Stephen S Whitehead

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

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Publications (92)474.27 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The incidence of infection with any of the four dengue virus serotypes (DENV 1-4) has increased dramatically in the last few decades, and the lack of a treatment or vaccine has contributed to significant morbidity and mortality worldwide. A recent comprehensive analysis of the human T cell response against wild-type DENV suggested an HLA-linked protective role for CD8(+) T cells. We have collected one-unit blood donations from study participants receiving the monovalent or tetravalent live attenuated DENV vaccine (DLAV), developed by the U.S. National Institutes of Health. PBMCs from these donors were screened in IFNγ ELISPOT assays with pools of predicted, HLA matched, class I binding peptides covering the entire DENV proteome. Here, we characterize for the first time CD8(+) T cell responses after live attenuated dengue vaccination and show that CD8(+) T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Interestingly, while broad responses to structural and non-structural (NS) proteins were observed after monovalent vaccination, T cell responses following tetravalent vaccination were, dramatically, focused towards the highly conserved NS proteins. Epitopes were highly conserved in a vast variety of field isolates and able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response will contribute to the identification of robust correlates of protection in natural immunity and following vaccination against DENV.
    Journal of virology. 10/2014;
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    ABSTRACT: The live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine, produced in primary hamster kidney cells, is safe and effective. Past attempts to adapt this virus to replicate in cells that are more favorable for vaccine production resulted in mutations that significantly reduced immunogenicity. In this study, 10 genetically distinct Vero cell-adapted JEV SA14-14-2 variants were isolated and a recombinant wild-type JEV clone, modified to contain the JEV SA14-14-2 polyprotein amino acid sequence, was recovered in Vero cells. A single capsid protein mutation (S66L) was important for Vero cell-adaptation. Mutations were also identified that modulated virus sensitivity to type I interferon-stimulation in Vero cells. A subset of JEV SA14-14-2 variants and the recombinant clone were evaluated in vivo and exhibited levels of attenuation that varied significantly in suckling mice, but were avirulent and highly immunogenic in weanling mice and are promising candidates for the development of a second-generation, recombinant vaccine.
    The American journal of tropical medicine and hygiene 10/2014; · 2.53 Impact Factor
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    ABSTRACT: The four serotypes of mosquito-borne dengue virus (DENV-1, -2, -3, and -4) that circulate in humans each emerged from an enzootic, sylvatic cycle in non-human primates. Herein, we present the first study of sylvatic DENV infection dynamics in a primate. Three African green monkeys were inoculated with 10(5) plaque-forming units (pfu) DENV-2 strain PM33974 from the sylvatic cycle, and one African green monkey was inoculated with 10(5) pfu DENV-2 strain New Guinea C from the human cycle. All four monkeys seroconverted (more than fourfold rise in 80% plaque reduction neutralization titer [PRNT80]) against the strain of DENV with which they were inoculated; only one (33%) of three monkeys infected with sylvatic DENV showed a neutralizing antibody response against human-endemic DENV. Virus was detected in two of three monkeys inoculated with sylvatic DENV at low titer (≤ 1.3 log10pfu/mL) and brief duration (≤ 2 days). Clinical signs included rash and elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels.
    The American journal of tropical medicine and hygiene 08/2014; · 2.53 Impact Factor
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    ABSTRACT: Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis worldwide and vaccination is one of the most effective ways to prevent disease. A suitable live-attenuated JEV vaccine could be formulated with a live-attenuated tetravalent dengue vaccine for the control of these viruses in endemic areas. Toward this goal, we generated chimeric virus vaccine candidates by replacing the precursor membrane (prM) and envelope (E) protein structural genes of recombinant dengue virus type 4 (rDEN4) or attenuated vaccine candidate rDEN4Δ30 with those of wild-type JEV strain India/78. Mutations were engineered in E, NS3 and NS4B protein genes to improve replication in Vero cells. The chimeric viruses were attenuated in mice and some elicited modest but protective levels of immunity after a single dose. One particular chimeric virus, bearing E protein mutation Q264H, replicated to higher titer in tissue culture and was significantly more immunogenic in mice. The results are compared with live-attenuated JEV vaccine strain SA14-14-2.
    Vaccine. 05/2014; 32(25):3010–3018.
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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: Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis worldwide and vaccination is one of the most effective ways to prevent disease. A suitable live-attenuated JEV vaccine could be formulated with a live-attenuated tetravalent dengue vaccine for the control of these viruses in endemic areas. Toward this goal, we generated chimeric virus vaccine candidates by replacing the precursor membrane (prM) and envelope (E) protein structural genes of recombinant dengue virus type 4 (rDEN4) or attenuated vaccine candidate rDEN4Δ30 with those of wild-type JEV strain India/78. Mutations were engineered in E, NS3 and NS4B protein genes to improve replication in Vero cells. The chimeric viruses were attenuated in mice and some elicited modest but protective levels of immunity after a single dose. One particular chimeric virus, bearing E protein mutation Q264H, replicated to higher titer in tissue culture and was significantly more immunogenic in mice. The results are compared with live-attenuated JEV vaccine strain SA14-14-2.
