Bio

Clinical Focus


  • Pathology
  • Transfusion Medicine

Academic Appointments


Administrative Appointments


  • Associate Chair for Academic Affairs, Stanford University School of Medicine - Pathology (2004 - Present)

Professional Education


  • Residency:Stanford University School of Medicine (1980) CA
  • Residency:UCSF Medical Center (1977) CA
  • Internship:San Francisco General Hospital (1976) CA
  • Medical Education:UCSD Medical Center (1975) CA

Research & Scholarship

Current Research and Scholarly Interests


The Foung laboratory is focused on the early events of hepatitis C virus infection- virus attachment and entry to susceptible cells. The approach is through the generation of human monoclonal antibodies (HMAbs) to the virus envelope proteins with an emphasis on antibodies to conformational epitopes. A large panel of HMAbs has been produced with many broadly reactive to different HCV isolates common in the US and elsewhere. Functional studies showed that the HCV envelope E2 glycoprotein is organized in distinct immunological and functional clusters with epitopes within each cluster or domain sharing similar functional and structural properties. At least three domains mediate virus neutralization and antibodies to epitopes within two domains inhibit virus binding to the virus receptor, CD81. Interestingly, some of these antibodies also block a second step in virus entry by inhibiting the low pH induced virus envelope conformational rearrangement that is necessary to trigger virus fusion with the endosomal membrane. These findings support the view that virus entry is mediated by only specific determinants on the virus surface and appear to be restricted to distinct immunogenic domains. This is in contrast for other viruses where the convention is that neutralization is the result of a critical number of any binding sites being occupied and preventing virus entry through steric hindrance. Studies are underway on expanding this model of the virus envelope glycoproteins, which will be important in linking structure and function.

Teaching

2013-14 Courses


Publications

Journal Articles


  • Structural basis of HCV neutralization by human monoclonal antibodies resistant to viral neutralization escape. PLoS pathogens Krey, T., Meola, A., Keck, Z., Damier-Piolle, L., Foung, S. K., Rey, F. A. 2013; 9 (5)

    Abstract

    The high mutation rate of hepatitis C virus allows it to rapidly evade the humoral immune response. However, certain epitopes in the envelope glycoproteins cannot vary without compromising virus viability. Antibodies targeting these epitopes are resistant to viral escape from neutralization and understanding their binding-mode is important for vaccine design. Human monoclonal antibodies HC84-1 and HC84-27 target conformational epitopes overlapping the CD81 receptor-binding site, formed by segments aa434-446 and aa610-619 within the major HCV glycoprotein E2. No neutralization escape was yet observed for these antibodies. We report here the crystal structures of their Fab fragments in complex with a synthetic peptide comprising aa434-446. The structures show that the peptide adopts an ?-helical conformation with the main contact residues F(442) and Y(443) forming a hydrophobic protrusion. The peptide retained its conformation in both complexes, independently of crystal packing, indicating that it reflects a surface feature of the folded glycoprotein that is exposed similarly on the virion. The same residues of E2 are also involved in interaction with CD81, suggesting that the cellular receptor binds the same surface feature and potential escape mutants critically compromise receptor binding. In summary, our results identify a critical structural motif at the E2 surface, which is essential for virus propagation and therefore represents an ideal candidate for structure-based immunogen design for vaccine development.

    View details for DOI 10.1371/journal.ppat.1003364

    View details for PubMedID 23696737

  • Contrasting Roles of Mitogen-Activated Protein Kinases in Cellular Entry and Replication of Hepatitis C Virus: MKNK1 Facilitates Cell Entry JOURNAL OF VIROLOGY Kim, S., Ishida, H., Yamane, D., Yi, M., Swinney, D. C., Foung, S., Lemon, S. M. 2013; 87 (8): 4214-4224

    Abstract

    The human kinome comprises over 800 individual kinases. These contribute in multiple ways to regulation of cellular metabolism and may have direct and indirect effects on virus replication. Kinases are tempting therapeutic targets for drug development, but achieving sufficient specificity is often a challenge for chemical inhibitors. While using inhibitors to assess whether c-Jun N-terminal (JNK) kinases regulate hepatitis C virus (HCV) replication, we encountered unexpected off-target effects that led us to discover a role for a mitogen-activated protein kinase (MAPK)-related kinase, MAPK interacting serine/threonine kinase 1 (MKNK1), in viral entry. Two JNK inhibitors, AS601245 and SP600125, as well as RNA interference (RNAi)-mediated knockdown of JNK1 and JNK2, enhanced replication of HCV replicon RNAs as well as infectious genome-length RNA transfected into Huh-7 cells. JNK knockdown also enhanced replication following infection with cell-free virus, suggesting that JNK actively restricts HCV replication. Despite this, AS601245 and SP600125 both inhibited viral entry. Screening of a panel of inhibitors targeting kinases that may be modulated by off-target effects of AS601245 and SP600125 led us to identify MKNK1 as a host factor involved in HCV entry. Chemical inhibition or siRNA knockdown of MKNK1 significantly impaired entry of genotype 1a HCV and HCV-pseudotyped lentiviral particles (HCVpp) in Huh-7 cells but had only minimal impact on viral RNA replication or cell proliferation and viability. We propose a model by which MKNK1 acts to facilitate viral entry downstream of the epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), both of which have been implicated in the entry process.

    View details for DOI 10.1128/JVI.00954-12

    View details for Web of Science ID 000316671000008

    View details for PubMedID 23365451

  • Characterization of Hepatitis C Virus Recombinants with Chimeric E1/E2 Envelope Proteins and Identification of Single Amino Acids in the E2 Stem Region Important for Entry JOURNAL OF VIROLOGY Carlsen, T. H., Scheel, T. K., Ramirez, S., Foung, S. K., Bukh, J. 2013; 87 (3): 1385-1399

    Abstract

    The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviable. After E2 exchange from three 1b isolates, long delays were observed before spread of infection. For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ~100-fold, apparently without influencing particle stability or cell binding although introducing slight decrease in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly/release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition assay indicated that the mutations influenced a late step of the HCV entry pathway. Overall, this study identified specific amino acids in the E2 stem region of importance for HCV entry and for production of infectious virus particles.

    View details for DOI 10.1128/JVI.00684-12

    View details for Web of Science ID 000313558100009

    View details for PubMedID 23152512

  • Cooperativity in Virus Neutralization by Human Monoclonal Antibodies to Two Adjacent Regions Located at the Amino Terminus of Hepatitis C Virus E2 Glycoprotein JOURNAL OF VIROLOGY Keck, Z., Wang, W., Wang, Y., Lau, P., Carlsen, T. H., Prentoe, J., Xia, J., Patel, A. H., Bukh, J., Foung, S. K. 2013; 87 (1): 37-51

    Abstract

    A challenge for hepatitis C virus (HCV) vaccine development is defining conserved epitopes that induce protective antibodies against this highly diverse virus. An envelope glycoprotein (E2) segment located at amino acids (aa) 412 to 423 contains highly conserved neutralizing epitopes. While polyclonal antibodies to aa 412 to 423 from HCV-infected individuals confirmed broad neutralization, conflicting findings have been reported on polyclonal antibodies to an adjacent region, aa 434 to 446, that may or may not interfere with neutralization by antibodies to aa 412 to 423. To define the interplay between these antibodies, we isolated human monoclonal antibodies (HMAbs) to aa 412 to 423, designated HC33-related HMAbs (HC33 HMAbs), and characterized their interactions with other HMAbs to aa 434 to 446. A subset of the HC33 HMAbs neutralized genotype 1 to 6 infectious cell culture-derived HCV virions (HCVcc) with various activities. Although nonneutralizing HC33 HMAbs were isolated, they had lower binding affinities than neutralizing HC33 HMAbs. These antibodies could be converted to neutralizing antibodies by affinity maturation. Unidirectional competition for binding to E2 was observed between HC33 HMAbs and HMAbs to aa 434 to 446. When HMAbs to aa 434 to 446, which mediated neutralization, were combined with neutralizing HC33 HMAbs, biphasic patterns in neutralization were observed. A modest degree of antagonism was observed at lower concentrations, and a modest degree of synergism was observed at higher concentrations. However, the overall effect was additive neutralization. A similar pattern was observed when these antibodies were combined to block E2 binding to the HCV coreceptor, CD81. These findings demonstrate that both of these E2 regions participate in epitopes mediating virus neutralization and that the antibodies to aa 412 to 423 and aa 434 to 446 do not hinder their respective virus-neutralizing activities.

    View details for DOI 10.1128/JVI.01941-12

    View details for Web of Science ID 000312455500004

    View details for PubMedID 23097455

  • Transient Activation of the PI3K-AKT Pathway by Hepatitis C Virus to Enhance Viral Entry JOURNAL OF BIOLOGICAL CHEMISTRY Liu, Z., Tian, Y., Machida, K., Lai, M. M., Luo, G., Foung, S. K., Ou, J. J. 2012; 287 (50): 41922-41930

    Abstract

    The PI3K-AKT signaling pathway plays an important role in cell growth and metabolism. Here we report that hepatitis C virus (HCV) transiently activates the PI3K-AKT pathway. This activation was observed as early as 15 min postinfection, peaked by 30 min, and became undetectable at 24 h postinfection. The activation of AKT could also be mediated by UV-inactivated HCV, HCV pseudoparticle, and the ectodomain of the HCV E2 envelope protein. Because antibodies directed against CD81 and claudin-1, but not antibodies directed against scavenger receptor class B type I or occludin, could also activate AKT, the interaction between HCV E2 and its two co-receptors CD81 and claudin-1 probably triggered the activation of AKT. This activation of AKT by HCV was important for HCV infectivity, because the silencing of AKT by siRNA or the treatment of cells with its inhibitors or with the inhibitor of its upstream regulator PI3K significantly inhibited HCV infection, whereas the expression of constitutively active AKT enhanced HCV infection. The PI3K-AKT pathway is probably involved in HCV entry, because the inhibition of this pathway could inhibit the entry of HCV pseudoparticle but not the VSV pseudoparticle into cells. Furthermore, the treatment of cells with the AKT inhibitor AKT-V prior to HCV infection inhibited HCV infection, whereas the treatment after HCV infection had no obvious effect. Taken together, our studies indicated that HCV transiently activates the PI3K-AKT pathway to facilitate its entry. These results provide important information for understanding HCV replication and pathogenesis and raised the possibility of targeting this cellular pathway to treat HCV patients.

    View details for DOI 10.1074/jbc.M112.414789

    View details for Web of Science ID 000312103000032

    View details for PubMedID 23095753

  • Toward a Hepatitis C Virus Vaccine: the Structural Basis of Hepatitis C Virus Neutralization by AP33, a Broadly Neutralizing Antibody JOURNAL OF VIROLOGY Potter, J. A., Owsianka, A. M., Jeffery, N., Matthews, D. J., Keck, Z., Lau, P., Foung, S. K., Taylor, G. L., Patel, A. H. 2012; 86 (23): 12923-12932

    Abstract

    The E2 envelope glycoprotein of hepatitis C virus (HCV) binds to the host entry factor CD81 and is the principal target for neutralizing antibodies (NAbs). Most NAbs recognize hypervariable region 1 on E2, which undergoes frequent mutation, thereby allowing the virus to evade neutralization. Consequently, there is great interest in NAbs that target conserved epitopes. One such NAb is AP33, a mouse monoclonal antibody that recognizes a conserved, linear epitope on E2 and potently neutralizes a broad range of HCV genotypes. In this study, the X-ray structure of AP33 Fab in complex with an epitope peptide spanning residues 412 to 423 of HCV E2 was determined to 1.8 ?. In the complex, the peptide adopts a ?-hairpin conformation and docks into a deep binding pocket on the antibody. The major determinants of antibody recognition are E2 residues L413, N415, G418, and W420. The structure is compared to the recently described HCV1 Fab in complex with the same epitope. Interestingly, the antigen-binding sites of HCV1 and AP33 are completely different, whereas the peptide conformation is very similar in the two structures. Mutagenesis of the peptide-binding residues on AP33 confirmed that these residues are also critical for AP33 recognition of whole E2, confirming that the peptide-bound structure truly represents AP33 interaction with the intact glycoprotein. The slightly conformation-sensitive character of the AP33-E2 interaction was explored by cross-competition analysis and alanine-scanning mutagenesis. The structural details of this neutralizing epitope provide a starting point for the design of an immunogen capable of eliciting AP33-like antibodies.

    View details for DOI 10.1128/JVI.02052-12

    View details for Web of Science ID 000310585300042

    View details for PubMedID 22993159

  • Mutations That Alter Use of Hepatitis C Virus Cell Entry Factors Mediate Escape From Neutralizing Antibodies GASTROENTEROLOGY Fofana, I., Fafi-Kremer, S., Carolla, P., Fauvelle, C., Zahid, M. N., Turek, M., Heydmann, L., Cury, K., Hayer, J., Combet, C., Cosset, F., Pietschmann, T., Hiet, M., Bartenschlager, R., Habersetzer, F., Doffoel, M., Keck, Z., Foung, S. K., Zeisel, M. B., Stoll-Keller, F., Baumert, T. F. 2012; 143 (1): 223-U429

    Abstract

    The development of vaccines and other strategies to prevent hepatitis C virus (HCV) infection is limited by rapid viral evasion. HCV entry is the first step of infection; this process involves several viral and host factors and is targeted by host-neutralizing responses. Although the roles of host factors in HCV entry have been well characterized, their involvement in evasion of immune responses is poorly understood. We used acute infection of liver graft as a model to investigate the molecular mechanisms of viral evasion.We studied factors that contribute to evasion of host immune responses using patient-derived antibodies, HCV pseudoparticles, and cell culture-derived HCV that express viral envelopes from patients who have undergone liver transplantation. These viruses were used to infect hepatoma cell lines that express different levels of HCV entry factors.By using reverse genetic analyses, we identified altered use of host-cell entry factors as a mechanism by which HCV evades host immune responses. Mutations that alter use of the CD81 receptor also allowed the virus to escape neutralizing antibodies. Kinetic studies showed that these mutations affect virus-antibody interactions during postbinding steps of the HCV entry process. Functional studies with a large panel of patient-derived antibodies showed that this mechanism mediates viral escape, leading to persistent infection in general.We identified a mechanism by which HCV evades host immune responses, in which use of cell entry factors evolves with escape from neutralizing antibodies. These findings advance our understanding of the pathogenesis of HCV infection and might be used to develop antiviral strategies and vaccines.

    View details for DOI 10.1053/j.gastro.2012.04.006

    View details for Web of Science ID 000305781500044

    View details for PubMedID 22503792

  • Human Monoclonal Antibodies to a Novel Cluster of Conformational Epitopes on HCV E2 with Resistance to Neutralization Escape in a Genotype 2a Isolate PLOS PATHOGENS Keck, Z., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J., Carlsen, T., Li, A. Y., Patel, A. H., Lemon, S. M., Bukh, J., Rey, F. A., Foung, S. K. 2012; 8 (4)

    Abstract

    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 relevant for vaccine design for this highly diverse virus.

    View details for DOI 10.1371/journal.ppat.1002653

    View details for Web of Science ID 000303444200043

    View details for PubMedID 22511875

  • Affinity Maturation to Improve Human Monoclonal Antibody Neutralization Potency and Breadth against Hepatitis C Virus JOURNAL OF BIOLOGICAL CHEMISTRY Wang, Y., Keck, Z., Saha, A., Xia, J., Conrad, F., Lou, J., Eckart, M., Marks, J. D., Foung, S. K. 2011; 286 (51): 44218-44233

    Abstract

    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 ?-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.

    View details for DOI 10.1074/jbc.M111.290783

    View details for Web of Science ID 000298402300062

    View details for PubMedID 22002064

  • Neutralizing Antibody Response to Hepatitis C Virus VIRUSES-BASEL Wang, Y., Keck, Z., Foung, S. K. 2011; 3 (11): 2127-2145

    Abstract

    A critical first step in a "rational vaccine design" approach for hepatitis C virus (HCV) is to identify the most relevant mechanisms of immune protection. Emerging evidence provides support for a protective role of virus neutralizing antibodies, and the ability of the B cell response to modify the course of acute HCV infection. This has been made possible by the development of in vitro cell culture models, based on HCV retroviral pseudotype particles expressing E1E2 and infectious cell culture-derived HCV virions, and small animal models that are robust tools in studies of antibody-mediated virus neutralization. This review is focused on the immunogenic determinants on the E2 glycoprotein mediating virus neutralization and the pathways in which the virus is able to escape from immune containment. Encouraging findings from recent studies provide support for the existence of broadly neutralization antibodies that are not associated with virus escape. The identification of conserved epitopes mediating virus neutralization that are not associated with virus escape will facilitate the design of a vaccine immunogen capable of eliciting broadly neutralizing antibodies against this highly diverse virus.

