Immunogenic variants of human cytomegalovirus glycoprotein b

Abstract

Agents useful for immunotherapy and prophylaxis of infection with human cytomegalovirus (hCMV) are provided. The agents are variant gB polypeptides (or genetic material encoding the polypeptides), wherein disulphide bridges are introduced in the amino acid sequence of gB so as to stabilize the pre-fusion conformation of the protein. The disulphide bridges are introduced via double substitutions in SEQ ID NO: 1 and are selected from the group consisting of 1) I103C and V645C; 2) A97C and A538C; and 3) G543C and K617C. Also disclosed are vaccine compositions as well as methods for their use.

Description

[0001]IMMUNOGENIC VARIANTS OF HUMAN CYTOMEGALOVIRUS GLYCOPROTEIN B FIELD OF THE INVENTION The present invention relates to immunotherapy and prophylaxis, in particular in the field of prophylactic immunization against infection with human cytomegalovirus (hCMV). BACKGROUND OF THE INVENTION Human cytomegalovirus (HCMV) glycoprotein B (gB) transitions irreversibly from a metastable pre-fusion conformation to a stable post-fusion conformation to facilitate fusion of the viral membrane with the human host cell membrane resulting in entry of the virus into the host cell to initiate infection. An existing strategy for devising gB protein variants, which are able to induce potent antibodies blocking the binding of gB protein to the human host cell membrane, is to stabilize the gB protein in its pre-fusion conformation. Similar approaches are known in the field of SARS-CoV-2 vaccines, where pre-fusion stabilized Spike protein has been employed as the antigenic component in the available regulatorily approved vaccines. For the gB protein, the strategy has been pursued as disclosed in WO 2021 / 260510, which relates to several mutations in the gB amino acid sequence in order to create disulphide bridges that stabilize the protein in its pre-fusion format. OBJECT OF THE INVENTION It is an object of embodiments of the invention to provide new effective vaccine agents that induce protective immunity against infection with hCMV. SUMMARY OF THE INVENTION It has been found by the present inventors that novel variants of hCMV gB with in-substituted cysteine pairs maintain the pre-fusion conformation of hCMV gB after being recombinantly expressed; it has also been found that the protective immune responses induced are superior when compared to those induced by a prior art gB variant protein disclosed in WO 2021 / 260510. So, in a first aspect the present invention relates to a variant of human cytomegalovirus glycoprotein B (gB) comprising 1, 2, or 3 double substitutions, each of said double substitutions corresponding to double substitutions in SEQ ID NO: 1 selected from the group consisting of 1) I103C and V645C,; 2) A97C and A538C; and 3) G543C and K617C, wherein said variant comprises a contiguous stretch of amino acids, which, when optimally aligned with residues 97-645 in SEQ ID NO: 1, comprises at least 470 identical amino acid residues. In a 2ndaspect, the present invention relates to a nucleic acid molecule encoding the variant of human gB of the first aspect of the invention (as well as any embodiment thereof disclosed herein in the context of the 1staspect of the invention). In a 3rdaspect, the present invention relates to a vector comprising the nucleic acid molecule of the 2ndaspect of the invention (as well as any embodiment thereof disclosed herein in the context of the 2ndaspect of the invention), such as a cloning vector or an expression vector. In a 4thaspect, the present invention relates to a genetically modified cell, which carries the nucleic acid molecule of the 2ndaspect of the invention (as well as any embodiment thereof disclosed herein in the context of the 2ndaspect of the invention) or carries the vector of the 3rdaspect of the invention (as well as any embodiment thereof disclosed herein in the context of the 3rdaspect of the invention). Part of this aspect is a cell line derived from the genetically modified cell. In a 5th aspect, the present invention relates to a composition comprising a variant gB polypeptide of the 1staspect of the invention (as well as any embodiment thereof disclosed herein in the context of the 1staspect of the invention) and an immunological adjuvant. In a 6th aspect, the present invention relates to a composition comprising the nucleic acid molecule of the 2ndaspect of the invention (as well as any embodiment thereof disclosed herein in the context of the 2nd aspect of the invention) or a vector of the 3rd aspect of the invention (as well as any embodiment thereof disclosed herein in the context of the 3rdaspect of the invention) in admixture with a pharmaceutically acceptable excipient, diluent, carrier, or vehicle, and optionally further comprising a nucleic acid fragment or vector, which encodes at least one further hCMV immunogen. In a 7thaspect, the present invention relates to a composition comprising the genetically modified cell of the 4thaspect of the invention (as well as any embodiment thereof disclosed herein in the context of the 4thaspect of the invention) in admixture with a pharmaceutically acceptable excipient, diluent, carrier, or vehicle. Finally, in an 8thaspect, the present invention relates to a method for inducing immunity in a human subject against infections caused by hCMV, the method comprising administering to the subject an effective amount of a variant of the gB polypeptide of the 1staspect of the invention (as well as any embodiment thereof disclosed herein in the context of the 1st aspect of the invention), the nucleic acid molecule of the 2ndaspect of the invention (as well as any embodiment thereof disclosed herein in the context of the 2nd aspect of the invention), the vector of the 3rdaspect of the invention (as well as any embodiment thereof disclosed herein in the context of the 3rdaspect of the invention), the genetically modified cell of the 4thaspect of the invention (as well as any embodiment thereof disclosed herein in the context of the 4thaspect of the invention), or the composition of the 5th-7thaspects of the invention (as well as any embodiment thereof disclosed herein in the context of the 5th-7thaspects of the invention). LEGENDS TO THE FIGURE Fig. 1: alignment of SEQ ID NO: 1 (UniProtKB accession no: P13201.1) with other full-length hCMV gB amino acid sequences (SEQ ID Nos: 8-122). The emphasized residues in Fig. 1 are those substituted as disclosed herein. Fig. 2: Schematic explanation of symbols used in vaccine designs shown in Fig. 3. Fig. 3: Schematic presentation of the different gB designs. A: gB control designs showing wt-gB design that is comparable to Sino-gB (cat.: 10202- VCCH1; Sino Biologicals) composed of both the ecto- and endodomain of gB, post_gB composed of gB ectodomain and post_gB_tri that contains the gB ectodomain and a trimerization domain. B: Construct comparable to the previously described pre-fusion gB design (WO2021 / 260510) with pre-fusion stabilizing cysteine mutations indicated in the brackets, herein referred to as gB2796-Ctag. C: Novel pre-fusion gB designs described herin with pre-fusion stabilizing cysteine mutations indicated in the brackets. Fig. 4: The pre-fusion (A) and post-fusion (B) structure of hCMV gB. Structures are obtained from the Protein Data Bank (H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne (2000) Nucleic Acids Research, 28: 235-242. https: / / doi.org / 10.1093 / nar / 28.1.235), PDB IDS: 7KDP and 7KDD. The location of the pre-fusion stabilizing disulfide bond mutations (G543C-L617C) are shown as black spheres (highlighted by arrows) in both conformations. Figures are made using PyMOL (Schrödinger, L. & DeLano, W. (2020). PyMOL. Retrieved from http: / / www.pymol.org / pymol). Fig. 5: The pre-fusion (A) and post-fusion (B) structure of hCMV gB. Structures are obtained from the Protein Data Bank (H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne (2000) Nucleic Acids Research, 28: 235-242. https: / / doi.org / 10.1093 / nar / 28.1.235), PDB IDS: 7KDP and 7KDD. The location of the pre- fusion stabilizing disulfide bond mutations (A97C-A538C) are shown as black spheres (highlighted by arrows) in both conformations. Figures are made using PyMOL. Fig. 6: The pre-fusion (A) and post-fusion (B) structure of hCMV gB. Structures are taken from the Protein Data Bank (Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne (2000) Nucleic Acids Research, 28: 235-242. https: / / doi.org / 10.1093 / nar / 28.1.235), PDB IDS: 7KDP and 7KDD. The location of the pre- fusion stabilizing disulfide bond mutations (I103C-V645C) are shown as black spheres (highlighted by arrows) in both conformations. Figures are made using PyMOL. Fig. 7: Western blot from transiently transfected S2 cells under reducing (+) and non- reducing (-) conditions. Lane M: molecular weight marker. Lane 6: Control; wild-type gB protein (SEQ ID NO: 1, residues 23-702) with C-terminal C- tag. Lane 7: Control; wild-type gB protein (SEQ ID NO: 1, residues 23-702) with C-terminal trimerization domain and C-tag. Lane 8: Comparative; prior art mutated gB protein (SEQ ID NO: 1, residues 23-646 with Cys substitutions D217C, Y589C, M371C, W506C) and the following further substitutions: YIH(155-157)GHR, W240A, and C246S; and a C-terminal C-tag. Lane 9: Mutated gB protein (SEQ ID NO: 2). Lane 10: Mutated gB protein (SEQ ID NO: 3). Lane 11: Mutated gB protein (SEQ ID NO: 4). Lane 13: Mutated gB protein (SEQ ID NO: 6). Lane 14: Mutated gB protein (SEQ ID NO: 7). Fig. 8: Size Exclusion HPLC overlays of EVX-pre_gB#2, EVX-pre_gB#3, EVX-pre_gB#5 and EVX-pre_gB#6. A Superdex200 column was used, and the Globular SEC marker has the following sizes: 670 kDa, 158 kDa, 44 kDa, 17 kDa and 1.3 kDa. (Cat. No: 151-1901, Biorad). The monodisperse SEC profile of pre_gB#2 and pre_gB3 evidences a trimeric form. Fig. 9: Size Exclusion HPLC overlay elution profiles. A: EVX-pre_gB#3 and WT-gB B: EVX-pre_gB#2 and WT-gB. A Superdex200 column was used, and the Globular SEC marker have the following sizes: 670 kDa, 158 kDa, 44 kDa, 17 kDa and 1.3 kDa. (Cat. No: 151-1901, Biorad). Fig. 10: Transmission electron microscopy (TEM) of wt_gB (A) and pre_gB#3 (B) confirm the ability of the mutations introduced to stabilize gB in a pre-fusion conformation. The protein samples were stained with 2% (w / v) phosphotungstic acid and viewed by electron microscopy. Scale bar (100 nm) is showm in the larger picture, with a zoom represented in the minor picture. Fig. 11: Cryo-electron microscopy confirming pre-fusion stabilized gB. Pictures were captured using a Titan Krios. Fig. 12: Bar graphs showing IgG response to wt_gB, post_gB and post_gB_tri. Dotted line represents limit of detection measured by the response againt wt_gB in placebo (AddaVax) immunized mice. Removing the endodomain or adding the trimerization domain does not impact the anti-gB antibody response. Fig. 13: Bar graphs showing immune responses A: IgG responses to wt_gB, gB2796-Ctag, pre_gB#2 and pre_gB#3 shows that pre_gB#2 induces significantly higher antibody responses than the wt_gB. B: Serum was diluted 1:3200 and the ability of the serum to neutralize virus infection of MRC5 cells was measured, showing that serum from gB_pre#3 immunized animals offered high level of protection against infection in vitro. Dotted line represents limit of detection measured by the response in placebo (AddaVax) immunized mice. Fig. 14: Bar graph shwoing IgG response to pre_gB#2, pre_gB#3, pre_gB#5 and pre_gB#6. It is demonstrated that both single mutational pairs in pre_gB#2 and pre_gB#3 and double mutational pairs in pre_gB#5 and pre_gB#6 provide high levels of antibodies. The dotted line represents limit of detection measured by the wt_gB response in placebo (AddaVax) immunized mice. Fig. 15: Bar graphs showing IgG responses in animals vaccinated with either wt_gB, post_gB, post_gB_tri, gB2796-Ctag, pre_gB#2 or pre_gB#3. A: Responses to wt_gB B: Responses to pre_gB#3. The dotted line represents limit of detection measured by the response in placebo (AddaVax) immunized mice. Fig. 16: Bar graph showing dose-dependent IgG response to pre_gB#3 in animals vaccinated with either 10 μg, 5 μg or 1 μg pre_gB#3. The titer remains high even at low doses. The dotted line represents