Immunogenic variants of human cytomegalovirus glycoprotein B

Abstract

Reagents useful for immunotherapy and prevention of human cytomegalovirus (hCMV) infection are provided. The reagents are variant gB polypeptides (or genetic material encoding said polypeptides) wherein disulfide bridges are introduced into the amino acid sequence of gB to stabilize the pre-fusion conformation of said protein. The disulfide bridges are introduced via double substitution as shown 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. Vaccine compositions and methods of using them are also disclosed.

Description

Technical Field

[0001] This invention relates to immunotherapy and prevention, particularly in the field of prophylactic immunization against human cytomegalovirus (hCMV) infection. Background Technology

[0002] Human cytomegalovirus (HCMV) glycoprotein B (gB) irreversibly transitions from a metastable pre-fusion conformation to a stable post-fusion conformation to promote the fusion of the viral membrane with the human host cell membrane, thereby enabling the virus to enter the host cell and initiate infection.

[0003] Existing strategies for designing gB protein variants capable of inducing potent antibodies that block the binding of the gB protein to the human host cell membrane involve stabilizing 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 proteins have been used as antigenic components in available, regulatory-approved vaccines.

[0004] For the gB protein, the strategy has been implemented, as disclosed in WO 2021 / 260510, which involves several mutations in the gB amino acid sequence to create disulfide bridges that stabilize the protein in its pre-fusion form.

[0005] Invention Objective

[0006] The objective of embodiments of the present invention is to provide new and effective vaccine reagents that induce protective immunity against hCMV infection. Invention Overview

[0008] The inventors discovered that a novel variant of hCMV gB with in-situ cysteine ​​pairs retains the pre-fusion conformation of hCMV gB after recombinant expression; they also found that the induced protective immune response is superior when compared with those induced by prior art gB variant proteins disclosed in WO 2021 / 260510.

[0009] Therefore, in a first aspect, the present invention relates to variants of human cytomegalovirus glycoprotein B (gB) comprising one, two, or three double permutations, each of which corresponds to a double permutation selected from the group consisting of the following in SEQ ID NO: 1:

[0010] 1) I103C and V645C;

[0011] 2) A97C and A538C; and

[0012] 3) G543C and K617C,

[0013] The variant comprises a continuous amino acid chain segment that, when optimally compared with residues 97-645 in SEQ ID NO:1, contains at least 470 identical amino acid residues.

[0014] In a second aspect, the present invention relates to nucleic acid molecules that encode variants of human gB of the first aspect of the invention (and any embodiments thereof disclosed herein in the context of the first aspect of the invention).

[0015] In a third aspect, the present invention relates to a vector comprising the nucleic acid molecule of the second aspect of the invention (and any embodiments thereof disclosed herein within the context of the second aspect of the invention), said vector being, for example, a cloning vector or an expression vector.

[0016] In a fourth aspect, the present invention relates to genetically modified cells carrying the nucleic acid molecule of the second aspect of the invention (and any embodiment thereof disclosed herein in the context of the second aspect of the invention) or carrying the vector of the third aspect of the invention (and any embodiment thereof disclosed herein in the context of the third aspect of the invention). A portion of this aspect is a cell line derived from said genetically modified cells.

[0017] In a fifth aspect, the present invention relates to compositions comprising a variant of the gB polypeptide of the first aspect of the invention (and any embodiments thereof disclosed herein within the context of the first aspect of the invention) and an immune adjuvant.

[0018] In a sixth aspect, the present invention relates to compositions comprising a nucleic acid molecule of the second aspect of the invention (and any embodiment thereof disclosed herein in the context of the second aspect of the invention) or a carrier of the third aspect of the invention (and any embodiment thereof disclosed herein in the context of the third aspect of the invention), and pharmaceutically acceptable excipients, diluents, carriers or loadings mixed therewith, and optionally further comprising a nucleic acid fragment or carrier encoding at least one further hCMV immunogen.

[0019] In a seventh aspect, the present invention relates to compositions comprising genetically modified cells of the fourth aspect of the invention (and any embodiments thereof disclosed herein within the context of the fourth aspect of the invention), and pharmaceutically acceptable excipients, diluents, carriers, or loadings mixed therewith.

[0020] Finally, in an eighth aspect, the present invention relates to a method for inducing immunity against an infection caused by hCMV in a human subject, the method comprising administering to the subject an effective amount of a variant of the gB polypeptide of the first aspect of the invention (and any embodiment thereof disclosed herein in the context of the first aspect of the invention), a nucleic acid molecule of the second aspect of the invention (and any embodiment thereof disclosed herein in the context of the second aspect of the invention), a vector of the third aspect of the invention (and any embodiment thereof disclosed herein in the context of the third aspect of the invention), a genetically modified cell of the fourth aspect of the invention (and any embodiment thereof disclosed herein in the context of the fourth aspect of the invention), or a composition of the fifth to seventh aspects of the invention (and any embodiment thereof disclosed herein in the context of the fifth to seventh aspects of the invention).

[0021] Legend of the attached figure

[0022] Figure 1 Alignment of SEQ ID NO: 1 (UniProtKB accession number: P13201.1) with other full-length hCMV gB amino acid sequences (SEQ ID NO: 8-122).

[0023] exist Figure 1 The residues emphasized here are those that have been substituted and disclosed herein.

[0024] Figure 2 : A schematic explanation of the symbols used in the vaccine design shown in Figure 3.

[0025] Figure 3: Schematic illustration of different gB designs.

[0026] A: gB control design, which shows the equivalent wt-gB design to Sino-gB (catalog number 10202-VCCH1; Sino Biologicals), which consists of both the extracellular and intracellular domains of gB, post_gB, which consists of the extracellular domain of gB, and post_gB_tri, which contains the extracellular domain of gB and the trimerized domain.

[0027] B: An equivalent of the previously described pre-fusion gB design (WO2021 / 260510) with the pre-fusion stabilized cysteine ​​mutation indicated in parentheses, referred to herein as gB2796-Ctag.

[0028] C: The novel pre-fusion gB design described in this paper has a pre-fusion stabilizing cysteine ​​mutation as indicated in parentheses.

[0029] Figure 4: Pre-fusion (A) and post-fusion (B) structures of hCMV gB. Structures were obtained from the protein database (HMBerman, J. Westbrook, Z. Feng, G. Gilliland, TN Bhat, H. Weissig, INShindyalov, PE Bourne (2000) Nucleic Acids Research, 28: 235-242. https: / / doi.org / 10.1093 / nar / 28.1.235), PDB IDS: 7KDP and 7KDD. The position of the pre-fusion stabilizing disulfide bond mutation (G543C-L617C) is shown as a black sphere in both conformations (highlighted with arrows). PyMOL was used for mapping (Schrödinger, L. & DeLano, W. (2020). PyMOL. Retrieved from http: / / www.pymol.org / pymol).

[0030] Figure 5: Pre-fusion (A) and post-fusion (B) structures of hCMV gB. Structures were obtained from the protein database (HMBerman, J. Westbrook, Z. Feng, G. Gilliland, TN Bhat, H. Weissig, INShindyalov, PE 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 mutation (A97C-A538C) is shown as a black sphere in both conformations (highlighted with arrows). PyMOL was used for plotting.

[0031] Figure 6: Pre-fusion (A) and post-fusion (B) structures of hCMV gB. Structures were obtained from the protein database (Berman, J. Westbrook, Z. Feng, G. Gilliland, TN Bhat, H. Weissig, IN Shindyalov, PE 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 mutation (I103C-V645C) is shown as a black sphere in both conformations (highlighted with arrows). PyMOL was used for plotting.

[0032] Figure 7 Western blots from transiently transfected S2 cells under reducing (+) and non-reducing (-) conditions.

[0033] Lane M: Molecular weight standard reference.

[0034] Lane 6: Control; Wild-type gB protein with a C-terminal C-tag (SEQ ID NO: 1, residues 23-702).

[0035] Lane 7: Control; Wild-type gB protein with C-terminal trimerized domain and C-tag (SEQ ID NO: 1, residues 23-702).

[0036] Lane 8: Comparative; prior art mutant 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 C-terminal C-tag.

[0037] Lane 9: Mutated gB protein (SEQ ID NO: 2).

[0038] Lane 10: Mutated gB protein (SEQ ID NO: 3).

[0039] Lane 11: Mutated gB protein (SEQ ID NO: 4).

[0040] Lane 13: Mutated gB protein (SEQ ID NO: 6).

[0041] Lane 14: Mutated gB protein (SEQ ID NO: 7).

[0042] Figure 8 Size exclusion HPLC coverage plots of EVX-pre_gB#2, EVX-pre_gB#3, EVX-pre_gB#5 and EVX-pre_gB#6.

[0043] Superdex 200 columns were used, and spherical SEC standard references were used with the following sizes: 670 kDa, 158 kDa, 44 kDa, 17 kDa, and 1.3 kDa (Catalogue No.: 151-1901, Biorad). Monodisperse SEC plots of pre_gB#2 and pre_gB3 confirmed the trimer form.

[0044] Figure 9: Size exclusion HPLC coverage elution chromatogram.

[0045] A: EVX-pre_gB#3 and WT-gB

[0046] B: EVX-pre_gB#2 and WT-gB.

[0047] Superdex 200 columns were used, and the spherical SEC standard references were available in the following sizes: 670 kDa, 158 kDa, 44 kDa, 17 kDa, and 1.3 kDa. (Catalogue No.: 151-1901, Biorad).

[0048] Figure 10: Transmission electron microscopy (TEM) of wt_gB (A) and pre_gB#3 (B) confirms the ability of the introduced mutation to stabilize gB in its pre-fusion conformation. Protein samples were stained with 2% (w / v) phosphotungstic acid and observed by electron microscopy. Scale bars (100 nm) are shown in the larger figures, with scaling shown in the smaller figures.

[0049] Figure 11 Cryo-electron microscopy confirmed the pre-fusion stabilization of gB.

[0050] Use Titan Krios to capture photos.

[0051] Figure 12 The bar chart shows the IgG response to wt_gB, post_gB, and post_gB_tri.

[0052] The dashed line represents the detection limit measured by the response to wt_gB in mice immunized with placebo (AddaVax). Removal of the intracellular domain or addition of the trimerizing domain does not affect the anti-gB antibody response.

[0053] Figure 13: Bar chart showing the immune response.

[0054] A: Regarding the IgG responses of wt_gB, gB2796-Ctag, pre_gB#2 and pre_gB#3, pre_gB#2 induced a significantly higher antibody response than wt_gB.

[0055] B: Serum was diluted 1:3200, and its ability to neutralize viral infection in MRC5 cells was measured, demonstrating that serum from animals immunized with gB_pre#3 provided a high level of protection against infection in vitro. The dashed line represents the limit of detection by response in mice immunized with placebo (AddaVax).

