Theodore C Pierson

National Institute of Allergy and Infectious Diseases, Maryland, United States

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Publications (76)531.13 Total impact

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    ABSTRACT: Flaviviruses are thought to sample an ensemble of structures at equilibrium. One consequence of a structurally dynamic virion is the observed time-dependent increases in neutralization sensitivity that can occur after prolonged incubation with antibody. Differences in how virus strains "breathe" may affect epitope exposure and contribute to the underlying mechanisms of strain-dependent neutralization sensitivity. Beyond the contribution of structural dynamics, flaviviruses exist as a structurally heterogeneous population due to an inefficient virion maturation process. Herein we investigate the interplay between virion maturation and structural dynamics that contribute to antibody-mediated neutralization. Using West Nile (WNV) and dengue (DENV) viruses produced under conditions that modify the extent of virion maturation, we investigated time-dependent changes in neutralization sensitivity associated with structural dynamics. Our results identify distinct patterns of neutralization against viruses that vary markedly with respect to the extent of virion maturation. Reducing the efficiency of virion maturation resulted in greater time-dependent changes in neutralization potency and a marked reduction in the stability of the particle at 37°C as compared to more mature virus. That neutralization sensitivity of WNV and DENV did not increase after prolonged incubation in the absence of antibody, regardless of virion maturation, suggests that the dynamic processes that govern epitope accessibility on infectious viruses are reversible. Against the backdrop of heterogeneous flavivirus structures, differences in the pathways by which viruses "breathe" represent an additional layer of complexity towards understanding maturation state-dependent patterns of antibody recognition.
    Journal of virology. 07/2014;
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    ABSTRACT: The production of neutralizing antibodies (nAbs) is a correlate of protection for many human vaccines, including currently licensed vaccines against flaviviruses. nAbs are typically measured using a plaque reduction neutralization test (PRNT). Despite its extensive use, parameters that impact the performance of the PRNT have not been investigated from a mechanistic perspective. The results of a recent phase IIb clinical trial of a tetravalent dengue virus (DENV) vaccine suggests that nAbs, as measured using a PRNT performed with Vero cells, do not correlate with protection. This surprising finding highlights the importance of understanding how well the PRNT captures the complexity of the nAb response to DENV. In this study, we demonstrate that the structural heterogeneity of flaviviruses arising from inefficient virion maturation impacts the results of neutralization assays in a cell type-dependent manner. Neutralization titers of several monoclonal antibodies were significantly reduced when assayed on Vero cells, as compared to Raji cells expressing DC-SIGNR. This pattern can be explained by differences in the efficiency with which partially mature flaviviruses attach to each cell type, rather than a differential capacity of antibody to block infection. Vero cells are poorly permissive to the fraction of virions that are most sensitive to neutralization. Analysis of sera from recipients of live-attenuated monovalent DENV vaccine candidates revealed a strong correlation between the sensitivity of serum antibodies to the maturation state of DENV and cell type-dependent patterns of neutralization. Cross-reactive patterns of neutralization may be under-represented by the "gold-standard" PRNT that employs Vero cells. Cell type-dependent patterns of neutralization describes a differential capacity of antibodies to inhibit virus infection when assayed on multiple cellular substrates. In this study, we establish a link between antibodies that neutralize infection in a cell type-dependent fashion and those sensitive to the maturation state of the flavivirus virion. We demonstrate that cell type-dependent neutralization reflects a differential capacity to measure neutralization of viruses that are incompletely mature. Partially mature virions that most efficiently bind maturation state-sensitive antibodies are poorly represented by assays typically used in support of flavivirus vaccine development. The selection of cellular substrate for neutralization assays may significantly impact evaluation of the neutralization potency of the polyclonal response. These data suggest that current assays do not adequately capture the full complexity of the neutralizing antibody response and may hinder the identification of correlates of protection following flavivirus vaccination.
