LU92999B1 - Means and methods for treating HSV - Google Patents

Abstract

The present invention relates to a vaccine composition comprising a multimeric complex of Herpes Simplex Virus (HSV) polypeptides for the treatment or vaccination against HSV. The present invention also relates to a vector comprising a polynucleotide encoding the HSV polypeptides and a host cell comprising the vector. The present invention further comprises a method for producing the vaccine composition.

Description

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Means and methods for treating HSV

FIELD OF INVENTION

[1] The present invention relates to a vaccine composition comprising a multimeric complex of Herpes Simplex Virus (HSV) polypeptides for the treatment or vaccination against HSV. The present invention also relates to a vector comprising a polynucleotide encoding the HSV polypeptides and a host cell comprising the vector. The present invention further comprises a method for producing the vaccine composition.

BACKGROUND

[2] Herpes simplex virus is a viral genus of the viral family known as Herpesviridae. The species that infect humans are commonly known as Herpes simplex virus 1 (HSV-1) and Herpes simplex virus 2 (HSV-2), wherein their formal names are Human herpesvirus 1 (HHV-1) and Human herpesvirus 2 (HHV-2), respectively. The initial infection with HSV-1 typically occurs during childhood or adolescence and persists lifelong. Infection rates with HSV-1 are between 40% and 80% worldwide, being higher among people of lower socialeconomic status. In many cases people exposed to HSV-1 demonstrate asymptomatic seroconversion. However, initial infection can also be severe, causing widespread 1 to 2 mm blisters associated with severe discomfort that interferes with eating and drinking to the point of dehydration, last 10 to 14 days, and occur 1 to 26 days after inoculation. Recurrent labial herpes affects roughly one third of the US population, and these patients typically experience 1 to 6 episodes per year. Papules on an erythematous base become vesicles within hours and subsequently progress through ulcerated, crusted, and healing stages within 72 to 96 hours (Cernik et al., 2008, Arch Intern Med., vol. 168, pp. 1137-1144). Global estimates in 2003 assume that 16.2% of the population are infected with HSV-2, being the major cause of genital herpes. The ability of the virus to successfully avoid clearance by the immune system by entering a non-replicating state known as latency leads to lifelong infection. Periodic reactivation from latency is possible and leads to viral shedding from the site of the initial infection. Genital lesions due to herpes are often very painful, and can lead to substantial psychological morbidity. The virus can also be passed from mother to child during birth. Without treatment, 80% of infants with disseminated disease die, and those who do survive are often brain damaged. In addition, genital herpes is associated with an increased risk of HIV acquisition by two- to threefold, HIV transmission on a per-sexual act basis by up to fivefold, and may account for 40-60% of new HIV infections in high HSV-2 prevalence populations (Looker et al., 2008, Bulletin of the World Health Organization, vol. 86, pp. 805-812).

[3] Currently, acyclovir, a synthetic acyclic purine-nucleoside analogue, is the standard therapy for HSV infections and has greatly helped control symptoms. Precursor drugs, valacyclovir (converted to acyclovir) and famciclovir (converted to penciclovir), have been licensed and have better orâ?i bioavailability than acyclovir and penciclovir, respectively. The available drugs have an excellent margin of safety because they are converted by viral thymidine kinase to the active drug only inside virally infected cells. However, HSV can develop resistance to acyclovir through mutations in the viral gene that encodes thymidine kinase by generation of thymidine-kinase-deficient mutants or by selection of mutants with a thymidine kinase unable to phosphorylate acyclovir. Most clinical HSV isolates resistant to acyclovir are deficient in thymidine kinase, although altered DNA polymerase has been detected in some. As HSV can lie latent in neurons for months or years before becoming active, such a therapy may be used to treat symptoms caused by HSV but cannot avoid the periodic reactivation of the virus.

[4] Accordingly, the most effective and economical way to fight HSV would be a vaccine preventing initial infection and/or periodic reactivation of the virus. A lot of effort has been put in the development of such a vaccine in the past several decades. However, attempts to develop a potent HSV vaccine have focused on a limited number of antigens that have shown poor performance in clinical trials. Accordingly, there is an urgent need of a vaccine against HSV.

DETAILED DESCRIPTION

Vaccine composition [5] The present invention addresses this need and provides a novel vaccine composition comprising a multimeric complex of Herpes Simplex Virus (HSV) polypeptides UL31 and UL34.