[0056] Figure 14 The bar chart shows the IgG response to pre_gB#2, pre_gB#3, pre_gB#5, and pre_gB#6.

[0057] The results showed that single mutation pairs in pre_gB#2 and pre_gB#3 and double mutation pairs in pre_gB#5 and pre_gB#6 both provided high levels of antibody.

[0058] The dashed line represents the detection limit measured by wt_gB response in mice immunized with placebo (AddaVax).

[0059] Figure 15: Bar chart showing the IgG response in animals vaccinated with wt_gB, post_gB, post_gB_tri, gB2796-Ctag, pre_gB#2, or pre_gB#3.

[0060] A: Response to wt_gB

[0061] B: Response to pre_gB#3.

[0062] The dashed line represents the detection limit measured by response in mice immunized with a placebo (AddaVax).

[0063] Figure 16 The bar chart shows the dose-dependent IgG response to pre_gB#3 in animals vaccinated with 10 μg, 5 μg, or 1 μg of pre_gB#3. Even at low doses, the titers remain high. The dashed line represents the detection limit as measured by response in mice immunized with a placebo (AddaVax).

[0064] Figure 17The line graph shows the dose-dependent IgG response to pre_gB#3 in animals vaccinated with 10 μg or 1 μg of pre_gB#3, measured after one dose (day 13), two doses (day 26), and three doses (day 42). The dashed line represents the response in mice immunized with placebo (AddaVax).

[0065] Detailed disclosure of the invention

[0066] definition

[0067] In the context of this application, the term "...corresponding to a substitution from the group consisting of substitution pairs selected from G543C and K617C; A97C and A538C; and I103C and V645C..." means a substitution in the gB amino acid sequence (typically selected from SEQ ID NO: 8-122) that best aligns with SEQ ID NO: 1, and wherein each substitution in the gB amino acid sequence different from SEQ ID NO: 1 is performed at a position aligned with the specified amino acid position in SEQ ID NO: 1. As from Figure 1 As can be seen, G543 of SEQ ID NO: 1 aligns with G541 in the gB protein with the amino acid sequence AMJ54594.1 and G545 in the gB protein with the amino acid sequence AZB53140.1. In this scenario, after performing a BLAST search starting from SEQ ID NO: 1, alignment is performed with proteins that have at least 93% sequence identity with SEQ ID NO: 1, where the algorithm parameters are:

[0068] Expected threshold: 0.05; word length: 5; matrix: BLOSUM62; gap penalty: 11 for existence, 1 for extension; end gap penalty: -5 for existence, -1 for extension.

[0069] In the context of this application, the term "peptide" refers to short peptides having 2-10 amino acid residues, oligopeptides having 11-100 amino acid residues, and polypeptides having more than 100 amino acid residues. Further, the term is also intended to include proteins, i.e., functional biomolecules containing at least one polypeptide; when containing at least two polypeptides, these can form a complex, covalently linked or non-covalently linked. The polypeptides in a protein can be glycosylated and / or lipotropic and / or contain a prosthetic group.

[0070] The term "subsequence" refers to any continuous chain segment having at least 3 amino acids or (when relevant) at least 3 nucleotides that is directly derived from a naturally occurring amino acid sequence or nucleic acid sequence.

[0071] The term "amino acid sequence" refers to the order in which amino acid residues linked by peptide bonds are located in the chain of a peptide or protein, from the free N-terminus to the free C-terminus.

[0072] An "immunogen" is a substance that can induce an adaptive immune response in a host whose immune system is exposed to it. Thus, an immunogen is a subset of a larger class of "antigens," which are substances that can be specifically recognized by the immune system (e.g., when bound by antibodies, or alternatively, when fragments of an antigen bound to an MHC molecule are recognized by T-cell receptors) but not necessarily induce immunity—however, an antigen can always elicit immunity, meaning that a host with established memory immunity against that antigen will launch a specific immune response against it.

[0073] "Adaptive immune response" is an immune response that responds to an antigen or immunogen, wherein the immune response is specific to the antigenic determinant of the antigen / immunogen. Examples of adaptive immune responses are the induction of antigen-specific antibody production or the antigen-specific induction / activation of T helper lymphocytes or cytotoxic lymphocytes.

[0074] A “protective adaptive immune response” is an antigen-specific immune response induced in a subject as a reaction to immunization with an antigen (artificial or natural), wherein the immune response protects the subject from subsequent attack with the antigen or a pathologically relevant agent including the antigen. Typically, prophylactic vaccination is intended to establish a protective adaptive immune response against one or more pathogens.

[0075] "Immune system stimulation" means that a substance or combination of substances exhibits a general, non-specific immunostimulatory effect. Many adjuvants and putative adjuvants (such as certain cytokines) share the ability to stimulate the immune system. The result of using immunostimulatory agents is increased "alertness" of the immune system, which means that simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to using the immunogen alone.

[0076] 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 refers to certain biological vectors useful for the same purpose, such as viral vectors and bacteriophages—both of which are infectious agents capable of introducing heterologous nucleic acid sequences.

[0077] The term "expression vector" refers to a vector containing at least a portion of a nucleic acid sequence that encodes a gene product that can be transcribed. In some cases, when the transcription product is an mRNA molecule, this is translated into a protein, polypeptide, or peptide.

[0078] A linker is an amino acid sequence that is introduced between two other amino acid sequences to spatially separate them. A linker can be "rigid," meaning it substantially does not allow the two amino acid sequences it links to to move freely relative to each other. Similarly, a "flexible" linker allows the two sequences linked via the linker to move substantially freely relative to each other. Both types of linkers are useful in the fusion proteins that are part of this invention, but flexible linkers are preferred.

[0079] An "immunogenic carrier" is a molecule or portion to which an immunogen or hapten can be conjugated to enhance or enable the induction of an immune response against said immunogen / hapten. In the classic case, immunogenic carriers are relatively large molecules (e.g., tetanus toxoid, KLH, diphtheria toxoid, etc.) that can be fused to or conjugated to immunogens / haptens that themselves do not possess sufficient immunogenicity. Typically, said immunogenic carriers can induce a strong T helper lymphocyte response against the combination of said immunogen and said immunogenic carrier, and this in turn provides an improved response against said immunogen via B lymphocytes and cytotoxic lymphocytes. Recently, large carrier molecules have been partially replaced by so-called non-species-selective T helper cell epitopes, i.e., shorter peptides that are recognized by most HLA haplotypes in the population and induce a T helper lymphocyte response.

[0080] The term "immune adjuvant" has its usual meaning in the field of vaccine technology, referring to a substance or composition of substances that 1) cannot, on its own, initiate a specific immune response against the immunogen of the vaccine, but 2) can still enhance the immune response against the immunogen. In other words, immunization with an adjuvant alone does not provide an immune response against the immunogen, immunization with the immunogen may or may not elicit an immune response against the immunogen, but combined vaccination with an immunogen and an adjuvant induces an immune response against the immunogen that is stronger than that induced by the immunogen alone.

[0081] Specific implementation scheme of the invention

[0082] Full-length gB proteins exist as many naturally occurring isotype variants from different strains of human cytomegalovirus. The gB variants disclosed in this application may, for example, be variants of any of the following proteins identified via their UniProtKB accession numbers:

[0083] P13201.1 (SEQ ID NO: 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, AHJ8514 5.1, AMJ52758.1, QTT58728.1, AHB20033.1, AQN69676.1, QTT59229.1, ALL26209.1, AKI13294.1, AKI13965.1, AKI23324.1, AQN70515.1, QBK8431 2.1, 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, AFR557 19.1, 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, AKI2470 4.1, QZX45917.1, QPZ44673.1, AHJ84975.1, AKI12129.1, AKI22656.1, AII80435.1, ADB92 600.1, 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.

[0084] It is noteworthy that the residues replaced by Cys in SEQ ID NO: 1 are 100% conserved in all these gB proteins, as shown in Figure 1 This is demonstrated in the comparison shown.

[0085] The first aspect of the present invention and its embodiments

[0086] As described above, a first aspect of the invention relates to variants of human cytomegalovirus glycoprotein B (gB) comprising one, two, or three double substitutions, each of which corresponds to a double substitution 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 the variant comprises a continuous amino acid chain containing at least 470 identical amino acid residues when optimally compared with residues 97-645 in SEQ ID NO: 1. Preferred double substitutions are selected from: 1) I103C and V645C, and 2) A97C and A538C, wherein double substitutions I103C and V645C are particularly preferred.

[0087] Such variants of the present invention exhibit a stable pre-fusion conformation, and genetically modified cells (particularly S2 cells) can provide the variants at high expression levels.

[0088] The at least 470 identical amino acid residues can be any higher number that allows for the introduction of the double substitutions discussed above, i.e., up to a maximum of 547 identical amino acid residues (in the case of only one double substitution in SEQ ID NO: 1). Therefore, the minimum number of identical amino acid residues compared to residues 97-645 in SEQ ID NO: 1 is 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, 50 6, 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 are the same amino acid residues. However, as in Figure 1 The sequence alignment shown in the data reveals certain variations among different gB protein isotypes from different viral isolates, which means that variants of the invention starting from a sequence different from SEQ ID NO: 1 will naturally include more residues different from SEQ ID NO: 1 than those non-uniformities indicated by the double substitution.

[0089] In some embodiments, the variant lacks one or both of the amino acid residue segments corresponding to the amino acid sequence defined by residues 1-22 and 703-907 of SEQ ID NO: 1, see, for example, the variants defined by SEQ ID NO: 2-7.

[0090] Since it has been proven that variants of the invention that include as few as one of the double permutations are conformationally stable, some preferred embodiments require that the variant include only one of the three double permutations defined in options 1-3 above.

[0091] However, the variants may include further biscysteine ​​substitutions, particularly those corresponding to those selected from the group consisting of the following in SEQ ID NO: 1: a) D217C and Y589C; and b) M371C and W506C.

[0092] All the variants discussed above may further include a substitution corresponding to YIH(155-157)GHR in SEQ ID NO: 1 and / or a substitution corresponding to C246S in SEQ ID NO: 1.

[0093] Furthermore, preferred variants of the invention incorporate and thereby include: i) a trimerizing domain, preferably located in the C-terminal portion of the variant, or ii) an affinity tag, or iii) a trimerizing domain and an affinity tag, optionally separated by a linker sequence, wherein the linker sequence is preferably flexible. The trimerizing domain preferably comprises or consists of a sequence of amino acid residues identical to or consisting of residues 681-704 in any of SEQ ID NO: 2-7. The affinity tag can be any suitable affinity tag (e.g., a His tag containing multiple H residues), but the preferred affinity tag (C-tag) comprises or consists of residues 709-712 in any of SEQ ID NO: 2-7.

