PREFUSION-STABILIZED HERPESVIRUS GLYCOPROTEIN-B

Abstract

The present invention generally relates to the field of herpesviruses and herpesvirus proteins that can be used for the production of antibodies or therapeutic agents, such as vaccines. More specifically, the present invention relates to a modified herpesvirus glycoprotein B that comprises one or more mutations at defined positions in the primary structure of the protein which retain a stable prefusion conformation which is highly advantageous for the production of antibodies or therapeutic agents, such as vaccines. The invention further relates to nucleic acid molecules which encode such a modified herpesvirus glycoprotein B. The invention further provides methods for producing antibodies or therapeutic agents, such as vaccines, which make use of the modified herpesvirus glycoprotein B or a nucleic acid molecule encoding it. Kits comprising the modified herpesvirus glycoprotein B are provided as well. Finally, the invention also relates to the use of the modified herpesvirus glycoprotein B or a nucleic acid molecule encoding same for drug screening.

Claims

1. A modified herpesvirus envelope glycoprotein-B, wherein the protein comprises a mutation in its amino acid sequence which stabilizes the prefusion confirmation of the protein by preventing the irreversible transition from the metastable prefusion to the postfusion conformation.

2. The modified herpesvirus envelope glycoprotein-B of claim 1, comprising a mutation of the residue in the position corresponding to position 516 of the unprocessed full-length Herpes simplex virus 1 (HSV-1) envelope glycoprotein-B set forth in SEQ ID NO:1.

3. The modified herpesvirus glycoprotein-B of claim 2, wherein the residue in the position corresponding to position 516 has been replaced by another amino acid residue.

4. The modified herpesvirus glycoprotein-B of claim 3, wherein the residue in the position corresponding to position 516 has been replaced by a proline residue.

5. The modified herpesvirus glycoprotein-B of claim 1, wherein the protein comprises or consists of (a) the amino acid sequence of any of SEQ ID NO:3, 4, 11, 12, 15, 16, 23, 24, 27, 28, 31, 32, 35 or 36; (b) an amino acid sequence which has at least 90% identity to the amino acid sequence of SEQ ID NO: 3, 4, 11, 12, 15, 16, 23, 24, 27, 28, 31, 32, 35 or 36; (c) an immunologically active fragment of any of (a) or (b).

6. The modified herpesvirus envelope glycoprotein-B of claim 1, comprising a mutation of the Ser residue in the position corresponding to position 392 and a mutation of the Gln residue in the position corresponding to position 532 of the unprocessed full-length Herpes simplex virus 1 (HSV-1) envelope glycoprotein-B set forth in SEQ ID NO: 1.

7. The modified herpesvirus glycoprotein-B of claim 6, wherein the Ser residue in the position corresponding to position 392 and the Gln residue in the position corresponding to position 532 of the unprocessed full-length Herpes simplex virus 1 (HSV-1) envelope glycoprotein-B have been replaced by a Cys residue.

8. The modified herpesvirus glycoprotein-B of claim 6, wherein the protein comprises or consists of (a) the amino acid sequence of SEQ ID NO: 5, 6, 17 or 18; (b) an amino acid sequence which has at least 90% identity to the amino acid sequence of SEQ ID NO:5, 6, 17 or 18; (c) an immunologically active fragment of any of (a) or (b).

9. The modified herpesvirus glycoprotein-B of claim 1, said protein comprising: (a) a mutation of the His residue in the position corresponding to position 516 of the unprocessed full-length Herpes simplex virus 1 (HSV-1) envelope glycoprotein-B set forth in SEQ ID NO:1, and (b) a mutation of the Ser residue in the position corresponding to position 392 and a mutation of the Gln residue in the position corresponding to position 532 of the unprocessed full-length Herpes simplex virus 1 (HSV-1) envelope glycoprotein-B set forth in SEQ ID NO: 1.

10. The modified herpesvirus glycoprotein-B of claim 9, wherein the protein comprises or consists of (a) the amino acid sequence of SEQ ID NO:7, 8, 19 or 20; (b) an amino acid sequence which has at least 90% identity to the amino acid sequence of SEQ ID NO:7, 8, 19 or 20; (c) an immunologically active fragment of any of (a) or (b).

11. A nucleotide sequence encoding the modified herpesvirus envelope glycoprotein-B of claim 1.

12. A plasmid comprising the nucleotide sequence of claim 11.

13. A herpesvirus or recombinant herpesvirus vector comprising the modified herpesvirus envelope glycoprotein-B of claim 1.

14. The herpesvirus or recombinant herpesvirus vector of claim 13, wherein said herpesvirus or recombinant herpesvirus vector is or is derived from HSV-1 or HSV-2.

