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. 2017 May 23:8:15411.
doi: 10.1038/ncomms15411.

Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope

Affiliations

Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope

Alexander Rouvinski et al. Nat Commun. .

Abstract

A problem in the search for an efficient vaccine against dengue virus is the immunodominance of the fusion loop epitope (FLE), a segment of the envelope protein E that is buried at the interface of the E dimers coating mature viral particles. Anti-FLE antibodies are broadly cross-reactive but poorly neutralizing, displaying a strong infection enhancing potential. FLE exposure takes place via dynamic 'breathing' of E dimers at the virion surface. In contrast, antibodies targeting the E dimer epitope (EDE), readily exposed at the E dimer interface over the region of the conserved fusion loop, are very potent and broadly neutralizing. We here engineer E dimers locked by inter-subunit disulfide bonds, and show by X-ray crystallography and by binding to a panel of human antibodies that these engineered dimers do not expose the FLE, while retaining the EDE exposure. These locked dimers are strong immunogen candidates for a next-generation vaccine.

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Conflict of interest statement

The EDE antibodies, EDE epitope and envelope protein dimers that induce EDE antibodies are the subject of a patent application by Imperial College and Institute Pasteur on which G.S., J.M., F.A.R., A.R., G.B.-S., P.G.-C., M.-C.V. and S.D. are named as inventors. The remaining authors declare no competing financial interests.

Figures

Figure 1
Figure 1. EDE mAbs can stabilize DENV envelope dimers.
(a) Binding of anti-FLE and anti-EDE mAbs to monomeric sE protein was determined by direct ELISA with sE coated to the ELISA plate. Results are expressed as mean of binding in arbitrary units (AU) from three independent experiments. The mAbs used in b are indicated by dots of the same colour. (b) The ability of selected mAbs to bind and assemble dimers was assessed by indirect ELISA. ELISA plates were coated with four anti-EDE mAbs and one anti-FLE mAb control, which binds monomeric sE. Plates were then incubated with a titration of soluble Strep-tagged sE monomer, bound sE was revealed using ALP-labelled StrepTactin. The data are shown as mean±s.e.m. from three independent experiments. (c) SEC/MALS analysis of isolated DENV2 sE, isolated Fab fragments and DENV2 sE with anti-EDE1 Fab C8 (left panel) and anti-EDE2 Fab A11 (right panel) mAbs. The molecular weight determined by MALS is indicated, corresponding to the y axis on the left. The ultraviolet absorbance was normalized such that the highest peak is set to 1 (y axis on the right).
Figure 2
Figure 2. Competition between anti-FLE and anti-EDE mAbs.
(a) Description of the schematic procedure of antibody replacement ELISA. (b,c) Competition for binding to dengue virions of anti-FLE mAb-B12, anti-EDE1 mAb-C10 and anti-EDE2 mAb A11. ELISA plates were coated with DENV2 virions produced in C6/36 cells (high prM) (b) and DC (low prM) (c) captured by murine mAb 2C8 which binds to EDIII of DENV2. Plates were then incubated with a pair of antibodies; one of which was biotinylated at a concentration of 1 μg ml−1 and a second antibody which was added in increasing concentrations. Binding of biotinylated antibody was revealed by ALP-conjugated Streptavidin. The data are shown as mean±s.e.m. from three independent experiments.
Figure 3
Figure 3. Engineering covalently linked E-dimers.
(a) Localization of the residues identified by MODIP susceptible to form inter-chain disulfide bonds upon mutation to cysteine. The sE dimer is coloured by subunit, with the MODIP residue pairs indicated in the corresponding colours. A disulfide bond is modelled and is shown as green sticks. The MODIP score, indicated for each residue pair, is a measure of favourability of the geometry of the selected amino acids for disulfide bond formation where A is best and D is worst. (b) Histogram showing the approximate yields in mg per litre of S2 cell culture of DENV2 FGA02 sE protein eluting as monomer, dimer and aggregates separated by SEC for wild type and for the four cysteine mutants presented in a. The yields of covalent dimers are shown in green bars (highlighted with green arrows when sufficient yields for further studies were obtained). (c) MALS analysis of DENV2 A259C sE (red trace). The fractions eluting as dimer in a first step of SEC (which eliminated monomers and aggregates) was re-run by SEC and then superposed to the elution profile of DENV2 WT sE (blue trace). The ultraviolet absorbance was normalized such that the highest peak of each run is set to 1 (y axis on the right). The molecular weight determined by MALS is indicated, corresponding to the y axis on the left. (d) SEC elution profile of L107C/A313C sE superposed to that of A259C sE, showing that the peaks are at the same elution volume, which corresponds to a dimer characterized by MALS in b. As in c the peaks corresponding to monomer and aggregates were eliminated in an initial SEC run. (e) Coomassie stained SDS-PAGE run of sE WT and sE mutants of DENV2 (in the absence (−) or presence (+) of reducing agent DTT). The black arrow indicates the bands of the disulfide stabilized sE dimer.
Figure 4
Figure 4. Structures of DENV2 sE FGA02 WT and mutants in complex with anti-EDE2 Fab A11.
(a) The previously determined structure of DENV2 sE WT in complex with EDE2 A11 Fab (PDB code 4UTB). The molecular two-fold axis is shown as a light-brown central rod, and the cysteines are displayed as green spheres. The heavy and light chains of Fab A11 are coloured in green and light grey, respectively. sE proteins are colour-coded by domains: domain I–red, domain II–yellow and domain III–blue. (b,c) Structures determined here of DENV2 sE A259C mutant in complex with anti-EDE2 A11 Fab (b) and DENV2 sE L107/A313C mutant in complex with anti-EDE2 A11 Fab (c). The constant domains of the two Fab A11 in DENV2 sE A259C complex were disordered in the final structure and thereby are shown in transparent ribbons. Lower b,c: zoom views of the engineered disulfides are shown respectively for DENV2 sE A259C in complex with anti-EDE2 A11 Fab and DENV2 sE L107/A313C in complex with anti-EDE2 A11 Fab.
Figure 5
Figure 5. Covalently linked sE dimers recapitulate the EDE and do not interact with liposomes.
(a,b) ELISA plates were coated with DENV2 either wild type monomeric sE (sE WT) or the two covalently linked sE dimers (a) A259C or (b) L107C/A313C and following incubation with panel of anti-EDE1 and anti-EDE2 mAbs (left panel) or anti-FLE at 1 μg ml−1 (right panel), binding was determined using ALP-conjugated anti-human IgG. (c) Results of co-flotation with liposomes in an Optiprep gradient at low pH (see Methods for lipid composition). Wild type, single and double mutants from DENV2 FGA02, DENV3 H86 and DENV4 1-0093 were incubated at pH 5.8 with liposomes and run in an Optiprep gradient. Insertion of the WT sE proteins in the liposome membrane results in its floatation to the low density top (T) fractions of the gradient (lanes 1). A fraction of the single mutants appears to still be able to float with the liposomes (lanes 3), whereas in the double mutants, there is no sE protein recovered from the top fraction (lanes 5), in line with the fact that the FLE is not exposed.

References

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