    View details for DOI 10.3390/v3112127

    View details for Web of Science ID 000297699200005

    View details for PubMedID 22163337

  • Mapping a Region of Hepatitis C Virus E2 That Is Responsible for Escape from Neutralizing Antibodies and a Core CD81-Binding Region That Does Not Tolerate Neutralization Escape Mutations JOURNAL OF VIROLOGY Keck, Z., Saha, A., Xia, J., Wang, Y., Lau, P., Krey, T., Rey, F. A., Foung, S. K. 2011; 85 (20): 10451-10463

    Abstract

    Understanding the interaction between broadly neutralizing antibodies and their epitopes provides a basis for the rational design of a preventive hepatitis C virus (HCV) vaccine. CBH-2, HC-11, and HC-1 are representatives of antibodies to overlapping epitopes on E2 that mediate neutralization by blocking virus binding to CD81. To obtain insights into escape mechanisms, infectious cell culture virus, 2a HCVcc, was propagated under increasing concentrations of a neutralizing antibody to isolate escape mutants. Three escape patterns were observed with these antibodies. First, CBH-2 escape mutants that contained mutations at D431G or A439E, which did not compromise viral fitness, were isolated. Second, under the selective pressure of HC-11, escape mutations progressed from a single L438F substitution at a low antibody concentration to double substitutions, L438F and N434D or L438F and T435A, at higher antibody concentrations. Escape from HC-11 was associated with a loss of viral fitness. An HCV pseudoparticle (HCVpp) containing the L438F mutation bound to CD81 half as efficiently as did wild-type (wt) HCVpp. Third, for HC-1, the antibody at a critical concentration completely suppressed viral replication and generated no escape mutants. Epitope mapping revealed contact residues for CBH-2 and HC-11 in two regions of the E2 glycoprotein, amino acids (aa) 425 to 443 and aa 529 to 535. Interestingly, contact residues for HC-1 were identified only in the region encompassing aa 529 to 535 and not in aa 425 to 443. Taken together, these findings point to a region of variability, aa 425 to 443, that is responsible primarily for viral escape from neutralization, with or without compromising viral fitness. Moreover, the region aa 529 to 535 is a core CD81 binding region that does not tolerate neutralization escape mutations.

    View details for DOI 10.1128/JVI.05259-11

    View details for Web of Science ID 000296254100003

    View details for PubMedID 21813602

  • Comprehensive linker-scanning mutagenesis of the hepatitis C virus E1 and E2 envelope glycoproteins reveals new structure-function relationships JOURNAL OF GENERAL VIROLOGY Rychlowska, M., Owsianka, A. M., Foung, S. K., Dubuisson, J., Bienkowska-Szewczyk, K., Patel, A. H. 2011; 92: 2249-2261

    Abstract

    Despite extensive research, many details about the structure and functions of hepatitis C virus (HCV) glycoproteins E1 and E2 are not fully understood, and their crystal structure remains to be determined. We applied linker-scanning mutagenesis to generate a panel of 34 mutants, each containing an insertion of 5 aa at a random position within the E1E2 sequence. The mutated glycoproteins were analysed by using a range of assays to identify regions critical for maintaining protein conformation, E1E2 complex assembly, CD81 receptor binding, membrane fusion and infectivity. The results, while supporting previously published data, provide several interesting new findings. Firstly, insertion at amino acid 587 or 596 reduced E1E2 heterodimerization without affecting reactivity with some conformation-sensitive mAbs or with CD81, thus implicating these residues in glycoprotein assembly. Secondly, insertions within a conserved region of E2, between amino acid residues 611 and 631, severely disrupted protein conformation and abrogated binding of all conformation-sensitive antibodies, suggesting that the structural integrity of this region is critical for the correct folding of E2. Thirdly, an insertion at Leu-682 specifically affected membrane fusion, providing direct evidence that the membrane-proximal 'stem' of E2 is involved in the fusion mechanism. Overall, our results show that the HCV glycoproteins generally do not tolerate insertions and that there are a very limited number of sites that can be changed without dramatic loss of function. Nevertheless, we identified two E2 insertion mutants, at amino acid residues 408 and 577, that were infectious in the murine leukemia virus-based HCV pseudoparticle system.

    View details for DOI 10.1099/vir.0.034314-0

    View details for Web of Science ID 000295856000003

    View details for PubMedID 21697343

  • The Hepatitis C Virus E1 Glycoprotein Undergoes Productive Folding but Accelerated Degradation When Expressed as an Individual Subunit in CHO Cells PLOS ONE Botti, V., Bianchi, A., Foung, S. K., Merola, M. 2011; 6 (8)

    Abstract

    Hepatitis C Virus E1E2 heterodimers are components of the viral spike. Although there is a general agreement on the necessity of the co-expression of both E1 and E2 on a single coding unit for their productive folding and assembly, in a previous study using an in vitro system we obtained strong indications that E1 can achieve folding in absence of E2. Here, we have studied the folding pathway of unescorted E1 from stably expressing CHO cells, compared to the folding observed in presence of the E2 protein. A DTT-resistant conformation is achieved by E1 in both situations, consistent with the presence of an E2-independent oxidative pathway. However, while the E1E2 heterodimer is stable inside cells, E1 expressed alone is degraded within a few hours. On the other hand, the oxidation and stability of individually expressed E2 subunits is dependent on E1 co-expression. These data are consistent with E1 and E2 assisting each other for correct folding via different mechanisms: E2 assists E1 by stabilizing a semi-native conformation meanwhile E1 drives E2 towards a productive folding pathway.

    View details for DOI 10.1371/journal.pone.0023838

    View details for Web of Science ID 000294121300100

    View details for PubMedID 21858229

  • The GB viruses: a review and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within the family Flaviviridae JOURNAL OF GENERAL VIROLOGY Stapleton, J. T., Foung, S., Muerhoff, A. S., Bukh, J., Simmonds, P. 2011; 92: 233-246

    Abstract

    In 1967, it was reported that experimental inoculation of serum from a surgeon (G.B.) with acute hepatitis into tamarins resulted in hepatitis. In 1995, two new members of the family Flaviviridae, named GBV-A and GBV-B, were identified in tamarins that developed hepatitis following inoculation with the 11th GB passage. Neither virus infects humans, and a number of GBV-A variants were identified in wild New World monkeys that were captured. Subsequently, a related human virus was identified [named GBV-C or hepatitis G virus (HGV)], and recently a more distantly related virus (named GBV-D) was discovered in bats. Only GBV-B, a second species within the genus Hepacivirus (type species hepatitis C virus), has been shown to cause hepatitis; it causes acute hepatitis in experimentally infected tamarins. The other GB viruses have however not been assigned to a genus within the family Flaviviridae. Based on phylogenetic relationships, genome organization and pathogenic features of the GB viruses, we propose to classify GBV-A-like viruses, GBV-C and GBV-D as members of a fourth genus in the family Flaviviridae, named Pegivirus (pe, persistent; g, GB or G). We also propose renaming 'GB' viruses within the tentative genus Pegivirus to reflect their host origin.

    View details for DOI 10.1099/vir.0.027490-0

    View details for Web of Science ID 000287638800001

    View details for PubMedID 21084497

  • Hepatitis C virus e2 protein ectodomain is essential for assembly of infectious virions. International journal of hepatology Bianchi, A., Crotta, S., Brazzoli, M., Foung, S. K., Merola, M. 2011; 2011: 968161-?

    Abstract

    The Hepatitis C virus E1 and E2 envelope proteins are the major players in all events required for virus entry into target cells. In addition, the recently developed HCV cell culture system has indicated that E1E2 heterodimer formation is a prerequisite for viral particle production. In this paper, we explored a new genetic approach to construct intergenotypic 2a/1b chimeras, maintaining the structural region of the infectious strain JFH1 and substituting the soluble portion of E1 and/or E2 proteins. This strategy provides useful information on the role of the surface-exposed domain of the envelope proteins in virus morphogenesis and allows comparative analysis of different HCV genotypes. We found that substituting the E2 protein ectodomain region abolishes the production of chimeric infectious particles. Our data indicate that the soluble part of the E2 protein is involved in a genotype-specific interplay with remaining viral proteins that affect the HCV assembly process.

    View details for DOI 10.4061/2011/968161

    View details for PubMedID 22007314

  • Hepatitis C Virus Hypervariable Region 1 Modulates Receptor Interactions, Conceals the CD81 Binding Site, and Protects Conserved Neutralizing Epitopes JOURNAL OF VIROLOGY Bankwitz, D., Steinmann, E., Bitzegeio, J., Ciesek, S., Friesland, M., Herrmann, E., Zeisel, M. B., Baumert, T. F., Keck, Z., Foung, S. K., Pecheur, E., Pietschmann, T. 2010; 84 (11): 5751-5763

    Abstract

    The variability of the hepatitis C virus (HCV), which likely contributes to immune escape, is most pronounced in hypervariable region 1 (HVR1) of viral envelope protein 2. This domain is the target for neutralizing antibodies, and its deletion attenuates replication in vivo. Here we characterized the relevance of HVR1 for virus replication in vitro using cell culture-derived HCV. We show that HVR1 is dispensable for RNA replication. However, viruses lacking HVR1 (Delta HVR1) are less infectious, and separation by density gradients revealed that the population of Delta HVR1 virions comprises fewer particles with low density. Strikingly, Delta HVR1 particles with intermediate density (1.12 g/ml) are as infectious as wild-type virions, while those with low density (1.02 to 1.08 g/ml) are poorly infectious, despite quantities of RNA and core similar to those in wild-type particles. Moreover, Delta HVR1 particles exhibited impaired fusion, a defect that was partially restored by an E1 mutation (I347L), which also rescues infectivity and which was selected during long-term culture. Finally, Delta HVR1 particles were no longer neutralized by SR-B1-specific immunoglobulins but were more prone to neutralization and precipitation by soluble CD81, E2-specific monoclonal antibodies, and patient sera. These results suggest that HVR1 influences the biophysical properties of released viruses and that this domain is particularly important for infectivity of low-density particles. Moreover, they indicate that HVR1 obstructs the viral CD81 binding site and conserved neutralizing epitopes. These functions likely optimize virus replication, facilitate immune escape, and thus foster establishment and maintenance of a chronic infection.

    View details for DOI 10.1128/JVI.02200-09

    View details for Web of Science ID 000277386700029

    View details for PubMedID 20357091

  • Mutations within a Conserved Region of the Hepatitis C Virus E2 Glycoprotein That Influence Virus-Receptor Interactions and Sensitivity to Neutralizing Antibodies JOURNAL OF VIROLOGY Dhillon, S., Witteveldt, J., Gatherer, D., Owsianka, A. M., Zeisel, M. B., Zahid, M. N., Rychlowska, M., Foung, S. K., Baumert, T. F., Angus, A. G., Patel, A. H. 2010; 84 (11): 5494-5507

    Abstract

    Cell culture-adaptive mutations within the hepatitis C virus (HCV) E2 glycoprotein have been widely reported. We identify here a single mutation (N415D) in E2 that arose during long-term passaging of HCV strain JFH1-infected cells. This mutation was located within E2 residues 412 to 423, a highly conserved region that is recognized by several broadly neutralizing antibodies, including the mouse monoclonal antibody (MAb) AP33. Introduction of N415D into the wild-type (WT) JFH1 genome increased the affinity of E2 to the CD81 receptor and made the virus less sensitive to neutralization by an antiserum to another essential entry factor, SR-BI. Unlike JFH1(WT), the JFH1(N415D) was not neutralized by AP33. In contrast, it was highly sensitive to neutralization by patient-derived antibodies, suggesting an increased availability of other neutralizing epitopes on the virus particle. We included in this analysis viruses carrying four other single mutations located within this conserved E2 region: T416A, N417S, and I422L were cell culture-adaptive mutations reported previously, while G418D was generated here by growing JFH1(WT) under MAb AP33 selective pressure. MAb AP33 neutralized JFH1(T416A) and JFH1(I422L) more efficiently than the WT virus, while neutralization of JFH1(N417S) and JFH1(G418D) was abrogated. The properties of all of these viruses in terms of receptor reactivity and neutralization by human antibodies were similar to JFH1(N415D), highlighting the importance of the E2 412-423 region in virus entry.

    View details for DOI 10.1128/JVI.02153-09

    View details for Web of Science ID 000277386700005

    View details for PubMedID 20237087

  • Antigen-specific Proteolysis by Hybrid Antibodies Containing Promiscuous Proteolytic Light Chains Paired with an Antigen-binding Heavy Chain JOURNAL OF BIOLOGICAL CHEMISTRY Sapparapu, G., Planque, S. A., Nishiyama, Y., Foung, S. K., Paul, S. 2009; 284 (36): 24622-24633

    Abstract

    The antigen recognition site of antibodies consists of the heavy and light chain variable domains (V(L) and V(H) domains). V(L) domains catalyze peptide bond hydrolysis independent of V(H) domains (Mei, S., Mody, B., Eklund, S. H., and Paul, S. (1991) J. Biol. Chem. 266, 15571-15574). V(H) domains bind antigens noncovalently independent of V(L) domains (Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989) Nature 341, 544-546). We describe specific hydrolysis of fusion proteins of the hepatitis C virus E2 protein with glutathione S-transferase (GST-E2) or FLAG peptide (FLAG-E2) by antibodies containing the V(H) domain of an anti-E2 IgG paired with promiscuously catalytic V(L) domains. The hybrid IgG hydrolyzed the E2 fusion proteins more rapidly than the unpaired light chain. An active site-directed inhibitor of serine proteases inhibited the proteolytic activity of the hybrid IgG, indicating a serine protease mechanism. The hybrid IgG displayed noncovalent E2 binding in enzyme-linked immunosorbent assay tests. Immunoblotting studies suggested hydrolysis of FLAG-E2 at a bond within E2 located approximately 11 kDa from the N terminus. GST-E2 was hydrolyzed by the hybrid IgG at bonds in the GST tag. The differing cleavage pattern of FLAG-E2 and GST-E2 can be explained by the split-site model of catalysis, in which conformational differences in the E2 fusion protein substrates position alternate peptide bonds in register with the antibody catalytic subsite despite a common noncovalent binding mechanism. These studies provide proof-of-principle that the catalytic activity of a light chain can be rendered antigen-specific by pairing with a noncovalently binding heavy chain subunit.

    View details for DOI 10.1074/jbc.M109.011858

    View details for Web of Science ID 000269380200071

    View details for PubMedID 19542217

  • CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells JOURNAL OF GENERAL VIROLOGY Witteveldt, J., Evans, M. J., Bitzegeio, J., Koutsoudakis, G., Owsianka, A. M., Angus, A. G., Keck, Z., Foung, S. K., Pietschmann, T., Rice, C. M., Patel, A. H. 2009; 90: 48-58

    Abstract

    Hepatitis C virus (HCV) infects cells by the direct uptake of cell-free virus following virus engagement with specific cell receptors such as CD81. Recent data have shown that HCV is also capable of direct cell-to-cell transmission, although the role of CD81 in this process is disputed. Here, we generated cell culture infectious strain JFH1 HCV (HCVcc) genomes carrying an alanine substitution of E2 residues W529 or D535 that are critical for binding to CD81 and infectivity. Co-cultivation of these cells with naïve cells expressing enhanced green fluorescent protein (EGFP) resulted in a small number of cells co-expressing both EGFP and HCV NS5A, showing that the HCVcc mutants are capable of cell-to-cell spread. In contrast, no cell-to-cell transmission from JFH1(DeltaE1E2)-transfected cells occurred, indicating that the HCV glycoproteins are essential for this process. The frequency of cell-to-cell transmission of JFH1(W529A) was unaffected by the presence of neutralizing antibodies that inhibit E2-CD81 interactions. By using cell lines that expressed little or no CD81 and that were refractive to infection with cell-free virus, we showed that the occurrence of viral cell-to-cell transmission is not influenced by the levels of CD81 on either donor or recipient cells. Thus, our results show that CD81 plays no role in the cell-to-cell spread of HCVcc and that this mode of transmission is shielded from neutralizing antibodies. These data suggest that therapeutic interventions targeting the entry of cell-free HCV may not be sufficient in controlling an ongoing chronic infection, but need to be complemented by additional strategies aimed at disrupting direct cell-to-cell viral transmission.

    View details for DOI 10.1099/vir.0.2008/006700-0

    View details for Web of Science ID 000262263000005

    View details for PubMedID 19088272

  • In vitro selection of a neutralization-resistant hepatitis C virus escape mutant PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gal-Tanamy, M., Keck, Z., Yi, M., McKeating, J. A., Patel, A. H., Foung, S. K., Lemon, S. M. 2008; 105 (49): 19450-19455

    Abstract

    Effective immunization against hepatitis C virus (HCV) infections is likely to require the induction of both robust T and B cell immunity. Although neutralizing antibodies may play an important role in control of infection, there is little understanding of the structure of the HCV envelope glycoproteins and how they interact with such antibodies. An additional challenge for vaccine design is the genetic diversity of HCV and the rapid evolution of viral quasispecies that escape antibody-mediated neutralization. We used a cell culture-infectious, chimeric HCV with the structural proteins of genotype 1a virus to identify envelope residues contributing to the epitope recognized by a broadly neutralizing, murine monoclonal antibody, AP33. By repetitive rounds of neutralization followed by amplification, we selected a population of viral escape mutants that resist stringent neutralization with AP33 and no longer bind the antibody. Two amino acid substitutions, widely separated in the linear sequence of the E2 envelope protein (N415Y and E655G), were identified by sequencing of cloned cDNA and shown by reverse genetics analysis to contribute jointly to the AP33 resistance phenotype. The N415Y mutation substantially lowered virus fitness, most likely because of a defect in viral entry, but did not reduce binding of soluble CD81 to immobilized HCV-pseudotyped retrovirus particles. The in vitro selection of an HCV escape mutant recapitulates the ongoing evolution of antigenic variants that contributes to viral persistence in humans and reveals information concerning the conformational structure of the AP33 epitope, its role in viral replication, and constraints on its molecular evolution.

