Novel immunomodulatory factor

Abstract

A chemokine mixture suitable for modulating an immune response, comprising a first chemokine, or an agonist or antagonist mutant thereof, suitable for binding to CCR4, and a second chemokine, or an agonist or antagonist mutant thereof, suitable for binding to CCR5 and / or CCR6.

Description

[Technical field]

[0001] The present invention relates to chemokine mixtures, polynucleotide mixtures and compositions suitable for modulating immune responses, as well as corresponding nucleic acid construct mixtures, nucleic acid constructs, host cells, pharmaceutical compositions and kits. The present invention also relates to the use of the chemokine mixtures, polynucleotide mixtures and compositions for the treatment of diseases and conditions, and to methods of treatment using the chemokine mixtures, polynucleotide mixtures and compositions of the invention. [Background technology]

[0002] Many diseases and conditions are associated with an excessive immune response, a dysregulated immune response, or an abnormal immune response. Examples of these are autoimmune diseases such as rheumatoid arthritis, inflammatory diseases, severe Covid or influenza, or infectious diseases associated with an excessive immune response such as antibody or cytokine storms as seen in chronic obstructive pulmonary disease, and conditions such as asthma and allergies. Currently, effective treatments and preventions of these diseases and conditions are often limited.

[0003] Immunotherapy using advanced therapeutic strategies holds great promise for the treatment of a wide range of pathologies including infectious diseases, cancer and autoimmune, inflammatory diseases and diseases or conditions associated with an abnormal or dysregulated immune system.

[0004] An appropriate immune response is central to the control of diseases, including infectious, inflammatory or neoplastic, as well as efficient immunization to prevent or treat infections or cancer. For this reason, a critical balance exists between conventional T cells, such as those that mediate adaptive immunity, and regulatory T cells (T-reg), which dampen induced responses to prevent autoimmunity (Mohr, Atif et al. 2019). In adaptive immunity, the therapeutic response recognizes damaged or infected cells and eliminates them through a complex interplay of innate and adaptive immune responses by antigen presentation to T cell subsets, resulting in specific memory and effector cells stimulated to eliminate the infected or damaged cells. Similarly, the regulation of this response is important, and after disabling and eliminating pathogen-infected or cancer cells, the immune response must be turned off, otherwise there is the possibility of pathogenic autoimmune responses, which can be equally fatal.

[0005] The focus for immunization against pathogens or treatment of cancer has been to boost the immune response using adjuvants. The focus for treatment of autoimmunity has been more limited, for example, to reduce the immune response using steroids such as dexamethasone for Covid runaway responses. Immunization for some pathogens, such as persistent infections such as herpes viruses or HIV that can establish latent infections, has not been successful. Furthermore, the durability of the response has been a concern, for example, new RNA vaccines against Covid have limited efficiency over time, lasting only a few months for an efficient antibody response and requiring booster vaccines (Bar-On, Goldberg et al. 2021, Eyre, Taylor et al. 2021). Immunization against cancer has also been similarly unsuccessful, as cancer can also evade the immune response or attract regulatory white blood cells. Meanwhile, limiting autoimmune responses such as runaway responses as seen in severe Covid, influenza cytokine storm, Crohn's disease, or sepsis has similarly not provided an efficient treatment.

[0006] New cancer therapies are directed towards strategies to "modulate regulators" by providing inhibitory antibody treatments against "checkpoints" of the immune response. However, these are only effective in a minority of patients. Nevertheless, a focus on regulating responses may be applicable to new solutions against autoimmunity as well as immunization as well. Central to this is the chemokine system, which can mediate the recruitment of T-reg populations and regulator leukocytes (Lu, Barbi et al. 2017; Luo and Li 2018; Mohr, Atif et al. 2019). Chemoattractant T-regs can dampen aberrant immune responses, e.g., late severe Covid or inflammatory conditions such as arthritis or skin inflammatory conditions (Ikebuchi, Fujimoto et al. 2019; Mohr, Atif et al. 2019). Conversely, blocking chemoattraction of T-regs can keep the immune-stimulating state in favor of the balance of conventional T-cell-mediated adaptive immunity, e.g., in cancer therapy (Ohue and Nishikawa 2019).

[0007] Previous studies have shown that blocking immune suppressors is a well-defined mechanism for the application of antibody molecules, i.e. "immune checkpoint inhibitors" in oncology. They target either receptors or ligand pairs of signaling molecules on immunosuppressive T lymphocytes that normally put a "brake" on immune signaling, for example to prevent the breakdown of immune tolerance and the release of unfavorable "autoimmune" responses. Although this strategy has been used successfully for patients with terminal cancer, it has inherent safety risks, works only in a minority of patients (less than 30%) (Ikebuchi, Fujimoto et al. 2019), and the success rate is not favorable for small molecule development. This strategy also requires additional resources for genetic profiling of patients and their cancers to determine sensitivity to this method via specific receptor expression on the tumor. Therefore, it is necessary to develop new ways to apply immunotherapy to more people and with a higher success rate.

[0008] Chemokines have been utilized as molecular adjuvants in experimental vaccine formulations and for the development of potential cancer immunotherapies (Mohan, Zhu et al. 2018, Ohue and Nishikawa 2019). Chemokines can activate and recruit immune cell subsets through chemoattraction to treat disease or enhance immune responses in immunization (Bobanga et al., 2013). Viral modifications of chemokines present a unique combination of properties that are useful as vaccine immunomodulators or in immunotherapy of diseases such as cancer or in autoimmunity (Vilgelm et al., 2019). Chemokines as adjuvants have been used to amplify protective immunity by their modulation of lymphocyte primer and effector functions and their development (Mohan et al., 2018).

[0009] Interestingly, chemokines can also chemoattract regulatory leukocytes to suppress immune responses, as opposed to enhancing responses. Thus, chemokines can modulate immune responses by enhancing or decreasing immune responses, depending on the chemokine receptors they present on interacting leukocyte subpopulations. T-regs have markers such as the transcription factor FOXP3, and subsets have been shown to express CC chemokine receptors CCR4, CCR5, CCR6, or CCR8 (Bayry et al., 2008; Schlecker et al., 2012; Barsheshet et al., 2017; Ohue and Nishikawa 2019; Snelgrove et al., 2019). Ligands for these receptors can chemoattract T-regs to dampen immune responses. CCR5 can indicate the activation state of T-reg subsets, for example for immunosuppression during pregnancy to prevent fetal rejection, or similarly in graft-versus-host disease during transplantation (Kallikourdis, Andersen et al. 2007; Schlecker, Stojanovic et al. 2012). Attraction of CCR5-expressing T-regs by ligand-secreting monocytic myeloid-derived suppressor cells thereby promoted tumor growth through evasion of conventional T cells (Schlecker, Stojanovic et al. 2012). Furthermore, CCR6-expressing T-regs can regulate trafficking to the thymus and similarly inflamed endothelium (Snelgrove, Abeynaike et al. 2019; Peligero-Cruz, Givony et al. 2020). Furthermore, CCR4- and CCR8-expressing T-regs can regulate cancer immunity by inhibiting anti-tumor immune responses, and CCR8-expressing T-regs regulate autoimmune encephalitis (Barsheshet, Wildbaum et al. 2017; Villarreal, L'Huillier et al. 2018; Ohue and Nishikawa 2019).WO2011 / 138785 discloses that CCL1 is specific for CCR8 on T-regs and treats inflammatory diseases, autoimmune diseases, neuroinflammatory diseases such as encephalitis, and transplant-related diseases, graft-versus-host disease.

[0010] The effector moiety of activated T-regs may contain numerous mechanisms to suppress the activation and function of other leukocytes (Lu, Barbi et al. 2017, Mohr, Atif et al. 2019). They can express co-inhibitory molecules such as CTLA4 and LAG3 or secrete anti-inflammatory cytokines, as well as starve growth factors such as IL-2 through interaction with CD25. For example, CCR4 has been shown to be expressed on CD4+CD25+ T-regs (Iellem, Mariani et al. 2001). T-regs play a role in dampening immune responses, allowing self-tolerance and preventing pathogenic over-responses to infection (Miyara and Sakaguchi 2007, Lu, Barbi et al. 2017, Luo and Li 2018). CCR4 binds both chemokines CCL17 and CCL22 secreted by dendritic cells (DCs) and can chemoattract T-regs, where they enhance the interaction between DCs and CCR4+ T cells (Tang and Cyster 1999; Iellem, Mariani et al. 2001; Katou, Ohtani et al. 2001; Wu, Fang et al. 2001; Bayry, Tchilian et al. 2008; Snelgrove, Abeynaike et al. 2019). Different subsets of T-regs exist with CCR4 alone or with CCR6 (Mohr 2018). On the other hand, CCR5 has been shown to mark activated "effector" T-regs that are effective in immune tolerance, for example with the fetus (Kallikourdis, Andersen et al. 2007) or that regulate inflamed skin (Ikebuchi, Fujimoto et al. 2019). T-regs may suppress DC maturation and costimulatory molecule expression, thereby reducing their role in T cell activation. Thus, chemoattraction of T-regs can inhibit immune responses, while antagonizing their recruitment during immunization can enhance immune responses induced by immunization.

[0011] Modified ligands for individual receptors, e.g., CCL1 for CCR8, have shown some utility in animal models in suppressing immune responses (Barsheshet, Wildbaum et al. 2017). However, due to redundancy in the system, other receptors on T-regs can still function, and novel treatments acting through this population of regulatory cells need to be developed.

[0012] Modified or mutated chemokines have also been developed and studied for their effects in treating diseases. For example, Met-CCL5 (also known as Met-RANTES) is an amino-terminally modified mature CCL5 protein in which a methionine residue is added to the amino terminus of the mature protein. Met-CCL5 was produced by recombinant expression of a cDNA encoding mature CCL5 in Escherichia coli (E. coli) (Proudfoot et al., 1996) and has been shown to antagonize T cell migration (Proudfoot et al., 1996) and antagonize monocyte migration and CCL5 / RANTES-induced chemotaxis (Proudfoot et al., 1999). This modification of the amino terminus of CCL5 interferes with its ability to fully activate certain signaling events without affecting other receptor activation states that lead to events such as receptor internalization (Proudfoot et al., 1999).

[0013] WO96 / 17935 discloses that modified forms of CCL5 / RANTES act as antagonists to CCL5 / RANTES due to the presence of one or more N-terminal amino acids that are not present at the corresponding positions in CCL5 / RANTES. The modified CCL5 / RANTES can be Met-RANTES, Leu-RANTES or Gln-RANTES.

[0014] CA2468790 discloses that mutants of CCL5 containing single non-conservative substitutions in a consensus sequence common to a subset of CC chemokines act as antagonists of CCL5. The human CC chemokines that share this consensus sequence are CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL11, CCL13 and CCL15.

[0015] WO2009 / 150433 discloses small molecule antagonists of CCR4 that enhance dendritic cell-mediated human T cell proliferation.

[0016] KR 20210003550 discloses compositions of one or more of CCL4, CCL5, CCL20 and CCL21 for treating or preventing infertility by improving endometrial cell proliferation and reducing the expression of endoplasmic reticulum stress-inducible proteins.

[0017] US2013344119 discloses an implantable composition comprising at least one of IL-8, MIP-3α (also known as CCL20) and their derivatives for treating tissue damage or promoting tissue regeneration through an immune response.

[0018] The above examples are of limited use, and therefore there is a need for the development of additional specific and more effective immunotherapies useful as treatments and vaccines against diseases by modulating immune responses. In particular, there is a need for the development of additional treatments and vaccines against a wide range of diseases and conditions associated with abnormal immune responses, such as cancer (Karin 2018, Ohue and Nishikawa 2019), HIV infection (Catusse, Parry et al. 2007), autoimmune and inflammatory diseases (Mohr, Atif et al. 2019), cytokine storm, sepsis (Dong, Wang et al., 2020), and diseases associated with runaway or excessive immune responses, such as allergies (Mikhak et al., 2009). Summary of the Invention

[0019] The inventors determined that by administering the appropriate mixture of agonist ligands for all T-regs, they could block the immune response, whereas, conversely, with antagonistic ligands, they could block T-regs and thereby stimulate the appropriate immune response. However, the exact mixture is unclear, considering that there are at least 23 chemokine receptors and 51 chemokine ligands in the CC chemokine family alone (Hughes and Nibbs 2018, Mohan, Zhu et al. 2018). Furthermore, there are multiple subsets of T-regs with different tissue expression and multiple chemokine receptor utilization, both of which mark different T-reg subsets and their activation states (Lu, Barbi et al. 2017, Luo and Li 2018, Mohr, Atif et al. 2019). To decipher this, the inventors studied the genome of herpes viruses. This is because these viruses can modulate the chemokine system with homologs to chemokines and chemokine receptors. Human betaherpesvirus 6A, HHV-6A, encodes a chemokine homolog, U83A, that binds and activates multiple chemokine receptors (Dewin, Catusse et al. 2006; Catusse, Parry et al. 2007). In US9,850,286 and US8,940,686, it was disclosed that the utility of full-length U83A is to enhance immune responses. Subsequent studies identify endogenous forms of this genome in human chromosomes integrated at the telomeres, an inherited human-adapted set of viral genes. These encode human-adapted forms of viral full-length chemokine homologs expressed in some people (Tweedy, Spyrou et al. 2015; Tweedy, Spyrou et al. 2016). The encoded chemokine receptor binding and signaling domains are conserved and combine activity for CCR4, CCR5, CCR6 and CCR8 (Dewin, Catusse et al. 2006; Catusse, Parry et al. 2007).A novel splicing form was identified by the inventors, the cDNA for which, iciU83A-N (SEQ ID NO: 16, referred to herein as VIT1), encodes the chemokine receptor binding domain but does not retain the signaling domain. Thus, VIT1 may act as an antagonist of these chemokine receptors. These receptors are present on immune-stimulating leukocytes as well as T-reg cells (Hughes and Nibbs 2018, Mohan, Zhu et al. 2018). Thus, the inventors tested a mixture of human chemokines to stimulate these receptors and compared the functional activity against novel humanized viral gene cDNA antagonists with these receptors. Surprisingly, when comparing in a preclinical model of immunization against HSV2, which involves combining chemokine preparations with glycoprotein gD, a well-established and known immunogen, the results with the novel antagonist VIT1 stimulated protective immunity, whereas the human chemokine agonist mixture (herein referred to as VTL3) inhibited immunity. This was reflected in the resulting specific antibody response: the novel antagonist VIT1 induced a specific antibody response, whereas the novel human chemokine agonist mixture VTL3 completely inhibited any antibody production. This is in line with recent results defining CCR4, CCR5, CCR6 and CCR8 for T-reg populations, and the combined activity against all these receptors may preclude the recruitment of all T-reg populations, thereby inhibiting helper and effector T cell proliferation-stimulating antibodies and effector cytotoxic T cell production. On the other hand, blocking T-reg chemoattraction with novel antagonists allows conventional adaptive T cell immunity to proceed uninhibited, thereby improving cellular immune responses. This efficient blockade of host cellular immunity with agonist mixtures can treat autoimmune or inflammatory diseases that involve runaway pathogenic immune responses.

[0020] By modulating interactions with regulatory leukocytes with novel chemokines or chemokine mixtures, it can control aberrant immune responses, for example in autoimmune conditions or inflammation. Conversely, if these interactions are inhibited, it can provide a transient stimulation by preventing regulatory leukocytes from dampening the immune response, with utility for vaccines against infectious diseases or for inducing immunological control of cancer.

[0021] Thus, it has now surprisingly been found that mixtures of chemokines or their agonist or antagonist variants that target CC chemokine receptors present on regulatory T cells or other regulatory leukocytes, such as monocytic myeloid-derived suppressor cells (MDSCs), can be used to regulate immune responses, thus resulting in the present invention.For example, MDSCs can secrete chemokines to attract T-regs (Schlecker et al., 2012).In particular, it has now surprisingly been found that mixtures that include CCR5 agonists and CCR4 and / or CCR6 agonists suppress immune responses.This is surprising, since it is not possible to predict how different chemokines and their variants will interact with each other with any reasonable expectation of success. To illustrate, various chemokine receptor chemokine interactions exist on various cancers, with individual chemokines being ligands for both activating and regulatory T cells (Korbecki, Grochans et al. 2020; Korbecki, Kojder et al. 2020; Hughes and Nibbs 2018; Mohan et al. 2018). As discussed above, human herpesviruses encode the chemokine homolog U83A, whose specificity for these receptors is interpreted as acting as an agonist, since it was demonstrated to effectively bind and signal to these receptors expressed separately (Dewin, Catusse et al. 2006; Catusse, Parry et al. 2007). In fact, the utility was based on its use as an agonist to stimulate immune responses, as disclosed in US9,850,286 B2 and US8,940,686. However, it has now surprisingly been found that a mixture of human chemokine agonists against these same sets of receptors results in immune suppression instead.Furthermore, a novel cDNA derived from the integrated human HHV-6A genome (VIT1) has only the binding domain and its utility has been shown to be the opposite, stimulating the immune response, along with a dominant activity of antagonizing the receptor on T-reg cells.Thus, combined targeting of these receptors present on T-regs with agonists chemoattracts the combined subpopulation, resulting in inhibition of the immune response, whereas antagonism results in the opposite, ie, immune stimulation.

[0022] The group of chemokine receptors targeted is present on the T-reg subset. CCR5 is present on activated T cell subsets, including regulatory T cells, while CCR4, CCR8 and CCR6 are present on regulatory T cell subsets. This set of chemokine receptors can be defined as those present as characterized on regulatory T cells or monocytic subpopulations, while these receptors are also present individually on conventional T cells (Hughes and Nibbs 2018, Mohan, Zhu et al. 2018). Thus, without being limited by theory, it is believed that the agonist or antagonist chemokine mixture of the present invention can modulate the immune response by stimulating receptors present on regulatory T cells, thereby recruiting regulatory T cells that act to attenuate or inhibit the immune response, or by antagonizing receptors present on regulatory T cells, thereby preventing the recruitment of regulatory T cells and causing the induction or enhancement of the immune response.