[0094] As mentioned above, variants of the present invention can be based on any hCMV wild-type sequence. Therefore, preferred variants comprise up to 15 distinct amino acid residues (i.e., a number selected from 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2) relative to the portion corresponding to amino acid sequences 97-645 in SEQ ID NO: 1, wherein said portion is found in amino acid sequences having the following 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, AKI25 042.1, AHJ85145.1, AMJ52758.1, QTT58728.1, AHB20033.1, AQN69676.1, QTT59229.1, ALL26209.1, AKI13294.1, AKI13965.1, AKI23324.1, AQ N70515.1, 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, AKI234 91.1, AAA45925.1, ACS92156.1, AMJ54426.1, AQN72370.1, AAA45926.1, WHN53998.1, AZB53148.1, AKI09288.1, AAA45923.2, AHV84013.1, AZB 53142.1, 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, UBQ 34153.1, AZB53164.1, ACS91991.1, AKI14299.1, AFR55216.1, AHJ85985.1, AKI22156.1, AAA45934.2, AKI24535.1, AHJ83292.1, AKI247 The following UniProtKB sequences are described in this paper: 04.1, 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 described herein as SEQ ID NO: 8-122.

[0095] A particularly preferred variant of gB in this invention is:

[0096] Variants containing residues 1-680 of SEQ ID NO: 4;

[0097] Variants containing residues 1-680 of SEQ ID NO: 3;

[0098] Variants containing residues 1-680 of SEQ ID NO: 2;

[0099] Variants containing SEQ ID NO: 4;

[0100] Variants containing SEQ ID NO: 3;

[0101] Variants containing SEQ ID NO: 2;

[0102] A variant consisting of residues 1-680 of SEQ ID NO: 4 or SEQ ID NO: 4;

[0103] A variant consisting of residues 1-680 of SEQ ID NO: 3 or SEQ ID NO: 3;

[0104] A variant consisting of residues 1-680 of SEQ ID NO: 2 or SEQ ID NO: 2;

[0105] Variants containing residues 1-680 of SEQ ID NO: 7;

[0106] Variants containing residues 1-680 of SEQ ID NO: 6;

[0107] Variants containing residues 1-680 of SEQ ID NO: 5;

[0108] Variants containing SEQ ID NO: 7;

[0109] Variants containing SEQ ID NO: 6;

[0110] Variants containing SEQ ID NO: 5;

[0111] A variant consisting of residues 1-680 of SEQ ID NO: 7 or SEQ ID NO: 7;

[0112] A variant consisting of residues 1-680 of SEQ ID NO: 6 or SEQ ID NO: 6; and

[0113] A variant consisting of residues 1-680 of SEQ ID NO: 5 or SEQ ID NO: 5.

[0114] Among these, the following variants are highly preferred:

[0115] Variants containing residues 1-680 of SEQ ID NO: 4;

[0116] Variants containing residues 1-680 of SEQ ID NO: 3;

[0117] Variants containing SEQ ID NO: 4;

[0118] Variants containing SEQ ID NO: 3;

[0119] A variant consisting of residues 1-680 of SEQ ID NO: 4 or SEQ ID NO: 4;

[0120] A variant consisting of residues 1-680 of SEQ ID NO: 3 or SEQ ID NO: 3;

[0121] Variants containing residues 1-680 of SEQ ID NO: 7;

[0122] Variants containing residues 1-680 of SEQ ID NO: 6;

[0123] Variants containing SEQ ID NO: 7;

[0124] Variants containing SEQ ID NO: 6;

[0125] A variant consisting of residues 1-680 of SEQ ID NO: 7 or SEQ ID NO: 7; and

[0126] A variant consisting of residues 1-680 of SEQ ID NO: 6 or SEQ ID NO: 6.

[0127] As indicated above, the most preferred variants are those that include substitutions for I103C and V645C. This, in turn, means that the highest priority is given to:

[0128] Variants containing residues 1-680 of SEQ ID NO: 4;

[0129] Variants containing SEQ ID NO: 4;

[0130] A variant consisting of residues 1-680 of SEQ ID NO: 4 or SEQ ID NO: 4;

[0131] Variants containing residues 1-680 of SEQ ID NO: 7;

[0132] Variants containing SEQ ID NO: 7; and

[0133] A variant consisting of residues 1-680 of SEQ ID NO: 7 or SEQ ID NO: 7.

[0134] Embodiment of the second aspect of the present invention

[0135] The nucleic acids encoding the variants of hCMV gB according to any one of the foregoing claims constitute the second aspect of the invention. These nucleic acids can be in the form of DNA or RNA and can be used both as in vivo pharmaceutical products (i.e., as vaccine agents to induce expression of the variants discussed above) and as ex vivo tools for recombinant generation of variants of the invention. In this scenario, the nucleic acid molecules of the invention are typically integrated into a vector backbone and thus fused to genetic elements that facilitate their use in each of these scenarios.

[0136] In an embodiment of a second aspect of the invention, the nucleic acid sequence is codon-optimized for expression in a host cell or host organism. Techniques for designing such codon-optimized sequences for a given host cell or organism are well known to those skilled in the art of molecular biology.

[0137] Embodiment of the third aspect of the present invention

[0138] Vectors containing nucleic acid molecules of the second aspect of the invention are typically provided as cloning vectors or expression vectors. In this context, the term "vector" broadly encompasses a carrier containing the nucleic acid molecule and capable of transferring genetic information to a host cell, which can be used to propagate the nucleic acid molecule or to express it.

[0139] Typical expression vectors are operatively linked and contain at least an expression control region in a 5'-3' orientation, the expression control region comprising an enhancer / promoter for driving the expression of the nucleic acid molecule of the present invention, optionally a signal peptide coding sequence, a nucleotide sequence of the second aspect of the present invention, and optionally a terminator. However, other genetic elements may be included, see the more general description below. Thus, such vectors constitute expression vectors useful for the production of polypeptides of the present invention in cells. Since the polypeptides of the present invention are virally derived, they can be recombinantly produced in a wide variety of host cells, and therefore the expression control region will (as appropriate) drive expression in prokaryotic cells such as bacteria, for example, *Escherichia coli*. E. coli The expression in a cell or in a eukaryotic cell (mammalian cell, insect or plant) or the expression in a plant should be adjusted to suit the specific purpose.

[0140] The vector may further comprise a sequence encoding a signal peptide, which may provide for the secretion or membrane integration of the expression product derived from the vector. For the purpose of nucleic acid vaccination, the encoded signal peptide is typically selected from those described in Williams J.A. Vaccines (Basel). 2013 Sep; 1(3): 225-249 and the references cited therein.

[0141] The expression control region drives expression in prokaryotic cells, such as bacteria, for example, in Escherichia coli, or drives expression in eukaryotic cells, such as in mammalian cells, insect cells, and plant cells.

[0142] If the vector is DNA, it can further encode a polyadenylation signal, which is useful for eukaryotic expression systems and DNA vaccination applications. Similarly, if the vector is RNA, it can contain a polyA tail, which is useful in eukaryotic expression, for example, in the context of RNA vaccination.

[0143] Some of the vectors of this invention are capable of autonomous replication, a feature of particular interest in cloning and propagation vectors.

[0144] Furthermore, some vectors (e.g., viral vectors, such as retroviral vectors) are able to integrate into the host cell genome, 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 particularly relevant for DNA vaccination or viral vector vaccination, where DNA integration into the genome of the vaccinated individual is highly undesirable.

[0145] As described in detail above, the vector can take various forms and shapes; typical vectors of the present invention are selected from the group consisting of viruses, such as attenuated viruses, bacteriophages, plasmids, microchromosomes, and granules.

[0146] Further details regarding the carrier of the present invention

[0147] The variant gB polypeptides disclosed herein can be encoded by nucleic acid molecules contained in a vector as shown. By their very nature, the nucleic acids in the context of this application are heterologous (because they are not naturally occurring), meaning they are foreign to the cells in which the vectors are introduced, and include sequences homologous to sequences in the cell but not typically present in the host cell. Vectors include naked DNA, RNA, plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art are fully capable of constructing vectors using standard recombination techniques (e.g., as described in the Biotechnology Handbook Sambrook et al., 2001; Ausubel et al., 1996). In addition to encoding the variant gB polypeptides of this invention, the vectors of this invention can also encode polypeptide sequences, such as tags or immunogenicity-enhancing peptides (e.g., immunogenic carriers or fusion partners (which stimulate the immune system), such as cytokines or their active fragments). Useful vectors encoding such fusion proteins include pIN vectors, vectors encoding histidine residue chains, and pGEX vectors, used to generate glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and separation or cleavage.

[0148] The vectors disclosed herein can be used in host cells to produce the polypeptides disclosed herein, which can then be purified for administration to a subject, or the vectors can be purified for direct administration to a subject to express the protein in the subject (as is the case when a nucleic acid vaccine is administered).

[0149] Expression vectors can contain a wide variety of "control sequences," which are nucleic acid sequences necessary for transcription and, possibly translation, of operatively linked coding sequences in a particular host organism. In addition to control sequences governing transcription and translation, vectors and expression vectors can contain nucleic acid sequences that also perform other functions and are described below.

[0150] 1. Promoters and enhancers

[0151] A promoter is a control sequence. A promoter is typically a region of a nucleic acid sequence in which the initiation and rate of transcription are controlled. It can contain genetic elements that regulate proteins and molecules (e.g., RNA polymerases and other transcription factors) to which they can bind. The phrases “effectively located,” “effectively connected,” “under control,” and “under transcriptional control” mean that the promoter is in the correct functional position and / or orientation relative to the nucleic acid sequence to control the initiation and expression of that sequence. Promoters may or may not be used together with “enhancers,” which refer to cis-regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0152] Promoters can be promoters naturally associated with a gene or sequence, such as those obtained by isolating a 5' non-coding sequence located upstream of a coding region or exon. Such promoters can be referred to as "endogenous." Similarly, enhancers can be enhancers naturally associated with a nucleic acid sequence, located downstream or upstream of that sequence. Alternatively, certain advantages can be obtained by placing 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. Recombinant or heterologous enhancers also refer to enhancers that are not normally associated with a nucleic acid sequence in their natural state. Such promoters or enhancers can include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic cells, viruses, or eukaryotic cells, as well as non-"naturally occurring" promoters or enhancers, i.e., different elements containing different transcriptional regulatory regions, and / or mutations altering expression. In addition to generating promoter and enhancer nucleic acid sequences synthetically, they can also be generated using recombinant cloning and / or nucleic acid amplification techniques (including PCR). TM (See U.S. Patent 4,683,202 and U.S. Patent 5,928,906) and the compositions disclosed herein to generate sequences.

