Stabilised herpesvirus antigens

Abstract

Stabilised Herpesvirus antigens The invention relates to stabilised Herpesvirus antigens, including Herpesvirus glycoprotein B (gB) antigens comprising a non-native Membrane Proximal Region (MPR), use of non-native MPR structures to stabilise the Herpesvirus gB in its pre-fusion form, and methods for designing non-native MPR structures. The invention further relates to a stabilised Herpesvirus gB for use as an immunogen, immune reagent or a vaccine.

Description

[0001] Stabilised Herpesvirus antigens

[0002]

[0003] Herpesviruses are a family of enveloped DNA viruses which cause disease in animals and humans. Viral entry into various human host cells is, among other mechanisms, mediated by a major structural rearrangement of herpesvirus glycoprotein B (gB) and subsequent membrane fusion.

[0004] gB is a class III fusion protein and serves as the primary fusogenic protein for Herpesviruses. It mediates membrane fusion events, enabling viral entry into host cells. Like all class III fusion proteins, it undergoes a conformational transition from a metastable “pre-fusion” state to a stable “post-fusion state”. The architecture of class III fusion proteins is conserved across multiple viral families, including Herpesviruses.

[0005] The gB forms a homotrimer, with each monomer comprising an ectodomain with five distinct extracellular domains (Dl-V), a membrane proximal region (MPR), and a transmembrane domain (TMD) (See Figure. 22). The core of the protein is an alpha-helical coiled-coil, formed by Dill. DI contains hydrophobic fusion loops that insert into the target cell membrane. In the pre-fusion conformation, DI interacts with the MPR.

[0006] The MPR is a short, conserved amino acid segment located immediately external to the TMD, adjacent to the viral envelope, and immediately preceding the rest of the gB ectodomain. The MPR has been proposed to act as regulatory interface connecting the ectodomain with the membrane anchor, helping coordinate conformational changes in gB to allow stability of the pre-fusion form, whilst facilitating transition to the post-fusion form during membrane fusion. The MPR is rich in hydrophobic and aromatic residues, which enable interaction with hydrophobic cell and virus membranes.

[0007] As a result of these hydrophobic surfaces on the MPR, simple truncation of the native gB by removing only the TMD does not result in expression of soluble gB with a functionally intact MPR. Rather, the expressed protein is low-yield and prone to aggregation. Truncation of the gB by removing TMD and MPR (i.e. expression of just the gB ectodomain), removes the MPR association with DI and the fusion loops, triggering the native truncated gB to adopt the postfusion conformation.

[0008] The gB is a key target for the development of therapeutic approaches. However, the complex conformational dynamics of gB make it a challenging target for the design of effective immunogens and discovery of antibody and small molecule therapeutics. Additionally, it has been proposed that the more biologically relevant target for effective antibody-mediated virusneutralisation is the metastable “pre-fusion” form of gB. Therefore, to develop effective vaccines and therapies against Herpesviruses, it is imperative to generate immunogens that closely resemble the native, pre-fusion form of gB.

[0009] Previous approaches for stabilising the gB in the pre-fusion form therefore involved truncating the MPR (due to its hydrophobic surfaces) and using core mutations to stabilise the gB (Sponholtz et al.), using a small molecule to bind and stabilise the gB in a pre-fusion-like conformation (Liu et al.), or expressing a gB bound to a vesicle-like particle (Vollmer et al). However, none of these approaches resulted in an antigen capable of triggering an effective neutralisation response.

[0010] In contrast, the present disclosure provides novel uses of non-native MPR structures to stabilise the Herpesvirus gB in its pre-fusion form, non-native MPR structures for use in the methods of the disclosure, and methods for designing such non-native MPR structures. The non-native MPRs provided herein stabilise the pre-fusion gB in a unique conformation which has not previously been characterised. The methods disclosed herein allow the critical structural and functional attributes of the native MPR to be maintained, whilst simultaneously, via the non-native design, enhancing expression of the soluble protein, and ensuring enhanced gB pre-fusion stability.

[0011] The MPR design strategies and non-native MPRs provided herein therefore preserve crucial structures and thereby yielding an effective vaccine antigen or small molecule target.

[0012] Summary of the invention

[0013] In one aspect, provided herein is the use of a non-native membrane proximal region (MPR) domain for stabilising a soluble Herpesvirus gB antigen in a pre-fusion conformation, wherein:

[0014] a) the gB antigen comprises the domains DI, Dll, Dill, DIV and DV and an MPR but does not comprise a transmembrane domain;

[0015] b) the non-native MPR replaces the native MPR domain; and

[0016] c) the non-native MPR comprises at least one amino acid substitution.

[0017] In a further aspect, provided herein is a method of increasing the solubility, expression yield, and / or stability of a soluble Herpesvirus gB, wherein the Herpesvirus gB comprises the domains DI, Dll, Dill, DIV and DV and an MPR, the method comprising:

[0018] a) replacing the native MPR with a non-native MPR engineered to interact with the DI domain of the gB; orb) introducing into the native MPR at least one amino acid substitution which increases the binding energy of the interaction between the MPR and the DI domain of the gB. In a further aspect, provided herein is an engineered Herpesvirus gB comprising at least the domains DI, Dll, Dill, DIV and DV and a MPR and not comprising a transmembrane domain, wherein the MPR is a non-native MPR and comprises at least one amino acid substitution which stabilises the tertiary structure of the gB in its pre-fusion conformation, and wherein the at least one amino acid substitution reduces the surface hydrophobicity of the non-native MPR relative to the native MPR and / or increases the binding energy of the interaction between the MPR and the DI domain of the gB.

[0019] One or more of the solvent facing amino acid residues in the native MPR may be substituted with amino acid residues which increase the polarity of the MPR.

[0020] The engineered Herpesvirus gB may show an increase in one of more of solubility, expression level, percentage of pre-fusion particles and aggregation temperature, and / or a decrease in Hydropathy index, instability index or Total Energy (dG), relative to a soluble Herpesvirus gB comprising a corresponding native MPR or no MPR.

[0021] The gB may further comprise a trimerisation domain.

[0022] The engineered Herpesvirus gB may be an alphaherpesvirus gB, a betaherpesvirus gB or a gammaherpesvirus gB. The engineered Herpesvirus gB may be a gB selected from the human Herpesviruses HSV-1, HSV-2, VZV, HCMV, HHV-6, HHV-7, EBV and KSHV / HHV8. The engineered Herpesvirus gB may be selected from an HCMV gB, an HSV-1 gB, an HSV-2 gB or an EBV gB.

[0023] The engineered Herpesvirus gB may be an HCMV gB and the substitution may be a substitution of one or more of amino acid residues 707 to 752 relative to SEQ ID NO:1 (strain Merlin / Towne).

[0024] The Herpesvirus gB may be an HCMV gB and the substitution may be a substitution of one or more of amino acid residues 706-751 relative to SEQ ID NO: 5 (strain AD169).

[0025] The engineered Herpesvirus gB may be HCMV gB and the substitution may be a substitution of one or more of amino acid residues 705-750 relative to SEQ ID NO: 9 (strain VR1814). The engineered Herpesvirus gB may be a HSV-1 gB and the substitution may be a substitution of one or more of amino acid residues relative to 725 to 775 of SEQ ID NO: 100 (strain KOS).The Herpesvirus gB may be a HSV-2 gB and the substitution may be a substitution of one or more of amino acid residues relative to 724 to 774 of any one of SEQ ID NOs:255-259 (strain HG52).

[0026] The engineered Herpesvirus gB may be an EBV gB and the substitution may be a substitution of one or more of amino acid residues 684 to 773 relative to SEQ ID NO: 108 (strain B95-8). The engineered Herpesvirus gB may be an HCMV gB and the MPR have a sequence of PPYLKGLDDLX1KX2LX3X4X5GX6X7EGVX8IGAX9FGKX1oX11X12KX13LX14X15EX16X17X18KN PF, wherein Xi is selected from I, or C; X2is selected from K, C, or I; X3is selected from W, or N; X4 is selected from F, or H; X5is selected from Y, or T; X6is selected from F, or N; X7is selected from A, C, or W; X8is selected from S, or C; X9is selected from E, V, or I; Xi0is selected from D, or C; Xu is selected from E, or F; X12is selected from W, or P; X13is selected from K, or P; X14is selected from S, or P; Xi5is selected from V, or P and Xi6is selected from T, or C (SEQ ID NO: 320).

[0027] The engineered Herpesvirus gB may be an HSV-1 or HSV-2 gB and the MPR may have a sequence of ADX1X2AX3X4X5X6X7X8X9X1OX11YX12X13X14GX15X16X17X18X19X2OGX21X22X23X24X25X26X27X28X29X3oX3iX32X33X34X35X36X3X38X39X4oAX4iX42PX43, wherein X4is selected from K, E, or A; X2is selected from I, K, R, or T; X3is selected from D, K, or A; X4 is selected from E, I, or L; X5is selected from F, Q, or L; X6is selected from A, R, or K; X7is selected from E, or A; X8is selected from L, or I; X9is selected from G, or A; Xi0is selected from R, K, or E; Xu is selected from F, K, or A; X12is selected from E, K, or A; X13is selected from A, K, or E; X14is selected from K, T, or G; Xi5is selected from P, A, or E; Xi6is selected from E, or A; X17is selected from G, or A; Xi8is selected from R, E, or F; X19is selected from A, or K; X20is selected from K, or A; X2iis selected from K, L, or R; X22is selected from V, or P; X23is selected from E, D, or A; X24is selected from M, L, or I; X25is selected from G, E, K, or A; X26is selected from E, K, or S; X27is selected from K, L, or T; X28is selected from G, or L; X29is selected from K, E, or A; X30is selected from V, or E; X3iis selected from V, M, or L; X32is selected from S, or E; X33is selected from A, or E; X34is selected from A, K, or Q; X35is selected from A, K, or E; X36is selected from T, A, or R; X37is selected from A, E, or Q; X38is selected from A, E, or Q; X39is selected from S, E, or A; X40 is selected from L, or R; X41 is selected from A, K, or L; X42is selected from D, or L and X43is selected from L, F, or Y (SEQ ID NO: 325).The engineered Herpesvirus gB may be an EBV gB and the MPR may have a sequence of NGX1X2X3X4VX5X6LX7X8X9X10X11X12X13X14X15X16X17QX18X19X20X21X22X23X24X25X26X27G X28X29X3OX3IX32X33X34X3SX36X3X38X39X4OX4IX42PX43, wherein X1is selected from R, or V; X2is selected from N, or L; X3is selected from Q, or A; X4 is selected from F, E, or L; X5is selected from D, or M; X6is selected from G, K, or D; X7is selected from G, or A; X8is selected from E, or D; X9is selected from L, or N; Xi0is selected from M, or Y; Xu is selected from D, or L; Xi2is selected from S, N, or R; X13is selected from L, or S; X14is selected from G, P, or D; Xis is selected from S, or P; Xi6is selected from V, A, S, or P; X17is selected from G, or E; Xis is selected from S, or R; X19is selected from I, A, or L; X20is selected from T, or A; X21is selected from N, or R; X22is selected from L, I, or A; X23is selected from V, or T; X24is selected from S, or N; X25is selected from T, or I; X26is selected from V, K, or I; X27is selected from G, or L; X28is selected from L, or K; X29is selected from F, E, I, or L; X30is selected from S, or E; X31is selected from S, or A; X32is selected from L, A, or F; X33is selected from V, R, or L; X34is selected from S, L, or E; X35is selected from G, A, or I; X36is selected from F, A, or L; X37is selected from I, E, or A; X38is selected from S, A, D, or R; X39is selected from F, A, or L; X^ is selected from F, V, or A; X41is selected from K, N, or A; X42is selected from N and S, L, or D; X43is selected from F, L, or S (SEQ ID NO: 329).

[0028] The engineered Herpesvirus gB may be an HCMV gB and the MPR may have a sequence selected from any one of SEQ ID NOs: 276-312. For example, the sequence may be selected from any one of SEQ ID NOs: 287, 297, 289 or 298.

[0029] The engineered Herpesvirus gB may be an HSV-1 or HSV-2 gB and the MPR may have a sequence selected from any one of SEQ ID NOs: 223-243. For example, the sequence may be selected from any one of SEQ ID NOs: 232, 243, 241 and 237.

[0030] The engineered Herpesvirus gB may be an EBV gB and the MPR may have a sequence selected from any one of SEQ ID NOs: 260-274. For example, the sequence may be selected from any one of SEQ ID NOs: 320, 325, 329.

[0031] The engineered Herpesvirus gB may be an HCMV gB and have a sequence selected from any one of SEQ ID NOs: 55 to 99. For example, the sequence may be selected from any one of SEQ ID NOs: 60, 61, 63 or 64.

[0032] The engineered Herpesvirus gB may be an HSV-1 gB and have a sequence selected from any one of SEQ ID NOs: 212-121. For example, the sequence may be selected from any one of SEQ ID NOs: 217, 219, 220 or 221.The engineered Herpesvirus gB may be an HSV-2 gB and have a sequence selected from any one of SEQ ID NOs: 255-259.

[0033] The engineered Herpesvirus gB may be an EBV gB and have a sequence selected from any one of SEQ ID NOs: 244-259. For example, the sequence may be selected from any one of SEQ ID NOs: 244, 246, 249 or 251.

[0034] The engineered Herpesvirus gB may further comprise one or more additional stabilising substitutions outside of the MPR.

[0035] The one or more additional stabilising substitutions may be cysteine mutations which introduce one or more disulfide bridges at the DI, Dll, MPR and / or core interfaces.

[0036] The engineered Herpesvirus gB may be a HCMV gB and the one or more additional stabilising substitutions may be selected from the list of A239C, A239W, A267V, A293P, A369L, A732C, A97V, C246S, D277C, D320L, D478L, E274I, E274V, E286F, E289P, E321I, E634C, E657C, E671C, E679C, F683C, G173C, G177C, G735C, H222C, H681C, I636C, I89C, I90C, K124V, K158C, K158I, K214F, K340P, K359P, K394-T452del, L288P, L484P, L641C, L678C, L709C, M677I, M677L, N132C, N132V, N341P, N635T, N657C, N658C, Q483P, Q501I, Q527C, Q669C, R180I, R180L, R291P, R429S, R431S, R432S, R457S, R460S, S164C, S244C, S269C, S362P, S367I, S631C, S641C, S641L, S739C, T100I, T225C, T225F, T292P, T343P, T572C, T630C, T659L, T676C, T676I, V342P, V363P, V480P, V684W, V728C, W240C, W356P, Y153C, Y155C, Y160C, Y242C, Y464-T498del, and / or Y481P relative to SEQ ID NO: 1.

[0037] The engineered Herpesvirus gB may be an HSV-1 gB and the one or more additional stabilising substitutions may be selected from the list of I154C, K158C, K158I, (WFGHR)174(YAYIH), W174N, F175H, Y179T, F182N, A239C, A239W, S244C, (AFH)261(WLY), E274V, E274I, D277C, E286F, W356P, K359P, S362P, V363P, Y464-T498del, T572C, L673(GCG), T676C, T676I, M677I, M677L, L678C, E679C, H681C, F683C, V684W and / or N709V relative to SEQ ID NO: 100.

[0038] The engineered Herpesvirus gB may be an HSV-2 gB and the one or more additional stabilising substitutions may be selected from the list of I149C, L670(GCG), T569C, E676C, N706V, W169N, F170H, Y174T and F177N relative to SEQ ID NO: 254.

[0039] The engineered Herpesvirus gB may be an EBV gB and the one or more additional stabilising substitutions may be selected from the list of I89C, I90C, T630C, (YNGWY)109(YAYIH), W112N, Y113T, K124V, G173C, G177C, R180I, R180L, T225C, T225F, L288P, E289P,T292P. A293P, K394-T452del, R429S, R431S, R432S, D478L, Q527C, L628(GCG), S631C, N635T, I636C, L641C, E634C, W196S and / or Y198D relative to SEQ ID NO: 108.

[0040] The pre-fusion conformation may be detected using an antibody specific for an epitope on the pre-fusion conformation which is not present in the post-fusion conformation.

[0041] In a further aspect, provided herein is a method of designing a non-native MPR domain, the method comprising the steps of:

[0042] a) obtaining an experimentally determined three-dimensional structure of a Herpesvirus gB or generating a three-dimensional structure of a Herpesvirus gB in silico from a Herpesvirus gB sequence;

[0043] b) using the obtained or generated three-dimensional structure as an input model;

[0044] c) energy minimising the input model using molecular dynamics simulations, wherein the energy minimisation reduces steric clashes and optimises side chain rotamers;

[0045] d) selecting target locations for potential pre-fusion stabilisation substitutions, wherein the target locations are located within the core domains of the gB and / or within the interface between the DI fusion loops and MPR domain;

[0046] e) predicting the impact of point mutations at the target locations in silico using a deep neural network and identifying favourable residue positions to be mutated;

[0047] f) applying residue masking at the residue positions identified in step (c) to generate a diverse set of non-native gB sequences;

[0048] g) generating in silico models of one or more of the non-native gB structures generated in step (f);

[0049] h) generating in silico data, for example, calculated properties based on the sets of diversified sequences and generated protein structures, and collating the data in one dataset for downstream selection purposes;

[0050] i) selecting one or more of the non-native gB structures having a pLDDT value above 80, a target RMSD match of below 2.0 A, a prediction RMSD match of below 3.0 A, a hydropathy index value of below 0.5, an instability index value of below 40, a shape complementary value of above 0.6, and / or an interface energy value above the mean of all designed sequences; or

[0051] j) ranking the sequences according to their total energy value selecting the top sequence designs; and

[0052] k) optionally repeating steps (a) to (j) two or more times to further optimise the non-native MPR designs.

[0053] The method may further comprise the steps of:l) generating one or more nucleic acid sequences, each encoding a gB comprising the modified MPR sequence of a selected structure;

[0054] m) expressing the one or more nucleic acid sequences in an expression system to produce a gB comprising the modified MPR; and

[0055] n) purifying the gB comprising the modified MPR to a purity of at least 80%.

[0056] The further characterisation may be in vitro characterisation, and the method may further comprise a step of assessing one or more biophysical and / or biochemical properties of the gB comprising the modified MPR. The biophysical and / or biochemical property may aggregation temperature, expression level, percentage of correctly folded protein, hydrodynamic radius, antigenicity, and / or negative-stain electron microscopy (nsEM) characteristics.

[0057] The further characterisation may be in vivo characterisation, and the method may further comprise the steps of:

[0058] o) administering the gB comprising the modified domain to a non-human subject to elicit an immune response;

[0059] p) isolating one or more antibodies from the non-human subject; and

[0060] q) assessing the binding activity and / or neutralising activity of the one or more antibodies against a live Herpesvirus corresponding to the species from which the gB protein is derived.

[0061] In a further aspect, provided herein is a gB trimer comprising three monomeric engineered Herpesvirus gBs of the disclosure.

[0062] In a further aspect, provided herein is the use of an engineered Herpesvirus gB of the disclosure for screening and identifying biologies and small molecules that inhibit or alter gB function. The biologic may be a peptide, a nanobody, a monoclonal antibody, or an aptamer. In a further aspect, provided herein is an engineered Herpesvirus gB of the disclosure conjugated to at least one further moiety or conjugation substrate. The further moiety may be selected from a detection moiety, a solid support, an antibody, an antibody fragment, or a binding molecule. The solid support may be a nanoparticle, chip, column, membrane, or viruslike particle, and the detection moiety may be a fluorescent tag, a magnetic tag, or a radiolabel. The virus-like particle may be an enveloped virus-like particle (comprising a lipid envelope), a non-enveloped virus-like particle (lacking a lipid envelope), or a chimeric virus-like particle. In a further aspect, provided herein is a method of screening antibodies specifically binding to the pre-fusion conformation of the engineered Herpesvirus gB, the method comprising:a) providing a sample containing B cells from a subject exposed to, or infected with, a Herpesvirus, a Herpesvirus vaccine or an engineered Herpesvirus gBs of any one of the disclosure;

[0063] b) culturing single B cells in individual wells such that the individual B cells proliferate and secrete a monoclonal antibody into the well;

[0064] c) detecting the monoclonal antibody in the well using a binding probe comprising a corresponding engineered Herpesvirus gB of the disclosure; and

[0065] d) identifying B cells that bind the binding probe; and / or

[0066] e) sequencing the immunoglobulin genes of the isolated B cells to identify monoclonal antibodies that bind specifically to the immune reagent.

[0067] In a further aspect, provided herein is a vaccine comprising the Herpesvirus gBs of the disclosure. The engineered gB may be displayed on nanoparticles.

[0068] In a further aspect, provided herein is a vaccine comprising an mRNA, DNA, or saRNA encoding a Herpesvirus gBs of the disclosure.

[0069] In a further aspect, provided herein is a Herpesvirus gB of the disclosure for use as a vaccine antigen.

[0070] In a further aspect, provided herein is a pharmaceutical composition comprising a Herpesvirus gB of the disclosure and a pharmaceutically acceptable carrier or adjuvant.

[0071] In a further aspect, provided herein is a Herpesvirus gB of the disclosure for use in the treatment or prevention of Herpesvirus infection, or of a disease or disorder associated therewith.

[0072] In a further aspect, provided herein is a non-native Herpesvirus gB MPR domain for use in a method of stabilising a soluble Herpesvirus gB in a pre-fusion conformation, wherein the nonnative MPR replaces the native MPR.

[0073] Brief Description of the Figures

[0074] Figure 1. gB stabilisation strategy aiming to fully conform the glycoprotein to a native prefusion conformation, (a) Domain layout of gB and models extracted from an all-atom molecular dynamics simulations representing a trajectory time span of 50 ns. (b) Free energy landscape of a 100 ns trajectory indicating a starting position of gB which was further minimised into a local energy well following a structural energy path, (c) Protein design strategy of the DI andMPR protein interface which aimed to improve the binding, stability, solubility and expression of gB constructs incorporating the MPR domain.

[0075] Figure 2. Evaluation of gB constructs with engineered MPR domains, (a) Sequence alignment of ten engineered M PR domains against the native M PR. The de novo protein design followed various strategies that varied the sequence conservation to the native MPR. Conserved residues as compared to the native sequence are highlighted, (b) Normalised Size Exclusion Chromatography (SEC) profiles of expressed pre-fusion constructs (OP63 (SEQ ID NO: 60), OP64 (SEQ ID NO: 61), OP66 (SEQ ID NO: 63), OP67 (SEQ ID NO: 64)) and a native postfusion gB construct. The arrow indicates the selected peak fraction for subsequent analyses of pre-fusion species, (c) Representative nsEM micrograph and top 2D classes of extracted particles from pre-fusion stabilised gB construct sample (OP67; SEQ ID NO: 64). nsEM particle datasets included >10,000 extracted particles for each construct, (d) 3D envelopes of selected constructs and fitted energy minimised pre-fusion model. Correlation coefficients (CCs) for each fitted model were calculated to 0.76 or above. Envelope diameters vary between 12.3 nm to 13.3 nm indicating conformational variability in the gB DI.

[0076] Figure 3. Sequences and representative 2D classes of pre-fusion particles from designed constructs (a) OP77 and (b) QP80 which contain an engineered MPR domain. These constructs do not contain any stabilising core interface mutations. Particle classes confirm the presence of pre-fusion stabilised particles and contrast with post-fusion particle classes which were averaged within the same data sets.

[0077] Figure 4. Molecular dynamics simulation properties along the 100 ns trajectory of gB. (a) The calculated radius of gyration (Rg) of gB changes minimally along the simulation which agrees with the (b) root-mean square deviation (RMSD) of the Co atoms where gB stabilises within the first 4 ns and minimally deviates with a mean RMSD of up to 0.54 nm. No severe or irregular conformational changes were observed in the trajectory, (c) Root-mean square fluctuations (RMSF) of gB residues highlight flexible loop regions, as well as the more flexible C-terminal MPR domain above the mean of 0.18 nm for residues 68-752.

[0078] Figure 5. Decomposed interchain interaction energies of the gB trimer as calculated through the MMPBSA method. Reported values represent the twenty lowest decomposed residue energies between (a) chain A to chain B and (b) chain A to chain C. Interaction energies were averaged from 20 ns to 60 ns which represented initially stabilised gB model states. These energy values indicate key interaction residues and regions within the pre-fusion gB trimer structure. Errors bars represent the standard deviation of the interaction energy for each residue along the specified trajectory.Figure 6. CryoEM collection results of gB OP53 (SEQ ID NO: 50) which contained core interfaces mutations as well as additional mutations to improve expression and stability. This construct did not contain an MPR domain, (a) Representative raw and denoised micrographs show appropriate particle distribution and density for cryoEM processing, (b) Protein particle map shows coverage of particle views in all directions which allowed for a map generation to a resolution of 3.02 A using 265,843 particles in a final local refinement job. (c) Final cryoEM density map of HCMV gB OP53 (SEQ ID NO: 50) in three directions. The local resolution colouring shows that the densities for the outer domains, which include the DI, Dll and connecting regions to the Dill, are less refined indicating increased domain flexibility.

[0079] Figure 7. MPR domain redesign strategy of 1000 sequences using ProteinMPNN at a sampling temperature of 0.1. (a) Theoretical GRAVY values calculated based on the Kyte-Doolittle method for each designed output sequence indicate a distinct range of hydropathy, (b) Instability index values for each designed output sequence, (c) Global ProteinMPNN scores and (d) a fused index score combining the GRAVY values, Instability index, and ProteinMPNN for final evaluation and selection of MPR sequences. The top 10% of sequences with the lowest combined scores were used to generate structural models using AlphaFold2.3. (e) Structural differences between generated models in different clusters, (f) Final selection of a promising redesigned MPR sequence to be included in a gB construct for protein production and examination. Model 17 was selected due to its optimal predicted structural properties to the target MPR structure as well as the evaluated sequence-based properties. The Model 17 MPR sequence was included in the gB OP63 (SEQ ID NO: 60) construct.

[0080] Figure 8. Antibody binding experiments between SM5-1 Fab antibody (Spindler et al.) and HCMV gB constructs. Samples represent various conformations of gB including (a) the postfusion state, (b) the pre-fusion state as stabilised only using core interface mutations and (c) the pre-fusion state as stabilised by core interface mutations and the addition of an engineered MPR domain. All binding experiments show binding between gB and the antibody indicating sound antigenicity for these three gB constructs. The SM5-1 Fab is non-specific to either conformational state of gB.

[0081] Figure 9. EBV gB multiple sequence alignment showing the conserved EBV amino acid sequence before the MPR region (boxed) for the non-native MPR designs OP117 (SEQ ID NO: 109), OP118 (SEQ ID NO: 110), OP119 (SEQ ID NO: 111), QP120 (SEQ ID NO: 112), OP121 (SEQ ID NO: 113), OP122 SEQ ID NO: 114), OP123 (SEQ ID NO: 115), relative to the wild type EBV gB from B95-8 strain virus genome (Genbank ID V01555.2;) and the corresponding gB protein sequence (Genbank ID CAA24806.1; SEQ ID NO: 108). Followingthe MPR the sequences diverge with the OP series containing the C-terminal foldon, TEV and purification tags rather than the wild type gB sequences.

[0082] Figure 10. HSV-1 gB multiple sequence alignment showing the conserved HSV-1 amino acid sequence before the MPR region (boxed) for the non-native MPR designs OP110 (SEQ ID NO: 101), OP111 (SEQ ID NO: 102), OP112 (SEQ ID NO: 103), OP113 (SEQ ID NO: 104), OP114 (SEQ ID NO: 105), OP115 (SEQ ID NO: 106), OP116 (SEQ ID NO: 107), relative to the wild type HSV-1 gB from the KOS strain virus (AAA45774.10) genome (Genbank ID KT899744.1) and the corresponding gB protein sequence (Genbank ID K01760.1; SEQ ID NO: 100). Following the MPR the sequences diverge with the OP series containing the C-terminal foldon, TEV and purification tags rather than the wild type gB sequences.

[0083] Figure 11. Human Herpesvirus gB multiple sequence alignment showing amino acid sequence conservation at the 70% or great level. The sequences are truncate after the end of the NPF amino acid triplet at the C terminus of the MPR region (boxed). The N terminal amino acid of the boxed MPR is relative to the HSV-1 gB (starting at amino acids “ADAN...”). The EBV MPR begins at amino acids “NGRN...” amino acids and the HCMV MPR begins at amino acids “LPPY...”

[0084] Figure 12. Input models for EBV gB de novo MPR design.

[0085] Figure 13. EBV gB de novo MPR design — Filtering Sequences. Generated sequences were evaluated based on the ProteinMPNN global score, an instability index, and GRAVY values (Kyte-Doolittle) which were normalised and combined into a weighted score. Sequences containing an N-glycan motif (N-glycan motif = 1) were removed and the top 10% of sequences based on the weighted score were selected for structure generation and further evaluation.

[0086] Figure 14. EBV gB de novo MPR design — MPR Structure Generation. Selected sequences were used as input for Alphafold2 structure generate on which allowed for RMSD comparison between model replicates (n=5) and to the target structure. The sequence-structure pairs with the lowest RMSD values, highest pLDDT values, and lowest hydropathy values were selected as promising MPR sequences for potential in vitro examination.

[0087] Figure 15. EBV gB de novo MPR design — Final Sequence Selection. The final selection of designed MPR sequences that showed favourable characteristics based on sequence and structural evaluation methods. The designed MPR sequences cover residue positions 684-733 of EBV gB (Strain B95-8; SEQ ID NO: 108). Design run IDs for each designed MPR are listed in Table 5.Figure 16. EBV gB de novo MPR design — Rosetta Docked Structures. Docked complex between the EBV gB DI and designed MPRs from the selected sequences. The docking procedure followed a standard Rosetta procedure with target alignment, side chair packing, and multiple rounds of FastRelax runs.

[0088] Figure 17. HSV-1 gB de novo MPR design — Input Models. Input models for the de novo design of the MPR domain against the DI. Generated 6000 MPR sequences against the DI fusion loop interface using ProteinMPNN.

[0089] Figure 18. Data: HSV-1 gB de novo MPR design — Filtering Sequences. Generated sequences were evaluated based on the ProteinMPNN global score, an instability index, and GRAVY values (Kyte-Doolittle) which were normalised and combined into a weighted score. Sequences containing an N-glycan motif (N-glycan motif = 1) were removed and the top 10% of sequences based on the weighted score were selected for structure generation and further evaluation.

[0090] Figure 19. HSV-1 gB de novo MPR design — MPR Structure Generation. Selected sequences were used as input for Alphafold2 structure generation which allowed for RMSD comparison between model replicates (n=5) and to the target structure. The sequence-structure pairs with the lowest RMSD values, highest pLDDT values, and lowest hydropathy values were selected as promising MPR sequences for potential in vitro examination.

[0091] Figure 20. HSV-1 gB de novo MPR design — Final Sequence Selection. The final selection of designed MPR sequences that showed favourable characteristics based on sequence and structural evaluation methods. The designed MPR sequences cover residue positions 725-775 of HSV-1 gB (Strain KOS). Design run IDs for each designed MPR are listed in Table 6.

[0092] Figure 21. HSV-1 gB - de novo MPR design — Input Models. Input models for the de novo design of the MPR domain against the DI. Generated 6000 MPR sequences against the DI fusion loop interface using ProteinMPNN (A) HSV-1 gB pre-fusion - AlphaFold2 model (B) HSV-1 gB DI / MPR input models for ProteinMPNN.

[0093] Figure 22. Domain structures of pre- and post-fusion gB. In its pre-fusion conformation, the gB forms a compact homotrimer where the domains DI, Dll, Dill, DIV, and DV are orientated so that Dill forms a trimer apex with Dll packing at the side of DI 11 and DIV located at the base of Dill. DI is located at the base of the trimeric structure, below Dll and packed alongside DIV and DV, with the fusion loops of DI located near the virus lipid envelope. The post-fusion form of gB has been visualised as an extended structure, where DIV now forms one end of the structure being located at the end of a rearranged Dill. DI is proposed to be in one of twolocations, either with the fusion loops embedded in the host cell lipid membrane as shown in this representation (and DV location is not defined), or in a structure where DI is associated with the virus membrane even though gB is now in the post-fusion conformation. Irrespective of this, the post-fusion gB conformation is defined by an elongated and rearranged Dill and the relocation of DIV away from DI. The Membrane Proximal Region (MPR) of pre-fusion gB is located next to the virus lipid membrane and interacting with DI.

[0094] Figure 23. HCMV phylogeny generated from a sequence alignment of 350 HCMV gBs available in GenBank. The unroot phylogeny clearly shows the five subtypes of HCMV, GB1, GB2, GB3, GB4, GB5 with representative HCMV species Merlin, Towne, Toledo, AD169, and C194A.

[0095] Figure 24. Post-fusion form of gB produced by expressing gB from HCMV Merlin where the fusion loops and furin cleavage sites are mutated as documented. The extended structure of the post-fusion form is clearly visible in the negative stain EM grids and through the reconstruction of the nsEM structure relative to the known structure obtained by X-ray crystallography (Ribbon diagrams).

[0096] Figure 25. Multiple sequence alignment of construct OP25 (SEQ ID NO: 23) which contains a native membrane proximal region (MPR) and no Furin cleavage site due to the mutations R457S & R460S (filled triangles) compared to OP58, 59, 60, 61, 62, 63, 64, 65, 66 and 67 (SEQ ID NOs: 55-64), also with no Furin cleavage site due to the mutations R457S & R460S (filled triangles). The MPR designs of OP58, 59, 60, 61, 62, 63, 64, 65, 66 and 67 (SEQ ID NOs: 55-64) are relative to OP25 (SEQ ID NO: 23) are highlighted in the boxed area. The position of the Fusion loop residues 1156, H157, W240 and Y242 are shown (filled circles), the position of the free cysteine mutation C246S shown (filled star) are shown. The position of the ‘Sponholtz mutations’ H222C / E657C, V134C / I653C and T100L / A267I are shown (unfilled circles)

[0097] Figure 26. Multiple sequence alignment of construct OP25 (SEQ ID NO: 23) which contains a native membrane proximal region (MPR) and no Furin cleavage site due to the mutations R457S & R460S (filled triangles) compared to: (i) OP52 (SEQ ID NO: 49) which is the Merlin gB containing the mutations H222C / E657C, V134C / I653C and T100L / A267I; (ii) OP53 (SEQ ID NO: 50) which is the Merlin gB containing the mutations H222C / E657C, V134C / I653C and T100L / A267I, the Furin cleavage site mutations and the fusion loop mutations I156H, H157R, W240N, Y242T; and (iii) OP58, 59, 60, 61, 62, 63, 64, 65, 66 and 67 (SEQ ID NOs: 55-64), also with no Furin cleavage site due to the mutations R457S & R460S (filled triangles). The MPR designs of OP58, 59, 60, 61, 62, 63, 64, 65, 66 and 67 (SEQ ID NOs: 55-64) are relativeto OP25, OP52 and OP53 (SEQ ID NOs: 23, 49 and 50) and are highlighted in the boxed area. The position of the Fusion loop residues 1156, H157, W240 and Y242 are shown (filled circles), the position of the free cysteine mutation C246S shown (filled star) are shown. The position of mutations H222C / E657C, V134C / I653C and T100L / A267I are shown as unfilled circles.