    View details for DOI 10.1073/pnas.0809879105

    View details for Web of Science ID 000261706600076

    View details for PubMedID 19052239

  • Hepatitis C virus (HCV)-induced immunoglobulin hypermutation reduces the affinity and neutralizing activities of antibodies against HCV envelope protein JOURNAL OF VIROLOGY Machida, K., Kondo, Y., Huang, J. Y., Chen, Y., Cheng, K. T., Keck, Z., Foung, S., Dubuisson, J., Sung, V. M., Lai, M. M. 2008; 82 (13): 6711-6720

    Abstract

    Hepatitis C virus (HCV) often causes persistent infection despite the presence of neutralizing antibodies against the virus in the sera of hepatitis C patients. HCV infects both hepatocytes and B cells through the binding of its envelope glycoprotein E2 to CD81, the putative viral receptor. Previously, we have shown that E2-CD81 interaction induces hypermutation of heavy-chain immunoglobulin (V(H)) in B cells. We hypothesize that if HCV infects antibody-producing B cells, the resultant hypermutation of V(H) may lower the affinity and specificity of the HCV-specific antibodies, enabling HCV to escape from immune surveillance. To test this hypothesis, we infected human hybridoma clones producing either neutralizing or non-neutralizing anti-E2 or anti-E1 antibodies with a lymphotropic HCV (SB strain). All of the hybridoma clones, except for a neutralizing antibody-producing hybridoma, could be infected with HCV and support virus replication for at least 8 weeks after infection. The V(H) sequences in the infected hybridomas had a significantly higher mutation frequency than those in the uninfected hybridomas, with mutations concentrating in complementarity-determining region 3. These mutations lowered the antibody affinity against the targeting protein and also lowered the virus-neutralizing activity of anti-E2 antibodies. Furthermore, antibody-mediated complement-dependent cytotoxicity with the antibodies secreted from the HCV-infected hybridomas was impaired. These results suggest that HCV infection could cause some anti-HCV-antibody-producing hybridoma B cells to make less-protective antibodies.

    View details for DOI 10.1128/JVI.02582-07

    View details for Web of Science ID 000256947300058

    View details for PubMedID 18417597

  • A point mutation leading to hepatitis C virus escape from neutralization by a monoclonal antibody to a conserved conformational epitope JOURNAL OF VIROLOGY Keck, Z., Olson, O., Gal-Tanamy, M., Xia, J., Patel, A. H., Dreux, M., Cosset, F., Lemon, S. M., Foung, S. K. 2008; 82 (12): 6067-6072

    Abstract

    A challenge in hepatitis C virus (HCV) vaccine development is defining conserved protective epitopes. A cluster of these epitopes comprises an immunodominant domain on the E2 glycoprotein, designated domain B. CBH-2 is a neutralizing human monoclonal antibody to a domain B epitope that is highly conserved. Alanine scanning demonstrated that the epitope involves residues G523, G530, and D535 that are also contact residues for E2 binding to CD81, a coreceptor required for virus entry into cells. However, another residue, located at position 431 and thus at a considerable distance in the linear sequence of E2, also contributes to the CBH-2 epitope. A single amino acid substitution at this residue results in escape from CBH-2-mediated neutralization in a genotype 1a virus. These results highlight the challenges inherent in developing HCV vaccines and show that an effective vaccine must induce antibodies to both conserved and more invariant epitopes to minimize virus escape.

    View details for DOI 10.1128/JVI.00252-08

    View details for Web of Science ID 000256453600041

    View details for PubMedID 18385242

  • Definition of a conserved immunodominant domain on hepatitis C virus E2 glycoprotein by neutralizing human monoclonal antibodies JOURNAL OF VIROLOGY Keck, Z., Li, T., Xia, J., Gal-Tanamy, M., Olson, O., Li, S. H., Patel, A. H., Ball, J. K., Lemon, S. M., Foung, S. K. 2008; 82 (12): 6061-6066

    Abstract

    Development of a successful hepatitis C virus (HCV) vaccine requires the definition of neutralization epitopes that are conserved among different HCV genotypes. Five human monoclonal antibodies (HMAbs) are described that cross-compete with other antibodies to a cluster of overlapping epitopes, previously designated domain B. Each HMAb broadly neutralizes retroviral pseudotype particles expressing HCV E1 and E2 glycoproteins, as well as the infectious chimeric genotype 1a and genotype 2a viruses. Alanine substitutions of residues within a region of E2 involved in binding to CD81 showed that critical E2 contact residues involved in the binding of representative antibodies are identical to those involved in the binding of E2 to CD81.

    View details for DOI 10.1128/JVI.02475-07

    View details for Web of Science ID 000256453600040

    View details for PubMedID 18400849

  • Antibody-dependent enhancement of hepatitis C virus infection JOURNAL OF VIROLOGY Meyer, K., Ait-Goughoulte, M., Keck, Z., Foung, S., Ray, R. 2008; 82 (5): 2140-2149

    Abstract

    Hepatitis C virus (HCV) often causes a persistent infection associated with hypergammaglobulinemia, high levels of antiviral antibody and circulating immune complexes, and immune complex disease. We previously reported that only a limited neutralizing activity to vesicular stomatitis virus or HCV pseudotype is generated in animals immunized with recombinant HCV envelope proteins and chronically infected HCV patient sera. Interestingly, when some of these neutralizing sera were diluted into a range of concentrations below those that reduced virus plaque number, an increase in pseudotype plaque formation was observed. Purified HCV E2-specific human monoclonal antibodies were used to further verify the specificity of this enhancement, and one- to twofold increases were apparent on permissive Huh-7 cells. The enhancement of HCV pseudotype titer could be inhibited by the addition of a Fc-specific anti-human immunoglobulin G Fab fragment to the virus-antibody mixture prior to infection. Treatment of cells with antibody to Fc receptor I (FcRI) or FcRII, but not FcRIII, also led to an inhibition of pseudotype titer enhancement in an additive manner. Human lymphoblastoid cell line (Raji), a poor host for HCV pseudotype infection, exhibited a four- to sixfold enhancement of pseudotype-mediated cell death upon incubation with antibody at nonneutralizing concentrations. A similar enhancement of cell culture-grown HCV infectivity by a human monoclonal antibody was also observed. Taken together, antibodies to viral epitopes enhancing HCV infection need to be taken into consideration for pathogenesis and in the development of an effective vaccine.

    View details for DOI 10.1128/JVI.01867-07

    View details for Web of Science ID 000253481000010

    View details for PubMedID 18094180

  • Broadly neutralizing human monoclonal antibodies to the hepatitis C virus E2 glycoprotein JOURNAL OF GENERAL VIROLOGY Owsianka, A. M., Tarr, A. W., Keck, Z., Li, T., Witteveldt, J., Adair, R., Foung, S. K., Ball, J. K., Patel, A. H. 2008; 89: 653-659

    Abstract

    The humoral response to hepatitis C virus (HCV) may contribute to controlling infection. We previously isolated human monoclonal antibodies to conformational epitopes on the HCV E2 glycoprotein. Here, we report on their ability to inhibit infection by retroviral pseudoparticles incorporating a panel of full-length E1E2 clones representing the full spectrum of genotypes 1-6. We identified one antibody, CBH-5, that was capable of neutralizing every genotype tested. It also potently inhibited chimeric cell culture-infectious HCV, which had genotype 2b envelope proteins in a genotype 2a (JFH-1) background. Analysis using a panel of alanine-substitution mutants of HCV E2 revealed that the epitope of CBH-5 includes amino acid residues that are required for binding of E2 to CD81, a cellular receptor essential for virus entry. This suggests that CBH-5 inhibits HCV infection by competing directly with CD81 for a binding site on E2.

    View details for DOI 10.1099/vir.0.83386-0

    View details for Web of Science ID 000253998700005

    View details for PubMedID 18272755

  • Structural elucidation of critical residues involved in binding of human monoclonal antibodies to hepatitis C virus E2 envelope glycoprotein BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS Iacob, R. E., Keck, Z., Olson, O., Foung, S. K., Tomer, K. B. 2008; 1784 (3): 530-542

    Abstract

    Human monoclonal antibodies derived from B cells of HCV-infected individuals provide information on the immune response to native HCV envelope proteins as they are recognized during infection. Monoclonal antibodies have been useful in the determination of the function and structure of specific immunogenic domains of proteins and should also be useful for the structure/function characterization of HCV E1 and E2 envelope glycoproteins. The HCV E2 envelope glycoprotein has at least three immunodistinctive conformation domains, designated A, B, and C. Conformational epitopes within domain B and C are neutralizing antibody targets on HCV pseudoparticles as well as from infectious cell culture virus. In this study, a combination of differential surface modification and mass spectrometric limited proteolysis followed by alanine mutagenesis was used to provide insight into potential conformational changes within the E2 protein upon antibody binding. The arginine guanidine groups in the E2 protein were modified with CHD in both the affinity bound and free states followed by mass spectrometric analysis, and the regions showing protection upon antibody binding were identified. This protection can arise by direct contact between the residues and the monoclonal antibody, or by antibody-induced conformational changes. Based on the mass spectrometric data, site-directed mutagenesis experiments were performed which clearly identified additional amino acid residues on E2 distant from the site of antibody interaction, whose change to alanine inhibited antibody recognition by inducing conformational changes within the E2 protein.

    View details for DOI 10.1016/j.bbapap.2007.12.015

    View details for Web of Science ID 000254780200012

    View details for PubMedID 18230369

  • Therapeutic control of hepatitis C virus: The role of neutralizing monoclonal antibodies HUMAN ANTIBODY THERAPEUTICS FOR VIRAL DISEASE Keck, Z. Y., Machida, K., Lai, M. M., Ball, J. K., Patel, A. H., Foung, S. K. 2008; 317: 1-38

    Abstract

    Liver failure associated with hepatitis C virus (HCV) accounts for a substantial portion of liver transplantation. Although current therapy helps some patients with chronic HCV infection, adverse side effects and a high relapse rate are major problems. These problems are compounded in liver transplant recipients as reinfection occurs shortly after transplantation. One approach to control reinfection is the combined use of specific antivirals together with HCV-specific antibodies. Indeed, a number of human and mouse monoclonal antibodies to conformational and linear epitopes on HCV envelope proteins are potential candidates, since they have high virus neutralization potency and are directed to epitopes conserved across diverse HCV genotypes. However, a greater understanding of the factors contributing to virus escape and the role of lipoproteins in masking virion surface domains involved in virus entry will be required to help define those protective determinants most likely to give broad protection. An approach to immune escape is potentially caused by viral infection of immune cells leading to the induction hypermutation of the immunoglobulin gene in B cells. These effects may contribute to HCV persistence and B cell lymphoproliferative diseases.

    View details for Web of Science ID 000251960900001

    View details for PubMedID 17990788

  • The neutralizing activity of anti-hepatitis C virus antibodies is modulated by specific glycans on the E2 envelope protein JOURNAL OF VIROLOGY Helle, F., Goffard, A., Morel, V., Duverlie, G., McKeating, J., Keck, Z., Foung, S., Penin, F., Dubuisson, J., Voisset, C. 2007; 81 (15): 8101-8111

    Abstract

    Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with up to 5 and 11 N-linked glycans on E1 and E2, respectively. Most of the glycosylation sites on HCV envelope glycoproteins are conserved, and some of the glycans associated with these proteins have been shown to play an essential role in protein folding and HCV entry. Such a high level of glycosylation suggests that these glycans can limit the immunogenicity of HCV envelope proteins and restrict the binding of some antibodies to their epitopes. Here, we investigated whether these glycans can modulate the neutralizing activity of anti-HCV antibodies. HCV pseudoparticles (HCVpp) bearing wild-type glycoproteins or mutants at individual glycosylation sites were evaluated for their sensitivity to neutralization by antibodies from the sera of infected patients and anti-E2 monoclonal antibodies. While we did not find any evidence that N-linked glycans of E1 contribute to the masking of neutralizing epitopes, our data demonstrate that at least three glycans on E2 (denoted E2N1, E2N6, and E2N11) reduce the sensitivity of HCVpp to antibody neutralization. Importantly, these three glycans also reduced the access of CD81 to its E2 binding site, as shown by using a soluble form of the extracellular loop of CD81 in inhibition of entry. These data suggest that glycans E2N1, E2N6, and E2N11 are close to the binding site of CD81 and modulate both CD81 and neutralizing antibody binding to E2. In conclusion, this work indicates that HCV glycans contribute to the evasion of HCV from the humoral immune response.

    View details for DOI 10.1128/JVI.00127-07

    View details for Web of Science ID 000248027400029

    View details for PubMedID 17522218

  • Immunogenic and functional organization of hepatitis C virus (HCV) glycoprotein E2 on infectious HCV virions JOURNAL OF VIROLOGY Keck, Z., Xia, J., Cai, Z., Li, T., Owsianka, A. M., Patel, A. H., Luo, G., Foung, S. K. 2007; 81 (2): 1043-1047

    Abstract

    Development of full-length hepatitis C virus (HCV) RNAs replicating efficiently and producing infectious cell-cultured virions, HCVcc, in hepatoma cells provides an opportunity to characterize immunogenic domains on viral envelope proteins involved in entry into target cells. A panel of immunoglobulin G1 human monoclonal antibodies (HMAbs) to three immunogenic conformational domains (designated A, B, and C) on HCV E2 glycoprotein showed that epitopes within two domains, B and C, mediated HCVcc neutralization, whereas HMAbs to domain A were all nonneutralizing. For the neutralizing antibodies to domain B (with some to conserved epitopes among different HCV genotypes), the inhibitory antibody concentration reducing HCVcc infection by 90%, IC90, ranged from 0.1 to 4 microg/ml. For some neutralizing HMAbs, HCVcc neutralization displayed a linear correlation with an antibody concentration between the IC50 and the IC90 while others showed a nonlinear correlation. The differences between IC50/IC90 ratios and earlier findings that neutralizing HMAbs block E2 interaction with CD81 suggest that these antibodies block different facets of virus-receptor interaction. Collectively, these findings support an immunogenic model of HCV E2 having three immunogenic domains with distinct structures and functions and provide added support for the idea that CD81 is required for virus entry.

    View details for DOI 10.1128/JVI.01710-06

    View details for Web of Science ID 000243350100061

    View details for PubMedID 17079294

  • High density lipoprotein inhibits hepatitis C virus-neutralizing antibodies by stimulating cell entry via activation of the scavenger receptor BI JOURNAL OF BIOLOGICAL CHEMISTRY Dreux, M., Pietschmann, T., Granier, C., Voisset, C., Ricard-Blum, S., Mangeot, P., Keck, Z., Foung, S., Vu-Dac, N., Dubuisson, J., Bartenschlager, R., Lavillette, D., Cosset, F. 2006; 281 (27): 18285-18295

    Abstract

    Hepatitis C virus (HCV) exploits serum-dependent mechanisms that inhibit neutralizing antibodies. Here we demonstrate that high density lipoprotein (HDL) is a key serum factor that attenuates neutralization by monoclonal and HCV patient-derived polyclonal antibodies of infectious pseudo-particles (HCVpp) harboring authentic E1E2 glycoproteins and cell culture-grown genuine HCV (HCVcc). Over 10-fold higher antibody concentrations are required to neutralize either HCV-enveloped particles in the presence of HDL or human serum, and less than 3-5-fold reduction of infectious titers are obtained at saturating antibody concentrations, in contrast to complete inhibition in serum-free conditions. We show that HDL interaction with the scavenger receptor BI (SR-BI), a proposed cell entry co-factor of HCV and a receptor mediating lipid transfer with HDL, strongly reduces neutralization of HCVpp and HCVcc. We found that HDL activation of target cells strongly stimulates cell entry of viral particles by accelerating their endocytosis, thereby suppressing a 1-h time lag during which cell-bound virions are not internalized and can be targeted by antibodies. Compounds that inhibit lipid transfer functions of SR-BI fully restore neutralization by antibodies in human serum. We demonstrate that this functional HDL/SR-BI interaction only interferes with antibodies blocking HCV-E2 binding to CD81, a major HCV receptor, reflecting its prominent role during the cell entry process. Moreover, we identify monoclonal antibodies targeted to epitopes in the E1E2 complex that are not inhibited by HDL. Consistently, we show that antibodies targeted to HCV-E1 efficiently neutralize HCVpp and HCVcc in the presence of human serum.

    View details for DOI 10.1074/jbc.M602706200

    View details for Web of Science ID 000238687300005

    View details for PubMedID 16675450

  • Preclinical evaluation of two neutralizing human monoclonal antibodies against hepatitis C virus (HCV): A potential treatment to prevent HCV reinfection in liver transplant patients JOURNAL OF VIROLOGY Eren, R., Landstein, D., Terkieltaub, D., Nussbaum, O., Zauberman, A., Ben-Porath, J., Gopher, J., Buchnick, R., Kovjazin, R., Rosenthal-Galili, Z., Aviel, S., Ilan, E., Shoshany, Y., Neville, L., Waisman, T., Ben-Moshe, O., Kischitsky, A., Foung, S. K., Keck, Z. Y., Pappo, O., Eid, A., Jurim, O., Zamir, G., Galun, E., Dagan, S. 2006; 80 (6): 2654-2664

    Abstract

    Passive immunotherapy is potentially effective in preventing reinfection of liver grafts in hepatitis C virus (HCV)-associated liver transplant patients. A combination of monoclonal antibodies directed against different epitopes may be advantageous against a highly mutating virus such as HCV. Two human monoclonal antibodies (HumAbs) against the E2 envelope protein of HCV were developed and tested for the ability to neutralize the virus and prevent human liver infection. These antibodies, designated HCV-AB 68 and HCV-AB 65, recognize different conformational epitopes on E2. They were characterized in vitro biochemically and functionally. Both HumAbs are immunoglobulin G1 and have affinity constants to recombinant E2 constructs in the range of 10(-10) M. They are able to immunoprecipitate HCV particles from infected patients' sera from diverse genotypes and to stain HCV-infected human liver tissue. Both antibodies can fix complement and form immune complexes, but they do not activate complement-dependent or antibody-dependent cytotoxicity. Upon complement fixation, the monoclonal antibodies induce phagocytosis of the immune complexes by neutrophils, suggesting that the mechanism of viral clearance includes endocytosis. In vivo, in the HCV-Trimera model, both HumAbs were capable of inhibiting HCV infection of human liver fragments and of reducing the mean viral load in HCV-positive animals. The demonstrated neutralizing activities of HCV-AB 68 and HCV-AB 65 suggest that they have the potential to prevent reinfection in liver transplant patients and to serve as prophylactic treatment in postexposure events.

