Ruklanthi de Alwis

University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

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Publications (7)43.89 Total impact

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    ABSTRACT: The four dengue virus (DENV) serotypes, DENV-1, -2, -3, and -4, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million acute infections annually. Infection confers long-term protective immunity against the infecting serotype, but secondary infection with a different serotype carries a greater risk of potentially fatal severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. The single most effective measure to control this threat to global health is a tetravalent DENV vaccine. To date, attempts to develop a protective vaccine have progressed slowly, partly because the targets of type-specific human neutralizing antibodies (NAbs), which are critical for long-term protection, remain poorly defined, impeding our understanding of natural immunity and hindering effective vaccine development. Here, we show that the envelope glycoprotein domain I/II hinge of DENV-3 and DENV-4 is the primary target of the long-term type-specific NAb response in humans. Transplantation of a DENV-4 hinge into a recombinant DENV-3 virus showed that the hinge determines the serotype-specific neutralizing potency of primary human and nonhuman primate DENV immune sera and that the hinge region both induces NAbs and is targeted by protective NAbs in rhesus macaques. These results suggest that the success of live dengue vaccines may depend on their ability to stimulate NAbs that target the envelope glycoprotein domain I/II hinge region. More broadly, this study shows that complex conformational antibody epitopes can be transplanted between live viruses, opening up similar possibilities for improving the breadth and specificity of vaccines for influenza, HIV, hepatitis C virus, and other clinically important viral pathogens.
    Proceedings of the National Academy of Sciences 01/2014; · 9.74 Impact Factor
  • Ruklanthi de Alwis, Aravinda M de Silva
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    ABSTRACT: Dengue virus (DENV) is an emerging virus that threatens over two-third of the world's population. The specific diagnosis of dengue infection by serology is based on assays that detect DENV-specific antibodies including neutralizing antibodies (Abs). Neutralizing Abs are an important, if not the main, mechanism of protection from natural dengue virus (DENV) infection as well. The current gold-standard assay for measuring neutralizing Ab responses against DENV is the plaque reduction neutralization assay (PRNT). However, this assay is slow and laborious and utilizes physiologically irrelevant cell lines. Here, we describe a relatively high-throughput, flow cytometry-based neutralization assay for DENV that has been optimized for use with a human monocytic suspension cell line, U937 + DC-SIGN, or the more commonly used adherent monkey kidney cells, Vero-81.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1138:27-39. · 1.29 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: Dengue is a mosquito-borne flavivirus that is spreading at an unprecedented rate and has developed into a major health and economic burden in over 50 countries. Even though infected individuals develop potent and long-lasting serotype-specific neutralizing antibodies (Abs), the epitopes engaged by human neutralizing Abs have not been identified. Here, we demonstrate that the dengue virus (DENV)-specific serum Ab response in humans consists of a large fraction of cross-reactive, poorly neutralizing Abs and a small fraction of serotype-specific, potently inhibitory Abs. Although many mouse-generated, strongly neutralizing monoclonal antibodies (mAbs) recognize epitopes that are present on recombinant DENV envelope (E) proteins, unexpectedly, the majority of neutralizing Abs in human immune sera bound to intact virions but not to the ectodomain of purified soluble E proteins. These conclusions with polyclonal Abs were confirmed with newly generated human mAbs derived from DENV-immune individuals. Two of three strongly neutralizing human mAbs bound to E protein epitopes that were preserved on the virion but not on recombinant E (rE) protein. We propose that humans produce Abs that neutralize DENV infection by binding a complex, quaternary structure epitope that is expressed only when E proteins are assembled on a virus particle. Mapping studies indicate that this epitope has a footprint that spans adjacent E protein dimers and includes residues at the hinge between domains I and II of E protein. These results have significant implications for the DENV Ab and vaccine field.
    Proceedings of the National Academy of Sciences 04/2012; 109(19):7439-44. · 9.74 Impact Factor
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    ABSTRACT: [This corrects the article on p. e1188 in vol. 5.].
    PLoS Neglected Tropical Diseases 08/2011; 5(8). · 4.57 Impact Factor
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    ABSTRACT: Humans who experience a primary dengue virus (DENV) infection develop antibodies that preferentially neutralize the homologous serotype responsible for infection. Affected individuals also generate cross-reactive antibodies against heterologous DENV serotypes, which are non-neutralizing. Dengue cross-reactive, non-neutralizing antibodies can enhance infection of Fc receptor bearing cells and, potentially, exacerbate disease. The actual binding sites of human antibody on the DENV particle are not well defined. We characterized the specificity and neutralization potency of polyclonal serum antibodies and memory B-cell derived monoclonal antibodies (hMAbs) from 2 individuals exposed to primary DENV infections. Most DENV-specific hMAbs were serotype cross-reactive and weakly neutralizing. Moreover, many hMAbs bound to the viral pre-membrane protein and other sites on the virus that were not preserved when the viral envelope protein was produced as a soluble, recombinant antigen (rE protein). Nonetheless, by modifying the screening procedure to detect rare antibodies that bound to rE, we were able to isolate and map human antibodies that strongly neutralized the homologous serotype of DENV. Our MAbs results indicate that, in these two individuals exposed to primary DENV infections, a small fraction of the total antibody response was responsible for virus neutralization.
    PLoS Neglected Tropical Diseases 06/2011; 5(6):e1188. · 4.57 Impact Factor
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    ABSTRACT: Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge "type specific" epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.
    PLoS Pathogens 01/2010; 6(3):e1000821. · 8.14 Impact Factor

Publication Stats

132 Citations
43.89 Total Impact Points

Institutions

  • 2010–2012
    • University of North Carolina at Chapel Hill
      • Department of Microbiology and Immunology
      Chapel Hill, NC, United States