Type- and Subcomplex-Specific Neutralizing Antibodies against Domain III of Dengue Virus Type 2 Envelope Protein Recognize Adjacent Epitopes

Department of Medicine, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110, USA.
Journal of Virology (Impact Factor: 4.44). 01/2008; 81(23):12816-26. DOI: 10.1128/JVI.00432-07
Source: PubMed


Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals. Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials.

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Available from: S. Kyle Austin, Sep 02, 2015
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    • "Structural studies of intact virions have shown that EDIII is exposed and accessible on the virion surface [8]. This domain, which is responsible for receptor binding, contains multiple type and subtype specific neutralizing epitopes [3] [9] [10]. Furthermore, recombinant EDIII protein [11,12] and EDIII-specific mAbs [13] can block virus infectivity. "
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    ABSTRACT: Dengue is a major international public health concern. There is no drug to treat dengue virus infections and a vaccine is yet to be licensed. The laboratory diagnosis of dengue virus infection has been greatly improved during the last decade; therefore, the main limiting factor is the production of recombinant viral antigens on a large scale. Domain III of dengue virus envelope protein contains multiplex conformation-dependent neutralizing epitopes, making it an attractive diagnostic candidate. In this work, we have demonstrated the expression of dengue virus type 1 envelope domain III protein (EDIII-D1) in methylotrophic yeast, Pichia pastoris GS115. The recombinant secreted protein (sEDIII-D1) was purified by affinity chromatography and characterized by SDS-PAGE. Purified protein was recognized in immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) with dengue-infected human serum samples. In conclusion, secreted expressions of domain III protein can be obtained in P. pastoris by methanol induction. This product has the potential to be used for the diagnosis of dengue infections.
    Protein Expression and Purification 08/2013; 92(1). DOI:10.1016/j.pep.2013.08.014 · 1.70 Impact Factor
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    • "The envelope (E) protein, the major surface protein on the virion, contains three distinct domains: I (EDI), II (EDII) and III (EDIII) (Modis et al., 2003). EDIII is exposed on the surface of the viral particle and contains DENV serotype-specific, subcomplex-specific and complex-specific epitopes as determined previously (Falconar, 1999, 2008; Gromowski & Barrett, 2007; Gromowski et al., 2008; Lisova et al., 2007; Matsui et al., 2010; Matsui et al., 2009; Pitcher et al., 2012; Roehrig et al., 1998; Sukupolvi-Petty et al., 2007, 2010). The majority of neutralizing mAbs against EDIII of DENV were raised through immunization of mice with DENV-1 or DENV-2 strains and localized to A-strand and/or lateral-ridge epitopes. "
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    ABSTRACT: Dengue virus (DENV) is a mosquito-borne virus that causes severe health burden. An effective tetravalent dengue vaccine candidate that can provide life-long protection simultaneously against all four DENV serotypes is highly anticipated. A better understanding of the antibody response to DENV envelope protein domain III (EDIII) may offer insights into vaccine development. Herein, we identified 25 DENV cross-reactive monoclonal antibodies (mAbs) from immunization with Pichia pastoris-expressed EDIII of a single or all four serotype(s) using prime/boost protocol, and through pepscan analysis found that 60% of them (15/25) specifically recognized the same highly conserved linear epitope 309-320 of EDIII. All 15 complex-reactive mAbs exhibited significant cross-reactivity with recombinant EDIII from all DENV serotypes and also with C6/36 cells infected with DENV-1, -2, -3, and -4. However, neutralization assay indicated that the majority of these 15 mAbs were either moderately or weakly neutralizing. Through further epitope mapping by yeast surface display, two residues in the AB loop, Q316 and H317, were discovered to be critical. Three-dimensional modeling analysis suggests that this epitope is surface-exposed on EDIII but less accessible on the surface of the E protein dimer and trimer, especially on the surface of the mature virion. It is concluded that EDIII as immunogen may elicit cross-reactive mAbs toward epitope that is not exposed on the virion surface, therefore contributing inefficiently to their neutralization potency. Therefore, prime-boost strategy of EDIII from a single serotype or four serotypes mainly elicited poorly neutralizing cross-reactive antibody response to conserved AB loop of EDIII.
    Journal of General Virology 07/2013; 94(Pt_10). DOI:10.1099/vir.0.055178-0 · 3.18 Impact Factor
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    • "MAb 3H5 binds to the DENV2 conserved FG loop AAs E383 and P384 as well as semi-conserved G304 and K305 on the neighboring A beta strand (Gromowski and Barrett, 2007; Gromowski et al., 2008; Roehrig et al., 1998; Sukupolvi-Petty et al., 2007). Binding of subcomplex-specific MAb 1A1D-2, which targets AAs on the A strand (K305/307/310), was not affected by mutation of the FG loop, demonstrating that the antigenic structure of the A strand was still intact (Lok et al., 2008; Roehrig et al., 1998; Sukupolvi-Petty et al., 2007). Evidence that a peptide containing the DENV2 FG loop sequence did not reduce binding of DENV2 DIII to mammalian cells (Hung et al., 2004) suggests that a common attachment site apart from the FG loop in DIII has a role in binding to vertebrate cells. "
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