[0023] Thus, in a first aspect of the present invention there is provided a chemokine mixture suitable for modulating an immune response comprising a first chemokine, or an agonist or antagonist variant thereof, suitable for binding to CCR4, and a second chemokine, or an agonist or antagonist variant thereof, suitable for binding to CCR5 and / or CCR6.

[0024] Preferably, the first chemokine, or an agonist or antagonist variant thereof, comprises CCL17, or an agonist or antagonist variant thereof.

[0025] Conveniently, the second chemokine, or an agonist or antagonist variant thereof, comprises CCL5 or CCL20, or an agonist or antagonist variant thereof.

[0026] Suitably, the first chemokine, or an agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:5, or SEQ ID NO:12, and / or the second chemokine, or an agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:26.

[0027] Preferably, the chemokine mixture comprises a third chemokine, or an agonist or antagonist variant thereof, wherein the third chemokine, or an agonist or antagonist variant thereof, is suitable for binding to one of CCR5 and CCR6 that is different from the second chemokine, or an agonist or antagonist variant thereof.

[0028] Suitably, the third chemokine, or an agonist or antagonist variant thereof, comprises CCL20, or an agonist or antagonist variant thereof.

[0029] Conveniently, the third chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:8 or SEQ ID NO:14.

[0030] Preferably, a) the first chemokine comprises CCL17, the second chemokine comprises CCL5 or SEQ ID NO:26, and the chemokine mixture comprises a third chemokine comprising CCL20; or b) the first chemokine comprises Met-CCL17, the second chemokine comprises Met-CCL5 or SEQ ID NO:20, and the chemokine mixture comprises a third chemokine that comprises Met-CCL20; or c) The first chemokine comprises CCL17, the second chemokine comprises Met-CCL5 or SEQ ID NO:20, and the chemokine mixture comprises a third chemokine that comprises CCL20.

[0031] Conveniently, a) the first chemokine comprises the amino acid sequence of SEQ ID NO:5, the second chemokine comprises the sequence of SEQ ID NO:2 or SEQ ID NO:26, and the chemokine mixture comprises a third chemokine comprising the amino acid sequence of SEQ ID NO:8; or b) the first chemokine comprises the amino acid sequence of SEQ ID NO:12, the second chemokine comprises the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:20, and the chemokine mixture comprises a third chemokine comprising the amino acid sequence of SEQ ID NO:14; or c) the first chemokine comprises the amino acid sequence of SEQ ID NO:5, the second chemokine comprises the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:20, and the third chemokine comprises the amino acid sequence of SEQ ID NO:8.

[0032] In a second aspect of the present invention, there is provided a polynucleotide mixture comprising a first polynucleotide and a second polynucleotide, wherein said first polynucleotide comprises a nucleic acid sequence encoding a first chemokine, or an agonist or antagonist variant thereof, suitable for binding to CCR4, and said second polynucleotide comprises a nucleic acid sequence encoding a second chemokine, or an agonist or antagonist variant thereof, suitable for binding to CCR5 and / or CCR6, and wherein said polynucleotide mixture is suitable for modulating an immune response.

[0033] Preferably, the first polynucleotide comprises a nucleic acid sequence encoding CCL17, or an agonist or antagonist variant thereof.

[0034] Suitably, the second polynucleotide comprises a nucleic acid sequence encoding CCL5, or an agonist or antagonist variant thereof, CCL20, or an agonist or antagonist variant thereof, or one of SEQ ID NO:20 and SEQ ID NO:26.

[0035] Conveniently, the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:6 and SEQ ID NO:13, and wherein the second polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:16 to SEQ ID NO:18 and SEQ ID NO:21 to SEQ ID NO:24.

[0036] Preferably, the second chemokine or a functional variant thereof is suitable for binding to CCR5, and the polynucleotide mixture comprises a third polynucleotide, wherein the third polynucleotide comprises a nucleic acid sequence encoding a third chemokine, or an agonist or antagonist variant thereof, wherein the third chemokine, or an agonist or antagonist variant thereof is suitable for binding to CCR6.

[0037] Suitably, the third chemokine, or an agonist or antagonist variant thereof, comprises CCL20, or an agonist or antagonist variant thereof.

[0038] Conveniently, the third polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:13 or SEQ ID NO:15.

[0039] Preferably, a) the first polynucleotide comprises a nucleic acid sequence encoding CCL17, the second polynucleotide comprises a nucleic acid sequence encoding CCL5 or having at least 70% sequence identity to one of SEQ ID NO:21 to SEQ ID NO:24, and the polynucleotide mixture comprises a third polynucleotide, wherein the third polynucleotide comprises a nucleic acid sequence encoding CCL20, or b) the first polynucleotide comprises a nucleic acid sequence encoding Met-CCL17, the second polynucleotide comprises a nucleic acid sequence encoding Met-CCL5 or having at least 70% sequence identity to one of SEQ ID NO: 16 to SEQ ID NO: 18, and the polynucleotide mixture comprises a third polynucleotide, wherein the third polynucleotide comprises a nucleic acid sequence encoding Met-CCL20, or c) the first polynucleotide comprises a nucleic acid sequence encoding CCL17, the second polynucleotide comprises a nucleic acid sequence encoding Met-CCL5 or having at least 70% sequence identity to one of SEQ ID NO:16-SEQ ID NO:18, and the polynucleotide mixture comprises a third polynucleotide, where the third polynucleotide comprises a nucleic acid sequence encoding CCL20.

[0040] Preferably, a) the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:3 and SEQ ID NO:21 to SEQ ID NO:24, and the polynucleotide mixture comprises a third polynucleotide, wherein the third polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9, or b) the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:13, the second polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:11 and SEQ ID NO:16 to SEQ ID NO:18, and the polynucleotide mixture comprises a third polynucleotide, wherein the third polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:15; or c) the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:11 and one of SEQ ID NOs:16-18, and the polynucleotide mixture comprises a third polynucleotide, wherein the third polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9.

[0041] Conveniently, a) the first polynucleotide comprises a nucleic acid sequence having the sequence of SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence having the sequence of SEQ ID NO:3, and the polynucleotide mixture comprises a third polynucleotide, wherein the third polynucleotide comprises a nucleic acid sequence having the sequence of SEQ ID NO:9; or b) the first polynucleotide comprises a nucleic acid sequence having the sequence of SEQ ID NO:13, the second polynucleotide comprises a nucleic acid sequence having the sequence of SEQ ID NO:11, and the polynucleotide mixture comprises a third polynucleotide, wherein the third polynucleotide comprises a nucleic acid sequence having the sequence of SEQ ID NO:15; or c) the first polynucleotide comprises a nucleic acid sequence having a sequence of SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence having a sequence of SEQ ID NO:11, and the polynucleotide mixture comprises a third polynucleotide, where the third polynucleotide comprises a nucleic acid sequence having a sequence of SEQ ID NO:9.

[0042] In a third aspect of the present invention, there is provided a composition suitable for modulating an immune response, comprising a first polynucleotide comprising a nucleic acid sequence encoding a first chemokine, or an agonist or antagonist variant thereof, suitable for binding to CCR5, CCR4 and / or CCR6, and a second chemokine, or an agonist or antagonist variant thereof, suitable for binding to a different one of CCR5, CCR4 and / or CCR6 from the first chemokine, or an agonist or antagonist variant thereof.

[0043] Preferably, one of the first and second chemokines, or agonist or antagonist variants thereof, is suitable for binding to CCR4.

[0044] Preferably, one of the first and second chemokines, or agonist or antagonist variants thereof, is suitable for binding to CCR4, and the other of the first and second chemokines, or agonist or antagonist variants thereof, is suitable for binding to CCR5 and / or CCR6.

[0045] Conveniently, the first polynucleotide encodes CCL5, CCL17, CCL20, or an agonist or antagonist variant thereof, or encodes an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO:20 and SEQ ID NO:26.

[0046] Suitably, the second chemokine, or agonist or antagonist variant thereof, comprises CCL5, CCL17, CCL20, or an agonist or antagonist variant thereof, or comprises an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO:20 and SEQ ID NO:26.

[0047] Preferably, the composition comprises a second polynucleotide comprising a nucleic acid sequence encoding a third chemokine, or an agonist or antagonist variant thereof, or the composition comprises a third chemokine, or an agonist or antagonist variant thereof, wherein the third chemokine, or an agonist or antagonist variant thereof, is suitable for binding to a different one of CCR5, CCR4 and / or CCR6 than each of the first and second chemokines, or their agonist or antagonist variants.

[0048] Suitably, the third chemokine, or agonist or antagonist variant thereof, comprises CCL5, CCL17, CCL20, or an agonist or antagonist variant thereof, or comprises an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO:20 and SEQ ID NO:26.

[0049] Conveniently, the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 to SEQ ID NO:18 and SEQ ID NO:21 to SEQ ID NO:24.

[0050] Preferably, the composition comprises a second polynucleotide, wherein the second polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15-SEQ ID NO:18, and SEQ ID NO:21-SEQ ID NO:24, which is different from the first polynucleotide.

[0051] Suitably, the first chemokine, or agonist or antagonist variant thereof, comprises CCL5, or an agonist or antagonist variant thereof, or comprises an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO:20 and SEQ ID NO:26, the second chemokine, or agonist or antagonist variant thereof, comprises CCL17, or an agonist or antagonist variant thereof, and the composition comprises a third chemokine, or agonist or antagonist variant thereof, comprising CCL20, or an agonist or antagonist variant thereof.

[0052] Preferably, the first chemokine, or an agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:26, the second chemokine, or an agonist or antagonist variant thereof, is CCL17, and the third chemokine, or a functional variant thereof, is CCL20.

[0053] Preferably, the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:21 to SEQ ID NO:24, the second chemokine, or an agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:5, and the third chemokine, or an agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:8.

[0054] Conveniently, the first chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:20, the second chemokine, or agonist or antagonist variant thereof, is Met-CCL17, and the third chemokine, or agonist or antagonist variant thereof, is Met-CCL20.

[0055] Preferably, the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:16 to SEQ ID NO:18, the second chemokine, or an agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:12, and the third chemokine, or an agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:14.

[0056] In a fourth aspect of the invention there is provided a chemokine or polynucleotide mixture for use in the treatment or prevention of a disease or disorder characterised by altered levels of CCR1, 4, 5, 6 and 8 or binding chemokines thereof, or a disease or disorder associated with a dysregulated immune response, or for use as an adjuvant, wherein the chemokine mixture comprises a first chemokine, or an agonist or antagonist variant thereof, and a second chemokine, or an agonist or antagonist variant thereof, or the polynucleotide mixture comprises a first polynucleotide comprising a nucleic acid sequence encoding the first chemokine, or an agonist or antagonist variant thereof, and a second chemokine, or an agonist or antagonist variant thereof, wherein the first chemokine or agonist or antagonist variant thereof is suitable for binding to a first CC chemokine receptor, the first CC chemokine receptor being a marker for regulatory T cells, and the second chemokine or agonist or antagonist variant thereof is suitable for binding to a second CC chemokine receptor, the second CC chemokine receptor being a marker for activated and / or regulatory T cells, and wherein the first and second chemokines or agonist or antagonist variants thereof are different from each other.

[0057] In a fifth aspect of the present invention there is provided a composition comprising a first polynucleotide comprising a nucleic acid sequence encoding a first chemokine, or an agonist or antagonist variant thereof, suitable for binding to a first CC chemokine receptor, and a second chemokine, or an agonist or antagonist variant thereof, suitable for binding to a second CC chemokine receptor, wherein the first CC chemokine receptor is a marker for regulatory and / or activated T cells and the second CC chemokine receptor is a marker for regulatory and / or activated T cells, and wherein the first and second chemokines are different from each other, for use in the treatment or prevention of a disease or disorder characterised by altered levels of CCR1, 4, 5, 6 and 8 or binding chemokines thereof, or a disease / condition associated with a dysregulated immune response, or for use as an adjuvant.

[0058] Preferably, the disease or disorder is cancer, a viral infection, Alzheimer's disease, an autoimmune disease, an inflammatory disease or condition, allergy or a disease or condition associated with an overactive immune system or an out-of-control immune response.

[0059] Suitably, in the fourth aspect, the first CC chemokine receptor is CCR4 and the second CC chemokine receptor is CCR5 or CCR6.

[0060] Conveniently, in the fourth aspect, the first chemokine comprises CCL17, or an agonist or antagonist variant thereof.

[0061] Preferably, in the fourth aspect, the second chemokine, or agonist or antagonist variant thereof, is CCL5 or CCL20, or an agonist or antagonist variant thereof, or comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:20 or SEQ ID NO:26.

[0062] Suitably, in the fourth aspect, the chemokine mixture comprises a third chemokine, or an agonist or antagonist variant thereof, or the polynucleotide mixture comprises a third polynucleotide comprising a nucleic acid sequence encoding a third chemokine, or an agonist or antagonist variant thereof, wherein the second CC chemokine receptor is a marker for activated T cells, and the third chemokine, or an agonist or antagonist variant thereof, is suitable for binding to the third CC chemokine receptor, wherein the third CC chemokine receptor is a marker for regulatory T cells and is different from the first CC chemokine receptor, respectively.

[0063] Conveniently, in a fourth embodiment, the second CC chemokine receptor is CCR5 and the third CC chemokine receptor is CCR6.

[0064] Preferably, in the fourth aspect, the second chemokine, or agonist or antagonist variant thereof, comprises CCL5, or an agonist or antagonist variant thereof, and the third chemokine, or agonist or antagonist variant thereof, comprises CCL20, or an agonist or antagonist variant thereof.

[0065] Suitably, in the fourth aspect, the first chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:5, and the second chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence comprising a sequence having at least 70% sequence identity to SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:26, and the disease or disorder is an autoimmune disease, an inflammatory disease or condition, an allergy or a disease or condition associated with an overactive immune system or an out-of-control immune response.

[0066] Conveniently, in a fourth aspect, the second chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or 26, wherein the chemokine mixture comprises a third chemokine, or agonist or antagonist variant thereof, and the polynucleotide mixture comprises a third polynucleotide comprising a nucleic acid sequence encoding the third chemokine, or agonist or antagonist variant thereof, wherein the third chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8.

[0067] Preferably, in a fourth aspect, a first chemokine, or an agonist or antagonist variant thereof, for use in the treatment or prevention of a disease or disorder selected from cancer, viral infection or Alzheimer's disease, or for use as an adjuvant, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 12, and a second chemokine, or an agonist or antagonist variant thereof, comprises an amino acid sequence comprising a sequence having at least 70% sequence identity to SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO: 20.

[0068] Suitably, in the fourth aspect, the second chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 20, respectively, and wherein the chemokine mixture comprises a third chemokine, or agonist or antagonist variant thereof, or the polynucleotide mixture comprises a third polynucleotide comprising a nucleic acid sequence encoding a third chemokine, or agonist or antagonist variant thereof, and wherein the third chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 14.

[0069] Conveniently, in a fourth aspect, a first chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:5 or SEQ ID NO:8, and a second chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence comprising a sequence having at least 70% sequence identity to SEQ ID NO:10 or SEQ ID NO:20, for use in the treatment or prevention of a disease or disorder selected from cancer, viral infection or Alzheimer's disease, or for use as an adjuvant.

[0070] Preferably, in the fourth aspect, the first chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:5, the second chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence comprising a sequence having at least 70% sequence identity to SEQ ID NO:10 or SEQ ID NO:20, the chemokine mixture comprises a third chemokine, or agonist or antagonist variant thereof, or the polynucleotide mixture comprises a third polynucleotide comprising a nucleic acid sequence encoding a third chemokine, or agonist or antagonist variant thereof, wherein the third chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:8.

[0071] Preferably, in the fourth aspect, the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:3 and one of SEQ ID NOs:21 to 24, and the third polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9.

[0072] Conveniently, in the fourth aspect, the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:13, the second polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:11 and one of SEQ ID NO:16 to SEQ ID NO:18, and the third polynucleotide comprises a nucleic acid molecule having at least 70% sequence identity to SEQ ID NO:15.

[0073] Preferably, in the fourth aspect, the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:11 and one of SEQ ID NOs:16 to 18, and the third polynucleotide comprises a nucleic acid molecule having at least 70% sequence identity to SEQ ID NO:9.

[0074] Preferably, in the fifth aspect, the first and second CC chemokine receptors are each independently CCR5, CCR4 or CCR6, wherein the first CC chemokine receptor is different from the second CC chemokine receptor.

[0075] Suitably, in the fifth aspect, the first CC chemokine receptor is CCR5 and the second CC chemokine receptor is CCR4 or CCR6.

[0076] Conveniently, in the fifth aspect, the first chemokine, or agonist or antagonist variant thereof, comprises CCL5, or an agonist or antagonist variant thereof, or comprises an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO:20 and SEQ ID NO:26, and the second chemokine, or agonist or antagonist variant thereof, comprises CCL17 or CCL20, or an agonist or antagonist variant thereof.

[0077] Preferably, in the fifth aspect, the composition comprises a second polynucleotide comprising a nucleic acid sequence encoding a third chemokine, or an agonist or antagonist variant thereof, or the composition comprises a third chemokine, or an agonist or antagonist variant thereof, wherein the third chemokine, or the agonist or antagonist variant thereof, is suitable for binding to a third CC chemokine receptor that is a marker of activated and / or regulatory T cells, and wherein the third CC chemokine receptor is distinct from each of the first and second CC chemokine receptors.

