Daved H Fremont

Publications (82)724.87 Total impact

• Article: Development of a Highly Protective Combination Monoclonal Antibody Therapy against Chikungunya Virus.

  Pankaj Pal, Kimberly A Dowd, James D Brien, Melissa A Edeling, Sergey Gorlatov, Syd Johnson, Iris Lee, Wataru Akahata, Gary J Nabel, Mareike K S Richter, Jolanda M Smit, Daved H Fremont, Theodore C Pierson, Mark T Heise, Michael S Diamond
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  ABSTRACT: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit infection of all three CHIKV genotypes. Four of 36 neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) provided complete protection against lethality as prophylaxis in highly susceptible immunocompromised mice lacking the type I IFN receptor (Ifnar(-/-) ) and mapped to distinct epitopes on the E1 and E2 structural proteins. CHK-152, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102+CHK-152 or CHK-166+CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 hours before CHIKV-induced death. Selected pairs of highly neutralizing MAbs may be a promising treatment option for CHIKV in humans.
  PLoS Pathogens 04/2013; 9(4):e1003312. · 9.13 Impact Factor
• Article: The mechanism of differential neutralization of dengue serotype 3 strains by monoclonal antibody 8A1.

  Yang Zhou, S Kyle Austin, Daved H Fremont, Boyd L Yount, Jeremy P Huynh, Aravinda M de Silva, Ralph S Baric, William B Messer
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  ABSTRACT: While previous studies have demonstrated that envelope (E) glycoprotein variation between dengue viruses (DENV) genotypes can influence antibody neutralization potency, the mechanisms of variable neutralization remain incompletely understood. Here we characterize epitope antibody interactions of a DENV-3 EDIII binding mouse mAb 8A1 which displays highly variable neutralizing activity against DENV-3 genotypes. Using a DENV-3 reverse genetics platform, we characterize ability of 8A1 to bind and neutralize naturally occurring DENV-3 E genotypic variant viruses. Introduction of single and multiple amino acid mutations into the parental clone background demonstrates that mutations at positions 301 and 383 on EDIII are responsible for 8A1 differential neutralization phenotypes. ELISA and surface plasmon resonance (SPR) studies indicate differences in binding are responsible for the variable neutralization. Variability at position 301 primarily determined binding difference through influencing antibody-EDIII dissociation rate. Our findings are relevant to many groups focusing on DENV EDIII as a vaccine target.
  Virology 02/2013; · 3.35 Impact Factor
• Article: Cowpox virus employs a two-pronged strategy to outflank MHCI antigen presentation.

  William H McCoy, Xiaoli Wang, Wayne M Yokoyama, Ted H Hansen, Daved H Fremont
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  ABSTRACT: Smallpox decimated humanity for thousands of years before being eradicated by vaccination, a success facilitated by the fact that humans are the only host of variola virus. In contrast, other orthopoxviruses such as cowpox virus can infect a variety of mammalian species, although its dominant reservoir appears to be rodents. This difference in host specificity suggests that cowpox may have developed promiscuous immune evasion strategies to facilitate zoonosis. Recent experiments have established that cowpox can disrupt MHCI antigen presentation during viral infection of both human and murine cells, a process enabled by two unique proteins, CPXV012 and CPXV203. While CPXV012 inhibits antigenic peptide transport from the cytosol to the ER, CPXV203 blocks MHCI trafficking to the cell surface by exploiting the KDEL-receptor recycling pathway. Our recent investigations of CPXV203 reveal that it binds a diverse array of classical and non-classical MHCI proteins with dramatically increased affinities at the lower pH of the Golgi relative to the ER, thereby providing mechanistic insight into how it works synergistically with KDEL receptors to block MHCI surface expression. The strategy used by cowpox to both limit peptide supply and disrupt trafficking of fully assembled MHCI acts as a dual-edged sword that effectively disables adaptive immune surveillance of infected cells.
  Molecular Immunology 01/2013; · 2.90 Impact Factor
• Article: Structural Mechanism of ER Retrieval of MHC Class I by Cowpox.