[0098] Figure 27. Herpesvirus phylogeny showing CMV, EBV and HSV as representatives for beta, gamma, alpha herpesvirus.

[0099] Figure 28. Computational approach for the design of core interface mutations and de novo MPR design based on an input model. The input model is initially energy minimised and target locations for potential pre-fusion stabilisation are selected. The selected sequences with specific residue positions are redesigned using ProteinMPNN and structures are predicted using AlphaFold. The output data based on sequence- and structure-based properties are used for a final selection of appropriate constructs designs for experimental validation.

[0100] Figure 29. Criteria and threshold values for the selection of viable mutations and sequences based on the computational design pipeline.

[0101] Figure 30. CryoEM densities of OP64 (SEQ ID NO: 61) and OP67 (SEQ ID NO: 64) showing the unique conformations of these pre-fusion designs. The resolved densities show that gB exists in two different conformations where one assumes an extended state and the other assumes a novel compact state, presumably stabilised by an interaction of the designed de novo MPR domains.

[0102] Figure 31. Prefusion HCMV gB (strains AD169 / VR1814; SEQ ID NO: 5 / 9) construct designs and prefusion particle abundance. Additional efforts to explore different core interface mutations show that prefusion stabilisation can be achieved using various residue locations within the core interfaces of gB. These designs include de novo MPR sequences from previous gB constructs (SEQ ID NOs: 90-99).

[0103] Figure 32. Translation of core mutations from the HCMV Merlin strain to AD169 and VR1814. The core interface mutations and designed MPR domains from OP63 (SEQ ID NO: 60), OP64 (SEQ ID NO: 61), and OP67 (SEQ ID NO: 64) were translated to the HCMV gB of two different strains. The designed constructs were shown to be stabilised in the pre-fusion conformation showcasing the ability to translate these mutations to other HCMV gB sequences (SEQ ID NOs: 90, 92, 93 and 95).

[0104] Figure 33. Mouse immunisation studies with HCMV pre-fusion gB candidates to assess ability of immunised mouse serum samples to neutralise live HCMV. (A / B) shows enhanced levelsof neutralising antibody response elicited by HCMV pre-fusion gB construct OP67 (SEQ ID NO: 64) over the post-fusion gB (OP16; SEQ ID NO: 16) and a gB with core mutations only (OP52; SEQ ID NO: 49). (C / D) shows enhanced levels of neutralising antibody response over post-fusion gB (OP16; SEQ ID NO: 16) elicited by pre-fusion gB constructs OP64 (SEQ ID NO: 61) and OP67 (SEQ ID NO: 64), as well as OP144 (SEQ ID NO: 161) and OP182 ('98) which contain different core mutations compared to OP64 (SEQ ID NO: 61) and OP67 (SEQ ID NO: 64).

[0105] Figure 34. Construct designs of HSV-1 gB (KOS strain; SEQ ID NOs: 154-167).

[0106] Figure 35. Construct designs of HSV-1 gB (KOS strain; SEQ ID NOs: 193-207) with resulting expression yields.

[0107] Figure 36. nsEM of selected gB constructs (OP141, OP143, OP145, OP189, OP191; SEQ ID NOs: 158, 160, 162, 206 and 208) showing a range of pre-fusion stabilised particle abundance. Multiple gB constructs were stabilised in the pre-fusion conformation using novel core interface mutations and stabilisation through de novo MPR domains.

[0108] Figure 37. Biophysical properties of HSV-1 gB (KOS strain; SEQ ID NOs 212-221) construct designs.

[0109] Figure 38. Biophysical properties of HSV-1 gB (KOS strain; SEQ ID NOs 212-221) construct designs.

[0110] Figure 39. nsEM micrographs of pre-fusion-stabilised and post-fusion HSV-1 gB constructs. Quantification and classification of particle conformations shows a high abundance of pre-fusion-stabilised population in the selected HSV-1 gB constructs (SEQ ID NOs 212-221). Figure 40. Sequence alignments of HSV-1 gB and HSV-2 gB show a high sequence identity. The identical sequences between HSV-1 gB and HSV-2 gB in the target regions enables direct translation of pre-fusion-stabilising mutations and designed MPR sequences (SEQ ID NOs: 330-335).

[0111] Figure 41. Construct designs of EBV gB (B95-8 strain) with resulting expression yields (SEQ ID NOs 244-254).

[0112] Figure 42. Biophysical properties of EBV gB (B95-8 strain) construct designs (SEQ ID NOs 123, and 244-254).

[0113] Figure 43. nsEM micrographs of pre-fusion-stabilised and post-fusion EBV gB constructs. Quantification and classification of particle conformations shows a high abundance of pre-fusion-stabilised population in the selected EBV gB constructs.Figure 44. List of selected HHV gB construct designs and their properties (SEQ ID NOs: 16, 61, 64, 161, 162).

[0114] Figure 45. Biophysical properties of constructs with varying core mutations. Additional efforts to explore different core interface mutations show that prefusion stabilisation can be achieved using various residue locations within the core interfaces of gB. These designs include de novo MPR sequences from previous gB constructs (SEQ ID NOs: 61 and 154-167, 199, 201, 206, 208, 217, 219, 220, 221, 123, 246, 249, 251).

[0115] Figure 46. Bar diagram showing the relative lengths of different protein segments in herpesvirus glycoprotein B. Signal sequences (signal), ectodomains (ectodomain) membrane proximal regions (MPR), transmembrane domains (TMD), and cytoplasmic domain (CTD). The exact amino acid number and amino length of the whole gB and therefore gene segment boundaries differ over individual herpesvirus strains, but the location of the MPR is always preceding the TM Domain.

[0116] Detailed description of the invention

[0117] Herpesviruses

[0118] Herpesviruses are a large group of DNA viruses. As of 2020, the order Herpesvirales is made up of three families: the Alloherpesvirdae, Orthoherpesvirdae and Malacoherpesvirdae. The Orthoherpesvirdae family, which infects amniotes including bird and mammals, comprises three subfamilies: the Alphaherpesvirinae, Betahersevirinae and the Gammaherpesvirinae. These subfamilies comprise 23 genera, for example, the Simplexviruses, the Cytomegaloviruses and the Lymphocryptoviruses, and 130 species, for example, Herpes simplex virus type 1 (HSV-1), a Simplex virus within the Alphaherpesvirus subfamily, Human cytomegalovirus (HCMV), a Cytomegalovirus within the Betaherpesvirus subfamily, and Epstein Barr virus (EBV), a lymphocryptovirus within the Gammaherpesvirus subfamily. The term “Herpesvirus” as used herein refers to members of the order Herpesvirales. In particular, the Herpesvirus may be a member of the Orthoherpesvirdae family, for example Alphaherpesvirinae (alphaherpesvirus), Betahersevirinae (betaherpesvirus) or Gammaherpesvirinae (gammaherpesvirus).

[0119] Herpesviruses typically establish latent infections, but may reactivate frequently, and represent a particular risk to children, pregnant and immunocompromised individuals. Members of the Herpesvirus family infecting humans include Herpes Simplex Virus (HSV-1 and HSV-2), causing oral and genital herpes; Varicella-Zoster Virus (VZV), causing chickenpox and shingles; Epstein-Barr Virus (EBV), which is linked to infectiousmononucleosis and certain cancers; Cytomegalovirus (CMV), which is linked to birth defects, including congenital hearing loss; and Human Herpesvirus 6 and 7 (HHV-6, HHV-7) which are associated with childhood illnesses, and opportunistic infections in immunosuppressed individuals.

[0120] Herpesviruses are also known to infect non-human mammals. Such viruses include Canine Herpesvirus, causing a severe, often fatal, disease of puppies; Equine Herpesvirus (EHV), also known as equine rhinopneumonitis, a family of highly contagious viruses found in horses worldwide and feline Herpesvirus type-1 (FHV-1) causing feline viral rhinotracheitis (FVR), an infectious disease of cats.

[0121] There are currently no approved vaccines for many of these viruses, including HSV, CMV and EBV. There is therefore a need for effective vaccines and therapies against Herpesviruses, as well as reagents for use in research and diagnostics.

[0122] The Herpesvirus glycoprotein B (gB) is a structurally conserved, class III membrane fusion protein. It catalyses the fusion of the virus with the cellular membrane. This requires significant structural rearrangements from the metastable “pre-fusion” conformation to a stable “postfusion” conformation.

[0123] The domains DI, Dll, Dill, DIV and DV and a Membrane Proximal Region are conserved across all virus class III fusion proteins, including all Herpesvirus gB proteins, as shown in e.g. Vollmer et al. (HSV-1), Oliver et al. 2020 (VZV), and Backovic et al. (EBV).

[0124] The term “Herpesvirus gB” as used herein is to be understood as referring to the native Herpesvirus gB polypeptide from any member of the Herpesvirus family, and in particular, any member of the Alphaherpesvirinae, Betahersevirinae and the Gammaherpesvirinae subfamilies.

[0125] The positions and substitutions referenced herein are identified by reference to the amino acid positions in the Towne (SEQ ID NO: 1; Full virus genome sequence GenBank: FJ616285.1) and Merlin strain (SEQ ID NO: 1; Full virus genome sequence GenBank: AY446894.2) of HCMV.

[0126] These strains have identical gB sequences and are therefore used interchangeably in reference to the gB of the present disclosure. All references to substitutions in the Towne and Merlin strains should be understood to encompass corresponding positions in other gBs, and the residue position should be adjusted accordingly, based on the relevant sequence alignments.Alternatively, the positions and substitutions referenced herein may be identified by reference to the amino acid positions in the Kos strain of HSV-1 (Full virus genome sequence GenBank: JQ673480; SEQ ID NO: 100) or the B95-8 strain of EBV (Full virus genome sequence GenBank: V01555; SEQ ID NO: 108).

[0127] The terms “engineered Herpesvirus gB” and “engineered gB” as used herein refer to the engineered Herpesvirus gBs of the present disclosure. Unless stated otherwise, such engineered Herpesvirus gBs should be understood to comprise at least one amino acid substitution which stabilises the tertiary structure of the gB in a pre-fusion conformation. The engineered Herpesvirus gB may comprise further modifications in accordance with the present disclosure. The engineered Herpesvirus gB may also comprise modifications not referenced in the present disclosure, so long as such modifications do not impair stabilisation of the tertiary structure of the gB in a pre-fusion conformation.

[0128] Unless otherwise specified, the term “post-fusion gB” as used herein refers to the corresponding native (i.e. non-modified) Herpesvirus gB in its naturally occurring post-fusion conformation, or to structurally equivalent variants, for example, soluble variants excluding the TMD, or both the TMD and MPR. Structurally equivalent variants may comprise modifications, so long as such modifications do not disrupt the post-fusion conformation. In particular, the post-fusion gB may comprise one or more modifications for the purpose of manufacture, purification or detection. Such modifications include affinity tags (e.g., His tags) fluorescent tags, and radiolabels, as well as the removal of intracellular domains, the addition of trimerisation domains, and the removal of manufacturing liabilities.

[0129] The engineered Herpesvirus gB comprises at least one amino acid substitution which stabilises the tertiary structure of the gB in a pre-fusion conformation.

[0130] The membrane-proximal region (MPR) of the herpesvirus gB is a short, conserved amino acid segment located immediately external to the gB TMD, adjacent to the viral envelope, and immediately preceding the rest of the gB ectodomain. This structural definition is also reflected in the gB gene coding sequence organisation (Figure 46). Structurally, the MPR is anchored in the upper region of the virus lipid bilayer, with two amphipathic helices forming the MPR. The MPR locates below gB Domain I (DI), with which it interacts. The MPR joins at one end to Domain V of the gB ectodomain, and at the other end connects to the TMD.

[0131] The MPR forms a surface at the base of the gB ectodomain. It interacts with the virus lipid membrane, connects to the gB TMD and the rest of the ectodomain, and interacts with DI and the gB fusion loops, highlighting the complexity of the MPR form and function. The MPR has been proposed to act as a regulatory interface connecting the ectodomain with the membraneanchor, helping to coordinate conformational changes in gB to allow stability of the pre-fusion form, whilst facilitating transition to the post fusion form during membrane fusion. The MPR is rich in hydrophobic and aromatic residues, features thought to enable membrane interactions that are critical for controlling the gB pre-fusion metastable state.

[0132] The MPR has a high degree of conservation with herpesvirus subfamilies. For example, approximately 40% of the amino acid residues within the MPR are conserved among alphaherpesviruses, and some of these residues are invariant among all herpesviruses. Mutation of this highly conserved subset of amino acids in HSV-1 gB yields viruses with negligible infectivity (Wanas etal 1999).

[0133] The MPR has an essential role in viral infection, often interacting with other glycoproteins like gH / gL for receptor-mediated entry triggering membrane fusion. Recent research, including structure determination of HSV-1 pre-fusion gB, reveals the MPR's dynamic involvement in conformational changes and that it has a role in preventing premature activation of the fusion machinery, acting as a conformational “brake” to ensure pre-fusion gB stability. It is likely that the MPR is a structurally critical control module between pre- and post-fusion gB.

[0134] Because the MPR has hydrophobic surfaces, simple truncation of the native gB after the MPR results in an expressed protein that is low yield, aggregation-prone and triggers into the postfusion form of gB. Truncation of the gB before the MPR (i.e. expression of just the gB ectodomains), avoids the detrimental impact of these hydrophobic surfaces on stability and expression, but also removes the MPR association with DI and the fusion loops, similarly triggering the gB to adopt a post-fusion conformation.

[0135] Previous approaches to stabilise the soluble gB in a pre-fusion conformation have therefore involved truncating the MPR, and either introducing core mutations to stabilise the gB in a pre-fusion-like conformation (e.g. Sponholtz et ai, using a small molecule to bind and stabilise the gB in a pre-fusion-like conformation (e.g. Liu et ai, or expressing gB bound to a vesicle like particle (Vollmer et al).

[0136] In contrast, the present disclosure provides non-native MPR structures, and methods for designing non-native MPR structures. The methods disclosed herein allow the critical structural and functional attributes of the native MPR to be maintained, whilst simultaneously enhancing expression of the soluble protein, and ensuring enhanced gB pre-fusion stability. The MPRs design strategies and non-native MPRs provided herein therefore preserve crucial functions whilst simultaneously yielding an effective vaccine antigen.Consequently, the present disclosure provides methods to redesign the MPRs of any Herpesvirus, whilst maintaining the properties of DI and fusion loop structural interaction, maintaining and enhancing the properties of pre-fusion stabilisation and removing the hydrophobic M PR patches to promote protein expression, expression yields and remove MPR driven aggregation.

[0137] In its pre-fusion conformation, gB associates with two other gBs to form a compact homotrimer where the domains DI, Dll, Dill, DIV, and DV are orientated so that Dill forms a trimer apex with Dll packing at the side of Dill and DIV located at the base of Dill (Figure 22). DI is located at the base of the trimeric structure, below Dll and packed alongside DIV and DV, with the fusion loops of DI located near the virus lipid envelope (Figure 22).

[0138] The post-fusion form of gB has been visualised as an extended structure, where DIV now forms one end of the structure being located at the end of a rearranged Dill (Figure 22). DI is proposed to be in one of two locations, either with the fusion loops embedded in the host cell lipid membrane (and DV location is not defined), or in a structure where DI is associated with the virus membrane, even though gB is now in the post-fusion conformation (Figure 22). Irrespective of this, the post-fusion gB conformation is defined by an elongated and rearranged Dill and the relocation of DIV away from DI (Figure 22). The MPR of pre-fusion gB is located next to the virus lipid membrane and interacting with DI.

[0139] The engineered gBs of the present disclosure may comprise a non-native MPR. The MPR is located at the C terminal end of the gB protein, and corresponds to amino acid residues 705-752 of the Towne and Merlin strains of HCMV (SEQ ID NO: 1), amino acid residues 725-775 of the KOS strain of HSV-1 (SEQ ID NO: 100), and amino acid residues 684-733 of the B95-8 strain of EBV (SEQ ID NO: 108). The locations of the MPRs of other Herpesvirus gBs are known in the art.

[0140] In one aspect, provided herein is a method of increasing the probability of a Herpesvirus gB remaining in a pre-fusion conformation, wherein the Herpesvirus gB comprises the domains DI, Dll, Dill, DIV and DV and an MPR, the method comprising:

[0141] a) replacing the native MPR with a non-native MPR; or

[0142] b) introducing into the native MPR at least one amino acid substitution.

[0143] The non-native MPR may be engineered to interact with the DI domain of the gB more strongly than the native MPR, or the at least one amino acid substitution may increase the strength of the interaction between the MPR and the DI domain of the gB.Alternatively, or in addition, the non-native MPR may be engineered to have reduced surface hydrophobicity relative to the native MPR or the at least one amino acid substitution may reduce the surface hydrophobicity relative to the native MPR.

[0144] The present disclosure further provides a method of increasing the probability of a Herpesvirus gB remaining in a pre-fusion conformation, wherein the Herpesvirus gB comprises the domains DI, Dll, Dill, DIV and DV and an MPR, the method comprising:

[0145] a) replacing the native MPR with a non-native MPR engineered to interact with the DI domain of the gB more strongly than the native MPR; or

[0146] b) introducing into the native MPR at least one amino acid substitution which increases the strength of the interaction between the MPR and the DI domain of the gB.

[0147] The MPR may further interact with the DIV and / or DV domain. The DI, Dll, Dill, DIV and / or DV domains may be stabilised in their pre-fusion conformation without interacting directly with the MPR. The at least one amino acid substitution may be a substitution of one or more hydrophobic residues at a solvent interface. The gB may further comprise one or more additional stabilising substitutions. The additional stabilising substitutions may be selected from cavity filling substitutions, electrostatic substitutions and / or H-bonding network substitutions. The one or more additional stabilising substitutions may be cysteine mutations, for example cysteine substitutions which introduce one or more disulfide bridges at the DI, Dll, MPR and / or core interfaces. The one or more of the disulfide bridges may be between DI and the MPR. The one or more of the additional stabilising substitutions may be outside of the MPR.

[0148] Further provided herein is an engineered Herpesvirus gB comprising at least the domains DI, Dll, Dill, DIV and DV and an MPR, wherein the MPR is a non-native MPR and comprises at least one amino acid substitution which stabilises the tertiary structure of the gB in its prefusion conformation.

[0149] The engineered gB provided herein may further comprise a trimerisation domain. The non-native MPR may interact with the DI domain. The non-native MPR may interact with the DI domain more strongly than the native MPR. One or more of the solvent-facing amino acid residues in the native MPR may be substituted with amino acid residues which increase the polarity of the MPR. The pre-fusion conformation may be detected using an antibody specific for an epitope on the pre-fusion conformation which may be not present in the post-fusion conformation. The engineered gB may have an increased probability of being in the pre-fusion conformation under physiological conditions.The pre-fusion conformation may be detected by nsEM, cryoEM or X-ray crystallography. The pre-fusion conformation may be detected using an antibody specific for an epitope on the prefusion conformation which may be not present on the post-fusion conformation. The MPR may further interact with the DIV and / or DV domain.

[0150] In a further aspect, provided herein is a conjugate comprising the engineered gB provided herein, and a further moiety or conjugation substrate.

[0151] The further moiety may be selected from a detection moiety, a solid support, an antibody, an antibody fragment, or a binding molecule. The solid support may be a nanoparticle, chip, column, membrane, or virus-like particle, or the detection moiety may be a fluorescent tag, a magnetic tag, or a radiolabel. The virus-like particle may be a virus-like particle with a lipid membrane (i.e. an enveloped virus-like particle) or a virus-like particle without a lipid membrane (i.e. a non-enveloped virus-like particle). For example, the virus-like particle may comprise only proteins.

[0152] The disclosure further provides an immune reagent comprising the engineered gB provided herein. The immune reagent may be used to screen and identify biologies that inhibit or alter gB function. The biologic may be a peptide, a nanobody, or an aptamer. The immune reagent may be used to screen and identify small molecule drugs that inhibit or alter gB function. Alternatively, the immune reagent may be used to screen and identify monoclonal antibodies that inhibit or alter gB function.

[0153] The disclosure further provides an engineered gB or a trimer for use as a vaccine antigen. The disclosure further provides an engineered gB or a trimer for use as an immune stimulatory molecule.

[0154] In a further aspect, provided herein is a vaccine comprising an engineered gB of the disclosure.

[0155] In a further aspect, provided herein is a vaccine comprising a nucleic acid encoding the engineered gB of the disclosure.

[0156] The engineered gB may be encoded by mRNA, DNA, or saRNA. The vaccine may comprise one or more further Herpesvirus components. The vaccine may produce a B-cell response and / or a T-cell response.

[0157] In a further aspect, provided herein is a pharmaceutical composition comprising an engineered gB of the disclosure and a pharmaceutically acceptable carrier or adjuvant.In a further aspect, provided herein is a pharmaceutical composition comprising an engineered gB of the disclosure and a pharmaceutically acceptable carrier or adjuvant for use in the treatment or prevention of a Herpesvirus infection or a disease associated therewith.

[0158] In a further aspect, provided herein is a an engineered gB of the disclosure for use in the treatment or prevention of a Herpesvirus infection or a disease associated therewith.

[0159] In a further aspect, provided herein is a pharmaceutical composition comprising the engineered gB and a pharmaceutically acceptable carrier or adjuvant for use in the treatment or prevention of Herpesvirus infection or a disease associated therewith.

[0160] In a further aspect, provided herein is a method of preventing Herpesvirus infection or a disease associated therewith in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition provided herein.

[0161] The engineered gB may be for use in screening and identifying biologies and small molecules that inhibit or alter gB function. The biologic may be a peptide, a nanobody, a monoclonal antibody, or an aptamer.

[0162] In a further aspect, provided herein is a method of designing a non-native MPR for stabilising a Herpesvirus gB in a pre-fusion conformation, the method comprising:

[0163] a) generating a plurality of modified MPR sequences;

[0164] b) generating two or more replicate structures from each modified MPR sequence;

[0165] c) determining the agreement between the generated structures (inter-Root Mean Square Deviation (RMSD));

[0166] d) determining the agreement between the generated MPR structures and the structure of the initial MPR sequence (target-RMSD);

[0167] e) clustering the modified MPR sequences on the basis of at least the inter-RMSD and target- RMSD values; and

[0168] f) selecting structures in clusters having the lowest target-RMSD in combination with the lowest inter-RMSD,

[0169] wherein the target-RMSD has a maximum value of 10 angstroms (A) and the inter-RMSD has a maximum value of 4 angstroms (A).

[0170] The target RMSD may be less than 2.0 A. The inter-RMSD may be less than 3.0 A. The modified MPR sequences may also be clustered on the basis of predicted local distance difference test (pLDDT) value and / or hydropathy. The modified MPR sequences may have a pLDDT value of more than 80 and / or a hydropathy value of between -0.5 and 0.5.The modified MPR sequences may also be clustered on the basis of one or more of the instability index, the interface shape complementarity (e.g. Rosetta Shape Complementarity or an equivalent metric from any suitable software) and / or the interface energy (dG) (e.g. Rosetta Interface Energy or an equivalent metric from any suitable software).

[0171] The modified MPR sequences may therefore be selected on the basis of their target RMSD, inter-RMSD, predicted Local Distance Difference Test (pLDDT) threshold, hydropathy, instability index, shape complementarity (e.g. Rosetta Shape Complementarity) and / or interface energy (dG) (e.g. Rosetta Interface Energy).

[0172] For example, the selected MPR sequences may have a predicted pLDDT value greater than 80, a hydropathy index less than 0.5, an instability index less than 40, a shape complementarity (e.g. Rosetta Shape Complementarity) greater than 0.6, and / or an interface energy (dG) (e.g. Rosetta Interface Energy) greater than the mean dG of all designed sequences.

[0173] The methods for designing a non-native MPR provided herein may be repeated in an iterative process to result in optimised engineered gB sequences containing optimised non-native MPRs that stabilise the engineered gB in a pre-fusion conformation. The method may be repeated two or more times. For example, the method may be repeated up to 3 times, up to 5 times, up to 10 times, up to 15 times or more than 15 times.

[0174] MPR domains

[0175] The engineered gBs of the present disclosure comprise a non-native MPR domain. The non-native MPR interacts with at least the DI domain to stabilise the gB in its pre-fusion form. The non-native MPR may further interact with the DIV and / or DV domain. Alternatively, or in addition, one or more of the DI, Dll, Dill, DIV and / or DV domains may be stabilised in their pre-fusion conformation via interactions which do not involve the MPR. For example, the DI domain may interact with the DIV and / or DV domain.

[0176] By increasing the strength of interaction, it is meant that the one or more substitutions introduce one or more new intermolecular interactions or increase the strength of one of more existing intermolecular interactions.

[0177] For example, the one or more substitutions may introduce a new or stronger covalent or non-covalent interaction or may replace a non-covalent interaction with a covalent interaction. The interaction may be a hydrogen bond, a salt bridge, an electrostatic bond, a Van der Waals Contact, a hydrophobic interaction, or a disulphide bond. The substitution may reduce the conformational flexibility or increase the contact surface between the two domains.A stronger interaction may be understood to refer to an inter-amino-acid interaction with a higher binding energy. The binding energies of inter-amino-acid interactions are determined by on side-chain-sidechain, side-chain-main-chain, and main-chain-main-chain interactions. Inter-amino-acid interaction may be defined by free energies of association (AGA) or binding affinity (KD) profiles, which are linked by the formula GA = RT In KD. Methods for measuring such interactions are provided in Du et al.

[0178] Alternatively, or in addition, the residues in the native MPR may be substituted with amino acid residues which increase the expression yields of the construct. In some instances, the substitutions may increase both the polarity of the MPR, and the expression yields of the construct.

[0179] Alternatively, or in addition, the residues in the native MPR may be substituted with amino acid residues which reduce the surface hydrophobicity of the MPR.

[0180] The substitutions may increase the expression yield of the gB. The substitutions increase the expression yield of gB to at least 0.01 mg / L, at least 0.02 mg / L, at least 0.03 mg / L, at least 0.04 mg / L, at least 0.05 mg / L, at least 0.06 mg / L, at least 0.07 mg / L, at least 0.08 mg / L, at least 0.09 mg / L, at least 0.1 mg / L, at least 0.2 mg / L, at least 0.3 mg / L, at least 0.4 mg / L, or at least 0.5 mg / L.

[0181] The substitutions may increase the percentage of soluble gB particles in the pre-fusion conformation. The substitutions may increase the percentage of soluble gB particles in the pre-fusion conformation to at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

[0182] The substitutions may increase the hydrodynamic radius (Rh). The hydrodynamic radius may be measured by any suitable assay known in the art, for example, a dynamic light scattering (DLS) assay. The substitutions may increase the Rh (nm) to at least 8 nm, at least 10 nm, at least 11 nm, at least 12 nm, or at least 13 nm. The hydrodynamic radius may be measured by any suitable assay known in the art. In particular instances, the hydrodynamic radius is measured using a dynamic light scattering (DLS) assay. Thus, the substitutions may increase the Rh (nm) to at least 8 nm, at least 10 nm, at least 11 nm, at least 12 nm, or at least 13 nm when measured using a dynamic light scattering (DLS) assay.

[0183] The substitutions may increase aggregation temperature (Tagg). The substitutions may increase Taggto at least 60°C, at least 65°C, at least 70°C, at least 75°C, or at least 80°C. The aggregation temperature may be measured by any suitable assay known in the art. In particular instances, the aggregation temperature is measured using a differential static lightscattering (DSLS) assay. Thus, the substitutions may increase Taggto at least 60°C, at least 65°C, at least 70°C, at least 75°C, or at least 80°C when measured using a differential static light scattering (DSLS) assay.

[0184] The pre-fusion conformation may be detected using an antibody. The antibody may be specific for the pre-fusion conformation, for example, nanobody Nb1 (Vollmer et al.) which is specific to the prefusion gB and neutralises HSV-1 and HSV-2. Other herpesvirus antibodies include the anti-Domain I antibodies, such as HDIT102 which recognises an epitope on DI of the HSV-1 gB and anti-Domain II antibodies, such as 3A3, which recognises an epitope on Dll of the EBV gB.

[0185] One or more of the solvent-facing amino acid residues in the native MPR may be substituted with amino acid residues which increase the polarity of the MPR. One or more hydrophobic solvent facing amino acid residues in the native MPR may be substituted with a polar or charged amino acid residues. The hydrophobic amino acid may be I, L, A, V or F and the substitution may be N, S, T, Q, Y, C, K, R or E. The hydrophobic amino acid may be L, A, V or F and / or the substitution may be K, R or E. K, R or E substitutions are particularly advantageous, due to their strong helical propensities. Alternatively, or in addition, one or more DI interfacing amino acid residues may be conserved.

[0186] The gB may be from any member of the Herpesvirus family. All references to substitutions within specific strains of a species, such as the HMCV strains Towne, Merlin, AD169 and VR1814, the HSV-1 strain KOS, or the EBV strain B95-8, should therefore be understood to encompass corresponding positions in other related Herpesvirus strains within a species, genera or subfamily, and the residue position should be adjusted accordingly, based on the relevant sequence. Appropriate adjustments to encompass corresponding positions in related herpesvirus strains, for example strains of the same species, can be determined using standard bioinformatics techniques or other methods known to the skilled person.

[0187] The Herpesvirus may be a member of the Herpesviridae family. The Herpesvirus may be an alphaherpesvirus, a betaherpesvirus or a gammaherpesvirus. The Herpesvirus may be a vertebrate infecting Herpesvirus. The Herpesvirus may infect humans. The Herpesvirus may be selected from the human Herpesviruses HSV-1, HSV-2, VZV, HCMV, HHV-6, HHV-7, EBV and KSHV / HHV8.

[0188] The Herpesvirus may infect non-human vertebrates. The non-human vertebrates may be domestic animals. The Herpesvirus may be a Herpesviruses that infects domesticated animals. Alternatively, the Herpesvirus may be a Herpesviruses that infects non-domesticated animals. Domesticated animals include pets, such as cats, dogs, rabbits, mice, rats, guineapigs, hamsters and birds, as well as domestic farm animals such as cows, pigs, horses, goats, chickens, ducks, geese, quails and turkeys.

[0189] Accordingly, the Herpesvirus may be selected from pseudorabies virus (causing Aujeszky's disease in pigs); bovine Herpesvirus 1 (causing bovine infectious rhinotracheitis and pustular vulvovaginitis); or Marek’s disease virus (causing Marek's disease ('MD' or 'fowl paralysis') causing disease in poultry species such as chickens, quails and turkeys); Canine Herpesvirus (an infection causing a severe, often fatal, disease of puppies); Equine Herpesvirus (EHV), also known as equine rhinopneumonitis, (a family of highly contagious viruses found in horses worldwide); orfeline Herpesvirus type-1 (FHV-1) (causing feline viral rhinotracheitis (FVR), an infectious disease of cats).

[0190] Alternatively, the Herpesvirus may be a Herpesviruses that infects humans. Herpesviruses are associated with a variety of diseases in humans, ranging from mild diseases such as cold sores and chickenpox to severe diseases such as encephalitis, cancer and birth defects. There are nine known human Herpesviruses. These are Herpes Simplex Virus 1 (HSV-1), which is most commonly associated with oral herpes (cold sores); Herpes Simplex Virus 2 (HSV-2), which is most commonly associated with genital herpes; Varicella-Zoster Virus (VZV / HHV-3) which is most commonly associated with chickenpox and shingles; Epstein-Barr Virus (EBV / HHV-4), which is most commonly associated with infectious mononucleosis, as well as being linked to certain cancers; Human Cytomegalovirus (HCMV / HHV-5), which typically causes mild flu-like illness but may cause severe illness in babies and immunocompromised individuals; Human Herpesvirus 6A (HHV-6A) which is associated with neuroinflammatory diseases; Human Herpesvirus 6B (HHV-6B) which is associated with roseola, a childhood rash; Human Herpesvirus 7 (HHV-7), which is associated with roseola as well as and febrile seizures, and Kaposi's Sarcoma-Associated Herpesvirus (KSHV / HHV-8) which has been linked to Kaposi's sarcoma and other cancers.

[0191] The Herpesvirus gB of the disclosure may be selected from an HSV-1 gB, an HSV-2 gB, a VZV gB, an EBV gB, an HCMV gB, an HHV-6A gB, an HHV-6B gB, an HHV-7 gB, and a KSHV / HHV-8 gB.

[0192] Alternatively, the Herpesvirus gB of the disclosure may be selected from any amniote infecting Herpesvirus. In particular, the gB may be an alphaherpesvirus gB, a betaherpesvirus gB or a gammaherpesvirus gB.In one aspect, there is provided a method of stabilising the tertiary structure of the Herpesvirus gB in a pre-fusion conformation, wherein the Herpesvirus gB comprises the domains DI, Dll, Dill, DIV and DV and a Membrane Proximal Region (MPR), the method comprising:

[0193] a) replacing the native MPR with a non-native MPR engineered to interact with the DI domain of the gB more strongly than the native MPR; or

[0194] b) introducing into the native MPR at least one amino acid substitution which increases the strength of the interaction between the DI domain of the gB and the MPR.

[0195] In one aspect, there is provided method of increasing the probability of a Herpesvirus gB remaining in a pre-fusion conformation, wherein the gB comprises the domains DI, Dll, Dill, DIV and DV and an MPR, the method comprising:

[0196] a) replacing the native MPR with a non-native MPR engineered to interact with at least one further DI domain more strongly than the native MPR; or

[0197] b) introducing into the native MPR at least one amino acid substitution which increases the strength of the interaction between the MPR and at least one further DI domain.