[6] The term “multimeric complex” or “complex” are used interchangeably herein and refer to a stable polypeptide complex composed of at least two polypeptide subunits along with any covalently attached molecules (such as lipid anchors or oligosaccharide) or non-protein prosthetic groups (such as nucleotides or metal ions). Prosthetic group in this context refers to a tightly bound cofactor. Accordingly, a multimeric complex may comprise two polypeptides (i.e. a dimer). A multimeric complex of the invention relates to a set of interacting proteins that has been shown to exist as a functional unit in vivo and the polypeptides of the multimeric complex of the invention can be co-purified using stringent protein purification methods. Such stringent protein purification methods make use of buffers and solutions that do not force unspecific and/or artificial protein interaction and thus result only in the purification of complexes that stay intact (i.e. no polypeptide of the complex is released) when subjected to stringent wash conditions. Therefore, methods that merely show an interaction of polypeptides, such as immunoprecipitation or pull-down experiments from cell extracts are not considered as suitable methods for purifying a complex of the invention. Likewise, methods that merely show the co-localization of polypeptides or the interaction of polypeptides are not indicative of a complex of the invention, in particular if such a method employs artificially modified polypeptides, such as e.g. yeast-2-hybrid systems. Accordingly, after purification a complex of the invention can be detected using a suitable method (e.g. A multimeric complex of the invention relates to a set of interacting proteins that has be^£( shown to exist as a functional unit in vivo and the polypeptides of the multimeric complex of the invention can be co-purified using stringent protein purification methods. Such stringent protein purification methods make use of buffers and solutions that do not force unspecific and/or artificial protein interaction and thus result only in the purification of complexes that stay intact (i.e. no polypeptide of the complex is released) when subjected to stringent wash conditions . Therefore, methods that merely show an interaction of polypeptides, such as immunoprecipitation or pull-down experiments from cell extracts are not considered as suitable methods for purifying a complex of the invention. Likewise, methods that merely show the co-localization of polypeptides or the interaction of polypeptides are not indicative of a complex of the invention, in particular if such a method employs artificially modified polypeptides, such as e.g. yeast-2-hybrid systems. Accordingly, after purification a complex of the invention can be detected using a suitable method (e.g. native polyacrylamide gel electrophoresis). Consequently, the mere presence of two or more polypeptides, which may have been shown to exist as a complex in vivo, in a composition are not considered as a complex of the invention as such a complex may form only using specific purification methods and conditions and may only be stable after purification under specific storage conditions. Thus, even if certain polypeptides have been shown to form a complex in vivo, said polypeptides may be present in solution as monomers.). Consequently, the mere presence of two or more polypeptides, which may have been shown to exist as a complex in vivo, in a composition are not considered as a complex of the invention as such a complex may form only using specific purification methods and conditions and may only be stable after purification under specific storage conditions. Thus, even if certain polypeptides have been shown to form a complex in vivo, said polypeptides may be present in solution as monomers.In a preferred embodiment the complex is a dimer comprising or consisting of HSV polypeptides UL31 and UL34. In the muitimeric complex of the invention, one or more of the proteins may comprise additional B-and/or T-cell epitopes. Said T-celi epitope can be a CD4 T-cell epitope or a CD8 T-cell epitope.

[7] The polypeptides of the vaccine composition may comprise a tag. A polypeptide tag as used herein is an amino acid sequence genetically fused with the recombinant polypeptide, conferring purification and/or detection of the polypeptide. The polypeptides of the vaccine composition may be fused to a HA-tag, Flag-tag, Myc-tag, V5-tag, Strep-tag, Strepll-tag, Sof-tag, His-Strep-Tag, Avi-tag, Calmodulin-tag, E-tag, S-tag, SBP-tag,TC-tag, VSV-tag, Xpress-tag, Ty-tag, Halo-tag, Nus-tag, Thioredoxin-tag, Fc-tag, chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), green fluorescent protein (GFP). In a preferred embodiment the polypeptides of the vaccine composition are fused to a polyhistidine-tag, which may be composed of 6 or 12 His-residues, with 8 His-residues being preferred. A polypeptide tag is preferably fused to the polypeptides of the vaccine composition via a polypeptide linker. A preferred combination of polypeptide linker and 8 His-tag is shown in SEQ ID NO: 3.

[8] A "polypeptide" refers to a molecule comprising a polymer of amino acids linked together peptide bonds. Said term is not meant herein to refer to a specific length of the molecule and is therefore herein interchangeably used with the term “protein”. When used herein, the term “polypeptide” or “protein” also includes a “polypeptide of interest” or “protein of interest” which is expressed by the expression cassettes or vectors or can be isolated from the host cells of the invention. A polypeptide comprises an amino acid sequence, and, thus, sometimes a polypeptide comprising an amino acid sequence is referred to herein as a “polypeptide comprising a polypeptide sequence”. Thus, herein the term “polypeptide sequence” is interchangeably used with the term “amino acid sequence”.

[9] The term "amino acid" or “aa” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[10] An “epitope” is the part of an antigen that is recognized by the immune system, e.g. B cells or T cells. The term encompasses both conformational and linear (or sequential) epitopes. Conformational epitopes comprise discontinuous sections of the antigen's amino acid sequence, whereas linear epitopes are composed of a continuous section of the antigen’s amino acid sequence. A conformational epitope may also comprise sections of two or more antigens’ amino acid sequences. The term further includes cryptotopes and neotopes. “Cryptotopes” are epitopes which are hidden in the naturally occurring antigen, e.g. virus, but can become accessible when the antigen is not present in its natural conformation. “Neotopes” are epitopes found only in quaternary structures of proteins, but not in protein monomers.