15. A method of treating a herpesvirus infection comprising administering to a subject in need thereof: (a) the modified herpesvirus glycoprotein-B of claim 1; (b) a nucleotide sequence encoding said modified herpesvirus envelope glycoprotein-B; or (c) a herpesvirus or a recombinant herpesvirus vector comprising said modified herpesvirus envelope glycoprotein-B or said nucleotide sequence.

16. A method of vaccinating a subject against herpesvirus infection comprising administering to a subject in need thereof: (a) the modified herpesvirus glycoprotein-B of claim 1; (b) a nucleotide sequence encoding said modified herpesvirus envelope glycoprotein-B; or (c) a herpesvirus or a recombinant herpesvirus vector comprising said modified herpesvirus envelope glycoprotein-B or said nucleotide sequence.

17. A cell comprising: (a) the modified herpesvirus glycoprotein-B of claim 1; (b) a nucleotide sequence encoding said modified herpesvirus envelope glycoprotein-B; or (c) a herpesvirus or a recombinant herpesvirus vector comprising said modified herpesvirus envelope glycoprotein-B or said nucleotide sequence.

18. A pharmaceutical composition or vaccine comprising: (a) the modified herpesvirus glycoprotein-B of claim 1; (b) a nucleotide sequence encoding said modified herpesvirus envelope glycoprotein-B; or (c) a herpesvirus or a recombinant herpesvirus vector comprising said modified herpesvirus envelope glycoprotein-B or said nucleotide sequence.

19. A method of preparing a vaccine comprising introducing (a) the modified herpesvirus glycoprotein-B of claim 1; (b) a nucleotide sequence encoding said modified herpesvirus envelope glycoprotein-B; or (c) a herpesvirus or a recombinant herpesvirus vector comprising said modified herpesvirus envelope glycoprotein-B or said nucleotide sequence, into a pharmaceutically acceptable carrier.

20. A method of screening for an antiviral compound comprising: incubating a candidate compound with (a) the modified herpesvirus glycoprotein-B of claim 1; (b) a nucleotide sequence encoding said modified herpesvirus envelope glycoprotein-B; (c) a herpesvirus or a recombinant herpesvirus vector comprising said modified herpesvirus envelope glycoprotein-B or said nucleotide sequence; or (d) or a cell comprising any of the foregoing (a)-(c) and detecting interaction of said candidate compound therewith, such as its ability to block viral binding.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0074] FIG. 1 shows the results from an SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie stain of extracellular vesicle purifications, obtained from cells transfected with the HSV-1 gB wild type (wt) and single-point mutants, and Western blot analysis of the cell lysate and extracellular vesicles. Mature gB protein is marked by a black arrowhead.

[0075] FIG. 2 shows the fusion activity of gB wt, a fusion-null construct, or a single-point mutant in combination with gH/gL and gD in a cell-cell fusion assay. Activities are normalized to wt fusion activity level.

[0076] FIG. 3 shows the analysis of purified extracellular vesicles formed by WT and gB His516Pro using cryo-EM. The long, postfusion form of gB is indicated by blue lines, and the short form is indicated by orange lines. Lower left images show the top views of gB trimers. Scale bars: 25 nm.

[0077] FIG. 4 shows the size distribution of vesicles found with short and long gB form on vesicles formed by wt gB (n=183) and gB His516Pro (n=114). Boxes indicate range from first to third quartile with whiskers showing ±1.5 interquartile range and outliers marked as points. *no range given, as only two vesicles were found displaying the long form of gB His516Pro. The red line marks the median diameter.

[0078] FIG. 5 shows the results from an SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie stain of extracellular vesicle purifications, obtained from cells transfected with the HSV-1 gB wild type (wt), a truncated construct missing the last 36 aa (868t) and a single-point mutant changing amino acid 898 from Tyrosine to Alanine.

[0079] FIG. 6 shows the Cryo-ET slices of gB mutant vesicles from different Herpesvirus species—HSV-1 and VZV. The gB protein is stabilised in its pre-fusion conformation by the corresponding mutations H516P and H527P.

[0080] FIG. 7 shows Cryo-ET slices of gB mutant vesicles. The gB protein is stabilised by the introduced disulphide bond or the disulphide bond combined with the previously described proline mutation in its pre-fusion conformation.

EXAMPLES

[0081] The present invention is further described in more detail by the following examples which are only provided for illustrating the invention and which are not to be construed as limiting the scope of the invention. The following material and methods were used in the Examples.

Example 1: Expression Plasmid Construction and Vesicle Preparation

[0082] The sequence for a 5xGS linker was added to the C terminus of the gB gene, followed by a 6xHis tag in the pEP98 plasmid. Single-point mutations were created using the Agilent QuikChange II Kit or NEB Q5 kit for site-directed mutagenesis.