[0153] Naturally, it may be important to employ promoters and / or enhancers that effectively direct the expression of DNA segments in the cell type or organism chosen for expression. Those skilled in the art of molecular biology are generally familiar with the use of combinations of promoters, enhancers, and cell type for protein expression (see Sambrook et al., 2001). The promoters employed can be constitutive, tissue-specific, or inducible, and in some embodiments, can direct high-level expression of the DNA segments introduced under specific conditions, such as large-scale production of recombinant proteins or peptides.

[0154] Examples of inducible elements (regions of nucleic acid sequences that can be activated in response to specific stimuli) include, but are not limited to, immunoglobulin heavy chains, immunoglobulin light chains, T-cell receptors, HLA DQα and / or DQβ, β-interferon, interleukin-2, interleukin-2 receptors, class II MHC 5, class II MHC HLA-DRα, β-actin, muscle creatine kinase (MCK), prealbumin (thyroxine), elastase I, metallothionein (MTII), collagenase, albumin, alpha-fetoprotein, γ-globin, β-globin, c-fos, c-HA-ras, insulin, neural cell adhesion molecule (NCAM), α1-antitrypsin, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), and troponin I (TN). I), Platelet-derived growth factor (PDGF), Dechena muscular dystrophy, SV40, polymorphonuclear leukemia, retrovirus, human papillomavirus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), IE, and gibberish leukemia virus.

[0155] Inducible elements and their inducers include the following pairs: MT II - phorbol ester (TFA) / heavy metals; MMTV (mouse mammary tumor virus) - glucocorticoids; β-interferon - poly(rl)x / poly(rc); adenovirus 5 E2 - ElA; collagenase - phorbol ester (TPA); matrix lysozyme - phorbol ester (TPA); SV40 - phorbol ester (TPA); murine MX gene - interferon, Newcastle disease virus; GRP78 gene - A23187; α-2-macroglobulin - IL-6; vimentin - serum; class I MHC gene H-2κb - interferon; HSP70 - E1A / SV40 large T antigen; proliferation protein - phorbol ester / TPA; tumor necrosis factor - PMA; and thyroid-stimulating hormone gene - thyroid hormone.

[0156] Dectin-1 and Dectin-2 promoters are also considered useful in this invention. Additionally, any promoter / enhancer combination (according to the eukaryotic promoter database EPDB) can also be used to drive the expression of structural genes encoding oligosaccharide processing enzymes, protein folding accessory proteins, selection marker proteins, or heterologous target proteins.

[0157] The specific promoter used to control the expression of the polynucleotide encoding the peptide or protein disclosed herein is not considered critical, as long as it can direct the expression of the polynucleotide in the target cell. When targeting human cells, it is preferable to place the polynucleotide coding region in proximity to and under the control of a promoter capable of expression in human cells. Generally, such promoters may include bacterial, human, or viral promoters.

[0158] In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat sequence can be used to obtain high-level expression of the polynucleotides related to this invention. The use of other viral, mammalian cell, or bacterial phage promoters well known in the art for polynucleotide expression is also considered.

[0159] Efficient promoters in insect cells are of particular interest; see, for example, heterozygous promoters and their associated S2 expression systems disclosed in WO 2009 / 150222.

[0160] In embodiments in which the vector is administered to a subject for protein expression, the desired promoter used with the vector is considered to be a promoter that is not downregulated by cytokines, or a promoter strong enough that, even if downregulated, would produce an effective amount of the protein / peptide of the invention in the subject to elicit an immune response. Non-limiting examples of these are CMV IE and RSV LTR. In other embodiments, promoters that are upregulated in the presence of cytokines are employed. The MHC I promoter increases expression in the presence of IFN-γ.

[0161] Tissue-specific promoters can be used, especially if expression occurs in cells where the antigen is to be expressed, such as dendritic cells or macrophages. Mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters. 2. Initiation Signal and Internal Ribosome Binding Site (IRES)

[0162] Efficient translation of the coding sequence may require specific start signals. These signals include the ATG start codon or adjacent sequences. Exogenous translation control signals, including the ATG start codon, may need to be provided. Those skilled in the art will readily be able to determine this and provide the necessary signals. It is well known that the start codon must "frame-match" the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translation control signals and start codons can be natural or synthetic and can be operable in bacterial or mammalian cells. Expression efficiency can be enhanced by including appropriate transcriptional enhancer elements.

[0163] In some embodiments disclosed herein, internal ribosome entry site (IRES) elements are used to create multigene or polycistronic information. IRES elements are able to bypass ribosome scanning models of 5' methylation-dependent translation and initiate translation at an internal site. IRES elements from two members of the Picornaviridae family (poliomyelitis and encephalomyocarditis), as well as IRES from mammalian information, have been described. IRES elements can be linked to heterologous open reading frames (OPFs). Multiple OPFs can be transcribed together, each separated by an IRES, thereby creating polycistronic information. With IRES elements, each OPF is ribosome-accessible for efficient translation. Multiple genes can be efficiently expressed by transcribing a single piece of information using a single promoter / enhancer (see U.S. Patents 5,925,565 and 5,935,819, which are incorporated herein by reference).

[0164] 2. Multiple cloning sites

[0165] The vector may include a multiple cloning site (MCS), which is a nucleic acid region containing multiple restriction enzyme sites, any one of which can be used in conjunction with standard recombinant techniques to digest the vector. Often, the vector is linearized or fragmented by using a restriction enzyme that cleaves within the MCS, allowing a foreign sequence to be ligated into the vector. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombinant technology.

[0166] 3. Splice site

[0167] Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcript. If relevant in the context of the vectors of this invention, vectors containing eukaryotic genomic sequences may require donor and / or recipient splicing sites to ensure proper processing of the transcript for protein expression.

[0168] 4. Termination signal

[0169] The vectors or constructs of the present invention will generally contain at least one termination signal. A “termination signal” or “terminator” consists of a DNA sequence involved in the specific termination of RNA transcripts via RNA polymerase. Therefore, in some embodiments, a termination signal is considered to end the production of RNA transcripts. A terminator may be necessary in vivo to achieve a desired level of information.

[0170] In eukaryotic systems, the terminator region may also contain a specific DNA sequence that allows for site-specific cleavage of the new transcript to expose the polyadenylation site. This signals a specialized endogenous polymerase to add a segment of approximately 200 A residues (poly A) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to be more stable and translated more efficiently. Therefore, in other embodiments involving eukaryotes, it is preferred that the terminator contains a signal regarding RNA cleavage, and more preferably, that the terminator signal promotes the polyadenylation of the information.

[0171] Terminators considered for use in this invention include any known transcription terminators described herein or known to those skilled in the art, including but not limited to, bovine growth hormone terminators or viral termination sequences such as the SV40 terminator. In some embodiments, the termination signal may lack a transcribed or translatable sequence, for example, due to sequence truncation.

[0172] 5. Polyadenylation signal

[0173] In expression, particularly in eukaryotic expression (as relevant in nucleic acid vaccination), polyadenylation signals will typically be included to achieve proper polyadenylation of the transcript. The nature of the polyadenylation signal is considered not critical to the successful implementation of the invention, and / or any such sequence can be used. Preferred embodiments include SV40 polyadenylation signals and / or bovine growth hormone polyadenylation signals, which are convenient and / or known to function well in a variety of target cells. Polyadenylation can increase transcript stability or promote cytoplasmic transport. Therefore, the corresponding encoded RNA fragment preferably contains a poly(A) tail.

[0174] 6. Copy the starting point

[0175] To propagate the vector within a host cell, it can contain one or more origin of replication sites (often called "ori"), which are specific nucleic acid sequences at which replication begins. Alternatively, if the host cell is yeast, an autonomous replication sequence (ARS) can be used.

[0176] 7. Select tags and filter tags

[0177] In some embodiments disclosed herein, cells containing the nucleic acid constructs of the present invention can be identified in vitro or in vivo by encoding screening or selection markers in an expression vector. When transcribed and translated, the markers confer identifiable changes to the cells, thereby allowing easy identification of cells containing the expression vector. Typically, a selection marker is a marker that confers a property that allows selection. A positive selection marker is a marker in which the presence of said marker allows selection, while a negative selection marker is a marker in which its presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0178] Typically, drug-selective markers are helpful for the cloning and identification of transformants; for example, markers conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, or histamine are useful selective markers. Besides markers that confer resistance allowing for condition-based differentiation of transformant phenotypes, other types of markers include selective markers, such as GFP, for colorimetric analysis. Alternatively, selectable enzymes, such as herpes simplex virus thymidine kinase (TK) or chloramphenicol acetyltransferase (CAT), can be used. Those skilled in the art will also know how to employ immunomarkers, which can be used in conjunction with FACS analysis. The markers used are not considered important, provided they can be co-expressed with the nucleic acid encoding the protein disclosed herein. Further examples of selective and selective markers are well known to those skilled in the art.

[0179] Embodiment of the fourth aspect of the present invention

[0180] Genetically modified cells can be used as production tools in the recombinant generation of variants of the present invention (for both the propagation and expression of genetic material), but in some cases they can also be used as vaccine reagents.

[0181] For example, in recombinant generation techniques, some genetically modified cells of the present invention are useful when they are capable of replicating nucleotide sequences encoding variants of the first aspect of the invention. However, more preferred genetically modified cells are those capable of expressing nucleotide sequences as defined above, which encode variants of the first aspect of the invention and any embodiments thereof disclosed herein; such cells can be prokaryotic or eukaryotic cells.

[0182] Among prokaryotic cells, bacterial cells used in expression systems for live or inactivated vaccine reagents are of particular interest. Such bacterial cells used for use as live vaccines are preferably non-pathogenic. In any case, bacteria useful in this aspect of the invention (i.e., whether for vaccination or for recombinant protein production) are typically selected from the genus *Escherichia* (*Escherichia*). Escherichia (e.g., Escherichia coli), Bacillus spp. ( Bacillus(For example, Bacillus subtilis ( Bacillus subtilis Salmonella species ()), Salmonella sp.) and mycobacteria ( Mycobacterium For example, Mycobacterium bovis ( M. bovis The group consisting of BCG.

[0183] Eukaryotic cells are primarily used for recombinant expression and can be, for example, mammalian cells, insect cells, or plant cells. Certain insect cells are preferred, particularly those derived from the Drosophila melanogaster (Drosophila dimerans). Drosophila melanogaster Cells such as S2 or S3 cells.

[0184] One interesting production system uses plants. For example, proteins can be produced in plants at low cost by using an Agrobacterium transfection system to genetically modify the plant to express a gene encoding a target protein. One commercially available platform is provided by iBio CMO LLC (8800 HSC Pkwy, Bryan, TX 77807, USA) and iBio, Inc (9 Innovation Way, Suite 100, Newark, DE 19711, USA) and those disclosed, for example, in EP 2 853599, EP 1 769 068, and EP 2 192 172. Therefore, in such systems, the vector is an Agrobacterium vector or other vectors suitable for transfecting plants.