[11] B cell epitope is a region of an antigen (e.g., a native protein) recognized by either a particular membrane-bound B-cell receptor (BCR) or an antibody. A number of methods are readily available to identify or select B-cell epitopes, including x-ray crystallography, array-based oligopeptide scanning, site-directed mutagenesis, mutagenesis mapping, and phage display, as well as computational methods as reviewed by Sun et al. Comput Math Methods Med. 2013; 2013: 943636. For example, suitable methods include as structure-based prediction models, which rely on the 3D structure of antigen and epitope-related propensity scales, including geometric attributes and specific physicochemical properties. Structure-based algorithms and web servers (programs) include, e.g., EPSVR & EPMeta (http://sysbio.unl.edu/services/), EPCES (http://sysbio.unl.edu/services/EPCES/), and Epitop^! (http://epitopia.tau.ac.il/). Mimotope-based prediction methods are combinatorial methods which require both antibody affinity-selected peptides and the 3D structure of antigen as input. Exemplary algorithms and programs based on mimotope-based prediction models include, e.g., MimoPro (http://informatics.nenu.edu.cn/MimoPro), PepSurf (http://pepitope.tau.ac.il and EpiSearch (http://curie.utmb.edu/episearch.html). Further, sequence-based prediction models are available which only rely on the primary sequence of an antigen, e.g. BEST and Zhang’s method as reviewed in Sun et al. Comput Math Methods Med. 2013; 2013: 943636. In addition, binding sites prediction models can be used which infer methods that that focus on binding sites prediction of protein-protein interaction the interaction of an antigen and an antibody, e.g. ProMate, ConSurf, PINUP, and PIER.

[12] T -cell epitopes are typically derived from processed protein antigens. A T cell epitope can be a CD4 T-cell epitope or a CD8 T-cell epitope. While cytotoxic (CD8) T-cells recognize intracellular peptides displayed by MHC class I molecules (CD8 T-cell epitopes), T helper cells recognize peptides that are taken up from the extracellular space and displayed by MHC class II molecules (CD4 T-cell epitopes). The peptide:MHC complex (pMHC) interacts with the T-cell receptor, leading to its activation and subsequent induction of a cellular immune response. A number of in silico methods for T cell epitope prediction and/or selection are available. For CD8+ T cell epitope prediction, NetCTL-1.2 (http://www.cbs.dtu.dk/services/NetCTL/), EpiJen (http://www.ddg- pharmfac.net/epijen/EpiJen/EpiJen.htm), or MAPPP (http://www.mpiib-berlin.mpg.de/MAPPP/), can be used, as reviewed in Larsen et al. BMC Bioinformatics 2007, 8:424. For CD4+ T cells, computational models for epitope prediction have been reviewed by Oyarzùn P et al. BMC Bioinformatics 2013, 14:52 and include data-driven methods which rely on peptide sequence comparisons to identify binding motifs, e.g. Rankpep (http://imed.med.ucm.es/Tools/rankpep.html), TEPITOPE, and NN-align (http://www.cbs.dtu.dk/services/NNAIign/), as well as structure-based methods which perform molecular modeling calculations in order to estimate the binding energies, thus offering independence from experimental binding data, e.g. NetMHCIIPan-2.0 (http://www.cbs.dtu.dk/services/NetMHCIIpan-2.0/), TEPITOPEpan (http://www.biokdd.fudan.edu.cn/Service/TEPITOPEpan/), and Predivac (http://predivac.biosci.uq.edu.au/).

[13] The term “Herpes Simplex Virus” and “HSV” are used interchangeably herein and refer generally to the viruses of the herpesviral Genus Simplexvirus, i.e. Ateline herpesvirus 1, Bovine herpesvirus 2, Cercopithecine herpesvirus 1, Cercopithecine herpesvirus 2, Cercopithecine herpesvirus 16, Human herpesvirus 1, Human herpesvirus 2, Macropodid herpesvirus 1, Macropodid herpesvirus 2, Saimiriine herpesvirus 1. Preferred viral species of the Genus Simplexvirus are viruses infecting humans. Even more preferred viral species are Herpes simplex virus 1 (HSV-1) and Herpes simplex virus 2 (HSV-2) which are also known as human herpesvirus 1 and 2 (HHV-1 and HHV-2), respectively.

[14] The term "vaccine composition" as used herein relates to a composition comprising the multimegg complex of the present invention which can be used to prevent or treat a pathological condition associated with HSV in a subject. The “vaccine composition" may or may not include one or more additional components that enhance the immunological activity of the active component or such as buffers, reducing agents, stabilizing agents, chelating agents, bulking agents, osmotic balancing agents (tonicity agents); surfactants, polyols, anti-oxidants; lyoprotectants; anti-foaming agents; preservatives; and colorants, detergents, sodium salts, and/or antimicrobials etc. The vaccine composition may additionally comprise further components typical to pharmaceutical compositions. The vaccine of the present invention is, preferably, for human and/or veterinary use. The vaccine composition may be sterile and/or pyrogen-free. The vaccine composition may be isotonic with respect to humans.

[15] The vaccine composition preferably comprises a therapeutically effective amount of the multimeric complex of the invention or obtainable by the method of the invention.

[16] The HSV polypeptide UL31 of the vaccine composition of the present invention preferably comprises an amino acid sequence which is 85% or more identical to the amino acid sequence of SEQ ID NO: 1, wherein said HSV polypeptide UL31 is capable of eliciting an immune response when administered in the form of a vaccine composition to a subject.

[17] The term “UL31” when used herein relates to the virion egress protein of HSV. SEQ ID NO: 1 depicts exemplarily an amino acid sequence of HSV-2 UL31, also deposited with NCBI GenBank under accession number AHG54695.1. However, the term “UL31” also encompasses UL31 polypeptides having an amino acid sequence which shares a certain degree of identity with the amino acid sequence shown in SEQ ID NO: 1 and also encompasses polypeptides having mutations relative to the reference sequence shown in SEQ ID NO: 1 as described herein. Accordingly, the term “UL31” encompasses polypeptides having an amino acid sequence identity of 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 84%, 83%, 82%, 81%, 80%, or preferably 85% or more compared to the amino acid sequence of SEQ ID NO: 1 or polypeptides having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, 60, 61 or preferably 46 amino acid substitutions, insertions and/or deletions compared to the amino acid sequence of SEQ ID NO: 1. Preferred UL31 proteins can form a dimer with UL34.

