of HIV-2 in association with Nab escape in the first years of infection in two children infected at birth. Results CD4+ T cells decreased from about 50% to below 30% in both children in the first five years of infection and the infecting R5 viruses were replaced by X4 viruses within the same period. With antiretroviral therapy, viral load in child 1 decreased to undetectable levels and CD4+ T cells recovered to normal levels, which have been sustained at least until the age of 12. In contrast, viral load increased in child 2 and she progressed to AIDS and death at age 9. Beginning in the first year of life, child 1 raised high titers of antibodies that neutralized primary R5 isolates more effectively than X4 isolates, both autologous and heterologous. Child 2 raised a weak X4-specific Nab response that decreased sharply as disease progressed. Rate of evolution, nucleotide and amino acid diversity, and positive selection, were significantly higher in the envelope of child 1 compared to child 2. Rates of R5-to-X4 tropism switch, of V1 and V3 sequence diversification, and of convergence of V3 to a β-hairpin structure were related with rate of escape from the neutralizing antibodies. Conclusion Our data suggests that the molecular and phenotypic evolution of the human immunodeficiency virus type 2 envelope are related with the dynamics of the

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

    PubMed

    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

    Differently from HIV-1, HIV-2 disease progression usually takes decades without antiretroviral therapy and the majority of HIV-2 infected individuals survive as elite controllers with normal CD4⁺ T cell counts and low or undetectable plasma viral load. Neutralizing antibodies (Nabs) are thought to play a central role in HIV-2 evolution and pathogenesis. However, the dynamic of the Nab response and resulting HIV-2 escape during acute infection and their impact in HIV-2 evolution and disease progression remain largely unknown. Our objective was to characterize the Nab response and the molecular and phenotypic evolution of HIV-2 in association with Nab escape in the first years of infection in two children infected at birth. 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. 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

  11. 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

  12. Development and characterization of a pre-treatment procedure to eliminate human monoclonal antibody therapeutic drug and matrix interference in cell-based functional neutralizing antibody assays.

    PubMed

    Xu, Weifeng; Jiang, Hao; Titsch, Craig; Haulenbeek, Jonathan R; Pillutla, Renuka C; Aubry, Anne-Françoise; DeSilva, Binodh S; Arnold, Mark E; Zeng, Jianing; Dodge, Robert W

    2015-01-01

    Biological therapeutics can induce an undesirable immune response resulting in the formation of anti-drug antibodies (ADA), including neutralizing antibodies (NAbs). Functional (usually cell-based) NAb assays are preferred to determine NAb presence in patient serum, but are often subject to interferences from numerous serum factors, such as growth factors and disease-related cytokines. Many functional cell-based NAb assays are essentially drug concentration assays that imply the presence of NAbs by the detection of small changes in functional drug concentration. Any drug contained in the test sample will increase the total amount of drug in the assay, thus reducing the sensitivity of NAb detection. Biotin-drug Extraction with Acid Dissociation (BEAD) has been successfully applied to extract ADA, thereby removing drug and other interfering factors from human serum samples. However, to date there has been no report to estimate the residual drug level after BEAD treatment when the drug itself is a human monoclonal antibody; mainly due to the limitation of traditional ligand-binding assays. Here we describe a universal BEAD optimization procedure for human monoclonal antibody (mAb) drugs by using a LC-MS/MS method to simultaneously measure drug (a mutant human IgG4), NAb positive control (a mouse IgG), and endogenous human IgGs as an indicator of nonspecific carry-over in the BEAD eluate. This is the first report demonstrating that residual human mAb drug level in clinical sample can be measured after BEAD pre-treatment, which is critical for further BEAD procedure optimization and downstream immunogenicity testing. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    1994-01-01

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

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

    PubMed

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

    2013-01-01

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

  15. 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

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

    PubMed Central

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

    2010-01-01

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

  17. Fully human monoclonal antibody directed to proteolytic cleavage site in severe acute respiratory syndrome (SARS) coronavirus S protein neutralizes the virus in a rhesus macaque SARS model.