    Vaccine 03/2014; · 3.77 Impact Factor
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    ABSTRACT: This study investigated whether a large dengue epidemic that struck Hanoi in 2009 also affected a nearby semirural area. Seroconversion (dengue virus-reactive immunoglobulin G enzyme-linked immunosorbent assay) was high during 2009 compared with 2008, but neutralization assays showed that it was caused by both dengue virus and Japanese encephalitis virus infections. The findings highlight the importance of continued Japanese encephalitis virus vaccination and dengue surveillance.
    The American journal of tropical medicine and hygiene 03/2014; · 2.53 Impact Factor
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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: WNV has become the leading vector-borne cause of meningoencephalitis in the United States. Although the majority of WNV infections result in asymptomatic illness, approximately 20% of infections result in West Nile fever and 1% in West Nile neuroinvasive disease (WNND), which causes encephalitis, meningitis, or flaccid paralysis. The elderly are at particular risk for WNND, with more than half the cases occurring in persons older than sixty years of age. There is no licensed treatment for WNND, nor is there any licensed vaccine for humans for the prevention of WNV infection. The Laboratory of Infectious Diseases at the National Institutes of Health has developed a recombinant live attenuated WNV vaccine based on chimerization of the wild-type WNV NY99 genome with that of the live attenuated DENV-4 candidate vaccine rDEN4Δ30. The genes encoding the prM and envelope proteins of DENV-4 were replaced with those of WNV NY99 and the resultant virus was designated rWN/DEN4Δ30. The vaccine was evaluated in healthy flavivirus-naïve adult volunteers age 18-50 years in two separate studies, both of which are reported here. The first study evaluated 10(3) or 10(4)PFU of the vaccine given as a single dose; the second study evaluated 10(5)PFU of the vaccine given as two doses 6 months apart. The vaccine was well-tolerated and immunogenic at all three doses, inducing seroconversion to WNV NY99 in 74% (10(3)PFU), 75% (10(4)PFU), and 55% (10(5)PFU) of subjects after a single dose. A second 10(5)PFU dose of rWN/DEN4Δ30 given 6 months after the first dose increased the seroconversion rate 89%. Based on the encouraging results from these studies, further evaluation of the candidate vaccine in adults older than 50 years of age is planned.
    Vaccine 08/2013; · 3.77 Impact Factor
  • Anna Durbin, Stephen S Whitehead
    The Journal of Infectious Diseases 07/2013; · 5.85 Impact Factor
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    ABSTRACT: There are currently no vaccines or therapeutics to prevent dengue disease which ranges in severity from asymptomatic infections to life-threatening illness. The National Institute of Allergy and Infectious Diseases (NIAID) Division of Intramural Research has developed live, attenuated vaccines to each of the four dengue serotypes (DENV-1-DENV-4). Two doses (10PFU and 1000PFU) of three monovalent vaccines were tested in human clinical trials to compare safety and immunogenicity profiles. DEN4Δ30 had been tested previously at multiple doses. The three dengue vaccine candidates tested (DEN1Δ30, DEN2/4Δ30, and DEN3Δ30/31) were very infectious, each with a human infectious dose 50%≤10PFU. Further, infectivity rates ranged from 90 to 100% regardless of dose, excepting DEN2/4Δ30 which dropped from 100% at the 1000PFU dose to 60% at the 10PFU dose. Mean geometric peak antibody titers did not differ significantly between doses for DEN1Δ30 (92±19 vs. 214±97, p=0.08); however, significant differences were observed between the 10PFU and 1000PFU doses for DEN2/4Δ30, 19±9 vs. 102±25 (p=0.001), and DEN3Δ30/31, 119±135 vs. 50±50 (p=0.046). No differences in the incidences of rash, neutropenia, or viremia were observed between doses for any vaccines, though the mean peak titer of viremia for DEN1Δ30 was higher at the 1000PFU dose (0.5±0 vs. 1.1±0.1, p=0.007). These data demonstrate that a target dose of 1000PFU for inclusion of each dengue serotype into a tetravalent vaccine is likely to be safe and generate a balanced immune response for all serotypes.