limit of detection measured by the response in placebo (AddaVax) immunized mice. Fig. 17: Line graphs showing dose-dependent IgG response to pre_gB#3 in animals vaccinated with either 10 μg, or 1 μg pre_gB#3 measured after one dose (study day 13), two doses (study day 26) and 3 doses (study day 42). The dotted line represents the response in placebo (AddaVax) immunized mice. DETAILED DISCLOSURE OF THE INVENTION Definitions The expression "… substitutions corresponding to substitution pairs selected from the group consisting of G543C and K617C; A97C and A538C; and I103C and V645C…" in the present context means substitutions in a gB amino acid sequence (typically selected among SEQ ID NOs: 8-122), which is optimally aligned with SEQ ID NO: 1, and where each substitution in a gB amino acid sequence different from SEQ ID NO: 1 is made in the position that aligns with the named amino acid position in SEQ ID NO: 1. As can be seen from Fig. 1, G543 of SEQ ID NO: 1 aligns with G541 in the gB protein having the amino acid sequence with UniProtKB Accession No. AMJ54594.1 and with G545 in the gB protein having the amino acid sequence with UniProtKB Accession No. AZB53140.1. In this context, the alignment is made with proteins that have at least 93% sequence identity with SEQ ID NO: 1 after a BLAST search setting out from SEQ ID NO: 1 with the algorithm parameters: Expect threshold: 0.05; Word size 5; Matrix: BLOSUM62; gap penalties: existence 11, extension 1; end gap penalties: existence -5, extension -1. The term "polypeptide" is in the present context intended to mean both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. Furthermore, the term is also intended to include proteins, i.e. functional biomolecules comprising at least one polypeptide; when comprising at least two polypeptides, these may form complexes, be covalently linked, or may be non-covalently linked. The polypeptide (s) in a protein can be glycosylated and / or lipidated and / or comprise prosthetic groups. The term "subsequence" means any consecutive stretch of at least 3 amino acids or, when relevant, of at least 3 nucleotides, derived directly from a naturally occurring amino acid sequence or nucleic acid sequence, respectively. The term “amino acid sequence” is the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins in the direction from the free N- terminus to the free C-terminus. An "immunogen" is a substance of matter, which is capable of inducing an adaptive immune response in a host, whose immune system is confronted with the immunogen. As such, immunogens are a subset of the larger genus "antigens", which are substances that can be recognized specifically by the immune system (e.g. when bound by antibodies or, alternatively, when fragments of the are antigens bound to MHC molecules are being recognized by T-cell receptors) but which are not necessarily capable of inducing immunity - an antigen is, however, always capable of eliciting immunity, meaning that a host that has an established memory immunity against the antigen will mount a specific immune response against the antigen. An "adaptive immune response" is an immune response in response to confrontation with an antigen or immunogen, where the immune response is specific for antigenic determinants of the antigen / immunogen – examples of adaptive immune responses are induction of antigen specific antibody production or antigen specific induction / activation of T helper lymphocytes or cytotoxic lymphocytes. A "protective, adaptive immune response" is an antigen-specific immune response induced in a subject as a reaction to immunization (artificial or natural) with an antigen, where the immune response is capable of protecting the subject against subsequent challenges with the antigen or a pathology-related agent that includes the antigen. Typically, prophylactic vaccination aims at establishing a protective adaptive immune response against one or several pathogens. "Stimulation of the immune system" means that a substance or composition of matter exhibits a general, non-specific immunostimulatory effect. A number of adjuvants and putative adjuvants (such as certain cytokines) share the ability to stimulate the immune system. The result of using an immunostimulating agent is an increased "alertness" of the immune system meaning that simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to isolated use of the immunogen. The term "vector" is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it can be replicated and expressed. The term further denotes certain biological vehicles useful for the same purpose, e.g., viral vectors and phage – both these infectious agents are capable of introducing a heterologous nucleic acid sequence The term "expression vector" refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, when the transcription product is an mRNA molecule, this is in turn translated into a protein, polypeptide, or peptide. A "linker" is an amino acid sequence, which is introduced between two other amino acid sequences in order to separate them spatially. A linker may be "rigid", meaning that it does substantially not allow the two amino acid sequences that it connects to move freely relative to each other. Likewise, a "flexible" linker allows the two sequences connected via the linker to move substantially freely relative to each other. In the fusion proteins, which are part of the present invention, both types of linkers are useful, but flexible linkers are preferred. "An immunogenic carrier" is a molecule or moiety to which an immunogen or a hapten can be coupled in order to enhance or enable the elicitation of an immune response against the immunogen / hapten. Immunogenic carriers are in classical cases relatively large molecules (such as tetanus toxoid, KLH, diphtheria toxoid etc.) which can be fused or conjugated to an immunogen / hapten, which is not sufficiently immunogenic in its own right – typically, the immunogenic carrier is capable of eliciting a strong T-helper lymphocyte response against the combined substance constituted by the immunogen and the immunogenic carrier, and this in turn provides for improved responses against the immunogen by B-lymphocytes and cytotoxic lymphocytes. More recently, the large carrier molecules have to a certain extent been substituted by so-called promiscuous T-helper epitopes, i.e. shorter peptides that are recognized by a large fraction of HLA haplotypes in a population, and which elicit T-helper lymphocyte responses. The term "immunological adjuvant" has its usual meaning in the art of vaccine technology, i.e. a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is 2) nevertheless capable of enhancing the immune response against the immunogen. Or, in other words, vaccination with the adjuvant alone does not provide an immune response against the immunogen, vaccination with the immunogen may or may not give rise to an immune response against the immunogen, but the combined vaccination with immunogen and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone. Specific embodiments of the invention The full-length gB protein exists as a number of naturally occurring isoform variants from different strains of human cytomegalovirus. The presently disclosed gB variant can for instance be a variant of any one of the following proteins, identified via their UniProtKB accession numbers: P13201.1 (SEQ ID NO: 1 in the present application), ABQ23592.1, UNW45129.1, AND81495.1, ACM48044.1, ACS32370.1, AKI25887.1, AFR55550.1, QPI35314.1, AKI19983.1, AFR55048.1, AKI19648.1, APG57425.1, AZB53165.1, YP_081514.1, APG57594.1, AAA45928.1, AQN72202.1, AMJ53258.1, QIA46047.1, AKI19483.1, AKI25042.1, AHJ85145.1, AMJ52758.1, QTT58728.1, AHB20033.1, AQN69676.1, QTT59229.1, ALL26209.1, AKI13294.1, AKI13965.1, AKI23324.1, AQN70515.1, QBK84312.1, ACS93398.1, AQN69508.1, AKI20319.1, APA45201.1, AHJ82619.1, AZB53139.1, AKI08783.1, ABV71586.1, QIA45035.1, AQN73719.1, AGL96655.1, AAB07485.1, QBF76487.1, AZV24343.1, AMJ54594.1, AKI24367.1, AHJ83628.1, AKI22824.1, AFR55885.1, AKI23491.1, AAA45925.1, ACS92156.1, AMJ54426.1, AQN72370.1, AAA45926.1, WHN53998.1, AZB53148.1, AKI09288.1, AAA45923.2, AHV84013.1, AZB53142.1, QIA45372.1, AQN70349.1, CAG7582570.1, QIA46216.1, AHJ82283.1, AII80437.1, AZB53140.1, AHJ84133.1, AAA45930.1, AZB53158.1, AKI17642.1, AQN70182.1, AFR55719.1, AKI12960.1, AFR54557.1, AHB19702.1, UNW44971.1, ACT81737.1, P06473.1, AZB79941.1, UBQ34153.1, AZB53164.1, ACS91991.1, AKI14299.1, AFR55216.1, AHJ85985.1, AKI22156.1, AAA45934.2, AKI24535.1, AHJ83292.1, AKI24704.1, QZX45917.1, QPZ44673.1, AHJ84975.1, AKI12129.1, AKI22656.1, AII80435.1, ADB92600.1, AFR54884.1, ADE88063.1, AZB53145.1, AKI20990.1, AHJ86153.1, AZB53175.1, ADD39116.1, ADV04383.1, AKI09624.1, CAH0494218.1, AHJ82785.1, AZB53172.1, and AQN71866.1. It is noted that the residues substituted with Cys in SEQ ID NO: 1 are 100% conserved across all these gB proteins as demonstrated in the alignment set forth in Fig. 1. 1staspect of the invention and embodiments thereof As set forth above, the 1staspect of the invention relates to a variant of human cytomegalovirus glycoprotein B (gB) comprising 1, 2, or 3 double substitutions, each of said double substitutions corresponding to double substitutions in SEQ ID NO: 1 selected from the group consisting of 1) I103C and V645C; 2) A97C and A538C; and 3) G543C and K617C, wherein said variant comprises a contiguous stretch of amino acids, which, when optimally aligned with residues 97-645 in SEQ ID NO: 1, comprises at least 470 identical amino acid residues. The preferred double substitutions are selected from 1) I103C and V645C and 2) A97C and A538C, with the double substitution I103C and V645C being particularly preferred. Such variants of the invention exhibit a stable pre-fusion conformation and genetically modified cells (in particular S2 cells) can provide the variants in high levels of expression. The at least 470 identical amino acid residues can be any higher number that allows for the introduction of the above-discussed double substitutions, i.e. up to a maximum of 547 identical amino acid residues (in a case where only one double substitution is carried out in SEQ ID NO: 1). The minimum number of identical amino acid residues compared to residues 97-645 in SEQ ID NO: 1 is hence selected from 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, and 547 identical amino acid residues. However, as shown in the sequence alignment in Fig. 1, a certain variation exists between different gB protein isoforms from different viral isolates, meaning that the variant of the invention setting out from a different sequence than SEQ ID NO: 1 naturally will include more residues non-identical with SEQ ID NO: 1 than those non- identities dictated by the double substitutions. In some embodiments, the variant lacks one or both amino acid residue stretches corresponding to the amino acid sequences defined by residues 1-22 and 703-907 of SEQ ID NO: 1, see for instance the variants defined by SEQ ID NOs: 2-7. Since it has turned out that variants of the invention including as few as 1 of the double substitutions are conformationally stable, some preferred embodiments entail that the variant comprises only one of the 3 double substitutions defined in options 1-3 above. However, the variant may comprise further double cysteine substitutions, in particular those corresponding to a double cysteine substitution in SEQ ID NO: 1 selected from the group consisting of a) D217C and Y589C; and b) M371C and W506C. All the above discussed variants may further comprise the substitutions corresponding to YIH(155-157)GHR in SEQ ID NO: 1 and / or the substitution corresponding to C246S in SEQ ID NO: 1. Also, preferred variants of the invention are fused to – and thereby comprises - i) a trimerization domain, which is preferably located in the C-terminal part of the variant, or ii) an affinity tag, or iii) a trimerization domain as well as an affinity tag, which optionally are separated by a linker sequence, where said linker sequence preferably is flexible. This trimerization domain preferably comprises or consists of a sequence of amino acid residues identical to residues 681-704 in any one of SEQ ID NOs: 2-7. The affinity tag can be any suitable affinity tag (such as a His tag comprising multiple H residues,) but a