A potent anti‐dengue human antibody preferentially recognizes the conformation of E protein monomers assembled on the virus surface
2014; Springer Nature; Volume: 6; Issue: 3 Linguagem: Inglês
10.1002/emmm.201303404
ISSN1757-4684
AutoresG. Fibriansah, Joanne L. Tan, Scott A. Smith, A. Ruklanthi de Alwis, Thiam‐Seng Ng, V.A. Kostyuchenko, Kristie D. Ibarra, Jiaqi Wang, Eva Harris, Aravinda de Silva, James E. Crowe, Shee‐Mei Lok,
Tópico(s)Viral Infections and Outbreaks Research
ResumoResearch Article14 January 2014Open Access A potent anti-dengue human antibody preferentially recognizes the conformation of E protein monomers assembled on the virus surface Guntur Fibriansah Guntur Fibriansah Program in Emerging Infectious Diseases, Duke–NUS Graduate Medical School, Singapore City, Singapore Centre for BioImaging Sciences, National University of Singapore, Singapore City, Singapore Search for more papers by this author Joanne L Tan Joanne L Tan Program in Emerging Infectious Diseases, Duke–NUS Graduate Medical School, Singapore City, Singapore Centre for BioImaging Sciences, National University of Singapore, Singapore City, Singapore Search for more papers by this author Scott A Smith Scott A Smith Department of Medicine, Vanderbilt University, Nashville, TN, USA The Vanderbilt Vaccine Center, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Adamberage R de Alwis Adamberage R de Alwis Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC, USA Search for more papers by this author Thiam-Seng Ng Thiam-Seng Ng Program in Emerging Infectious Diseases, Duke–NUS Graduate Medical School, Singapore City, Singapore Centre for BioImaging Sciences, National University of Singapore, Singapore City, Singapore Search for more papers by this author Victor A Kostyuchenko Victor A Kostyuchenko Program in Emerging Infectious Diseases, Duke–NUS Graduate Medical School, Singapore City, Singapore Centre for BioImaging Sciences, National University of Singapore, Singapore City, Singapore Search for more papers by this author Kristie D Ibarra Kristie D Ibarra Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, CA, USA Search for more papers by this author Jiaqi Wang Jiaqi Wang Program in Emerging Infectious Diseases, Duke–NUS Graduate Medical School, Singapore City, Singapore Centre for BioImaging Sciences, National University of Singapore, Singapore City, Singapore Search for more papers by this author Eva Harris Eva Harris Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, CA, USA Search for more papers by this author Aravinda de Silva Aravinda de Silva Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC, USA Search for more papers by this author James E Crowe Jr Corresponding Author James E Crowe Jr The Vanderbilt Vaccine Center, Vanderbilt University, Nashville, TN, USA Departments of Pediatrics and Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Shee-Mei Lok Corresponding Author Shee-Mei Lok Program in Emerging Infectious Diseases, Duke–NUS Graduate Medical School, Singapore City, Singapore Centre for BioImaging Sciences, National University of Singapore, Singapore City, Singapore Search for more papers by this author Guntur Fibriansah Guntur Fibriansah Program in Emerging Infectious Diseases, Duke–NUS Graduate Medical School, Singapore City, Singapore Centre for BioImaging Sciences, National University of Singapore, Singapore City, Singapore Search for more papers by this author Joanne L Tan Joanne L Tan Program in Emerging Infectious Diseases, Duke–NUS Graduate Medical School, Singapore City, Singapore Centre for BioImaging Sciences, National University of Singapore, Singapore City, Singapore Search for more papers by this author Scott A Smith Scott A Smith Department of Medicine, Vanderbilt University, Nashville, TN, USA The Vanderbilt Vaccine Center, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Adamberage R de Alwis Adamberage R de Alwis Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC, USA Search for more papers by this author Thiam-Seng Ng Thiam-Seng Ng Program in Emerging Infectious Diseases, Duke–NUS Graduate Medical School, Singapore City, Singapore Centre for BioImaging