[0198] The method may be a method of increasing the probability of a Herpesvirus gB remaining in a pre-fusion conformation in vitro. The method may be a method of increasing the probability of a Herpesvirus gB remaining in a pre-fusion conformation in vivo. The method may be a method of increasing the probability of a Herpesvirus gB remaining in a pre-fusion conformation both in vitro and in vivo. For example, the gB may be stabilised in a pre-fusion conformation for use as a vaccine antigen in vivo. Alternatively, the gB may be stabilised in a pre-fusion conformation for use as a reagent, e.g. an immune reagent, in vitro. The Herpesvirus gBs provided herein may elicit a robust neutralising immune response in vivo. The Herpesvirus gB may be in soluble form. The non-native MPR may comprise at least 2, at least 5, at least 10, at least 15, at least 20 or more than 20 substitutions, provided the non-native MPR retains the structural and functional attributes of the native MPR. The non-native MPT may enhance expression of the soluble protein and ensuring enhanced gB pre-fusion stability.

[0199] HCMV (Human cytomegalovirus)

[0200] In some instances, the engineered is a Human cytomegalovirus (HCMV) gB. Human cytomegalovirus (HCMV) is a Cytomegalovirus within the Betaherpesvirus subfamily. HCMV can be grouped into 5 clades based on sequence diversity, GB1, GB2, GB3, GB4 and GB5. Analysis of 350 gB sequences present in Genbank show the five clades are represented by defined strains GB1 (Towne / Merlin), GB2 (AD169), GB3 (VR1814), GB4 (C194A) and GB5(UKNEQAS1) (see Figure 23). Corresponding amino acid positions in any other HCMV strain can be determined by those of ordinary skill in the art using known information and by sequence alignment using readily available and well-known alignment algorithms (such as BLAST, using default settings; ClustalW2, using default settings).

[0201] HCMV is a double stranded DNA virus. Although it typically causes mild illness in healthy individuals, it is a leading cause of congenital defects, such as deafness and developmental delay following transmission in utero. It is also a common opportunistic infection in immunosuppressed patients and may be associated with serious illness. Despite extensive research, there are currently no licensed and approved vaccines for HCMV.

[0202] The HCMV genome contains several envelope glycoproteins, including glycoprotein B (gB). HCMV gB is encoded by the LIL55 gene of the HCMV genome. gB mediates virus entry into cells by facilitating virus and cell membrane fusion, making it an important target for neutralising antibodies and vaccine development. gB is the only known Herpesvirus fusion protein, meaning all receptor binding proteins must exert cell entry via gB. During viral entry, gB undergoes a series of conformational changes, to convert from its metastable “pre-fusion” conformation to a “post-fusion” form. The HMCV genome also includes a number of other envelope glycoproteins, including gH, gL, gM, gN and gO and other virus proteins including LIL128, LIL130 and LIL131A. These proteins form complexes to bind cell surface proteins, attaching the herpesvirus particle to the cell surface. gB is then essential for inducing virus and host cell membrane fusion.

[0203] The amino acid substitution may be a substitution at one or more of amino acid residues 705-752 of the gB, relative to SEQ ID NO: 1 (corresponding to the Merlin / Towne strain of HCMV). Specifically, the substitution may be a substitution at one or more of amino acid residues 713-750 of the gB, relative to SEQ ID NO: 1.

[0204] Alternatively, the amino acid substitution may be a substitution at one or more of amino acid residues 706-751 of the gB, relative to SEQ ID NO: 5 (corresponding to the AD169 strain of HCMV) or a substitution at one or more of amino acid residues 705-750, relative to SEQ ID NO: 9 (corresponding to the VR1814 strain of HCMV).

[0205] Unless specified otherwise, the MPR sequence defined herein is defined by reference to the gB of the Towne / Merlin strain of HCMV (SEQ ID NO:1).

[0206] The MPR may comprise one or more substitutions selected from A721C, D713C, S739C, L709C, Y708C, A732C, V728, G735C, M716C and G735C. The DI domain may comprise one or more substitutions selected from V197C, L162C, Y155C, Y160C, Y160C, W240C, Y242C,Y153C, Y242C and W240C. The MPR may comprise one or more substitutions selected from A721C, D713C, S739C, L709C, Y708C, A732C, V728, G735C, M716C and G735C and the DI domain may comprise one or more substitutions selected from V197C, L162C, Y155C, Y160C, Y160C, W240C, Y242C, Y153C, Y242C and W240C.

[0207] The non-native MPR may comprise a sequence selected from any one of SEQ ID Nos: 276-311.

[0208] The non-native MPR of the gB may comprise a sequence having at least 50% identity to SEQ ID NO: 1. The non-native MPR may comprise a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from SEQ ID NO 287, 297, 289 and 298.

[0209] The non-native MPR of the gB may comprise a sequence selected from any one of SEQ ID NOs: 287, 297, 289 and 298.

[0210] The engineered HCMV gB disclosed herein may comprise one or more additional stabilising substitutions outside of the MPR.

[0211] The engineered gB of the present disclosure may comprise substitutions at positions 153-157 and 239-242, or corresponding positions, relative to SEQ ID NO: 1 (the gB of the Towne strain of HCMV). The at least one amino acid substitution may be selected from I156H, H157R, W240N and Y242T relative to SEQ ID NO: 1, or an amino acid corresponding thereto. The at least one amino acid substitution may be a substitution at one or more of residue positions 705-752 of the gB (SEQ ID NO: 1).

[0212] The engineered HCMV gB may comprise substitutions in the fusion loops of the DI domain. For example, the engineered gB may comprise substitutions at one of more of positions 1156, H157, W240 and Y242 relative to SEQ ID NO: 1 (the gB of the Towne strain of HCMV), or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from I156H, H157R, W240N and Y242T relative to SEQ ID NO: 1 (the gB of the Towne strain of CMV), or corresponding substitutions in other HCMV gBs.

[0213] The one or more additional stabilising substitutions may be cysteine mutations which introduce one or more disulfide bridges at the DI, Dll, MPR and / or core interfaces. The MPR may comprise one or more substitutions selected from A721C, D713C, S739C, L709C, Y708C, A732C, V728, G735C, M716C and G735C relative to SEQ ID NO: 1 and / or the DI domain may comprise one or more cysteine substitutions selected from V197C, L162C, Y155C,Y160C, Y160C, W240C, Y242C, Y153C, Y242C and W240C relative to SEQ ID NO: 1, or substitutions corresponding thereto.

[0214] The gB may comprise one or more substitutions is selected from I156H, H157R, W240N and Y242T relative to SEQ ID NO: 1, or substitutions corresponding thereto.

[0215] The gB may comprise:

[0216] a) the substitution C246S relative to SEQ ID NO: 1; or

[0217] b) one or both of substitutions R457S and R460S relative to SEQ ID NO: 1.

[0218] The gB may comprise one or more pairs of substitutions selected from: (a) A721C and V197C; (b) D713C and L162C; (c) S739C and Y155C; (d) L709C and Y160C; (e) Y708C and Y160C; (f) a732C and W240C; (g) V728C and Y242C; (h) G735C and Y153C; (i) M716C and Y242C; and (j) G735C and W240C relative to SEQ ID NO: 1, or substitutions corresponding thereto. The engineered HCMV gB may comprise substitutions to eliminate free cysteines. For example, the gB may comprise a substitution at C246S or a corresponding position relative to SEQ ID NO: 1 Specifically, the gB may comprise the substitution C246S or a corresponding substitution relative to SEQ ID NO: 1.

[0219] The gB may therefore comprise substitutions at some or all of positions C246, R457, R460, or corresponding positions, relative to SEQ ID NO: 1. Specifically, the gB may comprise substitutions at C246S, R457S, R460S, or a corresponding substitution, relative to SEQ ID NO: 1.

[0220] The engineered gB may comprise substitutions to eliminate furin cleavage sites. For example, the gB may comprise one or both of substitutions R457S and R460S relative to SEQ ID NO: 1.

[0221] The gB may therefore comprise substitutions at some or all of positions C246S, R457S, R460S, relative to SEQ ID NO: 1. Alternatively or in addition, the gB may comprise one or more of the pairs of substitutions H222C and E657C, V134C and I653C, or T100L and A267I relative to SEQ ID NO: 1.

[0222] The gB may comprise substitutions one or more of the pairs of positions H222 and E65C, V134 and I653, or T100 and A267, or corresponding positions, relative to SEQ ID NO: 1. Specifically, the gB may comprise one or more of the pairs of substitutions H222C and E657C, V134C and I653C, or T100L and A267I, or corresponding substitutions, relative to SEQ ID NO: 1.The MPR may comprise one or more substitutions to introduce one or more disulfide bridges at the DI, Dll, MPR and / or core interfaces. In particular, the MPR may comprise one or more substitutions to introduce one or more disulfide bridges at the DI / MPR interface.

[0223] For example, the MPR may comprise one or more substitutions selected from A721C, D713C, S739C, L709C, Y708C, A732C, V728, G735C, M716C and G735C relative to SEQ ID NO: 1, or substitutions corresponding thereto.

[0224] The DI domain may comprise one or more substitutions selected from V197C, L162C, Y155C, Y160C, Y160C, W240C, Y242C, Y153C, Y242C and W240C relative to SEQ ID NO: 1, or substitutions corresponding thereto.

[0225] The MPR may comprise one or more substitutions selected from A721C, D713C, S739C, L709C, Y708C, A732C, V728, G735C, M716C and G735C and the DI domain may comprise one or more substitutions selected from V197C, L162C, Y155C, Y160C, Y160C, W240C, Y242C, Y153C, Y242C and W240C relative to SEQ ID NO: 1, or substitutions corresponding thereto.

[0226] The engineered HCMV gB may comprise one or more pairs of substitutions selected from: a) A721C and V197C;

[0227] b) D713C and L162C;

[0228] c) S739C and Y155C;

[0229] d) L709C and Y160C;

[0230] e) Y708C and Y160C;

[0231] f) A732C and W240C;

[0232] g) V728C and Y242C;

[0233] h) G735C and Y153C;

[0234] i) M716C and Y242C; and / or

[0235] j) G735C and W240C

[0236] relative to SEQ ID NO: 1, or substitutions corresponding thereto.

[0237] The engineered HCMV gB may comprise one or more pairs of substitutions selected from: a) S739C and Y155C;

[0238] b) L709C and Y160C;

[0239] c) A732C and W240C;

[0240] d) V728C and Y242C;

[0241] e) G735C and Y153C;

[0242] f) M716C and Y242C; and / or

[0243] g) G735C and W240Crelative to SEQ ID NO: 1, or substitutions corresponding thereto.

[0244] Disulphide bridges may also be introduced into other parts of the molecule, for example, between the DIII / DIV domains. A disulfide bridge between Dill and DV may involve substitutions at positions Q527C-E634C, or positions corresponding thereto, relative to SEQ ID NO: 1, or substitutions corresponding thereto.

[0245] The engineered HCMV gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from any one of SEQ ID NOs: 55 to 99.

[0246] The engineered HCMV gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from any one of SEQ ID NOs: 60, 61, 63, 64, 77, 78, 79 or 80. The gB may have a sequence of SEQ ID NO: 60, 61, 63, 64, 77, 78, 79 or 80.

[0247] The engineered HCMV gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% identity SEQ ID NOs: 60. The engineered HCMV gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% identity SEQ ID NOs: 61. The engineered HCMV gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% identity SEQ ID NOs: 63. The engineered HCMV gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% identity SEQ ID NOs: 64. The engineered HCMV gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% identity SEQ ID NOs: 77. The engineered HCMV gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% identity SEQ ID NOs: 78. The engineered HCMV gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% identity SEQ ID NOs: 79. The engineered HCMV gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% identity SEQ ID NOs: 80.

[0248] The engineered gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% identity to a sequence selected from any one of SEQ ID NOs: 60, 61, 63 or 64.The engineered gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% identity to a sequence selected from any one of SEQ ID NOs: 55 to 99.

[0249] The engineered gB may have a sequence of SEQ ID NOs: 60. The engineered gB may have a sequence of SEQ ID NOs: 61. The engineered gB may have a sequence of SEQ ID NOs: 63. The engineered gB may have a sequence of SEQ ID NOs: 64.

[0250] The engineered gB may have an MPR sequence selected from any one of SEQ ID NOs: 287, 297, 115 or 126.

[0251] The engineered HCMV gB may have a sequence of SEQ ID NO: 60. The engineered HCMV gB may have a sequence of SEQ ID NO: 61. The engineered HCMV gB may have a sequence of SEQ ID NO: 63. The engineered HCMV gB may have a sequence of SEQ ID NO: 64. The engineered HCMV gB may have a sequence of SEQ ID NO: 77. The engineered HCMV gB may have a sequence of SEQ ID NO: 78. The engineered HCMV gB may have a sequence of SEQ ID NO: 79. The engineered HCMV gB may have a sequence of SEQ ID NOs: 80. SEQ ID Nos: 77-80 correspond to SEQ ID Nos: 60, 61, 63 and 64 but further comprise the substitutions C246S, R457S and R460S.

[0252] The gB may also comprise DI / DII hinge stabilisation mutations, for example at position A293P relative to SEQ ID NO: 1, or substitutions corresponding thereto.

[0253] In alternative instances, the gB may comprise substitutions at some or all of positions A239, A239, A267, A293, A369, A732, A97, C246, D277, D320, D478, E274, E274, E286, E289, E321, E634, E657, E671, E679, F683, G173, G177, G735, H222, H681, I636, I89, I90, K124, K158, K158, K214, K340, K359, K394-T452, L288, L484, L641, L678, L709, M677, M677, N132, N132, N341, N635, N657, N658, Q483, Q501, Q527, Q669, R180, R180L, R291, R429, R431, R432, R457, R460, S164, S244, S269, S362, S367, S631, S641, S641, S739, T100, T225C, T225F, T292P, T343, T572C, T630, T659, T676, T676, V342, V363, V480, V684, V728, W240, W356, Y153, Y155, Y160, Y242, Y464-T498, Y481 and / or or corresponding positions, relative to SEQ ID NO: 1.

[0254] Specifically, the gB may comprise A239C, A239W, A267V, A293P, A369L, A732C, A97V, C246S, D277C, D320L, D478L, E274I, E274V, E286F, E289P, E321I, E634C, E657C, E671C, E679C, F683C, G173C, G177C, G735C, H222C, H681C, I636C, I89C, I90C, K124V, K158C, K158I, K214F, K340P, K359P, K394-T452del, L288P, L484P, L641C, L678C, L709C, M677I, M677L, N132C, N132V, N341P, N635T, N657C, N658C, Q483P, Q501I, Q527C, Q669C, R180I, R180L, R291P, R429S, R431S, R432S, R457S, R460S, S164C, S244C,S269C, S362P, S367I, S631C, S641C, S641L, S739C, T100I, T225C, T225F, T292P, T343P, T572C, T630C, T659L, T676C, T676I, V342P, V363P, V480P, V684W, V728C, W240C, W356P, Y153C, Y155C, Y160C, Y242C, Y464-T498del, Y481P and / or a corresponding substitution, relative to SEQ ID NO: 1.

[0255] The engineered gB may comprise substitutions at one or more of positions S269, E657, R457 and R460 or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from S269C, E657C, R457S and R460S.

[0256] The engineered gB may comprise substitutions at one or more of positions N132, S641, R457 and R460 or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from N132C, S641C, R457S and R460S.

[0257] The engineered gB may comprise substitutions at one or more of positions H222, E657, A97, T659, N132, S641, R457and R460 or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, A97V, T659L, N132V, S641L and R460S.

[0258] The engineered gB may comprise substitutions at one or more of positions H222, E657, T100, A267, N132, S641, R457 and R460 or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, T100I, A267V, N132V, S641L and R460S.

[0259] The engineered gB may comprise substitutions at one or more of positions H222, E657, T100, A267, A369, Q501, R457 and R460, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, T100I, A267V, A369L, Q501I and R460S.

[0260] The engineered gB may comprise substitutions at one or more of positions H222, E657, A97, T659, N132, S641, S367, R457 and R460 or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, A97V, T659L, N132V, S641L R457S and R460S.

[0261] The engineered gB may comprise substitutions at one or more of positions H222, E657, T100, A267, N132, S641, K340, N341, R457 and R460or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, T100I, A267V, N132V, S641L N341P, R457S and R460S.

[0262] The engineered gB may comprise substitutions at one or more of positions H222, E657, T100, A267, N132, S641, V342, T343, R457 and R460or corresponding positions in other HCMVgBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, T100I, A267V, N132V, S641L T343P, R457S and R460S.

[0263] The engineered gB may comprise substitutions at one or more of positions H222, N657, S164, Q669, T100, A267, R457 and R460, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, N657C, S164C, Q669C, T100I, A267V and R460S.

[0264] The engineered gB may comprise substitutions at one or more of positions H222, N657, S164, E671, T100, A267, R457 and R460, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, N657C, S164C, E671C, T100I, A267V and R460S.

[0265] The engineered gB may comprise substitutions at one or more of positions V480, Y481, R457 and R460, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from V480P, Y481P, R457S and R460S. The engineered gB may comprise substitutions at one or more of positions Q483, L484, R457 and R460, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from Q483P, L484P, R457S and R460S. The engineered gB may comprise substitutions at one or more of positions H222, E657, T100, A267, N132, S641, V480, Y481, R457 and R460 or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, T100I, A267V, N132V, S641L, Y481P, R457S, R460S.

[0266] The engineered gB may comprise substitutions at one or more of positions H222, E657, T100, A267, N132, S641, Q483, L484, R457 and R460 or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, T100I, A267V, N132V, S641L, L484P, R457S and R460S.

[0267] The engineered gB may comprise substitutions at one or more of positions S269, N658, T100, A267, A369, Q501, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from S269C, N658C, T100I, A267V, A369L, Q501I, R460S and C246S.

[0268] The engineered gB may comprise substitutions at one or more of positions S269, N658, T100, A267, N132, S641, V342, T343, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from S269C, N658C, T100I, A267V, N132V, S641L, T343P, R457S, R460S and C246S.The engineered gB may comprise substitutions at one or more of positions S269 N658, S164, Q669, T100, A267, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from S269C, N658C, S164C, Q669C, T100I, A267V, R460S and C246S.

[0269] The engineered gB may comprise substitutions at one or more of positions S269, N658, S164, E671, T100, A267, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from S269C, N658C, S164C, E671C, T100I, A267V, R460S and C246S.

[0270] The engineered gB may comprise substitutions at one or more of positions S269, N658, T100, A267, N132, S641, V480, Y481, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from S269C, N658C, T100I, A267V, N132V, S641L, Y481P, R457S, R460S and C246S. The engineered gB may comprise substitutions at one or more of positions H222, E657, T100, A267, A369, Q501, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, T100I, A267V, A369L, Q501I, R460S and C246S.

[0271] The engineered gB may comprise substitutions at one or more of positions H222, E657, T100, A267, N132, S641, V342, T343, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, T100I, A267V, N132V, S641L, T343P, R457S, R460S and C246S. The engineered gB may comprise substitutions at one or more of positions H222, E657, S164, Q669, T100, A267, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, S164C, Q669C, T100I, A267V, R460S and C246S.

[0272] The engineered gB may comprise substitutions at one or more of positions H222, E657, S164, E671, T100, A267, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, S164C, E671C, T100I, A267V, R460S and C246S.

[0273] The engineered gB may comprise substitutions at one or more of positions H222, E657, T100, A267, N132, S641, V480, Y481, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, T100I, A267V, N132V, S641L, Y481P, R457S, R460S and C246S.The engineered gB may comprise substitutions at one or more of positions S269, N658, T100, A267, A369, Q501, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from S269C, N658C, T100I, A267V, A369L, Q501I, R460S and C246S.

[0274] The engineered gB may comprise substitutions at one or more of positions S269, N658, T100, A267, N132, S641, V342, T343, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from S269C, N658C, T100I, A267V, N132V, S641L, T343P, R457S, R460S and C246S. The engineered gB may comprise substitutions at one or more of positions S269, N658, S164, Q669, T100, A267, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from S269C, N658C, S164C, Q669C, T100I, A267V, R460S and C246S.

[0275] The engineered gB may comprise substitutions at one or more of positions S269, N658, S164, E671, T100, A267, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from S269C, N658C, S164C, E671C, T100I, A267V, R460S and C246S.

[0276] The engineered gB may comprise substitutions at one or more of positions S269, N658, T100, A267, N132, S641, V480, Y481, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from S269C, N658C, T100I, A267V, N132V, S641L, Y481P, R457S, R460S and C246S. The engineered gB may comprise substitutions at one or more of positions H222, E657, T100, A267, N132, S641, V342, T343, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, T100I, A267V, N132V, S641L, T343P, R457S, R460S and C246S. The engineered gB may comprise substitutions at one or more of positions H222, E657, S164, Q669, T100, A267, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from H222C, E657C, S164C, Q669C, T100I, A267V, R460S and C246S.

[0277] The engineered gB may comprise substitutions at one or more of positions S269, N658, T100, A267, N132, S641, V342, T343, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from S269C, N658C, T100I, A267V, N132V, S641L, T343P, R457S, R460S and C246S.The engineered gB may comprise substitutions at one or more of positions S269 N658, S164, Q669, T100, A267, R457, R460 and C246, or corresponding positions in other HCMV gBs. Specifically, the engineered gB may comprise one or more substitutions selected from S269C, N658C, S164C, Q669C, T100I, A267V, R460S and C246S.

[0278] The amino acid substitutions may be at positions C246S, R457S, R460S, L709C and Y160C relative to SEQ ID NO: 1. The amino acid substitutions may be at positions C246S, R457S, R460S, S739C and Y155C relative to SEQ ID NO: 1. The amino acid substitutions may be at positions C246S, R457S, R460S, A732C and W240C relative to SEQ ID NO: 1. The amino acid substitutions may be at positions C246S, R457S, R460S, V728C and Y242C relative to SEQ ID NO: 1. The amino acid substitutions may be at positions C246S, R457S, R460S, G735C and Y153C relative to SEQ ID NO: 1. The amino acid substitutions may be at positions G177C, E634C, R429S, R431S and R432S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions I90C, T630C, R429S, R431S and R432S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions G177C. E634C, I90C, R429S, R431S, R432S and T630C relative to SEQ ID NO: 1. The amino acid substitutions may be at positions S269C, E657C, R457S and R460S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions N132C, S641C, R457S and R460S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions H222C, E657C, A97V, T659L, N132V, S641L, R457S and R460S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions H222C, E657C, T100I, A267V, N132V, S641L, R457S and R460S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions H222C, E657C, T100I, A267V, A369L, Q5011, R457S and R460S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions H222C, E657C, A97V, T659L, N132V, S641L, S367I, R457S and R460S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions H222C, E657C, T100I, A267V, N132V, S641L, K340P, N341P, R457S and R460S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions H222C, E657C, T100I, A267V, N132V, S641L, V342P, T343P, R457S and R460S relative to SEQ ID NO: 1.

[0279] The amino acid substitutions may be at positions H222C, N657C, S164C, Q669C, T100I, A267V, R457S and R460S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions H222C, N657C, S164C, E671C, T100I, A267V, R457S and R460S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions V480P, Y481P, R457S and R460S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions Q483P, L484P, R457S and R460S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions H222C, E657C, T100I, A267V, N132V, S641L, V480P, Y481P, R457S and R460S relative toSEQ ID NO: 1. The amino acid substitutions may be at positions H222C, E657C, T100I, A267V, N132V, S641L, Q483P, L484P, R457S and R460S relative to SEQ ID NO: 1.

[0280] The amino acid substitutions may be at positions G173C, L641C, R429S, R431S and R432S, relative to SEQ ID NO: 1. The amino acid substitutions may be at positions T225C, I636C, R429S, R431S and R432S, relative to SEQ ID NO: 1. The amino acid substitutions may be at positions G173C, L641C, T225C, E634C, R429S, R431S, R432S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions G173C, L641C, T225C, I636C, R429S, R431S, R432S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions G173C, L641C, I89C, S631C, R429S, R431S, R432S relative to SEQ ID NO: 1.

[0281] The amino acid substitutions may be at positions G173C, L641C, T225C, I636C, R180L, E321I, D478L, R429S, R431S, R432S and relative to SEQ ID NO: 1. The amino acid substitutions may be at positions G173C, L641C, R180I, T225F, N635T, D320L, D478L, R429S, R431S, R432S and relative to SEQ ID NO: 1. The amino acid substitutions may be at positions G173C, L641C, T225C, I636C, K214F, D320L, D478L, R429S, R431S, R432S and relative to SEQ ID NO: 1. The amino acid substitutions may be at positions G173C, L641C, T225C, I636C, K124V, D320L, D478L, R429S, R431S, R432S and relative to SEQ ID NO: 1. The amino acid substitutions may be at positions G173C, L641C, T225C, I636C, I89C, S631C, R180L, E321I, D478L, R429S, R431S and R432S relative to SEQ ID NO: 1.

[0282] The amino acid substitutions may be at positions G173C. L641C, T225C, I636C, L288P, E289P, R429S, R431S and R432S, relative to SEQ ID NO: 1. The amino acid substitutions may be at positions G173C, L641C, T225C, I636C, R291P, T292P, R429S, R431S and R432S, relative to SEQ ID NO: 1. The amino acid substitutions may be at positions K394-T452del, A293P, Q527C, E634C, R429S, R431S and R432S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions K158C, T676C, S244C and H681C relative to SEQ ID NO: 1. The amino acid substitutions may be at positions K158C, T676C, A239C and F683C relative to SEQ ID NO: 1. The amino acid substitutions may be at positions D277C, L678C, S244C and H681C relative to SEQ ID NO: 1. The amino acid substitutions may be at positions D277C, L678C, A239C and F683C relative to SEQ ID NO: 1. The amino acid substitutions may be at positions D277C, L678C, A239W, V684W, K158I and T676I relative to SEQ ID NO: 1. The amino acid substitutions may be at positions D277C, L678C, A239C, F683C and E274V relative to SEQ ID NO: 1. The amino acid substitutions may be at positions D277C, L678C, A239C, F683C, K158I and M677I, E274I relative to SEQ ID NO: 1.

[0283] The amino acid substitutions may be at positions D277C, L678C, A239W, V684W, K158I and M677I, E274I relative to SEQ ID NO: 1. The amino acid substitutions may be at positionsD277C, L678C, A239C, F683C, K158I, M677L, E274V and E286F relative to SEQ ID NO: 1. The amino acid substitutions may be at positions D277C, L678C, A239C, F683C, W356P, K359P relative to SEQ ID NO: 1. The amino acid substitutions may be at positions D277C, L678C, A239C, F683C, W356P, V363P relative to SEQ I D NO: 1. The amino acid substitutions may be at positions Y464-T498del, S362P and T572C relative to SEQ ID NO: 1. The amino acid substitutions may be at positions S269C, N658C, T100I, A267V, A369L, Q5011, R457S, R460S and C246S, relative to SEQ ID NO: 1. The amino acid substitutions may be at positions S269C, N658C, T100I, A267V, N132V, S641L, V342P, T343P R457S, R460S and C246S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions S269C, N658C, S164C, Q669C, T100I, A267V, R457S, R460S and C246S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions H222C, E657C, T100I, A267V, A369L, Q501I, R457S, R460S and C246S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions H222C, E657C, T100I, A267V, N132V, S641L, V342P, T343P, R457S, R460S and C246S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions H222C, E657C, S164C, Q669C, T100I, A267V, R457S, R460S and C246S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions H222C, E657C, S164C, E671C, T100I, A267V, R457S, R460S and C246S relative to SEQ ID NO: 1.

[0284] The amino acid substitutions may be at positions H222C, E657C, T100I, A267V, N132V, S641L, V480P, Y481P, R457S, R460S and C246S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions S269C, N658C, T100I, A267V, A369L, Q501I, R457S, R460S and C246S relative to SEQ ID NO: 1. The amino acid substitutions may be at positions S269C, N658C, T100I, A267V, N132V, S641L, V342P, T343P, R457S, R460S and C246S relative to SEQ ID NO: 1.

[0285] The engineered HCMV gB may comprise one or more pairs of substitutions selected from A239C, A239W, A267V, A293P, A369L, A732C, A97V, C246S, D277C, D320L, D478L, E274I, E274V, E286F, E289P, E321I, E657C, E671C, E679C, F683C, G173C, G735C, H222C, H681C, I636C, I89C, K124V, K158C, K158I, K214F, K340P, K359P, K394-T452del, L288P, L484P, L641C, L678C, L709C, M677I, M677L, N132C, N132V, N341P, N635T, N657C, N658C, Q483P, Q501I, Q527C, Q669C, R180I, R180L, R291P, R429S, R431S, R432S, R457S, R460S, S164C, S244C, S269C, S362P, S367I, S631C, S641C, S641L, S739C, T100I, T225C, T225F, T292P, T343P, T572C, T659L, T676C, T676I, V342P, V363P, V480P, V684W, V728C, W356P, Y464-T498del and / or Y481P relative to SEQ ID NO: 1, or substitutions corresponding thereto.Across all 350 sequences of HCMV gB, representing GB1-GB5, there are only 4 strains with a single amino acid substitution, strain BE / 4 / 2010 (GB2) has A738S, strain PAV1 (GB2) has L715F, strain PAV21 (GB2) has L715F and strain PAV26 (GB3) has K710R. Therefore, the MPR is conserved at the primary amino acid sequence level across all HCMV strains.

[0286] One or more DI interfacing amino acid residues in the MPR may be conserved. For example, one or more of the amino acid residues at positions 705 to 715, 719, 723, 727, 728, 730, 731, 732, 733, 736, 749 to 752 may be conserved, relative to SEQ ID NO: 1. Alternatively, all of positions 705 to 715, 719, 723, 727, 728, 730, 731, 732, 733, 736, 749 to 752 may be conserved, relative to SEQ ID NO: 1.

[0287] The engineered HCMV gB may be derived from the clade GB1, GB2, GB3, GB4 or GB5. Specifically, the engineered gB may be derived from the HCMV strain Merlin, Towne, AD169, VR1814, BE / 14 / 2011, Toledo, or UKNEQAS1. The engineered gB may derived from the clade GB1. Specifically, the engineered gB may be derived from the HCMV strain Merlin or Towne. Unless explicitly stated otherwise, references to the HCMV MPR in the context of the present disclosure refer to the gB of the Towne / Merlin strain of HCMV (SEQ ID NO:1).

[0288] Furthermore, unless explicitly stated otherwise, references to the HCMV MPR in the context of the present disclosure include the unstructured region between a7 and a8, and the unstructured region after a9. The Towne / Merlin MPR as defined herein therefore starts at position L705 of SEQ ID NO:1 and ends at position F752 of SEQ ID NO:1, i.e., SEQ ID NO: 275 (LPPYLKGLDDLMSGLGAAGKAVGVAIGAVGGA-VASVVEGVATFLKNPF).

[0289] The MPR is structurally defined as being C-terminal to domain V of gB, where the peptide backbone of each protomer diverges from the threefold axis to form the two glycine-rich amphipathic helices (a8 and a9). These helices structurally define the membrane-proximal region (MPR), with these two MPR helices form a hairpin in the plane of the virion membrane, the amphipathic helices (a8 and a9) being at K710 and end at K749. Here we have chosen to extend the structurally defined MPR region to encompass the unstructured region between a7 and a8, corresponding to amino acids L705, P706, P707, Y708 and L709 of the gB of the Towne / Merlin strain of HCMV (SEQ ID NO:1). The MPR sequence has also been extended to the unstructured region after a9 to include amino acid N750, P751 and F752 of Towne / Merlin HCMV gB, as these amino acids contacted the fusion inhibitor in Liu et al.

[0290] However, the HCMV MPR may alternatively be defined as beginning at any one of P706, P707, Y708, L709 in the native HCMV Towne / Merlin gB (SEQ ID NO:1) or at positions equivalent thereto in any non-native variant or alternative strain (including variants comprisingone or more substitutions at any of these positions), or as encompassing some or all of the amino acid residues between 705 to 709 of SEQ ID NO: 1, or positions equivalent thereto in any non-native variant or alternative strain.

[0291] The regions of the Towne / Merlin gB (or positions equivalent thereto in other HCMV strains) which correspond to the amino acid before a8 and after a9 respectively, may or may not be considered formally part of the MPR.

[0292] Thus, the MPR may alternatively be more strictly defined as corresponding to amino acid residues 707-752 of the Towne and Merlin strains of HCMV (SEQ ID NO: 1), 706-751 of the AD169 gB (SEQ ID NO:5), and 705-750 of the VR1814 gB (SEQ ID NO:9), or positions equivalent thereto in other HCMV strains. This definition excludes certain residues in the unstructured region.

[0293] It would be understood by the skilled person that any reference to replacement of the MPR should therefore be understood as resulting in an MPR which includes the amino acid residues 705 to 709 and 750 to 752, or positions equivalent thereto in other HCMV strains, in the equivalent portion of the gB molecule, such that positions 705 to 709 and 750 to 752, or positions equivalent thereto will always be present in the gB, irrespective of whether they are formally considered to be part of the MPR.

[0294] References to “positions equivalent thereto” refer to corresponding positions in different HCMV strains, taking into account insertions and / or deletions as determined by sequence alignment. Such equivalent positions can be determined by sequence alignment using standard bioinformatic methods. Appropriate methods are within the common general knowledge of the skilled person and include, but are not limited to, BLAST, using default settings; ClustalW2, using default settings, and use of sequence alignment algorithms such as the Needleman-Wunsch or Smith-Waterman algorithm.

[0295] Soluble gB constructs derived from the native Towne / Merlin gB, the native AD169 gB and the native VR1814 gB are shown below. Positions L705 to F752 of the native Towne / Merlin gB (SEQ ID NO:1) are underlined. Positions L707 to F751 of the native Towne / Merlin gB (SEQ ID NO:1) are indicated in bold.