    View details for DOI 10.1128/JVI.80.6.2654-2664.2006

    View details for Web of Science ID 000236131400007

    View details for PubMedID 16501075

  • Hepatitis C virus induces toll-like receptor 4 expression, leading to enhanced production of beta interferon and interleukin-6 JOURNAL OF VIROLOGY Machida, K., Cheng, K. T., Sung, V. M., Levine, A. M., Foung, S., Lai, M. M. 2006; 80 (2): 866-874

    Abstract

    Hepatitis C virus (HCV) induces inflammatory signals, leading to hepatitis, hepatocellular carcinomas, and lymphomas. The mechanism of HCV involvement in the host's innate immune responses has not been well characterized. In this study, we analyzed expression and regulation of the entire panel of toll-like receptors (TLRs) in human B cells following HCV infection in vitro. Among all of the TLRs (TLRs 1 to 10) examined, only TLR4 showed an altered expression (a three- to sevenfold up-regulation) after HCV infection. Peripheral blood mononuclear cells from HCV-infected individuals also showed a higher expression level of TLR4 compared with those of healthy individuals. HCV infection significantly increased beta interferon (IFN-beta) and interleukin-6 (IL-6) secretion from B cells, particularly after lipopolysaccharide stimulation. The increased IFN-beta and IL-6 production was mediated by TLR4 induction, since the introduction of the small interfering RNA against TLR4 specifically inhibited the HCV-induced cytokine production. Among all of the viral proteins, only NS5A caused TLR4 induction in hepatocytes and B cells. NS5A specifically activated the promoter of the TLR4 gene in both hepatocytes and B cells. In conclusion, HCV infection directly induces TLR4 expression and thereby activates B cells, which may contribute to the host's innate immune responses.

    View details for DOI 10.1128/JVI.80.2.866-874.2006

    View details for Web of Science ID 000234382900032

    View details for PubMedID 16378988

  • Analysis of a highly flexible conformational immunogenic domain a in hepatitis C virus E2 JOURNAL OF VIROLOGY Keck, Z. Y., LI, T. K., Xia, J. M., Bartosch, B., Cosset, F. L., Dubuisson, J., Foung, S. K. 2005; 79 (21): 13199-13208

    Abstract

    Hepatitis C (HCV) E2 glycoprotein is involved in virus attachment and entry, and its structural organization is largely unknown. Characterization of a panel of human monoclonal antibodies (HMAbs) to HCV by competition studies has led to an immunogenic organization model of E2 with three domains designated A, B, and C and epitopes in each domain having similar structural and functional properties. Domain A contains nonneutralizing epitopes, and domains B and C contain neutralizing epitopes. The isolation and characterization of three new HMAbs within domain A for a total of six provide support for this model. All six domain A HMAbs do not neutralize HCV retroviral pseudotype particle (HCVpp) infection on Huh-7 cells, and all six HMAbs have similar binding affinity and maximum binding, B(max), a relative indicator of epitope density, as other neutralizing HMAbs, suggesting that neutralization is epitope specific and not by binding to any surface epitope. The dose-dependent neutralizing activity of CBH-7, an HMAb to a domain C epitope in spatial proximity to domain A, and of CBH-5, a domain B HMAb to a more distant epitope, were tested in the presence and absence of each domain A HMAb. No enhancement or reduction in CBH-7 or CBH-5 neutralizing activity was observed, indicating that the potential induction of nonneutralizing antibodies should not be a central issue for HCV vaccine design. To assess whether domain A is involved in the structural changes as part of a pH-dependent virus envelope fusion process, changes in antibody binding patterns to normal pH and acid pH-treated HCVpp were measured. Antibody binding affinity of HMAbs to HCVpp was not affected by low pH. However, the B(max) values for low-pH-treated HCVpp with antibodies to domain A increased 46%, for domain C (CBH-7) they increased 23%, and for domain B (CBH-5) there was a decrease of 12%. Collectively, the organization and function of HCV E2 antigenic domains are roughly analogous to the large envelope glycoprotein E organizational structure for other flaviviruses with three distinct structural and functional domains.

    View details for DOI 10.1128/JVI.79.21.13199-13208.2005

    View details for Web of Science ID 000232666300001

    View details for PubMedID 16227243

  • Folding and dimerization of hepatitis C virus E1 and E2 glycoproteins in stably transfected CHO cells VIROLOGY Brazzoli, M., HELENIUS, A., Foung, S. K., Houghton, M., Abrignani, S., Merola, M. 2005; 332 (1): 438-453

    Abstract

    The recombinant E1E2 heterodimer of the hepatitis C virus is a candidate for a subunit vaccine. Folding analysis of E1 and E2 glycoproteins, stably expressed in CHO cells, showed that E1 folding was faster and more efficient than E2. The oxidized DTT-resistant conformation of E1 was completed within 2 h post-synthesis, while E2 not only required up to 6 h but also generated non-native species. Calnexin was found to assist E1 folding, whereas no chaperone association was found with E2. The assembly of E1 and E2 was assessed by co-immunoprecipitation and sedimentation velocity analysis. We found that the formation of native E1E2 heterodimers paralleled E2 oxidation kinetics, suggesting that E2 completed its folding process after association with E1. Once formed, sedimentation of the native E1E2 heterodimers was consistent with the absence of additional associated factors. Taken together, our data strongly suggest that productive folding of the major HCV spike protein E2 is assisted by E1.

    View details for DOI 10.1016/j.virol.2004.11.034

    View details for Web of Science ID 000226688500041

    View details for PubMedID 15661174

  • Antibody light chain-catalyzed hydrolysis of a hepatitis C virus peptide BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Taguchi, H., Keck, Z. Y., Foung, S. K., Paul, S., Nishiyama, Y. 2004; 14 (17): 4529-4532

    Abstract

    A panel of human monoclonal and recombinant antibody light chains was screened for cleavage of the synthetic peptide corresponding to a neutralizing epitope of hepatitis C virus (residues 192-205 of envelope glycoprotein E1). One of the 39 light chains studied hydrolyzed the Val197-Ser198 bond of the peptide with Km and kcat values of 223 +/- 7 microM and 0.087 +/- 0.001 min(-1).

    View details for DOI 10.1016/j.bmcl.2004.06.044

    View details for Web of Science ID 000223382000032

    View details for PubMedID 15357986

  • Hepatitis C virus E2 has three immunogenic domains containing conformational epitopes with distinct properties and biological functions JOURNAL OF VIROLOGY Keck, Z. Y., Op de Beeck, A., Hadlock, K. G., Xia, J. M., LI, T. K., Dubuisson, J., Foung, S. K. 2004; 78 (17): 9224-9232

    Abstract

    Mechanisms of virion attachment, interaction with its receptor, and cell entry are poorly understood for hepatitis C virus (HCV) because of a lack of an efficient and reliable in vitro system for virus propagation. Infectious HCV retroviral pseudotype particles (HCVpp) were recently shown to express native E1E2 glycoproteins, as defined in part by HCV human monoclonal antibodies (HMAbs) to conformational epitopes on E2, and some of these antibodies block HCVpp infection (A. Op De Beeck, C. Voisset, B. Bartosch, Y. Ciczora, L. Cocquerel, Z. Y. Keck, S. Foung, F. L. Cosset, and J. Dubuisson, J. Virol. 78:2994-3002, 2004). Why some HMAbs are neutralizing and others are nonneutralizing is looked at in this report by a series of studies to determine the expression of their epitopes on E2 associated with HCVpp and the role of antibody binding affinity. Antibody cross-competition defined three E2 immunogenic domains with neutralizing HMAbs restricted to two domains that were also able to block E2 interaction with CD81, a putative receptor for HCV. HCVpp immunoprecipitation showed that neutralizing and nonneutralizing domains are expressed on E2 associated with HCVpp, and affinity studies found moderate-to-high-affinity antibodies in all domains. These findings support the perspective that HCV-specific epitopes are responsible for functional steps in virus infection, with specific antibodies blocking distinct steps of virus attachment and entry, rather than the perspective that virus neutralization correlates with increased antibody binding to any virion surface site, independent of the epitope recognized by the antibody. Segregation of virus neutralization and sensitivity to low pH to specific regions supports a model of HCV E2 immunogenic domains similar to the antigenic structural and functional domains of other flavivirus envelope E glycoproteins.

    View details for DOI 10.1128/JVI.78.17.9224-9232.2004

    View details for Web of Science ID 000223386600026

    View details for PubMedID 15308717

  • Human monoclonal antibody to hepatitis C virus El glycoprotein that blocks virus attachment and viral infectivity JOURNAL OF VIROLOGY Keck, Z. Y., Sung, V. M., Perkins, S., Rowe, J., Paul, S., Liang, T. J., Lai, M. M., Foung, S. K. 2004; 78 (13): 7257-7263

    Abstract

    Human antibodies elicited in response to hepatitis C virus (HCV) infection are anticipated to react with the native conformation of the viral envelope structure. Isolation of these antibodies as human monoclonal antibodies that block virus binding and entry will be useful in providing potential therapeutic reagents and for vaccine development. H-111, an antibody to HCV envelope 1 protein (E1) that maps to the YEVRNVSGVYH sequence and is located near the N terminus of E1 and is able to immunoprecipitate E1E2 heterodimers, is described. Binding of H-111 to HCV E1 genotypes 1a, 1b, 2b, and 3a indicates that the H-111 epitope is highly conserved. Sequence analysis of antibody V regions showed evidence of somatic and affinity maturation of H-111. Finally, H-111 blocks HCV-like particle binding to and HCV virion infection of target cells, suggesting the involvement of this epitope in virus binding and entry.

    View details for DOI 10.1128/JVI.78.13.7257-7263.2004

    View details for Web of Science ID 000222153800057

    View details for PubMedID 15194801

  • Incomplete humoral immunity against hepatitis C virus is linked with distinct recognition of putative multiple receptors by E2 envelope glycoprotein JOURNAL OF IMMUNOLOGY Heo, T. H., Chang, J. H., Lee, J. W., Foung, S. K., Dubuisson, J., Kang, C. Y. 2004; 173 (1): 446-455

    Abstract

    Little is known about the role of the humoral immune response to hepatitis C virus (HCV). This study provides molecular evidence for the mechanism by which neutralizing Abs from the sera of chronic HCV patients have lower inhibitory activities against the binding of HCV E2 envelope protein to human hepatoma cell lines than to a lymphoma cell line. E2 binds to several putative receptors, specifically human CD81; human scavenger receptor, class B, type 1; and heparan sulfate. We have shown that E2 binds to target cells via these receptors in a noncompetitive manner. Thus, incomplete inhibition of one of the receptors leads to only a partial E2 blockade and, possibly, evasion of the host immune response. We demonstrated that the difference in and reduction of inhibition was closely related to impaired blockade of E2 binding to scavenger receptor, class B, type 1, and heparan sulfate. We have also shown that soluble E2 protein binds to multiple soluble receptors via separate binding domains on E2, providing further evidence for the distinct recognition of multiple cellular receptors by E2. This report suggests a novel finding that biased humoral immune responses to HCV E2 might provide an alternative mechanism for viral escape without the involvement of mutation. Additionally, our data give crucial consideration to the development of HCV vaccines that stimulate protective humoral immune responses.

    View details for Web of Science ID 000222170900057

    View details for PubMedID 15210804

  • Characterization of functional hepatitis C virus envelope glycoproteins JOURNAL OF VIROLOGY De Beeck, A. O., Voisset, C., Bartosch, B., Ciczora, Y., Cocquerel, L., Keck, Z., Foung, S., Cosset, F. L., Dubuisson, J. 2004; 78 (6): 2994-3002

    Abstract

    Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2, that assemble as a noncovalent heterodimer which is mainly retained in the endoplasmic reticulum. Because assembly into particles and secretion from the cell lead to structural changes in viral envelope proteins, characterization of the proteins associated with the virion is necessary in order to better understand how they mature to be functional in virus entry. There is currently no efficient and reliable cell culture system to amplify HCV, and the envelope glycoproteins associated with the virion have therefore not been characterized yet. Recently, infectious pseudotype particles that are assembled by displaying unmodified HCV envelope glycoproteins on retroviral core particles have been successfully generated. Because HCV pseudotype particles contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, should be in a mature conformation similar to that on the native HCV particles. In this study, we used conformation-dependent monoclonal antibodies to characterize the envelope glycoproteins associated with HCV pseudotype particles. We showed that the functional unit is a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. We did not observe any evidence of maturation by a cellular endoprotease during the transport of these envelope glycoproteins through the secretory pathway. These envelope glycoproteins were recognized by a panel of conformation-dependent monoclonal antibodies as well as by CD81, a molecule involved in HCV entry. The functional envelope glycoproteins associated with HCV pseudotype particles were also shown to be sensitive to low-pH treatment. Such conformational changes are likely necessary to initiate fusion.

    View details for DOI 10.1128/JVI.78.6.2994-3002.2004

    View details for Web of Science ID 000220043100034

    View details for PubMedID 14990718

  • DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2 JOURNAL OF BIOLOGICAL CHEMISTRY Lozach, P. Y., Lortat-Jacob, H., de Lavalette, A. D., Staropoli, I., Foung, S., Amara, A., Houles, C., Fieschi, F., Schwartz, O., Virelizier, J. L., Arenzana-Seisdedos, F., Altmeyer, R. 2003; 278 (22): 20358-20366

    Abstract

    The hepatitis C virus (HCV) genome codes for highly mannosylated envelope proteins, which are naturally retained in the endoplasmic reticulum. We found that the HCV envelope glycoprotein E2 binds the dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the related liver endothelial cell lectin L-SIGN through high-mannose N-glycans. Competing ligands such as mannan and an antibody directed against the carbohydrate recognition domains (CRD) abrogated binding. While no E2 interaction with distant monomeric CRDs on biosensor chips could be detected, binding is observed if CRDs are closely seeded (Kd = 48 nm) and if the CRD is part of the oligomeric-soluble extracellular domain of DC-SIGN (Kd = 30 nm). The highest affinity is seen for plasma membrane-expressed DC-SIGN and L-SIGN (Kd = 3 and 6 nm, respectively). These results indicate that several high-mannose N-glycans in a structurally defined cluster on E2 bind to several subunits of the oligomeric lectin CRD. High affinity interaction of viral glycoproteins with oligomeric lectins might represent a strategy by which HCV targets to and concentrates in the liver and infects dendritic cells.

    View details for DOI 10.1074/jbc.M301284200

    View details for Web of Science ID 000183078000100

    View details for PubMedID 12609975

  • Recognition of native hepatitis C virus E1E2 heterodimers by a human monoclonal antibody JOURNAL OF VIROLOGY Cocquerel, L., Quinn, E. R., Flint, M., Hadlock, K. G., Foung, S. K., Levy, S. 2003; 77 (2): 1604-1609

    Abstract

    The majority of hepatitis C virus (HCV)-infected individuals progress from acute to chronic disease, despite the presence of a strong humoral immune response to the envelope glycoproteins E1 and E2. When expressed in mammalian cells, E1 and E2 form both noncovalently linked E1E2 heterodimers, believed to be properly folded, and disulfide-linked, high-molecular-weight aggregates that are misfolded. Previously, we identified 10 human monoclonal antibodies (HMAbs) that bind E2 glycoproteins from different genotypes. Here we demonstrate that one of these HMAbs, CBH-2, is unique in its ability to distinguish between properly folded and misfolded envelope proteins. This HMAb recognizes HCV-E2 only when complexed with E1. The E1E2 complexes recognized by CBH-2 are noncovalently linked heterodimers and not misfolded disulfide-linked, high-molecular-weight aggregates. The E1E2 heterodimers seen by CBH-2 no longer associate with the endoplasmic reticulum chaperone calnexin and are likely to represent the prebudding form of the HCV virion.

    View details for DOI 10.1128/JVI.77.2.1604-1609.2003

    View details for Web of Science ID 000180166600080

    View details for PubMedID 12502876

  • Structural features of envelope proteins on hepatitis C virus-like particles as determined by anti-envelope monoclonal antibodies and CD81 binding VIROLOGY Triyatni, M., Vergalla, J., Davis, A. R., Hadlock, K. G., Foung, S. K., Liang, T. J. 2002; 298 (1): 124-132

    Abstract

    The envelope glycoprotein E2 of hepatitis C virus (HCV) is a major component of the viral envelope. Knowledge of its topologic features and antigenic determinants in virions is crucial in understanding the viral binding sites to cellular receptor(s) and the induction of neutralizing antibodies. The lack of a robust cell culture system for virus propagation has hampered the characterization of E2 presented on the virion. Here we report the structural features of hepatitis C virus-like particles (HCV-LPs) of the 1a and 1b genotypes as determined by various mouse and human monoclonal anti-envelope antibodies. Our results show that the E2 protein of HCV-LPs reacts with human monoclonal antibodies recognizing conformational determinants. Monoclonal antibodies (mAbs) specific for the hypervariable region 1 (HVR-1) sequence reacted strongly with HCV-LPs, suggesting that the HVR-1 is exposed on the viral surface. Several mAbs recognized both HCV-LPs with equally high affinity, indicating that the corresponding epitopes [amino acids (aa) 192-217 of E1 and aa 412-423, aa 522-531, and aa 640-653 of E2] are conserved in both genotypes and exposed on the surface of the HCV-LP. The E2 and E1/E2 dimers of 1a bound strongly to the recombinant large extracellular loop (LEL) of CD81 (CD81-LEL) of human and African green monkey, while the HCV-LP of 1a bound weakly to human CD81-LEL. E1/E2 dimers and the HCV-LPs of 1b did not bind CD81-LEL, consistent with the notion that CD81 recognition by E2 is strain-specific and does not correlate with permissiveness of infection. A model of the topology and exposed antigenic determinants of the envelope proteins of HCV is proposed.