  William H McCoy, Xiaoli Wang, Wayne M Yokoyama, Ted H Hansen, Daved H Fremont
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  ABSTRACT: One of the hallmarks of viral immune evasion is the capacity to disrupt major histocompatibility complex class I (MHCI) antigen presentation to evade T-cell detection. Cowpox virus encoded protein CPXV203 blocks MHCI surface expression by exploiting the KDEL-receptor recycling pathway, and here we show that CPXV203 directly binds a wide array of fully assembled MHCI proteins, both classical and non-classical. Further, the stability of CPXV203/MHCI complexes is highly pH dependent, with dramatically increased affinities at the lower pH of the Golgi relative to the endoplasmic reticulum (ER). Crystallographic studies reveal that CPXV203 adopts a beta-sandwich fold similar to poxvirus chemokine binding proteins, and binds the same highly conserved MHCI determinants located under the peptide-binding platform that tapasin, CD8, and natural killer (NK)-receptors engage. Mutagenesis of the CPXV203/MHCI interface identified the importance of two CPXV203 His residues that confer low pH stabilization of the complex and are critical to ER retrieval of MHCI. These studies clarify mechanistically how CPXV203 coordinates with other cowpox proteins to thwart antigen presentation.
  PLoS Biology 11/2012; 10(11):e1001432. · 11.45 Impact Factor
• Article: Subversion of cytokine networks by virally encoded decoy receptors.

  Megan L Epperson, Chung A Lee, Daved H Fremont
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  ABSTRACT: During the course of evolution, viruses have captured or created a diverse array of open reading frames, which encode for proteins that serve to evade and sabotage the host innate and adaptive immune responses that would otherwise lead to their elimination. These viral genomes are some of the best textbooks of immunology ever written. The established arsenal of immunomodulatory proteins encoded by viruses is large and growing, and includes specificities for virtually all known inflammatory pathways and targets. The focus of this review is on herpes and poxvirus-encoded cytokine and chemokine-binding proteins that serve to undermine the coordination of host immune surveillance. Structural and mechanistic studies of these decoy receptors have provided a wealth of information, not only about viral pathogenesis but also about the inner workings of cytokine signaling networks.
  Immunological Reviews 11/2012; 250(1):199-215. · 11.15 Impact Factor
• Article: Structure of the St. Louis encephalitis virus postfusion envelope trimer.

  Vincent C Luca, Christopher A Nelson, Daved H Fremont
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  ABSTRACT: St. Louis encephalitis virus (SLEV) is a mosquito borne flavivirus responsible for several human encephalitis outbreaks over the last 80 years. Mature flavivirus virions are coated with dimeric envelope (E) proteins that mediate attachment and fusion with host cells. E is a class II fusion protein, the hallmark of which is a distinct dimer-to-trimer rearrangement that occurs upon endosomal acidification and insertion of hydrophobic fusion peptides into the endosomal membrane. Herein, we report the crystal structure of SLEV E in the posfusion trimer conformation. The structure revealed specific features that differentiate SLEV E from trimers of related flavi- and alphaviruses. SLEV E fusion loops have distinct intermediate spacing such that they are positioned further apart than previously observed in flaviviruses but closer together than Semliki Forest Virus, an alphavirus. Domains II (DII) and III (DIII) of SLEV E also adopt different angles relative to domain I (DI) which suggests the DI-DII joint may accommodate spheroidal motions. However, trimer interfaces are well conserved amongst flaviviruses, so it is likely the differences observed represent structural features specific to SLEV function. Analysis of surface potentials revealed a basic platform underneath flavivirus fusion loops that may interact with the anionic lipid head groups found in membranes. Taken together, these results highlight variation in E structure and assembly that may direct virus-specific interactions with host determinants to influence pathogenesis.
  Journal of Virology 10/2012; · 5.40 Impact Factor
• Article: RANKL Employs Distinct Binding Modes to Engage RANK and the Osteoprotegerin Decoy Receptor.