    Journal of Virology 04/2014; · 5.08 Impact Factor
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    ABSTRACT: We recently described our most potently neutralizing monoclonal antibody, E106, which protected against lethal Dengue virus type 1 (DENV-1) infection in mice. To further understand its functional properties, we determined the crystal structure of E106 Fab in complex with domain III (DIII) of DENV-1 envelope (E) protein to 2.45 Å resolution. Analysis of the complex revealed a small antibody-antigen interface with the epitope on DIII composed of nine residues along the lateral ridge and A-strand regions. Despite strong virus neutralizing activity of E106 IgG at picomolar concentrations, E106 Fab exhibited a ∼20,000-fold decrease in virus neutralization and bound isolated DIII, E, or viral particles with only a micromolar monovalent affinity. In comparison, E106 IgG bound DENV-1 virions with nanomolar avidity. The E106 epitope appears readily accessible on virions, as neutralization was largely temperature-independent. Collectively, our data suggest that E106 neutralizes DENV-1 infection through bivalent engagement of adjacent DIII subunits on a single virion. The isolation of anti-flavivirus antibodies that require bivalent binding to inhibit infection efficiently may be a rare event due to the unique icosahedral arrangement of envelope proteins on the virion surface.
    PLoS Pathogens 04/2014; 10(4):e1004072. · 8.14 Impact Factor
  • Swati Mukherjee, Theodore C Pierson, Kimberly A Dowd
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    ABSTRACT: This chapter outlines methods for the production of dengue virus (DENV) reporter virus particles (RVPs) and their use in assays that measure antibody-mediated neutralization and enhancement of DENV infection. RVPs are pseudo-infectious virions produced by complementation of a self-replicating flavivirus replicon with the DENV structural genes in trans. RVPs harvested from transfected cells are capable of only a single round of infection and encapsidate replicon RNA that encodes a reporter gene used to enumerate infected cells. RVPs may be produced using the structural genes of different DENV serotypes, genotypes, and mutants by changing plasmids used for complementation. Further modifications are possible including generating RVPs with varying levels of uncleaved prM protein, which resemble either the immature or mature form of the virus. Neutralization potency is measured by incubating RVPs with serial dilutions of antibody, followed by infection of target cells that express DENV attachment factors. Enhancement of infection is measured similarly using Fc receptor-expressing cells capable of internalizing antibody-virus complexes.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1138:75-97. · 1.29 Impact Factor
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    ABSTRACT: Background Chikungunya virus—a mosquito-borne alphavirus—is endemic in Africa and south and southeast Asia and has recently emerged in the Caribbean. No drugs or vaccines are available for treatment or prevention. We aimed to assess the safety, tolerability, and immunogenicity of a new candidate vaccine. Methods VRC 311 was a phase 1, dose-escalation, open-label clinical trial of a virus-like particle (VLP) chikungunya virus vaccine, VRC-CHKVLP059-00-VP, in healthy adults aged 18–50 years who were enrolled at the National Institutes of Health Clinical Center (Bethesda, MD, USA). Participants were assigned to sequential dose level groups to receive vaccinations at 10 μg, 20 μg, or 40 μg on weeks 0, 4, and 20, with follow-up for 44 weeks after enrolment. The primary endpoints were safety and tolerability of the vaccine. Secondary endpoints were chikungunya virus-specific immune responses assessed by ELISA and neutralising antibody assays. This trial is registered with ClinicalTrials.gov, NCT01489358. Findings 25 participants were enrolled from Dec 12, 2011, to March 22, 2012, into the three dosage groups: 10 μg (n=5), 20 μg (n=10), and 40 μg (n=10). The protocol was completed by all five participants at the 10 μg dose, all ten participants at the 20 μg dose, and eight of ten participants at the 40 μg dose; non-completions were for personal circumstances unrelated to adverse events. 73 vaccinations were administered. All injections were well tolerated, with no serious adverse events reported. Neutralising antibodies were detected in all dose groups after the second vaccination (geometric mean titres of the half maximum inhibitory concentration: 2688 in the 10 μg group, 1775 in the 20 μg group, and 7246 in the 40 μg group), and a significant boost occurred after the third vaccination in all dose groups (10 μg group p=0·0197, 20 μg group p<0·0001, and 40 μg group p<0·0001). 4 weeks after the third vaccination, the geometric mean titres of the half maximum inhibitory concentration were 8745 for the 10 μg group, 4525 for the 20 μg group, and 5390 for the 40 μg group. Interpretation The chikungunya VLP vaccine was immunogenic, safe, and well tolerated. This study represents an important step in vaccine development to combat this rapidly emerging pathogen. Further studies should be done in a larger number of participants and in more diverse populations. Funding Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, and National Institutes of Health.