    View details for DOI 10.1006/viro.2002.1463

    View details for Web of Science ID 000176822900014

    View details for PubMedID 12093180

  • Epitope mapping of human monoclonal antibodies recognizing conformational epitopes within HTLV type 1 gp46, employing HTLV type 1/2 envelope chimeras AIDS RESEARCH AND HUMAN RETROVIRUSES Hadlock, K. G., Yang, Q., Rowe, J., Foung, S. K. 2002; 18 (1): 57-70

    Abstract

    The majority of the antibody response to HTLV-1 surface glycoprotein, gp46, is directed at conformational epitopes. However, the regions of HTLV-1 gp46 that contain conformational epitopes are poorly defined. We previously reported on human monoclonal antibodies (hMAbs) to conformational epitopes within the HTLV-1 surface glycoprotein (gp46) that inhibit HTLV-1-mediated syncytium formation (Hadlock KG, Rowe J, Perkins S, et al.: J Virol 1997;71:5828-5840). To localize the conformational epitopes recognized by these antibodies, chimeric envelope proteins were constructed in which selected regions of the HTLV-1 envelope were replaced with the corresponding sequences from other members of the HTLV family of retroviruses. The chimeras were tested for reactivity with three hMAbs to conformational epitopes in HTLV-1 gp46, PRH-7A, PRH-3, and PRH-4, and one hMAb to a linear epitope, 0.5alpha. hMAb PRH-3 was specifically nonreactive with a chimera that replaced amino acids 32-36 of HTLV-1 gp46 and exhibited sharply reduced reactivity with a chimera that replaced amino acids 224-251 of HTLV-1 with the corresponding HTLV-2 sequence. hMAb PRH-4 was specifically nonreactive with a construct replacing amino acids 1-162 of HTLV-1 gp46 with the corresponding HTLV-2 sequence. Thus, HTLV-1 gp46 contains multiple conformational epitopes located in the amino-terminal portion of the protein.

    View details for Web of Science ID 000173367400007

    View details for PubMedID 11804557

  • The B-cell receptor of a hepatitis C virus (HCV)-associated non-Hodgkin lymphoma binds the viral E2 envelope protein, implicating HCV in lymphomagenesis BLOOD Quinn, E. R., Chan, C. H., Hadlock, K. G., Foung, S. K., Flint, M., Levy, S. 2001; 98 (13): 3745-3749

    Abstract

    Hepatitis C virus (HCV) infection is associated with extrahepatic B-cell lymphoproliferative disorders. To determine whether a viral antigen drives this B-cell expansion, the B-cell receptors were cloned from HCV-associated lymphomas and were expressed as soluble immunoglobulins. The rescued immunoglobulins were then tested for their ability to bind the HCV-E2 envelope glycoprotein, an antigen that was previously implicated in the pathogenesis of HCV-associated B-cell diseases. One of 2 lymphoma immunoglobulin test cases bound the E2 protein in a manner identical to a bona fide human anti-E2 antibody. Moreover, it bound E2 from multiple viral genotypes, suggesting reactivity with a conserved E2 epitope. These findings support the hypothesis that some HCV-associated lymphomas originate from B cells that were initially activated by the HCV-E2 protein and might explain the association between HCV infection and some B-cell lymphoproliferative disorders.

    View details for Web of Science ID 000172604700032

    View details for PubMedID 11739181

  • Cross-reactivity and clinical impact of the antibody response to hepatitis C virus second envelope glycoprotein (E2) JOURNAL OF MEDICAL VIROLOGY Hadlock, K. G., Gish, R., Rowe, J., Rajyaguru, S. S., Newsom, M., Warford, A., Foung, S. K. 2001; 65 (1): 23-29

    Abstract

    The genotype of hepatitis C virus (HCV) can profoundly affect the success of antiviral therapy for HCV infection. A possible contributing factor is a varied immune response elicited by infection with different HCV genotypes. In this study, full-length E2 proteins of HCV genotypes 1a, 1b, 2a, and 2b were used to determine the fraction of the humoral immune response to HCV E2 that is genotype specific. Greater than 90% of all infected individuals had serum antibodies to the four E2 proteins. Overall, individuals infected with genotype 1a or 1b were characterized by variable immune responses to HCV E2 with relatively high amounts of cross-reactivity with other E2 proteins. Individuals infected with genotype 2a or 2b exhibited a strong preferential reactivity to genotype 2a and 2b E2 proteins. Individuals with elevated titers to HCV E2 were more likely to be infected with genotype 2a and had a significantly lower median viral load. These findings indicate that the antibody response to HCV E2 is affected by the genotype of the virus and that induction of a strong humoral immune response to HCV E2 may contribute to a decreased viral load.

    View details for Web of Science ID 000170257200004

    View details for PubMedID 11505439

  • V(H)1-69 gene is preferentially used by hepatitis C virus-associated B cell lymphomas and by normal B cells responding to the E2 viral antigen BLOOD Chan, C. H., Hadlock, K. G., Foung, S. K., Levy, S. 2001; 97 (4): 1023-1026

    Abstract

    Hepatitis C virus (HCV)-associated B cell lymphomas were previously shown to express a restricted repertoire of immunoglobulin V(H) and V(L) genes, V(H)1-69 and VkappaA27, respectively. Although this suggests a role for antigen selection in the pathogenesis of these lymphomas, the driving antigen involved in the clonal expansion has not been identified. B cell response to a viral antigen, the HCV envelope glycoprotein 2 (E2), was analyzed in an asymptomatic HCV-infected patient. Single B cells, immortalized as hybridomas and selected for binding E2, were analyzed for their V gene usage. Sequences of these V region genes demonstrated that each hybridoma expressed unique V(H) and V(L) genes. Remarkably, these anti-E2 hybridomas preferentially used the V(H)1-69 gene. Analysis of replacement to silent mutation ratios indicated that the genes underwent somatic mutation and antigenic selection. In a separate report, human anti-E2 antibodies were also shown to express the same V(H) gene. These data strengthen the hypothesis that the HCV-associated lymphomas are derived from clonally expanded B cells stimulated by HCV.

    View details for Web of Science ID 000166867200032

    View details for PubMedID 11159532

  • Human monoclonal antibodies that inhibit binding of hepatitis C virus E2 protein to CD81 and recognize conserved conformational epitopes JOURNAL OF VIROLOGY Hadlock, K. G., Lanford, R. E., Perkins, S., Rowe, J., Yang, Q., Levy, S., Pileri, P., Abrignani, S., Foung, S. K. 2000; 74 (22): 10407-10416

    Abstract

    The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.

    View details for Web of Science ID 000090083800016

    View details for PubMedID 11044085

  • The humoral immune response to human T-cell lymphotropic virus type 1 envelope glycoprotein gp46 is directed primarily against conformational epitopes JOURNAL OF VIROLOGY Hadlock, K. G., Rowe, J., Foung, S. K. 1999; 73 (2): 1205-1212

    Abstract

    Individuals infected with human T-cell lymphotropic virus type 1 (HTLV-1) develop a robust immune response to the surface envelope glycoprotein gp46 that is partially protective. The relative contribution of antibodies to conformation-dependent epitopes, including those mediating virus neutralization as part of the humoral immune response, is not well defined. We assess in this report the relationship between defined linear and conformational epitopes and the antibodies elicited to these domains. First, five monoclonal antibodies to linear epitopes within gp46 were evaluated for their ability to abrogate binding of three human monoclonal antibodies that inhibit HTLV-1-mediated syncytia formation and recognize conformational epitopes. Binding of antibodies to conformational epitopes was unaffected by antibodies to linear epitopes throughout the carboxy-terminal half and central domain of HTLV-1 gp46. Second, an enzyme-linked immunoadsorbent assay was developed and used to measure serum antibodies to native and denatured gp46 from HTLV-1-infected individuals. In sera from infected individuals, reactivity to denatured gp46 had an average of 15% of the reactivity observed to native gp46. Third, serum antibodies from 24 of 25 of HTLV-1-infected individuals inhibited binding of a neutralizing human monoclonal antibody, PRH-7A, to a conformational epitope on gp46 that is common to HTLV-1 and -2. Thus, antibodies to conformational epitopes comprise the majority of the immune response to HTLV-1 gp46, and the epitopes recognized by these antibodies do not appear to involve sequences in previously described immunodominant linear epitopes.

    View details for Web of Science ID 000078017500037

    View details for PubMedID 9882322

  • Large granular lymphocyte leukaemia occurring after renal transplantation BRITISH JOURNAL OF HAEMATOLOGY Gentile, T. C., Hadlock, K. G., Uner, A. H., Delal, B., Squiers, E., CROWLEY, S., Woodman, R. C., Foung, S. K., Poiesz, B. J., Loughran, T. P. 1998; 101 (3): 507-512

    Abstract

    Post-transplantation lymphoproliferative disorders (PTLD) are a clinicopathologically heterogeneous group of lymphoid proliferations. The majority are of B-cell origin and associated with Epstein-Barr virus (EBV) infection. In contrast, the development of T-cell PTLD is much less common and EBV does not appear to be involved in pathogenesis. In this report we describe three patients who developed large granular lymphocyte (LGL) leukaemia after renal transplantation. These patients had clonal expansion of CD3+, CD8+, CD57+, CD56- LGL. We were unable to detect CMV antigen or find evidence for EBV or human T-cell leukaemia/lymphoma virus genome in the LGL from these patients. These data show that LGL leukaemia should be included as one of the types of T-cell proliferations which can occur post transplant.

    View details for Web of Science ID 000074236100019

    View details for PubMedID 9633895

  • Epitope mapping of HTLV envelope seroreactivity in LGL leukaemia BRITISH JOURNAL OF HAEMATOLOGY Loughran, T. P., Hadlock, K. G., Perzova, R., Gentile, T. C., Yang, Q., Foung, S. K., Poiesz, B. J. 1998; 101 (2): 318-324

    Abstract

    Sera from approximately 50% of patients with large granular lymphocyte (LGL) leukaemia react with a recombinant human T-cell leukaemia/lymphoma virus (HTLV) transmembrane envelope protein, p21e. Two immunodominant epitopes within env p21e have been defined by reactivity against recombinant proteins GD21 and BA21. In this study sera from 41 patients with LGL leukaemia were examined for reactivity against these recombinant HTLV env proteins. Overall, 21/41 (51%) sera reacted to p21e. Only two sera reacted to GD21. The predominant immunoreactivity against p21e was directed against the BA21 epitope, with 19/41 (46%) sera being BA21 positive. Seroconversion to BA21 protein was also documented. PCR analyses confirmed the low incidence of protypical HTLV sequences (2/41, 5%). These data document an association between BA21 seroreactivity and LGL leukaemia. This finding raises the possibility that such BA21 seroreactivity could be due to cross-reactivity to a cellular or retroviral antigen sharing some amino acid homology with the transmembrane glycoprotein of HTLV.

    View details for Web of Science ID 000073738200016

    View details for PubMedID 9609528

  • GBV-C/HGV: A new virus within the Flaviviridae and its clinical implications TRANSFUSION MEDICINE REVIEWS Hadlock, K. G., Foung, S. K. 1998; 12 (2): 94-108

    View details for Web of Science ID 000073086100003

    View details for PubMedID 9566077

  • Experimental infection of chimpanzees with hepatitis G virus and genetic analysis of the virus JOURNAL OF INFECTIOUS DISEASES Bukh, J., Kim, J. P., Govindarajan, S., Apgar, C. L., Foung, S. K., Wages, J., Yun, A. J., Shapiro, M., Emerson, S. U., Purcell, R. H. 1998; 177 (4): 855-862

    Abstract

    Hepatitis G virus (HGV) was transmitted to 2 chimpanzees by inoculation with human plasma containing approximately 10(8) genome equivalents (GE) of HGV. The infection was characterized by the late appearance (weeks 10 and 11 after inoculation [pi]) of viremia that persisted throughout the 120-week follow-up. Serum HGV titer increased steadily until it plateaued at 10(6)-10(7) GE/mL. However, despite this relatively high titer, neither of the chimpanzees developed hepatitis. The sequence of the viral genome, recovered from each chimpanzee at week 77 pi, differed from that of the inoculum by 5 nt (2 aa) and 27 nt (2 aa). Two more chimpanzees were inoculated with a first-passage plasma pool. The chimpanzee inoculated with approximately 10(6.7) GE of HGV had viremia at week 1 pi. However, the viral titer increased with the same kinetics as observed in the first passage. The second chimpanzee inoculated with approximately 10(4.7) GE of HGV had late appearance (week 7 pi) of viremia.

    View details for Web of Science ID 000072719400004

    View details for PubMedID 9534956

  • Seroreactivity to an envelope protein of human T-cell leukemia/lymphoma virus in patients with CD3(-) (natural killer) lymphoproliferative disease of granular lymphocytes BLOOD Loughran, T. P., Hadlock, K. G., Yang, Q., Perzova, R., Zambello, R., Semenzato, G., Foung, S. K., Poiesz, B. J. 1997; 90 (5): 1977-1981

    Abstract

    Natural killer (NK) cells are CD3- large granular lymphocytes (LGL) responsible for immunity against viral infections. A chronic lymphoproliferative disorder of NK cells has been described in which the expanded NK cells display a restricted phenotype and cytotoxic activity. These data raise the hypothesis that proliferating LGL in these patients result from discrete expansions of NK cells responding to an unknown, perhaps viral, antigen. Recently, it was found that mice transgenic for the tax gene of human T-cell leukemia/lymphoma virus (HTLV) develop NK leukemia. Therefore, we studied 15 patients with chronic NK lymphoproliferative disorder for evidence of HTLV infection. Sera were tested using an HTLV-I/II-enzyme linked immunosorbent assay and a modified Western blot assay containing recombinant env proteins. None of the sera met conventional criteria for HTLV seroreactivity. However, sera from 11 patients (73%) reacted with the recombinant HTLV env protein p21E. The anti-p21E reactivity of these sera was then mapped employing the recombinant proteins GD21 and BA21. No reactivity to the immunodominant HTLV epitope GD21 was observed, suggesting that prototypical HTLV infection is unlikely in these patients. This was confirmed by finding no evidence for HTLV nucleic acids by PCR analyses employing primers specific for conserved regions in the env, pol, and pX genes. In contrast, 10 of the 15 sera reacted with the epitope BA21, documenting for the first time an association between a unique seroreactivity and disease. The high incidence of BA21 seroreactivity in these patients suggests that exposure to a protein containing homology to BA21 may be important in the pathogenesis of this lymphoproliferative disorder.

    View details for Web of Science ID A1997XX16600028

    View details for PubMedID 9292532

  • Neutralizing human monoclonal antibodies to conformational epitopes of human T-cell lymphotropic virus type 1 and 2 gp46 JOURNAL OF VIROLOGY 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; 71 (8): 5828-5840

    Abstract

    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.

    View details for Web of Science ID A1997XK15300016

    View details for PubMedID 9223472

  • A source of glycosylated human T-cell lymphotropic virus type 1 envelope protein: Expression of gp46 by the vaccinia virus T7 polymerase system JOURNAL OF VIROLOGY Arp, J., LEVATTE, M., Rowe, J., Perkins, S., King, E., LEYSTRALANTZ, C., Foung, S. K., Dekaban, G. K. 1996; 70 (11): 7349-7359

    Abstract

    Heterologous expression of the human T-cell lymphotropic virus type 1 (HTLV-1) envelope surface glycoprotein (gp46) in a vaccinia virus/T7 polymerase system resulted in the production of authentic recombinant gp46. Five differentially glycosylated forms of the surface envelope protein were produced by this mammalian system, as demonstrated by tunicamycin inhibition of N-glycosylation and N-glycan removal with endoglycosidase H and glycopeptidase F. These studies revealed that all four potential N-glycosylation sites in gp46 were used for oligosaccharide modification and that the oligosaccharides were mannose-rich and/or hybrid in composition. Conformational integrity of the recombinant HTLV-1 envelope protein was determined by the ability to bind to various HTLV-1-infected human sera and a panel of conformational-dependent human monoclonal antibodies under nondenaturing conditions. Furthermore, this recombinant gp46 was recognized by a series of HTLV-2-infected human sera and sera from a Pan paniscus chimpanzee infected with the distantly related simian T-cell lymphotropic virus STLVpan-p. Maintenance of highly conserved conformational epitopes in the recombinant HTLV-1 envelope protein structure suggests that it may serve as a useful diagnostic reagent and an effective vaccine candidate.

    View details for Web of Science ID A1996VL79200001

    View details for PubMedID 8892853

  • Molecular cloning and disease association of hepatitis G virus: A transfusion-transmissible agent SCIENCE Linnen, J., Wages, J., ZHANGKECK, Z. Y., Fry, K. E., KRAWCZYNSKI, K. Z., Alter, H., Koonin, E., Gallagher, M., Alter, M., Hadziyannis, S., Karayiannis, P., Fung, K., Nakatsuji, Y., SHIH, J. W., Young, L., Piatak, M., Hoover, C., Fernandez, J., Chen, S., Zou, J. C., Morris, T., Hyams, K. C., Ismay, S., Lifson, J. D., Hess, G., Foung, S. K., Thomas, H., Bradley, D., Margolis, H., Kim, J. P. 1996; 271 (5248): 505-508

    Abstract

    An RNA virus, designated hepatitis G virus (HGV), was identified from the plasma of a patient with chronic hepatitis. Extension from an immunoreactive complementary DNA clone yielded the entire genome (9392 nucleotides) encoding a polyprotein of 2873 amino acids. The virus is closely related to GB virus C (GBV-C) and distantly related to hepatitis C virus, GBV-A, and GBV-B. HGV was associated with acute and chronic hepatitis. Persistent viremia was detected for up to 9 years in patients with hepatitis. The virus is transfusion-transmissible. It has a global distribution and is present within the volunteer blood donor population in the United States.