preferred affinity tag (the C-tag) comprises or consists of residues 709-712 in any one of SEQ ID NOs: 2-7. As mentioned above, the variant of the invention could be based on any hCMV wild-type sequence. So preferred variants include at most 15 (that is: a number selected from 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2) non-identical amino acid residues compared to the part of the amino acid sequence corresponding to residues 97-645 in SEQ ID NO: 1, where said part is found in the amino acid sequences with the UniProtKB accession numbers P13201.1, ABQ23592.1, UNW45129.1, AND81495.1, ACM48044.1, ACS32370.1, AKI25887.1, AFR55550.1, QPI35314.1, AKI19983.1, AFR55048.1, AKI19648.1, APG57425.1, AZB53165.1, YP_081514.1, APG57594.1, AAA45928.1, AQN72202.1, AMJ53258.1, QIA46047.1, AKI19483.1, AKI25042.1, AHJ85145.1, AMJ52758.1, QTT58728.1, AHB20033.1, AQN69676.1, QTT59229.1, ALL26209.1, AKI13294.1, AKI13965.1, AKI23324.1, AQN70515.1, QBK84312.1, ACS93398.1, AQN69508.1, AKI20319.1, APA45201.1, AHJ82619.1, AZB53139.1, AKI08783.1, ABV71586.1, QIA45035.1, AQN73719.1, AGL96655.1, AAB07485.1, QBF76487.1, AZV24343.1, AMJ54594.1, AKI24367.1, AHJ83628.1, AKI22824.1, AFR55885.1, AKI23491.1, AAA45925.1, ACS92156.1, AMJ54426.1, AQN72370.1, AAA45926.1, WHN53998.1, AZB53148.1, AKI09288.1, AAA45923.2, AHV84013.1, AZB53142.1, QIA45372.1, AQN70349.1, CAG7582570.1, QIA46216.1, AHJ82283.1, AII80437.1, AZB53140.1, AHJ84133.1, AAA45930.1, AZB53158.1, AKI17642.1, AQN70182.1, AFR55719.1, AKI12960.1, AFR54557.1, AHB19702.1, UNW44971.1, ACT81737.1, P06473.1, AZB79941.1, UBQ34153.1, AZB53164.1, ACS91991.1, AKI14299.1, AFR55216.1, AHJ85985.1, AKI22156.1, AAA45934.2, AKI24535.1, AHJ83292.1, AKI24704.1, QZX45917.1, QPZ44673.1, AHJ84975.1, AKI12129.1, AKI22656.1, AII80435.1, ADB92600.1, AFR54884.1, ADE88063.1, AZB53145.1, AKI20990.1, AHJ86153.1, AZB53175.1, ADD39116.1, ADV04383.1, AKI09624.1, CAH0494218.1, AHJ82785.1, AZB53172.1, and AQN71866.1. These UniProtKB sequences are also set forth herein as SEQ ID NOs: 8-122. Particularly preferred variants of gB of the invention are: a variant, which comprises residues 1-680 of SEQ ID NO: 4; a variant, which comprises residues 1-680 of SEQ ID NO: 3; a variant, which comprises residues 1-680 of SEQ ID NO: 2; a variant, which comprises SEQ ID NO: 4; a variant which comprises SEQ ID NO: 3; a variant, which comprises SEQ ID NO: 2; a variant, which consists of SEQ ID NO: 4 or residues 1-680 of SEQ ID NO: 4; a variant, which consists of SEQ ID NO: 3 or residues 1-680 of SEQ ID NO: 3; a variant, which consists of SEQ ID NO: 2 or residues 1-680 of SEQ ID NO: 2; a variant, which comprises residues 1-680 of SEQ ID NO: 7; a variant, which comprises residues 1-680 of SEQ ID NO: 6; a variant, which comprises residues 1-680 of SEQ ID NO: 5; a variant, which comprises SEQ ID NO: 7; a variant, which comprises SEQ ID NO: 6; a variant, which comprises SEQ ID NO: 5; a variant, which consists of SEQ ID NO: 7 or residues 1-680 of SEQ ID NO: 7; a variant, which consists of SEQ ID NO: 6 or residues 1-680 of SEQ ID NO: 6; and a variant, which consists of SEQ ID NO: 5 or residues 1-680 of SEQ ID NO: 5. Of these the following variants are highly preferred: a variant, which comprises residues 1-680 of SEQ ID NO: 4; a variant, which comprises residues 1-680 of SEQ ID NO: 3; a variant, which comprises SEQ ID NO: 4; a variant which comprises SEQ ID NO: 3; a variant, which consists of SEQ ID NO: 4 or residues 1-680 of SEQ ID NO: 4; a variant, which consists of SEQ ID NO: 3 or residues 1-680 of SEQ ID NO: 3; a variant, which comprises residues 1-680 of SEQ ID NO: 7; a variant, which comprises residues 1-680 of SEQ ID NO: 6; a variant, which comprises SEQ ID NO: 7; a variant, which comprises SEQ ID NO: 6; a variant, which consists of SEQ ID NO: 7 or residues 1-680 of SEQ ID NO: 7; and a variant, which consists of SEQ ID NO: 6 or residues 1-680 of SEQ ID NO: 6. The most preferred variants are as indicated above those including the substitution pair I103C and V645C. In turn, this means that highest preference is given to: a variant, which comprises residues 1-680 of SEQ ID NO: 4; a variant, which comprises SEQ ID NO: 4; a variant, which consists of SEQ ID NO: 4 or residues 1-680 of SEQ ID NO: 4; a variant, which comprises residues 1-680 of SEQ ID NO: 7; a variant, which comprises SEQ ID NO: 7; and a variant, which consists of SEQ ID NO: 7 or residues 1-680 of SEQ ID NO: 7. Embodiments of the 2ndaspect of the invention Nucleic acids encoding the above-discussed variants of hCMV gB according to any one of the preceding claims constitute the 2ndaspect of the invention. These nucleic acids can be in the form of DNA or RNA and find use both as in vivo pharmaceutical products (i.e. as vaccine agents that cause expression of the above discussed variants) or as tools ex vivo for recombinant production of the variants of the invention. In this context the nucleic acid molecules of the invention are normally integrated into a vector backbone and thus fused to genetic elements that facilitate their use in each of these contexts. In embodiments of the 2ndaspect of the invention, the nucleic acid sequences are codon optimized for expression in a host cell or host organism. Technologies for devising such codon optimized sequences for a given host cell or organism are well-known to the person skilled in molecular biology Embodiments of the 3rdaspect of the invention Vectors comprising the nucleic acid molecule of the 2ndaspect of the invention are typically provided as cloning vectors or expression vectors. In this context, the term vector broadly covers a vehicle which contains the nucleic acid molecule and finds use for transferring the genetic information to host cells that may either be used to propagate the nucleic acid molecules or for to express them. A typical expression vector comprises at least in operable linkage and in the 5'-3' direction, an expression control region comprising an enhancer / promoter for driving expression of the nucleic acid molecule of the invention, optionally a signal peptide-coding sequence, a nucleotide sequence of the 2ndaspect of the invention, and optionally a terminator. However, other genetic elements may be included, cf. the more general description below. Hence, such a vector constitutes an expression vector useful for effecting production in cells of the polypeptide of the invention. Since the polypeptides of the invention are viral of origin, recombinant production can be brought about in a variety of host cells, so the expression control region will – according to circumstances - drive expression in a prokaryotic cell such as a bacterium, e.g. in E coli, or in eukaryotic cell (mammalian cell, insect or plant), the expression control region should be adapted to suit this particular use. The vector may further comprise a sequence encoding a signal peptide, which may provide for secretion or membrane integration of the expression product from said vector. For the purposes of nucleic acid vaccination, the signal peptides encoded are typically selected from those described in Williams J.A. Vaccines (Basel). 2013 Sep; 1(3): 225–249 as well as in the references cited therein. The expression control region and drive expression in a prokaryotic cell such as a bacterium, e.g. in E coli, or drive expression in a eukaryotic cell, such as in a mammalian cell, in an insect cell, and in a plant cell. If the vector is DNA , it may further encode a polyadenylation signal, useful for eukaryotic expression systems and DNA vaccination applications. Likewise, if the vector is RNA it can comprise a polyA tail, which is useful in eukaryotic expression such as in the context of RNA vaccination. Some vectors of the invention are capable of autonomous replication, a feature of particular interest in cloning and propagation vectors. Also, some vectors (e.g. viral, such as retroviral) are capable of being integrated into the genome of a host cell, which is particularly useful for obtaining stably modified cells for use in recombinant production. Other vectors do not integrate, at least not in mammalian cells, a feature of particular relevance for DNA vaccination or viral vector vaccination where DNA integration into the genome of the vaccinated individual is highly undesirable. As detailed above, the vector can be in various forms and shapes; a typical vector of the present invention is selected from the group consisting of a virus, such as an attenuated virus, a bacteriophage, a plasmid, a minichromosome, and a cosmid. Further details in of the vectors of the invention Variant gB polypeptides disclosed herein may as indicated be encoded by a nucleic acid molecule comprised in a vector. By nature, the nucleic acids in the present context are heterologous (since they do not occur naturally), which means that it is in a context foreign to the cell in which the vector is being introduced, which includes a sequence homologous to a sequence in the cell but in a position within the host cell where it is ordinarily not found. Vectors include naked DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (for example as set forth in the biotech handbooks Sambrook et al, 2001; Ausubel et al, 1996). In addition to encoding the variant gB polypeptides of this invention, a vector of the present invention may encode polypeptide sequences such as a tag or immunogenicity enhancing peptide (e.g. an immunogenic carrier or a fusion partner that stimulates the immune system, such as a cytokine or active fragment thereof). Useful vectors encoding such fusion proteins include pIN vectors, vectors encoding a stretch of histidinyl resodies, and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage. Vectors disclosed herein may be used in a host cell to produce a polypeptide disclosed herein that may subsequently be purified for administration to a subject or the vector may be purified for direct administration to a subject for expression of the protein in the subject (as is the case when administering a nucleic acid vaccine). Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. 1. Promoters and Enhancers A "promoter" is a control sequence. The promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and / or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon. Such a promoter can be referred to as "endogenous". Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural state. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Patent 4,683,202, U.S. Patent 5,928,906). Naturally, it may be important to employ a promoter and / or enhancer that effectively direct(s) the expression of the DNA segment in the cell type or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression (see Sambrook et al., 2001). The promoters employed may be constitutive, tissue-specific, or inducible and in certain embodiments may direct high level expression of the introduced DNA segment under specified conditions, such as large-scale production of recombinant proteins or peptides. Examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus, include but are not limited to Immunoglobulin Heavy Chain, Immunoglobulin Light Chain, T Cell Receptor, HLA DQα and / or DQβ, β- Interferon, Interleukin-2, Interleukin-2 Receptor, MHC Class II 5, MHC Class II HLA-DRα, β- Actin, Muscle Creatine Kinase (MCK), Prealbumin (Transthyretin), Elastase I, Metallothionein (MTII), Collagenase, Albumin, α-Fetoprotein, γ-Globin, β-Globin, c-fos, c-HA-ras, Insulin, Neural Cell Adhesion Molecule (NCAM), αl-Antitrypain, H2B (TH2B) Histone, Mouse and / or Type I Collagen, Glucose-Regulated Proteins (GRP94 and GRP78), Rat Growth Hormone, Human Serum Amyloid A (SAA), Troponin I (TN I), Platelet-Derived Growth Factor (PDGF), Duchenne Muscular Dystrophy, SV40, Polyoma, Retroviruses, Papilloma Virus, Hepatitis B Virus, Human Immunodeficiency Virus, Cytomegalovirus (CMV) IE, and Gibbon Ape Leukemia Virus. Inducible Elements and their inducers include the pairs MT II - Phorbol Ester (TFA) / Heavy metals; MMTV (mouse mammary tumour virus) - Glucocorticoids; β-Interferon - poly(rl)x / poly(rc); Adenovirus 5 E2 - ElA; Collagenase - Phorbol Ester (TPA); Stromelysin - Phorbol Ester (TPA); SV40 - Phorbol Ester (TPA); Murine MX Gene - Interferon, Newcastle Disease Virus; GRP78 Gene - A23187; α-2-Macroglobulin - IL-6; Vimentin - Serum; MHC Class I Gene H-2κb - Interferon; HSP70 - E1A / SV40 Large T Antigen; Proliferin - Phorbol Ester / TPA; Tumor Necrosis Factor - PMA; and Thyroid Stimulating Hormone Gene - Thyroid Hormone. Also contemplated as useful in the present invention are the dectin-1 and dectin-2 promoters. Additionally any promoter / enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of structural genes encoding oligosaccharide processing enzymes, protein folding accessory proteins, selectable marker proteins or a heterologous protein of interest. The particular promoter that is employed to control the expression of peptide or protein encoding polynucleotide disclosed herein is not believed to be critical, so long as it is capable of directing expression of the polynucleotide in a targeted cell. Where a human cell is targeted, it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a bacterial, human or viral promoter. In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat can be used to obtain high level expression of a related polynucleotide to this invention. The use of other viral or mammalian cellular or bacterial phage promoters, which are well known in the art, to achieve expression of polynucleotides is contemplated as well. Promoters effective in insect cells are of particular interest; cf. e.g. the hybrid promoters disclosed in WO 2009 / 150222 and their related S2 expression systems. In embodiments in which a vector is administered to a subject for expression of the protein, it is contemplated that a desirable promoter for use with the vector is one that is not down- regulated by cytokines or one that is strong enough that even if down-regulated, it produces an effective amount of the protein / polypeptide of the current invention in a subject to elicit an immune response. Non-limiting examples of these are CMV IE and RSV LTR. In other embodiments, a promoter that is up-regulated in the presence of cytokines is employed. The MHC I promoter increases expression in the presence of IFN-γ. Tissue specific promoters can be used, particularly if expression is in cells in which expression of an antigen is desirable, such as dendritic cells or macrophages. The mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters. 2. Initiation Signals and Internal Ribosome Binding Sites (IRES) A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic and may be operable in bacteria or mammalian cells. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements. In certain embodiments disclosed herein, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described, as well an IRES from a mammalian message. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, herein incorporated by reference). 2. Multiple Cloning Sites Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology. 3. Splicing Sites Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. If relevant in the context of vectors of the present invention, vectors containing genomic eukaryotic sequences may require donor and / or acceptor splicing sites to ensure proper processing of the transcript for protein expression. 4. Termination Signals The vectors or constructs of the present invention will generally comprise at least one termination signal. A "termination signal" or "terminator" is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels. In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (poly A) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message. Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the bovine growth hormone terminator or viral termination sequences, such as the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation. 5. Polyadenylation Signals In expression, particularly eukaryotic expression (as is relevant in nucleic acid vaccination), one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and / or any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and / or the bovine growth hormone polyadenylation signal, convenient and / or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport. Consequently, the corresponding encoded RNA fragment preferably comprises a poly(A) tail. 6. Origins of Replication In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "on"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast. 7. Selectable and Screenable Markers In certain embodiments disclosed herein, cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector. When transcribed and translated, a marker confers an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker. Usually, the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, markers that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin or histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP for colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers that can be used in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a protein disclosed herein. Further examples of selectable and screenable markers are well known to one of skill in the art. Embodiments of the 4thaspect of the invention Genetically modified cells find use as production tools in recombinant production of the variants of the invention (both for propagation of genetic material and of expression of the genetic material), but also in some cases as vaccine agents. For instance, in recombinant production technology some genetically modified cells of the invention are useful when capable of replicating the nucleotide sequence encoding a variant of the 1staspect of the invention. However, more preferred genetically modified cells are those capable of expressing the nucleotide sequence defined above, which encodes a variant of the first aspect of the invention and any embodiment thereof disclosed herein; such a cell can be a prokaryotic cell as well as a eukaryotic cell. Among prokaryotic cells, bacterial cells used in expression systems of as live or killed vaccine agents are of particular interest. Such a bacterial cell for use as a live vaccine is preferably non-pathogenic. At any rate, bacteria useful in this aspect of the invention (i.e. whether used for vaccination or recombinant protein production) are typically selected from the group consisting of Escherichia (such as E. coli), Bacillus (e.g. Bacillus subtilis), Salmonella sp., and Mycobacterium, e.g. M. bovis BCG Eukaryotic cells are primarily used for recombinant expression and can for instance be a mammalian cell, an insect cell, or a plant cell. Certain insect cells are preferred, in particular cell derived from Drosophila melanogaster, e.g. an S2 or S3 cell. An interesting production system is the use of plants. For instance, proteins can be produced at low cost in plants using an Agrobacterium transfection system to genetically modify plants to express genes that encode the protein of interest. One commercially available platform are those provided by iBio CMO LLC (8800 HSC Pkwy, Bryan, TX 77807, USA) and iBio, Inc (9 Innovatiin Way, Suite 100, Newark, DE 19711, USA) and disclosed in e.g. EP 2853599, EP 1 769068, and EP 2192172. Hence, in such systems the vector is an Agrobacterium vector or other vector suitable for transfection of plants. For recombinant production purposes, it is advantageous if the genetically modified cell of the present invention has the nucleotide sequence encoding the variant of the 1st aspect of the invention and embodiments thereof stably integrated into its genome. For recombinant production purposes, the availability of the expression product (i.e. the variant of the 1staspect of the present invention) in the fermentation medium is of relevance. It is generally an advantage if the expression product is secreted into the fermentation medium (thus requiring that the expression product includes relevant export signals), but alternatively, the expression product can be integrated into the membrane of the genetically modified cell (and thus be carried on its surface). In a bacteria, the expression product may also be exported into the periplasmic space or exported to appear in the cell wall / outer membrane. Finally, the genetically modified cell can conveniently be kept as a cell line (e.g. in a master cell bank). Further details on cells and cell lines Suitable cells for recombinant nucleic acid expression of the nucleic acid molecules of the present invention are prokaryotes and eukaryotes. Examples of prokaryotic cells include E. coli; members of the Staphylococcus genus, such as S. epidermidis; members of the Lactobacillus genus, such as L. plantarum; members of the Lactococcus genus, such as L. lactis; members of the Bacillus genus, such as B. subtilis; members of the Corynebacterium genus such as C. glutamicum; and members of the Pseudomonas genus such as Ps. fluorescens. Examples of eukaryotic cells include mammalian cells; insect cells; yeast cells such as members of the Saccharomyces genus (e.g. S. cerevisiae), members of the Pichia genus (e.g. P. pastoris), members of the Hansenula genus (e.g. H. polymorpha), members of the Kluyveromyces genus (e.g. K. lactis or K. fragilis) and members of the Schizosaccharomyces genus (e.g. S. pombe). As mentioned above, the nucleic acid sequence of the present invention can be appropriately codon optimized to facilitate effective expression from each of the genetically modified cells disclosed herein. Techniques for recombinant gene production, introduction into a cell, and recombinant gene expression are well known in the art. Examples of such techniques are provided in references such as Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-2002, and Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989. As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses. A host cell may be "genetically modified", for instance "transfected" or "transformed," which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell. A genetically modified cell includes the primary subject cell and its progeny. Host cells may be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells for replication of the vector or expression of part or all of the nucleic acid sequence(s). Numerous cell lines and cultures are available for use as a host cell, and they can e.g. be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials or from other depository institutions such as Deutsche Sammlung von Microorganismen und Zellkulturen (DSM). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryotic host cell for replication of many vectors or expression of encoded proteins. Bacterial cells used as host cells for vector replication and / or expression include Staphylococcus strains, DH5α, JMl 09, and KC8, as well as a number of commercially available bacterial hosts such as SURE(R) Competent Cells and SOLOP ACK(TM) Gold Cells (STRATAGENE®, La Jolla, CA). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses. Appropriate yeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastoris. Examples of eukaryotic host cells for replication and / or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12, S2, S3 cells. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector. Some vectors may employ control sequences that allow it to be replicated and / or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above-described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides. Expression Systems Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and / or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available. The insect cell / baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACKTMBaculovirus expression system from CLONTECH® In addition to the disclosed expression systems disclosed herein, other examples of expression systems include STRATAGENE®'s COMPLETE CONTROL™ Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide. Insect cell-based expression systems are i.a. described in WO 2009 / 150222. Embodiments of the 5thaspect of the invention The 5thaspect of the invention generally relates to compositions, such as compositions useful for immunization, and in particular vaccine compositions, which comprise the variant of the 1st aspect of the invention (or any embodiment thereof disclosed herein). In some embodiments, the composition comprises an immunological adjuvant (often useful in immunization / vaccination settings) and / or the composition comprises a pharmaceutically acceptable excipient, diluent, carrier, or vehicle. Details concerning the composition of vaccines and details pertaining to vaccination and immunization modes are detailed in the separate section below. However, a particularly interesting composition mode entails that the variant is formulated in, or forms part of, a virus-like particle (VLP). Apart from containing a variant of the 1staspect of the present invention, the composition can constitute a "cocktail", whereby it comprises at least one further immunogen derived from hCMV, thus enabling establishment of an immune response against several epitope- containing regions in hCMV. Embodiments of the 6thaspect of the invention This aspect relates generally to compositions where nucleic acids of the 2nd aspect or vectors of the 3rdaspect constitute a part; again, it is of particular interest to include the nucleic acid molecules or vectors as active ingredient in vaccines. Details about this type of composition and the related modes of immunization are detailed below. Generally, the nucleic acid molecule or vector is present in admixture with a pharmaceutically acceptable excipient, diluent, carrier, or vehicle, and – in analogy to the above – optionally also in admixture with a further nucleic acid molecule or vector, which encodes at least one further hCMV immunogen. Embodiments of the 7thaspect of the invention The embodiment relates generally to a composition comprising the genetically modified cell discussed in detail above, in particular in admixture with a pharmaceutically acceptable excipient, diluent, carrier, or vehicle; i.e. this aspect is of particular relevance when the genetically modified cells are used as immunization agents. General consideration concerning compositions of the invention, vaccines Pharmaceutical compositions, in particular vaccines, according to the invention may either be prophylactic (i.e. suited to prevent infection) or therapeutic (i.e. to treat disease after infection). In some embodiments disclosed herein, the pharmaceutical compositions such as vaccines include merely one single antigen, immunogen, polypeptide, protein, nucleic acid or vector of the invention, but in other embodiments, the pharmaceutical compositions comprise "cocktails" of the antigens or of the immunogens or of the polypeptides or of the protein or of the nucleic acids or of the vectors disclosed herein together with other hCMV-derived antigens or immunogens or polypeptides or proteins or nucleic acids or vectors or genetically modified cells, i.e. immunogenic agents as disclosed herein but not base on hCMV gB protein. These embodiments entail combinations of peptides / polypeptides which are admixed with each other. Alternatively, the same combinations of peptides / polypeptides can be constructed as fusion polypeptides, optionally connected via a linker as described above. Another alternative entails compositions where the immunogens are nucleic acids (DNA or RNA) encoding the peptide combinations or encoding such fusion polypeptides. In particular, RNA vaccines have attracted attention recently, with the Covid-19 RNA vaccines from Pfizer / BioNTech and Moderna being the first examples used in larger scale in humans. Vaccines disclosed herein typically comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid(s), usually in combination with "pharmaceutically acceptable carriers", which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition or targeting the protein / pathogen. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants"). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogen, cf. the description of immunogenic carriers supra. The pharmaceutical compositions disclosed herein thus typically contain an immunological adjuvant, which is commonly an aluminium based adjuvant or one of the other adjuvants described in the following: Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminium salts (alum), such as aluminium hydroxide, aluminium phosphate, aluminium sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immune stimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (WO 90 / 14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either micro-fluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM) ; (3) saponin adjuvants such as Stimulon™ (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumour necrosis factor (TNF), etc.; and (6) other substances that act as immune stimulating agents to enhance the effectiveness of the composition. Alum and MF59™ adjuvants are preferred. Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D- isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N- acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2"-2'-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc. As indicated in the examples, the glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE; developed by the Infectious Disease Research Institute, Seattle, WA) is one interesting adjuvant useful in the present invention. As an alternative to vaccine formulations described above, the variants of hCMV gB can be formulated with virus-like particles (VLPs): VLPs of interest also include capsid virus like particle (cVLP). VLPs of relevance for the present invention are i.a. discussed in Saghi Nooraei et al. Journal of Nanobiotechnology 19, Article number: 59 (2021) (doi.org / 10.1186 / s12951-021-00806-7). Other examples of particles of interest comprise the cVLPs presented by Liekniņa, I. et al., see specifically Fig. 2. The immunogenic compositions (e.g. the immunising antigen or immunogen or polypeptide or protein or nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers. Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By "immunologically effective amount", it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies or generally mount an immune response, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. However, for the purposes of protein vaccination, the amount administered per immunization is typically in the range between 0.5 μg and 500 mg (however, often not higher than 5,000 μg), and very often in the range between 10 and 200 μg. The immunogenic compositions are conventionally administered parenterally, eg, by injection, either subcutaneously, intramuscularly, or transdermally / transcutaneously (e.g. as disclosed in WO 98 / 20734). Additional formulations suitable for other modes of administration include oral, pulmonary and nasal formulations, suppositories, and transdermal applications. In the case of nucleic acid vaccination, also the intravenous or intraarterial routes may be applicable. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents. As an alternative to protein-based vaccines, DNA vaccination (also termed nucleic acid vaccination or gene vaccination) may be used (cf. e.g. Robinson & Torres (1997) Seminars in Immunol 9: 271-283; Donnelly et al. (1997) Annu Rev Immunol 15 : 617-648). Also and as also pointed out herein, vaccination with RNA (mRNA) is an interesting and highly promising technology, cf. the above-mentioned reference by Deering R.P. et al. Embodiments of the 8thaspect of the invention This aspect relates generally to immunization methods which utilise the variants of hCMV gB, the corresponding nucleic acids encoding the variants, vectors, or genetically modified cells as described above. In some embodiments a priming dosage is followed by one or more booster doses with an effective amount of a variant of the gB polypeptide of the first aspect or embodiments thereof described herein, the nucleic acid molecule of the 2ndaspect or embodiments thereof described herein, the vector of the third aspect or embodiments thereof described herein, the genetically modified cell of the 4thaspect or embodiments thereof described herein, or the compositions described herein comprising any of these. It is particularly relevant to boost a with protein-based vaccines or nucleic acid vaccines. It is also noted that priming made be undertaken with one type of immunization agent, followed by boosting with a different type of immunization agent – a DNA or RNA vaccine can be boosted with protein vaccination and vice versa, for instance. The vaccination methods described herein may be both prophylactic and therapeutic in nature. For instance, a subject having ongoing infection with hCMV can be subject to the method for immunization as can a subject having no prior exposure to hCMV as well as subjects that have subclinical infection (in this case, the treatment is essentially prophylaxis of re-emergence of disease). In other words, a vaccinated subject may have no ongoing infection with hCMV, and in this caser the subject may also be free of any previous or recent infection with hCMV as evidenced by being seronegative for anti-CMV antibodies. At any rate, the vaccination technology described herein in believed to reduce symptoms or prevent emergence of symptoms of disease caused by hCMV infection in the vaccinated subjects. Details relating the method of the invention The method o f the 7thaspect disclosed herein generally relates to induction of immunity and as such also entails method that relate to treatment, prophylaxis and amelioration of disease. When immunization methods entail that a hCMV gB variant disclosed herein or a composition comprising such a variant is administered the subject typically receives between 0.5 and 5,000 μg of the variant disclosed herein per administration. In preferred embodiments of this aspect, the immunization scheme includes that the animal (e.g. the human) receives a priming administration and one or more booster administrations. Preferred embodiments of this aspect disclosed herein comprise that the administration is for the purpose of inducing protective immunity against hCMV. In turn this means that the administration is a prophylactic or therapeutic treatment of diseases caused by hCMV. Vaccines disclosed herein induce humoral immunity, so it is preferred that the administration is for the purpose of inducing antibodies specific for hCMV. Pharmaceutical compositions can as mentioned above comprise polypeptides, antibodies, or nucleic acids disclosed herein. The pharmaceutical compositions will comprise a therapeutically effective amount thereof. The term "therapeutically effective amount" or "prophylactically effective amount" as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. Reference is however made to the ranges for dosages of immunologically effective amounts of polypeptides, cf. above. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician. For purposes of the present invention, an effective dose will be from about 0.01 mg / kg to 50 mg / kg or 0.05 mg / kg to about 10 mg / kg of DNA or RNA constructs in the individual to which it is administered. A pharmaceutical composition can as described herein also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991). Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier. As is apparent from the claims, the invention also relates to related aspect and embodiments to the treatment and prophylaxis disclosed herein: the invention also includes aspects and embodiments where - the variant hCMV gB polypeptide disclosed herein is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with hCMV; - the nucleic acid fragment disclosed herein or the vector disclosed herein is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with hCMV; - the genetically modified cell disclosed herein is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with hCMV. BIOLOGICIAL SEQUENCES The following gB and gB variant sequences are provided for better understanding of the invention. The reference sequence is SEQ ID NO: 1, which in Fig. 1 is aligned with multiple gB sequences. The substitutions marked with underlining in SEQ ID Nos: 2-7 can also be made in other gB amino acid sequences. As appears from Fig. 1, the aligned sequences are conserved with respect to the substituted residue, i.e. the same amino acid residue is found in both the reference sequence and all aligned sequences, with the exception of position 589 in SEQ ID NO: 1, which in 5 of the aligned sequences is constituted by H (histidine) instead of Y (tyrosine). In those 5 sequences, the substitution introduced with hence be H→C instead of Y→C. SEQ ID NO: 1 UniProtKB accession no: PI3201.1, where residues in underlined italics are deleted in variant sequences SEQ ID NO: 2-7 MESRIWCLVV CVNLCIVCLG AAVSSSSTRG TSATHSHHSS HTTSAAHSRS GSVSQRVTSS QTVSHGVNET IYNTTLKYGD VVGVNTTKYP YRVCSMAQGT DLIRFERNIV CTSMKPINED LDEGIMVVYK RNIVAHTFKV RVYQKVLTFR RSYAYIHTTY LLGSNTEYVA PPMWEIHHIN SHSQCYSSYS RVIAGTVFVA YHRDSYENKT MQLMPDDYSN THSTRYVTVK DQWHSRGSTW LYRETCNLNC MVTITTARSK YPYHFFATST GDVVDISPFY NGTNRNASYF GENADKFFIF PNYTIVSDFG RPNSALETHR LVAFLERADS VISWDIQDEK NVTCQLTFWE ASERTIRSEA EDSYHFSSAK MTATFLSKKQ EVNMSDSALD CVRDEAINKL QQIFNTSYNQ TYEKYGNVSV FETTGGLVVF WQGIKQKSLV ELERLANRSS LNLTHNRTKR STDGNNATHL SNMESVHNLV YAQLQFTYDT LRGYINRALA QIAEAWCVDQ RRTLEVFKEL SKINPSAILS AIYNKPIAAR FMGDVLGLAS CVTINQTSVK VLRDMNVKES PGRCYSRPVV IFNFANSSYV QYGQLGEDNE ILLGNHRTEE CQLPSLKIFI AGNSAYEYVD YLFKRMIDLS SISTVDSMIA LDIDPLENTD FRVLELYSQK ELRSSNVFDL EEIMREFNSY KQRVKYVEDK VVDPLPPYLK GLDDLMSGLG AAGKAVGVAI GAVGGAVASV VEGVATFLKN PFGAFTIILV AIAVVIIIYL IYTRQRRLCM QPLQNLFPYL VSADGTTVTS GNTKDTSLQA PPSYEESVYN SGRKGPGPPS SDASTAAPPY TNEQAYQMLL ALVRLDAEQR AQQNGTDSLD GQTGTQDKGQ KPNLLDRLRH RKNGYRHLKD SDEEENV SEQ ID NO: 2 gB variant, substituted in underlined positions and fused to trimerization domain (double underlining) and C-tag (bold italics) via a GSGS linker (dotted underline). VSSSSTRGTS ATHSHHSSHT TSAAHSRSGS VSQRVTSSQT VSHGVNETIY NTTLKYGDVV GVNTTKYPYR VCSMAQGTDL IRFERNIVCT SMKPINEDLD EGIMVVYKRN IVAHTFKVRV YQKVLTFRRS YAGHRTTYLL GSNTEYVAPP MWEIHHINSH SQSYSSYSRV IAGTVFVAYH RDSYENKTMQ LMPDDYSNTH STRYVTVKDQ WHSRGSTALY RETCNLNCMV TITTARSKYP YHFFATSTGD VVDISPFYNG TNRNASYFGE NADKFFIFPN YTIVSDFGRP NSALETHRLV AFLERADSVI SWDIQDEKNV TCQLTFWEAS ERTIRSEAED SYHFSSAKMT ATFLSKKQEV NMSDSALDCV RDEAINKLQQ IFNTSYNQTY EKYGNVSVFE TTGGLVVFWQ GIKQKSLVEL ERLANRSSLN LTHNRTKRST DGNNATHLSN MESVHNLVYA QLQFTYDTLR GYINRALAQI AEAWCVDQRR TLEVFKELSK INPSAILSAI YNKPIAARFM CDVLGLASCV TINQTSVKVL RDMNVKESPG RCYSRPVVIF NFANSSYVQY GQLGEDNEIL LGNHRTEECQ LPSLCIFIAG NSAYEYVDYL FKRMIDLSSI STVDSMIALD IDPLENTDFR VLELYSQKEL RSSNVFDLEE IMREFNSYKQ RVKYVEDKVV KIEEILSKQY HIENEIARIK KLIGGSGSEP EA SEQ ID NO: 3 gB variant, substituted in underlined positions and fused to trimerization domain (double underlining) and C-tag (bold italics) via a GSGS linker (dotted underline). VSSSSTRGTS ATHSHHSSHT TSAAHSRSGS VSQRVTSSQT VSHGVNETIY NTTLKYGDVV GVNTTKYPYR VCSMCQGTDL IRFERNIVCT SMKPINEDLD EGIMVVYKRN IVAHTFKVRV YQKVLTFRRS YAGHRTTYLL GSNTEYVAPP MWEIHHINSH SQSYSSYSRV IAGTVFVAYH RDSYENKTMQ LMPDDYSNTH STRYVTVKDQ WHSRGSTALY RETCNLNCMV TITTARSKYP YHFFATSTGD VVDISPFYNG TNRNASYFGE NADKFFIFPN YTIVSDFGRP NSALETHRLV AFLERADSVI SWDIQDEKNV TCQLTFWEAS ERTIRSEAED SYHFSSAKMT ATFLSKKQEV NMSDSALDCV RDEAINKLQQ IFNTSYNQTY EKYGNVSVFE TTGGLVVFWQ GIKQKSLVEL ERLANRSSLN LTHNRTKRST DGNNATHLSN MESVHNLVYA QLQFTYDTLR GYINRALAQI AEAWCVDQRR TLEVFKELSK INPSAILSAI YNKPICARFM GDVLGLASCV TINQTSVKVL RDMNVKESPG RCYSRPVVIF NFANSSYVQY GQLGEDNEIL LGNHRTEECQ LPSLKIFIAG NSAYEYVDYL FKRMIDLSSI STVDSMIALD IDPLENTDFR VLELYSQKEL RSSNVFDLEE IMREFNSYKQ RVKYVEDKVV KIEEILSKQY HIENEIARIK KLIGGSGSEP EA SEQ ID NO: 4 gB variant, substituted in underlined positions and fused to trimerization domain (double underlining) and C-tag (bold italics) via a GSGS linker (dotted underline). VSSSSTRGTS ATHSHHSSHT TSAAHSRSGS VSQRVTSSQT VSHGVNETIY NTTLKYGDVV GVNTTKYPYR VCSMAQGTDL CRFERNIVCT SMKPINEDLD EGIMVVYKRN IVAHTFKVRV YQKVLTFRRS YAGHRTTYLL GSNTEYVAPP MWEIHHINSH SQSYSSYSRV IAGTVFVAYH RDSYENKTMQ LMPDDYSNTH STRYVTVKDQ WHSRGSTALY RETCNLNCMV TITTARSKYP YHFFATSTGD VVDISPFYNG TNRNASYFGE NADKFFIFPN YTIVSDFGRP NSALETHRLV AFLERADSVI SWDIQDEKNV TCQLTFWEAS ERTIRSEAED SYHFSSAKMT ATFLSKKQEV NMSDSALDCV RDEAINKLQQ IFNTSYNQTY EKYGNVSVFE TTGGLVVFWQ GIKQKSLVEL ERLANRSSLN LTHNRTKRST DGNNATHLSN MESVHNLVYA QLQFTYDTLR GYINRALAQI AEAWCVDQRR TLEVFKELSK INPSAILSAI YNKPIAARFM GDVLGLASCV TINQTSVKVL RDMNVKESPG RCYSRPVVIF NFANSSYVQY GQLGEDNEIL LGNHRTEECQ LPSLKIFIAG NSAYEYVDYL FKRMIDLSSI STCDSMIALD IDPLENTDFR VLELYSQKEL RSSNVFDLEE IMREFNSYKQ RVKYVEDKVV KIEEILSKQY HIENEIARIK KLIGGSGSEP EA SEQ ID NO: 5 gB variant, substituted in underlined positions and fused to trimerization domain (double underlining) and C-tag (bold italics) via a GSGS linker (dotted underline). VSSSSTRGTS ATHSHHSSHT TSAAHSRSGS VSQRVTSSQT VSHGVNETIY NTTLKYGDVV GVNTTKYPYR VCSMAQGTDL IRFERNIVCT SMKPINEDLD EGIMVVYKRN IVAHTFKVRV YQKVLTFRRS YAGHRTTYLL GSNTEYVAPP MWEIHHINSH SQSYSSYSRV IAGTVFVAYH RDSYENKTMQ LMPDCYSNTH STRYVTVKDQ WHSRGSTALY RETCNLNCMV TITTARSKYP YHFFATSTGD VVDISPFYNG TNRNASYFGE NADKFFIFPN YTIVSDFGRP NSALETHRLV AFLERADSVI SWDIQDEKNV TCQLTFWEAS ERTIRSEAED SYHFSSAKMT ATFLSKKQEV NMSDSALDCV RDEAINKLQQ IFNTSYNQTY EKYGNVSVFE TTGGLVVFWQ GIKQKSLVEL ERLANRSSLN LTHNRTKRST DGNNATHLSN MESVHNLVYA QLQFTYDTLR GYINRALAQI AEAWCVDQRR TLEVFKELSK INPSAILSAI YNKPIAARFM CDVLGLASCV TINQTSVKVL RDMNVKESPG RCYSRPVVIF NFANSSCVQY GQLGEDNEIL LGNHRTEECQ LPSLCIFIAG NSAYEYVDYL FKRMIDLSSI STVDSMIALD IDPLENTDFR VLELYSQKEL RSSNVFDLEE IMREFNSYKQ RVKYVEDKVV KIEEILSKQY HIENEIARIK KLIGGSGSEP EA SEQ ID NO: 6 gB variant, substituted in underlined positions and fused to trimerization domain (double underlining) and C-tag (bold italics) via a GSGS linker (dotted underline). VSSSSTRGTS ATHSHHSSHT TSAAHSRSGS VSQRVTSSQT VSHGVNETIY NTTLKYGDVV GVNTTKYPYR VCSMCQGTDL IRFERNIVCT SMKPINEDLD EGIMVVYKRN IVAHTFKVRV YQKVLTFRRS YAGHRTTYLL GSNTEYVAPP MWEIHHINSH SQSYSSYSRV IAGTVFVAYH RDSYENKTMQ LMPDCYSNTH STRYVTVKDQ WHSRGSTALY RETCNLNCMV TITTARSKYP YHFFATSTGD VVDISPFYNG TNRNASYFGE NADKFFIFPN YTIVSDFGRP NSALETHRLV AFLERADSVI SWDIQDEKNV TCQLTFWEAS ERTIRSEAED SYHFSSAKMT ATFLSKKQEV NMSDSALDCV RDEAINKLQQ IFNTSYNQTY EKYGNVSVFE TTGGLVVFWQ GIKQKSLVEL ERLANRSSLN LTHNRTKRST DGNNATHLSN MESVHNLVYA QLQFTYDTLR GYINRALAQI AEAWCVDQRR TLEVFKELSK INPSAILSAI YNKPICARFM GDVLGLASCV TINQTSVKVL RDMNVKESPG RCYSRPVVIF NFANSSCVQY GQLGEDNEIL LGNHRTEECQ LPSLKIFIAG NSAYEYVDYL FKRMIDLSSI STVDSMIALD IDPLENTDFR VLELYSQKEL RSSNVFDLEE IMREFNSYKQ RVKYVEDKVV KIEEILSKQY HIENEIARIK KLIGGSGSEP EA SEQ ID NO: 7 gB variant, substituted in underlined positions and fused to trimerization domain (double underlining) and C-tag (bold italics) via a GSGS linker (dotted underline). VSSSSTRGTS ATHSHHSSHT TSAAHSRSGS VSQRVTSSQT VSHGVNETIY NTTLKYGDVV GVNTTKYPYR VCSMAQGTDL CRFERNIVCT SMKPINEDLD EGIMVVYKRN IVAHTFKVRV YQKVLTFRRS YAGHRTTYLL GSNTEYVAPP MWEIHHINSH SQSYSSYSRV IAGTVFVAYH RDSYENKTMQ LMPDCYSNTH STRYVTVKDQ WHSRGSTALY RETCNLNCMV TITTARSKYP YHFFATSTGD VVDISPFYNG TNRNASYFGE NADKFFIFPN YTIVSDFGRP NSALETHRLV AFLERADSVI SWDIQDEKNV TCQLTFWEAS ERTIRSEAED SYHFSSAKMT ATFLSKKQEV NMSDSALDCV RDEAINKLQQ IFNTSYNQTY EKYGNVSVFE TTGGLVVFWQ GIKQKSLVEL ERLANRSSLN LTHNRTKRST DGNNATHLSN MESVHNLVYA QLQFTYDTLR GYINRALAQI AEAWCVDQRR TLEVFKELSK INPSAILSAI YNKPIAARFM GDVLGLASCV TINQTSVKVL RDMNVKESPG RCYSRPVVIF NFANSSCVQY GQLGEDNEIL LGNHRTEECQ LPSLKIFIAG NSAYEYVDYL FKRMIDLSSI STCDSMIALD IDPLENTDFR VLELYSQKEL RSSNVFDLEE IMREFNSYKQ RVKYVEDKVV KIEEILSKQY HIENEIARIK KLIGGSGSEP EA In addition, Fig. 1 lists the following published sequences of gB isoforms from various sources and identifies the positions for modification analogous to the modifications introduced in SEQ ID NOs: 2-7 relative to SEQ ID NO: 1: Sequence Accession no SEQ ID NO: 8 : ABQ23592.1 SEQ ID NO: 9 : UNW45129.1 SEQ ID NO: 10 : AND81495.1 SEQ ID NO: 11 : ACM48044.1 SEQ ID NO: 12 : ACS32370.1 SEQ ID NO: 13 : AKI25887.1 SEQ ID NO: 14 : AFR55550.1 SEQ ID NO: 15 : QPI35314.1 SEQ ID NO: 16 : AKI19983.1 SEQ ID NO: 17 : AFR55048.1 SEQ ID NO: 18 : AKI19648.1 SEQ ID NO: 19 : APG57425.1 SEQ ID NO: 20 : AZB53165.1 SEQ ID NO: 21 : YP_081514.1 SEQ ID NO: 22 : APG57594.1 SEQ ID NO: 23 : AAA45928.1 SEQ ID NO: 24 : AQN72202.1 SEQ ID NO: 25 : AMJ53258.1 SEQ ID NO: 26 : QIA46047.1 SEQ ID NO: 27 : AKI19483.1 SEQ ID NO: 28 : AKI25042.1 SEQ ID NO: 29 : AHJ85145.1 SEQ ID NO: 30 : AMJ52758.1 SEQ ID NO: 31 : QTT58728.1 SEQ ID NO: 32 : AHB20033.1 SEQ ID NO: 33 : AQN69676.1 SEQ ID NO: 34 : QTT59229.1 SEQ ID NO: 35 : ALL26209.1 SEQ ID NO: 36 : AKI13294.1 SEQ ID NO: 37 : AKI13965.1 SEQ ID NO: 38 : AKI23324.1 SEQ ID NO: 39 : AQN70515.1 SEQ ID NO: 40 : QBK84312.1 SEQ ID NO: 41 : ACS93398.1 SEQ ID NO: 42 : AQN69508.1 SEQ ID NO: 43 : AKI20319.1 SEQ ID NO: 44 : APA45201.1 SEQ ID NO: 45 : AHJ82619.1 SEQ ID NO: 46 : AZB53139.1 SEQ ID NO: 47 : AKI08783.1 SEQ ID NO: 48 : ABV71586.1 SEQ ID NO: 49 : QIA45035.1 SEQ ID NO: 50 : AQN73719.1 SEQ ID NO: 51 : AGL96655.1 SEQ ID NO: 52 : AAB07485.1 SEQ ID NO: 53 : QBF76487.1 SEQ ID NO: 54 : AZV24343.1 SEQ ID NO: 55 : AMJ54594.1 SEQ ID NO: 56 : AKI24367.1 SEQ ID NO: 57 : AHJ83628.1 SEQ ID NO: 58 : AKI22824.1 SEQ ID NO: 59 : AFR55885.1 SEQ ID NO: 60 : AKI23491.1 SEQ ID NO: 61 : AAA45925.1 SEQ ID NO: 62 : ACS92156.1 SEQ ID NO: 63 : AMJ54426.1 SEQ ID NO: 64 : AQN72370.1 SEQ ID NO: 65 : AAA45926.1 SEQ ID NO: 66 : WHN53998.1 SEQ ID NO: 67 : AZB53148.1 SEQ ID NO: 68 : AKI09288.1 SEQ ID NO: 69 : AAA45923.2 SEQ ID NO: 70 : AHV84013.1 SEQ ID NO: 71 : AZB53142.1 SEQ ID NO: 72 : QIA45372.1 SEQ ID NO: 73 : AQN70349.1 SEQ ID NO: 74 : CAG7582570.1 SEQ ID NO: 75 : QIA46216.1 SEQ ID NO: 76 : AHJ82283.1 SEQ ID NO: 77 : AII80437.1 SEQ ID NO: 78 : AZB53140.1 SEQ ID NO: 79 : AHJ84133.1 SEQ ID NO: 80 : AAA45930.1 SEQ ID NO: 81 : AZB53158.1 SEQ ID NO: 82 : AKI17642.1 SEQ ID NO: 83 : AQN70182.1 SEQ ID NO: 84 : AFR55719.1 SEQ ID NO: 85 : AKI12960.1 SEQ ID NO: 86 : AFR54557.1 SEQ ID NO: 87 : AHB19702.1 SEQ ID NO: 88 : UNW44971.1 SEQ ID NO: 89 : ACT81737.1 SEQ ID NO: 90 : P06473.1 SEQ ID NO: 91 : AZB79941.1 SEQ ID NO: 92 : UBQ34153.1 SEQ ID NO: 93 : AZB53164.1 SEQ ID NO: 94 : ACS91991.1 SEQ ID NO: 95 : AKI14299.1 SEQ ID NO: 96 : AFR55216.1 SEQ ID NO: 97 : AHJ85985.1 SEQ ID NO: 98 : AKI22156.1 SEQ ID NO: 99 : AAA45934.2 SEQ ID NO: 100: AKI24535.1 SEQ ID NO: 101: AHJ83292.1 SEQ ID NO: 102: AKI24704.1 SEQ ID NO: 103: QZX45917.1 SEQ ID NO: 104: QPZ44673.1 SEQ ID NO: 105: AHJ84975.1 SEQ ID NO: 106: AKI12129.1 SEQ ID NO: 107: AKI22656.1 SEQ ID NO: 108: AII80435.1 SEQ ID NO: 109: ADB92600.1 SEQ ID NO: 110: AFR54884.1 SEQ ID NO: 111: ADE88063.1 SEQ ID NO: 112: AZB53145.1 SEQ ID NO: 113: AKI20990.1 SEQ ID NO: 114: AHJ86153.1 SEQ ID NO: 115: AZB53175.1 SEQ ID NO: 116: ADD39116.1 SEQ ID NO: 117: ADV04383.1 SEQ ID NO: 118: AKI09624.1 SEQ ID NO: 119: CAH0494218.1 SEQ ID NO: 120: AHJ82785.1 SEQ ID NO: 121: AZB53172.1 SEQ ID NO: 122: AQN71866.1 EXAMPLE 1 Engineering of pre-fusion stabilized gB constructs Human cytomegalovirus (hCMV) glycoprotein B (gB) transitions irreversibly from a metastable pre-fusion to a stable post-fusion conformation when fusing the viral envelope to the host cell membrane. Stabilizing the gB protein in its pre-fusion conformation could secure the generation of antibodies capable of blocking the binding of the gB protein to the host cell membrane. Similar strategies have been applied when developing vaccines against SARS-CoV-2, utilizing pre-fusion stabilized Spike protein as the antigenic component. Prior attempts to stabilize gB using mutations to create disulfide bonds that stabilize the protein in its pre-fusion format has also been investigated (patent WO 2021 / 260510 A1, and Sponholtz MR et al. 2024: https: / / doi.org / 10.1101 / 2024.02.10.579772). By inspecting and comparing the structures of wild type gB in pre- and post-fusion conformation (SEQ ID NO 1), potential sites for introducing a stabilizing disulfide bond were identified and evaluated. Three selected mutational pairs were selected to be introduced into gB: G543C-K617C (design construct pre_gB#1; SEQ ID NO 2), A97C-A538C (design construct pre_gB#2; SEQ ID NO 3), and I103C-V645C (design construct pre_gB#1; SEQ ID NO 4). These constructs also contains the mutations YIH(155-157)GHR and W240A which are known from literature to stabilize the construct, and the mutation C246S which removes a free cysteine to decrease aggregation and increase protein expression. In WO 2021 / 260510, the optimum construct was found to include the two mutations D217C- Y589C and M371C-W506C, in addition to the stabilizing mutations YIH(155-157)GHR, W240A and C246S in gB (V23-D646). A corresponding design was made, including a BIP secretion signal and a C-tag, produced in Drosophila melanogaster Schneider 2 cells and used as control. This construct is from here referred to as gB2796-Ctag. The introduction of a single mutation pair with D217C-Y589C has been shown to stabilize gB in a pre-fusion conformation, although this could only be maintained by a small molecule inhibitor WAY-174865 (WO 2021 / 260510 A1). Thus, for evaluation of double disulfide pairs, the three selected novel disulfide pairs described herein were evaluated