[0296] The equivalent positions are indicated for the native AD169 gB (SEQ ID NO. 5) and the native VR1814 gB SEQ ID NO. 9). Thus, positions 704-752 of the AD169 gB (SEQ ID NO:5), and 703-750 of the VR1814 gB (SEQ ID NO:9) are underlined, and positions 706-751 of the AD169 gB (SEQ ID NO:5), and 705-749 of the VR1814 gB (SEQ ID NO:9) are indicated in bold.Merlin / Towne HCMV gB (SEQ ID NO. 1) MESRIWCLVVCVNLCIVCLGAAVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSHGVNETIYNTTLKY GDVVGVNTTKYPYRVCSMAQGTDLIRFERNIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIH TTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMPDDYSNTHSTRYVTVKDQW HSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGR PNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSAL DCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNN ATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVL GLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSA YEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKVVDPL PPYLKGLDDLMSGLGAAGKAVGVAIGAVGGAVASVVEGVATFLKNPFGAFTIILVAIAVVIITYLIYTRQRRLCTQPLQ NLFPYLVSADGTTVTSGSTKDTSLQAPPSYEESVYNSGRKGPGPPSSDASTAAPPYTNEQAYQMLLALARLDAEQRA QQNGTDSLDGRTGTQDKGQKPNLLDRLRHRKNGYRHLKDSDEEENVSGSGENLYFQSGSGHHHHHHHHG

[0297] AD169 gB (SEQ ID NO. 5) MESRIWCLVVCVNLCIVCLGAAVSSSSTSHATSSTHNGSHTSRTTSAQTRSVYSQHVTSSEAVSHRANETIYNTTLKY GDVVGVNTTKYPYRVCSMAQGTDLIRFERNIICTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIYT TYLLGSNTEYVAPPMWEIHHINKFAQCYSSYSRVIGGTVFVAYHRDSYENKTMQLIPDDYSNTHSTRYVTVKDQWHS RGSTWLYRETCNLNCMLTITTARSKYPYHFFATSTGDVVYISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNA APETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCV RDEAINKLQQIFNTSYNQTYEKYGNVSVFETSGGLVVFWQGIKQKSLVELERLANRSSLNITHRTRRSTSDNNTTHLS SMESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASC VTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVD YLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKVVDPLPPYLK GLDDLMSGLGAAGKAVGVAIGAVGGAVASVVEGVATFLKNPFGAFTI I LVAIAVVI ITYLIYTRQRRLCTQPLQNLFPYL VSADGTTVTSGSTKDTSLQAPPSYEESVYNSGRKGPGPPSSDASTAAPPYTNEQAYQMLLALARLDAEQRAQQNGT DSLDGQTGTQDKGQKPNLLDRLRHRKNGYRHLKDSDEEENVSGSGENLYFQSGSGHHHHHHHHG

[0298] VR1814 HCMV gB (SEQ ID NO. 9) MESRIWCLVVCVNLCIVCLGAVVSSSSTSHATSSAHNGSHTSRTTSAQTRSVSSQHVTSSEAVSHRANETIYNTTLKY GDVVGVNTTKYPYRVCSMAQGTDLIRFERNIVCTPMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIH TTYLLGSNTEYVAPPMWEIHHINRHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMLDDYSNTHSTRYVTVKDQWH SRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNTSYFGENADKFFIFPNYTIVSDFGRA NSAPETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDPVLD CVRDQALNKLQQIFNASYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLLELERLANSSGVNSTRRTKRSTGNTTTL SLESESVRNVLYAQLQFTYDTLRSYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLA SCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFVNSSYVQYGQLGEDNEILLGNHRTEECQFPSLKIFIAGNSAYEY VDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKVVDPLPPY LKGLDDLMSGLGAAGKAVGVAIGAVGGAVASVVEGVATFLKNPFGAFTIILVAIAVVIIIYLIYTRQRRLCMQPLQNLFP YLVSADGTTVTSGNTKDTSLQAPPSYEESVYNSGRKGPGPPSSDASTAAPPYTNEQAYQMLLALARLDAEQRAQQN GTDSLDGQTGTQDKGQKPNLLDRLRHRKNGYRHLKDSDEEENVSGSGENLYFQSGSGHHHHHHHHG

[0299] The non-native MPR may comprise a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% to a sequence selected from SEQ ID NOs: 276-312.

[0300] The non-native MPR may comprise a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from SEQ ID NO: 287, 297, 289 or 298 or 306, 307, 309, 310.

[0301] The non-native MPR may comprise a sequence of SEQ ID NOs: 276-312.

[0302] The non-native MPR may comprise a sequence selected from any one of SEQ ID NOs: 287, 297, 289 or 298.The non-native MPR may comprise a sequence of SEQ ID NO: 287. The non-native MPR may comprise a sequence of SEQ ID NO: 297. The non-native MPR may comprise a sequence of SEQ ID NO: 289. The non-native MPR may comprise a sequence of SEQ ID NO: 298.

[0303] The non-native MPR may comprise a sequence of

[0304] PPYLKG LDDLXi KX2LX3X4X5GX3X7EG VX8I GAX9FGX10X11 Xi 2X13KX14X15X16Xi 7X18Xi 9X20X21 K NPF, wherein Xi is selected from V or M; X2is selected from K or L; X3 is selected from E, D or G; X4 is selected from E, K or A; X5 is selected from G or A; X8is selected from K, E or D; X7 is selected from E, K or G; Xs is selected from K or E; Xg is selected from K or E; X10 is selected from K or E; Xu is selected from K or E; X12 is selected from L or A; X13 is selected from A, E, R or S; X14 is selected from E or L; X15 is selected from L or I; Xi6is selected from A or K; X17 is selected from K or E; Xi8is selected from E or L; X19 is selected from A or K; X20 is selected from A or E, and X21 is selected from K or E. (SEQ ID NO: 319).

[0305] Alternatively, the non-native MPR comprises a sequence of:

[0306] PPYLKG 1X1X21X3X4X5X6X7X3X90X10X11X12X13X14X15IX16Xi 7X18FGXI 9X20X21 X22X23X24X25X26X 27X23X29X30X31X32X33 X34F, wherein Xi is selected from E or D; X2is selected from K or D; X3 is selected from M, K or V; X4 is selected from E, K or Q; X5 is selected from L, I, R or K; X8is selected from V or L; X7 is selected from D, E or G; X8is selected from K, E, A or Q; Xg is selected from G, K, A or E; X is selected from E, D, P or K; Xu is selected from E, A, N R, K or G; X12 is selected from E or D; X13 is selected from I or G; X14 is selected from E, A or V; X15 is selected from E, A or K; Xi6is selected from S or G; X17 is selected from R, K or A; Xi8is selected from K, E or Q; X19 is selected from K, E or R; X20 is selected from E, V, K or R; X21 is selected from L or A; X22is selected from E, A, R or S; X23 is selected from K, E or L; X24 is selected from I, K, V or E; X25 is selected from L, I or V; X28is selected from E, K or A; X27 is selected from E, K or R; X28is selected from E or L; X29 is selected from K, E or A; X30 is selected from K, E, A, or L; X31 is selected from L, K or E; X32 is selected from S, E or K; X33 is selected from E or N, and X34 is selected from K or P. (SEQ ID NO: 318).

[0307] Alternatively, the non-native MPR comprises a sequence of:

[0308] PPYLKGLDDLX1X2X3LX4X5X6GX7X3X9GVX10IGAX11X12GX13X14X15X16X17X18X19X20X21X22X23X 24X25KNPF wherein Xi is selected from V or M; X2is selected from S or K; X3 is selected from G, K or L; X4 is selected from E, D or G; X5 is selected from E, K or A; X8is selected from G or A; X7 is selected from K, E or D; Xs is selected from A, E, K or G; Xg is selected from E or V; X10 is selected from A, K or E; Xu is selected from V, K or E; X12 is selected from G or F;X13 is selected from A, K or E; X14 is selected from V, K or E;x is selected from L or A; X is selected from A, E, R or S; X17 is selected from V or K; X is selected from V, E or L; X19 is selected from E, L or I; X20 is selected from G, A or K; X21 is selected from V, K or E; X22 is selected from A, E or L; X23 is selected from T, A or K; X24 is selected from F, A or E, and X25 is selected from L, K or E (SEQ ID NO: 317).

[0309] Alternatively, the non-native MPR comprises a sequence of:

[0310] PPYLKG LX1X2LX3X4X5X6X7X8X9GX10X11X12X13X14X15IX16Xi 7X18Xi 9GX20X21X22X23X24X25X26X27 X28X29X30X31X32X33X34X35F, wherein Xi is selected from E or D; X2 is selected from K or D; X3 is selected from M, K or V; X4 is selected from S, E, K or Q; X5 is selected from G, L, I, R or K; Xe is selected from V or L; X7 is selected from D, E or G; Xs is selected from K, E, A or Q; Xg is selected from G, K, A or E; Xw is selected from E, D, P or K; Xu is selected from E, A, N R, K or G; X12 is selected from V, E or D; X13 is selected from I or G; X14 is selected from E, A or V; X15 is selected from E, A or K; X-5is, selected from S or G; X17 is selected from R, K or A; X is selected from V, K, E or Q; X19 is selected from G or F; X20 is selected from V, K, E or R; X21 is selected from A, E, V, K or R; X22 is selected from L or A; X23 is selected from E, A, R or S; X24 is selected from V, K, E or L; X25 is selected from I, K, V or E; X26 is selected from E, L, I or V; X27 is selected from G, E, K or A; X28 is selected from V, E, K or R; X29 is selected from A, E or L; X30 is selected from T, K, E or A; X31 is selected from F, K, E, A, or L; X32 is selected from L, K or E; X33 is selected from S, E or K; X34 is selected from E or N, and X35 is selected from K or P (SEQ ID NO: 316).

[0311] Alternatively, the non-native MPR may comprise a sequence of:

[0312] P PX1 X2G LX3X4 LX5X6X7X8X9X10X11 GX12X13X14X151 Xi eXi 7X13X19X20X21 X22X23X24X25X26X27X28X29 X30X31X32X33X34X35X36X37 ssF, wherein Xi is selected from C or Y; X2 is selected from C or L; X3 is selected from E or D; X4 is selected from K or D; X5 is selected from M, K, V, or C; Xe is selected from S, E, K or Q; X7 is selected from G, L, I, R or K; Xs is selected from V or L; Xg is selected from D, E or G; X10 is selected from A, K, E, Q or C; Xu is selected from A, G, K, or E; X12 is selected from K, E, D, or P; X is selected from A, E, N, R, K or G; X14 is selected from V, E or D; Xw is selected from I or G; X is selected from V, E, A or C; X17 is selected from A, E, or K; X is, selected from G or S; X is selected from A, R, K or C; X20 is selected from V, K, E or Q; X21 is selected from G or F; X22 is selected from G or C; X23 is selected from A, K, E or R; X24 is selected from A, E, V, K or R; X25 is selected from L or A; X26 is selected from S, E, A, R or C; X27 is selected from V, K, E or L; X28 is selected from V, I, K, or E; X29 is selected from E, L, I, V, or C; X30 is selected from G, E, K or A; X31 is selected from V, E, K or R; X32 is selected from A, E or L; X33 is selected from T, K, E or A;X34 is selected from F, K, E, A, or L; X35 is selected from L, K or E; X36 is selected from S, E or K; X37 is selected from E or N, and X38 is selected from K or P. (SEQ ID NO: 315).

[0313] PPYLKGLDDLX1KX2LX3X4X5GX6X7EGVX8IGAX9FGKX10X11X12KX13LX14X15EX16X17X18KN PF, wherein Xi is selected from I, or C; X2is selected from K, C, or I; X3is selected from W, or N; X4is selected from F, or H; X5is selected from Y, or T; X6is selected from F, or N; X7is selected from A, C, or W; X8is selected from S, or C; X9is selected from E, V, or I; Xi0is selected from D, or C; Xu is selected from E, or F; X12is selected from W, or P; X13is selected from K, or P; X14is selected from S, or P; Xi5is selected from V, or P; Xi6is selected from T, or C; X17is selected from is selected from A, or E and Xi8is selected from K, or E (SEQ ID NO: 320).

[0314] The non-native MPR may have more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 15, more than 20, more than 25, or more than 30 substitutions relative to the native MPR.

[0315] Human herpes simplex virus (HSV-1 and HSV-2)

[0316] In some instances, the engineered is a Human herpes simplex virus (HSV) gB. Human herpes simplex viruses are Simplexviruses within the Alphaherpesvirinae subfamily. There are two types of Human herpes simplex viruses, HSV-1 and HSV-2. Each virus can be grouped into multiple clades based on sequence diversity. Representative HSV-1 strains include 17, KOS, F, H129, and McKrae, while representative HSV-2 strains include HG52, MS, G, 333, and SD90. Corresponding amino acid positions in any HSV strain can be determined by those of ordinary skill in the art using established sequence information and routine sequence alignment techniques, such as BLAST (default settings) or ClustalW2 (default settings). HSV-1 and HSV-2 are double-stranded DNA viruses and among the most prevalent human pathogens worldwide. HSV-1 most commonly causes oral herpes, whereas HSV-2 is more frequently associated with genital herpes; however, both types can infect oral or genital sites and can cause keratitis, neonatal infection, and, more rarely, severe encephalitis. Following primary infection, both HSV-1 and HSV-2 establish lifelong latency in sensory neurons. Despite extensive research, no licensed vaccines are currently approved for either HSV-1 or HSV-2.

[0317] The genomes of HSV-1 and HSV-2 encode multiple envelope glycoproteins, including glycoprotein B (gB), which is encoded by the LIL27 gene. Additional envelope glycoproteins include gH, gC, gG, gD, gl, gE, and gM. These proteins form functional complexes that engagehost cell-surface receptors and mediate attachment and entry of the herpesvirus particle. They share high overall sequence identity, typically greater that 80% across the genome, with higher conservation in essential structural genes such as LIL27 (encoding gB).

[0318] The gB disclosed herein may be an HSV-1 gB. The engineered HSV-1 gB may be derived from the clade I, II, III, IV, V or VI of HSV-1. The engineered gB may be derived from an HSV-1 strain selected from 17, 134, CJ311, CJ360, CJ394, CJ970, CR38, E03, E06, E07, E08, E10, E11, E12, E13, E14, E15, E19, E22, E23, E25, E35, F, H129, KOS, OD4, R11, R62, S23, S25, TFT401, or HG52. The engineered gB may be derived from the HSV-1 KOS strain. Unless indicated otherwise, references to an HSV-1 MPR refer to the MPR of the KOS strain of HSV1 (SEQ ID NO: 100), which starts at position 725 of the HSV-1 gB according to SEQ ID NO: 1 and ends at position 775 of SEQ ID NO: 100, i.e.

[0319] ADANAAMFAGLGAFFEGMGDLGRAVGKVVMGIVGGVVSAVSGVSSFMSNPF (SEQ ID NO: 222).

[0320] The amino acid substitution may be a substitution at one or more of amino acid residues 725-775 of the HSV-1 gB, relative to SEQ ID NO: 100 (KOS strain).

[0321] The HSV-1 MPR may have a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to a sequence selected from SEQ ID NO: 223-243.

[0322] The HSV-1 MPR may have a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to SEQ ID NOs 232, 243, 241 or 237.

[0323] The HSV-1 MPR may have a sequence selected from any one of SEQ ID NOs: 223-243. The HSV-1 MPR may have a sequence selected from any one of SEQ ID NOs 232, 243, 241 and 237.

[0324] The HSV-1 MPR may have a sequence of SEQ ID NO: 232. The HSV-1 MPR may have a sequence of SEQ ID NO: 243. The HSV-1 MPR may have a sequence of SEQ ID NO: 241. The HSV-1 MPR may have a sequence of SEQ ID NO: 237.

[0325] The engineered HSV-1 gB disclosed herein may comprise one or more additional stabilising substitutions outside of the MPR.

[0326] The additional amino acid substitution may be a substitution at one or more of amino acid residues 1154, K158, K158, (WFGHR)174, W174, F175, Y179, F182, A239, A239, S244, (AFH)261, E274, E274, D277, E286, W356, K359, S362, V363, Y464-T498, T572, L673,T676, T676, M677, M677, L678, E679, H681, F683, V684, N709V of the HSV-1 gB, relative to SEQ ID NO: 100 (KOS strain), or substitutions corresponding thereto. The gB may comprise one or more pairs of substitutions selected from: 1154C and L673(GCG) orT572C and E679C, or substitutions corresponding thereto.

[0327] The engineered HSV-1 gB may comprise substitutions at one or more of positions 1154, K158, K158, (WFGHR)174, W174, F175, Y179, F182, A239, A239, S244, (AFH)261, E274, E274, D277, E286, W356, K359, S362, V363, Y464-T498, T572, L673, T676, T676, M677, M677, L678, E679, H681, F683, V684, N709V relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1), or corresponding positions in other Herpesvirus gB proteins. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from 1154C, K158C, K158I, (WFGHR)174(YAYIH), W174N, F175H, Y179T, F182N, A239C, A239W, S244C, (AFH)261(WLY), E274V, E274I, D277C, E286F, W356P, K359P, S362P, V363P, Y464-T498del, T572C, L673(GCG), T676C, T676I, M677I, M677L, L678C, E679C, H681C, F683C, V684W, N709V relative to SEQ ID NO: 100, or corresponding substitutions in other HSV-1 gBs.

[0328] The engineered HSV-1 gB may comprise one or more substitutions selected from 1154, L673, T572, E679 and N709 or corresponding positions, relative to SEQ ID NO: 100. Specifically, the gB may comprise substitutions at 1154C, L673(GCG), T572C, E679C and N709V, or a corresponding substitution, relative to SEQ ID NO: 100.

[0329] The engineered HSV-1 gB may comprise substitutions at some or all of positions 1154, L673, T572, E679, N709, W174, F175, Y179 and F182 or corresponding positions, relative to SEQ ID NO: 100. Specifically, the engineered HSV-1 gB may comprise the substitutions at I154C, L673(GCG), T572C, E679C, N709V, W174N, F175H, Y179T and F182N, or a corresponding substitution, relative to SEQ ID NO: 100.

[0330] The engineered HSV-1 gB may comprise substitutions at some or all of positions K158, M677, E274, F182, E274, and E286 or corresponding positions, relative to SEQ ID NO: 100. Specifically, the engineered HSV-1 gB may comprise the substitutions at K158I, M677I, E274I, F182N, E274V and E286F, or a corresponding substitution, relative to SEQ ID NO: 100. The amino acid substitutions may be 1154C, L673(GCG), T572C, E679C, (WFGHR)174(YAYIH) and (AFH)261(WLY) relative to SEQ ID NO: 1.

[0331] The amino acid substitutions may be 1154C, L673(GCG), T572C, E679C and N709V relative to SEQ ID NO: 100.The amino acid substitutions may be I154C, L673(GCG), T572C, E679C, N709V, W174N, F175H, Y179T and F182N relative to SEQ ID NO: 100.

[0332] The engineered HSV-1 gB may comprise one of more pairs of substitutions selected from: K158C and T676C,

[0333] S244C and H681C,

[0334] A239C and F683C,

[0335] D277C and L678C,

[0336] A239C and V684C,

[0337] W356P and K359P

[0338] or corresponding substitutions, relative to SEQ ID NO: 100.

[0339] The engineered HSV-1 gB may comprise substitutions at one of more of positions K158, T676, S244 and H681, relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from K158C, T676C, S244C and H681C, relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0340] The engineered HSV-1 gB may comprise substitutions at one of more of positions K158, T676, A239 and F683 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from K158C, T676C, A239C and F683C relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0341] The engineered HSV-1 gB may comprise substitutions at one of more of positions D277, L678, S244 and H681 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from D277C, L678C, S244C and H681C relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0342] The engineered HSV-1 gB may comprise substitutions at one of more of positions D277, L678, A239 and F683 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from D277C, L678C, A239C and F683C relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.The engineered HSV-1 gB may comprise substitutions at one of more of positions D277, L678, A239, V684, K158 and T676 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from D277C, L678C, A239W, V684W, K158I and T676I relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0343] The engineered HSV-1 gB may comprise substitutions at one of more of positions D277, L678, A239, F683 and E274 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from D277C, L678C, A239C, F683C and E274V relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0344] The engineered HSV-1 gB may comprise substitutions at one of more of positions D277, L678, A239, F683, K158, M677 and E274 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from D277C, L678C, A239C, F683C, K158I, M677I and E274I relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0345] The engineered HSV-1 gB may comprise substitutions at one of more of positions D277, L678, A239, V684, K158, M677 and E274 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from D277C, L678C, A239W, V684W, K158I, M677I and E274I relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0346] The engineered HSV-1 gB may comprise substitutions at one of more of positions D277, L678, A239, F683, K158, M677, E274 and E286 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from D277C, L678C, A239C, F683C, K158I, M677L, E274V and E286F relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0347] The engineered HSV-1 gB may comprise substitutions at one of more of positions D277, L678, A239, F683, W356 and K359 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from D277C, L678C, A239C, F683C, W356Pand K359P relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0348] The engineered HSV-1 gB may comprise substitutions at one of more of positions D277, L678, A239, F683, W356 and V363 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from D277C, L678C, A239C, F683C, W356P and V363P relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0349] The engineered HSV-1 gB may comprise substitutions at one of more of positions Y464-T498del, S362, T572 and E679 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from Y464-T498del, S362P, T572C and E679C relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0350] The engineered HSV-1 gB may comprise substitutions at one of more of positions 1154, L673, T572, E679, (WFGHR)174 and (AFH)261 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from 1154C, L673(GCG), T572C, E679C, (WFGHR)174(YAYIH) and(AFH)261(WLY) relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0351] The engineered HSV-1 gB may comprise substitutions at one of more of positions 1154, L673, T572, E679, (WFGHR)174 and (AFH)261 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from 1154C, L673(GCG), T572C, E679C, (WFGHR)174(YAYIH) and (AFH)261(WLY) relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0352] The engineered HSV-1 gB may comprise substitutions at one of more of positions 1154, L673, T572, E679, (WFGHR)174 and (AFH)261 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from 1154C, L673(GCG), T572C, E679C, (WFGHR)174(YAYIH) and (AFH)261(WLY) relative to SEQ IDNO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0353] The engineered HSV-1 gB may comprise substitutions at one of more of positions 1154, L673, T572, E679 and N709 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from I154C, L673(GCG), T572C, E679C and N709V relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0354] The engineered HSV-1 gB may comprise substitutions at one of more of positions 1154 and L673, T572, E679, N709 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from I154C, L673(GCG), T572C, E679C, N709V relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0355] The engineered HSV-1 gB may comprise substitutions at one of more of positions 1154 and L673, T572, E679, N709 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from I154C, L673(GCG), T572C, E679C and N709V relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0356] The engineered HSV-1 gB may comprise substitutions at one of more of positions 1154, L673, T572, E679 and N709 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from I154C, L673(GCG), T572C, E679C and N709V relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0357] The engineered HSV-1 gB may comprise substitutions at one of more of positions 1154, L673, T572, E679 and N709 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from I154C, L673(GCG), T572C, E679C and N709V relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.The engineered HSV-1 gB may comprise substitutions at one of more of positions 1154, L673, T572, E679 and N709 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from I154C, L673(GCG), T572C, E679C and N709V relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0358] The engineered HSV-1 gB may comprise substitutions at one of more of positions 1154, L673, T572, E679 and N709 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from I154C, L673(GCG), T572C, E679C and N709V relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0359] The engineered HSV-1 gB may comprise substitutions at one of more of positions 1154, L673, T572, E679, N709, W174, F175, Y179 and F182 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from 1154C, L673(GCG), T572C, E679C, N709V, W174N, F175H, Y179T and F182N relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0360] The engineered HSV-1 gB may comprise substitutions at one of more of positions 1154, L673, T572, E679, N709, W174, F175, Y179 andF182 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from 1154C, L673(GCG), T572C, E679C, N709V, W174N, F175H, Y179T and F182N relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.

[0361] The engineered HSV-1 gB may comprise substitutions at one of more of positions 1154, L673, T572, E679, N709, W174, F175, Y179 andF182 relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered HSV-1 gB may comprise one of more substitutions selected from 1154C, L673(GCG), T572C, E679C, N709V, W174N, F175H, Y179T and F182N relative to SEQ ID NO: 100 (the gB of the KOS strain of HSV-1) or corresponding substitutions in other HSV-1 gBs.The amino acid substitutions may be at position K158, T676, S244 and H681 relative to SEQ ID NO: 100. The amino acid substitutions may be at position K158, T676, A239 and F683 relative to SEQ ID NO: 100. The amino acid substitutions may be at position D277, L678, S244 and H681 relative to SEQ ID NO: 100. The amino acid substitutions may be at position D277, L678, A239 and F683 relative to SEQ ID NO: 100. The amino acid substitutions may be at position D277, L678, A239, V684, K158, T676 relative to SEQ ID NO: 100. The amino acid substitutions may be at position D277, L678, A239, F683, E274 relative to SEQ ID NO: 100. The amino acid substitutions may be at position D277, L678, A239, F683, K158, M677, E274 relative to SEQ ID NO: 100. The amino acid substitutions may be at position D277, L678, A239, V684, K158, M677, E274 relative to SEQ ID NO: 100. The amino acid substitutions may be at position D277, L678, A239, F683, K158, M677, E274, E286 relative to SEQ ID NO: 100. The amino acid substitutions may be at position D277, L678, A239, F683, W356, K359 relative to SEQ ID NO: 100. The amino acid substitutions may be at position D277, L678, A239, F683, W356, V363 relative to SEQ ID NO: 100. The amino acid substitutions may be at position Y464-T49, S362, T572, E679 relative to SEQ ID NO: 100. The amino acid substitutions may be at position 1154, L673, T572, E679, (WFGHR)174 and (AFH)261 relative to SEQ ID NO: 1. The amino acid substitutions may be at position 1154, L673, T572, E679 and N709 relative to SEQ ID NO: 100. The amino acid substitutions may be at position 1154, L673, T572, E679, N709, W174, F175, Y179 and F182 relative to SEQ ID NO: 100. The amino acid substitutions may be K158C, T676C, S244C and H681C, relative to SEQ ID NO: 100. The amino acid substitutions may be K158C, T676C, A239C and F683C relative to SEQ ID NO: 100. The amino acid substitutions may be D277C, L678C, S244C and H681C relative to SEQ ID NO: 100. The amino acid substitutions may be D277C, L678C, A239C and F683C relative to SEQ ID NO: 100. The amino acid substitutions may be D277C, L678C, A239Wand V684W, K158I, T676I relative to SEQ ID NO: 100. The amino acid substitutions may be D277C, L678C, A239C, F683C, E274V relative to SEQ ID NO: 100. The amino acid substitutions may be D277C, L678C, A239C and F683C, K158I, M677I, E274I relative to SEQ ID NO: 100. The amino acid substitutions may be D277C, L678C, A239W and V684W, K158I, M677I, E274I relative to SEQ ID NO: 100. The amino acid substitutions may be D277C, L678C, A239C and F683C, K158I, M677L, E274V, E286F relative to SEQ ID NO: 100. The amino acid substitutions may be D277C, L678C, A239C and F683C, W356P, K359P relative to SEQ ID NO: 100. The amino acid substitutions may be D277C, L678C, A239C and F683C, W356P, V363P relative to SEQ ID NO: 100. The amino acid substitutions may be Y464-T498del, S362P and T572C, E679C relative to SEQ ID NO: 100. The amino acid substitutions may be I154C, L673(GCG), T572C, E679C, (WFGHR)174(YAYIH) and (AFH)261(WLY) relative toSEQ ID NO: 1. The amino acid substitutions may be I154C, L673(GCG), T572C, E679C and N709V relative to SEQ ID NO: 100. The amino acid substitutions may be I154C, L673(GCG), T572C, E679C, N709V, W174N, F175H, Y179T and F182N relative to SEQ ID NO: 100. The engineered gB may have a sequence selected from any one of SEQ ID NOs: 127-133, and 181-221. The engineered gB may have a sequence selected from any one of SEQ ID NOs: 212-221.

[0362] The engineered gB may have a sequence selected from any one of SEQ ID NOs: 217, 219, 220 and 221. The HSV MPR may have a sequence selected from SEQ ID NOs: 223-243. The HSV-1 MPR may have a sequence selected from SEQ ID NOs 232, 243, 241 and 237.

[0363] The MPR may comprise one or more substitutions to introduce one or more disulfide bridges at the DI, Dll, MPR and / or core interfaces. In particular, the MPR may comprise one or more substitutions to introduce one or more disulfide bridges at the DI / MPR interface. Disulphide bridges may also be introduced into other parts of the molecule, for example, between the DIII / DIV domains. A disulfide bridge between Dill and DV may involve substitutions at positions G177C-E634C, I90C-T630C, or positions corresponding thereto, relative to SEQ ID NO: 108, or substitutions corresponding thereto.

[0364] The DI domain may comprise one or more substitutions selected from K158C, S244C and A239C, D277C and A239C relative to SEQ ID NO: 100 or corresponding substitutions.

[0365] The engineered HSV-1 gB may comprise one or more pairs of substitutions selected from: a) K158C and T676C,

[0366] b) S244C and H681C,

[0367] c) A239C and F683C,

[0368] d) D277C and L678C,

[0369] e) A239C and V684C and / or

[0370] f) W356P and K359P;

[0371] relative to SEQ ID NO: 100, or substitutions corresponding thereto.

[0372] The engineered HSV-1 gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from any one of SEQ ID NOs: 127-133, or 181-221.

[0373] The engineered HSV-1 gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from any one of SEQ ID NOs: 212-121.The engineered HSV-1 gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% identity SEQ ID NOs: 217, 219, 220 or 221.

[0374] The engineered HSV-1 gB may have a sequence selected from any one of SEQ ID NOs: 127-133, and 181-221. The engineered HSV-1 gB may have a sequence selected from any one of SEQ ID NOs: 212-121. The engineered HSV-1 gB may have a sequence selected from any one of SEQ ID NOs: 217, 219, 220 and 221.

[0375] The engineered HSV-1 gB may have a sequence of SEQ ID NO: 217. The engineered HSV-1 gB may have a sequence of SEQ ID NO: 219. The engineered HSV-1 gB may have a sequence of SEQ ID NO: 220. The engineered HSV-1 gB may have a sequence of SEQ ID NO: 221.

[0376] The engineered HSV-1 gB may comprise an amino acid sequence of MHQGAPSWGRRWFVVWALLGLTLGVLVASAAPTSPGTPGVAAATQAANGGPATPAPPPL GAAPTGDPKPKKNKKPKNPTPPRPAGDNATVAAGHATLREHLRDIKAENTDANFYVCPPPT GATVVQFEQPRRCPTRPEGQNYTEGIAVVFKENX1APYX2FKATMYYKDVTVSQVX3X4GHR X5SQX6MGIFEDRAPVPFEEVIDKINAKGVCRSTAKYVRNNLETTAFHRDDHETDMELKPAN XyATRTXgRGWHTTDLKYNPSRVEAFHRYGTTVNCIVXgEVXioARSVYPYDXnFVLATGDFV YMSPFYGYREGSHTEHTTYAADRFKQVDGFYARDLTTKARATAPTTRNLLTTPKFTVAWD

[0377] X12VPX13RPX14X15CTMTKWQEVDEMLRSEYGGSFRFSSDAISTTFTTNLTEYPLSRVDLGD CIGKDARDAMDRIFARRYNATHIKVGQPQYYQANGGFLIAYQPLLSNTLAELYVREHLREQS RKPPNPTPPPPGASANASVERIKTTSSIEFARLQFTYNHIQRHVNDMLGRVAIAWCELQNHE LTLWNEARKLNPNAIASVTVGRRVSARMLGDVMAVSX16CVPVAADNVIVQNSMRISSRPG ACYSRPLVSFRYEDQGPLVEGQLGENNELRLTRDAIEPCTVGHRRYFTFGGGYVYFEEYA YSHQLSRADITTVSTFIDGCG, wherein Xxis I, or C; X2is K, C, or I; X3is W, or N; X4 is F, or H; X5is Y, or T; X6is F, or N; X7is A, C, or W; X8is S, or C; X9is E, V, or I; Xi0is D, or C; Xu is E, or F; X12is W, or P; X13is K, or P; X14is S, or P; Xi5is V, or P and Xi6is T, or C (SEQ ID NO: 231).

[0378] The engineered HSV-1 gB may comprise an amino acid sequence of MHQGAPSWGRRWFVVWALLGLTLGVLVASAAPTSPGTPGVAAATQAANGGPATPAPPPL GAAPTGDPKPKKNKKPKNPTPPRPAGDNATVAAGHATLREHLRDIKAENTDANFYVCPPPT GATVVQFEQPRRCPTRPEGQNYTEGIAVVFKENCAPYKFKATMYYKDVTVSQVXiX. GHRX 3SQX4MGIFEDRAPVPFEEVIDKINAKGVCRSTAKYVRNNLETTAFHRDDHETDMELKPANA ATRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVYMSPF YGYREGSHTEHTTYAADRFKQVDGFYARDLTTKARATAPTTRNLLTTPKFTVAWDWVPKRPSVCTMTKWQEVDEMLRSEYGGSFRFSSDAISTTFTTNLTEYPLSRVDLGDCIGKDARDAM DRIFARRYNATHIKVGQPQYYQANGGFLIAYQPLLSNTLAELYVREHLREQSRKPPNPTPPP PGASANASVERIKTTSSIEFARLQFTYNHIQRHVNDMLGRVAIAWCELQNHELTLWNEARKL NPNAIASVTVGRRVSARMLGDVMAVSCCVPVAADNVIVQNSMRISSRPGACYSRPLVSFRY EDQGPLVEGQLGENNELRLTRDAIEPCTVGHRRYFTFGGGYVYFEEYAYSHQLSRADITTV STFIDGCGNITMLCDHEFVPLEVYTRHEIKDSGLLDYTEVQRRVQLHDLRFADIDTVIHAD wherein Xi is W, or N; X2is F, or H; X3is Y, or T and X4 is F, or N (SEQ ID NO: 322).