Sciences, National University of Singapore, Singapore City, Singapore Search for more papers by this author Victor A Kostyuchenko Victor A Kostyuchenko Program in Emerging Infectious Diseases, Duke–NUS Graduate Medical School, Singapore City, Singapore Centre for BioImaging Sciences, National University of Singapore, Singapore City, Singapore Search for more papers by this author Kristie D Ibarra Kristie D Ibarra Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, CA, USA Search for more papers by this author Jiaqi Wang Jiaqi Wang Program in Emerging Infectious Diseases, Duke–NUS Graduate Medical School, Singapore City, Singapore Centre for BioImaging Sciences, National University of Singapore, Singapore City, Singapore Search for more papers by this author Eva Harris Eva Harris Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, CA, USA Search for more papers by this author Aravinda de Silva Aravinda de Silva Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC, USA Search for more papers by this author James E Crowe Jr Corresponding Author James E Crowe Jr The Vanderbilt Vaccine Center, Vanderbilt University, Nashville, TN, USA Departments of Pediatrics and Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Shee-Mei Lok Corresponding Author Shee-Mei Lok Program in Emerging Infectious Diseases, Duke–NUS Graduate Medical School, Singapore City, Singapore Centre for BioImaging Sciences, National University of Singapore, Singapore City, Singapore Search for more papers by this author Author Information Guntur Fibriansah1,2,‡, Joanne L Tan1,2,‡, Scott A Smith3,4, Adamberage R Alwis5, Thiam-Seng Ng1,2, Victor A Kostyuchenko1,2, Kristie D Ibarra6, Jiaqi Wang1,2, Eva Harris6, Aravinda Silva5, James E Crowe 4,7 and Shee-Mei Lok 1,2 1Program in Emerging Infectious Diseases, Duke–NUS Graduate Medical School, Singapore City, Singapore 2Centre for BioImaging Sciences, National University of Singapore, Singapore City, Singapore 3Department of Medicine, Vanderbilt University, Nashville, TN, USA 4The Vanderbilt Vaccine Center, Vanderbilt University, Nashville, TN, USA 5Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC, USA 6Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, CA, USA 7Departments of Pediatrics and Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, USA ‡These authors contributed equally. *Corresponding author. Tel: +1 615 3438064; Fax: +1 615 3434456; E-mail: [email protected] *Corresponding author. Tel: +65 65165840; Fax: +65 62212529; E-mail: [email protected] EMBO Mol Med (2014)6:358-371https://doi.org/10.1002/emmm.201303404 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Dengue virus (DENV), which consists of four serotypes (DENV1-4), infects over 400 million people annually. Previous studies have indicated most human monoclonal antibodies (HMAbs) from dengue patients are cross-reactive and poorly neutralizing. Rare neutralizing HMAbs are usually serotype-specific and bind to quaternary structure-dependent epitopes. We determined the structure of DENV1 complexed with Fab fragments of a highly potent HMAb 1F4 to 6 Å resolution by cryo-EM. Although HMAb 1F4 appeared to bind to virus and not E proteins in ELISAs in the previous study, our structure showed that the epitope is located within an envelope (E) protein monomer, and not across neighboring E proteins. The Fab molecules bind to domain I (DI), and DI-DII hinge of the E protein. We also showed that HMAb 1F4 can neutralize DENV at different stages of viral entry in a cell type and receptor dependent manner. The structure reveals the mechanism by which this potent and specific antibody blocks viral infection. Synopsis Vaccine development against Dengue disease is complicated by the virus serotype priming to secondary infections. Structure of a potent human antibody 1F4 complexed with DENV serotype 1 highlights the hinge angle of the viral protein as critical for vaccine design. DI-DII hinge is a common epitope recognised by highly neutralising human antibodies. The conformation angle of the hinge is critical for 1F4 binding. 