[0078] Suitably, in the fifth aspect, the first CC chemokine receptor is CCR5, the second CC chemokine receptor is one of CCR4 and CCR6, and the third CC chemokine receptor is the other of CCR4 and CCR6.

[0079] Conveniently, in the fifth aspect, the first chemokine, or an agonist or antagonist variant thereof, comprises CCL5, or an agonist or antagonist variant thereof, or comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:20 or SEQ ID NO:26, the second chemokine comprises one of CCL17 and CCL20, or an agonist or antagonist variant thereof, and the third chemokine comprises the other of CCL17 and CCL20, or an agonist or antagonist variant thereof.

[0080] Preferably, in the fifth aspect, the first chemokine comprises Met-CCL5, or an agonist or antagonist variant thereof, or comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:20.

[0081] Preferably, in the fifth aspect, the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:10 and SEQ ID NO:16 to SEQ ID NO:18, the second chemokine comprises an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO:5 and SEQ ID NO:8, and the composition comprises a third chemokine having at least 70% sequence identity to the other of SEQ ID NO:5 and SEQ ID NO:8.

[0082] Conveniently, in the fifth aspect, the disease or disorder is selected from cancer, viral infection or Alzheimer's disease, or the composition is for use as an adjuvant.

[0083] In a sixth aspect of the invention there is provided a method for producing a nucleic acid sequence comprising the steps of: a first nucleic acid construct comprising a first polynucleotide of the second aspect; and a second nucleic acid construct comprising a second polynucleotide of the second aspect; a first nucleic acid construct comprising a first polynucleotide of the third aspect and a second nucleic acid construct comprising a third second polynucleotide; a first nucleic acid construct comprising a first polynucleotide of the fourth aspect and a second nucleic acid construct comprising a second polynucleotide of the fourth aspect; or A first nucleic acid construct comprising a first polynucleotide of the fifth aspect and a second nucleic acid construct comprising a second polynucleotide of the fifth aspect. A nucleic acid construct mixture is provided comprising:

[0084] Preferably, the nucleic acid construct mixture comprises: a first nucleic acid construct comprising a first polynucleotide of the second aspect, a second nucleic acid construct comprising a second polynucleotide of the second aspect and a third nucleic acid construct comprising a third polynucleotide of the second aspect; or A first nucleic acid construct comprising a first polynucleotide of the fourth aspect, a second nucleic acid construct comprising a polynucleotide of the fourth aspect, and a third nucleic acid construct comprising a polynucleotide of the fourth aspect. Includes.

[0085] In a seventh aspect of the present invention, the nucleic acid construct mixture comprises: a first polynucleotide of the second aspect and a second polynucleotide of the second aspect; a first polynucleotide of the third aspect and a second polynucleotide of claim 25 or 26; a first polynucleotide of the fourth aspect and a second polynucleotide of the fourth aspect; or A second nucleic acid construct comprising the first polynucleotide of the fifth aspect and the second polynucleotide of the fifth aspect. A nucleic acid construct comprising:

[0086] Preferably, the nucleic acid construct comprises a first polynucleotide of the second aspect, a second polynucleotide of the second aspect and a third polynucleotide of the second aspect; or The fourth aspect comprises a first polynucleotide, a second polynucleotide of the fourth aspect and a third polynucleotide of the fourth aspect.

[0087] In an eighth aspect of the present invention there is provided a host cell comprising a polynucleotide mixture of the second aspect, a polynucleotide mixture for use of the fourth aspect, a nucleic acid construct mixture of the sixth aspect or a nucleic acid construct according to the seventh aspect.

[0088] In a ninth aspect of the present invention there is provided a pharmaceutical formulation comprising a chemokine mixture of the first aspect, a polynucleotide mixture of the second aspect, a composition according to the third aspect, a chemokine mixture or a polynucleotide mixture for use according to the fourth aspect, a composition for use according to the fifth aspect, a nucleic acid construct mixture according to the sixth aspect, a nucleic acid construct of the seventh aspect or a host cell according to the eighth aspect and a pharma- ceutically acceptable carrier, excipient or diluent.

[0089] In a tenth aspect of the present invention there is provided a chemokine mixture of the first aspect, a polynucleotide mixture of the second aspect, a composition of the third aspect, a pharmaceutical composition comprising the chemokine mixture of the first aspect, the polynucleotide mixture of the second aspect or the composition of the third aspect together with a pharma- ceutically acceptable carrier, excipient or diluent, a nucleic acid construct mixture of the sixth aspect, a nucleic acid construct of the seventh aspect or a host cell of the eighth aspect for use in the treatment or prevention of a disease or disorder characterised by altered levels of CCR1, 4, 5, 6 and 8 or their binding chemokines, or a disease or disorder associated with an abnormal immune response, or for use as an adjuvant.

[0090] In an eleventh aspect of the present invention there is provided a kit comprising a chemokine mixture of the first aspect, a polynucleotide mixture of the second aspect, a composition of the third aspect, a chemokine mixture or a polynucleotide mixture for use of the fourth aspect, a composition for use of the fifth aspect, a nucleic acid construct mixture of the sixth aspect, a nucleic acid construct of the seventh aspect, a host cell of the eighth aspect or a pharmaceutical formulation of the ninth aspect.

[0091] In a twelfth aspect of the present invention there is provided the use of a chemokine mixture of the first aspect, a polynucleotide mixture of the second aspect, a composition of the third aspect, a nucleic acid construct mixture of the sixth aspect, a nucleic acid construct of the seventh aspect or a host cell of the eighth aspect in the manufacture of a medicament.

[0092] Preferably, the medicament is for the treatment of a disorder characterised by altered levels of CCR1, CCR4, CCR5, CCR6 and CCR8 or their binding chemokines, or a disease or disorder associated with an abnormal immune response.

[0093] In a thirteenth aspect of the present invention there is provided a method for treating or preventing a disease or disorder characterised by altered levels of CCR1, 4, 5, 6 and 8 or binding chemokines thereof or a disease or disorder associated with an abnormal immune response comprising administering to a patient in need of such treatment or prevention a chemokine mixture according to any one of claims 1 to 9, a polynucleotide mixture according to any one of claims 10 to 19, a composition according to any one of claims 20 to 33, a chemokine mixture or a polynucleotide mixture for use according to any one of claims 34 and 36 to 51, a composition for use according to any one of claims 35, 36 and 52 to 60, a nucleic acid construct mixture according to claim 61 or 62, a nucleic acid construct according to claim 63 or 64, a host cell according to claim 65 or a pharmaceutical formulation according to claim 66.

[0094] In a fourteenth aspect of the present invention there is provided the use of a chemokine mixture of the first aspect, a polynucleotide mixture of the second aspect, a composition of the third aspect, a chemokine mixture or a polynucleotide mixture for use of the fourth aspect, a composition for use of the fifth aspect, a nucleic acid construct mixture of the sixth aspect, a nucleic acid construct of the seventh aspect, a host cell of the eighth aspect or a pharmaceutical formulation of the ninth aspect as an adjuvant in a patient in need thereof.

[0095] definition The term "protein" as used herein refers to a polymeric form of amino acids of any length linked together by peptide bonds.

[0096] The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), naturally occurring, mutated, synthetic DNA or RNA molecules, and analogs of DNA or RNA produced using nucleotide analogs. It may be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, antisense sequences, and non-coding regulatory sequences that do not code for an mRNA or protein product. These terms also encompass genes. The terms "gene" or "gene sequence" are used broadly to refer to DNA nucleic acids associated with a biological function. Thus, a gene may include introns and exons as found in a genomic sequence, or may include only the coding sequence as found in a cDNA, and / or may include cDNA in combination with regulatory sequences.

[0097] The term "agonist variant," as used herein, refers to a variant of a compound that binds to and activates the same receptor as the compound, thereby mimicking the biological effect of the compound.

[0098] The term "antagonist variant," as used herein, refers to a variant of a compound that binds to the same receptor as the compound but does not activate it, thereby preventing or reducing the biological effect of the compound.

[0099] The term "mutant" as used herein refers to a mutant nucleotide or amino acid sequence, or a portion (e.g., a fragment) of that nucleotide or amino acid sequence. A mutant retains the ability to bind to the same receptor as the complete non-mutant sequence, but may have agonist or antagonist activity compared to the complete non-mutant. Also included are mutants that are substantially identical, i.e., have only some sequence variations, e.g., in non-conserved residues, compared to the wild-type sequence as shown herein, and retain the non-mutant binding activity. Changes in nucleic acid sequences that result in the production of different amino acids at a given site without affecting the functional properties of the encoded polypeptide are well known in the art. For example, a codon for the amino acid alanine, which is a hydrophobic amino acid, may be replaced by a codon that codes for another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, alterations resulting in the substitution of one negatively charged residue for another, e.g., aspartic acid for glutamic acid, or one positively charged residue for another, e.g., lysine for arginine, would also be expected to produce a functionally equivalent product. Variants may also include the addition or deletion of one or more amino acids to or from the non-variant amino acid sequence, and / or may include chemical modifications of the non-variant. Each of the proposed modifications is well within the routine skill of one of ordinary skill in the art, as is the determination of retention of biological activity of the encoded product.A variant may be at least 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%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1090%, 1091%, 1092%, 1093%, 1094%, 1095%, 1096%, 1097%, 1098%, 1099%, 1000%, 1010%, 1020%, 1030%, 1040%, 1050%, 1051%, 1060%, 1075%, 1086%, 1087%, 1098%, 1099%, 1090%, 1091%, 1092 The variants have an overall sequence identity of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. The variants have at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to the non-variant nucleic acid or amino acid sequence.

[0100] Alternatively, the variant may be a sequence that hybridizes to a nucleic acid or amino acid sequence under stringent conditions. "Stringent conditions" or "stringent hybridization conditions" are intended conditions under which a probe hybridizes to its target sequence detectably greater than to other sequences (e.g., at least 2-fold above background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences, so that a lower degree of similarity is detected (heterologous probing). In general, the probe is less than about 1000 nucleotides long, preferably less than 500 nucleotides long.

[0101] Typically, stringent conditions are those in which the salt concentration is less than about 1.5M Na ion, typically about 0.01-1.0M Na ion concentration (or other salt) at pH 7.0-8.3, and the temperature is at least about 30°C for short probes (e.g., 10-50 nucleotides) and at least about 60°C for long probes (e.g., more than 50 nucleotides). The duration of hybridization is generally less than about 24 hours, typically about 4-12 hours. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide.

[0102] The term "immune response" as used herein, in some aspects, refers to a T cell- or B cell-mediated immune response. A T cell-mediated immune response occurs upon presentation of peptides by major histocompatibility (MHC) molecules on the cell surface, and specifically refers to the activation of T cells upon presentation of peptides. A B cell-mediated immune response specifically refers to the production of antibodies in response to recognition of an antigen.

[0103] The percent "identity" between two sequences can be determined using the BLASTP algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402) using default parameters. In particular, the BLAST algorithm can be accessed on the Internet using the URL http: / / www.ncbi.nlm.nih.gov / blast / .

[0104] The term "pharmaceutical composition" as used herein means a pharmaceutical preparation suitable for administration to the intended human or animal subject for therapeutic purposes. [Brief description of the drawings]

[0105] [Figure 1] FIG. 1 shows the nucleic acid and corresponding amino acid sequences of the integrated iciHHV-6A iciU83A gene. [Diagram 2] Figure 3 shows the DNA sequence of spliced ​​integrated iciHHV-6A U83A. This figure demonstrates that splicing occurs via DR, direct repeat, TACC, and non-consensus splice donor / acceptor pairs as shown by cDNA analysis in transient gene expressing cells despite the mutation TGA-TGG proximal to the 3' splice site, and our unexpected finding that a non-synonymous SNP in the full-length gene transforms this spliced ​​product into a mutation of the original spliced ​​stop codon, unlike the circulating virus. This cDNA was not observed in samples from a series of donors and is therefore thought to disrupt non-consensus splicing (Tweedy et al., 2015, 2016), but our in vitro analysis in transfected cells (Figures 3 and 4) shows that it unexpectedly allows read-through encoding eight additional amino acids with hydrophobic interaction properties as shown here. [Diagram 3]Figure 1 shows in vitro expression of iciU83A in cells with splicing to iciU83A-N. The gene iciU83A was cloned into a plasmid expression construct and transfected into HEK293 cells. Lanes 1-3 are negative controls, reaction mix without oligonucleotide primers, reaction mix with oligonucleotide primers, and template water only. Lanes 4-7 are one-step RT-PCR reactions of total RNA extracted from transfected cells primed with primers from the plasmid vector pCMV (and also with primers amplifying the iciU83A gene, not shown). Lanes 5 and 7 are not treated with DNase and show residual DNA from transfection. Lanes 4 and 6 are treated with DNase. Lanes 4 and 5 contain reverse transcriptase. Lane 5 shows full-length DNA and lane 4 shows the expressed spliced ​​cDNA product iciU83A-N. [Figure 4] Figure 1 shows the in vitro expression of iciU83A-N cDNA (SEQ ID NO: 16) in cells. The cDNA of iciU83A-N was cloned into a plasmid expression construct and transfected into HEK293 cells. Two days after transfection, total RNA was extracted. RNA was treated with DNAse (lanes 2 and 3) followed by reverse transcriptase (lane 2) or was untreated (lane 3). The negative control in lane 1 is water template only. Lane 4 shows the DNA marker. [Diagram 5]Figure 1 shows the efficacy of immunization with (A) the iciU83A-N DNA construct designated VTL1 (also referred to herein as VIT1 or VIT), and (B) the VTL3 protein in an in vivo preclinical model of HSV2 evaluating protection from disease from acute infection up to 14 days after viral challenge. VTL1 or VTL3 were formulated with the known immunogen gD, either with VTL1 DNA (VTL1016; SEQ ID NO: 17) as DNA or with VTL3 protein (SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 8) as protein. Comparisons were made with negative controls (no vaccine) or positive controls (gD protein with mpl / alum adjuvant). The VTL1 DNA formulation showed nearly complete protection, while the VTL3 protein formulation eliminated the protection induced by the immunogen. [Figure 6] Figure 5 shows the efficacy of immunization with known immunogen gD formulated as seen in Figure 5 with (A) VIT1 DNA and (B) VTL3 protein in an in vivo preclinical model of HSV2 evaluating protection in individuals from the total severity of disease from acute infection. With VIT1 (A), the total mean acute lesions show total clearance for 75% of the animals, which is similar to the positive control (gD protein mpl / alum immunization). On the other hand, with VTL3 (SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8) (B), this effect is eliminated with only 25% of the animals clearing the acute lesions, which is not significantly different from the negative control (no vaccine). [Figure 7]Figure 7 shows the efficacy of immunization with formulations having (A) VIT1 DNA and (B) VTL3 protein in combination with the known immunogen gD as DNA and protein, respectively, in an in vivo preclinical model of HSV2 to evaluate the effect of immunization on protection from acute infection. Comparison was made with immunization with a positive control (gD protein + adjuvant MPL + alum). Figure 7a shows a significant reduction in viral load in individual animals after immunization and subsequent viral challenge with the positive control immunization compared to a negative control (no vaccine). Evaluation of immune modulators shows that VIT1 DNA (VTL1016; SEQ ID NO: 17) in combination with the known immunogen gD DNA was effective in reducing viral load. Figure 7b, in contrast, shows that immunization with VTL3 blocked this effect, resulting in a minimal reduction in viral load 2 days after infection compared to the positive control (gD subunit protein). [Figure 8] Figure 8a shows the efficacy of immunization with immunomodulators (A) VIT DNA and (B) VTL3 protein in combination with known immunogen gD evaluated in an in vivo preclinical model of HSV2 in protecting against recurrent disease. Figure 8a shows that VIT provided protection from recurrent disease, with a significant reduction in recurrent disease as indicated by the cumulative number of days of recurrent lesions in all animals following immunization and viral challenge. Protection from recurrence compared to the negative control (no vaccine treatment) was only shown with the combination of gD+VIT1 (SEQ ID NO: 17), but was abolished when VIT1 was omitted. Thus, VIT enhanced the immune modulation by gD immunization and provided protection from recurrent disease. Similarly, immunization with the positive control (gD protein with adjuvant MPL / Alum) also provided protection from recurrent disease. In contrast, Figure 8b shows that immunization of gD protein with VTL3 chemokine preparations (sequence number 2, sequence number 5 and sequence number 8) abrogated protection from recurrent disease compared to the positive control, where the response to the immunogen was similar to the negative control (no vaccine). [Figure 9]Figure 9 shows the efficacy of immunization with (A) VIT1 DNA and (B) VTL3 protein, combined in formulations with known immunogen gD, DNA or protein, respectively, and evaluated in an in vivo preclinical model of HSV2 in providing protection from recurrent disease. Figure 9a shows that immunization with VIT1 resulted in a significant reduction in recurrent disease in individual animals, as indicated by the total severity of lesions that recurred 15-63 days after immunization and viral challenge. Significant protection from recurrence compared to the negative control (no vaccine treatment) is only shown with the combination of gD+VIT1 (SEQ ID NO: 17), whereas omission of VIT1 shows a reduction in protection as immunization with gD immunogen alone. Figure 9b shows that immunization with gD protein together with VTL3 completely eliminated any reduction in recurrent disease, making it similar to the negative control (no vaccine). [Figure 10] Figure 10 shows the efficacy of immunization with immunomodulators (A) VIT1 DNA and (B) VTL3 protein, formulated with known immunogen gD as DNA or protein, respectively, and evaluated in an in vivo preclinical model of HSV2 in preventing asymptomatic recurrent shedding virus, as shown by total recurrence in individual animals, with the averages shown as measured by quantitative DNA PCR (qPCR). Figure 10a shows a reduction in virus shedding only by immunization with VIT1 DNA (VTL1016; SEQ ID NO: 17) together with gD DNA. In contrast, Figure 10b shows that immunization with VTL3 together with gD does not provide protection, with results similar to the negative control (no vaccine). [Figure 11]Figure 11 shows the efficacy of immunization with immunomodulators (A) VIT1 DNA (VTL1016; SEQ ID NO: 17) and (B) VTL3 protein, formulated with the known immunogen gD as DNA or protein, respectively, and evaluated in an in vivo preclinical model of HSV2 in providing protection from the establishment of latent infection in the dorsal root ganglia (DRG) of animals as measured by qPCR. Figure 11a shows that the VIT1 formulation provided significant protection from the establishment of latent infection in the DRG, halving that detected in the negative control, whereas Figure 11b shows that the VTL3 formulation did not provide any protection. [Figure 12] Figure 12 shows the efficacy of immunization with (A) VIT1 DNA (VTL1016; SEQ ID NO: 17) and (B) VTL3 protein, each formulated with the known immunogen gD and assessed in an in vivo preclinical model of HSV2 in providing protection from the establishment of latent infection in the DRG of individual animals as measured by qPCR. Figure 12a shows that VIT1 provided significant protection, with over half of the animals protected and DNA undetectable in 58% of the animals. Figure 12b shows that VTL3 formulated with gD did not provide protection from the establishment of latent infection in the DRG. [Figure 13] Figure 13 shows the efficacy of immunization with (A) VIT1 DNA (VTL1016; SEQ ID NO: 17) and (B) VTL3 protein, each formulated with the known immunogen gD and evaluated in an in vivo preclinical model of HSV2 in providing protection from the establishment of latent infection in the spinal cord of animals as measured by qPCR. Figure 13a shows that VIT1 provided significant protection, halving that detected in the negative control (no vaccine). Figure 13b shows that VTL3 formulated with gD did not provide any protection compared to the negative control (no vaccine). [Figure 14]Figure 14 shows the efficacy of immunization with (A) VIT1 DNA (VTL1016; SEQ ID NO: 17) and (B) VTL3 protein, each formulated with the known immunogen gD and evaluated in an in vivo preclinical model of HSV2 in providing protection from the establishment of latent infection in the spinal cord of individual animals as measured by qPCR. Figure 14a shows that VIT1 provided significant protection in individual animals compared to the negative control (no vaccine), with half the animals protected and DNA undetectable in 50% of the animals. Figure 14b shows that VTL3 did not provide any protection compared to the negative control (no vaccine). [Figure 15] Graph showing herpes simplex virus type 2 (HSV-2) neutralizing antibody titers in guinea pig serum after two intramuscular immunizations with the following vaccine formulations: gD protein combined with immunomodulator formulation VTL3; gD DNA with VIT1; gD protein combined with MPL and alum; gD DNA with CCL5 DNA and no vaccine treatment. Dashed lines indicate limit of detection. ***P<0.001 compared to no vaccine. Neutralizing antibodies were induced using gD combined with CCL5 or VIT1 compared to the negative control (no vaccine), but were completely inhibited by VTL3 protein and the immune response remained undetectable similar to the negative control (no vaccine). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0106] The present invention generally relates to a chemokine mixture comprising a first chemokine, or an agonist or antagonist variant thereof, and a second chemokine, or an agonist or antagonist variant thereof, which targets regulatory T cells. The present invention also relates to a polynucleotide mixture comprising a first polynucleotide comprising a nucleic acid sequence encoding a first chemokine, or an agonist or antagonist variant thereof, which targets regulatory T cells, and a second polynucleotide comprising a nucleic acid sequence encoding a second chemokine, or an agonist or antagonist variant thereof. The present invention further relates to a composition comprising a first polynucleotide comprising a nucleic acid sequence encoding a first chemokine, or an agonist or antagonist variant thereof, and a second chemokine, or an agonist or antagonist variant thereof, which targets regulatory T cells. The chemokine mixture, polynucleotide mixture and composition are each suitable for modulating an immune response. In each aspect of the invention, the first chemokine, or agonist or antagonist variant thereof, is suitable for binding to a first CC chemokine receptor, and the second chemokine, or agonist or antagonist variant thereof, is suitable for binding to a second CC chemokine receptor. If present, the third chemokine is suitable for binding to a third CC chemokine receptor. The CC chemokine receptors are markers of activated and / or regulatory T cells, and at least one of the CC chemokine receptors is a marker of regulatory T cells. The following description of the first and second chemokines, or agonist or antagonist variants thereof, as well as any further chemokines, or agonist or antagonist variants thereof, applies to chemokines encoded by any of, or any of, the chemokine mixtures, polynucleotide mixtures and compositions of the invention described herein. Thus, any combination of first and second, and optionally third and further chemokines, or agonist or antagonist variants thereof, applies to any of the chemokine mixtures, polynucleotide mixtures and compositions of the invention.