    View details for Web of Science ID A1996TR32200042

    View details for PubMedID 8560265

  • ENHANCED SPECIFICITY OF TRUNCATED TRANSMEMBRANE PROTEIN FOR SEROLOGIC CONFIRMATION OF HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE-1 (HTLV-1) AND HTLV-2 INFECTIONS BY WESTERN-BLOT (IMMUNOBLOT) ASSAY CONTAINING RECOMBINANT ENVELOPE GLYCOPROTEINS JOURNAL OF CLINICAL MICROBIOLOGY Varma, M., Rudolph, D. L., KNUCHEL, M., Switzer, W. M., Hadlock, K. G., Velligan, M., Chan, L., Foung, S. K., Lal, R. B. 1995; 33 (12): 3239-3244

    Abstract

    Immunoassays based on the highly immunogenic transmembrane protein of human T-cell lymphotropic virus type 1 (HTLV-1) (protein 21c) are capable of detecting antibodies in all individuals infected with HTLV-1 and HTLV-2. However, because of antigenic mimicry with other cellular and viral proteins, such assays also have a large proportion of false-positive reactions. We have recently identified an immunodominant epitope, designated GD21-I located within amino acids 361 to 404 of the transmembrane protein, that appears to eliminate such false positivity. This recombinant GD21-I protein was used in conjunction with additional recombinant HTLV type-specific proteins and a whole virus lysate to develop a modified Western blot (immunoblot) assay (HTLV WB 2.4). The sensitivity and specificity of this assay were evaluated with 352 specimens whose infection status was determined by PCR assay for the presence or absence of HTLV-1/2 proviral sequences. All HTLV-1-positive (n = 102) and HTLV-2-positive (n = 107) specimens reacted with GD21-1 in the HTLV WB 2.4 assay, yielding a test sensitivity of 100%. Furthermore, all specimens derived from individuals infected with different viral subtypes of HTLV-1 (Cosmopolitan, Japanese, and Melanesian) and HTLV-2 (IIa0, a3, a4, IIb1, b4, and b5) reacted with GD21-I in the HTLV WB 2.4 assay. More importantly, HTLV WB 2.4 analysis of 81 PCR-negative specimens, all of which reacted to recombinant protein 21e in the presence or absence of p24 and p19 reactivity in the standard WB assay, showed that only two specimens retained reactivity to GD21-I, yielding an improved test specificity for the transmembrane protein of 97.5%. None of 41 specimens with gag reactivity only or 21 HTLV-negative specimens demonstrated reactivity to GD21-I. In an analysis of additional specimens (n = 169) from different geographic areas for which PCR results were not available, a substantial increase in the specificity of GD21-I detection was demonstrated, with no effect on the sensitivity of GD21-I detection among specimens from seropositive donors. Thus, the highly sensitive, GD21-I-based HTLV WB 2.4 assay eliminates the majority of false-positive transmembrane results, thereby increasing the specificity for serologic confirmation of HTLV-1 and HTLV-2 infections.

    View details for Web of Science ID A1995TF01500033

    View details for PubMedID 8586709

  • DELINEATION OF AN IMMUNODOMINANT AND HUMAN T-CELL LYMPHOTROPIC VIRUS (HTLV)-SPECIFIC EPITOPE WITHIN THE HTLV-I TRANSMEMBRANE GLYCOPROTEIN BLOOD Hadlock, K. G., Goh, C. J., BRADSHAW, P. A., Perkins, S., Lo, J., Kaplan, J. E., Khabbaz, R., Foung, S. K. 1995; 86 (4): 1392-1399

    Abstract

    Antibody reactivity to the transmembrane region of human T-cell lymphotropic virus type I (HTLV-I) envelope, gp21, is observed in virtually all individuals infected with HTLV-I or HTLV-II. Recombinant proteins encoding selected portions of gp21 are described and used to define two immunogenic regions. The first epitope (designated GD21-I) contains amino acids 361 to 404 of the HTLV-I envelope and reacted with all of 54 sera from HTLV-I- and HTLV-II-infected individuals. The second epitope (designated BA21) expresses amino acids 397 to 430 of the HTLV-I envelope and was recognized by 33 of 54 HTLV antisera. To determine the specificity of GD21-I and BA21, sera from 17 HTLV-negative individuals with nonspecific reactivity to p21E were tested. None of these sera reacted with GD21-I, but 16 of 17 sera reacted with BA21. With virtually complete reactivity to sera from HTLV-infected individuals and no reactivity to sera from p21E-reactive uninfected individuals, GD21-I will be useful in immunoassays for the detection of HTLV infection.

    View details for Web of Science ID A1995RN46700022

    View details for PubMedID 7632947

  • Stabilizing antibody secretion of human Epstein Barr virus-activated B-lymphocytes with hybridoma formation by electrofusion. Methods in molecular biology (Clifton, N.J.) Perkins, S., FOUNG, S. K. 1995; 48: 295-307

    View details for PubMedID 8528401

  • LOCALIZATION OF ANTIGENIC SITES ON HUMAN CYTOMEGALOVIRUS VIRION STRUCTURAL PROTEINS ENCODED BY UL48 AND UL56 VIROLOGY BRADSHAW, P. A., DURANGUARINO, M. R., Perkins, S., ROWE, J. I., Fernandez, J., Fry, K. E., Reyes, G. R., Young, L., Foung, S. K. 1994; 205 (1): 321-328

    Abstract

    Two human cytomegalovirus (HCMV) virion structural proteins and their associated reading frames have been identified with two human-derived monoclonal antibodies (HMAbs), X2-16 and X-16. HMAb X2-16 identified recombinant protein expressing molecular clones that mapped to the open reading frame (ORF) of the UL48 gene of HCMV, between amino acids 584 and 646 (nucleotides 65,084 and 65,272, Chee et al., 1990, "Current Topics in Microbiology and Immunology," Vol. 154, pp. 125-169). The UL48 gene product has an apparent molecular weight of 216 kDa. HMAb X-16 identified clones derived from the UL56 ORF between amino acids 380 and 425 (nucleotides 84,733 and 84,870). On immunoblots, HMAb X-16 detected two HCMV proteins of 96 and 60 kDa. Both UL48 and UL56 are highly conserved among the human herpesviruses and their products have been predicted to have essential functions for virus production and maturation. These results confirm that UL48 and UL56 are functional genes encoding essential viral proteins which also generate an immune response in the immunocompetent host.

    View details for Web of Science ID A1994PQ62300036

    View details for PubMedID 7975228

  • IMMUNOGLOBULIN PROPHYLAXIS AGAINST HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE-II IN RABBITS JOURNAL OF INFECTIOUS DISEASES Morishita, N., Ishii, K., Tanaka, Y., Sawada, T., Hoshino, H., Foung, S. K., Miyoshi, I. 1994; 169 (3): 620-623

    Abstract

    Hyperimmune globulins, I-IgG and II-IgG, prepared from healthy persons seropositive for human T cell lymphotropic virus (HTLV) types I and II, respectively, were tested for their prophylactic effect against HTLV-II infection in rabbits. Three groups (A, B, and C) of 3 female rabbits each were used. Group A rabbits were inoculated intravenously with HTLV-II-carrying rabbit lymphoid cell line RII. Group B and C rabbits were immunized, respectively, with II-IgG and I-IgG and challenged with RII cells 24 h later. All group A and C rabbits seroconverted for HTLV-II after 2 weeks, but all group B rabbits were protected from HTLV-II infection. Gene amplification by polymerase chain reaction revealed the presence of HTLV-II provirus sequences in all group A and C rabbits but in none of the group B rabbits. These findings indicate that passive immunization with II-IgG is effective in preventing HTLV-II infection and that there is no cross-neutralization between HTLV-I and -II.

    View details for Web of Science ID A1994NP10900023

    View details for PubMedID 8158037

  • AMPLIFICATION AND DETECTION OF A SINGLE-MOLECULE OF HUMAN-IMMUNODEFICIENCY-VIRUS RNA VIRUS GENES Nakamura, S., Katamine, S., Yamamoto, T., Foung, S. K., Kurata, T., HIRABAYASHI, Y., Shimada, K., Hino, S., Miyamoto, T. 1993; 7 (4): 325-338

    Abstract

    Detection of plasma viremia in human immunodeficiency virus type 1 (HIV-1)-infected people is indispensable for the diagnosis of seronegative infection as well as for the evaluation of virus activities in vivo. The direct detection of HIV-1 RNA in circulation has been performed by means of reverse transcription followed by polymerase chain reaction (RT-PCR). As an attempt to establish a highly sensitive assay, we evaluated the effects of two-step amplification with nested primers and double priming of reverse transcription on the sensitivity of RT-PCR. The sensitivity of two-step amplification was 100 times higher than that of one-step amplification. The double priming of reverse transcription further increased the sensitivity of the following two-step amplification 100 times, which appeared to be enough to detect HIV-1 RNA from as little as a 2.2 x 10(-4) TCID50 unit equivalent of culture supernatant of HIV-1-infected cells and a single molecule of HIV-1 gag complementary RNA synthesized by in vitro transcription. By use of this most sensitive assay, we successfully detected HIV-1 RNA in serum or plasma from all 22 patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC) and 13 out of 14 untreated asymptomatic carriers. Of 43 asymptomatic carriers under the treatment with interferon-alpha or azidothymidine, 17 cases showed negative results, indicating that the virus activity was suppressed by the therapeutics. We also noted the inhibitory effect of heparin on RT-PCR.

    View details for Web of Science ID A1993MP63100002

    View details for PubMedID 8122393

  • HUMAN T-LYMPHOTROPIC VIRUS TYPE-I/II IN LAKE LINDU VALLEY, CENTRAL SULAWESI, INDONESIA JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES AND HUMAN RETROVIROLOGY Lal, R. B., LIPKA, J. J., Foung, S. K., Hadlock, K. G., Reyes, G. R., Carney, W. P. 1993; 6 (9): 1067-1068

    View details for Web of Science ID A1993MC82800016

    View details for PubMedID 8340898

  • ASSOCIATION OF (TROPICAL) ATAXIC NEUROPATHY WITH HTLV-II VIRUS RESEARCH Sheremata, W. A., Harrington, W. J., BRADSHAW, P. A., Foung, S. K., Raffanti, S. P., Berger, J. R., Snodgrass, S., Resnick, L., Poiesz, B. J. 1993; 29 (1): 71-77

    Abstract

    Jamaican Neuropathy of the ataxic type (tropical ataxic neuropathy [TAN] and spastic type (tropical spastic paraparesis [TSP]) have been recognized for over a century in Jamaica. The recent association of TSP with HTLV-I (TSP/HAM) is now well established. We now present evidence for a possible association between a TAN-like illness with HTLV-II in four females aged 34-49. All presented with ataxic gait and all four have prominent mental changes. Three of the four also have minor motor deficits with urinary frequency and two have nocturnal leg cramps. All have serum antibody and all had PCR evidence of HTLV-II infection. Antibody to HTLV-II is present in CSF from two subjects. The distinctive picture of prominent ataxia and altered mental status in these subjects contrasts with a predominantly myelopathic picture seen in TSP/HAM.

    View details for Web of Science ID A1993LQ24000006

    View details for PubMedID 8212851

  • ISOTYPIC AND IGG SUBCLASS RESTRICTION OF THE HUMORAL IMMUNE-RESPONSES TO HUMAN T-LYMPHOTROPIC VIRUS TYPE-I CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY Lal, R. B., Buckner, C., Khabbaz, R. F., Kaplan, J. E., Reyes, G., Hadlock, K., Lipka, J., Foung, S. K., Chan, L., Coligan, J. E. 1993; 67 (1): 40-49

    Abstract

    We have investigated the isotypic and IgG subclass profile of the antibody response to HTLV-I structural proteins (gag and env) in patients with HTLV-I-associated myelopathy (HAM; n = 20), adult T-cell leukemia (ATL; n = 15), and HTLV-I-positive asymptomatic carriers (ASY; n = 21). IgG, IgM, and IgA were the predominant antibody responses in all HTLV-I-infected individuals; minimal IgE response was detectable in the HAM and ATL groups. Among the IgG subclasses, IgG1 was the most predominant antibody detected in responses to HTLV-I antigens, followed by IgG3 and IgG2; IgG4 could not be detected in any patient group. Levels of both IgG1 and IgG3 were significantly higher in patients with HAM, when compared to ATL and ASY (P < 0.01 for both comparisons). In addition, Ig isotypes and IgG subclass antibody in patient sera reactive with purified viral proteins and several immunodominant epitopes, represented by synthetic peptides, Gag-1a102-117, Env-1(191-214), Env-5(242-257), and recombinant proteins, MTA-1(162-209) and r21e303-440, were examined to delineate specific epitopes responsible for inducing the host immune responses of each isotype and subclass to the structural proteins of HTLV-I. IgG, IgM, and IgA responses were directed against both the gag and env gene products. Among IgG subclasses, the IgG1 and IgG3 responses were directed against both the gag (p53, p24, p19, and Gag-1a) and env (recombinant MTA-1, r21e, and synthetic Env-1, Env-5) proteins; IgG2 responses were mainly restricted to gag proteins. The frequency profile of HTLV-I-specific antigen recognition in all four IgG subclasses were similar in all of the clinical groups. These results further define the fine specificity of anti-HTLV-I immune reaction for understanding the mechanism of pathogenesis in these individuals and suggest that factors other than the humoral immune responses may be associated with the clinical manifestation of the disease.

    View details for Web of Science ID A1993KT71700006

    View details for PubMedID 7680300

  • SPASTIC ATAXIA ASSOCIATED WITH HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE-II INFECTION ANNALS OF NEUROLOGY Harrington, W. J., Sheremata, W., Hjelle, B., Dube, D. K., Bradshaw, P., Foung, S. K., Snodgrass, S., TOEDTER, G., Cabral, L., Poiesz, B. 1993; 33 (4): 411-414

    Abstract

    Human T-cell lymphotropic virus type one (HTLV-1) is associated with tropical spastic paraparesis or HTLV-I--associated myelopathy. We report 2 women with a spastic ataxic illness similar to HTLV-I--associated myelopathy infected solely with HTLV-II. Identification of HTLV-II infection was made serologically, by polymerase chain reaction, and by viral culture (in 1 woman). One woman, treated with 200 mg of danazol orally, three times daily, had pronounced improvement in ambulation, nocturnal spasticity, and nighttime urinary frequency. It appears that infection with HTLV-II may cause an illness similar to HTLV-I--associated myelopathy, but distinguished by the presence of ataxia.

    View details for Web of Science ID A1993KW78100015

    View details for PubMedID 8489213

  • EVALUATION OF AN IMMUNOBLOT ASSAY FOR SEROLOGICAL CONFIRMATION AND DIFFERENTIATION OF HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE-I AND TYPE-II JOURNAL OF CLINICAL MICROBIOLOGY Roberts, B. D., Foung, S. K., LIPKA, J. J., Kaplan, J. E., Hadlock, K. G., Reyes, G. R., Chan, L., Heneine, W., Khabbaz, R. F. 1993; 31 (2): 260-264

    Abstract

    The confirmation of infection with human T-cell lymphotropic virus type I (HTLV-I) and type II (HTLV-II) currently involves multiple assays. These include Western blot (immunoblot) (WB) and/or radioimmunoprecipitation assay for detection of antibodies to HTLV-specific viral proteins and polymerase chain reaction and/or peptide-based enzyme immunoassays for differentiating between the two viruses. We undertook an evaluation of a modified WB assay that includes native HTLV-I viral proteins from MT-2 cells spiked with an HTLV-I transmembrane glycoprotein (recombinant p21e) and the HTLV-I- and HTLV-II-specific recombinant proteins MTA-1 and K55. The test panel consisted of well-characterized sera from U.S. blood donors, American Indians, intravenous drug users, and patients seen in sexually transmitted disease clinics. Of 158 HTLV-I/II-seropositive serum specimens tested, 156 (98.7%) were confirmed and typed as HTLV-I or HTLV-II. Of 82 HTLV-I/II-seroindeterminate or -seronegative serum specimens, only 1 was classified as HTLV-II positive: the sample had weak gag p19 and strong gag p24 and env p21e reactivity and was radioimmunoprecipitation assay negative for env gp61/68 but polymerase chain reaction positive for HTLV-II. The specificity of the modified WB for confirming and typing serum samples was therefore 100%. We conclude that this WB assay is useful for confirming and typing HTLV infection and can help simplify HTLV-I/II testing algorithms.

    View details for Web of Science ID A1993KH75100017

    View details for PubMedID 8432811

  • NEUTRALIZING ANTIBODY TO VESICULAR STOMATITIS-VIRUS (HTLV-I) PSEUDOTYPE IN INFANTS BORN TO SEROPOSITIVE MOTHERS JAPANESE JOURNAL OF CANCER RESEARCH Iwahara, Y., Sawada, T., Taguchi, H., Hoshino, H., Umemoto, M., Take, H., Foung, S., Miyoshi, I. 1993; 84 (2): 114-116

    Abstract

    Breast feeding is the major route of mother-to-child transmission of human T-cell leukemia virus type I (HTLV-I). Our experiments with rabbits have shown that passive immunization is capable of blocking cell-to-cell infection of HTLV-I by blood transfusion or breast feeding. In this study, sera were collected serially from 3 infants born to seropositive mothers and were tested for the presence of neutralizing antibody to vesicular stomatitis virus (HTLV-I) pseudotype as well as antibodies to viral structural proteins. There was a good correlation between neutralizing and viral antibody titers, both of which were detectable until 3-6 months after birth. Whether maternally transmitted neutralizing antibody is protective against perinatal infection of HTLV-I remains to be studied.