[0379] The non-native MPR may comprise an amino acid sequence of ADX1X2AX3X4X5X6X7X8X9X1OX11X12X13X14X15GX16X17X18X19X2OX21GX22X23X24X25X26X27X28X29X3oX3iX32X33X34X3sX36X37X38X39X4oX4iX42X43X44PX4s, wherein Xi is A, P, E, or K; X2is N, V, T, I, K, R, L, or E; X3is A, E, Q, D, or K; X4is M, F, A, Y, E, I, R, or L; X5is F, Q, M, or L; X6is A, R, Q, or K; X7is G, K, A, E, R, or S; X8is L, M, or I; X9is G, or A; X10is A, L, K, R, or E; Xu is F, Y, K, E, R, or A; X12is F, or Y; X13is E, A, R, or K; X14is G, A, S, E, or K; X15is M, L, Q, E, K, T, G, or N; Xi6is D, P, E, A, or L; X17is L, A, E, or K; Xi8is G, or A; X19is R, E, L, or F; X20is A, E, or K; X21is V, L, E, K, R, or A; X22is K, L, M, or R; X23is V, or P; X24is V, K, E, D, or A; X25is M, L, or I; X26is G, E, A, N, or K; X27is I, L, E, V, R, K, A, or S; X28is V, L, I, K, R, T, or F; X29is G, or L; X30is G, T, R, K, A, or E; X31is V, I, K, E, L, or R; X32is V, I, F, M, or L; X33is S, or E; X34is A, T, Q, L, R, or E; X35is V, L, A, I, Q, R, E, or K; X36is S, A, E, Q, R, T, L, M, or K; X37is G, E, T, S, D, F, A, or R; X38is V, Q, L, A, E, I, R, or K; X39is S, A, D, E, or Q; X40is S, E, or A; X41 is F, K, R, L, or A; X42is M, L, A, or E; X43is S, A, R, K, or L; X44 is N, D, H, or L and X45is F, E, Q, L, N, W, or Y (SEQ ID NO: 323).

[0380] The non-native MPR may comprise an amino acid sequence of ADX1X2AX3X4X5X6X7X8X9X1OX11X12X13X14X15GX16X17X18X19X2OX21GX22X23X24X25X26X27X28X29X3oX3iX32X33X34X35X36X37X38X39X4oX4iX42X43X44PX45, wherein Xi is P, E, A, or K; X2is V, T, N, I, K, R, L, or E; X3is E, A, Q, D, or K; X4 is F, A, Y, E, I, R, or L; X5is F, Q, M, or L; X6is R, A, Q, or K; X7is K, A, G, E, R, or S; X8is L, M, or I; X9is G, or A; Xi0is L, K, R, E, or A; Xu is F, Y, K, E, R, or A; X12is F, or Y; X13is E, A, R, or K; X14is A, S, G, E, or K; X15is L, Q, M, E, K, T, G, or N; X16is P, E, D, A, or L; X17is A, L, E, or K; X18is G, or A; X19is R, E, L, or F; X20is A, E, or K; X21is L, E, K, R, or A; X22is K, L, M, or R; X23is V, or P; X24is V, K, E, D, or A; X25is M, L, or I; X26is G, E, A, N, or K; X27is L, I, E, V, R, K, A, or S; X28is L, I, K, V, R, T, or F; X29is G, or L; X30is T, R, K, G, A, or E; X31is I, V, K, E, L, or R; X32is V, I, F, M, or L; X33is S, or E; X34is T, A, Q, L, R, or E; X35is L, A, I, Q, R, E, or K; X36is A, E, Q, S, R, T, L, M, or K; X37is E, T, G, S, D, F, A, or R; X38is Q, L, A, E, I, R, or K; X39is A, S, D, E, orQ; X40 is S, E, or A; X41 is K, R, L, or A; X42is L, A, or E; ><43 is A, S, R, K, or L; X44 is D, N, H, or L and X45is E, Q, F, L, N, W, or Y (SEQ ID NO: 324).

[0381] The non-native MPR may comprise an amino acid sequence of ADX1X2AX3X4X5X6X7X8X9X1OX11YX12X13X14GX15X16X17X18X19X2OGX21X22X23X24X25X26X27X28X29X3oX3iX32X33X34X3sX36X37X38X39X4oAX4iX42PX43, wherein Xi is K, E, or A; X2is I, K, R, or T; X3is D, K, or A; X4 is E, I, or L; X5is F, Q, or L; X6is A, R, or K; X7is E, or A; X8is L, or I; X9is G, or A; X10is R, K, or E; Xu is F, K, or A; X12is E, K, or A; X13is A, K, or E; X14is K, T, or G; X15is P, A, or E; X16is E, or A; X17is G, or A; X18is R, E, or F; X19is A, or K; X20is K, or A; X21is K, L, or R; X22is V, or P; X23is E, D, or A; X24is M, L, or I; X25is G, E, K, or A; X26is E, K, or S; X27is K, L, or T; X28is G, or L; X29is K, E, or A; X30is V, or E; X3iis V, M, or L; X32is S, or E; X33is A, or E; X34is A, K, or Q; X35is A, K, or E; X36is T, A, or R; X37is A, E, or Q; X38is A, E, or Q; X39is S, E, or A; X40 is L, or R; X41is A, K, or L; X42is D, or L and X43is L, F, or Y (SEQ ID NO: 325).

[0382] The non-native MPR may have more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 15, more than 20, more than 25, or more than 30 substitutions relative to the native MPR.

[0383] In some instances, the engineered gB is an HSV-2 gB. The gB disclosed herein may be an HSV-2 gB and the amino acid substitution may be a substitution at one or more of amino acid residues T569C, E676C, N706V, W169N, F170H, Y174T or F177N of the HSV-2 gB, relative to SEQ ID NO: 254 (HG2 strain).

[0384] The engineered HSV-2 gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from any one of SEQ ID NOs: 255-259.

[0385] The engineered HSV-2 gB may have a sequence selected from any one of SEQ ID NOs: 255-259.

[0386] The engineered HSV-2 gB may have an MPR sequence selected from any one of SEQ ID NOs: 223-243, optionally wherein the sequence is selected from any one of SEQ ID NOs: 232, 243, 241 and 237.

[0387] Further provided herein is an engineered HSV-1 and / or HSV-2 gB comprising at least the domains DI, Dll, Dill, DIV and DV, wherein at least one of the domains comprises an amino acid substitution which stabilises the tertiary structure of the gB in its pre-fusion conformation. The at least one amino acid substitution may be at one or more of positions 154, 158, 174, 175, 179, 182, 239, 244, 261, 274, 277, 286, 356, 359, 362, 363, 464-498, 572, 673, 676,Q77, 678, 679, 681, 683, 684 and 709, relative to SEQ ID NO: 1, or a substitution corresponding thereto.

[0388] The at least one amino acid substitution may be selected from I154C, K158C, K158I, (WFGHR)174(YAYIH), W174N, F175H, Y179T, F182N,a239C,a239W, S244C, (AFH)261(WLY), E274V, E274I, D277C, E286F, W356P, K359P, S362P, V363P, Y464-T498del, T572C, L673(GCG), T676C, T676I, M677I, M677L, L678C, E679C, H681C, F683C, V684W and N709V relative to SEQ ID NO: 1, or a substitution corresponding thereto.

[0389] Further provided herein is a method of increasing the probability of an HSV-1 and / or HSV-2 gB remaining in a pre-fusion conformation, wherein the HSV-1 and / or HSV-2 gB gB comprises the domains DI, Dll, Dill, DIV and DV, the method comprising introducing at least one amino acid substitution into one or more of the native domain DI, Dll, Dill, DIV and / or DV.

[0390] Further provided herein is a method of increasing the solubility, expression yield, and / or stability of an HSV-1 and / or HSV-2 gB, wherein the HSV-1 and / or HSV-2 gB gB comprises the domains DI, Dll, Dill, DIV and DV, the method comprising introducing at least one amino acid substitution into one or more of the native domains DI, Dll, Dill, DIV and / or DV.

[0391] The engineered gB domains provided herein may be engineered to interact with one or more other gB domains more strongly than the native domains. The at least one amino acid substitution may increase the strength of the interaction between domains of the gB. The amino acid substitutions may be cavity filling substitutions, electrostatic substitutions and / or H-bonding network substitutions. The amino acid substitutions may be cysteine mutations which introduce one or more disulfide bridges at the DI, Dll, MPR and / or core interfaces. Epstein-Barr virus (EBV)

[0392] In some instances, the engineered gB is an Epstein-Barr virus (EBV) gB. Epstein-Barr virus (EBV) is a Lymphocryptovirus within the Gammaherpesvirinae subfamily. EBV can be grouped into two main types, EBV type 1 (EBV-1) and EBV type 2 (EBV-2), based on sequence diversity, primarily in the EBNA-2 and EBNA-3 genes. Within these types, multiple strains have been identified including representative strains such as B95-8, AG876, and GD1. Corresponding amino acid positions in any other EBV strain can be determined by those of ordinary skill in the art using known information and by sequence alignment using readily available and well-known alignment algorithms (such as BLAST, using default settings; ClustalW2, using default settings).

[0393] EBV infects over 90% of the adult population worldwide. EBV primarily infects B cells and epithelial cells, causing infectious mononucleosis (glandular fever) during primary infectionand establishing lifelong latency in B cells. EBV is associated with several malignancies including Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma, and posttransplant lymphoproliferative disorders. Despite extensive research, there are currently no licensed and approved vaccines for EBV.

[0394] gB is encoded by the BALF4 gene of the EBV genome. gB mediates virus entry into cells by facilitating virus and cell membrane fusion, making it an important target for neutralizing antibodies and vaccine development. The EBV genome also includes a number of other envelope glycoproteins, including gH, gL, gp42, gp350 / 220, gN, and gM. These proteins form complexes to bind cell surface proteins, attaching the herpesvirus particle to the cell surface. The engineered gB of the invention may be an EBV gB. Unless indicated otherwise, references to an EBV MPR refer to the MPR of the B95-8 strain of EBV (SEQ ID NO: 108), which starts at position 684 of the EBV gB according to SEQ ID NO: 108 and ends at position 773 of SEQ ID NO: 108, i.e.

[0395] NGRNQFVDGLGELMDSLGSVGQSITNLVSTVGGLFSSLVSGFISFFKNPF (SEQ ID NO: 260).

[0396] The engineered EBV gB may be derived from any of the different virus strains within Type 1 and Type 2, identified by full EBV genome sequencing, or any EBV strains associated with diseases such as cancer, Multiple sclerosis, and autoimmune disease such as Lupus (Palser et al, 2015).

[0397] The non-native EBV MPR may comprise a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% to a sequence selected from SEQ ID NOs: 260-274.

[0398] The engineered EBV gB may have a sequence selected from any one of SEQ ID NOs: 244- 259.

[0399] The non-native EBV MPR may comprise a sequence selected from any one of SEQ ID NOs: 260-274.

[0400] The non-native EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 260. The non-native EBV MPR EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 261. The non-native EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 262. The non-native EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 263. The non-native EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 264. The non-native EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 265. The non-native EBV MPR may comprise the aminoacid sequence set forth in SEQ ID NO: 266. The non-native EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 267. The non-native EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 268. The non-native EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 269. The non-native EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 270. The non-native EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 271. The non-native EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 272. The non-native EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 273. The non-native EBV MPR may comprise the amino acid sequence set forth in SEQ ID NO: 274. The engineered EBV gB disclosed herein may comprise one or more additional stabilising substitutions outside of the MPR.

[0401] The amino acid substitution may be a substitution at one or more of amino acid residues I89, I90, T630C, (YNGWY)109, W112, Y113, K124, G173, G177, R180, R180, T225C, T225F, L288, E289, T292. A293, K394-T452I, R429, R431, R432, D478, Q527, L628, S631, N635, I636, L641, E634, W196, Y198 v of the gB, relative to SEQ ID NO: 108 (B95-8 strain). Specifically, the engineered EBV gB may comprise one of more substitutions selected from I89C, I90C, T630C, (YNGWY)109(YAYIH), W112N, Y113T, K124V, G173C, G177C, R180I, R180L, T225C, T225F, L288P, E289P, T292P. A293P, K394-T452del, R429S, R431S, R432S, D478L, Q527C, L628(GCG), S631C, N635T, I636C, L641C, E634C, W196S, Y198D relative to SEQ ID NO: 108, or corresponding substitutions in other HSV-1 gBs.

[0402] Alternatively, the amino acid substitution may be a substitution at one or more of amino acid residues 684-733 of the EBV gB, relative to SEQ ID NO: 108 (B95-8 strain).

[0403] The engineered EBV gB may comprise one of more substitutions selected from I89C, I90C, T630C, G173C, G177C, S631C, I636C, L641C, and / or E634C relative to SEQ ID NO: 108, or corresponding substitutions in other HSV-1 gBs.

[0404] The engineered EBV gB may comprise one or more pairs of substitutions at positions selected from G173C and L641C, T225C and E634C, G177C and E634C, G173C and L641C, T225C and I636C, and 90C and T630C, or corresponding positions, relative to SEQ ID NO: 108. The engineered EBV gB may comprise one or more pairs of substitutions at positions selected from I89C, L628(GCG), Q527C and E634C, or corresponding positions, relative to SEQ ID NO: 108.

[0405] The gB comprises one or more pairs of substitutions selected from: I89C and L628(GCG) or Q527C and E634C.The engineered EBV gB may comprise substitutions at one of more of R429, R431 and R432, or corresponding positions in other HSV-1 gBs. Specifically, the engineered gB may comprise one of more substitutions selected from R429S, R431S and R432S relative to SEQ ID NO: 108, or corresponding substitutions in other HSV-1 gBs.

[0406] The engineered EBV gB may comprise substitutions at one of more of I89, L628, Q527, E634, R429, R431 and R432, or corresponding positions in other HSV-1 gBs. Specifically, the engineered gB may comprise one of more substitutions selected from I89C, L628(GCG), Q527C, E634C, R429S, R431S and R432S relative to SEQ ID NO: 108, or corresponding substitutions in other HSV-1 gBs.

[0407] The engineered EBV gB may comprise substitutions at one of more of I89, L628, Q527, E634, R429, R431, R432, W112, Y113, W196, Y198 or corresponding positions in other HSV-1 gBs. Specifically, the engineered gB may comprise one of more substitutions selected I89C, L628(GCG), Q527C, E634C, R429S, R431S, R432S, W112N, Y113T, W196S, Y198D relative to SEQ ID NO: 108, or corresponding substitutions in other HSV-1 gBs.

[0408] The gB may comprise substitutions one or more of the combinations of positions G173 and L641; T225 and I636; T225 and E634; I89 and S631; L288 and E28P; R429, R431 and R432S; or corresponding positions, relative to SEQ ID NO: 108. Specifically, the gB may comprise one or more of the combinations of substitutions G173C and L641C; T225C and I636C; T225C and E634C; I89C and S631C; L288Pand E289P; R429S, R431S and R432S or corresponding substitutions, relative to SEQ ID NO: 108.

[0409] The engineered EBV gB may comprise substitutions at one of more of positions G173, L641, T225, I636, E634, I89, S631, R429, R431, R432, L288 or E289, relative to SEQ ID NO: 108 (the gB of the KOS strain of EBV) or corresponding substitutions in other HSV-1 gBs. Specifically, the engineered gB may comprise one of more substitutions selected from positions G173C, L641C, T225C, I636C, E634C, I89C, S631C, R429S, R431S, R432S, L288P or E289P, relative to SEQ ID NO: 108 (the gB of the KOS strain of EBV) or corresponding substitutions in other HSV-1 gBs.

[0410] The MPR may comprise one or more substitutions to introduce one or more disulfide bridges at the DI, Dll, MPR and / or core interfaces. In particular, the MPR may comprise one or more substitutions to introduce one or more disulfide bridges at the DI / MPR interface.

[0411] The engineered gB may comprise one or more combinations of substitutions selected from: a) G173C and L641C;

[0412] b) T225C and I636C;c) T225C and E634C;

[0413] d) I89C and S631C;

[0414] e) L288P and E289P; and / or

[0415] f) R429S, R431S and R432S

[0416] relative to SEQ ID NO: 108, or substitutions corresponding thereto.

[0417] Disulphide bridges may also be introduced into other parts of the molecule.

[0418] The engineered EBV gB may comprise substitutions at one of more of positions G177, E634, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from G177C, E634C, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0419] The engineered EBV gB may comprise substitutions at one of more of positions I90, T630, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from I90C, T630C, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0420] The engineered EBV gB may comprise substitutions at one of more of positions G177, E634, I90, T630, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from G177C, E634C, I90C, T630C, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0421] The engineered EBV gB may comprise substitutions at one of more of positions G173, L641, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from G173C, L641C, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0422] The engineered EBV gB may comprise substitutions at one of more of positions T225, I636, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may compriseone of more substitutions selected from T225C, I636C, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0423] The engineered EBV gB may comprise substitutions at one of more of positions G173C, L641C, T225C, E634C, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0424] Specifically, the engineered gB may comprise one of more substitutions selected from G173C, L641C, T225C, E634C, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0425] The engineered EBV gB may comprise substitutions at one of more of positions G173, L641, T225, I636, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from G173C, L641C, T225C, I636C, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0426] The engineered EBV gB may comprise substitutions at one of more of positions G173, L641, T225, I636, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from G173C, L641C, I89C, S631C, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0427] The engineered EBV gB may comprise substitutions at one of more of positions G173, L641, T225, I636, R180, E321, D478, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from G173C, L641C, T225C, I636C, R180L, E321I, D478L, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0428] The engineered EBV gB may comprise substitutions at one of more of positions G173, L641, T225, I636, R180, E321, D478, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from G173C, L641C, R180I, T225F, N635T, D320L, D478L, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.The engineered EBV gB may comprise substitutions at one of more of positions G173, L641, T225, I636, R180, E321, D478, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from G173C, L641C, T225C, I636C, K214F, D320L, D478L, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0429] The engineered EBV gB may comprise substitutions at one of more of positions G173, L641, T225, I636, R180, E321, D478, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from G173C, L641C, T225C, I636C, K124V, D320L, D478L, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0430] The engineered EBV gB may comprise substitutions at one of more of positions G173, L641, T225, I636, I89, S631, R180, E321, D478, R429S, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from G173C, L641C, T225C, I636C, I89C, S631C, R180L, E321I, D478L, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0431] The engineered EBV gB may comprise substitutions at one of more of positions G173, L641, T225, I636, L288, E289, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from G173C, L641C, T225C, I636C, L288P, E289P, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0432] The engineered EBV gB may comprise substitutions at one of more of positions G173, L641, T225, I636, L288, E289, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from G173C, L641C, T225C, I636C, R291P, T292P, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0433] The engineered EBV gB may comprise substitutions at one of more of positions K394-T452del, A293, Q527, E634, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, theengineered gB may comprise one of more substitutions selected from K394-T452del, A293P, Q527C, E634C, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0434] The engineered EBV gB may comprise substitutions at one of more of positions I89, L628, Q527, E634, (YNGWY)109, (WTY)196, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from I89C, L628(GCG), Q527C, E634C, (YNGWY)109(YAYIH), (WTY)196(WLY), R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0435] The engineered EBV gB may comprise substitutions at one of more of positions I89, L628, Q527, E634, (YNGWY)109, (WTY)196, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from I89C, L628(GCG), Q527C, E634C, (YNGWY)109(YAYIH), (WTY)196(WLY), R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0436] The engineered EBV gB may comprise substitutions at one of more of positions I89, L628, Q527, E634, (YNGWY)109, (WTY)196, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from I89C, L628(GCG), Q527C, E634C, (YNGWY)109(YAYIH), (WTY)196(WLY), R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0437] The engineered EBV gB may comprise substitutions at one of more of positions I89, L628, Q527, E634, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from I89C, L628(GCG), Q527C, E634C, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0438] The engineered EBV gB may comprise substitutions at one of more of positions I89, L628, Q527, E634, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from I89C, L628(GCG), Q527C, E634C, R429S,R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0439] The engineered EBV gB may comprise substitutions at one of more of positions I89, L628, Q527, E634, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from I89C, L628(GCG), Q527C, E634C, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0440] The engineered EBV gB may comprise substitutions at one of more of positions I89, L628, Q527, E634, R429, R431 and R432 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from I89C, L628(GCG), Q527C, E634C, R429S, R431S and R432S relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0441] The engineered EBV gB may comprise substitutions at one of more of positions I89, L628, Q527, E634, R429, R431, R432, W112, Y113, W196 and Y198 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from I89C, L628(GCG), Q527C, E634C, R429S, R431S, R432S, W112N, Y113T, W196S and Y198D relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0442] The engineered EBV gB may comprise substitutions at one of more of positions I89, L628, Q527, E634, R429, R431, R432, W112, Y113, W196 and Y198 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from I89C, L628(GCG), Q527C, E634C, R429S, R431S, R432S, W112N, Y113T, W196S and Y198D relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0443] The engineered EBV gB may comprise substitutions at one of more of positions I89, L628, Q527, E634, R429, R431, R432, W112, Y113, W196 and Y198 relative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs. Specifically, the engineered gB may comprise one of more substitutions selected from I89C, L628(GCG), Q527C, E634C, R429S, R431S, R432S, W112N, Y113T, W196S and Y198Drelative to SEQ ID NO: 108 (the gB of the B95-8 strain of EBV) or corresponding substitutions in other EBV gBs.

[0444] The amino acid substitutions may be at positions G173, L641, R429, R431 and R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position T225, I636, R429 and R431, R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position G173, L641, T225, E634, R429, R431 and R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position G173, L641, T225, I636, R429, R431 and R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position G173, L641, I89, S631, R429, R431 and R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position G173, L641, T225, I636, R180, E321, D478, R429, R431 and R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position G173, L641, R180, T225, N635, D320, D478, R429, R431, R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position G173, L641, T225, 1636, K214, D320, D478, R429, R431 and R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position G173, L641, T225, I636, K124, D320, D478, R429, R431 and R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position G173, L641, T225, I636, I89, S631, R180, E321, D47, R429, R431 and R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position G173, L641, T225, I636, L288, E289, R429, R431 and R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position G173, L641, T225, 1636, R291, T292, R429, R431 and R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position K394-T452, A293, Q527, E634, R429, R431, R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position I89, L628, Q527, E634, (YNGWY)109(YAYI), (WTY)196(WL), R429, R431 and R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position I89, L628, Q527, E634, R429, R431 and R432 relative to SEQ ID NO: 108. The amino acid substitutions may be at position I89, L628, Q527, E634, R429, R431, R432, W112, Y113, W196 and Y198 relative to SEQ ID NO: 108. The amino acid substitutions may be G173C, L641C, R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be T225C, I636C, R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be G173C, L641C, T225C, E634C, R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be G173C, L641C, T225C, I636C, R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be G173C, L641C, I89C, S631C, R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be G173C, L641C, T225C, I636C, R180L, E321I, D478L, R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be G173C, L641C, R180I, T225F, N635T, D320L, D478L, R429S, R431S and R432S relative toSEQ ID NO: 108. The amino acid substitutions may be G173C, L641C, T225C, I636C, K214F, D320L, D478L, R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be G173C, L641C, T225C, I636C, K124V, D320L, D478L, R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be G173C, L641C, T225C, I636C, I89C, S631C, R180L, E321I, D478L, R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be G173C, L641C, T225C, I636C, L288P, E289P, R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be G173C, L641C, T225C, I636C, R291P, T292P, R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be K394-T452del, A293P, Q527C, E634C, R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be I89C, L628(GCG), Q527C, E634C, (YNGWY)109(YAYIH), (WTY)196(WLY), R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be I89C, L628(GCG), Q527C, E634C, R429S, R431S and R432S relative to SEQ ID NO: 108. The amino acid substitutions may be I89C, L628(GCG), Q527C, E634C, R429S, R431S, R432S, W112N, Y113T, W196S and Y198D relative to SEQ ID NO: 108.

[0445] The engineered EBV gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from any one of SEQ ID NOs: 134-140 or 244-259.

[0446] The engineered EBV gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from any one of SEQ ID NOs: 244-259.

[0447] The engineered EBV gB may have a sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% identity to a sequence selected from any one of SEQ ID NOs: 244, 246, 249, 251,

[0448] The engineered EBV gB may have a sequence selected from any one of SEQ ID NOs: 134-140 or 244-259.

[0449] The engineered EBV gB may have a sequence selected from any one of SEQ ID NOs: 244-259.

[0450] The engineered EBV gB may have a sequence selected from any one of SEQ ID NOs: 244, 246, 249 and 251.

[0451] The engineered EBV gB may have a sequence of SEQ ID NO: 244. The engineered gB may have a sequence of SEQ ID NO: 246. The engineered gB may have a sequence of SEQ ID NO: 249. The engineered gB may have a sequence of SEQ ID NO: 251.The engineered EBV gB may comprise a sequence of MTRRRVLSVVVLLAALACRLGAQTPEQPAPPATTVQPTATRQQTSFPFRVCELSSHGDLFR FSSDIQCPSFGTRENHTEGLLMVFKDNX1X2PYSFKVRSYTKIVTNILIYNGX3X4ADSVTNRH EEX5FSVDSYETDQMDTIYQCYNAVKMTKDGLTRVYVDRDGVNITVNLKPTGX6LANX7VRX sYASQTELYDAPGXgLIXioTXuRTRTTVNCLITDMMAX^SNSPFDFFVTXisTGQTVEMSPFY DGKNKETFHERADSFHVRTNYKlVDYDNRGTNPQGERRAFLDKGTYTLSWKX^XigNXieXi7X18YCPLQHWQTFDSTIATETGKSIHFVTX19X20GTSSFVTNTTVGIELPDAFKCIEEQVNKT MHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELTTPTSSPPSSPSP PAPSAARGSTPAAVLSRRSSDAGNATTPVPPTAPGKSLGTLNNPATVQIQFAYDSLRRQIN RMLGX21LARAWCLEQKRQNMVLRELTKINPTTVMSSIYGKAVAAKRLGDVISVSX22CVPVN QATVTLRKSMRVPGSETMCYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQ YYFQSGNEIHVYNDYHHFKTIELDGIATLQTFISGCG, wherein Xxis I, or C; X2is I, or C; X3is N, or W; X4 is T, or Y; X5is K, or V; X6is G, or C; X7is C, or G; X8is R, L, or I; X9is S, or W; X10 is S, or W; Xu is T, Y, or D; X12is K, or F; Xi3is T, C, or F; X14is L, or P; Xi5is E, or P; Xi6is R, or P; X17is T, or P; Xi8is A, or P; X19is D, or L; X20is E, or I; X2iis D, or L and X22is Q, or C (SEQ ID NO: 326).

[0452] The engineered EBV gB may comprise a sequence of MTRRRVLSVVVLLAALACRLGAQTPEQPAPPATTVQPTATRQQTSFPFRVCELSSHGDLFR FSSDIQCPSFGTRENHTEGLLMVFKDNCIPYSFKVRSYTKIVTNILIYNGXiX. ADSVTNRHEE KFSVDSYETDQMDTIYQCYNAVKMTKDGLTRVYVDRDGVNITVNLKPTGGLANGVRRYAS QTELYDAPGWLIX3TX4RTRTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNKET FHERADSFHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQHWQTF DSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKTMHEKYEAVQDRYTKG QEAITYFITSGGLLLAWLPLTPRSLATVKNLTELTTPTSSPPSSPSPPAPSAARGSTPAAVLS RRSSDAGNATTPVPPTAPGKSLGTLNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQK RQNMVLRELTKINPTTVMSSIYGKAVAAKRLGDVISVSCCVPVNQATVTLRKSMRVPGSET MCYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNEIHVYNDYHH FKTIELDGIATLQTFISGCGNTSLICNIDFASLELYSRDEQRASNVFDLEGIFREYNFQAQNIA GLRKDLD, wherein Xi is W, or N; X2is Y, or T; X3is W, or S and X4 is Y, or D (SEQ ID NO: 327).

[0453] The engineered EBV MPR may comprise an amino acid sequence of NGX1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27X28X29X3OGX3IX32X33X34X35X36X37X38X39X4OX4IX42X43X44X45X46X47, wherein X4is I, V, R, or L; X2is L, N, or K; X3is E, A, Q, or L; X4 is K, E, F, or L; X5is V, or R; X6is A, M, D, or E; X7is E,K, V, A, L, I, D, N, or T; X8is L, or M; X9is A, or G; X10is S, D, Q, K, E, T, or R; Xu is N, I, V, or L; X12is Y, M, or Q; X13is L, D, or A; X14is N, R, or K; Xi5is L, V, or T; Xi6is P, or N; X17 is D, P, or N; Xi8is A, F, E, S, P, G, or L; X19is E, G, or L; X20is R, or Q; X2iis E, R, S, or I; X22is R, A, I, V, or L; X23is A, T, or L; X24is R, N, or Q; X25is E, I, V, L, or A; X26is E, T, I, or V; X27is T, N, S, or A; X28is L, I, T, W, or R; X29is K, I, V, or L; X30is L, or G; X31is Q, K, I, L, or A; X32is E, A, L, or I; X33is Q, E, S, or G; X34is H, A, S, R, or T; X35is A, I, L, V, or F; X36is A, R, or L; X37is L, S, A, or N; X38is E, A, T, D, N, L, or I; X39is A, I, or L; X40is A, E, L, V, or N; X^ is K, A, S, E, R, or D; X42is A, L, K, T, W, or S; X43is A, V, I, or L; X^ is G, K, S, L, R, T, or N; X^ is S, D, G, or L; X46is P, or S and X47 is L, D, H, F, W, G, Y, or Q (SEQ ID NO: 368).

[0454] The engineered EBV MPR may comprise an amino acid sequence of NGX1X2X3X4VX5X6LX7X8X9X10X11X12X13X14X15X16X17QX18X19X20X21X22X23X24X25X26X27G X28X29X3OX3IX32X33X34X3SX36X37X38X39X4OX4IX42PX43, wherein X4is R, or V; X2is N, or L; X3is Q, or A; X4is F, E, or L; X5is D, or M; X6is G, K, or D; X7is G, or A; X8is E, or D; X9is L, or N; Xi0is M, or Y; Xu is D, or L; X12is S, N, or R; X13is L, or S; X14is G, P, or D; Xi5is S, or P; Xi6is V, A, S, or P; X17is G, or E; Xi8is S, or R; X19is I, A, or L; X20is T, or A; X21is N, or R; X22is L, I, or A; X23is V, or T; X24is S, or N; X25is T, or I; X26is V, K, or I; X27is G, or L; X28is L, or K; X29is F, E, I, or L; X30is S, or E; X34is S, or A; X32is L, A, or F; X33is V, R, or L; X34is S, L, or E; X35is G, A, or I; X36is F, A, or L; X37is I, E, or A; X38is S, A, D, or R; X39is F, A, or L; X40is F, V, or A; X41is K, N, or A; X42is N, S, L, or D and X43is F, L, or S (SEQ ID NO: 329).

[0455] The non-native MPR may have more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 15, more than 20, more than 25, or more than 30 substitutions relative to the native MPR.

[0456] Engineered gBs

[0457] The terms “engineered Herpesvirus gB” and “engineered gB” as used herein refer to the engineered Herpesvirus gBs of a species within a genus of the present disclosure. Unless stated otherwise, such engineered Herpesvirus gBs should be understood to comprise at least one amino acid substitution which stabilises the tertiary structure of the gB in a pre-fusion conformation. The engineered Herpesvirus gB may comprise further modifications in accordance with the present disclosure. The engineered Herpesvirus gB may also comprise modifications not referenced in the present disclosure, so long as such modifications do notimpair stabilisation of the tertiary structure of the gB in a pre-fusion conformation. In particular, the engineered Herpesvirus gBs may comprise a non-native MPR.

[0458] The engineered Herpesvirus gBs provided herein may not comprise a transmembrane domain. In particular, the engineered gBs may be soluble gB variants that have been engineered to lack a transmembrane domain and are therefore not membrane bound. Such soluble gBs are suitable for use as immunogens and immune reagents of the disclosure, for example in vaccines, pharmaceutical compositions and the like, as well as in diagnostic and experimental assays of the disclosure.

[0459] An engineered Herpesvirus gB of a given species is most relevant to virus strains within the same species, for example an HCMV engineered Herpesvirus gB is most relevant for use in connection with HCMV infection, vaccination, diagnosis and therapy.

[0460] The terms “corresponding gB” and “corresponding Herpesvirus” as used herein refer to a gB and Herpesvirus from the same genus and associated viral diseases. For example, an engineered EBV gB of the disclosure would be intended for use in connection with detection, prevention and treatment of the EBV virus, and associated infections and diseases, an engineered HCMV gB would be intended for use in connection with detection, prevention and treatment of the HCMV virus, and associated infections and diseases, and an engineered HSV-1 gB would be intended for use in connection with detection, prevention and treatment of the HSV-1 virus, and associated infections and diseases.

[0461] In certain instances, the gB and Herpesvirus may be from the same strain. For example they may both be from the Towne strain of HCMV or the KOS strain of HSV-1.

[0462] In addition to the modifications to the MPR, the engineered gBs disclosed herein may comprise one or more additional stabilising mutations outside of the MPR. The one or more stabilising mutations may be cavity filling mutations, electrostatic mutations and / or H-bonding network mutations. The stabilising mutations may be located at one or more of the DI, Dll, MPR and / or core interfaces. The stabilising mutations may introduce one or more disulfide bridges at the DI, Dll, MPR and / or core interfaces.

[0463] The additional stabilising substitution may be a further substitution of one or more hydrophobic residues at a solvent interface. For example, one or more hydrophobic solvent facing amino acid residues in the native DI may have been substituted with a polar or charged amino acid residue. The hydrophobic amino acid may be I, L A, V or F and the substitution may be to N, S, T, Q, Y, C, K, R or E. The hydrophobic amino may be L, A, V or F and / or the substitution may be to K, R or E.The additional stabilising substitution may be a substitution ata Furin cleavage site. Exemplary Furin cleavage site mutations in the gB of HHV1-HHV8 are listed below:

[0464] Table 1. Furin cleavage site mutations.

[0465] Common Furin Cleavage Virus Strain Furin Cleavage Site

[0466] Name Substitutions HHV-1 HSV1 KOS None None

[0467] HHV-2 HSV2 HG52 None None

[0468] HHV-3 VZV Oka R491, R494 R491S, R494S R429S, R431S, HHV-4 EBV B95-8 R429, R431, R432

[0469] R432S HHV-5 CMV Merlin R457S, R460 R457S, R460S HHV-6A GS R396, R399 R396S, R399S HHV-6B Z29 R396, R399 R396S, R399S R395S, R396S, HHV-7 JI R395, R396, R398

[0470] R398S HHV-8 KSHV GK18 R439, R440 R439S, R440G

[0471]

[0472] The engineered gB may therefore comprise substitutions at one, two, or all of the relevant the furin cleavage sites provided in Table 1, or at corresponding positions. The engineered gB may comprise the one, two, or all of the relevant furin cleavage substitutions in Table 1, or at corresponding positions.

[0473] For example, an engineered HHV-3 (VZV) gB may comprise substitutions at positions R491 and / or R494, or positions corresponding thereto. Specifically, the substitutions may be R491S and / or R494S, or substitutions corresponding thereto.