[0107] Chemokine and CC chemokine receptors Chemokines are a type of signaling protein essential in cell migration through chemotaxis, especially in the recruitment of immune cells. Human chemokines have been classified into four main subfamilies according to their amino acid composition, in particular according to the first two cysteine ​​residues of a conserved tetracysteine ​​motif: CXC, CC, CX3C and C. However, there are other types of human chemokines, such as those derived from the chromosomally integrated human endogenous form of human herpesvirus (HHV-6A), referred to herein as iciHHV-6A. Chemokines also include virokines, which are virus-encoded proteins secreted from infected host cells and can act as chemokine agonists or antagonists.

[0108] Chemokines exert their effects by binding to chemokine receptors expressed on the surface of leukocytes. In humans, there are 23 known chemokine receptors, all of which are G protein-coupled receptors (GPCRs) that contain seven transmembrane domains. Receptors are classified into four families according to the type of chemokine they bind: CXC receptors (CXCRs) bind CXC chemokines, CC receptors (CCRs) bind CC chemokines, CX3C receptor 1 (CX3CR1) binds only CX3C chemokines (CX3CL1), and XC receptor 1 (XCR1) binds two C chemokines. In particular, the CC receptor family consists of CCR1-CCR10, whose ligands are listed in Table 1 (adapted from Mohan et al. 2018 and Hughes and Nibbs 2018):

[0109] [Table 1]

[0110] Modified chemokines Chemokines can also be modified to form agonist or antagonist variants. Protein modifications can include, among others, the addition, deletion or substitution of single amino acids, or the addition of chemical moieties. Met-CCL5 (Proudfoot et al., 1996; Proudfoot et al., 1999; WO96 / 17935), AOP-CCL5 (Proudfoot et al., 1999), Gln-RANTES and Leu-RANTES (WO96 / 17935) are examples of known modified chemokines. Met-CCL5, Gln-RANTES and Leu-RANTES all have amino acid additions at their N-terminus, while AOP-CCL5 is a chemical modification of CCL5.

[0111] Furthermore, the nucleotide sequence encoding the chemokine can be modified, for example, to enhance the expression of a functional protein, to modify the biological activity of the expressed protein, or to stabilize the expression of the nucleotide sequence. Modifications of nucleotide sequences are well known in the art, and specific examples of such modifications are shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 21 to SEQ ID NO: 24 of the present invention.

[0112] Therefore, any agonist mutant of a chemokine that is suitable for binding to and activating the CC chemokine receptor of interest, or any antagonist mutant of a chemokine that is suitable for binding to but does not activate the CC chemokine receptor of interest, can be used in the present invention.Methods are known for determining the binding affinity and biological activity of molecules using chemotaxis assays and receptor binding assays, such as those described in Proudfoot et al., 1996.Therefore, the agonist and antagonist mutants used in the present invention are not limited to the specific examples described herein.

[0113] mixture In the present invention, the first and second chemokines, or their agonist or antagonist variants, are suitable for binding to a CC chemokine receptor, where the first chemokine, or its agonist or antagonist variant, is suitable for binding to a different CC chemokine receptor than the second chemokine, or its agonist or antagonist variant. The first and second chemokines, or their agonist or antagonist variants, together target regulatory T cells and promote or prevent the recruitment of regulatory T cells, thereby modulating the immune response. The first chemokine, or its agonist or antagonist variant, may be suitable for binding to one of CCR5, CCR4 and / or CCR6, and the second chemokine, or its agonist or antagonist variant, may be suitable for binding to a different one of CCR5, CCR4 and / or CCR6. In some embodiments, one of the first and second chemokines, or agonist or antagonist variants thereof, is suitable for binding to CCR4 or CCR6. In some embodiments, the first chemokine, or agonist or antagonist variant thereof, is suitable for binding to CCR4 and the second chemokine, or agonist or antagonist variant thereof, is suitable for binding to CCR5 and / or CCR6. In other embodiments, the first chemokine, or agonist or antagonist variant thereof, is suitable for binding to CCR4 and the second chemokine, or agonist or antagonist variant thereof, is suitable for binding to CCR5.

[0114] In some embodiments, a chemokine, or agonist or antagonist variant thereof, that is suitable for binding to CCR4 is also suitable for binding to CCR8. This applies to any chemokine, or agonist or antagonist variant thereof, that is suitable for binding to CCR4 in any of the mixtures and compositions described below.

[0115] In some embodiments, the chemokine mixture comprises a third chemokine, or an agonist or antagonist variant thereof, the polynucleotide mixture comprises a third polynucleotide comprising a nucleic acid sequence encoding the third chemokine, or an agonist or antagonist variant thereof, and / or the composition comprises a second polynucleotide comprising a nucleic acid sequence encoding the third chemokine, or an agonist or antagonist variant thereof, or comprises a third chemokine, or an agonist or antagonist variant thereof, which is suitable for binding to a different one of CCR5, CCR4 and / or CCR6 than the first and second chemokines, or their agonist or antagonist variants, respectively. Thus, the first, second and third chemokines, or agonist or antagonist variants thereof, may be suitable for binding to CCR5, CCR4 and / or CCR6 in any of the combinations shown in Table 2:

[0116] [Table 2]

[0117] In a preferred embodiment, the first chemokine, or an agonist or antagonist variant thereof, is suitable for binding to CCR4, hi some embodiments, the first chemokine, or an agonist or antagonist variant thereof, is suitable for binding to CCR4 and CCR8, respectively.

[0118] In some embodiments, the chemokine, or agonist or antagonist variant thereof, suitable for binding to CCR5 comprises CCL5, Met-CCL5, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 25, or SEQ ID NO: 26, or agonist or antagonist variants thereof. In some embodiments, the chemokine, or agonist or antagonist variant thereof, suitable for binding to CCR4 comprises CCL17, Met-CCL17, or agonist or antagonist variants thereof. In some embodiments, the chemokine, or agonist or antagonist variant thereof, suitable for binding to CCR6 comprises CCL20, Met-CCL20, or agonist or antagonist variants thereof. Any of these chemokines, or agonist or antagonist variants thereof, can be used in any of the first and second chemokine, or agonist or antagonist variants thereof combinations, as well as in any of the combinations in Table 2. Thus, in some embodiments, the first chemokine, or agonist or antagonist variant thereof, comprises CCL17, Met-CCL17, or an agonist or antagonist variant thereof, and the second chemokine, or agonist or antagonist variant thereof, comprises CCL5, Met-CCL5, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 26, CCL20, or Met-CCL20, or an agonist or antagonist variant thereof. In some embodiments, the first chemokine, or agonist or antagonist variant thereof, comprises CCL17, Met-CCL17, or an agonist or antagonist variant thereof, the second chemokine, or agonist or antagonist variant thereof, comprises CCL5, Met-CCL5, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:25, or SEQ ID NO:26, or an agonist or antagonist variant thereof, and the third chemokine comprises CCL20, Met-CCL20, or an agonist or antagonist variant thereof.

[0119] In some embodiments, a chemokine, or agonist or antagonist variant thereof, suitable for binding to CCR5 also comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:25 and SEQ ID NO:26. In some embodiments, the amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:20 and SEQ ID NO:26. In some embodiments, the amino acid sequence has at least 90% sequence identity to one of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:20 and SEQ ID NO:26. In some embodiments, the amino acid sequence has at least 95% sequence identity to one of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:20, and SEQ ID NO:26. In some embodiments, the amino acid sequence has at least 99% sequence identity to one of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:20, and SEQ ID NO:26. In some embodiments, the amino acid sequence is identical to one of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:20, and SEQ ID NO:26.

[0120] In some embodiments, the chemokine, or agonist or antagonist variant thereof, suitable for binding to CCR4 also comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:12. In some embodiments, the amino acid sequence has at least 90% sequence identity to one of SEQ ID NO:5 and SEQ ID NO:12. In some embodiments, the amino acid sequence has at least 95% sequence identity to one of SEQ ID NO:5 and SEQ ID NO:12. In some embodiments, the amino acid sequence has at least 99% sequence identity to one of SEQ ID NO:5 and SEQ ID NO:12. In some embodiments, the amino acid sequence is identical to one of SEQ ID NO:5 and SEQ ID NO:12.

[0121] In some embodiments, the chemokine, or agonist or antagonist variant thereof, suitable for binding to CCR6 also comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:14. In some embodiments, the amino acid sequence has at least 90% sequence identity to one of SEQ ID NO:8 and SEQ ID NO:14. In some embodiments, the amino acid sequence has at least 95% sequence identity to one of SEQ ID NO:8 and SEQ ID NO:14. In some embodiments, the amino acid sequence has at least 99% sequence identity to one of SEQ ID NO:8 and SEQ ID NO:14. In some embodiments, the amino acid sequence is identical to one of SEQ ID NO:8 and SEQ ID NO:14.

[0122] Any of the above amino acid sequences and percent sequence identity thereto can be used in any of the first and second chemokine combinations, as well as in any of the combinations in Table 2. Thus, in some embodiments, the first chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:5 and SEQ ID NO:12, and the second chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20 and SEQ ID NO:26. In some embodiments, the first chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:5 and SEQ ID NO:12, and the second chemokine, or agonist or antagonist variant thereof, comprises one of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:20 and SEQ ID NO:26. and a third chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO: 8 and SEQ ID NO: 14. Preferably, the above amino acid sequence of each chemokine, or agonist or antagonist variant thereof, has at least 90%, 95% or 99%, more preferably 100%, sequence identity to the respective SEQ ID NO.

[0123] Agonist Chemokine Mixture In some embodiments, the first and second chemokines, or variants thereof, are each agonists of their respective CC chemokine receptors. In other words, in some embodiments, the first and second chemokines, or variants thereof, bind to and activate the relevant CC chemokine receptors, thereby recruiting regulatory T cells. Thus, in some embodiments, the first chemokine or variants thereof is a CCR4 agonist, and the second chemokine or variants thereof is a CCR5 or CCR6 agonist. In some embodiments, the first chemokine or variants thereof is a CCR4 agonist, the second chemokine or variants thereof is a CCR5 agonist, and the third chemokine or variants thereof is a CCR6 agonist. Methods for determining the binding affinity and biological activity of molecules using chemotaxis assays and receptor binding assays, such as those described in Proudfoot et al., 1996, are known. Thus, CC chemokine receptor agonists for use in the present invention are not limited to the specific examples described herein.

[0124] In some embodiments, the first chemokine, or agonist variant thereof, comprises CCL17 and the second chemokine, or agonist variant thereof, comprises CCL5, SEQ ID NO:26, CCL17 or CCL20. In some embodiments, the first chemokine, or agonist variant thereof, comprises CCL17 and the second chemokine, or agonist variant thereof, comprises CCL5 or CCL20. In some embodiments, the first chemokine, or agonist variant thereof, comprises CCL17 and the second chemokine, or agonist variant thereof, comprises SEQ ID NO:26 or CCL20. In some embodiments, the first chemokine, or agonist variant thereof, comprises CCL17, the second chemokine, or agonist variant thereof, comprises CCL5 or SEQ ID NO:26 and the third chemokine, or agonist variant thereof, comprises CCL20.

[0125] In some embodiments, the first chemokine, or agonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5, and the second chemokine, or agonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:2, SEQ ID NO:8 and SEQ ID NO:26. In some embodiments, the first chemokine, or agonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5, and the second chemokine, or agonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:2 and SEQ ID NO:8. In some embodiments, the first chemokine, or agonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5, and the second chemokine, or agonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:8 and SEQ ID NO:26.In some embodiments, the first chemokine, or agonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5; the second chemokine, or agonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:2 and SEQ ID NO:26; and the third chemokine, or agonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:8.

[0126] In some embodiments, the first chemokine, or agonist variant thereof, comprises the amino acid sequence of SEQ ID NO:5, and the second chemokine, or agonist variant thereof, comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, or SEQ ID NO:26. In some embodiments, the first chemokine, or agonist variant thereof, comprises the amino acid sequence of SEQ ID NO:5, and the second chemokine, or agonist variant thereof, comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:8. In some embodiments, the first chemokine, or agonist variant thereof, comprises the amino acid sequence of SEQ ID NO:25, and the second chemokine, or agonist variant thereof, comprises the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:26. In some embodiments, the first chemokine, or agonist variant thereof, comprises the amino acid sequence of SEQ ID NO:5, the second chemokine, or agonist variant thereof, comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:26, and the third chemokine, or agonist variant thereof, comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, the first chemokine consists of SEQ ID NO:5, the second chemokine consists of SEQ ID NO:2 or SEQ ID NO:26, and the third chemokine consists of SEQ ID NO:8. One embodiment in which the first chemokine consists of SEQ ID NO:5, the second chemokine consists of SEQ ID NO:2, and the third chemokine consists of SEQ ID NO:8 is referred to herein as "VTL3." In particular, Figures 5b, 6b, 7b, 8b, 9b, 10b, 11b, 12b, 13b, 14b, 15b, and 16b show that VTL3 abrogated any protection against viral challenge, and Figure 17 shows that VTL3 inhibited the antibody response.