[18] "Sequence identity" or “% identity” refers to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the NCBI BLAST program version 2.3.0 (Jan-13-2016) (Altschul et ä32( Nucleic Acids Res. (1997) 25:3389-3402). Sequence identity of two amino acid sequences can be determined with blastp set at the following parameters: Matrix: BLOSUM62, Word Size: 3; Expect value: 10; Gap cost: Existence = 11, Extension = 1; Compositional adjustments: Conditional compositional score matrix adjustment.

[19] The term “immune response" refers to the ability to induce a humoral and/or cell mediated immune response, preferably but not only in vivo. A humoral immune response comprises a B-cell mediated antibody response. A cell mediated immune comprises a T-cell mediated immune response, including but not limited to CD4+ T-cells and CD8+ T-cells. The ability of an antigen to elicit immune responses is called immunogenicity, which can be humoral and/or cell-mediated immune responses. An immune response of the present invention is preferably an immune response against HSV and even more preferably an immune response against a HSV infection in a subject.

[20] A variety of routes are applicable for administration of the vaccine composition of the present invention, including, but not limited to, orally, topically, transdermally, subcutaneously, intravenously, intraperitoneally, intramuscularly or intraocularly. However, any other route may readily be chosen by the person skilled in the art if desired.

[21] The exact dose of the vaccine composition of the invention which is administered to a subject may depend on the purpose of the treatment (e.g. treatment of acute disease vs. prophylactic vaccination), route of administration, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition, and will be ascertainable with routine experimentation by those skilled in the art. The administered dose is preferably an effective dose, i.e. effective to elicit an immune response.

[22] The vaccine composition of the present invention may be administered to the subject one or more times, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.

[23] The “subject” as used herein relates to an animal, preferably a mammal, which can be, for instance, a mouse, rat, guinea pig, hamster, rabbit, dog, cat, or primate. Preferably, the subject is a human. However, the term “subject” also comprises cells, preferably mammalian cells, even more preferred human cells. Such a cell may be an immune cell, preferably a lymphocyte.

[24] The HSV polypeptide UL34 of the vaccine composition the present invention preferably comprises an amino acid sequence which is 70% or more identical to the amino acid sequence of SEQ ID NO: 2, wherein said HSV polypeptide UL34 is capable of eliciting an immune response when administered in the form of a vaccine composition to a subject.

[25] The term “UL34” when used herein relates to the virion egress protein of HSV. SEQ ID NOg2 depicts exemplarily an amino acid sequence of HSV-2 UL34, also deposited with NCBI GenBank under accession number AHG54698.1. However the term “UL34” also encompasses UL34 polypeptides having an amino acid sequence which shares a certain degree of identity with the amino acid sequence shown in SEQ ID NO: 2 and also encompasses polypeptides having mutations relative to the reference sequence shown in SEQ ID NO: 2 as described herein. Accordingly, the term “UL34” encompasses polypeptides having an amino acid sequence identity of 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75% 74%, 73%, 72%, 71%, 69%, 68%, 67%, 66%, 65% or preferably 70% or more compared to the amino acid sequence of SEQ ID NO: 2 or polypeptides having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, or preferably 75 amino acid substitutions, insertions and/or deletions compared to the amino acid sequence of SEQ ID NO: 2. Preferred UL34 proteins can for a dimer with UL31.

[26] As stated, each protein of the invention, may contain mutations, such as insertions, deletions and substitutions relative to the reference sequences shown in SEQ ID NO: 1 (UL31), SEQ ID NO: 2 (UL34), as long as these mutations are not detrimental to the use of the proteins as antigens in the vaccine composition of the present invention. In addition, such mutations should not prevent the capacity of the proteins to form a multimeric complex of the invention. The formation of a multimeric complex of the invention can be tested by performing protein purification, and analyzing the proteins by e.g. nonreducing PAGE, Western blot and/or size exclusion chromatography. In particular, each protein may comprise a tag which, e.g., may facilitate detection, purification and/or enhances solubility.

[27] In a further preferred embodiment of the present invention the polypeptides of the multimeric complex of the vaccine composition of the present invention are HSV-1 polypeptides.

[28] In a further preferred embodiment of the present invention the polypeptides of the multimeric complex of the vaccine composition of the present invention are HSV-2 polypeptides.

[29] The vaccine composition of the invention may further comprise a pharmaceutically acceptable carrier or adjuvant.