  Christopher A Nelson, Julia T Warren, Michael W-H Wang, Steven L Teitelbaum, Daved H Fremont
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  ABSTRACT: Osteoprotegerin (OPG) and receptor activator of nuclear factor κB (RANK) are members of the tumor necrosis factor receptor (TNFR) superfamily that regulate osteoclast formation and function by competing for RANK ligand (RANKL). RANKL promotes osteoclast development through RANK activation, while OPG inhibits this process by sequestering RANKL. For comparison, we solved crystal structures of RANKL with RANK and RANKL with OPG. Complementary biochemical and functional studies reveal that the monomeric cytokine-binding region of OPG binds RANKL with ∼500-fold higher affinity than RANK and inhibits RANKL-stimulated osteoclastogenesis ∼150 times more effectively, in part because the binding cleft of RANKL makes unique contacts with OPG. Several side chains as well as the C-D and D-E loops of RANKL occupy different orientations when bound to OPG versus RANK. High affinity OPG binding requires a 90s loop Phe residue that is mutated in juvenile Paget's disease. These results suggest cytokine plasticity may help to fine-tune specific tumor necrosis factor (TNF)-family cytokine/receptor pair selectivity.
  Structure 10/2012; · 6.35 Impact Factor
• Article: Structural Basis of Differential Neutralization of DENV-1 Genotypes by an Antibody that Recognizes a Cryptic Epitope.

  S Kyle Austin, Kimberly A Dowd, Bimmi Shrestha, Christopher A Nelson, Melissa A Edeling, Syd Johnson, Theodore C Pierson, Michael S Diamond, Daved H Fremont
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  ABSTRACT: We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC' loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC' loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development.
  PLoS Pathogens 10/2012; 8(10):e1002930. · 9.13 Impact Factor
• Article: Evidence for a genetic and physical interaction between nonstructural proteins NS1 and NS4B that modulates replication of West Nile virus.

  Soonjeon Youn, Tuo Li, Broc T McCune, Melissa A Edeling, Daved H Fremont, Ileana M Cristea, Michael S Diamond
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  ABSTRACT: Flavivirus NS1 is a nonstructural glycoprotein that is expressed on the cell surface and secreted into the extracellular space. Despite its transit through the secretory pathway, NS1 is an essential gene linked to early viral RNA replication. How this occurs has remained a mystery given the disparate localization of NS1 and the viral RNA replication complex, as the latter is present on the cytosolic face of the endoplasmic reticulum (ER). We recently identified an N-terminal di-amino acid motif in NS1 that modulates protein targeting and affected viral replication. Exchange of two amino acids at positions 10 and 11 from dengue virus (DENV) into West Nile virus (WNV) NS1 (RQ10NK) changed its relative surface expression and secretion and attenuated infectivity. However, the phenotype of WNV containing NS1 RQ10NK was unstable, as within two passages heterogeneous plaque variants were observed. Here, using a mutant WNV encoding the NS1 RQ10NK mutation, we identified a suppressor mutation (F86C) in NS4B, a virally encoded transmembrane protein with loops on both the luminal and cytoplasmic sides of the ER membrane. Introduction of NS4B F86C specifically rescued RNA replication of mutant WNV but did not affect the wild-type virus. Mass spectrometry and coimmunoprecipitation studies established a novel physical interaction between NS1 and NS4B, suggesting a mechanism for how luminal NS1 conveys signals to the cytoplasm to regulate RNA replication.
  Journal of Virology 05/2012; 86(13):7360-71. · 5.40 Impact Factor
• Article: Complex phenotypes in mosquitoes and mice associated with neutralization escape of a Dengue virus type 1 monoclonal antibody.