    Lancet. 01/2014;
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    ABSTRACT: Dengue viruses are mosquito-borne flaviviruses that circulate in nature as four distinct serotypes (DENV1-4). These emerging pathogens are responsible for more than 100 million human infections annually. Severe clinical manifestations of disease are predominantly associated with a secondary infection by a heterotypic DENV serotype. The increased risk of severe disease in DENV-sensitized populations significantly complicates vaccine development, as a vaccine must simultaneously confer protection against all four DENV serotypes. Eliciting a protective tetravalent neutralizing antibody response is a major goal of ongoing vaccine development efforts. However, a recent large clinical trial of a candidate live-attenuated DENV vaccine revealed low protective efficacy despite eliciting a neutralizing antibody response, highlighting the need for a better understanding of the humoral immune response against dengue infection. In this study, we sought to identify epitopes recognized by serotype-specific neutralizing antibodies elicited by monovalent DENV1 vaccination. We constructed a panel of over 50 DENV1 structural gene variants containing substitutions at surface-accessible residues of the envelope (E) protein to match the corresponding DENV2 sequence. Amino acids that contribute to recognition by serotype-specific neutralizing antibodies were identified as DENV mutants with reduced sensitivity to neutralization by DENV1 immune sera, but not cross-reactive neutralizing antibodies elicited by DENV2 vaccination. We identified two mutations (E126K and E157K) that contribute significantly to type-specific recognition by polyclonal DENV1 immune sera. Longitudinal and cross-sectional analysis of sera from 24 participants of a phase I clinical study revealed a markedly reduced capacity to neutralize a E126K/E157K DENV1 variant. Sera from 77% of subjects recognized the E126K/E157K DENV1 variant and DENV2 equivalently (<3-fold difference). These data indicate the type-specific component of the DENV1 neutralizing antibody response to vaccination is strikingly focused on just two amino acids of the E protein. This study provides an important step towards deconvoluting the functional complexity of DENV serology following vaccination.
    PLoS Pathogens 12/2013; 9(12):e1003761. · 8.14 Impact Factor
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    ABSTRACT: Flavivirus-infected cells secrete a structurally heterogeneous population of viruses because of an inefficient virion maturation process. Flaviviruses assemble as non-infectious, immature virions composed of trimers of envelope (E) and precursor-membrane (prM) protein heterodimers. Cleavage of prM is a required process during virion maturation, although this often remains incomplete on infectious virus particles. Previous work demonstrated that the efficiency of virion maturation could impact antibody neutralization through changes in the accessibility of otherwise cryptic epitopes on the virion. In this study, we show that the neutralization potency of monoclonal antibody (mAb) E33 is sensitive to the maturation state of West Nile virus (WNV) despite recognizing an accessible epitope, the domain III lateral ridge (DIII-LR). Comprehensive epitope mapping studies using 166 E protein DIII-LR variants revealed that the functional footprint of mAb E33 on the E protein differs subtly from that of the well characterized DIII-LR mAb E16. Remarkably, aromatic substitutions at E protein residue 306 ablated the maturation-state sensitivity of E33 IgG, and the neutralization efficacy of E33 Fab fragments was not affected by changes in the virion maturation state. We propose that E33 IgG binding on mature virions orients the Fc region in a manner that impacts subsequent antibody binding to nearby sites. This Fc-mediated steric constraint is a novel mechanism by which the maturation state of a virion modulates the efficacy of the humoral immune response to flavivirus infection.