[30] The terms “carrier” and “excipient” are used interchangeably herein. Pharmaceutically acceptable carriers include, but are not limited to diluents (fillers, bulking agents, e.g. lactose, microcrystalline cellulose), disintegrants (e.g. sodium starch glycolate, croscarmellose sodium), binders (e.g. PVP, HPMC), lubricants (e.g. magnesium stearate), glidants (e.g. colloidal SiO2), solvents/co-solvents (e.g. aqueous vehicle, Propylene glycol, glycerol), buffering agents (e.g. citrate, gluconates, lactates), preservatives (e.g. Na benzoate, parabens (Me, Pr and Bu), BKC), anti-oxidants (e.g. BHT, BH$e< Ascorbic acid), wetting agents (e.g. polysorbates, sorbitan esters), anti-foaming agents (e.g. Simethicone), thickening agents (e.g. methylcellulose or hydroxyethylcellulose), sweetening agents (e.g. sorbitol, saccharin, aspartame, acesulfame), flavouring agents (e.g. peppermint, lemon oils, butterscotch, etc), humectants (e.g. propylene, glycol, glycerol, sorbitol). Further pharmaceutically acceptable carriers are (biodegradable) liposomes; microspheres made of the biodegradable polymer poly(D,L)-lactic-coglycolic acid (PLGA), albumin microspheres; synthetic polymers (soluble); nanofibers, protein-DNA complexes; protein conjugates; erythrocytes; or virosomes. Various carrier based dosage forms comprise solid lipid nanoparticles (SLNs), polymeric nanoparticles, ceramic nanoparticles, hydrogel nanoparticles, copolymerized peptide nanoparticles, nanocrystals and nanosuspensions, nanocrystals, nanotubes and nanowires, functionalized nanocarriers, nanospheres, nanocapsules, liposomes, lipid emulsions, lipid microtubules/microcylinders, lipid microbubbles, lipospheres, lipopolyplexes, inverse lipid micelles, dendrimers, ethosomes, multicomposite ultrathin capsules, aquasomes, pharmacosomes, colloidosomes, niosomes, discomes, proniosomes, microspheres, microemulsions and polymeric micelles. Other suitable pharmaceutically acceptable excipients are inter alia described in Remington's Pharmaceutical Sciences, 15th Ed., Mack Publishing Co., New Jersey (1991) and Bauer et al., Pharmazeutische Technologie, 5th Ed., Govi-Verlag Frankfurt (1997). The person skilled in the art will readily be able to choose suitable pharmaceutically acceptable carriers, depending, e.g., on the formulation and administration route of the pharmaceutical composition.

[31] The term “adjuvant” as used herein refers to a substance that enhances, augments or potentiates the host's immune response (antibody and/or cell-mediated) to an antigen or fragment thereof. Exemplary adjuvants for use in accordance with the present invention include inorganic compounds such as alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, the TLR9 agonist CpG oligodeoxynucleotide, the TLR4 agonist monophosphoryl lipid (MPL), the TLR4 agonist glucopyranosyl lipid (GLA), the water in oil emulsions Montanide ISA 51 and 720, mineral oils, such as paraffin oil, virosomes, bacterial products, such as killed bacteria Bordetella pertussis, Mycobacterium bovis, toxoids, nonbacterial organics, such as squalene, thimerosal, detergents (Quil A), cytokines, such as IL-1, IL-2, IL-10 and IL-12, and complex compositions such as Freund's complete adjuvant, and Freund's incomplete adjuvant. Generally, the adjuvant used in accordance with the present invention preferably potentiates the immune response to the multimeric complex of the invention and/or modulates it towards the desired immune responses.

[32] The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the multimeric complex according to the present invention.

Purification [33] “Purifying” in all its grammatical forms means removing undesirable compounds, e.g. cells, cell debris, culture medium, baculovirus, either intact or non-intact baculoviruses, etc. Suitable purification methods depending on the expression system, yield, etc. are readily available in the prior art. E.g., purification may include ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography and/or affinity chromatography, all of which have been described extensively before. As said, the purification step includes, inter alia, removing baculoviruses. Such baculoviruses may be contained in the culture medium and/or supernatant obtainable from host cells which were infected with a baculoviral vector or BacMam vector. It is preferred that such baculoviruses be removed when purifying a multimeric complex of the present invention. The present inventors found that in particular ion exchange chromatography, more particularly anion exchange chromatography may be applied to remove baculoviruses from the culture medium and/or supernatant obtainable from a host cell as described herein.

[34] Purifying as used herein also includes that host cells which co-express HSV proteins may be removed from the culture medium. Said culture medium comprises preferably a multimeric complex of the present invention, since said host cells may secrete said multimeric complex. Removing host cells from culture medium may be done by mechanical force, such as by centrifugation or by filtration. Filtration is preferably done by using filtration medium, such as microfiltration filters or on depth-filters. Microfiltration filters may be composed of polyethersulfone or regenerated cellulose. On depth-filters may be composed of polypropylene or glass fibers.

[35] However, it is also envisaged that said host cell do not necessarily have to secrete said multimeric complex. If so, then said host cells may be harvested. After harvest, said host cells may be broken up, e.g., enzymatically or mechanically in order to release a multimeric complex which may then be purified as described herein.

[36] After purification, it is envisaged that a chelating agent is added to the multimeric complex.

Storage [37] “Storing” in all its grammatical forms means preserving (for future use), preferably under conditions which maintain the multimeric complex of the invention in its intact or functional form, i.e. the multimeric complex preferably resembles its naturally occurring form. It is thus envisaged that storing conditions do not promote (or do even prevent) disintegration of the multimeric complex of the invention. The term “disintegration” is to be understood in its broadest sense herein and can mean “disassembly” and/or “dénaturation”. Storage of the multimeric complex of the invention is envisaged in a buffer solution comprising a chelating agent and/or a stabilizing agent.

[38] In general, any chelating agent and/or stabilizing agent is suitable as long as it enables storage of the muitimeric complex of the invention and does not promote its disintegration.