[0127] Surprisingly, it has been found that mixtures of chemokines that can target regulatory T cells have utility in inhibiting immune responses. This is surprising because, as discussed in Proudfoot et al., 2016, interactions between chemokines can have various unpredictable effects. Furthermore, individual agonists of individual receptors can be immunostimulatory, for example, by chemoattracting dendritic cells or helper T cell subsets TH17 (CCR6 with CCL20) or skin-homing effector TH2 cells (CCR4 or CCR8 with CCL17) (Hughes and Nibbs 2018, Mohan et al. 2018). Specifically with respect to VTL3, it has been shown herein that CCR5 agonists such as CCL5 induce immune responses, while combinations of agonist mixtures targeting CCR5, CCR4, CCR8 and CCR6 inhibit immune responses. Conversely, a novel antagonist molecule, VTL1 (also referred to herein as VIT1), which is specific for all of CCR5, CCR4, CCR8 and CCR6, induces an immune response, and therefore combinations of VTL1 with other antagonists are expected to have the opposite effect to agonist mixtures targeting these four CC chemokine receptors.

[0128] Antagonist Chemokine Mixture In some embodiments, the first and second chemokines, or variants thereof, are each antagonists of their respective CC chemokine receptors. In other words, in some embodiments, the first and second chemokines, or variants thereof, bind to but do not activate the relevant CC chemokine receptors, thereby inhibiting the recruitment of regulatory T cells and, as a result, permitting or enhancing immune responses. Thus, in some embodiments, the first chemokine or variants thereof is a CCR4 antagonist, and the second chemokine or variants thereof is a CCR5 antagonist or a CCR6 antagonist. In some embodiments, the first chemokine or variants thereof is a CCR4 antagonist, the second chemokine or variants thereof is a CCR5 antagonist, and the third chemokine or variants thereof is a CCR6 antagonist.

[0129] A mixture of antagonists of CC chemokine receptors that target activated regulatory T cells is expected to be effective in inducing, enhancing or condoning immune responses. In particular, as discussed above, the figure shows that the chemokine mixture VTL3, which is composed of CC chemokine receptor agonists that target both conventional and activated regulatory T cells, surprisingly inhibits immune responses. This mixture targets all T-reg subsets via combined chemokine receptor specificity, and the net effect is inhibitory. Thus, a mixture containing one or more antagonists of the chemokines contained in VTL3, especially a mixture containing antagonists of all the chemokines contained in VTL3, is expected to have the opposite effect, i.e., enhance or condone immune responses.

[0130] As discussed above, a number of CC chemokine receptor antagonists are known, such as Met-CCL5 (Proudfoot et al., 1996; Proudfoot et al., 1999; WO 96 / 17935), AOP-CCL5 (Proudfoot et al., 1999), Gln-RANTES and Leu-RANTES (WO 96 / 17935). These four specific CCL5 mutants are N-terminally modified, and since it is the N-terminus of the CC chemokine that interacts with the CC chemokine receptor (as discussed above), it is expected that N-terminal modification of a chemokine, particularly the Met N-terminal modification, will alter activity, shifting an agonist to an antagonist, as demonstrated by Met-CCL5. Thus, in the present invention, antagonist mutants of chemokines include N-terminally modified chemokines, such as terminal met-modified chemokines.

[0131] Furthermore, VTL1 (also referred to herein as VIT1) is shown herein to be an antagonist of CC chemokine receptors, specifically CCR5, along with CCR4, CCR6 and CCR8. This is because VTL1 retains the binding specificity but lacks any signaling domain. VTL1 is specific for the same receptor as VTL3. The encoded signaling domain has been previously characterized (Dewin et al., 2006; Catusse et al 2007). A novel cDNA encoding VTL1 has had the signaling domain removed by splicing but retains the binding domain. Furthermore, VTL1 has been shown to protect against HSV2 infection by vaccination with known immunogens (Figure 5a, Figure 6a, Figure 7a, Figure 8a, Figure 9a, Figure 10a, Figure 11a, Figure 12a, Figure 13a, Figure 14a, Figure 15). As mentioned above, this is because VTL1 retains binding domains that contain specificity for CCR5, as well as CCR4, CCR6 and CCR8, and thus may act as an antagonist by binding without signaling. Thus, VTL1 is expected to contain the same or similar CCR5 antagonist properties as Met-CCL5. Thus, VTL1, through combined chemokine receptor activity, may inhibit chemoattraction of the entire T-reg population, thereby shifting the balance of conventional T cell proliferation and resulting in more efficient antibody and effector T cell production.

[0132] Met-CCL17 and Met-CCL20 are also predicted to antagonize CCR4 and CCR6, respectively. In particular, CCL17 and CCL20 have a similar structure to CCL5, and as discussed above, it is the N-terminus of each of these chemokines that interacts with the relevant receptors. Met-CCL5 contains an additional methionine residue at its N-terminus compared to wild-type CCL (Proudfoot et al, 1996), and the same modification of CCL17 and CCL20 is predicted to have a similar effect as methionylation of CCL5, i.e., converting CCL17 and CCL20 into antagonists of CCR4 and CCR6, respectively.

[0133] Thus, any known CC chemokine receptor antagonist suitable for binding to the CC chemokine receptor of interest but not activating it may be used. Furthermore, methods are known for determining the binding affinity and biological activity of molecules using chemotaxis assays and receptor binding assays, such as those described in Proudfoot et al., 1996. Thus, the CC chemokine receptor antagonists used in the present invention are not limited to the specific examples described herein.

[0134] Furthermore, if the mixture includes a CCR5 antagonist, it is expected that the CCR5 antagonist can be combined with a CCR4 and / or CCR6 agonist and still antagonize, prevent or reduce the immune response. This is because CCR5 is a marker of activated T cells, such that the CCR5 antagonist prevents the recruitment of activated regulatory T cells. Thus, although CCR4 and / or CCR6 agonists promote the recruitment of regulatory T cells, they are not expected to be or become activated regulatory T cells, such that regulatory T cells are not effective at dampening the immune response.

[0135] The first and second chemokines, and an optional third chemokine or antagonist variant thereof, can be combined in any of the combinations shown in Table 3. Each of the chemokines or variants thereof comprises the specified sequence and may include further modifications.

[0136] [Table 3]

[0137] In some embodiments, the first chemokine, or antagonist variant thereof, comprises Met-CCL17 and the second chemokine, or antagonist variant thereof, comprises Met-CCL5 or SEQ ID NO: 20. In some embodiments, the first chemokine, or antagonist variant thereof, comprises CCL17 and the second chemokine, or antagonist variant thereof, comprises Met-CCL5 or SEQ ID NO: 20. In some embodiments, the first chemokine, or antagonist variant thereof, comprises Met-CCL17, the second chemokine, or antagonist variant thereof, comprises Met-CCL20 or SEQ ID NO: 20 and the third chemokine, or antagonist variant thereof, comprises Met-CCL20. In some embodiments, the first chemokine, or antagonistic variant thereof, is CCL17, the second chemokine, or antagonistic variant thereof, is Met-CCL5 or SEQ ID NO: 20, and the third chemokine, or antagonistic variant thereof, is CCL20.

[0138] In some embodiments, the first chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:12, and the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:10, SEQ ID NO:14 and SEQ ID NO:20. In some embodiments, the first chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12, and the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO: 10 and SEQ ID NO:20. In some embodiments, the first chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:12, and the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:14.

[0139] In some embodiments, the first chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5, and the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10 or SEQ ID NO:20.

[0140] In some embodiments, the first chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:12; the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10; and the third chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:14. In some embodiments, the first chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12; the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20; and the third chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14.

[0141] In some embodiments, the first chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5; the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10; and the third chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:14. In some embodiments, the first chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:12; the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10; and the third chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:8. In some embodiments, the first chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5; the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10; and the third chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:8.In some embodiments, the first chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5; the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:20; and the third chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:14. In some embodiments, the first chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12; the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20; and the third chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the first chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5; the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:20; and the third chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:8.

[0142] In some embodiments, the first chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 12 and the second chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO: 20. In some embodiments, the first chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 12 and the second chemokine, or antagonist thereof, comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 20. In some embodiments, the first chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 12 and the second chemokine, or antagonist thereof, comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments of the above mixtures, each of the chemokines, or antagonist variants thereof, comprises the specified sequence.

[0143] In some embodiments, the first chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 5, and the second chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 20. In some embodiments of the above mixtures, each of the chemokines, or antagonist variants thereof, consists of the designated sequences.

[0144] In some embodiments, the first chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 12, the second chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 10, and the third chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the first chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 12, the second chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 20, and the third chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments of the above mixtures, each of the chemokines, or antagonist variants thereof, comprises the designated sequence.

[0145] In some embodiments, the first chemokine, or antagonist variant thereof, comprises the amino acid sequence consisting of SEQ ID NO:5, the second chemokine, or antagonist variant thereof, comprises the amino acid sequence consisting of SEQ ID NO:10, and the third chemokine, or antagonist variant thereof, comprises the amino acid sequence consisting of SEQ ID NO:14. In some embodiments, the first chemokine, or antagonist variant thereof, comprises the amino acid sequence consisting of SEQ ID NO:5, the second chemokine, or antagonist variant thereof, comprises the amino acid sequence consisting of SEQ ID NO:20, and the third chemokine, or antagonist variant thereof, comprises the amino acid sequence consisting of SEQ ID NO:14. In some embodiments, the first chemokine, or antagonist variant thereof, comprises the amino acid sequence consisting of SEQ ID NO:12, the second chemokine, or antagonist variant thereof, comprises the amino acid sequence consisting of SEQ ID NO:10, and the third chemokine, or antagonist variant thereof, comprises the amino acid sequence consisting of SEQ ID NO:8. In some embodiments, the first chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 12, the second chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 20, and the third chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 5, the second chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 10, and the third chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 5, the second chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 20, and the third chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments of the above mixtures, each of the chemokines, or antagonist variants thereof, consists of a designated sequence.

[0146] In some embodiments, the first chemokine consists of SEQ ID NO: 12, the second chemokine consists of SEQ ID NO: 10, and the third chemokine consists of SEQ ID NO: 14. In some embodiments, the first chemokine consists of SEQ ID NO: 5, the second chemokine consists of SEQ ID NO: 10, and the third chemokine consists of SEQ ID NO: 8.

[0147] Polynucleotide mixture The mixture of chemokines, or agonist or antagonist variants thereof, may be encoded by a nucleic acid sequence contained in a polynucleotide. Thus, the present invention also provides a polynucleotide mixture comprising a first and a second, and optionally a third polynucleotide encoding any of the above combinations of the first and the second, and optionally the third, chemokines, and their agonist or antagonist variants, as shown in Table 3 and described above. The polynucleotides may be DNA, cDNA or RNA, and in some embodiments, the polynucleotides are preferably cDNA. In some embodiments, the polynucleotides may also each encode a signal sequence required for correct processing of the respective translated protein into a mature functional form inside a cell, such as a human cell. For example, if the protein is produced in bacteria, it does not require a signal sequence and the mature protein may be produced in that expression system. For example, the amino acid sequences of CCL5, CCL17 and CCL20, including their signal sequences, are shown in SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 7, respectively, and the nucleic acid sequences encoding these proteins are shown in SEQ ID NO: 27 to SEQ ID NO: 29, respectively. The amino acid sequences of the mature forms of CCL5, CCL17 and CCL20 are shown in SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8. However, the signal sequence used in the present invention does not have to be a wild-type signal sequence and may be replaced with a signal sequence from other secreted or membrane-bound proteins, including from other species. As discussed in Nielsen et al., 2019, signal sequences are well known and can be predicted, for example, using the program SIgnalP (Bendtsen et al., 2004). Furthermore, it is well known in the art that signal sequences can be replaced with signal sequences from other proteins and / or other species, such as signal sequences from HSV gD or IgE.

[0148] Thus, the present invention also provides a polynucleotide mixture comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises a nucleic acid sequence encoding a first chemokine, or an agonist or antagonist variant thereof, suitable for binding to CCR4, and the second polynucleotide comprises a nucleic acid sequence encoding a second chemokine, or an agonist or antagonist variant thereof, suitable for binding to CCR5 and / or CCR6, wherein the polynucleotide mixture is suitable for modulating an immune response. In some embodiments, the first polypeptide encodes CCL17, or an agonist or antagonist variant thereof, and the second polynucleotide encodes CCL5 or CCL20, or an agonist or antagonist variant thereof, or SEQ ID NO:20 or SEQ ID NO:26.

[0149] In some embodiments, the polynucleotide mixture comprises a first polynucleotide comprising a nucleic acid sequence encoding a first chemokine, or an agonist or antagonist variant thereof, suitable for binding to CCR4, a second polynucleotide comprising a nucleic acid sequence encoding a second chemokine, or an agonist or antagonist variant thereof, suitable for binding to CCR5, and a third polynucleotide comprising a nucleic acid sequence encoding a third chemokine, or an agonist or antagonist variant thereof, suitable for binding to CCR20. In some embodiments, the first polypeptide encodes CCL17, or an agonist or antagonist variant thereof, the second polynucleotide encodes CCL5, or an agonist or antagonist variant thereof, or SEQ ID NO:20 or SEQ ID NO:26, and the third polypeptide encodes CCL20, or an agonist or antagonist variant thereof.

[0150] In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6 or SEQ ID NO:13, and the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NOs:15-18, and SEQ ID NOs:21-24. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6 or SEQ ID NO:13, and the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:3, SEQ ID NO:11, SEQ ID NOs:16-18, and SEQ ID NOs:21-24. In some embodiments, the first polynucleotide comprises a nucleic acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:6 or SEQ ID NO:13, and the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:9 or SEQ ID NO:15.

[0151] In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6 or SEQ ID NO:13; the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:3, SEQ ID NO:11, SEQ ID NOs:16-18, and SEQ ID NOs:21-24; and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:9 or SEQ ID NO:15.

[0152] Agonist polynucleotide mixture In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6, and the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:3. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6, and the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:9. In some embodiments, the first polynucleotide comprises a nucleic acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6, and the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs:21-24.

[0153] In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:3 and one of SEQ ID NOs:21-24, and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:9. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6; the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:3; and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:9. In some embodiments, the first polynucleotide comprises a nucleic acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs:21-24, and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:9.

[0154] In some embodiments, the first polynucleotide comprises a nucleic acid sequence comprising SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence comprising SEQ ID NO:3, and the third polynucleotide comprises a nucleic acid sequence comprising SEQ ID NO:9. In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:3, and the third polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:9.

[0155] Antagonist Polynucleotide Mixture In some embodiments, a first polynucleotide encodes Met-CCL17 and a second polynucleotide encodes Met-CCL20, Met-CCL5, or SEQ ID NO: 20. In some embodiments, a first polynucleotide encodes CCL17 and a second polynucleotide encodes Met-CCL5 or SEQ ID NO: 20. In some embodiments, a first polynucleotide encodes Met-CCL17, a second polynucleotide encodes Met-CCL5 or SEQ ID NO: 20, and a third polynucleotide encodes Met-CCL20.

[0156] In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13, and the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:11 and SEQ ID NO:15 to SEQ ID NO:18. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13, and the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:11 and SEQ ID NO:16-SEQ ID NO:18. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13, and the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:11. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13, and the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:15. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13, and the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:17.

[0157] In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6, and the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:11 and SEQ ID NO:16-SEQ ID NO:18. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6, and the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13 or SEQ ID NO:17.

[0158] In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13; the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:11; and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:15. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13, the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:16 to 18, and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:15. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13; the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:17; and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:15.

[0159] In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6; the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:11; and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:15. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13; the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:11; and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:9. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6; the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:11; and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:9.In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:16 to 18, and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:15. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6; the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:17; and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:15. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13, the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:16 to 18, and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:9.In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13, the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:17, and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:9. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:16 to 18, and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:9. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6; the second polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:17; and the third polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:9.

[0160] In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 13, and the second polynucleotide comprises a nucleic acid sequence consisting of any one of SEQ ID NO: 11 and SEQ ID NO: 15 to SEQ ID NO: 18. In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 13, and the second polynucleotide comprises a nucleic acid sequence consisting of any one of SEQ ID NO: 16 to SEQ ID NO: 18, preferably SEQ ID NO: 17. In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 13, and the second polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 15.

[0161] In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:6, and the second polynucleotide comprises a nucleic acid sequence consisting of any one of SEQ ID NO:11 and SEQ ID NO:16 to SEQ ID NO:18. In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:6, and the second polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:11 or SEQ ID NO:17.

[0162] In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 13, the second polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 11, and the third polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 15. In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 13, the second polynucleotide comprises a nucleic acid sequence consisting of any one of SEQ ID NOs: 16 to 18, preferably SEQ ID NO: 17, and the third polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 15.

[0163] In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:11, and the third polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:15. In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence consisting of any one of SEQ ID NO:16 to SEQ ID NO:18, preferably SEQ ID NO:17, and the third polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:15. In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:13, the second polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:11, and the third polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:9. In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:13, the second polynucleotide comprises a nucleic acid sequence consisting of any one of SEQ ID NO:16 to SEQ ID NO:18, preferably SEQ ID NO:17, and the third polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:9. In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 6, the second polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 11, and the third polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 9. In some embodiments, the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 6, the second polynucleotide comprises a nucleic acid sequence consisting of any one of SEQ ID NOs: 16 to 18, preferably SEQ ID NO: 17, and the third polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 9.