    PubMed

    Miyoshi-Akiyama, Tohru; Ishida, Isao; Fukushi, Masaya; Yamaguchi, Keina; Matsuoka, Yusuke; Ishihara, Takashi; Tsukahara, Masayoshi; Hatakeyama, Seisuke; Itoh, Norikazu; Morisawa, Aki; Yoshinaka, Yoshiyuki; Yamamoto, Naoki; Lianfeng, Zhang; Chuan, Qin; Kirikae, Teruo; Sasazuki, Takehiko

    2011-06-01

    There is still no effective method to prevent or treat severe acute respiratory syndrome (SARS), which is caused by SARS coronavirus (CoV). In the present study, we evaluated the efficacy of a fully human monoclonal antibody capable of neutralizing SARS-CoV in vitro in a Rhesus macaque model of SARS. The antibody 5H10 was obtained by vaccination of KM mice bearing human immunoglobulin genes with Escherichia coli-producing recombinant peptide containing the dominant epitope of the viral spike protein found in convalescent serum samples from patients with SARS. 5H10, which recognized the same epitope that is also a cleavage site critical for the entry of SARS-CoV into host cells, inhibited propagation of the virus and pathological changes found in Rhesus macaques infected with the virus through the nasal route. In addition, we analyzed the mode of action of 5H10, and the results suggested that 5H10 inhibited fusion between the virus envelope and host cell membrane. 5H10 has potential for use in prevention and treatment of SARS if it reemerges. This study represents a platform to produce fully human antibodies against emerging infectious diseases in a timely and safe manner.

  18. Neutral glycosphingolipids in human blood: a precise mass spectrometry analysis with special reference to lipoprotein-associated Shiga toxin receptors[S

    PubMed Central

    Schweppe, Christian H.; Hoffmann, Petra; Nofer, Jerzy-Roch; Pohlentz, Gottfried; Mormann, Michael; Karch, Helge; Friedrich, Alexander W.; Müthing, Johannes

    2010-01-01

    Shiga toxin (Stx)-producing Escherichia coli are the leading cause of hemorrhagic colitis and life-threatening extraintestinal complications in humans. Stx1 and Stx2 are transferred by yet to be delineated mechanisms from the intestine to the circulation where they injure microvascular endothelial cells. The resulting vascular lesions cause renal failure and brain damage. Because lipoproteins are potential carriers of Stx through the circulation, we investigated human lipoprotein-associated neutral glycosphingolipids (GSLs) with emphasis on high (globotriaosylceramide) and low (globotetraosylceramide) affinity Stx-receptors. TLC overlay employing Stx1, Stx2, and anti-GSL antibodies demonstrated preferential distribution of globo-series GSLs to very low- and low-density lipoproteins compared with minor association with high-density lipoproteins. Electrospray ionization quadrupole time-of-flight mass spectrometry portrayed C24:0/C24:1 and C16:0 as the major fatty acid of the ceramide moieties of Stx-receptors carrying nonvarying d18:1 sphingosine. This structural heterogeneity was also found in precursor lactosylceramide, glucosylceramide, and galactosylceramide, the last showing an exceptionally high degree of hydroxylated C24 fatty acids. Our findings provide the basis for exploring the functional role of lipoprotein-associated Stx-receptors in human blood. PMID:20444989

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    1997-10-01

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

  1. 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.

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

    PubMed

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

    2016-03-01

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

  3. 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.

  4. 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.

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

    PubMed Central

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

    2009-01-01

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

  6. 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

  7. Global CNS Gene Delivery and Evasion of Anti-AAV Neutralizing Antibodies by Intrathecal AAV Administration in Non-Human Primates

    PubMed Central

    Gray, Steven J.; Kalburgi, Sahana Nagabhushan; McCown, Thomas J.; Samulski, R. Jude