[39] The buffer solution in accordance with the present invention may comprise Tris buffer, NaCI, KCI, PBS, HEPES buffer.

Use of the vaccine composition [40] The present invention also pertains to the use of the vaccine composition in a method of inducing an immune response against HSV in a subject.

[41] In a preferred embodiment of the present invention the vaccine composition is used for the treatment, prevention or amelioration of HSV infection or preventing reactivation of HSV.

[42] Accordingly, the vaccine composition may be used in fighting diseases caused by HSV and/or related symptoms. It is also envisaged that the vaccine composition of the present invention may be used for clearing the virus in a subject, i.e. after treatment no HSV can be detected in a suitable sample obtained from the subject using suitable methods known to those of ordinary skill in the art, e.g. PCR, ELISA etc. Thus, the vaccine composition of the present invention may be used to block primary infection, stop primary disease, block virus reactivation and re-infection, and to block latency.

[43] To reduce the chance of genital herpes a prophylactic vaccine to prevent the first HSV infection of the mother is desirable, whereas an effective therapy is needed in the case a mother is diagnosed with an active HSV infection. A multimeric complex of the present invention may be applied as a prophylactic vaccine, e.g. for expectant mothers or children, or as a therapeutic vaccine in seropositive women to prevent subclinical reactivation at the time of delivery.

[44] In a further preferred embodiment of the present invention the vaccine composition is used in a method for inducing an immune response against HSV-1 or HSV-2 in a subject.

Vector [45] The present invention further pertains to a vector comprising a polynucleotide encoding UL31 and UL34. Generally, the genes encoding the HSV proteins can also be present on more than one vector, e.g. on two vectors. Preferably, however, said genes are present on a single vector. Genes may also be present in polygenic form (EP1945773).

[46] The term "vector" as used herein refers to a nucleic acid sequence into which an expression cassette comprising a gene encoding the protein of interest may be inserted or cloned. Furthermore, the vector may encode an antibiotic resistance gene conferring selection of the host cell. Preferably, the vector is an expression vector.

[47] The vector can contain elements for propagation in bacteria (e.g. E. coli), yeast (e.g. §£< cerevisiae), insect cells and/or mammalian cells. Preferably, said vector is a Baculovirus vector or a Baculovirus BacMam vector. The vector may have a linear, circular, or supercoiled configuration and may be complexed with other vectors or other material for certain purposes.

[48] In the BacMam system, baculovirus vectors are used to deliver genes into mammalian cells. The BacMam system can be used for gene delivery to a broad range of cell lines and primary cells as host cells, an exemplary list of which is included elsewhere herein. The unmodified baculovirus is able to enter mammalian cells, however its genes are not expressed unless a mammalian recognizable promoter is incorporated upstream of a gene of interest. Thus, it is envisaged that the BacMam vector of the invention comprises a mammalian promoter upstream the genes encoding the proteins of the multimeric complex of the invention. The vector may comprise additional elements as described elsewehere herein, e.g. antibiotic resistance genes, elements for propagation in E.coli, S. cerevisiae etc.

[49] The vector may contain one or more further elements, including, e.g., an origin of replication, promoters, cloning sites, genetic markers, antibiotic resistance genes, epitopes, reporter genes, targeting sequences and/or protein purification tags. The person skilled in the art will readily know which elements are appropriate for a specific expression system.

[50] In particular, the vector in accordance with the invention may further contain elements for propagation in bacteria (E. coli), yeast (S. cerevisiae), insect cells and/or mammalian cells, such as origin of replication, selection markers, etc.

[51] It is envisaged that the vector comprises a promoter for gene expression. Each of the gene encoding the proteins of the invention described herein is driven by a promoter. The promoters are preferably selected from the group consisting of polh, p10 and ρχιν very late baculoviral promoters, vp39 baculoviral late promoter, vp39polh baculoviral late/very late hybrid promoter, pca/polh, pcna, etl, p35, egt, da26 baculoviral early promoters; CMV-IE1, UBc. EF-1, RSVLTR, MT, Simian virus 40 promoter, CAG promoter (beta-actin promoter with CMV-IE1 enhancer), hepatitis B virus promoter/enhancer, human ubiquitin C promoter, hybrid neuronal promoter, pds47, Ac5, and Pgal and Padh. Each of the genes is followed by a terminator sequence such as HSVtk terminator, SV40 terminator, or bovine growth hormone (BGH) terminator.

[52] The terms “polynucleotide”, "nucleotide sequence" or "nucleic acid molecule" are used interchangeably herein and refer to a polymeric form of nucleotides which are usually linked from one deoxyribose or ribose to another. The term "polynucleotide" preferably includes single and double stranded forms of DNA or RNA. A nucleic acid molecule of this invention may include both sense and antisense strands of RNA (containing ribonucleotides), cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non- d Ω natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Sugfe modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

[53] In this regard, a nucleic acid being an expression product is preferably a RNA, whereas a nucleic acid to be introduced into a cell is preferably DNA or RNA, e.g. synthetic DNA, genomic DNA or cDNA.