    View details for Web of Science ID A1993KN14300003

    View details for PubMedID 8385083

  • IMMUNE RESPONSIVENESS TO THE IMMUNODOMINANT RECOMBINANT ENVELOPE EPITOPES OF HUMAN T-LYMPHOTROPIC VIRUS TYPE-1 AND TYPE-2 IN DIVERSE GEOGRAPHIC POPULATIONS JOURNAL OF INFECTIOUS DISEASES Buckner, C., ROBERTS, C. R., Foung, S. K., Lipka, J., Reyes, G. R., Hadlock, K., Chan, L., GONGORABIACHI, A., Hjelle, B., Lal, R. B. 1992; 166 (5): 1160-1163

    Abstract

    The heterogeneity of immune responsiveness to the immunodominant epitopes of human T lymphotropic virus (HTLV) types I (MTA-1(162-209)) and II (K-55(162-205)) were determined in natural infections with HTLV-I and -II from diverse geographic areas (n = 285). Of the HTLV-I specimens confirmed by polymerase chain reaction (PCR), all North American (n = 37) and Peruvian (n = 19) specimens reacted with MTA-1. Of HTLV-II specimens confirmed by PCR, 44 (96%) of 46 from North American blood donors, 28 (97%) of 29 from native Americans, and all from intravenous drug users (n = 29) reacted with K-55. Specimens from other geographic areas (Peru, 30; Brazil, 4; Mexico, 10; Italy, 5; Somalia, 13; Ethiopia, 17; Japan, 32; and Jamaica, 15) all reacted either with MTA-1 or K-55. By synthetic peptide-based serologic typing, all of these specimens could be typed as HTLV-I or -II. In addition to the direct implications of these findings for diagnostic purposes, these data provide indirect evidence for the conservation of immunodominant HTLVenv epitopes in diverse geographic populations.

    View details for Web of Science ID A1992JV01500031

    View details for PubMedID 1383353

  • CLONING AND ANALYSIS OF A RECOMBINANT ANTIGEN CONTAINING AN EPITOPE SPECIFIC FOR HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE-II BLOOD Hadlock, K. G., LIPKA, J. J., Chow, T. P., Foung, S. K., Reyes, G. R. 1992; 79 (10): 2789-2796

    Abstract

    An immunodominant HTLV-I-specific epitope in the HTLV-I envelope glycoprotein (GP) 46 has been described. To determine if the analogous region of HTLV-II contains a similarly immunogenic and specific epitope, the polymerase chain reaction (PCR) was used to amplify HTLV-II DNA fragments encoding various portions of the putative epitope. The synthesized DNAs were cloned into lambda-phage gt11 and screened for production of immunoreactive fusion protein using sera from HTLV-II- or HTLV-I-infected individuals. Antisera from HTLV-II-infected individuals identified three of four recombinant clones when tested in a plaque immunoassay. Fusion protein from one of the clones, GH2-K15, was purified and analyzed by Western blot against a panel of HTLV-I and HTLV-II antisera. Twenty-one of 22 HTLV-II-infected sera were reactive with the GH2-K15 epitope. Sera from HTLV-I-infected and HTLV-I-uninfected individuals did not cross-react with GH2-K15. Western blot analysis of recombinant proteins encoding portions of the HTLV-II sequences in the Gh2-K15 antigen localized the HTLV-II-specific epitope to a 17-amino acid sequence. Recombinant antigens containing this epitope should be useful for type-specific serologic diagnosis of HTLV-II infection.

    View details for Web of Science ID A1992HW61800041

    View details for PubMedID 1375112

  • SEROLOGIC CONFIRMATION OF SIMIAN T-LYMPHOTROPIC VIRUS TYPE-I INFECTION BY USING IMMUNOASSAYS DEVELOPED FOR HUMAN T-LYMPHOTROPIC VIRUS-ANTIBODY DETECTION JOURNAL OF CLINICAL MICROBIOLOGY Rudolph, D. L., Yee, J., Mone, J., Foung, S. K., LIPKA, J. J., Reyes, G. R., Hadlock, K., Chan, L., Villinger, F., Lairmore, M. D., Sinha, S., Lal, R. B. 1992; 30 (4): 858-861

    Abstract

    Serum specimens from diverse species of Old World monkeys, categorized as seropositive (n = 97) or seronegative (n = 23) for human T-lymphotropic virus (HTLV) infection, were tested by using recombinant env-spiked Western immunoblot assays and synthetic peptide assays for simultaneous detection and discrimination of simian T-lymphotropic virus (STLV) infection. Of the 97 seropositive specimens, 93 reacted with the recombinant transmembrane (r21env) protein and 90 reacted with a recombinant, MTA-1, derived from the central region of the external glycoprotein of HTLV-I (rgp46env), thus yielding test sensitivities of 96 and 93%, respectively. While 1 of the 23 negative monkey specimens reacted with r21env, none reacted with rgp46env, for overall specificities of 96 and 100%, respectively. Analysis of synthetic peptide-based immunoassays demonstrated that while 85 of 97 (88%) seropositive specimens reacted with HTLV-I-specific epitope (p19gag), none of the specimens reacted with HTLV-II-specific epitope (gp52env). These results show that recombinant envelope-spiked Western blots provide a simple means for serologic confirmation of STLV-I infection and that type-specific synthetic peptides can be used to confirm the virus type in seropositive monkey specimens.

    View details for Web of Science ID A1992HJ48700018

    View details for PubMedID 1349306

  • ANTIBODIES TO HEPATITIS-C VIRUS IN LOW-RISK BLOOD-DONORS - IMPLICATIONS FOR COUNSELING POSITIVE DONORS GASTROENTEROLOGY Hsu, H. H., Gonzalez, M., Foung, S. K., Feinstone, S. M., Greenberg, H. B. 1991; 101 (6): 1724-1727

    Abstract

    The significance of antibodies to hepatitis C virus (HCV) found by screening enzyme-linked immunoassay testing in a low-risk blood donor population is unclear. The rate of false positivity in this group as well as the usefulness of supplemental testing were examined by correlating the results of two screening enzyme-linked immunoassays (Ortho Diagnostics, Raritan, NJ, and Abbott Laboratories, North Chicago, IL) with supplemental antibody testing by the recombinant immunoblot assays (1 and 2) (Ortho) and neutralization assay (Abbott). Polymerase chain reaction was used to detect HCV genomic RNA to confirm viremia. Among 11.243 volunteer donors who were screened for the presence of antibodies to HCV by enzyme-linked immunoassay, 60 (0.53%) sera were repeatedly reactive. Twenty-five of these 60 sera were available for further testing. Seven sera were reactive by both screening enzyme-linked immunoassays, as well as by both recombinant immunoblot and neutralization assays. Six of these seven sera had detectable HCV genomic RNA by polymerase chain reaction. Among the remaining 18 sera, none were reactive by either recombinant immunoblot assays, whereas two sera were reactive by the neutralization assay only. None of the 18 samples had detectable HCV genomic RNA. Five of the six sera with elevated aminotransferase levels were among the seven sera reactive by all immunoassays. It is concluded that there is a significant false positivity rate associated with screening enzyme-linked immunoassay testing in a low-risk blood donor population. Supplemental testing correlates well with detection of hepatitis C genomic material by polymerase chain reaction and identifies donors who are truly infected.

    View details for Web of Science ID A1991GT00700035

    View details for PubMedID 1720106

  • MODIFIED WESTERN-BLOT ASSAY FOR CONFIRMATION AND DIFFERENTIATION OF HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE-I AND TYPE-II JOURNAL OF INFECTIOUS DISEASES LIPKA, J. J., Santiago, P., Chan, L., Reyes, G. R., Samuel, K. P., Blattner, W. A., Shaw, G. M., Hanson, C. V., Sninsky, J. J., Foung, S. K. 1991; 164 (2): 400-403

    Abstract

    Disease association studies of human T cell lymphotropic virus (HTLV) types I and II are hindered by the need for multiple assays to confirm and differentiate between the viruses. A modified Western blot assay has been developed using HTLV-I viral lysate and unique (MTA-4) and shared (p21E) HTLV recombinant proteins. By defining confirmation of infection as the presence of antibodies to p24 gag protein and to p21E, all 56 HTLV-I and 49 HTLV-II antisera were confirmed by this modified Western blot alone. Differentiation was determined by reactivity to MTA-4. All HTLV-I antisera reacted with MTA-4 and all HTLV-II antisera did not react with MTA-4. These findings indicate the utility of selected HTLV-I recombinant proteins in a single assay format to confirm and differentiate infections with HTLV-I and HTLV-II.

    View details for Web of Science ID A1991FY10000025

    View details for PubMedID 1856487

  • Parameters to enhance human hybridoma formation with hypoosmolar electrofusion. Human antibodies and hybridomas Perkins, S., Zimmermann, U., FOUNG, S. K. 1991; 2 (3): 155-159

    Abstract

    Hypoosmolar conditions have permitted the development of electrofusion techniques capable of producing human hybridomas from as few as 10(5) B cells. A hybridoma formation efficiency of one hybrid for each 125 input B cells has been achieved with Epstein-Barr virus--activated B cells and mouse-human heteromyelomas. This is at least 100-fold higher in efficiency than with polyethylene glycol-induced cell fusion, as well as a 50- to 100-fold decrease in the required number of human B cells. The ability to fuse a small number of input B cells should lead to a greater success rate in immortalizing the rare antigen-specific B cells. The critical parameters include fusion voltages, the composition and number of wash steps used in cell preparation, the composition and duration of exposure to hypoosmolar fusion medium, fusion ratio, plating density, the use of growth medium without pH indicator, and the use of an irradiated human fibroblast feeder layer. By manipulating these parameters, a high hybrid yield can be achieved with different mouse-human heteromyelomas and Epstein-Barr virus-activated B cells.

    View details for PubMedID 1651789

  • HTLV-I-ASSOCIATED MYELOPATHY IN A CALIFORNIAN - DIAGNOSIS BY REACTIVITY TO A VIRAL RECOMBINANT ANTIGEN NEUROLOGY Honig, L. S., LIPKA, J. J., Young, K. Y., Weiss, J. H., Foung, S. K. 1991; 41 (3): 448-450

    Abstract

    A male patient developed leg numbness and weakness, and bowel, bladder, and erectile dysfunction. Examination revealed an isolated thoracic myelopathy, with lower-extremity spasticity, decreased vibration and position sense, hyperreflexia, and Babinski's signs. Serum and CSF showed antibody reactivity to human T-cell lymphotropic virus type I or II (HTLV-I/II), suggesting HTLV-I-associated myelopathy. Antibody reactivity to a unique HTLV-I recombinant protein provided definitive diagnosis of HTLV-I infection.

    View details for Web of Science ID A1991FD56600028

    View details for PubMedID 1706492

  • SIGNIFICANCE OF HUMAN T-LYMPHOTROPIC VIRUS TYPE-I INDETERMINANT SEROLOGICAL FINDINGS AMONG HEALTHY-INDIVIDUALS VOX SANGUINIS LIPKA, J. J., Young, K. K., Kwok, S. Y., Reyes, G. R., Sninsky, J. J., Foung, S. K. 1991; 61 (3): 171-176

    Abstract

    Follow-up studies on 67 blood donors with indeterminant serological findings for human T-lymphotropic virus (HTLV) type I by standard immunoassays showed no evidence of infection by polymerase chain reaction analysis for HTLV-I or HTLV-II nucleic acids or by antibody reactivity to a unique HTLV-I recombinant envelope protein, MTA-4. Among HTLV-I- or -II-infected individuals, a history of blood transfusion, past residence in established HTLV-I endemic areas or some association with intravenous drug use were common. In contrast, 85% of indeterminant cases had none of these risk factors. These observations suggest that healthy individuals with indeterminant serology for HTLV-I should not require additional studies.

    View details for Web of Science ID A1991GR19300003

    View details for PubMedID 1725466

  • DEVELOPMENT OF MICROFUSION TECHNIQUES TO GENERATE HUMAN HYBRIDOMAS JOURNAL OF IMMUNOLOGICAL METHODS Foung, S., Perkins, S., Kafadar, K., Gessner, P., Zimmermann, U. 1990; 134 (1): 35-42

    Abstract

    The rarity of antigen-specific B cells in peripheral blood and lymphoid tissues is a major limitation in the production of human monoclonal antibodies. This has led to a requirement for techniques capable of fusing small numbers of cells and achieving a higher hybridoma formation efficiency than currently is possible. The approach used in these studies to generate human hybridomas is based on the observation that under hypo-osmolar conditions electric field induced cell fusion or electrofusion is facilitated. Electrofusion parameters have been defined in strongly hypo-osmolar solutions which have resulted in a hybridoma formation efficiency greater than 5 X 10(-3) under optimal conditions. Furthermore, this has been accomplished with total input B cells of 1-2 X 10(5). This is a ten-fold reduction in the required number of input B cells and is associated with a hybridoma formation efficiency at least equal to that achieved with a higher input B cell number. An important factor in the development of this microfusion technique appears to be the duration of exposure to the hypo-osmolar solution by B cells to be immortalized. Other parameters which may affect hybridoma yield include the electrical field strength used for cell alignment and membrane breakdown, ratio of human B cells to fusion partner, washing procedure, post-fusion incubation time, and the elimination of toxic molecules.

    View details for Web of Science ID A1990EG52200003

    View details for PubMedID 2172386

  • EFFICIENT HYBRIDIZATION OF MOUSE-HUMAN CELL-LINES BY MEANS OF HYPOOSMOLAR ELECTROFUSION JOURNAL OF IMMUNOLOGICAL METHODS Zimmermann, U., Gessner, P., Schnettler, R., Perkins, S., Foung, S. K. 1990; 134 (1): 43-50

    Abstract

    The fusion of a mouse-human heteromyeloma with a mouse hybridoma is used as a model to define parameters to generate human hybridomas. Electrofusion of these cells in 300 mosM and 75 mosM solutions showed that strong hypo-osmolar conditions resulted in a dramatic increase in the efficiency of hybridoma formation. In contrast to iso-osmolar electrofusion, a high hybrid yield could be obtained by injection of only a single field pulse. The field strength was adjusted in proportion to the increased size of the cells in hypo-osmolar solutions. Hypo-osmolar electrofusion allowed the generation of approximately 0.45% hybrids at a suspension density of 1.75 X 10(5) mouse-human cells/ml corresponding to an input number of 3.5 X 10(4) mouse-human cells. A further increase in the efficiency of hybridoma formation to about 0.6% was achieved by cell alignment in an alternating field of modulated field strength. Experiments in which the total cell number per fusion chamber was decreased at constant optimum suspension density showed that a further increase in the efficiency of hybridoma formation in hypo-osmolar solution was not possible because of the increasing influence of the heterogeneity of the cell lines with decreasing cell number. The results allow the conclusion that hypo-osmolar electrofusion is a potential tool to enhance successful immortalisation of human B lymphocytes.

    View details for Web of Science ID A1990EG52200004

    View details for PubMedID 2230148

  • LOW INCIDENCE OF HTLV INFECTIONS IN RANDOM BLOOD-DONORS WITH INDETERMINATE WESTERN-BLOT PATTERNS TRANSFUSION Kwok, S., LIPKA, J. J., McKinney, N., KELLOGG, D. E., Poiesz, B., Foung, S. K., Sninsky, J. J. 1990; 30 (6): 491-494

    Abstract

    Peripheral blood mononuclear cells (PBMCs) were recovered from platelet units of 61 blood donors who were HTLV-I positive and 3 blood donors who were HTLV-I negative on enzyme-linked immunosorbent assay (ELISA). Western blot analyses were performed on the sera and DNA was prepared from the PBMCs and analyzed by the polymerase chain reaction (PCR). Of the 61 repeatably reactive samples, 2 were positive, 26 were negative, and 33 were interpreted as indeterminate on Western blot. HTLV-II sequences were detected by PCR in one of the Western blot-positive samples, as well as in one Western blot-indeterminate sample that showed reactivity to p24 only. HTLV-I sequences were detected in the second Western blot-positive sample. HTLV sequences were not detected in the remaining samples, which suggested that the majority of individuals with indeterminate results on Western blots that used one set of commercially available reagents are not infected with HTLV. It is demonstrated in this study that PCR can be used not only to resolve the infection status of individuals with indeterminate Western blots but also to distinguish between HTLV-I and HTLV-II.

    View details for Web of Science ID A1990DR32700004

    View details for PubMedID 1974089

  • GENERATION OF A HUMAN HYBRIDOMA PRODUCING A PURE ANTI-HLA-A2 MONOCLONAL-ANTIBODY HUMAN IMMUNOLOGY WALLACE, E. F., Foung, S. K., Bradbury, K., PASK, S. L., GRUMET, F. C. 1990; 28 (1): 65-69

    Abstract

    We report the production and characterization of a human monoclonal IgM hybridoma antibody recognizing antigen HLA-A2. B lymphocytes obtained from the peripheral blood of a multiparous volunteer 1 week postpartum were transformed in vitro by Epstein-Barr virus, screened by a microlymphocytotoxicity assay, and electrofused with the heterohybridoma fusion partner, K6H6/B5. A specifically anti-A2 secreting hybridoma cell line. MBW1, was then identified and cloned. The cytotoxic IgM antibody produced showed complete correlation (r = 1.00) with the A2 antigen on a large panel of unrelated donors' lymphocytes, and no cross-reactivity with A28, Aw68, or Aw69 antigens was observed.