    Journal of Virology 10/2013; · 5.08 Impact Factor
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    ABSTRACT: Dengue virus (DENV) is a mosquito-transmitted flavivirus that can cause severe disease in humans and is considered a re-emerging pathogen of significant importance to public health. The DENV capsid (C) protein functions as a structural component of the infectious virion, however, it may have additional functions in the virus replicative cycle. Here we show that the DENV C protein interacts and co-localizes with the multi-functional host protein nucleolin (NCL). Furthermore, we demonstrate that this interaction can be disrupted by the addition of a NCL binding aptamer (AS1411). Knockdown of NCL with siRNA, or treatment of cells with AS1411 results in a significant reduction of viral titers after DENV infection. Western blot and qRT-PCR analysis revealed no differences in viral RNA or protein levels at early time points post-infection, suggesting a role for NCL in viral morphogenesis. We support this hypothesis by showing that treatment with AS1411 alters the migration characteristics of the viral capsid as visualized by native electrophoresis. Here we identify a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics.
    Journal of Virology 09/2013; · 5.08 Impact Factor
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    ABSTRACT: Although prior studies have characterized the neutralizing activity of monoclonal antibodies (MAbs) against DENV serotypes 1, 2, and 3 (DENV-1, DENV-2, and DENV-3), few reports have assessed the activity of MAbs against DENV-4. Here, we evaluated the inhibitory activity of 81 new mouse anti-DENV-4 MAbs. We observed strain and genotype-dependent differences in neutralization of DENV-4 by MAbs mapping to epitopes on domains II (DII) and III (DIII) of the envelope (E) protein. Several anti-DENV-4 MAbs inefficiently inhibited at least one strain and/or genotype, suggesting that the exposure or sequence of neutralizing epitopes varies within isolates of this serotype. Remarkably, flavivirus cross-reactive MAbs, which bound to the highly conserved fusion loop in DII and inhibited infection of DENV-1, DENV-2, and DENV-3, more weakly neutralized five different DENV-4 strains encompassing the genetic diversity of the serotype after pre-incubation at 37°C. However, increasing the time of pre-incubation at 37°C or raising the temperature to 40°C enhanced the potency of DII fusion loop-specific MAbs and some DIII-specific MAbs against DENV-4 strains. Prophylaxis studies in two new DENV-4 mouse models showed that neutralization titers of MAbs after pre-incubation at 37°C correlated with activity in vivo. Our studies establish the complexity of MAb recognition against DENV-4, and suggest that differences in epitope exposure relative to other DENV serotypes affects antibody neutralization and protective activity.
    Journal of Virology 06/2013; · 5.08 Impact Factor
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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. · 8.14 Impact Factor
  • Theodore C Pierson, Margaret Kielian
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    ABSTRACT: Flaviviruses are small spherical virus particles covered by a dense icosahedral array of envelope (E) proteins that mediate virus attachment to cells and the fusion of viral and cellular membranes. Our understanding of the mechanism by which flavivirus E proteins orchestrate entry into cells has been advanced by studies of E structure and arrangement on the virion at different steps of the virus entry/membrane fusion process. When combined with an increasingly clear (albeit still incomplete) view of the cell biology of virus entry, these advances suggest new antiviral strategies. Indeed, inhibitors that target cellular and viral processes involved in entry show promise as powerful tools to study this critical step of the viral lifecycle, and with luck, may ultimately lead to therapeutic advances.