[0164] Polypeptide + Chemokine Compositions In some embodiments, a composition is provided that comprises at least one polynucleotide and at least one chemokine, or an agonist or antagonist variant thereof, where the composition corresponds to the chemokine mixture and polynucleotide mixture described above. Thus, a composition is provided that comprises a first polynucleotide that comprises a nucleic acid sequence encoding a first chemokine, or an agonist or antagonist variant thereof, suitable for binding to one of CCR5, CCR4 and / or CCR6, and a second chemokine, or an agonist or antagonist variant thereof, suitable for binding to one of CCR5, CCR4 and / or CCR6 different from the second chemokine, or an agonist or antagonist variant thereof, where the composition is suitable for modulating an immune response. The first polynucleotide may be any of the polynucleotides described above, and the second chemokine, or an agonist or antagonist variant thereof, may be any of the chemokine mixtures described above, or an agonist or antagonist variant thereof. The composition may also comprise a second polynucleotide comprising a nucleic acid sequence encoding a third chemokine, or an agonist or antagonist variant thereof, or the composition may comprise a third chemokine, or an agonist or antagonist variant thereof, which is suitable for binding to a different one of CCR5, CCR4 and / or CCR6 than the first and second chemokines, or agonist or antagonist variants thereof. The second polynucleotide may be any of the polynucleotides described above, and the third chemokine, or agonist or antagonist variant thereof, may be any of the chemokines described above, or agonist or antagonist variants thereof.

[0165] Thus, in some embodiments, the composition comprises a first and a second polynucleotide, each encoding a first and a second chemokine, or an agonist or antagonist variant thereof, respectively, and includes those chemokines, or an agonist or antagonist variant thereof, hi other embodiments, the composition comprises a first polynucleotide encoding a first chemokine, or an agonist or antagonist variant thereof, and includes a second and a third chemokine, or an agonist or antagonist variant thereof.

[0166] In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine, or an agonist or antagonist variant thereof, and a second chemokine, or an agonist or antagonist variant thereof, according to any combination shown in Table 4. The first polynucleotide corresponds to any of the related polynucleotides described above, and the second chemokine, or an agonist or antagonist variant thereof, corresponds to any of the related chemokines, or agonist or antagonist variants thereof, described above.

[0167] [Table 4]

[0168] In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine, or an agonist or antagonist variant thereof, a second chemokine, or an agonist or antagonist variant thereof, and a second polynucleotide encoding a third chemokine, or an agonist or antagonist variant thereof, according to any of the combinations shown in Table 5 (wherein column 3 is labeled "Third chemokine, or agonist or antagonist variant thereof," which includes a chemokine encoded by a second polynucleotide). The first and second polynucleotides independently correspond to any of the above-mentioned polynucleotides, and each of the chemokines, or agonist or antagonist variants thereof, independently corresponds to any of the above-mentioned chemokines, or agonist or antagonist variants.

[0169] [Table 5]

[0170] In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine, or an agonist or antagonist variant thereof, a second chemokine, or an agonist or antagonist variant thereof, and a third chemokine, or an agonist or antagonist variant thereof. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 95%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:3, SEQ ID NO:11, SEQ ID NOs:16-18, and SEQ ID NOs:21-24, and the second chemokine, or an agonist or antagonist variant thereof, is selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6. 12, and a third chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:8 or SEQ ID NO:14.

[0171] Agonist Compositions In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine or agonist variant thereof, a second chemokine or agonist variant thereof, and a third chemokine or agonist variant thereof. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:21-24; the second chemokine or agonist variant thereof comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5; and the third chemokine or agonist variant thereof comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:8.

[0172] In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine or agonist variant thereof, where the first polynucleotide comprises a nucleic acid sequence comprising any one of SEQ ID NO:21-SEQ ID NO:24, the second chemokine or agonist variant thereof comprises the amino acid sequence of SEQ ID NO:5, and the third chemokine or agonist variant thereof comprises the amino acid sequence of SEQ ID NO:8.

[0173] In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine or agonist variant thereof (wherein the first polynucleotide comprises a nucleic acid sequence consisting of any one of SEQ ID NO:21 to SEQ ID NO:24), a second chemokine or agonist variant thereof consisting of SEQ ID NO:5, and a third chemokine or agonist variant thereof consisting of SEQ ID NO:8.

[0174] Antagonist Compositions In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine, or an antagonist variant thereof, a second chemokine, or an antagonist variant thereof, and a third chemokine, or an antagonist variant thereof. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:16-18; the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:12; and the third chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:14. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:16-18; the second chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5; and the third chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:8.

[0175] In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:17, the second chemokine, or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:12, and the third chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:14. In some embodiments, the first polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:17; the second chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5; and the third chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:8.

[0176] In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine, or an antagonist variant thereof, wherein the first polynucleotide comprises a nucleic acid sequence comprising any one of SEQ ID NOs: 16-18, the second chemokine, or an antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 12, and the third chemokine, or an antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine, or an antagonist variant thereof, wherein the first polynucleotide comprises a nucleic acid sequence comprising any one of SEQ ID NOs: 16-18, the second chemokine, or an antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 5, and the third chemokine, or an agonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine, or antagonist variant thereof, where the first polynucleotide comprises a nucleic acid sequence comprising SEQ ID NO: 17, the second chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 12, and the third chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine, or antagonist variant thereof, where the first polynucleotide comprises a nucleic acid sequence comprising SEQ ID NO: 17, the second chemokine, or antagonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 5, and the third chemokine, or agonist variant thereof, comprises the amino acid sequence of SEQ ID NO: 8.

[0177] In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine, or an antagonist variant thereof, where the first polynucleotide comprises a nucleic acid sequence consisting of any one of SEQ ID NO:16-SEQ ID NO:18, a second chemokine, or an antagonist variant thereof consisting of SEQ ID NO:12, and a third chemokine, or an agonist variant thereof consisting of SEQ ID NO:14. In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine, or an antagonist variant thereof, where the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO:16-SEQ ID NO:18, a second chemokine, or an antagonist variant thereof consisting of SEQ ID NO:5, and a third chemokine, or an agonist variant thereof consisting of SEQ ID NO:8. In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine, or antagonist variant thereof, where the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 17, a second chemokine, or antagonist variant thereof, consisting of SEQ ID NO: 12, and a third chemokine, or agonist variant thereof, consisting of SEQ ID NO: 14. In some embodiments, the composition comprises a first polynucleotide encoding a first chemokine, or antagonist variant thereof, where the first polynucleotide comprises a nucleic acid sequence consisting of SEQ ID NO: 17, a second chemokine, or antagonist variant thereof, consisting of the amino acid sequence of SEQ ID NO: 5, and a third chemokine, or agonist variant thereof, consisting of the amino acid sequence of SEQ ID NO: 8.

[0178] Nucleic acid construct mixture Also provided herein are nucleic acid construct mixtures corresponding to the above-mentioned polynucleotide mixtures and compositions. In particular, the nucleic acid construct mixtures of the present invention comprise nucleic acid constructs corresponding to the polynucleotides in the polynucleotide mixtures and compositions of the present invention, respectively. For example, when the polynucleotide mixture comprises a first and a second polynucleotide, the nucleic acid construct mixture comprises a first nucleic acid construct comprising the first polynucleotide, and a second nucleic acid construct comprising the second polynucleotide. When the polynucleotide mixture also comprises a third polynucleotide, the nucleic acid construct mixture comprises a third nucleic acid construct comprising the third polynucleotide. Similarly, when the composition comprises a first and a second polynucleotide, the nucleic acid construct mixture comprises a first nucleic acid construct comprising the first polynucleotide, and a second nucleic acid construct comprising the second polynucleotide.

[0179] nucleic acid construct Also provided herein are nucleic acid constructs each encoding all of the polynucleotides in the polynucleotide mixture or composition of the present invention.Thus, when the polynucleotide mixture of the present invention comprises a first and a second polynucleotide, the nucleic acid construct comprises both the first and the second polynucleotide.When the polynucleotide mixture of the present invention comprises a first, a second and a third polynucleotide, the nucleic acid construct comprises the first, the second and the third polynucleotide.When the composition of the present invention comprises a first polynucleotide, the nucleic acid construct comprises the first polynucleotide.When the composition of the present invention comprises a first and a second polynucleotide, the nucleic acid construct comprises the first and the second polynucleotide.

[0180] Each of the above-mentioned nucleic acid constructs in the nucleic acid construct mixture may contain additional elements required for the expression of the polynucleotide(s) contained therein. For example, each of the nucleic acid constructs may contain a promoter for initiating expression operably linked to the polynucleotide, and / or a polyadenylation site for terminating expression. When a nucleic acid construct contains more than one polynucleotide, there may be a promoter operably linked to each of the polynucleotides, or there may be a single promoter operably linked in such a way as to allow expression of all of the polynucleotides.

[0181] host cell In some embodiments, a host cell is provided that comprises the polynucleotide mixture, nucleic acid construct mixture or nucleic acid construct described above. The host cell may be prokaryotic or eukaryotic and may comprise a bacterial cell, a fungal cell such as yeast, a plant cell, an insect cell, or a mammalian cell. In a preferred embodiment, the mammalian cell is a human cell. Preferably, the host cell expresses the polynucleotide(s) contained therein.

[0182] Pharmaceutical Compositions In some embodiments, a pharmaceutical composition is provided that includes the chemokine mixture, polynucleotide mixture, composition, nucleic acid construct mixture, nucleic acid construct, or host cell of the present invention. The pharmaceutical composition further includes a pharma- ceutically acceptable carrier, excipient, or diluent. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences", by EW Martin, 1995. Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions, emulsions, etc.) composition for oral, topical, or parenteral administration.

[0183] In some embodiments, the pharmaceutical composition further comprises a therapeutic or prophylactic agent, and / or an adjuvant. The adjuvant may be any adjuvant known in the art, such as GM-CSF or G-CSF.

[0184] use The chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, nucleic acid constructs, host cells and pharmaceutical compositions of the present invention are for use as immunostimulants or immunosuppressants. In particular, the chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, nucleic acid constructs, host cells and pharmaceutical compositions of the present invention target regulatory T cells that regulate immune responses. In some embodiments, the chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, nucleic acid constructs, host cells and pharmaceutical compositions of the present invention are for use in the treatment or prevention of diseases or disorders characterized by altered levels of CCR1, 4, 5, 6 and 8 or their binding chemokines, or diseases or disorders associated with dysregulated immune responses. In some embodiments, the treatment includes using the chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, nucleic acid constructs, host cells and pharmaceutical compositions as adjuvants.

[0185] In some embodiments, the disease or disorder is cancer, a viral infection, Alzheimer's disease, an autoimmune disease, an inflammatory disease or condition, allergy, or a disease or condition associated with a dysregulated immune response.

[0186] The viral infection may be an HIV infection, HIV / AIDS, an HSV infection, e.g., HSV1 or HSV2, influenza, a coronavirus infection, e.g., a human coronavirus infection such as SARS-CoV-2 or a SARS-like virus, human cytomegalovirus, Epstein-Barr virus, a rhinovirus infection, or hepatitis B or C virus.

[0187] The cancer may be a solid tumor, such as prostate cancer, breast cancer or lymphoma.

[0188] The autoimmune disease may be arthritis, such as rheumatoid arthritis, Crohn's disease or chronic obstructive pulmonary disease.

[0189] Dysregulation of immune response may include conditions that are considered to have characteristics of an innate response, such as Alzheimer's disease or multiple sclerosis, or an over-response to an infectious disease, such as infectious mononucleosis. Diseases and conditions associated with dysregulation of immune response may also include diseases and conditions associated with runaway immune response, such as cytokine storm, such as SARS-CoV-2. Diseases and conditions associated with dysregulation of immune response may also include rheumatoid arthritis or CNS or pulmonary inflammatory diseases.

[0190] Allergies may include cat allergies, dust mite allergies, and hay fever. In some embodiments, the chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, nucleic acid constructs, host cells, and pharmaceutical compositions may be injected with an allergen, such as a cat hair allergen, to induce tolerance to the allergen or reduce the immune response to the allergen.

[0191] In some embodiments, the chemokine mixture, polynucleotide mixture, composition, nucleic acid construct mixture, nucleic acid construct, host cell and pharmaceutical composition of the present invention are used as immunosuppressants to reduce or prevent immune responses. In these embodiments, the chemokine mixture, polynucleotide mixture or composition targets activated regulatory T cells, which in turn downregulates immune responses such as T cell responses or antibody responses. Thus, the above-mentioned agonist mixtures and compositions can be used as such immunosuppressants by targeting activated T cells and recruiting them. In these embodiments, the disease and / or condition to be treated is preferably one associated with an excessive or out-of-control immune response or is an autoimmune disease, an inflammatory disease and an allergy. Specific examples of these diseases are described above.

[0192] In some embodiments, the chemokine mixture, polynucleotide mixture, composition, nucleic acid construct mixture, nucleic acid construct, host cell and pharmaceutical composition of the present invention are used as immunostimulants. In these embodiments, the chemokine mixture, polynucleotide mixture or composition targets regulatory T cells but blocks the recruitment of activated regulatory T cells. This can be by antagonizing CC chemokine receptors, which are markers of activated regulatory T cells, or by stimulating CC chemokine receptors (e.g., CCR4, CCR8 and CCR6), which are markers of regulatory T cells, but antagonizing CC chemokine receptors (e.g., CCR5), which are markers of activated T cells. In this latter embodiment, regulatory T cells are recruited, but they are not activated, so that they do not suppress the immune response. This blocking of regulatory T cells allows an immune response, such as a T cell response or an antibody response. Thus, the antagonist mixtures and compositions described above can be used as immunostimulants by targeting and blocking regulatory T cells. In these embodiments, the disease and / or condition to be treated is preferably cancer, a viral infection or Alzheimer's disease, specific examples of which are described above. These antagonist mixtures and compositions may also be used as adjuvants, thereby enhancing the immune response induced by a therapeutic or prophylactic treatment.

[0193] Treatment In some embodiments, a method of treating a patient in need of treatment is provided, wherein the method comprises administering to the patient a chemokine mixture, polynucleotide mixture, composition, nucleic acid construct mixture, nucleic acid construct, host cell or pharmaceutical composition as described above according to the present invention. In some embodiments, the patient is a human or a non-human animal, preferably a human. In some embodiments, the chemokine mixture, polynucleotide mixture, composition, nucleic acid construct mixture, nucleic acid construct, host cell and pharmaceutical composition are administered as a therapeutic or prophylactic treatment. In other embodiments, the chemokine mixture, polynucleotide mixture, composition, nucleic acid construct mixture, nucleic acid construct, host cell and pharmaceutical composition are administered as an adjuvant. The treatment may be a disease or disorder characterized by altered levels of CCR1, 4, 5, 6 and 8 or their binding chemokines, or a disease or disorder associated with an abnormal immune response, as described above. Thus, in some embodiments, the chemokine mixture, polynucleotide mixture, composition, nucleic acid construct mixture, nucleic acid construct, host cell or pharmaceutical composition stimulates regulatory T cells to suppress or prevent an immune response. In other embodiments, the chemokine mixture, polynucleotide mixture, composition, nucleic acid construct mixture, nucleic acid construct, host cell or pharmaceutical composition antagonizes regulatory T cells and tolerates or enhances an immune response.

[0194] Administration The chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, nucleic acid constructs, host cells and pharmaceutical compositions of the present invention may be administered by any suitable method known to those skilled in the art. For example, administration may be accomplished orally or parenterally. Methods of parenteral delivery include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intracerebroventricular, intravenous, intraperitoneal, mucosal or intranasal administration. In some embodiments, for example, in the treatment of arthritis, the chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, nucleic acid constructs, host cells and pharmaceutical compositions may be injected directly into the joint to be treated. In some embodiments, when the chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, nucleic acid constructs, host cells and pharmaceutical compositions are for the treatment or prevention of allergies, the chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, nucleic acid constructs, host cells and pharmaceutical compositions may be injected subcutaneously in the style of an epipen. In embodiments where the chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, nucleic acid constructs, host cells and pharmaceutical compositions are for the treatment of respiratory diseases or conditions associated with cytokine storm (e.g., SARS-CoV-2 and influenza), the chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, nucleic acid constructs, host cells and pharmaceutical compositions may be administered to the lungs using an inhaler.

[0195] When the chemokine mixture, polynucleotide mixture, composition, nucleic acid construct mixture, nucleic acid construct, host cell and pharmaceutical composition of the present invention is used as a prophylactic treatment, it may be administered at least once, at least twice or at least three times, each administration may be at least one week apart, at least two weeks apart or at least three weeks apart. Preferably, the chemokine mixture, polynucleotide mixture, composition, nucleic acid construct mixture, nucleic acid construct, host cell or pharmaceutical composition is administered twice, each administration may be three weeks apart.

[0196] When the chemokine mixture, polynucleotide mixture, composition, nucleic acid construct mixture, nucleic acid construct, host cell and pharmaceutical composition is used as a therapeutic treatment, it may be administered once every week, once every two weeks, once every three weeks, or once every month. For example, the chemokine mixture, polynucleotide mixture, composition, nucleic acid construct mixture, nucleic acid construct, host cell and pharmaceutical composition may be administered upon onset or diagnosis of a disease or condition, and then administered every two weeks on a maintenance schedule.