[0083] Vesicles were subsequently prepared as described (Zeev-Ben-Mordehai et al. (2014). In brief, BHK-21 cells were grown in GMEM (Glasgow's Minimal Essential Medium) supplemented with 20 mM Hepes (pH 7.4), 2% (v/v) TPB (tryptose phosphate broth), and 2% (v/v) fetal bovine serum. At around 70% confluency, cells were transiently transfected. Cells were grown for an additional 48 hours with a media exchange to serum-free GMEM after 24 hours. Vesicles were harvested from the supernatant by differential centrifugation and resuspended in 20 mM Hepes (pH 8) and 150 mM NaCl.

[0084] Vesicle preparations were tested in SDS—polyacrylamide gel electrophoresis (PAGE) followed by Coomassie staining or Western blotting with a rabbit anti-His6 antibody (Abcam) followed by anti-rabbit horseradish peroxidase (HRP) (Sigma-Aldrich Chemie GmbH). After supernatants were removed for vesicle preparations, cells were washed with cold phosphate-buffered saline (PBS) and detached using cell scrapers. Cells were pelleted by centrifugation (5 min, 4500 g, 4° C.), transferred into 1.5-ml tubes, and washed in cold PBS again before resuspension in radioimmunoprecipitation buffer, 100 μl per T175 flask [50 mM Tris (pH 8), 1% NP-40, 0.1% SDS, 150 mM NaCl, 0.5% sodium deoxycholate, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride]. Samples were shaken at 4° C. for 30 min before being spun at 500 g for 10 min. Supernatants were mixed in SDS sample buffer and run in parallel with vesicle samples in SDS-PAGE. For loading control, Western blots were re-probed using a mouse anti-glyceraldehyde-3-phosphate dehydrogenase antibody (Sigma-Aldrich Chemie GmbH), followed by anti-mouse HRP (Sigma-Aldrich Chemie GmbH).

[0085] Results: Helix-breaking point mutations to proline were introduced individually at residues 515 to 517 of gB. It can be seen in FIG. 1 that a wild type (WT)-like expression of full-length mutant gB was obtained only with a proline at position 516. Mutations in adjacent positions resulted in low or no expression of the mature protein and absence of vesicles. The amount and ratio of fully to partly glycosylated gB on extracellular vesicles, detectable as a single or double band, respectively (Sommer & Courtney, 1991), were not altered by the His516Pro mutation, suggesting that expression and posttranslational modifications were not affected (FIG. 1).

Example 2: Transient Transfection-Based Cell-Cell Fusion Assay

[0086] Fusion activity of the different HSV-1 gB constructs was determined after transient transfection of RK13 cells as described (Vallbracht, et al., 2017a). Briefly, cells were transfected with 200 ng each of the expression plasmids for enhanced green fluorescent protein (EGFP) (pEGFP-N1; Clontech), nectin-1, and HSV-1 glycoproteins gD, gL, gH, and gB or mutant gB in 100 μl of Opti-MEM using 1 μl of Lipofectamine 2000. Twenty-four hours after transfection, the cells were fixed with 3% paraformaldehyde and analyzed using an Eclipse Ti-S fluorescence microscope and NIS-Elements Imaging Software (Nikon). Fusion activity was determined by multiplication of the number of syncytia by the mean syncytia area within 10 fields of view (5.5 mm.sup.2 each). Each experiment was repeated four times, and average percent values of positive control transfections as well as standard deviations were calculated.

[0087] Results: When testing the fusion activity of gB His516Pro in a cell-cell fusion assay (Vallbracht, et al. 2017b) in the presence of gD, gH, and gL. All four glycoproteins are essential and sufficient for HSV-1 membrane fusion and entry (Davis-Poynter et al., 1994; Rogalin & Heldwein, 2016). Compared to wt gB, the cell-cell fusion activity of gB His516Pro was reduced to 6.5% (SD: 3.2%) (FIG. 2), only slightly above the background level [1.6% (SD: 1.4%)] of a fusion-null construct (Cai et al., 1988; Chowdary & Heldwein, 2010). This fusion inhibition is consistent with a block in the pre- to postfusion state transition, functionally locking the protein.

Example 3: Electron Cryomicroscopy (Cryo-EM) to Determine Vesicle Size Distribution

[0088] It has been reported that wt gB is present in two major forms on extracellular vesicles (Zeev-Ben-Mordehai et al., 2016): an extended one (of about 16 nm in length) corresponding to the postfusion conformation and mostly found on small vesicles (approximate median diameter=59 nm) and a compact one (of about 12 nm in length) corresponding to the prefusion state, the latter of which is found on larger vesicles (approximate median diameter=98 nm). For wt gB, about 30% of vesicles predominantly presented the extended form, while 70% predominantly presented the compact form (n=183), similar to previous observations (Zeev-Ben-Mordehai et al., 2016).