[0474] An engineered HHV-4 (EBV) gB may comprise substitutions at positions R429, R431 and / or R432, or positions corresponding thereto. Specifically, the substitutions may be R429S, R431S and / or R432S, or substitutions corresponding thereto.

[0475] An engineered HHV-5 (CMV) gB may comprise substitutions at positions R457S and / or R460, or positions corresponding thereto. Specifically, the substitutions may be R457S and / or R460S, or substitutions corresponding thereto.An engineered HHV-6A gB may comprise substitutions at positions R396 and / or R399, or positions corresponding thereto. Specifically, the substitutions may be R396S and / or R399S, or substitutions corresponding thereto.

[0476] An engineered HHV-6B gB may comprise substitutions at positions R396, R399, or positions corresponding thereto. Specifically, the substitutions may be R396S and / or R399S, or substitutions corresponding thereto.

[0477] An engineered HHV-7 gB may comprise substitutions at positions R395, R396 and / or R398, or positions corresponding thereto. Specifically, the substitutions may be R395S, R396S and / or R398S, or substitutions corresponding thereto.

[0478] An engineered HHV-8 (KSHV) gB may comprise substitutions at positions R439 and / or R440, or positions corresponding thereto. Specifically, the substitutions may be R439S and / or R440G, or substitutions corresponding thereto.

[0479] The engineered gB may comprise one or both of substitutions R457S and R460S relative to SEQ ID NO: 1.

[0480] “Corresponding positions” as used herein refers to analogous positions in other Herpesvirus gB proteins. The identification of such analogous positions based on e.g. sequence alignments is known in the art. Sequence alignments indicating correspondence between the gB sequences of an exemplary selection of Herpesviruses are provided in Figure 11.

[0481] “Corresponding substitutions” as used herein refers a substitution at corresponding position of the same initial amino acid residue for the same substituted amino acid residue. For example, a corresponding substitution for C246S in the HCMV (Towne) gB protein would be a substitution of a cysteine at an analogous position in another Herpesvirus strain for a serine. “Functionally equivalent substitutions” as used herein refer to substitutions at a corresponding position, wherein the original amino acid residue is alternatively replaced with an amino residue having the same or corresponding physicochemical properties and / or performing a substantially similar structural or functional role to the amino acid residue specified. For example, a functionally equivalent substitution for T100L would include substitution of the threonine residue (T) with another amino acid which is functionally equivalent to leucine (L), and therefore capable of providing similar stabilising interactions within the three-dimensional structure of the protein to the T100L substitition.

[0482] The gB may further comprise a Foldon domain to promote trimerisation. The Foldon domain may be at the C-terminus, after the MPR. The gB may further comprise one or morepurification tags, such as a His-tag and / or a Step-tag. Such tags may be excluded from the gB when used as a vaccine antigen.

[0483] Where sequences of the disclosure are shown to comprise a purification tag, e.g., a His-tag such as “HHHHHHHH” or any other tags, it should be understood that the disclosure is also intended to encompass corresponding sequences lacking such tags. For example, and any or all of the sequence elements after the engineered gB of the invention may be optionally excluded and or replaced with alternative sequence elements. Thus, any or all of the sequence elements after the MPR of any one of OP1-OP232 (SEQ ID NOs: 1-221, 244-259 or 363-377) or may be excluded or replaced.

[0484] MPR Design

[0485] In a further aspect, there is provided a method of designing a non-native MPR for stabilising a Herpesvirus gB in a pre-fusion conformation, the method comprising:

[0486] a) generating a plurality of modified MPR sequences;

[0487] b) generating two or more replicate structures from each modified MPR sequence;

[0488] c) calculating the agreement between the generated structures (inter-Root Mean Square Deviation (RMSD));

[0489] d) calculating the agreement between the generated MPR structures and the structure of the initial MPR sequence (target-RMSD);

[0490] e) clustering the modified MPR sequences on the basis of at least the inter-RMSD and target- RMSD values; and

[0491] selecting structures in clusters having the lowest target-RMSD in combination with the lowest inter-RMSD, wherein the target-RMSD has a maximum value of 10 Angstroms and the inter-RMSD has a maximum value of 4 Angstroms.

[0492] The method may be a method of designing a non-native MPR for stabilising a Herpesvirus gB in a pre-fusion conformation, the method comprising:

[0493] a) providing a three-dimensional molecular structure of a pre-fusion stabilised gB;

[0494] b) preparing an input model of the pre-fusion stabilised gB from the three-dimensional molecular structure;

[0495] c) energy minimising the input model;

[0496] d) generating a plurality of modified MPR sequences;

[0497] e) generating two or more replicate structures from each modified MPR sequence;

[0498] f) determining the agreement between the generated structures (inter-Root Mean Square Deviation (RMSD));g) determining the agreement between the generated MPR structures and the structure of the initial MPR sequence (target-RMSD);

[0499] h) clustering the modified MPR sequences on the basis of at least the inter-RMSD and target-RMSD values; and

[0500] i) selecting structures in clusters having the lowest target-RMSD in combination with the lowest inter-RMSD for further characterisation in vivo and / or in vitro',

[0501] wherein the target-RMSD has a maximum value of 10 angstroms (A) and the inter-RMSD has a maximum value of 4 angstroms (A).

[0502] The method may further comprise the steps of:

[0503] j) generating one or more nucleic acid sequences, each encoding a gB comprising the modified MPR sequence of a selected structure;

[0504] k) expressing the one or more nucleic acid sequences in an expression system to produce a gB comprising the modified MPR; and

[0505] l) purifying the gB comprising the modified MPR to a purity of at least 80%.

[0506] Where the further characterisation is in vitro, the method may further comprise a step of assessing one or more biophysical and / or biochemical properties of the gB comprising the modified MPR. The biophysical and / or biochemical properties may be selected from the aggregation temperature, expression level, percentage of correctly folded protein, hydrodynamic radius, antigenicity, and / or negative-stain electron microscopy (nsEM) characteristics.

[0507] Where the further characterisation is in vivo, the method may further comprise the steps of: m) administering the gB comprising the modified domain to a non-human subject to elicit an immune response;

[0508] n) isolating one or more antibodies from the non-human subject; and

[0509] o) assessing the binding activity and / or neutralising activity of the one or more antibodies against a live Herpesvirus corresponding to the species from which the gB is derived. The method may further comprise the steps of:

[0510] m) administering the gB comprising the modified domain to a non-human subject to elicit an immune response;

[0511] n) isolating one or more antibodies from the non-human subject; and

[0512] o) assessing the binding activity and / or neutralising activity of the one or more antibodies against a live Herpesvirus corresponding to the species from which the gB is derived. The three-dimensional molecular structure may be generated experimentally and / or computationally. For example, molecular structure may be generated by X-raycrystallography, cryo-electron microscopy, homology modelling, or in-silico prediction, or by a combination of such methods.

[0513] The modified MPR sequences may also be clustered on the basis of predicted local distance difference test (pLDDT) and / or hydropathy. The selected structures may have a pLDDT value of more than 80 and / or a hydropathy value of between -0.5 and 0.5.

[0514] The target RMSD may be less than 2.0 A. The inter-RMSD may be less than 3.0 A.

[0515] The modified MPR sequences may also be clustered on the basis of predicted local distance difference test (pLDDT) value and / or hydropathy. The modified MPR sequences may have a pLDDT value of more than 80 and / or a hydropathy value of between -0.5 and 0.5.

[0516] The modified MPR sequences may also be clustered on the basis of one or more of the instability index, the interface shape complementarity (e.g. Rosetta Shape Complementarity or equivalent metrics using any suitable software) and / or the interface energy (dG) (e.g. Rosetta Interface Energy or equivalent metrics from any suitable software.)

[0517] The Rosetta ShapeComplementarity and Rosetta Interface Energy can be calculated using the Rosetta software, for example, Rosetta 3.14 or Rosetta 3.15, available from http: / / www.rosettacommons.org). The Rosetta ShapeComplementarity filters on the shape complementarity of interface (Lawrence & Colman 1993, incorporated herein by reference). Details are provided at

[0518] https: / / docs.rosettacommons.org / docs / latest / scripting documentation / RosettaScripts / Filters / f ilter pages / ShapeComplementarityFilter). The mathematical models and physical concepts underlie Rosetta energy calculations are set out in e.g. Alford et al and Leman et al., incorporated herein by reference.

[0519] The modified MPR sequences may therefore be selected on the basis of their target RMSD, inter-RMSD, predicted Local Distance Difference Test (pLDDT) threshold, hydropathy, instability index, Shape Complementarity and / or Interface Energy (dG) (e.g. Rosetta Shape Complementarity and / or a Rosetta Interface Energy).

[0520] For example, the selected MPR sequences may have a predicted pLDDT value greater than 80, a hydropathy index less than 0.5, an instability index less than 40, a shape complementarity greater than 0.6, and / or an interface energy (dG) greater than the mean dG of all designed sequences.

[0521] Preferably, more than two replicate structures will be generated. For example, three, four, five, or more than five replicate structures may be generated. In particular, five replicate structures may be generated.In a further aspect, provided herein non-native Herpesvirus gB MPR domain for use in a method of stabilising a Herpesvirus gB in a pre-fusion conformation, wherein the non-native MPR replaces the native MPR.

[0522] The Herpesvirus gB may be a soluble gB. The non-native gB may show an increase in one or more properties selected from the list of solubility, expression level, percentage of pre-fusion particles and aggregation temperature, and / or a decrease in one or more properties selected from the list of hydropathy index, instability index or total energy (dG) (e.g. Rosetta Total Energy), relative to a soluble Herpesvirus gB comprising a corresponding native MPR or no MPR.

[0523] Where the non-native gB is designed computationally, it may have a pLDDT value above 80, a target RMSD match of below 2.0 A, a prediction RMSD match of below 3.0 A, a hydropathy index value of the designed MPR domain below 0.5, an instability index value of below 40, a shape complementary value of above 0.6, and / or an interface energy value above the mean of all designed sequences.

[0524] In a further aspect, provided herein is a soluble Herpesvirus gB comprising a non-native MPR domain, wherein the non-native MPR domain comprises at least one amino acid substitution, and wherein the Herpesvirus gB shows an increase in one of more of solubility, expression level, percentage of pre-fusion particles and aggregation temperature, and / or a decrease in hydropathy index, instability index or total energy (dG) (e.g. Rosetta Total Energy), relative to a soluble Herpesvirus gB comprising a corresponding native MPR or no MPR.

[0525] The non-native MPR domain comprises one or more substitutions which decreases the surface hydrophobicity of the MPR relative to the corresponding native MPR.

[0526] The non-native MPR may comprise at least 2, at least 5, at least 10, at least 15, at least 20 or more than 20 substitutions, provided the non-native MPR retains the structural and functional attributes of the native MPR. The non-native MPR may enhance expression of the soluble protein and ensuring enhanced gB pre-fusion stability.

[0527] One or more hydrophilic solvent-interfacing amino acid residues may be substituted with hydrophobic amino-acid residues relative to the corresponding wildtype MPR domain.

[0528] In a further aspect, provided herein is a soluble Herpesvirus gB comprising a non-native MPR for stabilising the Herpesvirus gB in a pre-fusion conformation, wherein the non-native MPR comprises at least one amino acid substitution relative to the native-MPR which increases the strength of the interaction between the MPR and the DI domain relative to the native-MPR of the gB, or reduces the hydrophobicity of the surface of the MPR relative to the native-MPR.The residue positions in the MPR domain may be selected based on their hydropathy value. For example, the amino acids substituted may be amino acids with hydrophobic side chains such as Leu, lie, Vai, and Phe.

[0529] In one aspect, provided herein is a soluble Herpesvirus gB comprising a means for stabilising the Herpesvirus gB in a pre-fusion conformation.

[0530] The stabilising the Herpesvirus gB may comprise a means for stabilising the Herpesvirus gB in a pre-fusion conformation, such as, for example a non-native MPR and one or more core interface mutations. Soluble Herpesvirus gBs comprising means for stabilising the Herpesvirus gB include equivalents of the structural features of the engineered the Herpesvirus gBs described herein, for example, equivalents of the non-native MPR described herein and core interface mutations described herein, as well as equivalents of the functional features described herein. In particular, the non-native MPR may comprise one or more functionally equivalent substitutions to any of the substitutions described herein.

[0531] In a further aspect, provided herein is a method of designing a soluble pre-fusion stabilised Herpesvirus gB. Such methods allow for the generation of M PR-based sequences and core interface mutations that would stabilise the DI / MPR interaction in the pre-fusion conformation. The method may comprise the steps of:

[0532] a) obtaining an experimentally determined three-dimensional structure of a Herpesvirus gB or generating a three-dimensional structure of a Herpesvirus gB in silico from a Herpesvirus gB sequence;

[0533] b) using the obtained or generated thee-dimensional structure as an input model;

[0534] c) energy minimising the input model using molecular dynamics simulations, wherein the energy minimisation reduces steric clashes and optimises side chain rotamers;

[0535] d) selecting target locations for potential pre-fusion stabilisation substitutions, wherein the target locations are located within the core of the protein and / or within the interface between the DI fusion loops and MPR domain;

[0536] e) predicting the impact of point mutations at the target locations in silico using a deep neural network and identifying favourable residue positions to be mutated;

[0537] f) applying residue masking at the residue positions identified in step (c) to generate a diverse set of non-native gB sequences;

[0538] g) generating in silico models of one or more of the non-native gB structures generated in step (f);h) generating in silica data, for example, calculated properties based on the sets of diversified sequences and generated protein structures, and collating the data in one dataset for downstream selection purposes;

[0539] i) selecting one or more of the non-native gB structures having a pLDDT value above 80, a target RMSD match of below 2.0 A, a prediction RMSD match of below 3.0 A, a hydropathy index value of below 0.5, an instability index value of below 40, a shape complementary value of above 0.6, and / or an interface energy value above the mean of all designed sequences; or

[0540] j) ranking the sequences according to their total energy value selecting the top sequence designs.

[0541] The method may further comprise examining the sequence designs experimentally. The method may further comprise repetition of the above steps in an iterative process to arrive at an optimised engineered gB that is stabilised in a pre-fusion conformation that can elicit neutralizing antibodies.

[0542] The residue positions in the MPR domain may be specifically chosen based on their hydropathy value. For example, residues to be mutated include amino acids with hydrophobic side chains such as Leu, lie, Vai, and Phe or functionally equivalent substitutions.

[0543] The non-native MPR may increase the stability and expression of a soluble gB relative to a corresponding native MPR. The non-native MPR may retain the function of the native MPR in stabilising the gB in a pre-fusion state.

[0544] The MPR design may have a predicted Local Distance Difference Test pLDDT threshold of > 80.

[0545] The MPR design may have a target Root Mean Square Deviation (RMSD) of < 2.0 A.

[0546] The MPR design may have a prediction Root Mean Square Deviation (RMSD) of < 3.0 A. The MPR design may have in hydropathy index of < 0.5.

[0547] The MPR design may have an instability index of < 40.

[0548] The MPR design may have a shape complementarity (e.g. Rosetta Shape Complementarity) of > 0.6.

[0549] The MPR design may have a total energy (dG) (e.g. Rosetta Interface Energy) of < than the mean dG of all designed sequences.The MPR design may be free of amino acid repeat sequences (i.e., comprising three or more amino acids of the same time sequentially), N-glycosylation sites, and repeating subsequence amino acids.

[0550] The methods provided herein may also be used to design stabilised post-fusion constructs. In such instances, the methods comprise selecting target locations for potential post-fusion stabilisation substitutions. Further provided herein are stabilised post-fusion constructs. Such constructs include SEQ ID NOs. 122, 124, 125, and 126.

[0551] In a further aspect, there is provided an engineered Herpesvirus gB comprising at least the domains DI, Dll, Dill, DIV and DV and an MPR, wherein the MPR is a non-native MPR, and comprises at least one amino acid substitution which stabilises the tertiary structure of the gB in its pre-fusion conformation.

[0552] The non-native MPR may fold into an alpha helical hairpin structure. The gB may further comprise a trimerisation domain located C-terminal to the non-native MPR such as the T4 foldon domain. The gB may further comprise one or more protein purification tags located C-terminal to the non-native MPR such as a His-tag and a Strep-tag. The gB may further comprise a protease cleavage site between the foldon domain and the purification tags and located C-terminal to the non-native MPR such as a TEV cleavage site.

[0553] The non-native MPR may interact with the DI domain. The non-native MPR may interact with the DI domain more strongly than the native MPR.

[0554] The engineered gB may have increased expression in vitro or may not have reduced expression in vitro compared to a corresponding non-engineered gB.

[0555] In a further aspect, there is provided an engineered Herpesvirus gB comprising at least the domains DI, Dll, Dill, DIV, DV and an MPR, wherein the engineered gB has a Root Mean Square Fluctuation (RMSF) range from 0 Angstroms to the mean RMSF plus 2 standard deviations compared to a corresponding native gB or a homology model of the native gB generated computationally.

[0556] The negative stain electron microscopy (nsEM) envelope of the pre-fusion gB may have a correlation coefficient (CC) value of more than 0.6 as correlated to an electron density map of the pre-fusion gB model. For example, the CC value may be more than 0.60, more than 0.65, or more than 0.70.

[0557] The engineered gB may have an aggregation temperature of least 65°C, at least 70 °C, at least 75 °C, or at least 80 °C when measured using a differential static light scattering (DSLS) assay.The engineered gB may have a hydrodynamic radius between 7 and 13nM measured using a dynamic light scattering (DLS) assay. For example, the hydrodynamic radius may be about 7nm, about 8nm, about 9nm, about 10nm, about 11nm, about 12nm or about 13nm.

[0558] The engineered gB may have a polydispersity value below 10%, below 20%, or below 30%, when measured using a dynamic light scattering (DLS) assay.

[0559] The ratio of pre-fusion gB: post-fusion gB in vitro may be about 50:50, about 60:40, about 70:30, about 80:20, about 90: 10, about 95:5 or about 1. The percentage of gB particles in the pre-fusion state may be at least 80%, or between 80% and 100% when measured using a nsEM imaging assay, where imaged particles are extracted, classified into conformational classes, counted, and represented as a fraction of the total number of particles.

[0560] Prior to purification, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 97%, more than 99% or 100% of gB molecules in vitro may be in the pre-fusion conformation. Following purification, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or 100% of gB molecules in vitro may be in the pre-fusion conformation.

[0561] The percentage of gB molecules in the pre-fusion conformation may be at least 80%, or between 80% and 100% when measured using a nsEM imaging assay.

[0562] The expression yield of gB molecules in the pre-fusion conformation may be least 0.01 mg / L, at least 0.02 mg / L, at least 0.03 mg / L, at least 0.04 mg / L, at least 0.05 mg / L, at least 0.06 mg / L, at least 0.07 mg / L, at least 0.08 mg / L, at least 0.09 mg / L, at least 0.1 mg / L, at least 0.2 mg / L, at least 0.3 mg / L, at least 0.4 mg / L, at least 0.5 mg / L, at least 1 mg / L, at least 2 mg / L, at least 3 mg / L, or at least 4 mg / L, when measured using a spectrophotometric assay. In certain embodiments, the expression yield may be in the range of from about 0.02 mg / L to about 4 mg / LL when measured using a spectrophotometric assay.

[0563] In a further aspect, there is provided a trimer comprising three monomeric gB proteins disclosed herein. The pre-fusion conformation of the gB may comprise a downward orientated DI and / or a Dill apex.

[0564] The pre-fusion conformation may comprise a compact trimer where the domains DI, Dll, Dill, DIV, and DV are orientated so that Dill forms a trimer apex with Dll packing at the side of Dill and DIV located at the base of Dill. DI is located at the base of the trimer, below Dll and packed alongside DIV and DV, with the fusion loops of DI adjacent to the MPR.

[0565] The pre-fusion conformation may be detected by nsEM, cryoEM, and X-ray crystallography.Alternatively, the pre-fusion conformation may be detected using an antibody specific for an epitope on the pre-fusion conformation which is not present in the post-fusion conformation. The engineered gB may comprise one or more amino acid substitutions outside the MPR which stabilises the tertiary structure of the gB in a pre-fusion conformation. For example, the gB may comprise at least one amino acid substitution in one or more of DI, Dll, Dill, DIV, and DV. The stabilising mutations provided herein may be used combination with the non-native MPRs provided herein. However, use of the stabilising mutations provided herein is not limited to the gB constructs provided herein. The stabilising mutations provided herein may alternatively be use with gB constructs which do not comprise the non-native MPRs provided herein. Thus, gB sequences comprising the stabilising mutations provided herein may, in certain instances, not comprise a non-native MPR provided herein.

[0566] Further provided herein is an engineered HCMV gB comprising SEQ ID NO: 49 or 50, or having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO: 49 or 50.

[0567] Alternatively, the engineered gB may comprise one or more stabilising substitutions in combination with a non-native MPR. The engineered gB may comprise one or more additional stabilising substitutions outside the MPR.

[0568] The one or more stabilising substitutions may be cavity filling substitutions, electrostatic substitutions and / or H-bonding network substitutions. The stabilising substitutions may be located at one or more of the DI, Dll, MPR and / or core interfaces. The stabilising substitutions may introduce one or more disulfide bridges at the DI, Dll, MPR and / or core interfaces.

[0569] By the term “core interactions” or “core interface”, it is meant that the interfaces between the DI, Dll, and / or MPR to the Dill, DIV, and / or DV.

[0570] The stabilising substitutions may be in the DI domain. For example, one or more hydrophobic solvent facing amino acid residues in the native DI may be substituted with a polar or charged amino acid residue. The hydrophobic solvent facing amino acid may be A, V or F and the substitution is to N, S, T, Q, Y, C, K, R or E or a functionally equivalent substitution. Specifically, the substitution may be to K, R or E or a functionally equivalent substitution. Immunogens and antibodies

[0571] In a further aspect, there is provided an engineered gB of the present disclosure for use as an immunogen.In a further aspect, there is provided an engineered gB of the present disclosure for use as a vaccine antigen.

[0572] In a further aspect, provided herein is a method of characterising an immune response to Herpesvirus infection, Herpesvirus vaccination, or an engineered pre-fusion gB of the disclosure, the method comprising:

[0573] a) providing a sample containing lymphocytes and / or antibodies from a subject exposed to or infected with Herpesvirus, a Herpesvirus vaccine or an engineered pre-fusion gB provided herein;

[0574] b) contacting the sample with an immune reagent comprising a corresponding engineered pre-fusion gB; and

[0575] c) detecting binding of the immune reagent to lymphocytes and / or antibodies in the sample; wherein lymphocytes and / or antibodies binding to the immune reagent are identified as binding to the pre-fusion conformation of Herpesvirus gB and / of having specificity for the prefusion conformation of Herpesvirus gB.

[0576] The characterisation may include determining the specificity of the immune response to prefusion gB epitopes over post-fusion gB epitopes or other herpesvirus proteins. Such a method may comprise:

[0577] a) providing a sample containing lymphocytes and / or antibodies from a subject exposed to or infected with Herpesvirus, a Herpesvirus vaccine or an engineered pre-fusion gB provided herein;

[0578] b) contacting the sample with a first immune reagent comprising an engineered pre-fusion gB provided herein; and

[0579] c) simultaneously or separately contacting the sample with at least a second immune reagent comprising a corresponding post-fusion gB or non-gB Herpesvirus protein as a comparator;

[0580] d) simultaneously or separately detecting binding of at least the first and second immune reagents to lymphocytes and / or antibodies in the sample;

[0581] wherein lymphocytes and / or antibodies binding selectively to the first immune reagent are identified as being specific for the pre-fusion conformation of Herpesvirus gB.

[0582] In some instances, the lymphocytes and / or antibodies may bind only to the first immune reagent. In some instances, the lymphocytes and / or antibodies may bind more strongly to the first immune reagent, for example, they may bind to the first immune reagent with an affinitythat is at least 2-fold, 5-fold, 10-fold, 50-fold or 100-fold greater than the affinity for the second immune reagent.

[0583] Three or more immune reagents may be tested. For example, the method may involve contacting the sample with a further immune reagent comprising a post-fusion gB, and / or one or more further immune reagents comprising non-gB Herpesvirus proteins.

[0584] The lymphocytes may include T cells, B cells, natural killer (NK) cells, regulatory T cells (Tregs), helper T cells (CD4+ T cells), cytotoxic T cells (CD8+ T cells), memory B cells, plasma cells, gamma delta (yd) T cells, innate lymphoid cells (ILCs), natural killer T (NKT) cells. In particular, the lymphocytes may include B cells.

[0585] The sample may contain one type of lymphocyte or multiple types of lymphocyte. The sample may also contain non-lymphocyte cells, such as neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells, mast cells, erythrocytes, platelets, or epithelial cells. The lymphocytes and / or antibodies binding to the immune reagent may be further identified as lymphocytes and / or antibodies which do not bind to the post-fusion conformation of the corresponding Herpesvirus gB.

[0586] The method may further comprise a step of contacting the sample with an immune reagent comprising a corresponding native gB and identifying lymphocytes that (i) bind to the immune reagent comprising an engineered gB according to the present disclosure and (ii) do not bind to the immune reagent comprising a corresponding native gB.

[0587] The sample may be a sample containing lymphocytes / and or antibodies from a subject exposed to or infected with a Herpesvirus, a Herpesvirus vaccine or a pre-fusion gB of the present disclosure, wherein the subject is determined to have an immune response to the prefusion conformation.

[0588] The method may further comprise a step of contacting the sample with a corresponding native gB (i.e. a gB of the same genus or strain which is stable in the post-fusion conformation) and detecting binding, wherein lymphocytes and / or antibodies binding to the immune reagent of the present disclosure but not the native gB are identified as being specific for to the pre-fusion conformation of Herpesvirus.

[0589] The sample may be a cell free sample comprising antibodies from a subject exposed to or infected with a Herpesvirus, a Herpesvirus vaccine or an engineered Herpesvirus gB of the present disclosure. The sample may be a body fluid. The sample may be blood, serum, plasma, saliva or other fluid.In a further aspect, there is provided a method of characterising an antibody response to Herpesvirus infection, Herpesvirus vaccination, or an engineered gB of the present disclosure, the method comprising:

[0590] a) providing a sample comprising cell-free fluid comprising antibodies from a subject exposed to, or infected with, a Herpesvirus, a Herpesvirus vaccine or an engineered gB of the present disclosure;

[0591] b) contacting the sample with an immune reagent comprising a corresponding engineered gB of the present disclosure; and

[0592] c) detecting binding of the immune reagent to antibodies produced in the sample; wherein antibodies binding to the immune reagent are identified as binding to the pre-fusion conformation of the corresponding Herpesvirus gB.

[0593] The cell free fluid may be blood, serum, plasma, saliva or other another fluid.

[0594] The method may further comprise a step of contacting the sample with an immune reagent comprising a comprising a corresponding post-fusion gB and identifying antibodies that (i) bind to the immune reagent comprising a corresponding engineered gB according to the present disclosure and (ii) do not bind to the immune reagent comprising a post-fusion gB.

[0595] In a further aspect, provided herein is a method of isolating monoclonal antibodies specifically binding to the pre-fusion conformation of the Herpesvirus gB, the method comprising: a) providing a sample containing B cells from a subject exposed to, or infected with, a corresponding Herpesvirus, a corresponding Herpesvirus vaccine, or a corresponding engineered gB of the present disclosure;

[0596] b) contacting the B cells with the immune reagent provided herein comprising a corresponding engineered pre-fusion gB;

[0597] c) isolating B cells bound to the immune reagent; and

[0598] d) culturing the isolated B cells to produce monoclonal antibodies; and / or

[0599] e) sequencing the immunoglobulin genes of the isolated B cells to identify monoclonal antibodies.

[0600] In a further aspect, provided herein is a method of generating monoclonal antibodies specifically binding to the pre-fusion conformation of the Herpesvirus gB, the method comprising:

[0601] a) obtaining a sample containing B cells from a subject exposed to, or infected with, a corresponding Herpesvirus, a corresponding Herpesvirus vaccine, or a corresponding engineered gB of the present disclosure; orb) providing a sample containing B cells from a subject exposed to, or infected with, a corresponding Herpesvirus, a corresponding Herpesvirus vaccine, or a corresponding engineered gB of the present disclosure;

[0602] c) contacting the B cells with the immune reagent provided herein a corresponding engineered gB;

[0603] d) isolating B cells that bind to the immune reagent; and

[0604] e) culturing the isolated B cells to produce monoclonal antibodies; and / or

[0605] f) sequencing the immunoglobulin genes of the isolated B cells to identify monoclonal antibodies.

[0606] The isolated B cells may bind only to the immune reagent, but not other proteins such as postfusion gB.

[0607] In a further aspect, there is provided a method of generating monoclonal antibodies specifically binding to the pre-fusion conformation of Herpesvirus gB, the method comprising:

[0608] a) providing a sample containing B cells from a subject exposed to, or infected with, the Herpesvirus, a corresponding Herpesvirus vaccine or an engineered gB of the present disclosure;

[0609] b) contacting the B cells with an immune reagent a corresponding engineered gB of the present disclosure comprising a corresponding engineered gB;

[0610] c) isolating B cells that bind to the immune reagent; and

[0611] d) culturing the isolated B cells to produce monoclonal antibodies; and / or

[0612] e) sequencing the immunoglobulin genes of the isolated B cells to identify monoclonal antibodies.

[0613] Prior to step (a), the method may further comprise:

[0614] i. exposing a subject to a Herpesvirus vaccine or an engineered gB of the present disclosure;

[0615] ii. obtaining a sample containing B cells from the subject exposed to a Herpesvirus vaccine or a pre-fusion gB of the present disclosure.

[0616] The subject may be a human a non-human animal.

[0617] Also provided herein are antibodies specifically binding to the engineered gB of the present disclosure. In some aspects, the monoclonal antibodies are identified as antibodies binding to the immune reagent of the present disclosure using the methods provided herein.

[0618] The antibodies disclosed herein may be any isotype. For example, they may be IgG, IgA, IgM, IgE or IgD. In particular, the antibodies may be IgG antibodies. The antibodies disclosed hereinmay be any suitable format. For example, the antibodies may be Fab, scFab, F(ab’)2, Fv, scFv, dsFv, sdAb, VHH, bispecific antibodies, multi-specific antibodies, diabodies, tribodies, TandAbs, nanobodies, and BITEs, DARTs, or functional fragments thereof.

[0619] The antibodies may be IgG, IgA, IgM, IgE or IgD antibodies. Preferably, the antibodies are IgG antibodies.

[0620] The monoclonal antibodies identified may bind specifically to the pre-fusion conformation of gB and may not bind to the post-fusion form of gB, or may display reduced, altered, or weakened binding to the post-fusion form of gB.

[0621] The monoclonal antibodies may exhibit lower affinity for the post-fusion form of gB, for example, the monoclonal antibodies may bind to the pre-fusion conformation of gB with a binding affinity that is at least 10-fold, at least 100-fold, or at least 1000-fold greater than binding affinity to the post-fusion form of gB. The monoclonal antibodies may bind to the postfusion conformation of gB with a Kd greater than 1x10-4, greater than 1x10-5, or greater than 1x10-6, and may bind to the pre-fusion conformation of gB with a Kd less than 1x107, less than 1x108, less than 1x109, or less than 1x1010. Binding to the post-fusion conformation may be negligible or non-detectable under standard assay conditions. The antibodies may bind preferentially to the pre-fusion conformation of gB, for example as demonstrated in a competitive or displacement assay indicative of conformational selectivity.

[0622] The subject may be a vertebrate. For example, the subject may be a human subject. Alternatively, the subject may be a non-human subject. For example, a non-human mammal. The non-human mammal may be a mouse, a rat, a rabbit, a guinea pig, a hamster, a dog, a cat, a pig, a llama, a sheep, a goat, a cow, a horse, a chicken, a quail, a turkey or a non-human primate, such as a cynomolgus macaque or a rhesus macaque.

[0623] In a further aspect, there is provided a method of screening antibodies specifically binding to the pre-fusion conformation of the Herpesvirus gB, the method comprising:

[0624] a) providing a sample containing B cells from a subject exposed to, or infected with, a Herpesvirus, a Herpesvirus vaccine or an engineered gB of the present disclosure; b) culturing single B cells individually, such that the individual B cells proliferate and secrete a monoclonal antibody into the well;

[0625] c) detecting the monoclonal antibody in the well using a binding probe comprising an immune reagent comprising a corresponding engineered gB according to the present disclosure; and

[0626] d) identifying B cells that bind the immune reagent; and / ore) sequencing the immunoglobulin genes of the isolated B cells to identify monoclonal antibodies that bind specifically to the immune reagent.

[0627] Cell culture may be carried out in individual wells of a microtitre plate or microfluidics system. The method may further comprise a step of detecting the monoclonal antibody in the well using a one or more further binding probes comprising a corresponding post-fusion gB and / or another Herpesvirus protein. The monoclonal antibodies identified may therefore bind only to the immune reagent, but not other proteins such as post-fusion gB.

[0628] In a further aspect, provided herein is a method of screening antibodies specifically binding to the pre-fusion conformation of a Herpesvirus gB, the method comprising:

[0629] a) providing a sample containing B cells from a subject exposed to, or infected with, a corresponding Herpesvirus, a corresponding Herpesvirus vaccine, or a corresponding engineered gB of the present disclosure;

[0630] b) screening B cells in the sample with at least two binding probes using Fluorescence Activated Cell Sorting (FACS), wherein at least one binding probe is an immune reagent comprising a corresponding engineered gB according to the present disclosure, and at least a second binding probe is an immune reagent comprising a corresponding postfusion gB;

[0631] c) identifying B cells that (i) bind to the immune reagent comprising a corresponding engineered gB according to the present disclosure and (ii) do not bind to the immune reagent comprising a post-fusion gB;

[0632] d) culturing the isolated B cells identified in step (c) to produce monoclonal antibodies; and / or e) sequencing the immunoglobulin genes of the B cells identified in step (c) to identify monoclonal antibodies that bind specifically to the immune reagent.

[0633] The isolated B cells may bind only to the immune reagent, but not other proteins such as a gB in post-fusion conformation.

[0634] Two binding probes may be used, wherein one is an immune reagent comprising an engineered gB of the present disclosure, and the second is an immune reagent comprising a corresponding native gB. The B cells which bind the engineered gB of the present disclosure may not bind to the corresponding native gB. The monoclonal antibodies which bind the engineered gB of the present disclosure may not bind to the native gB.