    Vaccine 06/2013; · 3.77 Impact Factor
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    ABSTRACT: The immunopathogenesis of severe dengue is poorly understood, but there is concern that induction of cross-reactive non-neutralizing antibodies by infection or vaccination may increase the likelihood of severe disease during a subsequent infection. We generated a total of 63 new human mAbs to compare the B cell response of subjects who received the NIH live attenuated DENV1 vaccine (rDEN1Δ30) to that of subjects following symptomatic primary infection with DENV1. Both infection and vaccination induced serum neutralizing antibodies and DENV1-reactive peripheral blood B cells, but to a lesser magnitude in vaccinated individuals. Serotype cross-reactive weakly neutralizing antibodies dominated the response in both vaccinated and naturally-infected subjects. Antigen specificities were very similar, a slightly greater percentage of antibodies targeted E protein domain I-II than domain III. These data shed light on the similarity of human B cell response to live attenuated DENV vaccine or natural infection.
    The Journal of Infectious Diseases 03/2013; · 5.85 Impact Factor
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    ABSTRACT: Background. Dengue virus (DENV) causes hundreds of millions of infections annually. Four dengue serotypes exist and previous infection with one serotype increases the likelihood of severe disease with a second, heterotypic DENV infection.Methods. In a randomized, placebo-controlled study, the safety and immunogenicity of four different admixtures of a live attenuated tetravalent (LATV) dengue vaccine were evaluated in 113 flavivirus-naïve adults. Serum neutralizing antibody levels to all four dengue viruses were measured on days 0, 28, 42, and 180. Clinicaltrials.gov: NCT01072786Results. A single dose of each LATV admixture induced a trivalent or better neutralizing antibody response in 75% to 90% of vaccinees. There was no significant difference in the incidence of adverse events between vaccinees and placebo-recipients other than rash. A trivalent or better response correlated with rash and with non-Black race (p<0.0001). Black race was significantly associated with a reduced incidence of vaccine viremia.Conclusions. TV003 induced a trivalent or greater antibody response in 90% of flavivirus-naïve vaccinees and is a promising candidate for the prevention of dengue. Race was identified as a factor influencing the infectivity of the LATV viruses, reflecting observations of the effect of race on disease severity in natural dengue infection.
    The Journal of Infectious Diseases 01/2013; · 5.85 Impact Factor
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    ABSTRACT: Dysregulated immune responses may contribute to the clinical complications that occur in some patients with dengue. In Vietnamese pediatric dengue cases randomized to early prednisolone therapy, 81 gene-transcripts (0.2% of the 47,231 evaluated) were differentially abundant in whole-blood between high-dose (2 mg/kg) prednisolone and placebo-treated patients two days after commencing therapy. Prominent among the 81 transcripts were those associated with T and NK cell cytolytic functions. Additionally, prednisolone therapy was not associated with changes in plasma cytokine levels. The inability of prednisolone treatment to markedly attenuate the host immune response is instructive for planning future therapeutic strategies for dengue.
    PLoS Neglected Tropical Diseases 01/2013; 7(12):e2592. · 4.57 Impact Factor
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    ABSTRACT: The genus Flavivirus includes both vector-borne and no known vector (NKV) species, but molecular determinants of transmission mode are not known. Conserved sequence differences between the two groups occur in 5' and 3' untranslated regions (UTRs). To investigate the impact of these differences on transmission, chimeric genomes were generated in which UTRs, UTRs + capsid, or upper 3' UTR stem loop of mosquito-borne dengue virus (DENV) were replaced with homologous regions from NKV Modoc virus (MODV); the conserved pentanucleotide sequence (CPS) was also deleted from the DENV genome. Virus was not recovered following transfection of these genomes in three different cell types. However, DENV genomes in which the CPS or variable region (VR) of the 3' UTR was replaced with MODV sequences were recovered and infected Aedes aegypti mosquitoes with similar efficiencies to DENV. These results demonstrate that neither vector-borne CPS nor VR is required for vector-borne transmission.
    Journal of General Virology 12/2012; · 3.13 Impact Factor
  • Anna P Durbin, Stephen S Whitehead
    The Journal of Infectious Diseases 12/2012; · 5.85 Impact Factor
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    ABSTRACT: Dengue is a systemic arthropod-borne viral disease of major global public health importance. At least 2.5 billion people who live in areas of the world where dengue occurs are at risk of developing dengue fever (DF) and its severe complications, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Repeated reemergences of dengue in sudden explosive epidemics often cause public alarm and seriously stress healthcare systems. The control of dengue is further challenged by the lack of effective therapies, vaccines, and point-of-care diagnostics. Despite years of study, even its pathogenic mechanisms are poorly understood. This article discusses recent advances in dengue research and identifies challenging gaps in research on dengue clinical evaluation, diagnostics, epidemiology, immunology, therapeutics, vaccinology/clinical trials research, vector biology, and vector ecology. Although dengue is a major global tropical pathogen, epidemiologic and disease control considerations in this article emphasize dengue in the Americas.