    Current opinion in virology. 01/2013;
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    ABSTRACT: Neutralization of flaviviruses requires engagement of the virion by antibodies with a stoichiometry that exceeds a required threshold. Factors that modulate the number of antibodies bound to an individual virion when it contacts target cells impact neutralization potency. However, the contribution of cellular factors to the potency of neutralizing antibodies has not been explored systematically. Here we investigate the relationship between expression level of a viral attachment factor on cells and the neutralizing potency of antibodies. Analysis of the attachment factor DC-SIGNR on cells in neutralization studies failed to identify a correlation between DC-SIGNR expression and antibody-mediated protection. Furthermore, neutralization potency was equivalent on a novel Jurkat cell line induced to express DC-SIGNR at varying levels. Finally, blocking virus-attachment factor interactions had no impact on neutralization activity. Altogether, our studies suggest that cellular attachment factor expression is not a significant contributor to the potency of neutralizing antibodies to flaviviruses.
    Virology 01/2013; · 3.35 Impact Factor
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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. · 8.14 Impact Factor
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    Theodore C Pierson, Jonathan W Yewdell
    Proceedings of the National Academy of Sciences 08/2012; 109(37):14724-5. · 9.81 Impact Factor
  • Theodore C Pierson, Michael S Diamond
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    ABSTRACT: Flaviviruses are small enveloped virions that enter target cells in a pH-dependent fashion. Virus attachment, entry, and membrane fusion are orchestrated by the envelope (E) and pre-membrane (prM) proteins, the two structural proteins displayed on the surface of virions. Flaviviruses assemble as an immature non-infectious form onto which prM and E form trimeric spikes. During egress from infected cells, flaviviruses undergo dramatic structural changes characterized by the formation of a herringbone arrangement of E proteins that lie flat against the surface of the virion and cleavage of the prM protein by the cellular protease furin. The result is a relatively smooth, infectious mature virion. This dynamic process is now understood in structural detail at the atomic level. However, recent studies indicate that many of the virions released from cells share structural features of both immature and mature virus particles. These mosaic partially mature virions are infectious and interact uniquely with target cells and the host immune response. Here, we will discuss recent advances in our understanding of the biology and significance of partially mature flaviviruses.
    Current opinion in virology. 03/2012; 2(2):168-75.
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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
  • Theodore C Pierson, Richard J Kuhn
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    ABSTRACT: The four serotypes of dengue virus present a formidable challenge for the development of efficacious human vaccines. Cockburn and colleagues, in this issue of Structure, describe the structural basis of a cross-reactive neutralizing antibody, providing greater insight into immune protection and pathogenesis.
    Structure 02/2012; 20(2):200-2. · 5.99 Impact Factor
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    ABSTRACT: Molecular clone technology has proven to be a powerful tool for investigating the life cycle of flaviviruses, their interactions with the host, and vaccine development. Despite the demonstrated utility of existing molecular clone strategies, the feasibility of employing these existing approaches in large-scale mutagenesis studies is limited by the technical challenges of manipulating relatively large molecular clone plasmids that can be quite unstable when propagated in bacteria. We have developed a novel strategy that provides an extremely rapid approach for the introduction of mutations into the structural genes of West Nile virus (WNV). The backbone of this technology is a truncated form of the genome into which DNA fragments harboring the structural genes are ligated and transfected directly into mammalian cells, bypassing entirely the requirement for cloning in bacteria. The transfection of cells with this system results in the rapid release of WNV that achieves a high titer (∼10(7) infectious units/ml in 48 h). The suitability of this approach for large-scale mutagenesis efforts was established in two ways. First, we constructed and characterized a library of variants encoding single defined amino acid substitutions at the 92 residues of the "pr" portion of the precursor-to-membrane (prM) protein. Analysis of a subset of these variants identified a mutation that conferred resistance to neutralization by an envelope protein-specific antibody. Second, we employed this approach to accelerate the identification of mutations that allow escape from neutralizing antibodies. Populations of WNV encoding random changes in the E protein were produced in the presence of a potent monoclonal antibody, E16. Viruses resistant to neutralization were identified in a single passage. Together, we have developed a simple and rapid approach to produce infectious WNV that accelerates the process of manipulating the genome to study the structure and function of the structural genes of this important human pathogen.