[0635] Prior to step (a), the above methods may further comprise the steps of:

[0636] i. exposing a subject to a Herpesvirus vaccine or an engineered gB of the present disclosure;ii. obtaining a sample containing B cells from the subject exposed to a Herpesvirus vaccine or a pre-fusion gB of the present disclosure.

[0637] In a further aspect, provided herein is a method for purifying an engineered gB according to the present disclosure, the method comprising:

[0638] a) transfecting mammalian cells with a plasmid comprising a nucleic acid sequence encoding an engineered gB construct of the present disclosure;

[0639] b) collecting the expression media from the transfected cells;

[0640] c) carrying out Strep-Tag affinity purification on the collected expression media to generate Strep-Tag affinity elution fractions;

[0641] d) collecting Strep-Tag affinity elution fractions;

[0642] e) performing a size-exclusion chromatography on the eluted fractions;

[0643] f) collecting the peak elution fraction; and

[0644] g) isolating engineered gB particles from the peak elution fraction.

[0645] Immune reagents and conjugates

[0646] The term “immune reagent” as used herein refers to a molecule used in in vitro, in vivo and ex vivo immunological research or diagnostic applications to detect, measure, or manipulate components of the immune system, such as antigens, antibodies, cytokines, T cells or B cells. References to immune reagents of the present disclosure refer to engineered Herpesvirus gBs according to the disclosure, as well as variants thereof which have been modified for use in research or diagnosis. For example, the term “immune reagent” encompasses engineered Herpesvirus gBs which have been conjugated to further moiety or substate, including engineered gBs conjugated to a binding molecule such as an antibody or antibody fragment, a solid support, such as a bead, nanoparticle, chip, column or membrane, or a detection moiety, such as a fluorescent label, a radiolabel or a magnetic label.

[0647] The immune reagent may be used to screen and identify small molecule drugs that inhibit or alter gB function. The immune reagent may be used to screen and identify monoclonal antibodies that inhibit or alter gB function. The immune reagent may be used to screen and identify biologies that inhibit or alter gB function. The biologic may be a peptide, a nanobody, or an aptamer.

[0648] Also provided is an engineered gB of the present disclosure for use as an immune reagent. In a further aspect, there is provided a conjugate comprising the engineered gB disclosed herein conjugated to at least one further moiety or conjugation substrate.The conjugate may comprise the engineered gB of the present disclosure conjugated to conjugation substrate or peptide providing a structural element to the conjugate. Alternatively, the conjugate may comprise the engineered gB of the present disclosure and a further molecule having biological activity.

[0649] The conjugation substrate may be a peptide or protein sequence, or another biologically active molecule, such as an enzyme, an antibody or a binding molecule.

[0650] Alternatively, the further moiety may be a detection moiety. Any suitable moiety known in the art may be used. For example, the detection moiety may be a fluorescent tag, a dye, a magnetic tag or a radiolabel.

[0651] The further moiety or substrate may be a solid support. For example, the solid support may be a column, a plate, a well, a microchip, a bead, a nanoparticle, or a membrane. Exemplary solid supports include Sepharose, agarose, silica, glass, silicon, polystyrene, polypropylene, nitrocellulose, cellulose, gold and quantum dots. The nanoparticle may be a Ferritin nanoparticle.

[0652] The engineered gB may be conjugated to the substrate via a covalent bond. Alternatively, the conjugation may also be via adsorptive or hydrophobic / hydrophilic interaction.

[0653] Nucleic acids

[0654] In a further aspect, there is provided a nucleic acid encoding the engineered gB of the present disclosure. The nucleic acid may be DNA or RNA. The RNA may be mRNA or saRNA. The nucleic acid may be codon optimised. The nucleic acid comprises one or more chemically modified bases.

[0655] The nucleic acid may include modifications for improving the stability, translational efficiency, half-life and / or immunogenicity of the vaccine. For example, the nucleic acid may include modified nucleosides, such as pseudouridine ('+'), N1 -methylpseudouridine (ml^P), and 5-methylcytidine (m5C). Suitable modifications are known in the art.

[0656] The nucleic acid may be codon optimised to preferentially use codons that are translated efficiently in the relevant host cells, for example in mammalian or human cells. The sequence may avoid rare codons and / or preferentially use with optimal codons that match the abundant tRNAs in the host cells. The sequence may also avoid codons which contribute to RNA secondary structures that could impede ribosome access and adjust the GC content to ensure RNA stability. Strategies for codon optimisation are known in the art.In a further aspect, there is provided a host cell expressing the nucleic acid of the present disclosure.

[0657] The engineered gB may be used as a vaccine antigen. Accordingly, one aspect, there is provided an engineered gB, nucleic acid or vector encoding a gB of the present disclosure for use in a method of immunisation.

[0658] Vaccines

[0659] In a further aspect, there is provided a vaccine comprising a gB of the present disclosure. The vaccine may be a protein vaccine, an mRNA vaccine, a DNA vaccine, or an saRNA vaccine. The vaccine may comprise the Herpesvirus gB of the present disclosure as a protein, for example a recombinant protein or a protein conjugate. Alternatively, the vaccine may be an mRNA vaccine comprising an mRNA encoding the Herpesvirus gB of the present disclosure. The vaccine may be a DNA vaccine comprising DNA encoding the Herpesvirus gB of the present disclosure. The vaccine may be a saRNA vaccine comprising saRNA encoding the Herpesvirus gB of the present disclosure.

[0660] The vaccine may be displayed on nanoparticles. The particles may be ferritin-based nanoparticles. For example, the particles may be HP-Ferritin nanoparticles. The particles may be virus-like particles (VLPs), viral nanoparticles, bacteriophage-based nanoparticles, extracellular vesicles, biomimetic nanoparticles, self-assembling nanoparticles, immune stimulating complexes (ISCOMs), lipid nanoparticles (LNPs), liposomes, polymeric nanoparticles, PLGA nanoparticles, PEGylated nanoparticles, dendrimers, polysaccharide-based nanoparticles, albumin nanoparticles, vault nanoparticles, inorganic nanoparticles, gold nanoparticles, silica nanoparticles, calcium phosphate nanoparticles, iron oxide nanoparticles, hybrid or composite nanoparticles. The VLPs may be enveloped, non-enveloped, or chimeric. Enveloped VLPs comprise a lipid membrane. Non-enveloped VLPs lack a lipid membrane and instead consist of structural proteins. Chimeric VLPs comprise structural components derived from two or more different viruses, or incorporate heterologous proteins or antigens, thereby forming hybrid particles.

[0661] The protein vaccine may be mixed with an adjuvant compatible with human use to boost the immune response in humans. Alternatively, the protein vaccine may be mixed with an adjuvant compatible with animal use to boost the immune response in animals.

[0662] The vaccine may comprise the Herpesvirus gB of the present disclosure as a protein, for example a recombinant protein or a protein conjugate. Alternatively, the vaccine may be an mRNA vaccine comprising an mRNA encoding the Herpesvirus gB of the present disclosure.The vaccine may be a DNA vaccine comprising DNA encoding the Herpesvirus gB of the present disclosure. The vaccine may be a saRNA vaccine comprising saRNA encoding the Herpesvirus gB of the present disclosure.

[0663] The vaccine may comprise the Herpesvirus gB of the present disclosure displayed on nanoparticles. The nanoparticles are ferritin-based nanoparticles. For example, the nanoparticles may be HP-Ferritin nanoparticles.

[0664] The vaccine may comprise one or more further Herpesvirus components. For example, the vaccine may comprise an HCMV gB, and one or more further HCMV components, such as Herpesvirus trimers (gH / gL / gO), Herpesvirus pentamers (gH / gL / UL128 / UL130 / UL131a), pp65, gH and / or gL, virus like particles, or any other Herpesvirus proteins.

[0665] The vaccine may comprise one or more further HSV-1 and / or HSV-2 components. For example, the vaccine may comprise an HCMV gB, and one or more further HCMV components, such as HSV-1 and / or HSV-2 trimers (gH / gL / gO), HSV-1 and / or HSV-2 pentamers (gH / gL / UL128 / UL130 / UL131a), pp65, gH and / or gL, virus like particles, or any other HSV-1 and / or HSV-2 proteins, or combinations thereof.

[0666] The vaccine may comprise one or more further EBV components. For example, the vaccine may comprise an HCMV gB, and one or more further HCMV components, such as HSV-1 and / or HSV-2 trimers (gH / gL / gO), HSV-1 and / or HSV-2 pentamers (gH / gL / UL128 / UL130 / UL131a), pp65, gH and / or gL, virus like particles, or any other HSV-1 and / or HSV-2 proteins, or combinations thereof.

[0667] The vaccine may comprise two or more gB antigens as disclosed herein. The two or more Herpesvirus gBs may be derived from different Herpesvirus species. For example, the vaccine may comprise two or more Herpesvirus gBs selected from an HCMV gB, an HSV-1 gB, an HSV-2 gB, a varicella-zoster virus (HHV-3) gB, an EBV gB, an HHV-6A gB, an HHV-6B gB, an HHV-7 gB, an HHV-8 gB, or any combination thereof. Alternatively, the two or more Herpesvirus gBs may be derived from the same Herpesvirus species. The vaccine may further comprise one or more additional components. For example, the vaccine may comprise adjuvants, stabilisers, preservatives, emulsifiers, surfactants, diluents, antibiotics, buffering agents, residual inactivating agents, residual cell culture materials, residual yeast proteins, cryoprotectants, suspending agents.

[0668] The vaccine of may produce a B-cell response. Alternatively, or in addition, the vaccine may produce a T cell response. The vaccine may produce humoral immunity and / or cellular immunity. For example, the vaccine may activate B cells and plasma cells, resulting in theproduction of antibodies specifically binding to the gB of the present disclosure. Alternatively, or in addition, the vaccine may activate cytotoxic T cells, resulting in the production of T cells which specifically recognise a fragment of the gB of the present disclosure bound to an MHC. The vaccine may further comprise an adjuvant. For example, the vaccine may further comprise Aluminium salts such as aluminium hydroxide, aluminium phosphate and aluminium potassium sulphate. The vaccine may further comprise oil in water emulsion. The oil in water emulsion may further comprise squalene and vitamin E. The vaccine may further comprise a combination of QS-21, cholesterol and monophosphoryl lipid A. The vaccine may further comprise a combination of monophosphoryl lipid A and aluminium based formulations. The vaccine may further comprise CpG, nucleic-acid based adjuvant that stimulates the immune system through Toll-like receptor 9 (TLR9) and retinoic acid-inducible gene-l-like receptors (RLRs).

[0669] The adjuvants suitable for use with the vaccines disclosed herein are not limited to the above. The vaccine may comprise any suitable adjuvant known in the art. It would be within the common general knowledge of the skilled person to select a suitable vaccine.

[0670] Also provided herein are engineered gB molecules of the present disclosure for use in eliciting an immune response.

[0671] Also provided herein are engineered gB molecules of the present disclosure for use as an immunostimulatory molecule.

[0672] Also provided herein is a method immunising a subject against Herpesvirus infection, the method comprising administering to the subject an effective dose of a vaccine comprising an engineered gB molecules of the present disclosure.

[0673] Also provided herein is a method of reducing the risk of Herpesvirus infection in a subject, the method comprising administering to the subject an effective dose of a vaccine comprising an engineered gB molecules of the present disclosure.

[0674] Also provided herein is a method of eliciting an immune response against the pre-fusion gB of Herpesvirus in subject, the method comprising administering to the subject an effective dose of a vaccine comprising an engineered gB molecules of the present disclosure.

[0675] The Herpesvirus infection may be caused by any Herpesvirus. For example, the Herpesvirus infection may be caused by HCMV, HSV-1, HSV-1 or EBV.Accordingly, also provided herein is a method immunising a subject against HCMV, HSV-1, HSV-2 and / or EBV infection, the method comprising administering to the subject an effective dose of a vaccine comprising one or more engineered gB molecules of the present disclosure. Also provided herein is a method of reducing the risk of HCMV, HSV-1, HSV-2 and / or EBV infection in a subject, the method comprising administering to the subject an effective dose of a vaccine comprising one or more engineered gB molecules of the present disclosure.

[0676] Also provided herein is a method of eliciting an immune response against the pre-fusion gB of HCMV, HSV-1, HSV-2 and / or EBV, in subject, the method comprising administering to the subject an effective dose of a vaccine comprising one or more engineered gB molecules of the present disclosure.

[0677] The vaccine may comprise at least an HCMV gB, at least an HSV-1 gB, at least an HSV-2 gB and / or at least an EBV gB. The vaccine may comprise at least an HCMV gB and an HSV-1. The vaccine may comprise at least an HCMV gB and an HSV-2. The vaccine may comprise at least an HCMV gB and an EBV gB. The vaccine may comprise at least an HSV-1 gB and an EBV gB. The vaccine may comprise at least an HSV-2 gB and an EBV gB. The vaccine may comprise at least an HSV-1 gB and an HSV-2 gB. The vaccine may comprise at least an HCMV gB, at least an HSV-1, at least an HSV-2 and at least an EBV gB. The vaccine may further comprise one or more other Herpesvirus proteins.

[0678] The vaccine may further include a pharmaceutically acceptable carrier and / or adjuvant. Suitable carriers and adjuvants are known in the art.

[0679] In a further aspect, there is provided a pharmaceutical composition comprising the gB of the present disclosure, or a nucleic acid encoding the gB of the present disclosure and a pharmaceutically acceptable carrier.

[0680] Pharmaceutical compositions

[0681] The pharmaceutical composition may be a vaccine composition or a composition for use in a method of vaccination.

[0682] The pharmaceutical composition may comprise a gB of the present disclosure, and at least one other pharmaceutically acceptable ingredient. Such compositions may comprise any suitable and pharmaceutically acceptable carrier, diluent, adjuvant or buffer solution. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, liposomes, water, glycerol, ethanol and combinations thereof. The composition may comprise a further active agent. The composition may comprise a further active agent or be administered in combination with one or more additional active agents.The pharmaceutical composition may comprise one or more further components of the Herpesvirus.

[0683] The pharmaceutical composition may comprise one or more further components of HCMV. For example, the composition may comprise an HCMV gB, and one or more further HCMV components, such as glycoprotein complexes involved in viral entry and fusion, including trimers (gO / gL / gO) and pentamers (gH / gL / UL128 / UL130 / UL131a).

[0684] The pharmaceutical composition may comprise one or more further components of HSV-1 and / or HSV-2. For example, the composition may comprise an HSV-1 and / or HSV-2 gB, and one or more further HSV-1 and / or HSV-2 components, such as glycoprotein complexes involved in viral entry and fusion, including gO / gL, gH / gL / gO and / or gD, as well as gH and / or gL.

[0685] The pharmaceutical composition may comprise one or more further components of Epstein-Barr virus (EBV). For example, the composition may comprise an EBV gB, and one or more further EBV components, such as glycoprotein complexes involved in viral entry and fusion, including gH / gL and gH / gL / gp42, as well as envelope glycoproteins gp350 and / or gp220. The pharmaceutical composition may comprise an HSV1 or HSV2 gB, and one or more further HSV1 or HSV2 components, such as gD, gp350 and / or gHgL The pharmaceutical composition may comprise an EBV gB, and one or more further HCMV components, such as gp350 and / or gH / gL The pharmaceutical composition may comprise one or more further gBs, for example, the vaccine may comprise one or more of an HCMV, an HSV1 and / or an EBV gB.

[0686] The additional agents may be therapeutic compounds, e.g. anti-inflammatory drugs, cytotoxic agents, cytostatic agents or antibiotics. Such additional agents may be present in a form suitable for administration to patient in need thereof and such administration may be simultaneous, separate or sequential. The components may be prepared in the form of a kit which may comprise instructions as appropriate.

[0687] The pharmaceutical compositions may be administered in any convenient manner effective for treating or preventing disease, or reducing the risk or severity of disease, including, but not limited to administration by oral, topical, intravenous, intramuscular, intranasal, or intradermal routes among others. In particular, the composition may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic. Alternatively, the active agent may be administered as a capsule or tablet.

[0688] Suitable doses for vaccination are known in the art. The physician in any event will determine the actual dosage which will be most suitable for an individual which will be dependent onfactors including the age, weight, sex and response of the individual. The above dosages are exemplary of the average case. There can, of course, be instances where higher or lower dosages are merited, and such are within the scope of this disclosure.

[0689] The present disclosure also provides an engineered gB for use in medicine, and in particular, for use as a vaccine. Accordingly, also provided herein is an engineered gB for use in the manufacture of a medicament. In particular, the engineered gB may be for use in the manufacture of a Herpesvirus vaccine.

[0690] Medical use

[0691] In a further aspect, there is provided a method of preventing Herpesvirus infection or a disease associated therewith in a subject, comprising administering to the subject an effective amount of the vaccine of the present disclosure.

[0692] Also provided a method of inducing an immune response in a subject, comprising administering to the subject an effective amount of the engineered gB of the present disclosure.

[0693] Further provided are methods of preventing or reducing the risk and / or severity of a disease or disorder associated with Herpesvirus in a subject, comprising administering to the subject an effective amount of the vaccine of the present disclosure.

[0694] Also provided is an engineered gB of the disclosure for use as an antigen. The antigen may be used to elicit an immune response in a subject. The subject may be a mammalian subject, for example, a human or a non-human mammal.

[0695] Further provided is an engineered gB of the disclosure for use in the manufacture of a medicament for eliciting an immune response in a subject.

[0696] Further provided is an engineered gB of the disclosure for use in the manufacture of a vaccine for use in preventing Herpesvirus infection or a disease associated therewith in a subject. Further provided is an engineered gB of the disclosure for use in a method of eliciting an immune response in a subject, the method comprising administering an effective amount to of the engineered gB subject at risk of Herpesvirus infection.

[0697] Further provided is an engineered gB of the disclosure for use in a method of preventing Herpesvirus infection or a disease associated therewith in a subject, the method comprising administering an effective amount to of the engineered gB subject at risk of Herpesvirus infection.

[0698] The subject may be a mammalian subject, for example a human or a non-human mammal.The disease may be oral herpes, genital herpes, neonatal herpes, meningitis, chickenpox, shingles, postherpetic neuralgia, infectious mononucleosis, keratitis, encephalitis, Burkitt's lymphoma, Hodgkin's lymphoma, Oral hairy leukoplakia, congenital HCMV infection, retinitis, pneumonitis, roseola, febrile seizures, encephalitis, Kaposi's sarcoma, primary effusion lymphoma or multicentric Castleman disease. In particular, the disease may be Oral herpes, genital herpes, neonatal herpes, infectious mononucleosis or congenital HCMV infection. The disease may be congenital HCMV infection, retinitis, pneumonia, hepatitis, gastroenteritis / colitis, encephalitis, mononucleosis, esophagitis, adrenalitis, or cancer, for example breast cancer, glioblastoma or colorectal cancer.

[0699] In a further aspect, there is provided a pharmaceutical composition of the present disclosure for use in the treatment or prevention of Herpesvirus infection or a disease associated therewith.

[0700] Further provided is a pharmaceutical composition of the present disclosure for use in the treatment or prevention of HCMV infection or a disease associated therewith.

[0701] The subject may be a subject at particular risk from HCMV infection. For example, the subject may be immunocompromised or pregnant. In the context of pregnancy, preventing HCMV infection or a disease associated therewith and reducing the risk and / or severity of a disease or disorder associated with HCMV in a subject also encompasses the risk of vertical transmission to the child, including transmission in utero, during delivery and after birth. Further provided is a pharmaceutical composition of the present disclosure for use in the treatment or prevention of HSV-1 and / or HSV-2 infection or a disease associated therewith. The disease may be oral herpes, genital herpes, neonatal herpes, keratitis, meningitis, encephalitis, or disseminated herpes simplex virus infection. The subject may be a subject at particular risk from HSV-1 and / or HSV-2 infection. For example, the subject may be immunocompromised, pregnant, or a neonate.

[0702] Further provided is a pharmaceutical composition of the present disclosure for use in the treatment or prevention of EBV infection or a disease associated therewith.

[0703] The disease may be infectious mononucleosis, chronic active Epstein-Barr virus infection, oral hairy leukoplakia, encephalitis, hepatitis, or lymphoproliferative disease, including Burkitt’s lymphoma, Hodgkin’s lymphoma, post-transplant lymphoproliferative disorder, primary effusion lymphoma, or nasopharyngeal carcinoma. The subject may be a subject at particular risk from EBV infection. For example, the subject may be immunocompromised, a transplant recipient, or undergoing immunosuppressive therapy.Such uses also encompass methods for immunising patients in need thereof, the methods comprising administering to the patient an effective dosage of a vaccine as defined herein, comprising an engineered gB of the present disclosure.

[0704] The vaccine may be co-administered with another therapy or vaccine. The vaccine may be administered only once, or on multiple occasions separated by a period of time. The period of time may be minutes, hours, day, weeks or months.

[0705] As used herein, the terms “treatment” and “therapy” includes any regime that can benefit a human or a non-human animal. The treatment of “non-human animals” in veterinary medicine extends to the treatment of domestic animals, including horses and companion animals (e.g. cats and dogs) and farm / agricultural animals including members of the ovine, caprine, porcine, bovine and equine families.

[0706] The Herpesvirus may be a member of the order Herpesviridae, for example a member of the Orthoherpesvirdae family. The Herpesvirus may be an alphaherpesvirus, a betaherpesvirus or a gammaherpesvirus. The Herpesvirus may be a vertebrate infecting Herpesvirus. The Herpesvirus may infect humans. For example, the Herpesvirus may be selected from HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6A, HHV-6B, HHV-7, and KSHV / HHV-8.

[0707] The Herpesvirus may infect domestic animals. For example, the Herpesvirus may be selected from pseudorabies virus, and bovine Herpesvirus 1, or Marek’s disease virus, Canine Herpesvirus, Equine Herpesvirus (EHV; also known as equine rhinopneumonitis) and feline Herpesvirus type-1 (FHV-1)

[0708] Also included within the present disclosure are variants, analogues, derivatives and fragments of the gB having the amino acid sequence of the protein in which several e.g. about 5 to 10, or about 1 to 5, or about 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the gB of the present disclosure. Also especially preferred in this regard are conservative substitutions where the stability of the gB of the present disclosure in the pre-fusion form is preserved.

[0709] Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:SUS-WSSES AwiRC I Acidic add, Skkamsc scid

[0710] Sasic Nocicydsc: Arsons, Lys&ie; Cydic: Histidicss

[0711] Charts As^artte add. Arinin®, Lyslrss, Hssddim*

[0712] A& Nre, Weww, fWHn®

[0713] wWkk, SWwstns, CysW®, Thtese?

[0714] Hyckc-phafeic Tys'sjsine, Vislics,. Lsidste. HeShSanififc, Pheny^assine,.

[0715] TryptaphifC

[0716] Aromatk Tryptepdacs, Tyrosisia, PhaoyPaianirns

[0717] JtesMaes that Wksence Siyersa and Pcolists

[0718]

[0719] The engineered gB of the present disclosure may maintain its conformation at physiological pH, for example between pH 6.5 and pH 7.5, between pH 7.0 and pH 7.5, or between pH 7.35 and pH 7.45. The engineered gB of the present disclosure may further maintain its conformation at a pH above or below physiological pH. For example, below pH 6, below pH 5.5 and / or below pH 5. The engineered gB may maintain its conformation at a pH between 3 and 7. The engineered gB may maintain its conformation at a pH between 4 and 6. The engineered gB may maintain its conformation at a pH between 5 and 6. Alternatively or in addition, the engineered gB may maintain its conformation at a pH above pH 7, above pH 7.5 and / or above pH 8. The engineered gB may maintain its conformation at a pH between 7.5 and 9. The engineered gB may maintain its conformation a pH between 7.5 and 8.

[0720] The engineered gB of the present disclosure may maintain its conformation at physiological temperature, for example between 35.5°C and 38.5°, or between 36 °C and 37.5°C. The engineered gB of the present disclosure may further maintain its conformation at a temperature above or below physiological temperature. For example, below 35°C, below 30°C, below 20°C, below 10°C, or below 5°C. Alternatively or in addition, the engineered gB may maintain its conformation at a temperature above 38°C, above 40°C, above 50°C, above 60°C, or above 70°C. For example, the engineered gB may maintain its conformation at a temperature between 0°C and 70°C, 10°C and 60°C, 20°C and 50°C, or 30°C and 40°C. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. For the purposes of the present disclosure, the following terms are defined below.Definitions

[0721] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0722] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0723] Further, the terms "about" and "approximate", as used herein when referring to a measurable value such as an amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length and the like, is meant to encompass variations of ± 15%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length and the like. In instances in which the terms "about" and "approximate" are used in connection with the location or position of regions within a reference polypeptide, these terms encompass variations of ± up to 20 amino acid residues, ± up to 15 amino acid residues, ± up to 10 amino acid residues, ± up to 5 amino acid residues, ± up to 4 amino acid residues, ± up to 3 amino acid residues, ± up to 2 amino acid residues, or even ± 1 amino acid residue.

[0724] The term “antibody” refers to an intact immunoglobulin of any isotype, or fragments thereof, and includes, for instance, chimeric, humanised, fully human, and bispecific antibodies. Antibodies according to the present disclosure include IgG, IgA, IgM, IgE, IgD, Fab, scFab, F(ab’)2, Fv, scFv, dsFv, sdAb, VHH, bispecific antibodies, multi-specific antibodies, diabodies, tribodies, TandAbs, nanobodies, and BITEs, DARTs. The fragments may be functional fragments, for example, they may compete with the intact antibody for specific binding to the target antigen or comprise all three CDRs of the heavy chain and / or light chain of the intact antibody.

[0725] As used herein, the term "antigen" refers to a compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor.

[0726] As used herein, the term "specifically binding" refers to a binding reaction which is determinative of the presence of a pre-fusion gB of the present disclosure in the presence of a heterogeneous population of molecules including macromolecules such as proteins and other biologies.Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0727] As used herein, the terms "conjugated", "fused" or "fusion" and their grammatical equivalents, in the context of joining together of two more elements or components or domains by whatever means including chemical conjugation or recombinant means (e.g., by genetic fusion) are used interchangeably. Suitable methods of conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art.

[0728] The term "host" refers to any organism, or cell thereof, whether eukaryotic or prokaryotic into which a construct of the present disclosure can be introduced. In particular embodiments, the term "host" refers to eukaryotes, including unicellular eukaryotes such as yeast and fungi as well as multicellular eukaryotes such as animals non-limiting examples of which include invertebrate animals (e.g., insects, cnidarians, echinoderms, nematodes, etc.); eukaryotic parasites (e.g., malarial parasites, such as Plasmodium falciparum, helminths, etc.); vertebrate animals (e.g., fish, amphibian, reptile, bird, mammal); and mammals (e.g., rodents, primates such as humans and non-human primates). Thus, the term "host cell" suitably encompasses cells of such eukaryotes as well as cell lines derived from such eukaryotes. The terms "patient", "subject", "individual" used interchangeably herein. The subject may be a vertebrate subject. In particular, a mammalian subject.

[0729] Suitable subjects include primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomolgus monkeys such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatta) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such aschimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish.

[0730] The term "nucleic acid" as used herein designates mRNA, RNA, cRNA, cDNA, saRNA or DNA. The term typically refers to polymeric forms of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

[0731] " Polypeptide", "peptide", and "protein" are used interchangeably herein to refer to molecules comprising or consisting of a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0732] “Identity” as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness (homology) between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polypeptide or two polynucleotide sequences, methods commonly employed to determine identity are codified in computer programs. Preferred computer programs to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, et al., Nucleic acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul eta!., J. Molec. Biol. 215, 403 (1990).

[0733] The terms "wild-type", "native" and "naturally occurring" are used interchangeably herein to refer to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild type, native or naturally occurring gene or gene product (e.g., a polypeptide) is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene or gene product. The term “non-native” refers to a gene or gene product which has changed characteristics from the native equivalent.

[0734] “Cell”, “cell line”, and “cell culture” are used interchangeably (unless the context indicates otherwise) and such designations include all progeny of a cell or cell line. Thus, for example,terms like “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent substitutions. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

[0735] “Soluble” as used herein, refers to a protein that can be expressed in a cell-based expression system (for example, a eukaryotic cell) and that remains correctly folded and dispersed in an aqueous solution, rather than forming insoluble aggregates.

[0736] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

[0737] In certain instances, the sequences provided herein are shown with tags, such as a polyhistidine (poly-His) tag, to facilitate expression and / or purification. The skilled person will appreciate that such tags are not required to form part of the gB molecules of the invention and may therefore be substituted with alternative tags or removed entirely. Accordingly, the sequences provided herein should be understood to also encompass the corresponding sequences without such tags.

[0738] Examples

[0739] Materials and methods

[0740] Initial molecular examination of the pre-fusion glycoprotein B (gB) was done using an all-atom molecular dynamics simulation through GROMACS 2024.2 1,2. The starting model was based on a small molecule-based pre-fusion stabilised cryoEM model (Residues 86-752, PDB ID: 7KDP3). The model was prepared manually by inserting missing side chains and removing added ligands after which topology and parameter files were generated using the CHARM36 force field 4. The model was then solvated in a triclinic periodic box using a SPC / E water model and a minimal distance to the box border of 10 A. The system was then neutralised with sodium ions at a minimum salt concentration.

[0741] After solvation, the system was energy minimised over 5,000 steps using the steepest descend minimisation method and heating it from 0 to 300 K for 100 ps (NVT ensemble) with harmonic restraint force constants placed on the system at 1000 kJ mol-1 nm-2. The system pressure was then equilibrated in an isothermal ensemble (NPT ensemble) to a constant pressure of 105 Pa over 100 ps. After these steps, an MD simulation run was carried out along a 100 ns trajectory using time integration steps of 2 fs, where the short-range columbic interactions forces were cut off at 12 A following the particle mesh Ewald method 5.The simulation trajectory was processed by removing the periodic boundary condition and centring the system which allowed for calculations of biophysical properties including the radius of gyration (Rg), root-mean-square deviation (RMSD), and root-mean-square fluctuation (RMSF) values along the trajectory (Figure 4). The trajectory was also used to perform a covariance analysis of atomic coordinates and their vectors to capture reduced protein dynamic vectors.

[0742] Combining this with delta Gibb’s free energy calculations allowed for a free energy landscape representation of gB structural models within the 100 ns MD simulation. Additionally, a Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis using the GROMACS and APBS packages was performed 6. Decomposed interaction energies for residue positions involved in interchain interactions were averaged between 20 ns to 60 ns and reported (Figure 5). Finally, structural models representing local energy minima and various conformational states were extracted from the trajectory. Additional details can be found in Table 3.

[0743] Example 1: Computational Construct Design

[0744] Initial protein design strategies, focusing on the Domain I (DI) and Membrane Proximal Region (MPR) domains (Figure 1a), were based on an energy minimised model from the MD simulation. This served as the input model for multiple protein design runs to optimise the DI / MPR interface while also optimizing physicochemical properties of the MPR domain through ProteinMPNN 7. Designed positions of the gB base sequence included residues 151-161, 238-242 and 712-748. Multiple design runs were performed to generate 1000 sequences per run at sampling temperatures ranging between 0.1 -0.4 while keeping the model version with 48 edges and a 0.1 A noise value constant.

[0745] After the structure-based sequence prediction, sequences were filtered using parameters including ProteinMPNN prediction scores, predicted isoelectric points, grand average of hydropathy (GRAVY) values 8, instability index values 9, and removal of sequences containing predicted N-glycosylation patterns. The top 100 scoring sequences for each prediction run were then used to generate five AlphaFold2 models per sequence using the AlphaFold2.3 Multimer model with five model recycles 10,11. Final selection of sequences included comparing the intra backbone RMSD between the five predicted structures and comparing the backbone RMSD to the DI / MPR target structure. Combining the sequence analysis with the structural prediction analysis then allowed for the selection of the final sequences to be evaluated in test expressions.Example 2: Protein Expression and Purification

[0746] pcDNA3.4 plasmids (GeneArt, Thermo Fisher Scientific) encoding the selected gB constructs were transfected and expressed in a Freestyle™ 293 Expression System using mammalian HEK 293-F cells which were cultured in suspension using Gibco™ Freestyle™ 293 Medium (Life Technologies). Expression culture conditions were set at 37 °C, 70 % humidity, and 8 % CO2 while shaking at 130 rpm using a Multitron Pro Shaker (Infors AG). Expression media was collected after six days, centrifuged at 6000 x g for 15 min and filtered through a 0.2 pm glass fibre filter (MilliporeSigma). Then, filtered media was passed through a 5 ml StrepTrap column (Cytiva) equilibrated in 20 mM TRIS, pH 8.0, 150 mM NaCI, 2 mM CaCI2 on an AKTA Start system (Cytiva), after which bound proteins were eluted with 20 mM TRIS, pH 8.0, 150 mM NaCI, 2 mM CaCI2, 5 mM d-Desthiobiotin. Collected fractions were then applied to a size exclusion chromatography (SEC) Superose 6 Increase 10 / 300 GL column (Cytiva) equilibrated in 20 mM TRIS, pH 8.0, 150 mM NaCI, 2 mM CaCI2 on an AKTA Pure system (Cytiva). Selected elution fractions were concentrated using an Amicon® Ultra Centrifugal Filters (MilliporeSigma) with a molecular weight cut-off of 30 kDa and then stored at 4 °C. A list of selected and expressed gB constructs is available Table 4.

[0747] A fragment antigen-binding (Fab) region of a gB-specific antibody (SM5-1) was expressed in a Freestyle™ 293 Expression System (Life Technologies) following the same protocol as above. Expression media was then collected, centrifuged at 6000 x g for 15 min and filtered through a 0.2 pm glass fibre filter (MilliporeSigma). The filtered media was applied to a HiTrap LambdaSelect on an AKTA Start system (Cytiva) based on the antibody’s light chain type. The columns were equilibrated in phosphate-buffered saline (PBS), pH 7.4 buffer and bound protein was eluted using a 100 mM Glycine, pH 2.2 buffer. Collected fractions were then applied to a MonoS 5 / 50 GL column (SigmaAldrich) on an AKTA Pure system (Cytiva) for ionexchange chromatography. The column was equilibrated prior in 20 mM Sodium Acetate, pH 5.6 and bound protein was eluted after sample application with 20 mM Sodium Acetate, pH 5.6, 1M KCI on an increasing gradient ranging from 0 to 100 %. Selected Fab antibody elution fractions were collected and concentrated using an Amicon® Ultra Centrifugal Filters (MilliporeSigma) with a molecular weight cut-off of 10 kDa and then stored at 4 °C.