  Bimmi Shrestha, S Kyle Austin, Kimberly A Dowd, Abhishek N Prasad, Soonjeon Youn, Theodore C Pierson, Daved H Fremont, Gregory D Ebel, Michael S Diamond
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  ABSTRACT: DENV1-E106 is a monoclonal antibody (MAb) with strong neutralizing activity against all five DENV-1 genotypes and therapeutic activity in mice. Here, we evaluated the potential for DENV-1 to escape neutralization by DENV1-E106. A single mutation in domain III of the envelope protein (T329A) emerged, which conferred resistance to DENV1-E106. However, the T329A variant virus had differing phenotypes in vitro and in vivo with attenuation in cell culture yet increased infectivity in Aedes aegypti mosquitoes. Mice infected with this T329A variant still were protected against lethal infection by DENV1-E106 even though much of the neutralizing activity was lost. This study reveals the complex dynamics of neutralization escape of an inhibitory MAb against DENV, and suggests that evaluation of therapeutic MAbs requires detailed investigation in relevant hosts.
  Virology 03/2012; 427(2):127-34. · 3.35 Impact Factor
• Article: Crystal structure of the Japanese encephalitis virus envelope protein.

  Vincent C Luca, Jad AbiMansour, Christopher A Nelson, Daved H Fremont
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  ABSTRACT: Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-Å resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.
  Journal of Virology 12/2011; 86(4):2337-46. · 5.40 Impact Factor
• Article: Hepatitis C virus epitope exposure and neutralization by antibodies is affected by time and temperature.

  Michelle C Sabo, Vincent C Luca, Stuart C Ray, Jens Bukh, Daved H Fremont, Michael S Diamond
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  ABSTRACT: A recent study with flaviviruses suggested that structural dynamics of the virion impact antibody neutralization via exposure of ostensibly cryptic epitopes. To determine whether this holds true for the distantly related hepatitis C virus (HCV), whose neutralizing epitopes may be obscured by a glycan shield, apolipoprotein interactions, and the hypervariable region on the E2 envelope protein, we assessed how time and temperature of pre-incubation altered monoclonal antibody (MAb) neutralization of HCV. Notably, several MAbs showed increased inhibitory activity when pre-binding was performed at 37°C or after longer pre-incubation periods, and a corresponding loss-of-neutralization was observed when pre-binding was performed at 4°C. A similar profile of changes was observed with acute and chronic phase sera from HCV-infected patients. Our data suggest that time and temperature of incubation modulate epitope exposure on the conformational ensembles of HCV virions and thus, alter the potency of antibody neutralization.
  Virology 11/2011; 422(2):174-84. · 3.35 Impact Factor
• Article: Neutralizing monoclonal antibodies against hepatitis C virus E2 protein bind discontinuous epitopes and inhibit infection at a postattachment step.

  Michelle C Sabo, Vincent C Luca, Jannick Prentoe, Sharon E Hopcraft, Keril J Blight, Minkyung Yi, Stanley M Lemon, Jonathan K Ball, Jens Bukh, Matthew J Evans, Daved H Fremont, Michael S Diamond
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  ABSTRACT: The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To characterize the structural and functional basis of HCV neutralization, we generated a novel panel of 78 monoclonal antibodies (MAbs) against E2 proteins from genotype 1a and 2a HCV strains. Using high-throughput focus-forming reduction or luciferase-based neutralization assays with chimeric infectious HCV containing structural proteins from both genotypes, we defined eight MAbs that significantly inhibited infection of the homologous HCV strain in cell culture. Two of these bound E2 proteins from strains representative of HCV genotypes 1 to 6, and one of these MAbs, H77.39, neutralized infection of strains from five of these genotypes. The three most potent neutralizing MAbs in our panel, H77.16, H77.39, and J6.36, inhibited infection at an early postattachment step. Receptor binding studies demonstrated that H77.39 inhibited binding of soluble E2 protein to both CD81 and SR-B1, J6.36 blocked attachment to SR-B1 and modestly reduced binding to CD81, and H77.16 blocked attachment to SR-B1 only. Using yeast surface display, we localized epitopes for the neutralizing MAbs on the E2 protein. Two of the strongly inhibitory MAbs, H77.16 and J6.36, showed markedly reduced binding when amino acids within hypervariable region 1 (HVR1) and at sites ∼100 to 200 residues away were changed, suggesting binding to a discontinuous epitope. Collectively, these studies help to define the structural and functional complexity of antibodies against HCV E2 protein with neutralizing potential.
  Journal of Virology 07/2011; 85(14):7005-19. · 5.40 Impact Factor
• Article: Endogenous MHC-related protein 1 is transiently expressed on the plasma membrane in a conformation that activates mucosal-associated invariant T cells.