[0185] For the purpose of recombination, it is advantageous if the genetically modified cells of the present invention have nucleotide sequences that are stably integrated into their genome, encoding variants of the first aspect of the present invention and embodiments thereof.

[0186] For the purposes of recombinant production, the availability of the expression product (i.e., a variant of the first aspect of the invention) in the fermentation medium is of great significance. It is generally advantageous if the expression product is secreted into the fermentation medium (and therefore requires that the expression product include the associated output signal); alternatively, the expression product can be integrated into the membrane of a genetically modified cell (and thus carried on its surface). In bacteria, the expression product can also be exported into the periplasmic space or exported to appear in the cell wall / outer membrane.

[0187] Finally, the genetically modified cells can be conveniently preserved as cell lines (e.g., in a master cell bank).

[0188] Further details regarding cells and cell lines

[0189] Suitable cells for the recombinant nucleic acid expression of the nucleic acid molecules used in this invention are prokaryotes and eukaryotes.

[0190] Examples of prokaryotic cells include Escherichia coli; Staphylococcus spp. Staphylococcus Members of, for example, Staphylococcus epidermidis ( S. epidermidis ); Lactobacillus ( Lactobacillus Members, such as Lactobacillus plantarum ( L. plantarum ); Lactococcus spp. ( Lactococcus Members, such as Lactococcus lactis ( L. lactis ); Bacillus spp. ( Bacillus Members of, such as Bacillus subtilis ( Bacillus subtilis Corynebacterium spp. Corynebacterium Members, such as Corynebacterium glutamicum ( C. glutamicum ); and Pseudomonas spp. ( Pseudomonas Members, such as Pseudomonas fluorescens ( Ps. fluorescens ).

[0191] Examples of eukaryotic cells include mammalian cells; insect cells; and yeast cells, such as those in the genus *Saccharomyces*. Saccharomyces Members of (e.g., brewer's yeast) S. cerevisiae ), Pichia pastoris ( Pichia Members of (e.g., Pichia pastoris) P. pastoris ), Hansenula genus ( Hansenula Members of (e.g., Hansenula polymorpha) H. polymorpha Kluyveromyces ( )), Kluyveromyces ( Kluyveromyces Members of (e.g., Kluyveromyces lactis) K. lactis ) or Kluyveromyces brittle-walled ( K. fragilis )) and the genus *Fissionyomyces* ( Schizosaccharomyces Members of (e.g., *Schizosaccharomyces cerevisiae*) S. pombe As mentioned above, the nucleic acid sequences of the present invention can be appropriately codon-optimized to promote efficient expression in each of the genetically modified cells disclosed herein.

[0192] Techniques for generating, introducing into cells, and expressing recombinant genes are well known in the art. Examples of such techniques are provided in the references, such as Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987–2002, and Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989.

[0193] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably. All these terms also include their progeny, which are any and all subsequent generations. It should be understood that all progeny may not be identical due to intentional or unintentional 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 capable of replicating a vector or expressing a heterologous gene encoded by the vector. Host cells can and have been used as recipients of vectors or viruses. Host cells can be “genetically modified,” such as “transfected” or “transformed,” referring to the process by which a foreign nucleic acid, such as a recombinant protein-coding sequence, is transferred or introduced into a host cell. Genetically modified cells include primary test cells and their progeny.

[0194] Host cells can be derived from prokaryotes or eukaryotes, including bacterial, yeast, insect, and mammalian cells, for vector replication or partial or complete expression of nucleic acid sequences. Many cell lines and cultures are available for use as host cells, and they can be obtained, for example, from the American Type Culture Collection (ATCC), an organization that acts as an archive of living cultures and genetic material, or from other collections such as the German Microbiological Collection (DSM). A suitable host can be determined by those skilled in the art based on the vector backbone and the desired outcome. For example, plasmids or granules can be introduced into prokaryotic host cells to replicate many vectors or express the encoded proteins. Bacterial cells used as host cells for vector replication and / or expression include Staphylococcus spp. strains DH5α, JM1 09, and KC8, as well as many commercially available bacterial hosts such as SURE® competent cells and SOLOP ACK™ Gold cells (STRATAGENE®, La Jolla, CA). Alternatively, bacterial cells such as *Escherichia coli* LE392 can be used as host cells for bacteriophage viruses. Suitable yeast cells include *Saccharomyces cerevisiae* and *Saccharomyces cerevisiae*. Saccharomyces pombe ) and Pichia pastoris.

[0195] Examples of eukaryotic host cells used for vector replication and / or expression include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12, S2, and S3 cells. Many host cells from various cell types and organisms are available and will be known to those skilled in the art. Similarly, viral vectors can be used in conjunction with eukaryotic or prokaryotic host cells, particularly those that allow for vector replication or expression.

[0196] Some vectors may employ control sequences that allow them to replicate and / or be expressed in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions under which all the host cells described above are incubated to sustain them and allow vector replication. It is also understood and known that the techniques and conditions that will allow for the large-scale production of vectors and the production of nucleic acids encoded by the vectors, as well as their associated polypeptides, proteins, or peptides, will also be employed.

[0197] Expression system

[0198] Many expression systems exist that comprise at least some or all of the compositions discussed above. Systems based on prokaryotes and / or eukaryotes can be used in this invention to generate nucleic acid sequences, or their associated polypeptides, proteins, and peptides. Many such systems are commercially and widely available.

[0199] Insect cell / baculovirus systems can produce high levels of protein expression of heterologous nucleic acid segments, as described, for example, in U.S. Patents 5,871,986 and 4,879,236 (both of which are incorporated herein by reference), and are available, for example, from INVITROGEN® under the name MAXBAC® 2.0 and under the name BACPACK. TM The baculovirus expression system was purchased from CLONTECH®.

[0200] In addition to the expression systems disclosed herein, other examples of expression systems include STRATAGENE®'s COMPLETE CONTROL. TM Inducible mammalian expression systems involving synthetic ecdysone-inducible receptors, or their pET expression systems, are a type of *E. coli* expression system. Another example of an inducible expression system is INVITROGEN®, which carries T-REX. TM (Tetracycline-regulated expression) system, an inducible mammalian expression system using a full-length CMV promoter. INVITROGEN® also offers a yeast expression system called the Pichia pastoris expression system, which is designed for use in the methyltrophic yeast Pichia pastoris (Tetracycline-regulated expression). Pichia methanolica This results in high-level production of recombinant proteins. Those skilled in the art will know how to express vectors (e.g., expression constructs) to produce nucleic acid sequences or their associated polypeptides, proteins, or peptides.

[0201] Insect cell-based expression systems are particularly described in WO 2009 / 150222.

[0202] Embodiment of the fifth aspect of the present invention

[0203] The fifth aspect of the invention generally relates to compositions, such as compositions useful for immunization, particularly vaccine compositions, which comprise variations of the first aspect of the invention (or any embodiments thereof disclosed herein). In some embodiments, the composition comprises an immune adjuvant (which is often useful in immunization / vaccination scenarios) and / or the composition comprises a pharmaceutically acceptable excipient, diluent, carrier, or loading.

[0204] Details regarding the composition of the vaccine, as well as details regarding the vaccination and immunization patterns, are described in separate sections below. However, a particularly interesting compositional pattern requires that the variant be formulated in or constitute part of a virus-like particle (VLP).

[0205] In addition to variations incorporating the first aspect of the invention, the composition may constitute a "mixture" in which it contains at least one further immunogen derived from hCMV, thereby enabling the establishment of an immune response against several regions containing epitopes in hCMV.

[0206] Embodiment of the sixth aspect of the present invention

[0207] This aspect generally relates to compositions in which the nucleic acid of the second aspect or the carrier of the third aspect constitutes a portion; again, of particular interest are compositions comprising the nucleic acid molecule or carrier as an active ingredient in a vaccine. Details regarding this type of composition and associated immunization modalities are described in detail below. Typically, the nucleic acid molecule or carrier is present in combination with pharmaceutically acceptable excipients, diluents, carriers, or feedstocks, and – similar to the above – optionally also in combination with further nucleic acid molecules or carriers encoding at least one further hCMV immunogen.

[0208] Embodiment of the seventh aspect of the present invention

[0209] This embodiment generally relates to compositions comprising the genetically modified cells discussed in detail above, particularly mixed with pharmaceutically acceptable excipients, diluents, carriers or loadings; that is, this aspect is particularly relevant when the genetically modified cells are used as an immunization agent.

[0210] General considerations regarding the compositions and vaccines of the present invention

[0211] The pharmaceutical compositions according to the invention, particularly vaccines, can be preventive (i.e. suitable for preventing infection) or therapeutic (i.e. for treating disease after infection).

[0212] In some embodiments disclosed herein, the pharmaceutical composition, such as a vaccine, comprises only a single antigen, immunogen, polypeptide, protein, nucleic acid, or carrier of the present invention. However, in other embodiments, the pharmaceutical composition comprises a “mixture” of the antigens or immunogens or polypeptides or proteins or nucleic acids or carriers disclosed herein, as well as other hCMV-derived antigens or immunogens or polypeptides or proteins or nucleic acids or carriers, or genetically modified cells, i.e., immunogenic agents disclosed herein but not based on the hCMV gB protein.

[0213] These implementations require combinations of peptides / peptides that are mixed with each other. Alternatively, the same combination of peptides / peptides can be constructed as a fusion polypeptide, optionally linked via a linker as described above. Another alternative requires a composition in which the immunogen is a nucleic acid (DNA or RNA) encoding the combination of said peptides or encoding such a fusion polypeptide. In particular, RNA vaccines have recently attracted attention, with the Covid-19 RNA vaccines from Pfizer / BioNTech and Moderna being the first examples of large-scale use in humans.

[0214] The vaccines disclosed herein typically comprise immunogenic antigens, immunogens, peptides, proteins, or nucleic acids, usually in combination with a pharmaceutically acceptable carrier, which includes any carrier that does not itself induce antibodies harmful to the individual receiving the composition or that target the protein / pathogen. Suitable carriers are typically large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates (e.g., oil droplets or liposomes), and inactive viral particles.

[0215] Such carriers are well known to those skilled in the art. Furthermore, these carriers can act as immunostimulatory agents (“adjuvants”). Further, the antigen or immunogen can be conjugated to bacterial toxoids, such as those derived from diphtheria, tetanus, cholera, or Helicobacter pylori. H. pylori For toxoids of pathogens such as , see the description of immunogenic carriers above.