[0197] Use in pharmaceutical manufacturing In some embodiments, the chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, nucleic acid constructs, host cells or pharmaceutical compositions of the invention are used in the manufacture of a medicament for use in the treatment or prevention of a disease or disorder characterized by altered levels of CCR1, 4, 5, 6, and 8 or their binding chemokines, or a disease or disorder associated with an abnormal immune response, as described above, or for use as an immunostimulant or immunosuppressant.

[0198] kit In some embodiments, kits are provided comprising the chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, nucleic acid constructs, host cells or pharmaceutical compositions of the invention, which may be for use in the treatment or prevention of diseases or disorders characterized by altered levels of CCR1, 4, 5, 6 and 8 or their binding chemokines, or diseases or disorders associated with an abnormal immune response, as described above, or for use as an immunostimulant or immunosuppressant.

[0199] In some embodiments, the kits include pre-formed chemokine mixtures, polynucleotide mixtures, compositions, nucleic acid construct mixtures, or pharmaceutical compositions of the invention. In other embodiments, the kits include separate chemokines, polynucleotides, or nucleic acid constructs in separate containers. The mixtures and compositions of the invention are then prepared from the kits by mixing together appropriate amounts of each of the chemokines, polynucleotides, or nucleic acid constructs. The kits may also include pharma- ceutically acceptable carriers, diluents, or excipients that can be combined with the mixture or composition in appropriate amounts to form a pharmaceutical composition. EXAMPLES

[0200] Example 1 The human herpesviruses HHV-6A and HHV-6B can integrate in the telomeric regions of the host genome, integrating in approximately 1% of the human population at roughly equal rates: HHV-6A 0.2% and HHV-6B 0.4% (Tweedy, 2016 #18). These appear to be ancient events, linked to Mendelian genetic lineages, and therefore these integrations appear to be endogenous viral genomes. The chromosomally integrated inherited human HHV-6B (iciHHV-6B) genome appears to co-segregate with the circulating viral HHV-6B, while the chromosomally integrated inherited HHV-6A (iciHHV-6A) genome appears to be separate (Tweedy, 2015, Tweedy, 2016, Greninger, 2018). Our analysis by deep next generation sequencing shows one of the oldest lineages related to integration events at the telomere of chromosome 17p.Surprisingly, our analysis of the genome shows that all genes are intact, and that the known cis-acting sequences required for viral replication are present.As described herein, the encoded immunomodulatory genes include those with novel spliced ​​products with unexpectedly distinct characteristics (as shown in Figure 2).

[0201] In circulating HHV-6A virus, the U83A gene encodes a chemokine-like molecule that can mediate immune cell chemotaxis with unique specificity through interaction with a series of human chemokine receptors (Catusse, 2009; Catusse 2007; Clark, 2013; Dewin, 2006). This specificity was different from that of any other human chemokines or microbial peptides. We hypothesized that if the integrated genome was derived from an ancestral virus that infected Homo sapiens or the ancestors of hominidae, the immune-modulating genes may be distinct. While we found that the gene structure was maintained across the genome in the immune-modulating gene, chemokine U83A, we unexpectedly found that in iciHHV-6A, iciU83A (Figure 1), the transcripts were different (cDNA, Figure 2) and encoded altered protein products (Figure 2).

[0202] The circulating viral U83A gene is spliced ​​using a non-canonical splice donor and acceptor site contained within the aberrant direct repeat sequence CT-AC within TACC (French, 1999; Tweedy, 2015). In the iciHHV-6A genome, these cis-acting elements are maintained, but a separate non-synonymous SNP is present proximal to the tentative splice acceptor site, which is positioned to affect the consensus site of the acceptor splicing sequence and thus disrupt the aberrant splicing event (Figure 1). Furthermore, our previous results provide evidence for a full-length unspliced ​​transcript and integrated iciHHV-6A expressed in two individuals with heart disease (Tweedy, 2015). Therefore, it was unclear whether any splicing events had taken place at this locus in iciHHV-6A. This is shown in our annotation of the first genome sequence for iciHHV-6A, where only the full-length gene product is shown (NCBI NC_001664.4 reference genome sequence for HHV-6A and KT895199.1 for iciHHV-6A). Surprisingly, in the virus, full-length U83A encodes a complete signal sequence, resulting in a mature secretory product, which is in fact rarely expressed in circulating viruses. This is due to a mutation in the polyT tract that disrupts expression of the gene by conferring a frameshift mutation, so that the signal sequence is not made and the product is not secreted. However, in the iciHHV-6A genome, the iciU83A gene is genetically fixed and encodes a complete signal sequence with a fixed length polyT tract (Tweedy et al., 2015). Furthermore, it was not clear whether any splicing took place in this ancestral iciU83A gene.Transcriptomic analysis using RNAseq on circulating viral gene expression identified only spliced ​​antisense transcripts mapping to the U83 locus or restricted gene expression in integrated iciHHV-6A / B genomes, consisting of immediate early genes involved in gene regulation, with no further description of U83A (Peddu;, 2019). Previous reviews on viral chemokines noted restricted gene expression and also the lack of spliced ​​U83A in transcripts identified in people carrying iciHHV-6A genomes, and refer to our previous genomic analysis, which refers to two iciHHV-6 integrated genomes, where full-length transcript expression of U83A was only detected in two patient-donors (Pontejo et al., 2018; Tweedy et al., 2015).

[0203] By studying individual gene expression in transfected cells in vitro, we have shown that the integrated U83A gene (iciU83A) can be expressed and spliced ​​as described herein. It was identified by using genomic prediction and by cDNA analysis of the ciU83A gene in an expression vector transfected into cells, and then characterized using RT-PCR followed by sequencing. This shows that, despite previous in vivo and cellular characterization of iciHHV-6A individuals, and despite a SNP proximal to the splice donor site, it is able to utilize non-consensus splicing (Figure 2). This mutation causes a coding mutation of GAT (Asp) to GGT (Gly) in the integrated full-length human iciU83A gene in the virus (Figures 1 and 2). Moreover, we surprisingly found that this same coding mutation from a splicing-induced frameshift precisely disrupted the spliced-out stop codon that led to a truncated version of the viral U83A gene, U83A-N, since it now contains only the encoded N-terminal half of the molecule (Fig. 2 ).

[0204] In the viral genome, U83A and U83A-N respectively code for full-length and truncated protein products from the spliced ​​gene (Dewin et al., 2006). They function as agonist and antagonist forms of the U83A chemokine pair and are central to orchestrating immune cell attraction to viral replication sites for viral spread or immune evasion. Surprisingly, in chromosomally integrated human HHV-6A (iciU83A), the spliced ​​product cannot be cleaved at this site. Instead, it now extends to the downstream stop codon, resulting in the extended cleavage product iciU83A-N (Figure 2), which now bears a hydrophobic tag of eight amino acids (Figure 2). It can act to bind to membranes or mediate multimerization, causing changes in stabilization and presentation.

[0205] The spliced ​​products have distinct functional domains. We have previously demonstrated that the encoded N-terminal domain determines the specificity of chemokine receptor interactions (Dewin et al., 2006). This can be delineated into a 17 amino acid peptide region, where the determinant of specificity between the interactions of CCR2 and CCR5 is determined by a single arginine residue (Clark et al., 2013). In the iciU83A-N molecule, this N-terminal domain remains intact (Figures 1 and 2), representing the receptor specificity as previously defined. Indeed, in a comparison of the spliced ​​viral U83A-N molecule with the full-length U83A, all receptor specificity was maintained, and only the C-terminal signaling domain was abrogated in the spliced ​​truncated molecule. This functional N-terminal domain is also conserved in the iciU83A-N molecule, where the encoded N-terminal binding domain is completely intact, but the C-terminal signaling domain has been removed. Surprisingly, however, the iciU83A-N molecule is not only truncated, but it also has a C-terminal extension of eight amino acids that constitute a hydrophobic extension (Figure 2). This is composed of an aromatic residue-rich domain, which, via multiple tryptophan residues, can increase membrane binding and specific multimerization of the molecule. Such tryptophan "tagging" experimentally increased the stability and multimerization of the covalent peptide, and also disrupted lipid membrane interactions, enhanced bactericidal activity, and promoted multimerization (Kamei et al., 2018; Singh et al., 2017; Yau et al., 1998). Thus, this hydrophobic C-terminal tag in the iciU83A-N encoded molecule provides a distinct activity superior to other virus-encoded chemokine molecules.

[0206] Reported specificities derived from the conserved N-terminal domain include targeting CCR1, 4, 5, 6 and 8 receptors (Catusse et al., 2009, Catusse et al., 2007, Dewin et al., 2006). This unique combination allows targeting of immunosuppressive T-regulatory factor lymphocytes, specifically via CCR4 and CCR6. Human CCR6 is monospecific for CCL20. Thus, the expansion of receptor interactions to include CCR6 is a unique property of the iciU83A-N molecule. The unique utility of iciU83A-N lies in its ability to act as an antagonist of these receptors, due to the absence of a C-terminal signaling portion. Antagonism of CCR4 in particular has been demonstrated as a novel mechanism to boost immunity against target antigens (Bayry et al., 2008) and is also useful in elucidating immune reactivity against tumors by altering the tumor microenvironment (Vilgelm et al., 2019).

[0207] The iciU83A and iciU83A-N genes have some unusual cis-acting features. We have previously shown that the viral U83A gene has a poly-T motif towards the N-terminal coding region. This results in instability at the 5' end of the gene, as variations in the number of T bases can cause frameshift mutations, which lead to premature termination of the encoded peptide, thereby controlling the expression of the U83A gene (Figure 1) (Dewin et al., 2006; Tweedy et al., 2015). This is likely controlled by a herpesvirus DNA editing mechanism, which promotes mutations in many human herpesviruses. For example, in herpes simplex virus, human alphaherpesvirus type 1 and type 2, DNA editing occurs in polyC or G tracts, which may occur in this GC-rich genome, leading to homopolymer frameshift mutations (HFMs) arising due to individual differences in homopolymer tracts, whereas in HHV-6, which has an AT-rich genome, HFMs now occur in polyT tracts (Tweedy et al., 2016, Tweedy et al., 2017, Dewin et al., 2006, Tweedy et al., 2015). Only with an in-frame set of polyT bases can the complete gene be expressed, which then encodes an N-terminal signal sequence required for co-translational insertion into the endoplasmic reticulum, followed by cleavage and processing for secretion of the mature U83A chemokine-like molecule. In circulating viruses, most of them disrupt the U83A gene via a polyT tract, so this is a rare variant, and evidence has been presented that this may change during monoinfection (Dewin et al., 2006; Tweedy et al., 2015). However, unlike viruses, only the integrated genome of iciHHV-6A, the ciU83A genome integrated at the 17p locus in the subtelomeric / telomeric region, has a polyT tract that allows the full-length ciU83A molecule to be produced.Deep sequencing shows that this is distinct from the circulating virus and is the predominant or only form (Tweedy et al., 2015, Tweedy et al., 2015, Tweedy et al., 2016, Tweedy., 2017). This form iciU83A has only two nonsynonymous SNPs, resulting in coding changes compared to the circulating virus U83A (Figure 2). One of these is in the N-terminal region, but is part of the natural variation in the exogenous virus (Clark et al., 2013). The second is a mutation proximal to the splice acceptor site (Figure 2). As the N-terminal region is maintained compared to the viral U83A, this indicates shared receptor specificity. Only the C-terminal signaling domain has this single nonsynonymous mutation.

[0208] Analysis of the length of the polyT tract in strain mutants and other integrated viral genomes shows that the full-length gene can be stabilized only when the polyT tract is disrupted. Therefore, we mutated this polyT tract while retaining the same codon usage and coding capacity to fix the gene in a full-length encoded functional form. We found that this fixed the gene in a functional form and encoded a signal sequence so that the mature product could be secreted (Figure 1), which we now introduced for function with iciU83A and iciU83A-N (SEQ ID NO: 17, SEQ ID NO: 21 and SEQ ID NO: 23, Figure 6a).

[0209] U83A and iciU83A share two other novel features in their gene structures that affect gene expression. First, both contain the direct repeat TACC, which is novel to this gene. Furthermore, the TACC motif forms part of a non-consensus splice donor and acceptor pair CT-AC, which we previously identified in the disrupted smaller gene product (French et al., 1999). However, the splice donor / acceptor pair in the circulating viral gene U83A is recognized by the cellular splicing machinery, probably via the minor spliceosome, since it is spliced ​​when expressed individually in human cell lines (French et al., 1999; Lin et al., 2010). Although both of these features are present in the iciU83A gene, the predicted splicing effects are completely different. The U83A gene is characteristically spliced ​​to introduce a stop codon at the splice site, resulting in a truncated U83A-N product that is half the size of the full-length product, however this stop codon, TGA, is mutated to TGG in iciU83A-N (Figures 1 and 2) (while in frame with the full-length iciU83A gene, this results in the coding mutation Asp-Gly, Figure 2), which is proximal to the splice acceptor site and may therefore disrupt splicing.

[0210] We cloned this product using a plasmid DNA expression vector containing the human cytomegalovirus IE gene promoter and the SV40 virus polyadenylation site, and transduced it into the cell line HEK293 using a transfection reagent. RNA was extracted and then analyzed by reverse transcription polymerase chain reaction (RT-PCR). The results showed that this splice donor site was utilized, and the further spliced ​​product read through the site of the previous stop codon, unusually extending the coding region by 8 amino acids as described above (Figures 3 and 4).

[0211] In circulating viruses, the encoded full-length U83A is rare due to control by both poly-T tracts that disrupt the gene, as well as non-consensus cellular splicing that cleaves the full-length product. At later stages of infection, splicing can be suppressed, resulting in read-through of the full-length gene product (Dewin et al., 2006; Tweedy et al., 2015). To simulate this effect in the absence of control exerted by circulating viral gene expression, both the direct repeat and the splice donor / acceptor pair can be mutated. This was done for the iciU83A gene while maintaining both codon usage and coding capacity as shown herein. In SEQ ID NO:21-SEQ ID NO:23, we have modified full-length iciU83A to stabilize expression for the uses described herein. SEQ ID NO:21 removes N-terminal heterogeneity, SEQ ID NO:22 prevents splicing, and SEQ ID NO:23 removes N-terminal heterogeneity and prevents splicing. Thus, these products are now uniquely locked to remove heterogeneity (SEQ ID NO:21 and SEQ ID NO:23) or to lock in full length (SEQ ID NO:22 and SEQ ID NO:23) for immune stimulatory utility as outlined in Pontejo et al. 2018 and shown below.

[0212] As mentioned above, the integrated iciHHV-6A genome retains an AT composition bias that is different from that of the human host. Increased protein expression has been demonstrated for other viral genes when the biased composition is matched to that of the human host. To do this, we followed the latest compilation and prediction programs and changed the codon usage of both U83A and iciU83A, and U83A-N and iciU83A-N cDNAs to match that of the human genome (including using public databases such as HIVE db). These were then further modified to accommodate the polyT tract, as well as any remaining parts of the TACC motif and non-consensus donor / acceptor sites. If optimal expressed protein concentrations are required for use as described herein (Figure 6a), for example, these gene constructs can be used in SEQ ID NO: 24 (SEQ ID NO: 24 shows iciU83A mutated to prevent N-terminal heterogeneity, disrupt the TACC direct repeat, and remove splice donor / acceptor sites to lock in gene expression in a stable full-length agonist form with maximized human codon usage (including using HIVE db)).

[0213] Example 2 To evaluate the efficacy of novel virokines derived from iciU83A-N (referred to herein as "VTL1", "VIT1" or "VIT") and a novel chemokine cocktail including CCL5, CCL17 and CCL20 (referred to herein as "VTL3") as immunomodulators and immunotherapeutics, they were tested in preclinical models of infectious disease to distinguish their effects on inducing or inhibiting protective immunity.

[0214] To assess the utility of the chemokine cocktail VTL3 (i.e., a mixture of mature CCL5 (SEQ ID NO:2), mature CCL17 (SEQ ID NO:5) and mature CCL20 (SEQ ID NO:8)) in modulating acute disease, latency, relapse and response to viral infection, assays were performed using a guinea pig model.

[0215] 2.1 Materials and Methods virus The challenge virus was HSV-2 strain MS (ATCC-VR540) propagated at low passage in primary rabbit kidney cells as described (Bernstein 1986) and subsequently titrated on rabbit kidney cell monolayers (Gudnadottir 1964).

[0216] vaccine The HSV2 subunit protein vaccine consisted of a truncated gD2 as formulated by G. Cohen (University of Pennsylvania) from Sf9 (Spodoptera frugiperda) cells (GIBCO BRL) infected with a recombinant baculovirus expressing the gD2 protein as described in Willis 1998. Five micrograms of gD2 protein was mixed with 50 micrograms of the adjuvant MPL (Sigma-Aldrich L6895), made in an aqueous formulation (Ballridge 1999) and stored at 4°C until added to the gD2 protein, and adsorbed to 500 micrograms of aluminum hydroxide and Alhydrogel (Accurate Chemical & Scientific), as previously described in Bourne 2003. The gD2 protein was also formulated with the chemokines CCL5, CCL17 and CCL20 (Peprotech) in sterile saline. This mixture of CCL5, CCL17 and CCL20 is referred to herein as VTL3.

[0217] For the VTL1 vaccine, 200 μg of gD DNA (referred to herein as VTL2gD) was formulated with 100 μg of VTL1 (SEQ ID NO: 17). A DNA construct encoding VTL1 (mutated iciU83A-N, SEQ ID NO: 17) was formulated with 0.25% bupivacaine as described (Bernstein, 1999 #120).