[0748] Example 3: Biophysical Characterisation

[0749] Expressed proteins were first assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm expected molecular weights. gB construct samples were then examined for their size distribution and sample homogeneity using dynamic light scattering. Duplicate samples at a concentration of 1 mg / mL in 20 mM TRIS, pH 8.0, 150 mMNaCI, 2 mM CaCI2 were centrifuged at 10,000 x g and placed into a 386-well plate at a 20 pL volume. Dynamic light scattering profiles were then measured using a DynaPro DLS Plate Reader III (Wyatt Technology) at 20 °C which collected ten representative scattering profiles over ten seconds each for data averaging. Resulting data was processed and analysed with the DYNAMICS™ software package.

[0750] Additionally, thermal stabilities of the gB constructs were assessed using a StarGazer-2 (Epiphyte3) through a temperature gradient denaturation assay using differential static light scattering (DSLS). Duplicate gB samples at a volume of 10 pL and a concentration of 1 mg / mL in 20 mM TRIS, pH 8.0, 150 mM NaCI, 2 mM CaCI2 were heated at a ramp rate of 1 °C / min from 25 °C to 95 °C. Resulting data was then processed and analysed with the Magellanic software package.

[0751] To confirm antigenic functionality, gB constructs were examined in Biolayer Interferometry (BLI) assays for anti-gB specific antibody binding. As gB constructs also contained a C-terminal His6x tag, binding experiments were carried out on an Octet RED96 Biolayer Inferometer using Ni-NTA biosensors (Sartorius) in an assay buffer containing 20 mM TRIS, pH 8.0, 150 mM NaCI, 2 mM CaCI2, 1 mg / mL BSA. Assay conditions were set at a temperature of 25 °C and a shake speed of 1000 rpm. First, gB samples were loaded onto Ni-NTA biosensors at a concentration of 300 nM in assay buffer. Biosensors were quenched using a quenching reagent containing 1 M ethanolamine, pH 8.5 and then placed into assay buffer over 60 s for baseline generation. Then an SM5-1 Fab antibody concentration series ranging from 12.5 nM to 400 nM in assay buffer was prepared. Association steps of immobilised gB into SM5-1 Fab antibody samples were carried out for 300 s after which antibody dissociation was measured for an additional 300 s in assay buffer. Biosensors were regenerated using multiple wash steps in wash buffer containing 750 mM imidazole and assay buffer.

[0752] Example 4: Electron Microscopy

[0753] Negative stain electron microscopy (nsEM) experiments were performed to assess the conformational states and quality of various gB constructs. gB samples at 150 nM were stained on glow discharged Formvar / Carbon 300 mesh TEM grids (Ted Pella) using 2 % (w / v) uranyl formate. The grid was then imaged on a HT7800 TEM (Hitachi) at an accelerating voltage of 100 kV and at a 50,000-x magnification which corresponds to a micrograph pixel size of 2.884 A / pixel. Micrographs were processed and analysed using CryoSPARC v4.5 (Structura Biotechnology).

[0754] Cryo-electron microscopy (cryoEM) experiments were performed to gain high resolution information of selected gB constructs. First, holey gold grids were prepared according to apreviously established protocol 12. The holey gold grids were glow discharged for 15 s using a Pelco easiGlow glow discharger (Ted Pella). Then, gB samples prepared at 2.0 mg / mL in 20 mM TRIS, pH 8.0, 150 mM NaCI, 2 mM CaCI2 were vitrified on the prepared holey gold grids using a Vitrobot Mark IV (Thermo Fisher Scientific) for 10 s with a blotting force of -5 at 4 °C and approximately 100% relative humidity before being plunge-frozen in liquid ethane at below -170 °C. Grids were imaged on a Titan Krios G3 electron microscope (Thermo Fisher Scientific), operating at an accelerating voltage of 300 kV and equipped with a Falcon 4i camera. Movies were acquired using the EPU software in electron event representation format with 30 micrograph frames per movie. Collection parameters were set at a 92,000-x magnification corresponding to 1.03 A / pixel, 10.3 e- / pixel / sec and a total exposure of ~45 e- / A2.

[0755] All the collected data was processed using CryoSPARC v4.5 (Structura Biotechnology). Movies for each gB construct were aligned with the patch motion correction job and the contrast transfer function (CTF) parameters were estimated with the patch CTF job. Resulting micrographs were then denoised using the denoiser job which allowed for effective particle picking using 30 evenly directional particle templates generated from an imported gB model. The imported models corresponded to energy minimised models from MD simulations associated with the appropriate gB sample sequence. Picked particles were extracted using a 256x256 pixel box size and classified in a 2D classification job. Noisy and non-particle like 2D classes were removed from the data set after which four ab-initio models were generated. The ab-initio class with the highest number of particles was further refined in a non-uniform refinement job in C3 symmetry and subsequent particle rebalancing allowed for an improvement of particle distributions in the dataset. A second non-uniform refinement job based on the rebalanced particles was performed in C3 symmetry and a local refinement allowed for a final 3D map generation. Models were built into the 3D maps using multiple iterations of ISOLDE 13 and Phenix real space refinement (Phenix 1.20.1) 14. In addition to structural model building of gB, protein dynamics were explored through the 3D flex refinement process trained on the map and particles of the final 3D map using default parameters.

[0756] Example 5: gB construct design for pre-fusion stabilisation

[0757] Conformational stabilisation of glycoproteins has been achieved by introducing stabilising mutations at the core interfaces of dynamic protein regions or domains 15-17. Since the native HCMV pre-fusion glycoprotein B (gB) is in a metastable state, the soluble gB ectodomain primarily adopts the post-fusion conformation 3. Initial attempts, such as including the native MPR, were unsuccessful in stabilising the pre-fusion state. Indeed, addition of the native MPRdomain resulted in low yields, and recovered fractions consisted predominantly of soluble aggregates, probably due to the high content of non-polar residues in the domain. Therefore, more advanced protein engineering strategies were necessary to develop a well-behaved gB construct in its pre-fusion state. To initiate the protein design strategies, previously determined core interface mutations that partially prevented the full conformational change of the prefusion to the post-fusion structure were examined 18. These included stabilising mutations along the buried interfaces of the DI and Dll to the core domains which include disulfide pairs H222C / E657C and V134C / I653C. These constructs also contained cavity filling mutations T100L and A267I, the removal of a free cysteine, C246S, and the removal of the Furin cleavage site by R457S and R460S mutations.

[0758] Initial experiments showed that this construct was able to stabilise gB in its pre-fusion conformation. However, as this construct was lacking the MPR domain, the lower part of the DI showed an extended and highly flexible conformation in nsEM and cryoEM data analyses conforming only a partially stabilised gB pre-fusion structure (Figure 6). As such, it was hypothesised that a more near native pre-fusion conformation would be achieved by the addition of a soluble and stable M PR-like domain. First, an all-atom molecular dynamics simulation was performed that aimed to reduce potential energetic strains on the available pre-fusion gB model which contained residues 86-752 (Figure. 1a). The resulting trajectories allowed for mapping out a free energy landscape from which energy minimised models of gB were extracted for a multi-state protein design strategy (Figure. 1b). The mapped principal components represented 53.2% of the total protein conformational dynamics across the simulation trajectory. The subsequent protein binder design was then performed on the MPR domain which aimed to improve on the binding interaction to the DI while also improving overall stability and solubility of the MPR domain itself (Figure. 1c). To achieve a set of promising sequences to be expressed, multiple ProteinMPNN design runs were initiated. Design runs varied the temperature sampling parameters ranging from 0.1 to 0.4 and residue positions of the MPR which ranged from complete redesign to selected solvent facing residues for solubility improvements. The deep learning-based sequence design runs generated around 8000 different sequences which were then examined and filtered to the top 10% using a combined filter score. This filter score consisted of combined values of the ProteinMPNN score, an instability index and a hydropathy index based on the Kyte-Doolittle method (Figure 7). The selected sequences were then used to generate structure models using AlphaFold2 to recapitulate the MPR structural fold which were filtered against pLDDT mean values and RMSD scores to the target MPR domain structure. The combination of these two values for filtering significantly reduced the number of available sequences as predicted models withhigh pLDDT confidence values tended towards single helix fold predictions that did not resemble the MPR domain. However, after structural filtering and visual examination, multiple sequences were selected for investigation as pre-fusion gB constructs. Comparing the selected sequences, we observed that residues interfacing the DI through mostly non-polar interactions, showed a high sequence conservation with the native MPR. Although some design runs specifically focused on only changing the solvent-accessible residues, this general trend was observed for the final selected as well as for other promising MPR sequences. This contrasted with residues 736-748 which showed low sequence conservation but were designed to reduce the number of non-polar amino acids (Figure. 2a).

[0759] Example 6. Evaluation of pre-fusion stabilised gB constructs

[0760] To examine the ability of an engineered MPR domain to fully stabilise the pre-fusion gB state, the selected MPR sequences were added to the partially stabilised gB construct with previously identified stabilising core interface mutations. These new constructs also contained a C246S mutation and removal of the Furin cleavage site which were intended to improve expression levels, stability and homogeneity. Surprisingly, all selected constructs resulted in significant expression levels which was not observed for the gB construct containing the native MPR sequence. SEC profiles showed the presence of two main peaks where the low molecular weight peak was ascribed as the individual gB trimer species (Figure. 2b). The expression products had various aggregation temperatures (Tagg) ranging from 34.8±5.1 °C to 90.0±2.6 °C. This high temperature variation was attributed to inappropriate protein folding and the occurrence of larger trimer aggregates. This was also supported by the corresponding hydrodynamic radii (Rh-1) values for each sample where more thermostable constructs corresponded to more homogeneous species distributions and featured radii in the 8.7 nm to 9.8 nm range. To then examine the functionality of our gB constructs, antibody binding experiments were performed which confirmed antigenicity of the gB protein against a previously established antibody (Figure 8). Finally, to get visual confirmation of pre-fusion particles, nsEM imaging on all constructs was performed.

[0761] Initial datasets containing 10,000 or more selected particles were collected to accurately assess the frequency of pre- fusion particles after protein expression, revealing that the highest individual pre-fusion trimer species exceeded 55% (Table 2). However, after careful fractionation of the low molecular weight peak from the SEC purification, the frequency of stable pre-fusion particles improved up to 95.7%. Based on the biophysical data collected to this point, four constructs were chosen for further examination. First, the low molecular weight peak fractions were carefully isolated and additional negatively stained micrographs werecollected. Examining these micrographs allowed for the generation of 2D classes and low-resolution electron density maps that matched the pre-fusion gB model. The final fitted correlation coefficients (CC) for the selected samples were above 0.76. Interestingly, the density maps showed additional density for the C-termini of the gB constructs and also showed that the DI was in a downward conformation, presumably stabilised on the interfacing MPR domain. This data suggests that the addition of the engineered MPR domains decreased flexibility across the lower part of the DI which also stabilising gB in a near-native pre-fusion conformation.

[0762] Example 7. Engineering MPR domains stabilise pre-fusion gB without core interface stabilising mutations

[0763] Since further stabilisation of gB was possible through the addition of the designed MPR based domains, their stabilisation capacity in the absence of core interface stabilising mutations were chosen for further examination. To do this, constructs with modified MPR domains were designed based on designs OP63 (SEQ ID NO: 60) and OP67 (SEQ ID NO: 64). In addition to the modified MPR sequence, mutations in these constructs still included C246S, R457S, and R460S to improve expression yields and sample homogeneity. Protein expression and subsequent examination of nsEM micrographs showed the presence of pre-fusion-stabilised gB particles (Figure 3), confirming the capability of the engineered MPR domain to stabilise gB in its pre-fusion conformation, presumably through stabilising interactions with the DI. Thus far, the engineering efforts of the MPR domain have resulted in four constructs that display improved biophysical and structural characteristics of the pre-fusion gB as compared to previously described constructs (Sponholz etal.). The addition of a soluble MPR generated a pre-fusion stabilised conformation which closely resembled the native pre-fusion gB where the DI appears in a downward conformation. This indicates potential interactions with the MPR for additional stabilisation effects. Based on the presented results, these novel constructs already offer an improved immunogen alternative to currently available HCMV gB constructs due to their stable and more near-native pre-fusion conformation. Additional confirmation and closer structural examination of the DI / MPR interaction through higher resolution techniques such as cryoEM would allow for further improvements of engineering efforts and immunogen design.

[0764] Example 8. Engineering MPR domains for EBV

[0765] Input models were generated for the de novo design of the EBV MPR domain against the DI.

[0766] 6000 MPR sequences were generated against the DI fusion loop interface using ProteinMPNN (Figure 12). The generated sequences were evaluated based on the ProteinMPNN globalscore, an instability index, and GRAVY values (Kyte-Doolittle) which were normalised and combined into a weighted score. The sequences containing an N-glycan motif (N-glycan motif = 1) were removed and the top 10% of sequences based on the weighted score were selected for structure generation and further evaluation (Figure 13).

[0767] The selected sequences were used as input for Alphafold2 structure generation which allowed for RMSD comparison between model replicates (n=5) and to the target structure. The sequence-structure pairs with the lowest RMSD values, highest pLDDT values, and lowest hydropathy values were selected as promising MPR sequences for potential in vitro examination (Figure 14).

[0768] The final selection of designed MPR sequences that showed favourable characteristics based on sequence and structural evaluation methods (Figure 15). The designed MPR sequences cover residue positions 684-733 of EBV gB (Strain B95-8). (Table 5).

[0769] Docked complex between the EBV gB DI and designed MPRs from the selected sequences are shown in Figure 16. The docking procedure followed a standard Rosetta procedure with target a / ignment, side chain packing, and multiple rounds of FastRelax runs.

[0770] Example 9. Engineering MPR domains for HSV-1

[0771] Input models were for the de novo design of the HSV-1 MPR domain against the DI. 6000 MPR sequences were generated against the DI fusion loop interface using ProteinMPNN (Figure 17). The generated sequences were evaluated based on the ProteinMPNN global score, an instability index, and GRAVY values (Kyte-Doolittle) which were normalised and combined into a weighted score. Sequences containing an N-glycan motif (N-glycan motif = 1) were removed and the top 10% of sequences based on the weighted score were selected for structure generation and further evaluation (Figure 18).

[0772] The selected sequences were used as input for Alphafold2 structure generation which allowed for RMSD comparison between model replicates (n=5) and to the target structure. The sequence-structure pairs with the lowest RMSD values, highest pLDDT values, and lowest hydropathy values were selected as promising MPR sequences for potential in vitro examination (Figure 19)

[0773] The final selection of designed MPR sequences that showed favourable characteristics based on sequence and structural evaluation methods (Figure 20). The designed MPR sequences cover residue positions 725-775 of HSV-1 gB (Strain KOS) (Table 6).Docked complex between the HSV-1 gB DI and designed MPRs from the selected sequences are shown in Figure 21. The docking procedure followed a standard Rosetta procedure with target a / ignment, side chain packing, and multiple rounds of FastRelax runs.

[0774] Example 10. Computational design of core interface mutations and de novo MPR A computational approach (Figure 28) was used for the design of core interface mutations and de novo MPR design based on an input model of gB. For this approach, the input model is generated or collected through experimental structure determination or in silico structure generation. The input model is initially energy minimised through molecular dynamics simulations which aims to reduce steric clashes and optimise side chain rotamers. After generating a confident input model, the target locations for potential pre-fusion stabilisation are selected. For the gB proteins of interest, this includes interfaces within the core of the protein as well as the target interface between the DI fusion loops and MPR domain. Favourable residue positions to be mutated are then identified through ThermoMPNN which suggests stabilising amino acid mutations in the regions of interest. In addition, residue positions in the MPR domain are specifically chosen based on their hydropathy value. Example residues to be mutated include amino acids with hydrophobic side chains such as Leu, lie, Vai, and Phe. Various strategies for residue masking are then employed to generate a diverse set of sequences. Using the target structure models, ProteinMPNN is used as an inverse protein folding model to generate plausible amino sequences that would fold into the desired structure. To confirm these sequences, in silico structures are generated using AlphaFold and further energy minimised using molecular dynamics simulations. The output of this approach is a large dataset of sequence- and structure-based properties that allow for down-selection of promising sequence designs that can be examined experimentally. Final selection properties include pLDDT values above 80, a target RMSD match of below 2.0 A, a prediction RMSD match of below 3.0 A, a hydropathy index value of the designed MPR domain below 0.5, an instability index value of below 40, a shape complementary value of above 0.6, and an interface energy value above the mean of all designed sequences. After filtering, sequences are ranked according to their total energy value and the top sequence designs are selected.

[0775] Example 11. Structural characterisation of pre-fusion stabilised HCMV gB constructs To structurally examine the designed pre-fusion stabilised HCMV gB constructs, cryoEM datasets were collected for OP64 (SEQ ID NO: 61) and OP67 (SEQ ID NO: 64) (Figure 29). These constructs included core interface mutations as well as t...

Claims

1. Claims1. Use of a non-native membrane proximal region (MPR) domain for stabilising a soluble Herpesvirus gB antigen in a pre-fusion conformation, wherein:a) the gB antigen comprises the domains DI, Dll, Dill, DIV and DV and an MPR but does not comprise a transmembrane domain,b) the non-native MPR replaces the native MPR domain, andc) the non-native MPR comprises at least one amino acid substitution.

2. A method of increasing the solubility, expression yield, and / or stability of a soluble Herpesvirus gB, wherein the Herpesvirus gB comprises the domains DI, Dll, Dill, DIV and DV and a MPR, the method comprising:a) replacing the native MPR with a non-native MPR engineered to interact with the DI domain of the gB; orb) introducing into the native MPR at least one amino acid substitution which increases the binding energy of the interaction between the MPR and the DI domain of the gB.

3. An engineered Herpesvirus gB comprising at least the domains DI, Dll, Dill, DIV and DV and a MPR and not comprising a transmembrane domain, wherein the MPR is a non- native MPR and comprises at least one amino acid substitution which stabilises the tertiary structure of the gB in its pre-fusion conformation, and wherein the at least one amino acid substitution reduces the surface hydrophobicity of the non-native MPR relative to the native MPR and / or increases the binding energy of the interaction between the MPR and the DI domain of the gB.

4. The use of claim 1, the method of claim 2, or the engineered Herpesvirus gB of claim 3, wherein one or more of the solvent facing amino acid residues in the native MPR have been substituted with amino acid residues which increase the polarity of the MPR.

5. The use of claim 1 or 4, the method of claim 2 or 4, or the engineered Herpesvirus gB of claim 3 or 4, wherein the non-native MPR domain comprises at least one amino acid substitution, and wherein the engineered Herpesvirus gB shows an increase in one or more of solubility, expression level, percentage of pre-fusion particles and aggregation temperature, and / or a decrease in Hydropathy index, instability index or Total Energy (dG), relative to a soluble Herpesvirus gB comprising a corresponding native MPR or no MPR.

6. The use of claim 1 or any one of claims 4-5, the method of claim 2 or any one of claims 4-5, or the engineered Herpesvirus gB of claim 3 or any one of claims 4-5, wherein the gB further comprising a trimerisation domain.

7. The use of claim 1 or any one of claims 4-6, the method of claim 2 or any one of claims 4-6, or the engineered Herpesvirus gB of claim 3 or any one of claims 4-6, wherein the engineered Herpesvirus gB is an alphaherpesvirus gB, a betaherpesvirus gB or a gammaherpesvirus gB.

8. The use, method, or engineered Herpesvirus gB of claim 7, wherein the Herpesvirus gB is selected from an HCMV gB, an HSV-1 gB, an HSV-2 gB or an EBV gB.

9. The use, method or engineered Herpesvirus gB of claim 8, wherein:a) the Herpesvirus gB is an HCMV gB and the substitution is a substitution of one or more of amino acid residues 707 to 752 relative to SEQ ID NO:1 (strain Merlin / Towne);b) the Herpesvirus gB is an HCMV gB and the substitution is a substitution of one or more of amino acid residues 706-751 relative to SEQ ID NO: 5 (strain AD169); c) the Herpesvirus gB is an HCMV gB and the substitution is a substitution of one or more of amino acid residues 705-750 relative to SEQ ID NO: 9 (strain VR1814); d) the Herpesvirus gB is an HSV-1 gB and the substitution is a substitution of one or more of amino acid residues relative to 725 to 775 of SEQ ID NO: 100 (strain KOS); e) the Herpesvirus gB is an HSV-2 gB and the substitution is a substitution of one or more of amino acid residues relative to 724 to 774 of any one of SEQ ID NOs:255- 259 (strain HG52); orf) the Herpesvirus gB is an EBV gB and the substitution is a substitution of one or more of amino acid residues 684 to 773 relative to SEQ ID NO: 108 (strain B95-8).

10. The use, method or engineered Herpesvirus gB of claim 9, wherein:a) the Herpesvirus gB is an HCMV gB and the MPR has a sequence of PPYLKGLDDLX1KX2LX3X4X5GX6X7EGVX8IGAX9FGKX1oX11X12KX13LX14X15EX16X17XI8KNPF, wherein Xi is selected from I, or C; X2is selected from K, C, or I; X3is selected from W, or N; X4 is selected from F, or H; X5is selected from Y, or T; X6is selected from F, or N; X7is selected from A, C, or W; X8is selected from S, or C; X9is selected from E, V, or I; Xi0is selected from D, or C; Xu is selected from E, or F; X12 is selected from W, or P; X13is selected from K, or P; X14is selected from S, or P; Xis is selected from V, or P and Xi6is selected from T, or C (SEQ ID NO: 320); b) the Herpesvirus gB is selected from an HSV-1 or HSV-2 gB and the MPR has a sequence of ADX1X2AX3X4X5X6X7X8X9X10X11YX12X13X14GX15X16X17X18X19X20GX21X22X23X24X25X26X27X28X29X3OX3IX32X33X34X35X36X37X38X39X4OAX4IX42PX43, wherein X1is selected from K, E, or A; X2is selected from I, K, R, orT; X3is selected from D, K, or A; X4is selected from E, I, or L; X5is selected from F, Q, or L; X6is selected from A, R, or K; X7is selected from E, or A; X8is selected from L, or I; X9is selected from G, or A; Xi0is selected from R, K, or E; Xu is selected from F, K, or A; X12is selected from E, K, or A; X13is selected from A, K, or E; X14is selected from K, T, or G; Xi5is selected from P, A, or E; Xi6is selected from E, or A; X17is selected from G, or A; Xi8is selected from R, E, or F; X19is selected from A, or K; X20is selected from K, or A; X24is selected from K, L, or R; X22is selected from V, or P; X23is selected from E, D, or A; X24is selected from M, L, or I; X25is selected from G, E, K, or A; X26is selected from E, K, or S; X27is selected from K, L, or T; X28is selected from G, or L; X29is selected from K, E, or A; X30is selected from V, or E; X34is selected from V, M, or L; X32is selected from S, or E; X33is selected from A, or E; X34is selected from A, K, or Q; X35is selected from A, K, or E; X36is selected from T, A, or R; X37is selected from A, E, or Q; X38is selected from A, E, or Q; X39is selected from S, E, or A; X^ is selected from L, or R; X41is selected from A, K, or L; X42is selected from D, or L and X43is selected from L, F, or Y (SEQ ID NO: 325); orc) the Herpesvirus gB is an EBV gB and the MPR has a sequence of NGX1X2X3X4VX5X6LX7X8X9X10X11X12X13X14X15X16X17QX18X19X20X21X22X23X24X25X26X2GX28X29X3OX3IX32X33X34X35X36X3X38X39X4OX4IX42PX43, wherein X1is selected from R, or V; X2is selected from N, or L; X3is selected from Q, or A; X4is selected from F, E, or L; X5is selected from D, or M; X6is selected from G, K, or D; X7is selected from G, or A; X8is selected from E, or D; X9is selected from L, or N; X10 is selected from M, or Y; Xu is selected from D, or L; X12is selected from S, N, or R; X13is selected from L, or S; X14is selected from G, P, or D; Xi5is selected from S, or P; Xi6is selected from V, A, S, or P; X17is selected from G, or E; Xi8is selected from S, or R; X19is selected from I, A, or L; X20is selected from T, or A; X24is selected from N, or R; X22is selected from L, I, or A; X23is selected from V, or T;X24is selected from S, or N; X25is selected from T, or I; X26is selected from V, K, or I; X27is selected from G, or L; X28is selected from L, or K; X29is selected from F, E, I, or L; X30is selected from S, or E; X3iis selected from S, or A; X32is selected from L, A, or F; X33is selected from V, R, or L; X34is selected from S, L, or E; X35is selected from G, A, or I; X36is selected from F, A, or L; X37is selected from I, E, or A; X38is selected from S, A, D, or R; X39is selected from F, A, or L; X40is selected from F, V, or A; X41is selected from K, N, or A; X42is selected from N, S, L, or D and X43is selected from F, L, or S (SEQ ID NO: 329).

11. The use, method or engineered Herpesvirus gB of claim 9, wherein:a) the engineered Herpesvirus gB is an HCMV gB and the MPR has a sequence selected from any one of SEQ ID NOs: 276-312, optionally wherein the sequence is selected from any one of SEQ ID NOs: 287, 297, 289 or 298;b) the engineered Herpesvirus gB is an HSV-1 or HSV-2 gB and the MPR has a sequence selected from any one of SEQ ID NOs: 223-243, optionally wherein the sequence is selected from any one of SEQ ID NOs: 232, 243, 241 and 237; or c) the engineered Herpesvirus gB is an EBV gB and the MPR has a sequence selected from any one of: 260-274, optionally wherein the sequence is selected from any one of SEQ ID NOs: 320, 325, 329.

12. The use, method or engineered Herpesvirus gB of claim 10 or 11, wherein:a) the engineered Herpesvirus gB is an HCMV gB and has a sequence selected from any one of SEQ ID NOs: 55 to 99, optionally wherein the sequence is selected from any one of SEQ ID NOs: 60, 61, 63 or 64;b) the engineered Herpesvirus gB is an HSV-1 gB and has a sequence selected from any one of SEQ ID NOs: 212-121 optionally wherein the sequence is selected from any one of SEQ ID NOs: 217, 219, 220 or 221;c) the engineered Herpesvirus gB is an HSV-2 gB and has a sequence selected from any one of SEQ ID NOs: 255-259; ord) the engineered Herpesvirus gB is an EBV gB and has a sequence selected from any one of SEQ ID NOs: 244-259, optionally wherein the sequence is selected from any one of SEQ ID NOs: 244, 246, 249 or 251.

13. The use of claim 1 or any one of claims 4-12, the method of claim 2 or any one of claims 4-12, or the engineered Herpesvirus gB of claim 3 or any one of claims 4-12, wherein theengineered Herpesvirus gB further comprises one or more of the additional stabilising substitutions are outside of the MPR.

14. The use, method or engineered Herpesvirus gB of claim 13, wherein the one or more additional stabilising substitutions are cysteine mutations which introduce one or more disulfide bridges at the DI, Dll, MPR and / or core interfaces.

15. The use, method or engineered Herpesvirus gB of claim 14, wherein:a) the Herpesvirus gB is an HCMV gB and the one or more additional stabilising substitutions are selected from the list of A239C, A239W, A267V, A293P, A369L, A732C, A97V, C246S, D277C, D320L, D478L, E274I, E274V, E286F, E289P, E321I, E634C, E657C, E671C, E679C, F683C, G173C, G177C, G735C, H222C, H681C, I636C, I89C, I90C, K124V, K158C, K158I, K214F, K340P, K359P, K394- T452del, L288P, L484P, L641C, L678C, L709C, M677I, M677L, N132C, N132V, N341P, N635T, N657C, N658C, Q483P, Q501I, Q527C, Q669C, R180I, R180L, R291P, R429S, R431S, R432S, R457S, R460S, S164C, S244C, S269C, S362P, S367I, S631C, S641C, S641L, S739C, T100I, T225C, T225F, T292P, T343P, T572C, T630C, T659L, T676C, T676I, V342P, V363P, V480P, V684W, V728C, W240C, W356P, Y153C, Y155C, Y160C, Y242C, Y464-T498del and Y481P relative to SEQ ID NO: 1;b) the Herpesvirus gB is an HSV-1 gB and the one or more additional stabilising substitutions are selected from the list of I154C, K158C, K158I, (WFGHR)174(YAYIH), W174N, F175H, Y179T, F182N, A239C, A239W, S244C, (AFH)261(WLY), E274V, E274I, D277C, E286F, W356P, K359P, S362P, V363P, Y464-T498del, T572C, L673(GCG), T676C, T676I, M677I, M677L, L678C, E679C, H681C, F683C, V684Wand N709V relative to SEQ ID NO: 100; orc) the Herpesvirus gB is an HSV-2 gB and the one or more additional stabilising substitutions are selected from the list of I149C, L670(GCG), T569C, E676C, N706V, W169N, F170H, Y174T and F177N relative to SEQ ID NO: 254; or d) the Herpesvirus gB is an EBV gB and the one or more additional stabilising substitutions are selected from the list of I89C, I90C, T630C, (YNGWY)109(YAYIH), W112N, Y113T, K124V, G173C, G177C, R180I, R180L, T225C, T225F, L288P, E289P, T292P, A293P, K394-T452del, R429S, R431S, R432S, D478L, Q527C, L628(GCG), S631C, N635T, I636C, L641C, E634C, W196S and Y198D relative to SEQ ID NO: 108.

16. The Herpesvirus gB of claim 3 wherein the pre-fusion conformation is detected using an antibody specific for an epitope on the pre-fusion conformation which is not present in the post-fusion conformation.

17. A method of designing a non-native MPR domain, the method comprising the steps of:a) obtaining an experimentally determined thee-dimensional structure of a Herpesvirus gB or generating three-dimensional structure of a Herpesvirus gB in silico from a Herpesvirus gB sequence;b) using the obtained or generated three-dimensional structure as an input model; c) energy minimising the input model using molecular dynamics simulations, wherein the energy minimisation reduces steric clashes and optimises side chain rotamers; d) selecting target locations for potential pre-fusion stabilisation substitutions, wherein the target locations are located within the core domains of the gB and / or within the interface between the DI fusion loops and MPR domain;e) predicting the impact of point mutations at the target locations in silico using a deep neural network and identifying favourable residue positions to be mutated;f) applying residue masking at the residue positions identified in step (c) to generate a diverse set of non-native gB sequences;g) generating in silico models of one or more of the non-native gB structures generated in step (f);h) generating in silico data, for example, calculated properties based on the sets of diversified sequences and generated protein structures, and collating the data in one dataset for downstream selection purposes;i) selecting one or more of the non-native gB structures having a pLDDT value above 80, a target RMSD match of below 2.0 A, a prediction RMSD match of below 3.0 A, a hydropathy index value of below 0.5, an instability index value of below 40, a shape complementary value of above 0.6, and / or an interface energy value above the mean of all designed sequences; orj) ranking the sequences according to their total energy value selecting the top sequence designsk) optionally repeating steps (a) to (j) two or more times to further optimise the non- native MPR designs.

18. The method of claim 17, wherein the method further comprises the steps of:l) generating one or more nucleic acid sequences, each encoding a gB comprising the modified MPR sequence of a selected structure;m) expressing the one or more nucleic acid sequences in an expression system to produce a gB comprising the modified MPR; andn) purifying the gB comprising the modified MPR to a purity of at least 80%.

19. The method of claim 18, whereini. the further characterisation is in vitro characterisation, and the method further comprises a step of assessing one or more biophysical and / or biochemical properties of the gB comprising the modified MPR, optionally wherein the biophysical and / or biochemical properties are selected from the aggregation temperature, expression level, percentage of correctly folded protein, hydrodynamic radius, antigenicity, and / or negative-stain electron microscopy (nsEM) characteristics; and / orii. the further characterisation is in vivo characterisation, and the method further comprises the steps of:o) administering the gB comprising the modified domain to a non-human subject to elicit an immune response;p) isolating one or more antibodies from the non-human subject; andq) assessing the binding activity and / or neutralising activity of the one or more antibodies against a live Herpesvirus corresponding to the species from which the gB protein is derived.

20. A gB trimer comprising three monomeric Herpesvirus gBs of any one of claims 3-15.

21. Use of the Herpesvirus gBs of any one of claims 3-15 for screening and identifying biologies and small molecules that inhibit or alter gB function, optionally wherein the biologic is a peptide, a nanobody, a monoclonal antibody or an aptamer.

22. A conjugate comprising the Herpesvirus gB of any one of claims 3-15, conjugated to at least one further moiety or conjugation substrate, optionally wherein the further moiety is selected from a detection moiety, a solid support, an antibody, an antibody fragment, or a binding molecule, further optionally wherein the solid support is a nanoparticle, chip, column, membrane, or virus like particle, or the detection moiety is a fluorescent tag, a magnetic tag, or a radiolabel.

23. A method of screening antibodies specifically binding to the pre-fusion conformation of the Herpesvirus gB, the method comprising:a) providing a sample containing B cells from a subject exposed to, or infected with, a Herpesvirus, a Herpesvirus vaccine or a Herpesvirus gBs of any one of claims 3-15; b) culturing single B cells in individual wells such that the individual B cells proliferate and secrete a monoclonal antibody into the well;c) detecting the monoclonal antibody in the well using a binding probe comprising a corresponding Herpesvirus gB of any one of claims 3-15; andd) identifying B cells that bind the binding probe; and / ore) sequencing the immunoglobulin genes of the isolated B cells to identify monoclonal antibodies that bind specifically to the immune reagent.

24. A vaccine comprising the Herpesvirus gBs of any one of claims 3-15, optionally wherein the engineered gB is displayed on nanoparticles.

25. A vaccine comprising an mRNA, DNA, or saRNA encoding the Herpesvirus gB of any one of claims 3-15.

26. The Herpesvirus gBs of any one of claims 3-15 for use as a vaccine antigen.

27. A pharmaceutical composition comprising the Herpesvirus gBs of any one of claims 3-15 and a pharmaceutically acceptable carrier or adjuvant.

28. The Herpesvirus gB of any one of claims 3-15 for use in the treatment or prevention of Herpesvirus infection, or a disease or disorder associated therewith.

29. A non-native Herpesvirus gB MPR domain for use in a method of stabilising a soluble Herpesvirus gB in a pre-fusion conformation, wherein the non-native MPR replaces the native MPR.

Images