[0216] Therefore, the pharmaceutical compositions disclosed herein typically contain an immune adjuvant, which is usually an aluminum-based adjuvant or one of the other adjuvants described below:

[0217] Preferred adjuvants for enhancing the efficacy of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulatory agents such as muramyl peptides (see below) or bacterial cell wall components), such as (a) MF59 (WO 90 / 14837; Chapter 10 of Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, PlenumPress 1995), which contains 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (which optionally contains various amounts of MTP-PE (see below), although not required), formulated into submicron particles using a microfluidizer such as a 110Y microfluidizer (Microfluidics, Newton, MA); (b) SAF, which contains 10% squalene, 0.4% Tween 80, 5% pluronic block polymer L121 and thr-MDP (see below), microfluidized into submicron emulsions or vortexed to generate larger particle-size emulsions, and (c) the Ribi adjuvant system (RAS) (Ribi Immunochem, Hamilton, MT), comprising 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL), trehalose dimethicone ester (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (Detox™); (3) a saponin adjuvant, such as Stimulon, may be used. TM (Cambridge Bioscience, Worcester, MA), or particles derived therefrom, such as ISCOM (immunostimulatory complex); (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), tumor necrosis factor (TNF), etc.; and (6) other substances that act as immunostimulatory agents to enhance the efficacy of the composition. Alum and MF59 TM Adjuvants are preferred.

[0218] Cellular acyl peptides include, but are not limited to, N-acetyl-cellular acyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-norcellyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetyl-cellular acyl-L-alanyl-D-isoglutamine acyl-L-alanine-2"-2'-dipalmitoyl-sn-glycerol-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

[0219] As shown in the examples, the glucopyranosyl lipid adjuvant-stabilized emulsion (GLA-SE; developed by the Infectious Disease Research Institute, Seattle, WA) is an adjuvant of interest that is useful in this invention.

[0220] As an alternative to the vaccine formulation described above, variants of hCMV gB can be formulated using virus-like particles (VLPs): target VLPs also include capsid virus-like particles (cVLPs). VLPs relevant to this invention are discussed in particular in SaghiNooraei et al., Journal of Nanobiotechnology 19, Article number: 59 (2021) (doi.org / 10.1186 / s12951-021-00806-7).

[0221] Other examples of particles of interest include those by Liekni cVLP proposed by a, I. et al., see details. Figure 2 .

[0222] The immunogenic composition (e.g., the immunogenic antigen or immunogen or peptide or protein or nucleic acid, a pharmaceutically acceptable carrier, and an adjuvant) will typically contain a diluent, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting agents or emulsifiers, pH buffers, etc., may be present in such carriers.

[0223] Typically, the immunogenic composition is prepared as an injection, as a liquid solution or suspension; it can also be prepared in a solid form suitable for dissolving or suspending in a liquid carrier prior to injection. As discussed above under pharmaceutically acceptable carriers, the preparation may also be emulsified or encapsulated in liposomes for enhanced adjuvant effect.

[0224] Immunogenic compositions used as vaccines contain an immunologically effective amount of an antigenic or immunogenic polypeptide, and any other components mentioned above, if desired. An “immunologically effective amount” means that the amount administered to an individual, either as a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending on the health and physical condition of the individual to be treated, the individual’s taxonomy (e.g., non-human primates, primates, etc.), the individual’s immune system’s ability to synthesize antibodies or normally initiate an immune response, the desired level of protection, the vaccine formulation, the attending physician’s assessment of the medical condition, and other relevant factors. It is expected that this amount will fall within a relatively wide range that can be determined through routine testing. However, for the purpose of protein vaccination, the amount administered per immunization is typically in the range of 0.5 μg to 500 mg (however, often not exceeding 5,000 μg), and very often in the range of 10 to 200 μg.

[0225] The immunogenic compositions are routinely administered parenterally, such as by injection, subcutaneously, intramuscularly, or percutaneously (e.g., as disclosed in WO 98 / 20734). Other formulations suitable for other administration methods include oral, pulmonary, and nasal preparations, suppositories, and percutaneous application. In the case of nucleic acid vaccination, intravenous or intra-arterial routes are also applicable.

[0226] The administration regimen can be a single-dose regimen or a multiple-dose regimen. The vaccine can be administered together with other immunomodulatory agents.

[0227] As an alternative to protein-based vaccines, DNA vaccination (also known as nucleic acid vaccination or gene vaccination) can be used (see, for example, Robinson & Torres (1997) Seminars in Immunol 9: 271-283; Donnelly et al., (1997) Annu Rev Immunol 15: 617-648). Furthermore, as also noted herein, RNA (mRNA) vaccination is an interesting and promising technique; see the references above by Deering RP et al.

[0228] Embodiment of the eighth aspect of the present invention

[0229] This aspect generally relates to immunization methods that utilize variants of hCMV gB described above, corresponding nucleic acids encoding the variants, vectors, or genetically modified cells.

[0230] In some embodiments, following the initial stimulation dose, one or more booster doses are administered with an effective amount of a variant of the gB peptide of the first aspect or an embodiment thereof as described herein, a nucleic acid molecule of the second aspect or an embodiment thereof as described herein, a vector of the third aspect or an embodiment thereof as described herein, a genetically modified cell of the fourth aspect or an embodiment thereof as described herein, or a composition comprising any of these as described herein. Of particular significance is booster administration with protein-based or nucleic acid vaccines. It should also be noted that, for example, initial stimulation can be performed with one type of immunizing agent, followed by booster administration with a different type of immunizing agent – ​​for example, a DNA or RNA vaccine can be boosted with a protein vaccine, and vice versa.

[0231] The vaccination methods described herein can be both preventative and therapeutic in nature. For example, subjects with an ongoing hCMV infection may receive the methods used for immunization, as may subjects without prior hCMV exposure and those with subclinical infection (in which case the treatment is essentially a prevention of disease recurrence). In other words, vaccinated subjects may not have an ongoing hCMV infection, and in this case, the subject may also not have any prior or recent hCMV infection, as demonstrated by seronegativity regarding anti-CMV antibodies. In any case, the vaccination techniques described herein are believed to reduce or prevent the onset of symptoms of disease caused by hCMV infection in vaccinated subjects.

[0232] Details of the method of the present invention

[0233] The methods disclosed in the seventh aspect generally relate to the induction of immunity, and thus necessarily include methods for the treatment, prevention and improvement of diseases.

[0234] When an immunization method requires the administration of the hCMV gB variant disclosed herein or a composition containing such a variant, the subject typically receives 0.5 to 5,000 μg of the variant disclosed herein per administration.

[0235] In a preferred embodiment of this aspect, the immunization regimen includes the animal (e.g., a human) receiving an initial stimulant administration and one or more booster administrations.

[0236] Preferred embodiments of this aspect disclosed herein include administration intended to induce protective immunity against hCMV. This further implies that the administration is a preventative or therapeutic treatment for diseases caused by hCMV.

[0237] The vaccines disclosed herein induce humoral immunity, and therefore preferably, the administration is for the purpose of inducing antibodies specific to hCMV.

[0238] As mentioned above, pharmaceutical compositions may contain peptides, antibodies, or nucleic acids disclosed herein. The pharmaceutical compositions will contain therapeutically effective amounts of those.

[0239] As used herein, the terms "therapeutic effective dose" or "preventive effective dose" refer to the amount of a therapeutic agent used to treat, improve, or prevent a desired disease or condition, or to demonstrate a detectable therapeutic or preventive effect. This effect can be detected, for example, by chemical labeling or antigen levels. Therapeutic effects also include the reduction of physical symptoms, such as a decrease in body temperature. The precise effective dose for a subject will depend on the subject's body size and health, the nature and severity of the condition, and the therapeutic agent or combination of therapeutic agents selected for administration. Therefore, specifying an exact effective dose in advance is not helpful. However, the dose range of immunologically effective doses of peptides can be referenced above.

[0240] However, the effective quantity for a given situation can be determined through routine experiments and is within the judgment of clinicians.

[0241] For the purposes of this invention, in individuals to whom the DNA or RNA construct is administered, the effective dose will be from about 0.01 mg / kg to 50 mg / kg or from 0.05 mg / kg to about 10 mg / kg of the DNA or RNA construct.

[0242] As described herein, pharmaceutical compositions may also comprise pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent (e.g., an antibody or peptide, gene, and other therapeutic agents). This term refers to any such pharmaceutical carrier that, in itself, does not induce antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive viral particles. Such carriers are well known to those skilled in the art.

[0243] Pharmaceutically acceptable salts may be used, such as inorganic acid salts, such as hydrochlorides, hydrobroms, phosphates, sulfates, etc.; and salts of organic acids, such as acetates, propionates, malonates, benzoates, etc. A detailed discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0244] Pharmaceutically acceptable carriers in therapeutic compositions may comprise liquids such as water, saline, glycerol, and ethanol. Additionally, excipients such as wetting agents or emulsifiers, pH buffers, etc., may be present in such carriers. Typically, the therapeutic compositions are prepared as injections, as liquid solutions or suspensions; they may also be prepared in solid forms suitable for dissolving or suspending in a liquid carrier prior to injection. Liposomes are included within the definition of pharmaceutically acceptable carriers.

[0245] As is clear from the claims, the invention also relates to aspects and embodiments of treatment and prevention disclosed herein: the invention further includes aspects and embodiments in which...

[0246] - The variant hCMV gB peptide disclosed herein is intended for use as a medicine, particularly for use as a medicine in the treatment, prevention or improvement of hCMV infection;

[0247] - The nucleic acid fragments or vectors disclosed herein are intended for use as drugs, particularly for use as drugs in the treatment, prevention or improvement of hCMV infection;

[0248] - The genetically modified cells disclosed herein are intended for use as drugs, particularly as drugs for the treatment, prevention, or improvement of hCMV infection.

[0249] Biological sequence

[0250] The following gB and gB variant sequences are provided to better understand the present invention. The reference sequence is SEQ ID NO: 1, which is in Figure 1 The sequence was compared with multiple gB sequences. The substitutions marked with underline in SEQ ID NO: 2-7 can also be performed in other gB amino acid sequences.

[0251] like Figure 1 As shown, the aligned sequences are conserved with respect to the substituted residues; that is, the same amino acid residues are found in the reference sequence and all aligned sequences, except for position 589 in SEQ ID NO: 1, which is composed of H (histidine) instead of Y (tyrosine) in five of the aligned sequences. In these five sequences, the introduced substitution is therefore H→C instead of Y→C.

[0252] SEQ ID NO: 1

[0253] UniProtKB accession number: PI3201.1, wherein the residues shown in underlined italics are missing in the variant sequences SEQ ID NO: 2-7.

[0254]

[0255] SEQ ID NO: 2

[0256] The gB variant is substituted at the underlined position and fused to the trimerized domain (double underline) and the C-tag (bold italic) (via the GSGS linker (dot underline)).

[0257]

[0258] SEQ ID NO: 3

[0259] The gB variant is substituted at the underlined position and fused to the trimerized domain (double underline) and the C-tag (bold italic) (via the GSGS linker (dot underline)).