[54] Also envisaged is a vaccine composition comprising a nucleic acid or a vector encoding the multimeric complex as disclosed herein. Said nucleic acid or vector can be DNA- or RNA-based. Suitable vectors for use in accordance with the vaccine composition include DNA-based vectors such as baculovirus vectors, BacMam vectors, adenovirus vectors, lentiviral vectors, AAV vectors, herpesvirus vectors, poxvirus vectors, and Epstein-Barr virus (EBV) vectors. The use of naked DNA; e.g. in the form of a plasmid, and optionally complexed and/or in stabilized form (e.g. lipoplexes, polypiexes, dendrimers, virosomes and complexes with inorganic nanoparticles) is also envisaged. Suitable RNA-based vectors include retroviral vectors, Semliki forest virus (SFV), Sindbis virus (SIN) and Venezuelan equine encephalitis virus (VEE) vectors.

[55] The vaccine composition comprising the multimeric complex and the vaccine composition comprising the nucleic acid or the vector encoding the multimeric complex may be used in a prime boost regimen. In the prime boost regimen, a prime/boost vaccine is used which is composed of two or more types of vaccine including a vaccine used in primary immunization (prime or priming) and a vaccine used in booster immunization (boost or boosting). The vaccine used in primary immunization and the vaccine used in booster immunization may differ from each other. Primary immunization and boosting immunization may be performed sequentially, this is, however, not mandatory. The prime/boost regimen includes, without limitation, e.g. DNA prime/protein boost. However, the boosting composition can also be used as priming composition and said priming composition is used as boosting composition.

Host cell [56] The present invention further pertains to a host cell comprising a vector comprising a polynucleotide encoding UL31 and UL34.

[57] The host cell may be an insect cell or mammalian cell. However, the host cell may also be bactega (e.g. E. coli) or yeast (e.g. S. cerevisiae). Generally, any host cell that is suitable to express nucleic acid molecules to produce the multimeric complex of the invention may be used. The host cell used in accordance with the invention may be an insect cell, such as Sf9, Sf21, Super Sf9-1 (VE-1), Super Sf9-2 (VE-2), Super Sf9-3 (VE-3), Hi-5, Express Sf+, and S2 Schneider cells, with Hi-5 being preferred [Oxford Expression Technologies, Cat. No. 600103, Oxford, UK; Fath-Goodin et al. (2006), Adv. Virus Res. 68, 75-90; Kraemer et al. (2006), J. Virol. 80(24), 12291-12228 and US20060134743.]. Exemplary mammalian host cells that may be used are known in the art and include immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, HEK293, HEK293F, CHO, HeLa, HUVEC, HUAEC, Huh7, HepG2, BHK, MT-2, Cos-7, Cos-1, C127, 3T3, human foreskin fibroblasts (HFF), bone-marrow fibroblasts, Bowes melanoma, primary neural cells, or epithelial cells. In the BacMam system, baculovirus expression vectors are used to deliver genes to mammalian cells.

Method for production [58] The present invention further provides a method for producing the vaccine composition comprising the multimeric complex, comprising (i) culturing a host cell of the present invention; (ii) obtaining a multimeric complex; (iii) and admixing said multimeric complex with a pharmaceutically acceptable carrier or adjuvant.

[59] It is to be noted that the embodiments described in the context of the multimeric complex of the invention also apply to the method of the invention, mutatis mutandis.

[80] The multimeric complex may be expressed in a host cell, preferably insect cell or mammalian cell, by using baculovirus, e.g., a Baculovirus expression system or BacMam expression system. An “expression vector” is defined herein as vehicle used to transfer genetic material to a target host cell where the genetic material can be expressed. An “expression system” is the combination of an expression vector, and the host cell for the vector that provide a context to allow foreign gene expression in the host cell. The complex of the present invention may be expressed transiently or stably.

[61] The baculovirus expression system is typically based on the introduction of a foreign gene into a nonessential viral genome region, e.g. via homologous recombination with a transfer vector containing a target gene. The resulting recombinant baculovirus may lack one of the nonessential genes (e.g. polh, v-cath, chiA) replaced with a foreign gene encoding the heterologous protein which can be expressed in a suitable host cell. These techniques are generally known to those skilled in the art and have been reviewed e.g. by Kosta et al. Nat Biotechnol. 2005; 23(5):567-75. A specific approach for preparing recombinant baculovirus vectors is the Bac-to-Bac® baculovirus system (Invitrogen).

[62] The recombinant baculovirus expression vector may be capable of replication in a host cell and' optionally in a prokaryotic cell such as E. coli. According to the present invention, any baculovirus expression vector derived from a baculovirus commonly used for the recombinant expression of proteins may be used. For example, the baculovirus vector may be derived from, e.g., AcMNPV, Bombyx mori (Bm)NPV, Helicoverpa armigera (Hear) NPV) or Spodoptera exigua (Se) MNPV. The baculovirus vector may be a bacmid.

[63] It must be noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “an expression cassette” includes one or more of the expression cassettes disclosed herein and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[64] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or sometimes when used herein with the term “having”.

[65] When used herein “consisting of' excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms "comprising", "consisting essentially of and "consisting of' may be replaced with either of the other two terms.

[66] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes also the concrete number, e.g., about 20 includes 20.