1F4 recognises E protein monomers on the virus surface but not recombinant E proteins due to the conserved hinge angle of endogenous viral E proteins. Introduction Dengue virus (DENV), a member of the family Flaviviridae, is transmitted to humans by Aedes mosquitoes. Other flaviviruses that are important human pathogens include West Nile virus (WNV), yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus. DENV targets susceptible populations residing in tropical and sub-tropical regions of the globe. An estimated 400 million people worldwide are infected with DENV annually, leading to approximately 100 million cases of dengue and 21 000 deaths (Thomas & Endy, 2011; Bhatt et al, 2013). DENV consists of four distinct serotypes (DENV1–4), and the amino acid sequence variation of the polyprotein between serotypes is about 25–40% (Holmes & Twiddy, 2003; Vasilakis & Weaver, 2008). People infected with DENV can be asymptomatic or develop symptoms that range from a mild fever to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). A dengue-naïve individual exposed to a primary infection develops long-lasting protective immunity only to the infecting serotype (Imrie et al, 2007). A second infection with a new serotype increases the risk of developing DHF/DSS. The presence of cross-reactive but weakly neutralizing antibodies (Abs) induced following the primary infection have been hypothesized to be a cause of DHF or DSS through a mechanism known as Ab-dependent enhancement (Halstead, 2003). Experts in the field reason that a safe and effective vaccine against DENV will likely need to be tetravalent (Raviprakash et al, 2008), since the induction of Abs that neutralize a single serotype by monovalent vaccines may predispose individuals to Ab-enhanced disease. DENV is an enveloped virus, in which the nucleocapsid core is surrounded by a lipid bilayer membrane. On the lipid envelope, there are 180 copies each of membrane (M) and envelope (E) proteins (Kuhn et al, 2002; Zhang et al, 2013a). These M and E proteins are arranged with icosahedral symmetry with each asymmetric unit consisting of three pairs of M and E proteins. The three individual E proteins in an asymmetric unit each have a different local chemical environment. The 180 copies of E protein are arranged into 90 head-to-tail homodimers. Three of these homodimers lie parallel to each other forming a raft. Together, the 30 E protein rafts on the DENV surface form a herringbone pattern (Kuhn et al, 2002; Zhang et al, 2013a). Crystal structures of the ectodomain part of the E protein showed that it consists of three distinct domains, designated DI, DII and DIII. E protein also likely exists as a head-to-tail oriented homodimer in solution (Modis et al, 2003, 2005; Zhang et al, 2004). This molecule is critical for viral entry into cells, as it mediates binding to cellular receptors and also fusion between the virus and endosomal membranes. In addition, E protein is the major target for neutralizing Abs (Roehrig, 2003). Studies with mouse monoclonal Abs (MAbs) showed that the most potent neutralizing Abs are serotype-specific and bind to DIII (Gromowski & Barrett, 2007; Sukupolvi-Petty et al, 2007; Shrestha et al, 2010). In contrast, a large fraction of potent neutralizing anti-DENV Abs produced in humans does not appear to bind to DIII (Wahala et al, 2009, 2012; Costin et al, 2013). These human MAbs (HMAbs) bind only to whole virus particles but not to recombinant E (rE) protein, suggesting that they recognize quaternary structure-dependent epitopes (Beltramello et al, 2010; De Alwis et al, 2012; Teoh et al, 2012). One such HMAb is 1F4, a DENV1-specific Abs (De Alwis et al, 2012). Here, we have solved the cryo-electron microscopy (cryo-EM) structure of Fab 1F4 complexed with DENV1 to 6 Å resolution. Surprisingly, unlike other HMAbs recognizing quaternary structure-dependent epitopes (Kaufmann et al, 2010; Teoh et al, 2012), 1F4 does not bind across neighboring E proteins. Results HMAb 1F4 exhibits potent neutralizing activity in vitro and in vivo, and it inhibits virus infection at different stages of viral entry depending on the cell type and receptor A neutralization assay conducted with HMAb 1F4 and DENV1 in human monocytic U937 cells