[0260]

[0261]

[0262] SEQ ID NO: 4

[0263] The gB variant is substituted at the underlined position and fused to the trimerized domain (double underline) and the C-tag (bold italic) (via the GSGS linker (dot underline)).

[0264]

[0265] SEQ ID NO: 5

[0266] The gB variant is substituted at the underlined position and fused to the trimerized domain (double underline) and the C-tag (bold italic) (via the GSGS linker (dot underline)).

[0267]

[0268] SEQ ID NO: 6

[0269] The gB variant is substituted at the underlined position and fused to the trimerized domain (double underline) and the C-tag (bold italic) (via the GSGS linker (dot underline)).

[0270]

[0271] SEQ ID NO: 7

[0272] The gB variant is substituted at the underlined position and fused to the trimerized domain (double underline) and the C-tag (bold italic) (via the GSGS linker (dot underline)).

[0273]

[0274] in addition, Figure 1 The following are published sequences of gB isotypes from various sources, and the positions of modifications similar to those introduced into SEQ ID NO: 2-7 relative to SEQ ID NO: 1 were identified:

[0275]

[0276]

[0277]

[0278] Example 1

[0279] Engineering of pre-fusion stabilized gB builds

[0280] When the viral envelope fuses with the host cell membrane, the human cytomegalovirus (hCMV) glycoprotein B (gB) irreversibly changes from a metastable pre-fusion conformation to a stable post-fusion conformation.

[0281] Stabilizing the gB protein in its pre-fusion conformation ensures the generation of antibodies that can block the binding of the gB protein to the host cell membrane.

[0282] A similar strategy has been applied when developing vaccines against SARS-CoV-2, in which the pre-fusion stabilized spike protein is used as an antigenic component. Previous attempts to stabilize gB by using mutations to create disulfide bonds that stabilize the protein in its pre-fusion pattern have also been investigated (Patent WO 2021 / 260510 A1; and Sponholtz MR et al., 2024: https: / / doi.org / 10.1101 / 2024.02.10.579772).

[0283] By examining and comparing the structures of wild-type gB in pre-fusion and post-fusion conformations (SEQ ID NO: 1), potential sites for introducing stabilizing disulfide bonds were identified and evaluated. Three selected mutation pairs were chosen for introduction into gB:

[0284] G543C-K617C (Design builder pre_gB#1; SEQ ID NO: 2),

[0285] A97C-A538C (designed constructor pre_gB#2; SEQ ID NO: 3), and

[0286] I103C-V645C (Design builder pre_gB#1; SEQ ID NO: 4).

[0287] These constructs also include the known mutations YIH(155-157)GHR and W240A used in the literature to stabilize the constructs, as well as the mutation C246S which removes free cysteine ​​to reduce aggregation and increase protein expression.

[0288] In WO 2021 / 260510, the optimal construct was found in gB (V23-D646) to include not only the stabilizing mutations YIH(155-157)GHR, W240A, and C246S, but also two mutations D217C-Y589C and M371C-W506C.

[0289] The corresponding design, including BIP secretion signaling and C-tag, was performed and generated in Drosophila melanogaster Schneider 2 cells and used as a control. This construct is referred to here as gB2796-Ctag.

[0290] Introducing a single mutant pair with D217C-Y589C has been shown to stabilize gB in the pre-fusion conformation, although this can only be maintained by the small molecule inhibitor WAY-174865 (WO 2021 / 260510 A1). Therefore, to evaluate the disulfide pairs, the three selected novel disulfide pairs described herein will be evaluated in combination with D217C-Y589C, resulting in the following design:

[0291] G543C-K617C; D217C-Y589C (designed constructor pre_gB#4; SEQ ID NO: 5),

[0292] A97C-A538C; D217C-Y589C (designed constructor pre_gB#5; SEQ ID NO: 6), and

[0293] I103C-V645C; D217C-Y589C (Design builder pre_gB#6; SEQ ID NO: 7).

[0294] To establish a baseline, three distinct wild-type gB designs were derived: wt_gB, which is designed to be equivalent to commercially available gB proteins, including a BIP secretion signal and a C-terminal C-tag (catalog: 10202-VCCH1; SinoBiologicals); a post_gB design, which excludes the intracellular domain (residues 23-702 of SEQ ID NO: 1, which has a BIP secretion signal and a C-terminal C-tag); and post_gB_tri, in which a trimerizing domain is added to the post_gB design (GCN4CC tri2).

[0295] exist Figure 2 Figures 3 and 4 provide a schematic overview of all the tested designs. Figures 4-6 show the banded structures, which depict the positions of the three mutation pairs described in the pre-fusion and post-fusion gB conformations.

[0296] Example 2

[0297] Recombination of C-tagged gB proteins and variants

[0298] Genes encoding wild-type gB proteins (post_gB and post_gB-tri; residues 23-702 of SEQ ID NO: 1, having a BIP secretion signal, a C-terminal C-tag, and a trimerization domain) and recombinant C-tagged gB protein variants (SEQ ID NO: 1 with prior art modifications, and SEQ ID NO: 2-4 and 6-7, see Figure 3 for a schematic overview of the design) were obtained from Twist Biosciences or GeneArt as synthetic DNA fragments and subsequently cloned into the proprietary expression vector pExpreS2-1 to produce transfected ready-to-use DNA. pExpresS2-1 was available commercially from Expres2ion Biotechnologies (Agern Allé 1, 2970 Hørsholm, Denmark) under catalogue 175360 under research terms. The DNA sequence of pExpresS2-1 is shown in SEQ ID NO: 123.

[0299] These plasmids were transiently expressed in Drosophila melanogaster Schneider 2 cells, and their expression was analyzed by Western blotting. Figure 7 ( ), in which anti-C-tag antibodies are used for detection.

[0300] exist Figure 7Lane 8 shows the expression of the prior art protein gB2796-Ctag (SEQ ID NO: 1, residues 23-646, with cysteine ​​substitutions D217C, Y589C, M371C, and W506C, and further modified with YIH(155-157)GHR, W240A, and C246S; and a C-terminal C-tag) in transiently transfected cells. Under non-reducing conditions, Western blot showed a high molecular weight band for gB2796-Ctag, which was absent for wild-type gB (lane 6), indicating stable trimerization. Furthermore, Western blot showed the expression and trimerization of all designed constructs (SEQ ID NO: 2-7, lanes 9-11 and 13-14) in transiently transfected cells.

[0301] A stable polyclonal pool of the gB variant was established by transfecting Drosophila melanogaster S2 cells with an expression vector and subjecting the cells to a three-week selection process using the antibiotic selection marker zeocin. Cells were expanded, secreting proteins into a supernatant, and harvested by centrifugation. Subsequently, ultrafiltration and diafiltration using tangential flow filtration were performed to transfer the proteins to a buffer solution.

[0302] C-tagged proteins were loaded onto equilibrated CaptureSelect CtagXL columns. The column was washed, and the proteins were eluted using a four-step gradient (12.5%, 25%, 50%, and 100%) of elution buffer containing 25 mM Tris, 150 mM NaCl, and 2 M MgCl2 at pH 7.5. The elution fractions were analyzed by SDS-PAGE, and those fractions containing the target protein were pooled, concentrated on a 10 kDa MWCO Amicon rotary filter, and purified by gel filtration on a Superdex 20016 / 60 column. The purified proteins were analyzed by SDS-PAGE and size exclusion HPLC.

[0303] Figure 8 Coverage of size exclusion HPLC chromatograms from four gB variants successfully expressed in stable cell lines is shown.

[0304] The data shows the monodisperse spectra of EVX-pre-gB#2 (SEQ ID NO: 3) and EVX-pre-gB#3 (SEQ ID NO: 4) at the trimer size. 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 trimer size, but both spectra exhibit "shoulder peaks," indicating that a portion of the protein in these spectra is a heterogeneous solution with a higher molecular weight. By comparing the size-resistance HPLC spectra of EVX-pre-gB#2 (SEQ ID NO: 3) and EVX-pre-gB#3 (SEQ ID NO: 4) with those of WT-gB (Figure 9), it was observed that both gB variants exhibited sharper and more symmetrical peaks than WT-gB, indicating a more homogeneous solution than WT-gB.

[0305] Example 3

[0306] Transmission electron microscopy analysis

[0307] Transmission electron microscopy (TEM) analysis was performed by applying a small droplet of protein sample onto a carbon-coated mica sheet. Excess liquid was gently blotted from the grid using filter paper. The grid was immediately transferred to a 2% (w / v) phosphotungstic acid solution for negative staining. To enhance grid adhesion, the grid was exposed to a Sellotape sheet placed in chloroform. The stained grid was immediately examined using a Philips CM100 TEM microscope.

[0308] result

[0309] With from Figure 7 Consistent with the observations of Western blots, TEM analysis of the wt_gB protein sample revealed a heterogeneous sample, in which most proteins appeared as extended “bars” containing three distinct domains (approximately 20.3–23.03 nm in size), which corresponded well to the expected post-fusion structure of gB. Figure 10A A few longer proteins (approximately 27.32–39.12 nm in size) were also observed, and these are thought to represent two gB molecules. A few shorter and rounder protein structures (15.85–17.49 nm) were also observed, which most likely represent a smaller pre-fusion structural group or simply reflect the post-fusion conformation viewed from a different angle.

[0310] Conversely, the pre_gB#3 sample exhibits a very uniform image composed of small triangular protein structures (approximately 7.5–9.21 nm in size), indicating the pre-fusion structure of gB ( Figure 10B).

[0311] Similar results were observed in the preliminary cryo-electron microscopy of the sample, see [link to previous section]. Figure 11 .

[0312] Example 4

[0313] Immunogenicity of gB variants

[0314] The immunogenicity of the mutant gB protein disclosed in this application was evaluated and compared with the wild-type gB protein in mice. Three different wild-type gB protein constructs were evaluated: wt_gB (Sino-gB comparator), post_gB (extracellular domain of gB), and post_gB_tri (extracellular domain of gB with trimerized domains). The prior art gB protein, gB2796, was included in this study as a comparator. The mutant variants disclosed in this application tested were pre_gB#2, pre_gB#3, pre_gB#5, and pre_gB#6. For a detailed overview of the design, see [link to relevant documentation]. Figure 2 And 3.

[0315] BALB / c mice were immunized with a total dose of 10 μg protein / 100 μL in TRIS buffer prepared 1:1 with AddaVax (InvivoGen). TRIS buffer prepared 1:1 with AddaVax without protein served as a placebo. Mice received three intramuscular (im) injections of 50 μL each, administered at two-week intervals, into the tibialis anterior muscle of each hind leg.

[0316] Nine (9) groups (groups #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.

[0317] Serum was separated from tail vein blood collected on day 26 of the study and from retro-orbital blood collected two weeks after the last immunization at the end of the study.