[67] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present invention are generally performed according to conventional methods well-known in the art. Generally, nomenclatures used in connection with techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[68] The methods and techniques of the present invention are generally performed according conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e. g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001); Ausubel et al., Current Protocols in Molecular Biology, J, Greene Publishing Associates (1992, and Supplements to 2002); Handbook of Biochemistry: Section A Proteins, Vol I 1976 CRC Press; Handbook of Biochemistry: Section A Proteins, Vol II 1976 CRC Press. The nomenclatures used in connection with, and the laboratory procedures and techniques of, molecular and cellular biology, protein biochemistry, enzymology and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

DESCRIPTON OF THE FIGURES

[69] Fig. 1 : Amino acid sequences of HSV proteins of the present invention [70] Fig. 2: Example of a SDS-PAGE and a western blot showing the UL31-His/UL34 dimer [71] Fig. 3: Example of a SDS-PAGE and a western blot showing the UL31/UL34-His dimer [72] Fig. 4: Example of a SDS-PAGE and a western blot showing the UL31/UL34-His dimer

d C

EXAMPLES

The following Examples illustrate the invention, but are not to be construed as limiting the scope of the invention.

[73] Example 1 :

The UL31-HÎS/UL34 dimer was expressed in Hi-5 insect cells and released from cell pellets after proper lysis. The dimer was subsequently purified using IMAC and a 0-500 mM imidazole continuous gradient buffer system (50 mM Hepes, 500 mM NaCI, pH 7.0, 1 mM TCEP). Fig.2 (A) An example of an SDS-PAGE is shown. One of the two most concentrated fractions is marked with an asterisk. Fig.2 (B) An example of the western blot performed using an anti-His antibody to detect UL31-His is shown. Labeling in both examples: 1. Standards, 2. Supernatant, 3. Filtrated supernatant, 4. Cell pellet, 5. Flowthrough, 6. Wash of unbound protein, 7-13. Fractions A8, A10, B4, B8, B11, C2, C7.

[74] Example 2:

The UL31/UL34-HÎS dimer was expressed in Hi-5 insect cells and released from cell pellets after proper lysis. The dimer was subsequently purified using IMAC and a 25-500 mM imidazole continuous gradient buffer system (50 mM Hepes, 500 mM NaCI, pH 7.0, 1 mM TCEP). Fig.3 (A) An example of an SDS-PAGE is shown. One of the two most concentrated fractions is marked with an asterisk. Fig.3 (B) An example of the western blot performed using an anti-His antibody to detect UL34-His is shown. Labeling in both examples: 1. Standards, 2. Supernatant, 3. Filtrated supernatant, 4. Cell pellet, 5. Flowthrough, 6. Wash of unbound protein, 7-13. Fractions A8, A10, B4, B8, B11, C2, C7.

[75] Example 3:

The UL31/UL34-HÏS dimer was expressed in Hi-5 insect cells and released from cell pellets after proper lysis. The dimer was subsequently purified using IMAC and a 0-500 mM imidazole buffer system (50 mM Hepes, 500 mM NaCI, pH 7.0, 1 mM TCEP, 10% glycerol). Impurities were washed out in two steps by applying 50 mM and 75 mM imidazole to the column. The dimer was then eluted with 350 mM imidazole, followed by dialysis in Hepes buffer without imidazole (50 mM Hepes, 500 mM NaCI, pH 7.0, 0.5 mM TCEP, 10% glycerol). Fig.4 (A) An example of an SDS-PAGE is shown. Fig.4 (B) An example of the western blot performed using an anti-His antibody to detect UL34-His is shown. Labeling in both examples: 1. Standards, 2. Culture cell pellet, 3. Lysate, 4. Supernatant, 5. Crude pellet, 6. Filtrated supernatant, 7. Flowthrough, 8-16. Fractions B12, C2, C5, C7, C11 and F1 17. Pool of fractions C3-C7, 18. Dialysis material total, 19. Supernatant after dialysis, 20. Pellet after dialysis, 21. Filtrated protein.

Claims (14)

A vaccine composition comprising a multimeric complex of herpes simplex virus (HSV) polypeptides UL31 and UL34. The vaccine composition of claim 1, wherein the HSV polypeptide UL31 comprises an amino acid sequence that is 85% or more identical to the amino acid sequence of SEQ ID NO: 1, wherein the HSV polypeptide UL31 is administered to a subject as a vaccine composition , can cause an immune response. The vaccine composition of claim 1, wherein the HSV polypeptide UL34 comprises an amino acid sequence that is 70% or more identical to the amino acid sequence of SEQ ID NO: 2, wherein the HSV polypeptide UL34 is administered to a subject as a vaccine composition , can cause an immune response. A vaccine composition according to any one of the preceding claims, wherein the polypeptides are HSV-1 polypeptides. A vaccine composition according to any one of the preceding claims, wherein the polypeptides are HSV-2 polypeptides. A vaccine composition according to any one of the preceding claims which is encoded by a nucleic acid. A vaccine composition according to any one of the preceding claims, further comprising a pharmaceutically acceptable carrier or adjuvant. A vaccine composition according to any one of the preceding claims, for use in a method of inducing an immune response to HSV in a subject. A vaccine composition for use according to claim 8 which is for the treatment, prevention or amelioration of HSV infection or for the prevention of HSV reactivation. A vaccine composition for use according to claim 8 or 9, wherein the HSV is HSV-1. The vaccine composition for use according to claim 8 or 9, wherein the HSV is HSV-2. 12. Vector comprising a polynucleotide encoding UL31 and UL34. 13. A host cell comprising the vector of claim 12. 14. A method of preparing the vaccine composition according to any one of claims 1 to 17, comprising (i) cultivating a host cell according to claim 13; (ii) obtaining a multimeric complex; (iii) and mixing the multimeric complex with a pharmaceutically acceptable carrier or adjuvant.