expressing dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) demonstrated that it is a highly potent Ab with a Neut50 value of 0.03 μg/ml (Fig 1A). In addition, the Fab fragment of HMAb 1F4 also neutralized the virus, although the potency of the Fab was reduced by approximately 4-fold (Neut50 value of 0.11 μg/ml) compared with full-length IgG (Fig 1B). Figure 1. The mechanism of HMAb 1F4 neutralization of DENV1 (Western Pacific 74 strain) in Vero or DC-SIGN expressing U937 cell lines A, B. Both HMAb 1F4 IgG (A) and its Fab fragment (B) neutralized DENV1 infection in U937 cells expressing DC-SIGN although the Fab fragment required a 4-fold higher concentration. C. HMAb 1F4 inhibited a post-attachment step of virus infection in Vero cells. HMAb 1F4 had similar neutralization activities when exposed to virus pre- (FRNT50 = 0.41 μg/ml) or post-attachment (FRNT50 = 0.37 μg/ml) to Vero cells. The two experiments were done with two replicates. Error bars represent standard deviations. Pre and post-attachment neutralization curves are not significantly different by a 2-way repeated measures analysis of variance (RM ANOVA). D. HMAb 1F4 prevented virus infection of DC-SIGN-expressing U937 cells by blocking both virus attachment and also a post-attachment step. Error bar represented standard deviations. Neutralization curves of pre and post-attachment groups are significantly different by 2-way RM ANOVA, with a P < 0.001. Download figure Download PowerPoint In mice that had been given 20 μg of HMAb 1F4 prior to infection with a sub-lethal dose of DENV1, a significant reduction in the viral genomic RNA copy number as compared to the isotype control was observed in serum and bone marrow (Fig 2A and B). Figure 2. Prophylactic efficacy of HMAb 1F4 in DENV1-inoculated AG129 mice A, B. Mice were administered 20 μg of HMAb 1F4 or 50 μg IgG1 isotype control 24 h prior to a sub-lethal dose of 5 × 106 pfu of DENV1 Western Pacific 74. Serum viremia (A) and bone marrow viral load (B) were determined 3 days post-infection by qRT-PCR. One representative experiment of two is shown, with n = 4 mice per group. DENV1 limit of detection is indicated by the dashed line. *P = 0.0209 compared to IgG1 control, as determined using a 2-tailed Wilcoxon Rank Sum test. Download figure Download PowerPoint To test whether HMAb 1F4 inhibits receptor binding in Vero and DC-SIGN-expressing U937 cells, we compared the neutralization profile of HMAb 1F4 to DENV before and after the virus was allowed to attach to cells. In Vero cells, HMAb 1F4 was just as effective in neutralizing virus when the Ab was added to virus prior to exposure to cells (FRNT50 of 0.41 μg/ml) as when added after attachment to cells (FRNT50 of 0.37 μg/ml) (Fig 1C). This suggests that HMAb 1F4 neutralizes virus infection in Vero cells by inhibiting a step after virus attachment to cells. In experiments where the virus was exposed to HMAb 1F4 after attachment to cells, DC-SIGN-expressing U937 cells showed a similar neutralization profile as Vero cells (Neut50 of 0.40 μg/ml) (Fig 1D). However, when virus was exposed to Ab before attachment to DC-SIGN-expressing cells, the neutralizing concentration was approximately 6 fold lower (Neut50 of 0.07 μg/ml) (Fig 1D). These data suggest that the Ab can prevent virus from attaching to DC-SIGN-expressing cells in addition to neutralizing the virus after attachment to the cells. Cryo-EM structure of DENV-1 complexed with Fab 1F4 For the cryoEM reconstruction of the Fab 1F4-DENV1 complex, the DENV1 primary isolate strain PVP159 was used, and neutralization tests showed that the Ab neutralized this virus strain with high potency (supplementary Fig 1). A micrograph of an untreated control DENV1 (PVP159) (supplementary Fig 2), which was grown at 28°C in C6/36 cells and kept at 4°C, showed that the sample consisted of mainly smooth mature virus particles, with about 15% spiky immature particles and a few broken particles. DENV2 particles have been shown to exhibit a change from a smooth to a bumpy surface when incubated at 37°C for 30 min (Fibriansah et al, 2013; Zhang et al, 2013b). Incubation of DENV1 (PVP159) at 37°C