  Wei-Jen Chua, Sojung Kim, Nancy Myers, Shouxiong Huang, Lawrence Yu, Daved H Fremont, Michael S Diamond, Ted H Hansen
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  ABSTRACT: The development of mucosal-associated invariant T (MAIT) cells is dependent upon the class Ib molecule MHC-related protein 1 (MR1), commensal bacteria, and a thymus. Furthermore, recent studies have implicated MR1 presentation to MAIT cells in bacteria recognition, although the mechanism remains undefined. Surprisingly, however, surface expression of MR1 has been difficult to detect serologically, despite ubiquitous detection of MR1 transcripts and intracellular protein. In this article, we define a unique mAb capable of stabilizing endogenous mouse MR1 at the cell surface, resulting in enhanced mouse MAIT cell activation. Our results demonstrated that under basal conditions, endogenous MR1 transiently visits the cell surface, thus reconciling the aforementioned serologic and functional studies. Furthermore, using this approach, double-positive thymocytes, macrophages, and dendritic cells were identified as potential APCs for MAIT cell development and activation. Based on this pattern of MR1 expression, it is intriguing to speculate that constitutive expression of MR1 may be detrimental for maintenance of immune homeostasis in the gut and/or detection of pathogenic bacteria in mucosal tissues.
  The Journal of Immunology 03/2011; 186(8):4744-50. · 5.79 Impact Factor
• Article: Identification and sequencing of a novel rodent gammaherpesvirus that establishes acute and latent infection in laboratory mice.

  Joy Loh, Guoyan Zhao, Christopher A Nelson, Penny Coder, Lindsay Droit, Scott A Handley, L Steven Johnson, Punit Vachharajani, Hilda Guzman, Robert B Tesh, David Wang, Daved H Fremont, Herbert W Virgin
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  ABSTRACT: Gammaherpesviruses encode numerous immunomodulatory molecules that contribute to their ability to evade the host immune response and establish persistent, lifelong infections. As the human gammaherpesviruses are strictly species specific, small animal models of gammaherpesvirus infection, such as murine gammaherpesvirus 68 (γHV68) infection, are important for studying the roles of gammaherpesvirus immune evasion genes in in vivo infection and pathogenesis. We report here the genome sequence and characterization of a novel rodent gammaherpesvirus, designated rodent herpesvirus Peru (RHVP), that shares conserved genes and genome organization with γHV68 and the primate gammaherpesviruses but is phylogenetically distinct from γHV68. RHVP establishes acute and latent infection in laboratory mice. Additionally, RHVP contains multiple open reading frames (ORFs) not present in γHV68 that have sequence similarity to primate gammaherpesvirus immunomodulatory genes or cellular genes. These include ORFs with similarity to major histocompatibility complex class I (MHC-I), C-type lectins, and the mouse mammary tumor virus and herpesvirus saimiri superantigens. As these ORFs may function as immunomodulatory or virulence factors, RHVP presents new opportunities for the study of mechanisms of immune evasion by gammaherpesviruses.
  Journal of Virology 03/2011; 85(6):2642-56. · 5.40 Impact Factor
• Article: Structural and biophysical analysis of BST-2/tetherin ectodomains reveals an evolutionary conserved design to inhibit virus release.