    The Journal of Infectious Diseases 07/2012; 206(7):1121-7. · 5.85 Impact Factor
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    ABSTRACT: The four dengue virus serotypes (DENV-1-DENV-4) have a large impact on global health, causing 50-100 million cases of dengue fever annually. Herein, we describe the first kinetic T cell response to a low-dose DENV-1 vaccination study (10 PFU) in humans. Using flow cytometry, we found that proinflammatory cytokines, IFNγ, TNFα, and IL-2, were generated by DENV-1-specific CD4(+) cells 21 days post-DENV-1 exposure, and their production continued through the latest time-point, day 42 (p<0.0001 for all cytokines). No statistically significant changes were observed at any time-points for IL-10 (p = 0.19), a regulatory cytokine, indicating that the response to DENV-1 was primarily proinflammatory in nature. We also observed little T cell cross-reactivity to the other 3 DENV serotypes. The percentage of multifunctional T cells (T cells making ≥ 2 cytokines simultaneously) increased with time post-DENV-1 exposure (p<0.0001). The presence of multifunctional T cells together with neutralizing antibody data suggest that the immune response generated to the vaccine may be protective. This work provides an initial framework for defining primary T cell responses to each DENV serotype and will enhance the evaluation of a tetravalent DENV vaccine.
    PLoS Neglected Tropical Diseases 07/2012; 6(7):e1742. · 4.57 Impact Factor
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    ABSTRACT: A vaccine to prevent dengue disease is urgently needed. Fortunately, a few tetravalent candidate vaccines are in the later stages of development and show promise. But, if the cost of these candidates is too high, their beneficial potential will not be realized. The price of a vaccine is one of the most important factors affecting its ultimate application in developing countries. In recent years, new vaccines such as those for human papilloma virus and pneumococcal disease (conjugate vaccine) have been introduced with prices in developed countries exceeding $50 per dose. These prices are above the level affordable by developing countries. In contrast, other vaccines such as those against Japanese encephalitis (SA14-14-2 strain vaccine) and meningitis type A have prices in developing countries below one dollar per dose, and it is expected that their introduction and use will proceed more rapidly. Because dengue disease is caused by four related viruses, vaccines must be able to protect against all four. Although there are several live attenuated dengue vaccine candidates under clinical evaluation, there remains uncertainty about the cost of production of these tetravalent vaccines, and this uncertainty is an impediment to rapid progress in planning for the introduction and distribution of dengue vaccines once they are licensed. We have undertaken a detailed economic analysis, using standard industrial methodologies and applying generally accepted accounting practices, of the cost of production of a live attenuated vaccine, originally developed at the US National Institutes of Health (National Institute of Allergy and Infectious Diseases), to be produced at the Instituto Butantan in Sao Paulo, Brazil. We determined direct costs of materials, direct costs of personnel and labor, indirect costs, and depreciation. These were analyzed assuming a steady-state production of 60 million doses per year. Although this study does not seek to compute the price of the final licensed vaccine, the cost of production estimate produced here leads to the conclusion that the vaccine can be made available at a price that most ministries of health in developing countries could afford. This conclusion provides strong encouragement for supporting the development of the vaccine so that, if it proves to be safe and effective, licensure can be achieved soon and the burden of dengue disease can be reduced.
    Vaccine 03/2012; 30(32):4892-6. · 3.77 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

Publication Stats

3k Citations
474.27 Total Impact Points

Institutions

  • 1996–2014
    • National Institute of Allergy and Infectious Diseases
      • Laboratory of Immunoregulation
      Maryland, United States
  • 2013
    • National Institute of Allergy and Infectious Disease
      Maryland, United States
  • 2008–2013
    • Johns Hopkins Bloomberg School of Public Health
      • Department of International Health
      Baltimore, Maryland, United States
  • 2012
    • International Vaccine Institute
      Sŏul, Seoul, South Korea
  • 1999–2012
    • National Institutes of Health
      • • Laboratory of Viral Diseases
      • • Laboratory of Infectious Diseases
      Bethesda, MD, United States
  • 2009–2010
    • Oxford University Clinical Research Unit
      Thành phố Hồ Chí Minh, Ho Chi Minh City, Vietnam
  • 2000–2008
    • Vanderbilt University
      • Department of Pediatrics
      Nashville, MI, United States
    • Friedrich Loeffler Institute
      • Institute of Molecular Biology
      Griefswald, Mecklenburg-Vorpommern, Germany
  • 2001
    • Walter Reed Army Institute of Research
      Silver Spring, Maryland, United States
    • Johns Hopkins University
      • Center for Immunization Research
      Baltimore, MD, United States