for 30 min, on the other hand, did not show any increase in the number of bumpy particles compared to the sample that was never exposed to 37°C (supplementary Fig 2). This indicated that the DENV1 strain PVP159 did not undergo similar structural changes when incubated at 37°C for 30 min as had been observed in DENV2. However, we cannot eliminate the possibility of small local domain movements of the E proteins on DENV1 surface. The expansion of the virus structure as observed in DENV2, does not seem to enhance infectivity in mammalian cells. Fibriansah et al (2013) showed that DENV2 titres were similar at both 37 and 28°C. This implied that both structural forms are equally infectious to mammalian cells. This indicates there may not be a strong selection pressure for the virus to adopt the expanded structure. Cryo-EM reconstruction of Fab 1F4 complexed with DENV1 strain PVP159 when incubated at 4 or 37°C resulted in similar maps. Hence, further structural analysis was done using the complex formed at 4°C as the viral components and Fab 1F4 were likely to be less mobile, thus allowing us to achieve higher resolution. The E protein shell of the cryo-EM map of the Fab-virus complex was solved to 6 Å resolution (Fig 3A–D). At this resolution, we were able to observe densities of the helical ridges (Fig 3C, left) of the E protein transmembrane region. On the other hand, the densities corresponding to the Fab molecules are poorer in resolution (Fig 3D). Resolutions of the Fab variable and constant regions were about 7.7 and 12 Å, respectively. The difference in resolution between the Fab variable and constant regions suggests high flexibility of the elbow angle between these domains (Fig 3B and D). Figure 3. The cryo-EM structure of Fab 1F4 complexed with DENV1 Cryo-EM map of Fab 1F4 complexed with DENV1 showed 120 copies of Fab (blue) bound to the virus surface (cyan). White triangle indicates an icosahedral asymmetric unit and the numbers represents the vertices. Cross-section of a quarter of a cryo-EM map. The resolution of the cryoEM map is 6 Å. Regions of the density map corresponding to trans-membrane α-helices (left) and β-strands (right). The density map corresponding to Fab 1F4. The density of the constant region (indicated by arrow) is much poorer than the variable region indicating the constant region is flexible. Download figure Download PowerPoint Fitting of E protein and Fab molecules into the cryo-EM density map showed that the Fab molecules bind in an identical way to two of the three individual E proteins (molecules A and B) in an asymmetric unit (Fig 4A and B). Since the resolution of the map did not permit observation of side chain densities, interacting residues between Fab and E protein were identified by observing pairs of Cα atoms of less than approximately 8 Å in distance. The possibility of hydrogen bonding and hydrophobic interactions between the side chains of these residues was also taken into consideration. The footprint of the Fab 1F4 molecule on an E protein is approximately 1340 Å2, which is bigger than that of a typical Ab footprint on antigen (900–1000 Å2) (Davies et al, 1990; Lok et al, 2008; Austin et al, 2012), but smaller than that of MAb E16 on WNV E protein (1550 Å2) (Nybakken et al, 2005). One Fab 1F4 molecule binds to an E protein monomer and not across E proteins. The majority of the footprint of Fab 1F4 is on E protein DI, with some interactions with the DI-DII hinge region (Fig 4B). The epitope, consisting of 26 amino acids, is located on the Do strand and the downstream Doa loop (46–52), Eo strand (136–138), part of the EoFo loop, Fo strand (155–165), Go strand, the following GoHo loop (170–177), and kl loop (272–276) (Fig 4C). Comparison of the epitope residues between DENV serotypes (Fig 4C) revealed significant variation, which likely explains the serotype specificity of HMAb 1F4. A total of 27 interacting residues are located on the complementarity determining regions (CDRs) of the Fab molecule: 14 residues from the light chain and 13 residues from the heavy chain (Fig 5A and B). Also, two other non-CDR related interactions were observed. The side