in combination with D217C-Y589C, resulting in the following designs: G543C-K617C; D217C-Y589C (design construct pre_gB#4; SEQ ID NO 5), A97C-A538C; D217C-Y589C (design construct pre_gB#5; SEQ ID NO 6), and I103C-V645C; D217C-Y589C (design construct pre_gB#6; SEQ ID NO 7). For benchmarking, three different wild-type gB designs were produce, including wt_gB which is comparable in design to the commercially available gB protein, including a BIP secretion signal and a C-terminal C-tag (cat.: 10202-VCCH1; Sino Biologicals), the post_gB design in which the endo-domain was excluded (residues 23-702 of SEQ ID NO: 1 with a BIP secretion signal and a C-terminal C-tag), and the post_gB_tri in which a trimerization domain was added to the post_gB design (GCN4 CC tri2). Schematic overviews of all designs tested are provided in Figs. 2 and 3. Ribbon structures depicting the locations of the three mutational pairs in pre-fusion and post-fusion gB confirmations are shown in Figs. 4-6. EXAMPLE 2 Recombinant production of C-tagged gB protein and variants The genes encoding wildtype gB protein (post_gB and post_gB-tri; residues 23-702 of SEQ ID NO: 1 with a BIP secretion signal, a C-terminal C-tag and a trimerization domain) as well as recombinant C-tagged gB protein variants (SEQ ID NO: 1 with prior art modifications and SEQ ID NOs: 2-4 and 6-7, cf. Fig. 3 for a schematic overview of the designs) were procured as synthetic DNA fragments from Twist Biosciences or GeneArt and subsequently cloned into the proprietary expression vector pExpreS2-1 to yield transfection-ready DNA. pExpresS2-1 is available from Expres2ion Biotechnologies (Agern Allé 1, 2970 Hørsholm, Denmark) under commercial terms and from Addgene (490 Arsenal Way, Suite 100, Watertown, MA 02472, USA) under research terms as catalog # 175360.The DNA sequence of pExpresS2-1 is set forth in SEQ ID NO: 123. These plasmids were transiently expressed in Drosophila melanogaster Schneider 2 cells and the expression was analysed by western blot (Fig. 7) using an anti-C-tag antibody for detection. The expression by the transiently transfected cells of prior art protein, gB2796-Ctag from WO 2021 / 260510 with 4 cysteine substitutions (SEQ ID NO: 1, residues 23-646 with cysteine substitutions D217C, Y589C, M371C, and W506C, and with the further modifications: YIH(155-157)GHR, W240A, and C246S; and a C-terminal C-tag) is shown in lane 8 in Fig. 7. Under non-reducing conditions the western blot shows high molecular weight bands for gB2796-Ctag, which are not present for the wild type gB (lane 6), indicative of stable trimerization. Moreover, the western blot shows expression and trimerization of all the designed constructs SEQ ID NOs: 2-7 (lanes 9-11 and 13-14) in the transiently transfected cells. Stable polyclonal pools of the gB variants were established by transfecting Drosophila melanogaster S2 cells with the expression vectors and subjecting the cells to a three-week selection process utilizing the antibiotic selection marker zeocin. The cells were expanded, the proteins were secreted into the supernatant, and the harvest was carried out by centrifugation. Following this, ultra- and diafiltration using tangential flow filtration were performed to transfer the proteins into a buffered solution. The C-tagged proteins were loaded on an equilibrated CaptureSelect CtagXL column. The column was washed, and the protein was eluted using a 4-step gradient (12.5%, 25%, 50% and 100%) of an elution buffer containing 25 mM Tris, 150 mM NaCl, 2 M MgCl2pH 7.5. Elution fractions were analysed on SDS-PAGE and those containing the protein of interest were pooled, concentrated on a 10 kDa MWCO Amicon spin filter and polished by gel filtration on a Superdex20016 / 60 column. The purified proteins were analysed by SDS-PAGE and size exclusion HPLC. Fig. 8 shows overlays of chromatograms from size exclusion HPLC of the four gB variants that successfully expressed in the stable cell lines. The data shows a monodisperse profile of EVX-pre-gB#2 (SEQ ID NO: 3) and EVX-pre-gB#3 (SEQ ID NO: 4) at the size of the trimer. The main peaks of EVX-pre-gB#5 (SEQ ID NO: 6) and EVX-pre-gB#6 (SEQ ID NO: 7) are also at the size of the trimer, but both these profiles have a “shoulder” indicating a heterogeneous solution where a part of the protein have a higher molecular weight. By comparing the size exclusion HPLC profile of the EVX-pre-gB#2 (SEQ ID NO: 3) and EVX-pre-gB#3 (SEQ ID NO: 4) with the WT-gB (Fig. 9), it is observed that both gB variants exhibit a sharper and more symmetrical peak than does the WT-gB indicating a more homogeneous solution than the WT-gB. EXAMPLE 3 Transmission electron microscopy analysis Transmission electron microscopy (TEM) analysis was performed applying a droplet of protein sample onto carbon-coated mica sheets. Excess liquid was gently blotted from the grid using filter paper. The grids were immediately transferred to a 2% (w / v) phosphotungstic acid solution for negative staining. To enhance grid adhesion, the grids were exposed to a piece of Sellotape placed in chloroform. The stained grids were immediately examined using a Philips CM100 Transmission Electron Microscope. Results In line with the observations from the western blot in Fig. 7, TEM analysis of the wt_gB protein sample showed a heterogeneous sample, with most proteins being presented as extended “rods” containing three distinct domains (approximate size of 20.3-23.03 nm) corresponding well to the expected post-fusion structure of gB (Fig. 10A). A few longer proteins (approximate size of 27.32-39.12 nm) were also seen and are believed to represent two gB molecules. A few shorter and more round protein structures were also seen (15.85- 17.49 nm), these most likely represent a minor population of pre-fusion structures or just reflect the post-fusion conformation viewed from a different angle. In contrast, the pre_gB#3 sample exhibited a very uniform picture consisting of small triangular-shaped protein structures (approximate size of 7.5-9.21 nm), indicative of a prefusion structure of gB (Fig. 10B). A similar result was observed in preliminary cryogenic electron microscopy of samples, see Fig. 11. EXAMPLE 4 Immunogenicity of gB variants The immunogenicity of the mutated gB proteins disclosed in the current application was evaluated and compared to wild-type gB protein in mice. Three different wild-type gB protein constructs were evaluated; wt_gB (Sino-gB comparator), post_gB (ectordomain of gB) and post_gB_tri (ectordomain of gB with trimerization domain). The prior art gB protein, gB2796, was included in the study as comparator. The presently disclosed mutation variants tested were pre_gB#2, pre_gB#3, pre_gB#5 and pre_gB#6. See Figs. 2 and 3 for a detailed overview of the designs. BALB / c mice were immunized with a total dose of 10 μg protein / 100 μL in TRIS buffer formulated 1:1 with AddaVax (InvivoGen). TRIS buffer formulated 1:1 with AddaVax without protein was used as placebo. The mice received three intramuscular (i.m.) injections each of 50 μL in the tibialis anterior muscles of each hindleg at two-week intervals. Nine (9) groups (group #1-9) of mice were assigned to the following immunizations: 1. AddaVax (placebo); 2. wt_gB; 3. post_gB; 4. post_gB_tri; 5. gB2796; 6. pre_gB#2, 7. pre_gB#3; 8. pre_gB#5; 9. pre_gB#6. Sera were isolated from tail vein blood collected at study day 26 and from retro-orbital blood collected at study termination two weeks after the last immunization. ELISA was used to determine the level of antigen-specific IgG. In short, ELISA, MaxiSorp microtiter plates (Thermo scientific) were coated with the specific gB variant used for each immunization group. Cross-reactions were evaluated by coating the ELISA plates with either wt_gB or pre_gB#3. gB-specific total IgG was detected by HRP-conjugated polyclonal rabbit anti-mouse IgG (Sigma-Aldrich) using TMB EASY (Kementec) for development. Antibody binding was analyzed using the number of absorbance values (OD450 nm minus OD620 nm). Specifically, endpoint titers were calculated as the highest serum dilution at which the OD values were above the cutoff determined by sera from placebo immunized mice. The ability of sera from vaccinated animals to neutralize virus infection were evaluated using a live-virus neutralization assay. The assay relies on the cytopathogenic effect (CPE) readout evaluated with a fluorescence microscope, in which the survival of virus-infected cells is determined in the presence of immune serum at different concentrations. In brief, a dilution series of immune sera was incubated with the hCMV Merlin strain in equal volumes for one hour. Following this initial incubation period, the serum-virus mixture was used to infect a confluent monolayer of MRC-5 cells in a 96-well plate. Plates were incubated at 37°C with 5% CO2 for eight days. MRC-5 cell death as a result of infection with hCMV was visualized with Sytox viability dye and analyzed using florescence microscopy and automated counting of stained cells. Results No difference in titers could be seen between the wt_gB (Sino-gB), the post_gB and the post_gB_tri, indicating that excluding the endo-domain and adding a trimerization domain does not impact immunogenicity (Fig. 12). Immunogenicity analysis of the novel variants of hCMV gB with in-substituted cysteine pairs induced IgG levels comparable or higher (pre_gB#2) to the wild-type gB protein as well as to the prior art gB, gB2796 (Fig. 13A). Immunization with pre_gB#3 induced antibodies with higher neutralizing capacity against live human CMV virus compared to wt_gB and gB2796 (Fig. 13B). No difference in antibody titers could be observed between pre_gB#2 and pre_gB#5, and pre_gB#3 and pre_gB#6. In line with the observations for expression (Fig. 7), this indicates that the identified mutations ALA97C-ALA538C and ILE103C-VAL645C are alone sufficient for expression, trimeric stabilization and induction of high level of functional antibodies (Fig. 14). When evaluating the cross-construct IgG response across the immunization groups it was noted that all immunized mice raised comparable levels of IgG recognizing the wt_gB (Fig. 15A). On the contrary, when analyzing the response across groups to the pre_gB#3 protein, both pre_gB#2 and especially pre_gB#3 gave higher antibody levels compared to the other groups tested. Differences in antibody response against pre_gB#3 across the different vaccination groups confirmed that the conformations of pre_gB#2 and pre_gB#3 were different than protein found in post-fusion confirmation (wt_gB, post_gB, post_gB-tri) and the gB2796 protein. Thus the conformations of pre_gB#2 and pre_gB#3 likely expose other epitopes that could explain the increased IgG titer and protection shown for pre_gB#2 and pre_gB#3, respectively (Fig. 15B). EXAMPLE 5 Dose response study The dose-dependent antibody response to the pre-fusion stabilized pre_gB#3 disclosed herein was evaluated by immunogenicity testing in mice. BALB / c mice were immunized with a total dose of 1-10 μg protein / 100 μL in TRIS buffer formulated 1:1 with AddaVax (InvivoGen). TRIS buffer formulated 1:1 with AddaVax without protein was used as placebo. The mice received three intramuscular (i.m.) injections of 50 μL in the tibialis anterior muscles of each hind leg at two-week intervals. Six (6) groups (group #1-6) of mice were assigned to the following immunizations: 1. AddaVax (placebo) 2. pre_gB#3; 10 μg / dose 3. pre_gB#3; 7.5 μg / dose 4. pre_gB#3; 5 μg / dose 5. pre_gB#3; 2.5 μg / dose 6. pre_gB#3; 1 μg / dose Sera were isolated from tail vein blood collected at study day 13, study day 26 and from retro-orbital blood collected at study termination two weeks after the last immunization. ELISA was used to determine the level of pre_gB#3-specific IgG as described in Example 3. Results Immunogenicity analysis of the novel variants of hCMV gB with in-substituted cysteine pairs revealed that I103C-V645C induced high levels of IgG even at 1 μg doses (Fig. 16). The IgG levels in response to one and two doses of 1 μg pre_gB#3 were comparable to using 10 μg / dose, while a higher boosting effect of the antibody response was seen with the third dose of 1 μg compared to mice receiving three doss of 10 µg (Fig. 17). This highlights the immunological potency of this novel variant of gB.