    View details for Web of Science ID A1990CZ82300007

    View details for PubMedID 2160447

  • HUMAN MONOCLONAL-ANTIBODIES TO HUMAN CYTOMEGALO-VIRUS JOURNAL OF INFECTIOUS DISEASES Foung, S. K., Perkins, S., Bradshaw, P., Rowe, J., RABIN, L. B., Reyes, G. R., Lennette, E. T. 1989; 159 (3): 436-443

    Abstract

    Human monoclonal antibodies (HMAbs) to human cytomegalovirus (HCMV) have been developed by using electric field-induced cell fusion of human B lymphocytes to the human-mouse cell line SBC-H20. By this procedure, multiple hybridomas have been produced that secrete IgG 1 HMAbs with distinct patterns of indirect immunofluorescence on HCMV-infected cells. HMAbs Z01 and X20 immunoprecipitated a major protein at 64 kDa. HMAb Z02 immunoprecipitated a major protein of 48-50 kDa. HMAb Z10 identified a single protein at 65 kDa and HMAb X16 identified proteins at 100, 65, and 36-38 kDa. The HMAbs demonstrated varying degrees of virus-neutralizing activity. The production of HMAbs to HCMV provides an important approach to studying the human host response to HCMV by elucidating biologically relevant antigens and epitopes. In addition, HMAbs are a potentially unlimited source of relevant human antibodies for treating life-threatening HCMV infection.

    View details for Web of Science ID A1989T266800011

    View details for PubMedID 2536787

  • ELECTRIC FIELD-INDUCED CELL-FUSION AND HUMAN MONOCLONAL-ANTIBODIES JOURNAL OF IMMUNOLOGICAL METHODS Foung, S. K., Perkins, S. 1989; 116 (1): 117-122

    Abstract

    A persistent problem in the generation of antigen-specific human monoclonal antibodies is the rarity of appropriate B cells in human blood or splenic tissues. In order to immortalize the rare antigen-specific cells that are available, an electric field-induced cell fusion technique has been shown to markedly increase the fusion efficiency in comparison to polyethylene glycol-induced cell fusion using Epstein-Barr virus (EBV) or pokeweed mitogen activated B cells. Fusion efficiency of 10(-3)-10(-4) has been achieved by this process with as low as 1 X 10(6) input EBV-activated B cells. A panel of human monoclonal antibodies to human cytomegalovirus has subsequently been produced using this technique. This improvement should enable wider therapeutic and diagnostic applications of human monoclonal antibodies.

    View details for Web of Science ID A1989R695200014

    View details for PubMedID 2536773

  • INITIAL IDENTIFICATION AND MAPPING OF DISTINCT RH0 (D) EPITOPES REVUE FRANCAISE DE TRANSFUSION ET IMMUNO-HEMATOLOGIE Rodriguez, L., WINN, L., Foung, S. K. 1988; 31 (2): 223-231

    View details for Web of Science ID A1988T651900013

    View details for PubMedID 2463657

  • THE EFFICACY OF PLA1-NEGATIVE PLATELET TRANSFUSION THERAPY IN POSTTRANSFUSION PURPURA WESTERN JOURNAL OF MEDICINE Lippman, S. M., LIZAK, G. E., Foung, S. K., GRUMET, F. C. 1988; 148 (1): 86-88

    View details for Web of Science ID A1988L724600026

    View details for PubMedID 3341139

  • HUMAN MONOCLONAL-ANTIBODIES TO RH0(D) VOX SANGUINIS Foung, S. K., Blunt, J. A., Wu, P. S., AHEARN, P., WINN, L. C., Engleman, E. G., GRUMET, F. C. 1987; 53 (1): 44-47

    Abstract

    B lymphocytes from Rh negative donors with serum anti-D antibodies were isolated and fused with the mouse-human heteromyeloma, SBC-H20, to produce hybridomas secreting IgM or IgG1 human monoclonal antibodies to D antigen. The IgM antibody in hybridoma supernatant agglutinates all normal D positive cells at the immediate spin phase of reactivity. Using concentrated IgM hybridoma supernatant of approximately 50 micrograms/ml, Du cells were also agglutinated. The IgG1 antibody reacts by indirect hemagglutination with all D and Du cells. Against Rh mosaics, different reactivity was noted for each antibody. Furthermore, D positive cells precoated with the IgG1 antibody inhibit the IgM direct hemagglutination, suggesting that the antibodies identify closely associated epitopes. These human monoclonal antibodies will be useful diagnostic reagents and, ultimately, should be useful in the prevention of Rh hemolytic disease of the newborn.

    View details for Web of Science ID A1987J436100009

    View details for PubMedID 3116771

  • HUMAN MONOCLONAL ANTI-T-CELL ANTIBODY FROM A PATIENT WITH JUVENILE RHEUMATOID-ARTHRITIS JOURNAL OF IMMUNOLOGY ALPERT, S. D., Turek, P. J., Foung, S. K., Engleman, E. G. 1987; 138 (1): 104-108

    Abstract

    Antibody JRAI is a human monoclonal IgM antibody derived from a patient with juvenile rheumatoid arthritis that is cytotoxic to a subpopulation of normal T lymphocytes. JRAI recognizes approximately 80% of normal peripheral blood T cells, 90% of CD4+ cells, and 75% of CD8+ cells, as determined by complement-mediated cytotoxicity. Within the CD8+ population, JRAI preferentially spares OKM1+ and Leu-11+ cells. These CD8+ cells retain suppressor-effector potential and show enriched natural killer cell activity. Within the CD4+ population, JRAI preferentially kills cells within the Leu-8+ subset, which contains suppressor-inducer cells, and spares the Leu-8- subset, which contains the helpers for immunoglobulin synthesis. JRAI appears to recognize a previously undefined human lymphocyte surface molecule expressed differentially on phenotypically and functionally distinct subsets of human T cells.

    View details for Web of Science ID A1987F377900018

    View details for PubMedID 3097140

  • EARLY IMMUNE-RESPONSE IN HEALTHY AND IMMUNOCOMPROMISED SUBJECTS WITH PRIMARY VARICELLA-ZOSTER VIRUS-INFECTION JOURNAL OF INFECTIOUS DISEASES Arvin, A. M., Koropchak, C. M., Williams, B. R., GRUMET, F. C., Foung, S. K. 1986; 154 (3): 422-429

    Abstract

    Events in pathogenesis and immunity during primary varicella-zoster virus (VZV) infection were examined in 64 healthy subjects and 21 immunocompromised patients. Activation of the interferon system and activation of circulating T lymphocytes were early immune responses that occurred during the incubation period in some healthy subjects. Elevated levels of 2-5A synthetase in peripheral blood mononuclear cells and detection of serum alpha interferon (IFN-alpha) and gamma interferon (IFN-gamma) were present in the majority of healthy subjects who had acute primary VZV infection. Expression of HLA-DR antigen occurred on circulating T lymphocytes from subjects with acute VZV infection. The early production of VZV-specific IgG or IgM antibodies did not correlate with the severity of the clinical infection, but the detection of T lymphocyte proliferation to VZV antigen within three days after the appearance of the varicella exanthem was associated with milder illness. The mean VZV-specific lymphocyte transformation for subjects with less than 100 lesions/m2 was 7.5 +/- 10.43 SD compared with 1.4 +/- 1.85 SD for those with greater than 400 lesions/m2 (P less than .05). Only one (7.7%) of 13 immunocompromised patients had early VZV-specific lymphocyte transformation compared with 19 (42%) of 45 healthy subjects (P less than .05). The rapid host response to primary VZV infection was associated with rapid termination of viremia in healthy subjects; VZV was isolated from only 11% of peripheral blood mononuclear cell samples cultured within 48 hr after the appearance of the exanthem.

    View details for Web of Science ID A1986D760000006

    View details for PubMedID 3016110

  • A MONOCLONAL-ANTIBODY AGAINST HLA-A11 AND A24 HUMAN IMMUNOLOGY Foung, S. K., Taidi, B., Ness, D., GRUMET, F. C. 1986; 15 (3): 316-319

    Abstract

    A monoclonal IgG antibody was produced from a mouse immunized with an A11, A24; B27, B44 Epstein-Barr virus transformed B lymphoblastoid cell line. The antibody, A11.1M, by standard lymphocytotoxicity assay, reacts with all cells expressing HLA-A11 and -A24. Absorption studies with both A11+, A24- and A11-, A24+ platelets removed antibody reactivity against A11 and A24 lymphocytes. The shared antigenic determinant between A11 and A24, as defined by this antibody, A11.1M, represents a new "supertypic" determinant.

    View details for Web of Science ID A1986A381800006

    View details for PubMedID 2420768

  • A HUMAN MONOCLONAL-ANTIBODY TO RHG VOX SANGUINIS Foung, S. K., Blunt, J., Perkins, S., WINN, L., GRUMET, F. C. 1986; 50 (3): 160-163

    Abstract

    Hybridomas secreting a human monoclonal IgG1 antibody against a variant of the rhG antigen were produced from B lymphocytes of an Rh-negative donor with serum antibodies to D and G [correction of C] antigens. The antibody reacts by indirect hemagglutination with nearly all C- or D-positive cells, confirming the strong association in the expression of G with D or C antigens. The lack of antibody reactivity to C-negative Du cells suggests a particular epitope on the D complex associated with the G antigen.

    View details for Web of Science ID A1986A969300008

    View details for PubMedID 2424176

  • GENERATION OF HUMAN MONOCLONAL-ANTIBODIES BY FUSION OF EBV-ACTIVATED B-CELLS TO A HUMAN MOUSE HYBRIDOMA METHODS IN ENZYMOLOGY Foung, S. K., Engleman, E. G., GRUMET, F. C. 1986; 121: 168-174

    View details for Web of Science ID A1986D161200013

    View details for PubMedID 3014266

  • HUMAN MONOCLONAL-ANTIBODIES NEUTRALIZING VARICELLA-ZOSTER VIRUS JOURNAL OF INFECTIOUS DISEASES Foung, S. K., Perkins, S., KOROPCHAK, C., Fishwild, D. M., Wittek, A. E., Engleman, E. G., GRUMET, F. C., Arvin, A. M. 1985; 152 (2): 280-285

    Abstract

    Hybridomas secreting human monoclonal antibodies to varicella-zoster virus were produced by fusing B cells of a patient recovering from acute varicella infection with a human-mouse cell line. Two hybrid lines have continued to secrete IgG1, one with kappa and the other with lambda chains, for at least 12 months. Each antibody neutralizes virus infectivity between 1-5 micrograms of partially purified immunoglobulin/ml, each shows a different pattern of immunofluorescent staining of virus-infected cells, and one identifies three viral proteins with molecular weights of 60,000, 95,000, and 97,000.

    View details for Web of Science ID A1985AQZ7900006

    View details for PubMedID 2993433

  • HUMAN MONOCLONAL-ANTIBODIES AGAINST GROUP-A RED BLOOD-CELLS VOX SANGUINIS Raubitschek, A., SENYK, G., LARRICK, J., LIZAK, G., Foung, S. 1985; 48 (5): 305-308

    Abstract

    A human monoclonal antibody with anti-A specificity was generated by Epstein-Barr virus transformation of lymphocytes isolated from splenic tissue after in vitro stimulation with group A red blood cells. This antibody is of the IgM class and directly agglutinates group A red blood cells. Type A1, A2, Aint, A3, AX, Aend and A5 cells were agglutinated by the reagent indicating a single determinant is shared by these A subgroups.

    View details for Web of Science ID A1985AFM5900006

    View details for PubMedID 2986353

  • A NEW APPROACH TO THE DIAGNOSIS OF ACTIVE RH0(D) IMMUNIZATION IN PASSIVELY IMMUNIZED PREGNANT-WOMEN VOX SANGUINIS Foung, S. K., Perkins, S., MCCALLUM, W. D., Tom, S., Engleman, E. G., GRUMET, F. C. 1985; 49 (3): 206-210

    Abstract

    An assay has been developed to distinguish active from passive Rh0(D) immunization in a patient who had recently received hyperimmune anti-Rh0(D) immunoglobulin therapy. Isolated peripheral B lymphocytes from a pregnant woman at 32 weeks gestation were co-cultured with Epstein-Barr virus in a liquid growth medium. After 7 days, anti-Rh0(D) antibodies produced in vitro by the transformed lymphocytes were detected in culture supernatants, thereby proving active immunization and indicating the potential of hemolytic disease of the newborn in the current pregnancy. This assay was also performed with peripheral B lymphocytes from three groups of individuals: mothers known to be Rh0(D) immunized and who recently delivered Rh-positive infants, women with longstanding Rh0(D) immunization, and women who were treated with anti-Rh0(D) globulin. In the first group, anti-Rh0(D) antibodies were again detected after in vitro viral stimulation. In the latter two groups, essentially no anti-Rh0(D) activity was detected.

    View details for Web of Science ID A1985APS1800005

    View details for PubMedID 2996227

  • RESCUE OF HUMAN MONOCLONAL-ANTIBODY PRODUCTION FROM AN EBV-TRANSFORMED B-CELL LINE BY FUSION TO A HUMAN-MOUSE HYBRIDOMA JOURNAL OF IMMUNOLOGICAL METHODS Foung, S. K., Perkins, S., Raubitschek, A., LARRICK, J., LIZAK, G., Fishwild, D., Engleman, E. G., GRUMET, F. C. 1984; 70 (1): 83-90

    Abstract

    A human-mouse cell line that is hypoxanthine-aminopterin-thymidine sensitive and ouabain resistant was derived from a fusion between human B lymphocytes and a mouse myeloma line. This new mutant, when fused to a relatively unstable EBV-transformed B cell secreting a human monoclonal anti-A (red blood cell antigen) antibody, resulted in stable hybridomas capable of long term production of the specific human monoclonal antibody. Furthermore, some of the hybrid clones secreted antibody in far greater titer than the original EBV cell line. We conclude that fusion to this human-mouse line is an efficient approach to the production of human monoclonal alloantibodies and an effective method of 'rescuing' secretion of desired antibody from EBV cell lines.

    View details for Web of Science ID A1984SR32200010

    View details for PubMedID 6201562

  • MONOCLONAL-ANTIBODY (B27M2) SUBDIVIDING HLA-B27 HUMAN IMMUNOLOGY GRUMET, F. C., Fendly, B. M., Fish, L., Foung, S., Engleman, E. G. 1982; 5 (1): 61-72

    View details for Web of Science ID A1982PH53300005

    View details for PubMedID 6981637

  • PRODUCTION OF FUNCTIONAL HUMAN T-T-HYBRIDOMAS IN SELECTION MEDIUM LACKING AMINOPTERIN AND THYMIDINE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Foung, S. K., Sasaki, D. T., GRUMET, F. C., Engleman, E. G. 1982; 79 (23): 7484-7488

    Abstract

    The production of hybridomas between immunologically activated T cells and malignant T-cell lines offers a potentially unlimited source of soluble T-cell-derived products. Recently, human T-T hybrids have been described; however, their use has been hampered by slow growth and chromosomal instability due at least in part to the presence of thymidine in the traditional hypoxanthine/aminopterin/thymidine (HAT) selection medium. In this report, we describe the development of a rapidly growing hypoxanthine phosphoribosyltransferase-deficient human T-cell line designated J3R7, the use of azaserine/hypoxanthine (AH) medium as an alternative selection medium to HAT medium, and the production of functional T-T hybrids by using the J3R7 line and the AH selection technique. Hybrids selected in AH medium were 4-fold greater in number and 3-fold faster in growth rate than hybrids grown in HAT medium. No stable clones were obtained from HAT cultures whereas AH-derived hybrids could be readily cloned by the method of limiting dilution. Evidence for hybridization included (i) the presence of approximately twice the number of chromosomes in hybrids than in J3R7 cells; (ii) the presence on hybrid cells of the Leu-3a surface antigen, present on normal helper T cells but not on J3R7 cells; (iii) the expression of HLA antigens of both the normal T-cell partner and the J3R7 line; and (iv) the constitutive secretion of interleukin 2 from multiple hybrid clones but not from the J3R7 cell line. Thus far, these clones have maintained their rapid growth, chromosome number, surface phenotype, and constitutive secretion of interleukin 2 for 4 months.

    View details for Web of Science ID A1982PT40700084

    View details for PubMedID 6984190

  • EOSINOPHILIA IN CHILDREN PEDIATRIC ANNALS Foung, S., Glader, B. E. 1979; 8 (6): 39-?

    View details for Web of Science ID A1979GY10400003

    View details for PubMedID 471550

  • EFFECTS OF STARVATION AND REFEEDING ON MUSCLE PROTEIN-SYNTHESIS AND CATABOLISM IN YOUNG RAT JOURNAL OF NUTRITION Ogata, E. S., Foung, S. K., Holliday, M. A. 1978; 108 (5): 759-765

    Abstract

    We studied the effects of acute starvation and refeeding on muscle protein synthesis and degradation in young rats. As measures of synthesis, we determined muscle RNA concentration and the rate of incorporation of [14C]leucine into skeletal muscle protein (Sm). As an estimate of nitrogen retention we measured urea production (UrP). Starvation reduced these variables significantly. One refeeding period returned Sm to control values, only partially restored RNA concentration, and increased UrP. We determined the urinary excretion rate of 3-methylhistidine (3-MH) as a measure of the rate of myofibrillar protein degradation. Excretion of 3-MH was lowest in control and highest in starved rats. Refeeding decreased 3-MH excretion to a level midway between control and starved animals. Growth was attended by high rates of synthesis and low rates of degradation. Starvation depressed synthesis and increased degradation. With refeeding, synthesis increased and degradation decreased, compared with the starved state.

    View details for Web of Science ID A1978EZ25200005

    View details for PubMedID 641592

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