of the Fab molecule interacted with the N153 glycosylation site on the same E protein molecule (Fig 6, left) and also the N67 glycosylation site of a neighboring dimer-related E protein (Fig 6, right). Figure 4. The epitope bound by Fab 1F4 Densities of Fab 1F4 on E proteins in two icosahedral asymmetric units. Fab 1F4 molecules bind to molecules A and B in each asymmetric unit. Open-book representation of Fab 1F4 binding to E protein. The E protein DI, DII and DIII are colored in red, yellow and blue (top), respectively, whereas the Fab 1F4 heavy and light chains are colored in green and cyan (bottom), respectively. The epitope bound by Fab 1F4 is located on the DI and DI-II hinge regions (top). The epitope on E protein bound by the heavy and light chain are colored in green and cyan, respectively (top). Also the epitope on DI that interacted with both heavy and light chains are colored in purple (top). The paratope on heavy and light chain (bottom) are colored in its corresponding interacting residues on the DI (red) and DII (yellow) E protein, paratope binding to both DI and II on the E protein is colored in pink. Ribbon representation of the HMAb 1F4 epitope on E protein (top) and also sequence alignment of the epitope region between all dengue virus serotypes (bottom). The β-strand and loop of the epitope are colored in orange and black, respectively (top). In the bottom panel, the secondary structures are shown above the amino acid sequence. The amino acid residues that interact with Fab 1F4 are highlighted in the same coloring scheme as in (B). The amino acid sequences are derived from DENV1 strain PVP159, DENV2 strain S16803, DENV3 strain Thailand 1995 and DENV4 strain Dominica 1981. Download figure Download PowerPoint Figure 5. Interaction interface between Fab 1F4 and DENV1 E protein Open-book representation showing the electrostatic potential of the interaction interface on the E protein (left) and the Fab 1F4 (right). The blue and red colors indicate positive and negative charges, respectively, whereas the white color shows neutral charge. The dotted lines indicate the border of the footprint between heavy and light chains on the E protein epitope (left) and also the corresponding border on the antibody paratope (right). Residues on E protein identified in the neutralization escape mutants (K47 and G274) are indicated, together with other residues that have opposite charges in other serotypes. The corresponding interacting residues on the Fab 1F4 light and heavy chains are indicated with cyan and green colored fonts, respectively. Table of the list of putative interactions between DENV1 and Fab 1F4. Download figure Download PowerPoint Figure 6. Fab 1F4 also has non-CDR related interactions with glycan chains on N153 on the same E protein and the N67 of a neighboring E proteinLeft and right panels show different views of these interactions.The heavy and light chains of Fab 1F4 are colored in green and cyan, respectively, whereas the E protein molecules A and C' are in beige and gray, respectively (see Fig 4A for reference). The oxygen and nitrogen atoms of the glycan chains are colored in red and blue, respectively. Download figure Download PowerPoint Fab 1F4 is unable to bind to the E proteins near the 3-fold vertices (molecule C) (Fig 4A). Superposition of the three individual E protein molecules did not show significant structural differences, suggesting that the lack of binding of Fab to molecule C may be due to steric hindrance caused by the neighboring E proteins (Fig 7A). Comparison of the accessibility of the epitopes on the three E proteins in an asymmetric unit showed that the epitopes on molecules A and B are completely exposed (Fig 7B and C), while the epitope on C molecule (near to 3-fold vertices) is partially covered by DIII of a neighboring E protein (Fig 7D). Figure 7. Analysis of the accessibility of the epitope on all three individual E protein molecules in an asymmetric unit A. Superposition of the three individual E protein molecules in an asymmetric unit (molecules A–C). The epitope location is indicated by a rectangular box. Molecules A, B and C in each