[0089] For grid preparation, 3.5 μl of vesicles were mixed with 10-nm gold fiducials on Quantifoil R2/1 grids and plunge-frozen in a propane/ethane mixture using a manual plunge freezer. Microscopy was performed using a Tecnai F30 “Polara” microscope (FEI Thermo Fisher Scientific) at 300 kV equipped with a Quantum 964 post-column energy filter (Gatan) operated in zero-loss imaging mode. Images were recorded on a 4k×4k K2 Summit electron detector with a calibrated pixel size of 0.14 nm at the specimen level. Transmission images were recorded using SerialEM (49) at a −3-μm defocus. Vesicle diameters were measured in 3dmod.

[0090] Results: The results of the cryo-EM are depicted in FIG. 3. For gB His516Pro, total vesicle production was similar to wt gB (FIG. 1), but the ratio was drastically shifted to 98.2% (n=112) of vesicles displaying solely the compact form and only 1.8% displaying the extended form (n=2), with comparable size distributions as the wt vesicles (approximate median diameter=53 nm for extended form; approximate median diameter=90 nm for compact form) (FIG. 4). Thus, introducing the His516Pro mutation apparently inhibited the transition of gB from the compact to the extended form. The small number of vesicles displaying the extended form of gB His516Pro parallels the remaining residual fusion activity, which is marginally above background levels (FIG. 2).

Example 4: Recombinant Expression and Crystallization of Modified gB

[0091] The synthetic gene encoding residues 31 to 730 of HSV-1 gB including the His516Pro mutation was codon-optimized for protein expression in insect cells and cloned into the pT350 vector (Krey et al., 2010) between the vector-encoded Bip signal peptide that drives protein secretion and a double strep tag. The gB modified ectodomain was produced in S2 Drosophila cells using standard methods (Backovic & Krey, 2016). The protein was purified on a Strep-Tactin affinity resin and by size exclusion chromatography using Superdex 200 16/60 column and 10 mM Tris (pH 8) and 50 mM NaCl as running buffer. The protein was concentrated to 6.4 mg/ml and crystallized in 0.1 M Tris (pH 8), 18% ethanol at the Institut Pasteur core facility for crystallization (Weber et al., 2019). They were flash-frozen in liquid nitrogen in cryosolution containing 0.1 M Tris (pH 8), 20% ethanol.

[0092] Results: The results of the crystallisation followed by X-ray diffraction and density map acquisition showed that the ectodomain comprising the single His516Pro mutation and lacking the transmembrane region still adopts the postfusion conformation. Therefore, a stabilisation of the prefusion conformation by the His516Pro mutation is only achieved in combination with a full-length gB protein (aa 1-904), natively embedded in a membrane.

Example 5: Vesicle Production Test

[0093] SDS-PAGE analysis (Coomassie stained) of HSV-1 vesicles from BHK-21 cells transfected with either wild-type gB, a truncated construct ending at residue 868 or gB with a Y889A point mutation. Vesicles were harvested 48 h post transfection. Vesicle samples were mixed in a 2:1 ratio with SDS sample buffer and were loaded in two volumes— 10 μL on the left, 2.5 μL on the right.

[0094] Results: It was found that removal or mutation of the c-terminal endocytosis motif increases the yield of vesicles that can be purified from the supernatant of transfected cells.

Example 6: Cryo-EM to Determine gB Conformation on the Vesicle Surfaces

[0095] Vesicles were produced as and prepared as described (Zeev-Ben-Mordehai et al., 2014) using plasmids encoding gB of HSV-1 or VZV and comprising two kinds of mutations to stabilise the gB protein in prefusion conformation either alone or in combination. In addition, the gB genes all contained an additional mutation in the C-terminal Tyr-X-X-Z endocytosis motif that leads to an increased release of gB containing vesicles. Vesicles were analysed by cryo-EM after plunge freezing on grids (see Example 3). Tomographic imaging was done using a Titan Krios microscope (Thermo Fisher Scientific) at 300 kV with a 70 μm C2 aperture and post-column Quantum energy filter operated in zero-loss mode using an energy slit of 20 eV and K2 Summit direct electron detector in counting mode (Gatan). SerialEM was used for automated data collection. IMOD software suite was used to reconstruct the tomograms.

[0096] Results: Example images (slices of tomograms) of vesicles displaying mutated forms of gB are shown in FIGS. 6 and 7. Introduction of the His516Pro mutation in HSV-1 gB stabilises the protein in pre-fusion conformation. The same effect is seen when the corresponding residue is mutated to proline in VZV gB (His527Pro) (FIG. 6). A similar stabilisation is achieved by introduction of two mutations S392C and Q532C in HSV-1 gB. By introduction of the same amino acid changes in the corresponding residues of VZV gB S397C and Q543C a similar stabilisation of the protein in prefusion conformation is achieved. The two mutations can also be combined with the helix breaking mutation for an additional stabilisation of the prefusion conformation (FIG. 7).

LITERATURE

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