    2012-01-01

    Injection of AAV into the cerebrospinal fluid (CSF) offers a means to achieve widespread transgene delivery to the central nervous system, where the doses can be readily translated from small to large animals. In contrast to studies with other serotypes (AAV2, AAV4, AAV5) in rodents, we report that a naturally-occurring capsid (AAV9) and rationally-engineered capsid (AAV2.5) are able to achieve broad transduction throughout the brain and spinal cord parenchyma following a single injection into the CSF (via cisterna magna or lumbar cistern) in non-human primates (NHP). Using either vector at a dose of ~2×1012 vg per 3-6 kg animal, approximately 2% of the entire brain and spinal cord was transduced, covering all regions of the CNS. AAV9 in particular displayed efficient transduction of spinal cord motor neurons. The peripheral organ biodistribution was highly reduced compared to intravascular delivery, and the presence of circulating anti-AAV neutralizing antibodies up to a 1:128 titer had no inhibitory effect on CNS gene transfer. Intra-CSF delivery effectively translates from rodents to NHPs, which provides encouragement for the use of this approach in humans to treat motor neuron and lysosomal storage diseases. PMID:23303281

  8. A Non-VH1-69 Heterosubtypic Neutralizing Human Monoclonal Antibody Protects Mice against H1N1 and H5N1 Viruses

    PubMed Central

    Solforosi, Laura; Moreno, Guisella J.; Sun, Xiangjie; Tumpey, Terrence M.; Gubareva, Larisa V.; Mishin, Vasiliy; Clementi, Massimo; Burioni, Roberto

    2012-01-01

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

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

    PubMed

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

    2014-07-18

    Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.

  10. Human cytomegalovirus hyperimmune globulin not only neutralizes HCMV infectivity, but also inhibits HCMV-induced intracellular NF-kappaB, Sp1, and PI3-K signaling pathways.

    PubMed

    Andreoni, Kenneth A; Wang, Xin; Huang, Shu-Mei; Huang, Eng-Shang

    2002-05-01

    Inhibition of virus-induced intracellular signaling pathways and viral infectivity are our ultimate goals in the development of effective antiviral agents to control human cytomegalovirus (HCMV) infections. The HCMV hyperimmune globulin may meet such criteria. In a human embryonic lung (HEL) fibroblast culture model, pretreatment of Towne strain HCMV with HCMV hyperimmune globulin was shown to inhibit viral infectivity successfully, as measured by a standard plaque assay. The extracellular viral titers and extracellular viral DNA, as measured by plaque assay and PCR, respectively, were also decreased. In addition, the HCMV hyperimmune globulin prevented HCMV from inducing the intracellular activation of NF-kappaB, Sp-1, and PI3-K signaling pathways. The PI3-K pathway was examined by following phosphorylation (activation) of two of its downstream kinases, Akt and p70S6K. HCMV hyperimmune globulin also prevented the production of immediate early, early, and late viral proteins. These studies show that HCMV hyperimmune globulin neutralization of HCMV prevents the earliest known events observed after viral envelope glycoproteins bind their cell membrane receptors, i.e., NF-kappaB, Sp-1 and PI3-K activation. This suggests that HCMV hyperimmune globulin not only can inhibit viral infectivity, but can also prevent the abnormal cellular signaling that may induce unwanted cellular proliferation or cytokine synthesis. Copyright 2002 Wiley-Liss, Inc.

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

    PubMed

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

    2017-01-01

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

  12. 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.

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

    PubMed

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

    1999-01-01

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

  14. Impact of naturally occurring variation in the human papillomavirus (HPV) 33 capsid proteins on recognition by vaccine-induced cross-neutralizing antibodies.

    PubMed

    Godi, Anna; Bissett, Sara L; Miller, Elizabeth; Beddows, Simon

    2017-07-01

    We investigated naturally occurring variation within the major (L1) and minor (L2) capsid proteins of human papillomavirus (HPV) genotype 33. Pseudoviruses (PsV) representing HPV33 lineages A1, A2, A3, B and C exhibited comparable particle-to-infectivity ratios and morphology but demonstrated a decreased sensitivity (A2, A3, B and C) to cross-neutralization by HPV vaccine antibodies compared to the A1 sublineage. Chimeric PsVs demonstrated that these differences in sensitivity were due to polymorphisms in the L1 protein, with little or no influence from variation within the L2 protein. Site-directed mutagenesis of the L1 gene identified the DE loop residue 133 and the FG residue 266 as being critical for conferring this differential sensitivity. The use of HPV33 homology models based upon the HPV16 crystal structure suggested that they are likely to act independently on more than one antibody epitope. These data improve our understanding of the potential impact of natural capsid variation on recognition by vaccine antibodies.