asymmetric unit are colored in yellow, red and gray, respectively. The E proteins have similar conformation indicating that the lack of Fab binding to molecule C is mainly due to the blockage of the epitope by a neighboring E protein molecule. B–D. Molecules A, B, and C in each asymmetric unit are colored in yellow, red and gray, respectively. The epitope is colored in the same coloring scheme as in Fig 4B. The epitopes on molecules A (B) and B (C) (circled) are fully accessible. (D) In contrast, the part of the epitope on molecule C is blocked by DIII of a neighboring E protein (indicated by arrow). For clarity, one of the E proteins (left) is represented as a transparent surface to show the hidden part of an adjacent epitope (marked by*). Download figure Download PowerPoint Discussion HMAb 1F4 is a strongly neutralizing Ab against DENV1, as shown in vitro (Fig 1) and in the AG129 mouse model (Fig 2). As observed previously (Beltramello et al, 2010; De Alwis et al, 2012), potent HMAbs usually bind to quaternary structure-dependent epitopes, as determined by the ability of the Ab to bind to whole virus particles and not rE protein in ELISAs. HMAb 1F4 is unique compared to the previously published HMAbs (Kaufmann et al, 2010; Teoh et al, 2012) that bind to quaternary structure-dependent epitopes, as HMAb 1F4 does not bind across the interface between separate E proteins. Instead, it recognizes E protein monomers that adopt a conformation that only exists when the protein is displayed on virus particles. The cryo-EM structure of another strongly neutralizing DENV1-specific HMAb, 14c10, in complex with DENV1 had been solved previously (Teoh et al, 2012). Comparison of the HMAb 1F4 and 14c10 epitopes showed that HMAb 1F4 binds to an E protein monomer whereas 14c10 binds across two E proteins (Fig 8). Both HMAbs 1F4 and 14c10 bind mainly to DI and the hinge between DI and DII. In addition, HMAb 14c10 also binds to the DIII of a neighboring E protein. There is another report of the crystal structure of a chimpanzee Fab 5H2 complexed with DENV4 E protein (Cockburn et al, 2012). The MAb 5H2 is a DENV4 specific antibody that binds to DI (Lai et al, 2007). The complex structure showed that the MAb 5H2 epitope is located on β-strands Fo, Go,Ho and the loops between them as well as the loop downstream of β-strand Io (DI-DIII linker). Similar interactions between Fab and amino residues on β-strands Fo and Go and the loop FoGo were also found in the complex structure of Fab 14c10 (Teoh et al, 2012) and 1F4 (Fig 4 and 8), but interactions with DI-II hinge region was not observed in the Fab 5H2 complex structure. Since the Fab 5H2 complex structure is a crystal structure, its ability to bind across E proteins and also the level of occupancy on the DENV particle surface are unknown. However, analysis of the epitopes on all three E protein molecules in an asymmetric unit suggests that the epitope would be occluded in the E protein near the three-fold vertices (molecule C) (Fig 8). The observation that the epitopes bound by HMAbs 14c10 and 1F4 overlap at the DI-DII hinge and it is not recognized by chimpanzee MAb 5H2 suggests that this region could be important in eliciting type-specific neutralizing antibody responses in humans. Figure 8. Comparison of epitopes bound by HMAb 1F4 (left), HMAb14c10 (middle) and 5H2 (right)The epitopes are colored in green. Download figure Download PowerPoint Although Fab 1F4 binds to E protein monomers on the virus, it was reported that HMAb 1F4 only binds to intact virus but not to rE protein (De Alwis et al, 2012). This indicates that Ab binding may requires a certain E protein conformation that is only present when the E protein is assembled on the virus. Part of the Fab 1F4 epitope is located on the kl loop (273–276) in the DI-II hinge (Fig 9A). Notably, residue 274 in the kl loop was reported to be important for HMAb 1F4 binding, since one escape mutant virus containing a substitution at this position (G274E) completely abolished Ab binding (De Alwis et al, 2012). When DI of the crystal structures of the DENV2 and DENV3
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