[0318] ELISA was used to determine the level of antigen-specific IgG. In short, the ELISA, MaxiSorp microtiter plates (Thermo Scientific), were coated with a specific gB variant for each immunization group. Cross-reactivity was evaluated by coating the ELISA plates with either wt_gB or pre_gB#3. gB-specific total IgG was detected using HRP-conjugated polyclonal rabbit anti-mouse IgG (Sigma-Aldrich), with TMB EASY (Kementec) used for colorimetric development. Antibody binding was analyzed using the number of absorbance values ​​(OD450 nm minus OD620 nm). Specifically, the endpoint titer was calculated as the highest serum dilution at which the OD value exceeded the cutoff value determined from serum from mice immunized with placebo.

[0319] A live virus neutralization assay was used to evaluate the ability of serum from vaccinated animals to neutralize viral infection. The assay relied on cytopathic effect (CPE) readout evaluated by fluorescence microscopy, in which the survival of virus-infected cells was determined in the presence of different concentrations of immune serum. Briefly, a series of diluted immune serum were incubated with the hCMV Merlin strain in equal volumes for one hour. After this initial incubation period, confluent monolayers of MRC-5 cells in 96-well plates were infected using the serum-virus mixture. The plates were incubated at 37°C for eight days with 5% CO2. MRC-5 cell death due to hCMV infection was visualized using Sytox viability dye, and analysis was performed using fluorescence microscopy and automated counting of the stained cells.

[0320] result

[0321] No difference in titer was observed between wt_gB (Sino-gB), post_gB, and post_gB_tri, indicating that excluding the intracellular domain and adding the trimerizing domain did not affect immunogenicity. Figure 12 ).

[0322] Immunogenicity analysis of a novel variant of hCMV gB with in-situ transposed cysteine ​​pairs induced IgG levels comparable to or higher than those of wild-type gB protein and the prior art gB, gB2796 (pre_gB#2). Figure 13A Immunization with pre_gB#3 induced antibodies with higher neutralizing capacity against live human CMV virus compared to wt_gB and gB2796. Figure 13B ).

[0323] No differences in antibody titers were observed between pre_gB#2 and pre_gB#5, or between pre_gB#3 and pre_gB#6. This is consistent with observations regarding expression. Figure 7 This indicates that the identified mutants ALA97C-ALA538C and ILE103C-VAL645C are sufficient on their own for expression, trimer stabilization, and induction of high levels of functional antibodies. Figure 14 ).

[0324] When evaluating cross-constructed IgG responses among immunized groups, it was noted that all immunized mice produced comparable levels of IgG recognizing wt_gB. Figure 15A Conversely, when analyzing responses to the pre_gB#3 protein across groups, pre_gB#2 and, especially, pre_gB#3, both showed higher antibody levels compared to the other groups tested. The differences in antibody responses to pre_gB#3 between different vaccination groups confirmed that the conformations of pre_gB#2 and pre_gB#3 differed from those of the proteins found in their post-fusion conformations (wt_gB, post_gB, post_gB-tri) and gB2796 proteins. Therefore, the conformations of pre_gB#2 and pre_gB#3 may expose other epitopes, which could explain the increased IgG titers and protective effects shown for pre_gB#2 and pre_gB#3, respectively. Figure 15B ).

[0325] Example 5

[0326] Dose-response study

[0327] The dose-dependent antibody response to the pre-fusion stabilized pre_gB#3 disclosed herein was evaluated by immunogenicity testing in mice.

[0328] BALB / c mice were immunized with a total dose of 1–10 μg protein per 100 μL in TRIS buffer prepared 1:1 with AddaVax (InvivoGen). TRIS buffer prepared 1:1 with AddaVax without protein served as a placebo. Mice received three intramuscular (im) injections of 50 μL each at two-week intervals into the tibialis anterior muscle of each hind leg.

[0329] Six (6) groups (groups #1-6) of mice were assigned to the following immunization programs:

[0330] 1. AddaVax (placebo)

[0331] 2. pre_gB#3; 10 μg / dose

[0332] 3. pre_gB#3; 7.5 μg / dose

[0333] 4. pre_gB#3; 5 μg / dose

[0334] 5. pre_gB#3; 2.5 μg / dose

[0335] 6. pre_gB#3; 1 μg / dose.

[0336] Serum was isolated from tail vein blood collected on day 13 and day 26 of the study, and from retro-orbital blood collected two weeks after the last immunization at the end of the study.

[0337] As described in Example 3, ELISA was used to determine the level of pre_gB#3 specific IgG.

[0338] result

[0339] Immunogenicity analysis of a novel variant of hCMV gB with in-situ cysteine ​​pairs revealed that I103C-V645C induced high levels of IgG even at a dose of 1 μg. Figure 16 IgG levels in response to one and two doses of 1 μg pre_gB#3 were comparable to those with 10 μg / dose, while a significantly enhanced antibody response was observed with a third dose of 1 μg compared to mice receiving three doses of 10 μg. Figure 17 This highlights the immunogenicity of this new variant of gB.

Claims

1. A variant of human cytomegalovirus glycoprotein B (gB) comprising one, two, or three double substitutions, each of which corresponds to a double substitution selected from the group consisting of the following in SEQ ID NO: 1: 1) I103C and V645C; 2) A97C and A538C; and 3) G543C and K617C, The variant comprises a continuous amino acid chain segment containing at least 470 identical amino acid residues when optimally compared with residues 97-645 in SEQ ID NO:

1.

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

1.

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

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

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

1.

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

1.

7. A variation according to any one of the preceding claims, further comprising: i) A trimerization domain, preferably located in the C-terminal portion of the variant, or ii) Affinity labels, or iii) Trimerization domains and affinity tags, which are optionally separated by linker sequences.

8. The variant according to claim 7, wherein the trimerizing domain comprises or is composed of residues 681-704 in any one of SEQ ID NO: 2-7.

9. The variant according to claim 7 or 8, wherein the affinity tag comprises or is composed of residues 709-712 in any one of SEQ ID NO: 2-7.

10. A variant according to any one of the preceding claims, which, when optimally aligned, comprises at most 15 distinct amino acid residues relative to the portion of the amino acid sequence corresponding to residues 97-645 in SEQ ID NO: 1, said portion being found in amino acid sequences having the following 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, AHB20 033.1, AQN69676.1, QTT59229.1, ALL26209.1, AKI13294.1, AKI13965.1, AKI23324.1, AQN70515.1, QBK84312.1, ACS93398.1, AQN69508.1, AK I20319.1, 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, AQN723 70.1, AAA45926.1, WHN53998.1, AZB53148.1, AKI09288.1, AAA45923.2, AHV84013.1, AZB53142.1, QIA45372.1, AQN70349.1, CAG7582570.1, Q IA46216.1, 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, AKI 14299.1, AFR55216.1, AHJ85985.1, AKI22156.1, AAA45934.2, AKI24535.1, AHJ83292.1, AKI24704.1, QZX45917.1, QPZ446 73.1, 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 of claim 1, comprising residues 1-680 of SEQ ID NO:

2.

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

3.

13. The variant of claim 1, comprising residues 1-680 of SEQ ID NO:

4.

14. The variant of claim 1, comprising SEQ ID NO:

2.

15. The variant of claim 1, comprising SEQ ID NO:

3.

16. The variant of claim 1, comprising SEQ ID NO:

4.

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

2.

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

3.

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

4.

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

5.

21. The variant of claim 1, comprising residues 1-680 of SEQ ID NO:

6.

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

7.

23. The variant of claim 1, comprising SEQ ID NO:

5.

24. The variant of claim 1, comprising SEQ ID NO:

6.

25. The variant of claim 1, comprising SEQ ID NO:

7.

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

5.

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

6.

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

7.

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

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

31. A vector comprising a nucleic acid molecule according to claim 29 or 30, said vector being, for example, a cloning vector or an expression vector.

32. The vector of claim 31, wherein it is operatively linked and comprises an expression control region in a 5'-3' orientation, the expression control region comprising an enhancer / promoter for driving the expression of a nucleic acid molecule as defined in claim 29, optionally a signal peptide coding sequence, a nucleotide sequence as 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 cells, such as bacteria, for example, *Escherichia coli*. E. coli The expression in ).

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

35. The carrier according to claim 34, wherein, When it is DNA, it encodes a polyadenylation signal, and when it is RNA, it contains a polyA tail.

36. The carrier according to 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 integrating into the genome of a host cell.

38. The vector according to any one of claims 31-36, which cannot be integrated into the genome of a mammalian host cell.

39. The vector according to any one of claims 31-38, wherein the vector is selected from the group consisting of viruses, such as attenuated viruses, bacteriophages, plasmids, microchromosomes, and kinases.

40. A genetically modified cell carrying a nucleic acid molecule according to any one of claims 29-30 or a vector according to any one of claims 31-39.

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

42. The genetically modified cell according to claim 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, wherein the cell is selected from prokaryotic cells and eukaryotic cells.

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

45. The genetically modified cell according to any one of claims 40-44, which is stably genetically modified by stably integrating the nucleotide sequence defined in claim 29 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 of claim 46, wherein the cell is a bacterium and is secreted into the interstitial space.

48. A cell line derived from genetically modified cells 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 immune adjuvant.

50. The composition of claim 49, further comprising at least one further hCMV immunogen.

51. The composition according to claim 49 or 50, further comprising a pharmaceutically acceptable excipient, diluent, carrier, or loading agent.

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

53. A composition comprising a nucleic acid molecule according to claim 29 or 30 or a carrier according to any one of claims 31-39, and a pharmaceutically acceptable excipient, diluent, carrier or loading thereof, and optionally further comprising a nucleic acid fragment or carrier encoding at least one further hCMV immunogen.

54. A composition comprising the genetically modified cells according to any one of claims 40-47, and pharmaceutically acceptable excipients, diluents, carriers, or loadings mixed therewith.

55. A method for inducing immunity against an infection caused by hCMV in a human subject, 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, a nucleic acid molecule according to claim 29 or 30, a vector according to any one of claims 31-39, a genetically modified cell according to any one of claims 40-47, or a composition according to any one of claims 49-54.

56. The method of claim 55, wherein one or more booster doses are administered after the initial stimulation dose, using an effective amount of a variant of the gB polypeptide according to any one of claims 1-28, a nucleic acid molecule according to claim 29 or 30, a vector according to any one of claims 31-39, a genetically modified cell according to any one of claims 40-47, or a composition according to any one of claims 49-54.

57. The method of claim 55 or 56, wherein the subject has an ongoing hCMV infection.

58. The method of claim 55 or 56, wherein the subject has no ongoing hCMV infection, and wherein the subject preferably has no prior or recent hCMV infection, as demonstrated by evidence that the anti-CMV antibody is seronegative.

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

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