  Melissa Swiecki, Suzanne M Scheaffer, Marc Allaire, Daved H Fremont, Marco Colonna, Tom J Brett
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  ABSTRACT: BST-2/tetherin is a host antiviral molecule that functions to potently inhibit the release of enveloped viruses from infected cells. In return, viruses have evolved antagonists to this activity. BST-2 traps budding virions by using two separate membrane-anchoring regions that simultaneously incorporate into the host and viral membranes. Here, we detailed the structural and biophysical properties of the full-length BST-2 ectodomain, which spans the two membrane anchors. The 1.6-Å crystal structure of the complete mouse BST-2 ectodomain reveals an ∼145-Å parallel dimer in an extended α-helix conformation that predominantly forms a coiled coil bridged by three intermolecular disulfides that are required for stability. Sequence analysis in the context of the structure revealed an evolutionarily conserved design that destabilizes the coiled coil, resulting in a labile superstructure, as evidenced by solution x-ray scattering displaying bent conformations spanning 150 and 180 Å for the mouse and human BST-2 ectodomains, respectively. Additionally, crystal packing analysis revealed possible curvature-sensing tetrameric structures that may aid in proper placement of BST-2 during the genesis of viral progeny. Overall, this extended coiled-coil structure with inherent plasticity is undoubtedly necessary to accommodate the dynamics of viral budding while ensuring separation of the anchors.
  Journal of Biological Chemistry 11/2010; 286(4):2987-97. · 4.77 Impact Factor
• Article: Basic and translational applications of engineered MHC class I proteins.

  Ted H Hansen, Janet M Connolly, Keith G Gould, Daved H Fremont
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  ABSTRACT: Major histocompatibility complex (MHC) class I molecules can be engineered as single chain trimers (SCTs) that sequentially incorporate all three subunits of the fully assembled proteins, namely peptide, β2 microglobulin, and heavy chain. SCTs have been made with many different MHC-peptide complexes and are used as novel diagnostic and therapeutic reagents, as well as probes for diverse biological questions. Here, we review the recent and diverse applications of SCTs. These applications include new approaches to enumerate disease-related T cells, DNA vaccines, eliciting responses to pre-assembled MHC-peptide complexes, and unique probes of lymphocyte development and activation. Future applications of SCTs will be driven by their further engineering and the ever-expanding identification of disease-related peptides using chemical, genetic and computational approaches.
  Trends in Immunology 10/2010; 31(10):363-9. · 10.40 Impact Factor
• Article: Genotype-specific neutralization and protection by antibodies against dengue virus type 3.

  James D Brien, S Kyle Austin, Soila Sukupolvi-Petty, Katie M O'Brien, Syd Johnson, Daved H Fremont, Michael S Diamond
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  ABSTRACT: Dengue viruses (DENV) comprise a family of related positive-strand RNA viruses that infect up to 100 million people annually. Currently, there is no approved vaccine or therapy to prevent infection or diminish disease severity. Protection against DENV is associated with the development of neutralizing antibodies that recognize the viral envelope (E) protein. Here, with the goal of identifying monoclonal antibodies (MAbs) that can function as postexposure therapy, we generated a panel of 82 new MAbs against DENV-3, including 24 highly neutralizing MAbs. Using yeast surface display, we localized the epitopes of the most strongly neutralizing MAbs to the lateral ridge of domain III (DIII) of the DENV type 3 (DENV-3) E protein. While several MAbs functioned prophylactically to prevent DENV-3-induced lethality in a stringent intracranial-challenge model of mice, only three MAbs exhibited therapeutic activity against a homologous strain when administered 2 days after infection. Remarkably, no MAb in our panel protected prophylactically against challenge by a strain from a heterologous DENV-3 genotype. Consistent with this, no single MAb neutralized efficiently the nine different DENV-3 strains used in this study, likely because of the sequence variation in DIII within and between genotypes. Our studies suggest that strain diversity may limit the efficacy of MAb therapy or tetravalent vaccines against DENV, as neutralization potency generally correlated with a narrowed genotype specificity.
  Journal of Virology 10/2010; 84(20):10630-43. · 5.40 Impact Factor
• Article: A short N-terminal peptide motif on flavivirus nonstructural protein NS1 modulates cellular targeting and immune recognition.