Claims

CLAIMS 1. A variant of human cytomegalovirus glycoprotein B (gB) comprising 1, 2, or 3 double substitutions, each of said double substitutions corresponding to double substitutions in SEQ ID NO: 1 selected from the group consisting of 1) I103C and V645C,; 2) A97C and A538C; and 3) G543C and K617C, wherein said variant comprises a contiguous stretch of amino acids, which, when optimally aligned with residues 97-645 in SEQ ID NO: 1, comprises at least 470 identical amino acid residues.

2. The variant according to claim 1, which lacks one or both amino acid residue stretches corresponding to the amino acid sequences defined by residues 1-22 and 703-907 of SEQ ID NO:

1.

3. The variant according to claim 1 or 2, which comprises only one of the 3 double substitutions defined in options 1-3.

4. The variant according to any one of the preceding claims, which comprises at least one further double substitution corresponding to a double substitution in SEQ ID NO: 1 selected from the group consisting of a) D217C and Y589C; and b) M371C and W506C.

5. The variant according to any one of the preceding claims, which further comprises a substitution corresponding to YIH(155-157)GHR in SEQ ID NO:

1.

6. The variant according to any one of the preceding claims, which further comprises a substitution corresponding to C246S in SEQ ID NO:

1.

7. The variant according to any one of the preceding claims, which further comprises i) a trimerization domain, which is preferably located in the C-terminal part of the variant, or ii) an affinity tag, or iii) a trimerization domain as well as an affinity tag, which optionally are separated by a linker sequence.

8. The variant according to claim 7, wherein the trimerization domain comprises or consists of residues 681-704 in any one of SEQ iD NOs: 2-7.

9. The variant according to claim 7 or 8, wherein the affinity tag comprises or consists of residues 709-712 in any one of SEQ ID Nos: 2-7.

10. The variant according to any one of the preceding claims, which, when optimally aligned, includes at most 15 non-identical amino acid residues compared to the part of the amino acid sequence corresponding to residues 97-645 in SEQ ID NO: 1, where said part is found in the amino acid sequences with the UniProtKB accession numbers P13201.1, ABQ23592.1, UNW45129.1, AND81495.1, ACM48044.1, ACS32370.1, AKI25887.1, AFR55550.1, QPI35314.1, AKI19983.1, AFR55048.1, AKI19648.1, APG57425.1, AZB53165.1, YP_081514.1, APG57594.1, AAA45928.1, AQN72202.1, AMJ53258.1, QIA46047.1, AKI19483.1, AKI25042.1, AHJ85145.1, AMJ52758.1, QTT58728.1, AHB20033.1, AQN69676.1, QTT59229.1, ALL26209.1, AKI13294.1, AKI13965.1, AKI23324.1, AQN70515.1, QBK84312.1, ACS93398.1, AQN69508.1, AKI20319.1, APA45201.1, AHJ82619.1, AZB53139.1, AKI08783.1, ABV71586.1, QIA45035.1, AQN73719.1, AGL96655.1, AAB07485.1, QBF76487.1, AZV24343.1, AMJ54594.1, AKI24367.1, AHJ83628.1, AKI22824.1, AFR55885.1, AKI23491.1, AAA45925.1, ACS92156.1, AMJ54426.1, AQN72370.1, AAA45926.1, WHN53998.1, AZB53148.1, AKI09288.1, AAA45923.2, AHV84013.1, AZB53142.1, QIA45372.1, AQN70349.1, CAG7582570.1, QIA46216.1, AHJ82283.1, AII80437.1, AZB53140.1, AHJ84133.1, AAA45930.1, AZB53158.1, AKI17642.1, AQN70182.1, AFR55719.1, AKI12960.1, AFR54557.1, AHB19702.1, UNW44971.1, ACT81737.1, P06473.1, AZB79941.1, UBQ34153.1, AZB53164.1, ACS91991.1, AKI14299.1, AFR55216.1, AHJ85985.1, AKI22156.1, AAA45934.2, AKI24535.1, AHJ83292.1, AKI24704.1, QZX45917.1, QPZ44673.1, AHJ84975.1, AKI12129.1, AKI22656.1, AII80435.1, ADB92600.1, AFR54884.1, ADE88063.1, AZB53145.1, AKI20990.1, AHJ86153.1, AZB53175.1, ADD39116.1, ADV04383.1, AKI09624.1, CAH0494218.1, AHJ82785.1, AZB53172.1, and AQN71866.

1.

11. The variant according to claim 1, which comprises residues 1-680 of SEQ ID NO:

2.

12. The variant according to claim 1, which comprises residues 1-680 of SEQ ID NO:

3.

13. The variant according to claim 1, which comprises residues 1-680 of SEQ ID NO:

4.

14. The variant according to claim 1, which comprises SEQ ID NO:

2.

15. The variant according to claim 1, which comprises SEQ ID NO:

3.

16. The variant according to claim 1, which comprises SEQ ID NO: 4.

17. The variant according to claim 1, which consists of SEQ ID NO: 2 or residues 1-680 of SEQ ID NO:

2.

18. The variant according to claim 1, which consists of SEQ ID NO: 3 or residues 1-680 of SEQ ID NO:

3.

19. The variant according to claim 1, which consists of SEQ ID NO: 4 or residues 1-680 of SEQ ID NO:

4.

20. The variant according to claim 1, which comprises residues 1-680 of SEQ ID NO:

5.

21. The variant according to claim 1, which comprises residues 1-680 of SEQ ID NO:

6.

22. The variant according to claim 1, which comprises residues 1-680 of SEQ ID NO:

7.

23. The variant according to claim 1, which comprises SEQ ID NO:

5.

24. The variant according to claim 1, which comprises SEQ ID NO:

6.

25. The variant according to claim 1, which comprises SEQ ID NO:

7.

26. The variant according to claim 1, which consists of SEQ ID NO: 5 or residues 1-680 of SEQ ID NO:

5.

27. The variant according to claim 1, which consists of SEQ ID NO: 6 or residues 1-680 of SEQ ID NO:

6.

28. The variant according to claim 1, which consists of SEQ ID NO: 7 or residues 1-680 of SEQ ID NO:

7.

29. A nucleic acid molecule comprising a nucleotide sequence encoding the variant of hCMV gB according to any one of the preceding claims.

30. The nucleic acid molecule according to claim 29, which is a DNA fragment or an RNA fragment.

31. A vector comprising the nucleic acid molecule according to claim 29 or 30, such as a cloning vector or an expression vector.

32. The vector according to claim 31, which comprises in operable linkage and in the 5'-3' direction, an expression control region comprising an enhancer / promoter for driving expression of the nucleic acid molecule defined in claim 29, optionally a signal peptide coding sequence, a nucleotide sequence defined in claim 29, and optionally a terminator.

33. The vector according to claim 31 or 32, wherein the expression control region drives expression in prokaryotic cell such as a bacterium, e.g. in E coli.

34. The vector according to claim 31 or 32, wherein the expression control region drives expression in a eukaryotic cell, such as in a mammalian cell, in an insect cell, and in a plant cell.

35. The vector according to claim 34, wherein, when being DNA, encodes a polyadenylation signal, and wherein, when being RNA, comprises a polyA tail.

36. The vector according to claim any one of claims 31-35, which is capable of autonomous replication.

37. The vector according to any one of claims 31-36, which is capable of being integrated into the genome of a host cell.

38. The vector according to any one of claims 31-36, which is incapable of being integrated into the genome of a mammalian host cell.

39. The vector according to any one of claims 31-38, which is selected from the group consisting of a virus, such as an attenuated virus, a bacteriophage, a plasmid, a minichromosome, and a cosmid.

40. A genetically modified cell, which carries the nucleic acid molecule according to any one of claims 29-30 or the vector according to any one of claims 31-39.

41. The genetically modified cell according to claim 40, which is capable of replicating the nucleotide sequence defined in claim 29.

42. The genetically modified cell according to claim or 40 or 41, which is capable of expressing the nucleotide sequence defined in claim 29.

43. The genetically modified cell according to any one of claims 40-42, which is selected from a prokaryotic cell and a eukaryotic cell.

44. The genetically modified cell according to any one of claims 40-43, which is a bacterial cell, which is preferably non-pathogenic, selected from the group consisting of Escherichia (such as E. coli), Bacillus (e.g. Bacillus subtilis), Salmonella, and Mycobacterium, e.g. M. bovis BCG, or which is a eukaryotic cell such as a mammalian cell, an insect cell or a plant cell.

45. The genetically modified cell according to any one of claims 40-44, which is stably genetically modified by having the nucleotide sequence defined in claim 29 stably integrated into its genome.

46. The genetically modified cell according to any one of claims 40-45, which secretes or carries on its surface the variant according to any one of claims 1-28.

47. The genetically modified cell according to claim 46, wherein the cell is a bacterium and secretion is into the periplasmic space.

48. A cell line derived from a genetically modified cell according to any one of claims 40- 47.

49. A composition comprising a variant gB polypeptide according to any one of claims 1- 28 and an immunological adjuvant.

50. The composition according to claim 49, which further comprises at least one further hCMV immunogen.

51. The composition according to claim 49 or 50, which also comprises a pharmaceutically acceptable excipient, diluent, carrier, or vehicle.

52. The composition according to any one of claims 49-51, wherein the variant is formulated in or forms part of a virus-like particle (VLP).

53. A composition comprising the nucleic acid molecule according to claim 29 or 30 or a vector according to any one of claims 31-39 in admixture with a pharmaceutically acceptable excipient, diluent, carrier, or vehicle, and optionally further comprising a nucleic acid fragment or vector, which encodes at least one further hCMV immunogen.

54. A composition comprising the genetically modified cell according to any one of claims 40-47 in admixture with a pharmaceutically acceptable excipient, diluent, carrier, or vehicle.

55. A method for inducing immunity in a human subject against infections caused by hCMV, the method comprising administering to the subject an effective amount of a variant of the gB polypeptide according to any one of claims 1-28, the nucleic acid molecule according to claim 29 or 30, the vector according to any one of claims 31-39, the genetically modified cell according to any one of claims 40-47, or the composition according to any one of claims 49-54.

56. The method according to claim 55, wherein a priming dosage is followed by one or more booster doses with an effective amount of a variant of the gB polypeptide according to any one of claims 1-28, the nucleic acid molecule according to claim 29 or 30, the vector according to any one of claims 31-39, the genetically modified cell according to any one of claims 40-47, or the composition according to any one of claims 49-54.

57. The method according to claim 55 or 56, wherein the subject has ongoing infection with hCMV.

58. The method according to claim 55 or 56, wherein the subject has no ongoing infection with hCMV, and wherein the subject preferably has had no previous or recent infection with hCMV as evidenced by being seronegative for anti-CMV antibodies.

59. The method according to any one of claims 55-58, which reduces symptoms or prevents emergence of symptoms of disease caused by hCMV infection.

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