    Journal of Virology 01/2012; 86(7):3501-12. · 5.08 Impact Factor
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    ABSTRACT: The human antibody response to flavivirus infection is dominantly directed against a cross-reactive epitope on the fusion loop of domain II (DII-FL) of the envelope (E) protein. Although antibodies against this epitope fail to recognize fully mature West Nile virus (WNV) virions and accordingly neutralize infection poorly in vitro, their functional properties in vivo remain less well understood. Here, we show that while passive transfer of poorly neutralizing monoclonal antibodies (MAb) and polyclonal antibodies against the DII-FL epitope protect against lethal WNV infection in wild-type mice, they fail to protect mice lacking activating Fcγ receptors (FcγR) and the complement opsonin C1q. Consistent with this, an aglycosyl chimeric mouse-human DII-FL MAb (E28) variant that lacks the ability to engage FcγR and C1q also did not protect against WNV infection in wild-type mice. Using a series of immunodeficient mice and antibody depletions of individual immune cell populations, we demonstrate that the nonneutralizing DII-FL MAb E28 does not require T, B, or NK cells, inflammatory monocytes, or neutrophils for protection. Rather, E28 treatment decreased viral load in the serum early in the course of infection, which resulted in blunted dissemination to the brain, an effect that required phagocytic cells, C1q, and FcγRIII (CD16). Overall, these studies enhance our understanding of the functional significance of immunodominant, poorly neutralizing antibodies in the polyclonal human anti-flavivirus response and highlight the limitations of current in vitro surrogate markers of protection, such as cell-based neutralization assays, which cannot account for the beneficial effects conferred by these antibodies.
    Journal of Virology 09/2011; 85(22):11567-80. · 5.08 Impact Factor
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    ABSTRACT: Cleavage of the flavivirus prM protein by a cellular furin-like protease is a hallmark of virion maturation. While this cleavage is a required step in the viral life cycle, it can be inefficient. Virions that retain uncleaved prM may be infectious. We investigated whether cleavage by furin of prM on partially mature West Nile virus (WNV) during virus entry contributes to infectivity. Using quantitative assays of WNV infection, we found that virions incorporating considerable amounts of uncleaved prM protein were insensitive to treatment of cells with a potent inhibitor of furin activity. Thus, partially mature WNV does not require furin-like proteases for infectivity.
    Journal of Virology 08/2011; 85(22):12067-72. · 5.08 Impact Factor

Publication Stats

5k Citations
531.13 Total Impact Points

Institutions

  • 2008–2014
    • National Institute of Allergy and Infectious Diseases
      • Laboratory of Immunoregulation
      Maryland, United States
  • 2013
    • Georgetown University
      Washington, Washington, D.C., United States
  • 2007–2013
    • National Institutes of Health
      • Laboratory of Viral Diseases
      Maryland, United States
  • 2005–2012
    • University of Washington Seattle
      • • Department of Medicine
      • • Department of Immunology
      Seattle, WA, United States
  • 2010
    • University of California, Berkeley
      • Department of Infectious Diseases and Vaccinology
      Berkeley, California, United States
  • 2007–2009
    • Washington University in St. Louis
      • • Department of Pathology and Immunology
      • • Department of Molecular Microbiology
      Saint Louis, MO, United States
  • 2003–2007
    • Hospital of the University of Pennsylvania
      • Department of Microbiology
      Philadelphia, Pennsylvania, United States
  • 2000–2007
    • University of Pennsylvania
      • • Department of Microbiology (Medicine)
      • • Department of Pathology and Laboratory Medicine
      Philadelphia, PA, United States
  • 1997–2002
    • Johns Hopkins University
      • Department of Medicine
      Baltimore, MD, United States
  • 2001
    • Molecular and Cellular Biology Program
      Seattle, Washington, United States