  Soonjeon Youn, Hyelim Cho, Daved H Fremont, Michael S Diamond
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  ABSTRACT: Flavivirus NS1 is a versatile nonstructural glycoprotein, with intracellular NS1 functioning as an essential cofactor for viral replication and cell surface and secreted NS1 antagonizing complement activation. Even though NS1 has multiple functions that contribute to virulence, the genetic determinants that regulate the spatial distribution of NS1 in cells among different flaviviruses remain uncharacterized. Here, by creating a panel of West Nile virus-dengue virus (WNV-DENV) NS1 chimeras and site-specific mutants, we identified a novel, short peptide motif immediately C-terminal to the signal sequence cleavage position that regulates its transit time through the endoplasmic reticulum and differentially directs NS1 for secretion or plasma membrane expression. Exchange of two amino acids within this motif reciprocally changed the cellular targeting pattern of DENV or WNV NS1. For WNV, this substitution also modulated infectivity and antibody-induced phagocytosis of infected cells. Analysis of a mutant lacking all three conserved N-linked glycosylation sites revealed an independent requirement of N-linked glycans for secretion but not for plasma membrane expression of WNV NS1. Collectively, our experiments define the requirements for cellular targeting of NS1, with implications for the protective host responses, immune antagonism, and association with the host cell sorting machinery. These studies also suggest a link between the effects of NS1 on viral replication and the levels of secreted or cell surface NS1.
  Journal of Virology 09/2010; 84(18):9516-32. · 5.40 Impact Factor
• Article: Structure and function analysis of therapeutic monoclonal antibodies against dengue virus type 2.

  Soila Sukupolvi-Petty, S Kyle Austin, Michael Engle, James D Brien, Kimberly A Dowd, Katherine L Williams, Syd Johnson, Rebeca Rico-Hesse, Eva Harris, Theodore C Pierson, Daved H Fremont, Michael S Diamond
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  ABSTRACT: Dengue virus (DENV) is the most prevalent insect-transmitted viral disease in humans globally, and currently no specific therapy or vaccine is available. Protection against DENV and other related flaviviruses is associated with the development of antibodies against the viral envelope (E) protein. Although prior studies have characterized the neutralizing activity of monoclonal antibodies (MAbs) against DENV type 2 (DENV-2), none have compared simultaneously the inhibitory activity against a genetically diverse range of strains in vitro, the protective capacity in animals, and the localization of epitopes. Here, with the goal of identifying MAbs that can serve as postexposure therapy, we investigated in detail the functional activity of a large panel of new anti-DENV-2 mouse MAbs. Binding sites were mapped by yeast surface display and neutralization escape, cell culture inhibition assays were performed with homologous and heterologous strains, and prophylactic and therapeutic activity was evaluated with two mouse models. Protective MAbs localized to epitopes on the lateral ridge of domain I (DI), the dimer interface, lateral ridge, and fusion loop of DII, and the lateral ridge, C-C' loop, and A strand of DIII. Several MAbs inefficiently inhibited at least one DENV-2 strain of a distinct genotype, suggesting that recognition of neutralizing epitopes varies with strain diversity. Moreover, antibody potency generally correlated with a narrowed genotype and serotype specificity. Five MAbs functioned efficiently as postexposure therapy when administered as a single dose, even 3 days after intracranial infection of BALB/c mice. Overall, these studies define the structural and functional complexity of antibodies against DENV-2 with protective potential.
  Journal of Virology 09/2010; 84(18):9227-39. · 5.40 Impact Factor