[0218] DNA immunization included a preparation of the HSV2 glycoprotein gene encoding gD, and the human CCL5 gene was synthesized, sequence verified, and expressed from a separate plasmid constructed in the pCMV6neo expression plasmid vector, which contains the human CMV transcription 5' enhancer, promoter, and start site, and the SV40 3' polyadenylation site (Origene).

[0219] animal Pathogen-free female Hartley guinea pigs (Charles River Laboratories) were used (250-350 g).

[0220] immunization When evaluating the prophylactic vaccine VTL3, cohorts of n=12 mice / group were used as follows: Group 1 - no vaccine, Group 2 - 5 μg gD2 protein + MPL / alum, Group 3 - 5 μg gD2 protein + VTL3 (5 μg each of CCL5, CCL17 and CCL20, endotoxin-free, Peprotech), Groups 4 and 5 - DNA formulation of expression plasmid containing full-length HSV2 gD DNA (200 μg VTL2gD) with 100 μg VTL1 DNA (Group 4) or CCL5 DNA (Group 5). All DNA immunizations were formulated in 0.25% bupivacaine as described in Bernstein, Tepe et al., 1999. All immunizations were injected intramuscularly, i.m., followed by an identical boost 3 weeks later.

[0221] Preclinical models of viral challenge The day before virus challenge, animals were bled by clipping the toenails and the isolated serum was subsequently stored at -20°C. Three weeks after the second immunization, animals were challenged with the virus intravaginally, i.vag., as described in Bernstein 2010. Viral challenge was examined for evidence of induction or inhibition of immunity from the vaccine formulation as assayed by effects on acute and recurrent disease, acute and recurrent viral replication, establishment of latent infection and development of neutralizing antibodies using calcium alginate moistened cotton swabs. 6 Animals were inoculated with virus through rupture of the vaginal closure by dosing 0.1 ml of a virus suspension of 1 plaque-forming unit (PFU). Cervicovaginal secretions were collected by swabbing on days 1, 2, 3, and 8 postinoculation (PI) as described by Stanberry 1987, and then stored for assay of viral PFU on rabbit kidney cells cultured in BME (GIBCO) and 10% FBS (Hyclone, Thermo Fisher Scientific).

[0222] Each guinea pig was examined daily and scored for primary genital skin disease. The scoring scale was 0 to 4, with 0 being no disease, 1 being redness or swelling, 2 being 1-3 small vesicles, 3 being more than 3 large confluent lesions, and 4 being several large ulcers with maceration. Animals were also evaluated from days 14-63 after virus challenge to detect any recurrent herpetic lesions by scoring and summing the number of days of lesions, as well as to assess recurrent shedding of virus by vaginal swabs three times a week. Swabs were stored at -80°C until processed for PCR analysis as a marker for viral shedding. At the completion of the study, guinea pigs were sacrificed and dorsal root ganglia (DRG) were aseptically removed and stored at -80°C until DNA extraction for evaluation by PCR for evidence of latent viral infection.

[0223] Neutralizing antibody assay Serum samples were prepared in serial two-fold dilutions at 50 μl per well in 96-well microtiter plates. Then, 50 μl of HSV-2 strain MS containing 3 log10 PFU was added to each well, followed by incubation with 0.1 ml of a 5 log10 suspension of BHK cells at 37° C. for 1 h. Plates were incubated at 37° C. in a 5% CO2 incubator for 3 days. Culture medium was removed and cells were stained with crystal violet, washed, and examined for viral plaques. Effective neutralization titers were determined as the reciprocal of the highest serum dilution without viral plaques indicating 100% protection from CPE, cytopathic effect.

[0224] Quantification of viral DNA by PCR DNA quantification by PCR was performed on vaginal swabs and DRG DNA. DNA extraction was performed using QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's protocol, using tissue homogenized on ice in 500 μl of 2% FBS BME and swabs in vaginal swab medium as described in Bernstein et al. 2010. To detect viral replication, the gB gene was amplified by PCR with two sets of primers for 35 cycles, using 50 ng of purified DNA, 100 pmol of each primer, and Promega Master mix (PROMEGA) in a reaction volume of 25 μl in each PCR reaction, as described in Jerome 2002 and Bernstein 2010.

[0225] statistics Statistics were performed with one-way ANOVA using Dunnett's test for multiple comparisons for the different vaccine treatments versus no vaccine, and for all in vivo cases using Graphpad Prism. In case of non-Gaussian distributions, non-parametric comparisons using Wilcoxon test were used. Significance was determined at a P value <0.05 ( * ), <0.01( ** ), <0.001( ***) Immunization was compared with a non-vaccinated control. Fisher's exact test was used for incidence data with two-sided comparisons.

[0226] 2.2 Results Incidence and severity of acute illnesses Figures 5 and 6 show the daily mean lesion scores and total mean lesion scores, respectively. Figure 5a shows that the total lesions experienced were significantly reduced or eliminated in VTL1 immunized animals compared to the negative control. Those without immunization had a mean total lesion score in terms of severity of 8.29 (SD 6.57) compared to 0.67 (SD 1.48) for the positive control gD protein vaccine and 0.33 (SD 0.62) for the immunogenic formulation VTL2gD+VTL1 immunomodulator VTL1 vaccine (4-14 days after inoculation). These results were better than the gD subunit protein positive control immunization, showing highly significant and almost complete protection compared to the negative control (p<0.0001). The gD subunit protein has been in clinical trials as a vaccine and showed partial protection, therefore this positive control is used to compare all immunization formulations. These were also marked improvements over previous experiments using full-length HSV2 gD2 plasmids expressed alone using a similar protocol, which showed a total lesion score of 2.7 (+ / - 0.7) compared with 5.9 (+ / - 0.5) for the negative control in that experiment (Strasser et al. 2000). Figure 6a also shows a significantly reduced total mean lesion score for animals immunized with VTL1 compared with the negative control, indicating that the VIT1 vaccine confers near complete protection. Thus, the VTL1 DNA vaccine, which contains chromosomally integrated virally encoded cDNA for a human chemokine-like molecule, virokine, provided efficient protection that exceeded that of the adjuvanted subunit protein vaccines used previously in clinical trials.

[0227] Figures 5b and 6b show that VTL3 does not significantly reduce daily or total mean lesions in immunized animals. Thus, VTL3 blocks the immunity induced by gD2 (positive control) against acute infection. VTL3 vaccine treatment eliminated the gD-induced immune response as shown by the significantly reduced scores associated with the VTL3 formulation with gD compared to the gD-only protein vaccine.

[0228] Effect on vaginal replication of the virus Results regarding the effect of gD immunization with VTL1 and VTL3 formulations with gD on lesion development after viral challenge were compared to the effect on viral shedding during primary disease. Analysis of significantly reduced vaginal viral load for VTL1 correlated with disease protection demonstrated and was close to the log reduction in viral shed similar to the gD-only protein formulation (positive control). Positive control (gD2 / MPL-Alum protein formulation). By 8 days after viral challenge, the VIT1 vaccine formulation significantly reduced viral shedding to undetectable levels in nearly all animals (p<0.01) (Figure 7a).

[0229] In contrast, even 2 days after virus challenge, the VTL3 vaccine completely blocked the reduced viral shedding effect observed in the positive control (gD alone, formulated with mpl / alum) (Figure 7b), blocking the immunity induced against gD to prevent viral replication.

[0230] Efficacy for recurrent disease The in vivo preclinical HSV2 model extended follow-up to 63 days after virus challenge after a two-dose immunization schedule with the vaccine formulation. In this guinea pig preclinical model of HSV2 infection, after clearance of the acute primary infection, the virus can reactivate from latency and cause recurrent disease as seen in humans. Assays performed on samples included DNA PCR of vaginal shedding swabs and DNA PCR of sites of latent infection, i.e., dorsal root ganglion (DRG) and spinal cord. Efficacy endpoints were effects on recurrent disease, asymptomatic shedding, and latent viral load. Detection limits were marked and measured for virus quantification at 0.7 log pfu / mL and undetectable qPCR below a detection limit of 0.5 log microgram copies DNA / ml. Thus, VTL3 was able to block protective immunity that can be induced by gD protein, but now its ability to also block immunity against recurrent infection was examined and compared to VIT (also referred to herein as "VTL1").

[0231] Effect on recurrent lesions and number of days of lesions The effect of vaccine (VTL3) treatment on disease recurrence was analyzed from days 15 to 63 after challenge with HSV2 virus. Cumulative daily lesions were plotted and total mean lesion scores per individual were compared.

[0232] This demonstrated that the VTL2gD DNA vaccine was only able to prevent recurrent disease when combined with VIT1, demonstrating the utility of VIT1 (i.e., VTL1) as a therapeutic agent to induce immunity to prevent recurrent disease. Notably, the addition of VIT1 chemokine DNA to VTL2gD DNA immunization induced effective control of recurrent lesions (p<0.05). This was similar to the positive control gD protein subunit vaccine formulation (p<0.01), demonstrating clinical benefit (Figures 8a and 9a). Both VTL2gD DNA+VIT1 and the gD protein subunit vaccine formulation (positive control) reduced the number of days with lesions in those with disease (Figure 8a), while most animals were completely protected from any disease recurrence (7 / 12, 58%, Figure 9a).

[0233] In contrast, Figures 8b and 9b show that immunization with the VTL3+gD formulation eliminated this effect, as no immunity was induced to reduce the number of lesion days or significantly reduce the number of recurrent lesions compared to the negative control (no vaccine), despite the presence of the gD immunogen, and in contrast, an increase in the number of lesion days was observed compared to the negative control (no vaccine treatment) (Figure 8b).

[0234] Effect on viral reactivation as indicated by asymptomatic shedding The efficacy of vaccine treatment was examined for reduction in recurrent viral shedding following evidence of viral reactivation 20 days after viral challenge. To do this, the amount of DNA assayed in vaginal swabs by quantitative PCR was used as a surrogate for viral secretion.

[0235] Analysis of reactivated virus and total mean load in recurrent shedding events in vaccinated animals compared to unvaccinated animals was performed. Whereas the gD protein subunit vaccine was ineffective, a trend towards reduced virus shedding was observed for the VTL2gD DNA+VIT formulation, with VIT1 vaccinated animals having almost half the total load (p=0.1) and one third of the shedding events (20% reduced to 14 recurrences). This reduction was significant compared to the positive control (gD only protein vaccine) (Figure 10a).

[0236] In contrast to the VIT1+gD immunization effect, immunization with VTL3+gD had no effect on virus shedding, as this vaccine showed no significant alteration in recurrent shedding compared to the negative control (no vaccine, FIG. 10b).

[0237] Effect on latent viral load The effect of immunization with VIT1+gD and VTL3+gD formulations on the establishment of latency at sites in the dorsal root ganglion (DRG) and spinal cord was assayed. At the end of the study, 63 days after virus challenge, DNA present was quantified at these sites of latency using qPCR. Analysis of total mean DRG load, as well as trends in virus secretion, showed that the positive control and VTL2gD DNA+VIT1 vaccine significantly induced immunity and reduced levels compared to the negative control, with the VTL2gD DNA+VIT1 vaccine halving the load at p<0.01 (Figure 11a). More than half of animals treated with gD DNA+VIT1 were protected from detectable DNA in the DRG compared to less than 20% of animals not given the vaccine (Figure 12a).

[0238] In contrast, immunization with the VTL3+gD vaccine formulation did not significantly reduce DRG latent DNA load (Figure 11b), and VTL3 did not induce immunity to reduce detectable DNA in DRG (Figure 12b). Even the negative controls showed that some animals were protected from latency after natural infection without immunization, and this effect was abolished by immunization with VTL3.

[0239] Analysis of latent DNA detected in the spinal cord showed similar effects, with both the positive control (gD protein subunit) and the VIT1+gD vaccine formulation significantly inducing immunity to reduce the amount of latent DNA in the spinal cord (p<0.05, FIG. 13a) as well as the number of animals harboring latent viral DNA (FIG. 14a).

[0240] In contrast, VTL3 completely abrogated the significant reduction in latent DNA load in the spinal cord seen in the positive control (p<0.05, FIG. 13b). Moreover, as shown by latent infection DRG analysis, all animals treated with VTL3 established the same latent infection as the negative control (no vaccine), despite exposure to the known efficient immunogen gD2 protein (FIG. 14b). Thus, formulation of the gD2 immunogen with VTL3 completely inhibited all induction of immunity in this preclinical model.

[0241] Effect on induction of neutralizing antibodies VIT1 and VTL3 gD immunization had opposite effects on induction of immunity in guinea pig virus challenge experiments, as assayed by protection from acute and recurrent disease and inhibition of acute or recurrent viral replication. Therefore, we further directly tested the ability of VTL1+gD and VTL3+gD formulations to induce or inhibit antibody production. gD protein immunogens (truncated gD proteins with the transmembrane domain deleted) have previously been shown to effectively induce antibodies, while the addition of CpG, alum or mpl / alum induces neutralizing antibodies as well (Bourne et al., 2003; Awasthi et al., 2017; Ghiasi et al., 1994). Induction of neutralizing antibodies has been shown as a correlate of protection in previous clinical trials of a gD protein formulation with mpl / alum (referred to as ASO4), which showed partial protection (Belshe et al., 2014). It was used here as a positive control in an animal model, where it has previously been shown to induce neutralizing antibodies (Bourne et al. 2003). In a previous comparison of immunizations in the same guinea pig model, gD alone and gD with mpl / alum induced similar levels of neutralizing antibodies (Bernstein et al. 2010). In immunizations with the formulations analyzed here, the gD protein + mp / alum formulation was used as a positive control, which induced effective neutralizing antibodies as previously demonstrated, while the negative control (no vaccine) did not induce detectable virus-neutralizing antibodies (Figure 15).

[0242] The efficacy of immunization with VTL1+gD and VTL3+gD formulations was evaluated in comparison to the known immunogen gD (positive control) as described in the Materials and Methods section above.

[0243] The results (Figure 15) showed high levels of neutralizing antibodies after immunization with either the positive control (gD2) or gD2 DNA formulated with VIT1 (i.e., VTL1). Strikingly, the VTL3 formulation completely abrogated the antibody response. No antibody induction was observed with VTL3 treatment, which resulted in undetectable levels similar to the negative control (no vaccine treatment). This demonstrates that VTL3 acts as a potent immune modulator and can prevent antibody stimulation even in the presence of a potent immunogen. This appears to be a specific inhibitor as a mixture, since immunization with only CCL5, of the three chemokines in gD and VTL3, still results in an immunostimulatory effect. Only the trivalent cytokine formulation VTL3 showed complete abrogation of the immune response. Furthermore, the VTL1 antagonist molecule blocks the chemokine receptor to which VTL3 binds, leading to sustained immune stimulation.

[0244] Such potent immunomodulator formulations have clear applications in conditions of dysregulated or abnormal immune responses, such as those seen in autoimmune diseases, in patients with conditions exacerbated by autoimmune antibody responses, such as rheumatoid arthritis, or after chronic infections such as COVID-19, chronic obstructive pulmonary disease exacerbated by rhinovirus, or other inflammatory conditions. Other potential applications include the treatment or prevention of allergic reactions to allergens, where allergens can be used with the VTL3 formulation in a vaccine to induce a type of immune tolerance to eliminate the immune response to the allergen. Therapeutic formulations for these conditions fill a growing unmet medical need, which the availability of this new formulation can address.

[0245] 2.3 Summary Positive controls showed protection from virus challenge and efficiently induced neutralizing antibodies. It is well established in preclinical animal models that gD2 functions efficiently as an immunogen by itself, either as protein or DNA, and that its immunogenicity can be moderately enhanced with adjuvants such as MPL and alum (Bernstein et al. 1999, Bourne et al. 2003, Bernstein et al. 2010). In clinical trials, gD subunit protein vaccines with MPL and alum showed partial protection (Belshe, Leone et al. 2012).

[0246] In contrast, the evaluation herein showed that the human chemokine cocktail VTL3 has a blocking effect on these activities of gD2. VTL3 was formulated to contain the natural chemokine ligands of chemokine receptors CCR5, CCR4 and CCR6, namely CCL5 (SEQ ID NO:2), CCL17 (SEQ ID NO:5) and CCL20 (SEQ ID NO:8). Thus, VTL3 chemoattracts T regulatory subsets by activating rather than blocking these cognate chemokine receptors. Instead, the results show a complete blockade of all protective effects stimulated by the known immunogenic gD protein.

[0247] In contrast, the VTL1 formulation was highly effective against acute primary disease and viral replication. The VTL1 vaccine was effective against reactivation of viral infection and reduced recurrent viral shedding. This was not seen with the protein subunit vaccine positive control, which had previously had some efficacy in clinical trials, but required greater activity, which is provided herein by the VTL1 formulation. There was also a reduction in both primary and recurrent disease not seen in the absence of VTL1, as well as a significant reduction in the detection of latent load, with over half of the animals being fully protected. There were no adverse side effects from immunization, with only one animal dying from the effects of the viral infection itself in this study, and only one animal dying in the negative control (no vaccine) group, with two additional animals in this negative control (no vaccine) group suffering from severe viral infections that prevented sample collection. In comparison, the VTL1 and VTL3 formulated vaccines were safe and either protected against infection and disease or eliminated induced immunity without adverse side effects.

[0248] The above results are summarized in Table 6.

[0249] [Table 6]

[0250] The cellular recruitment provided by the virokine (VTL1) shows an enhanced effect on relapse. It is well established that cellular immunity controls herpes virus latency. In this case, immunization with the cellular immunomodulator VLT1 with the known immunogen gD enhanced the immunostimulatory effect, which is consistent with the ability of the VTL1 molecule to antagonize all chemokine receptors on T-regs, thereby preventing their recruitment to dampen the immune response. This mechanism of action can block receptors present on regulatory T cell subsets, thus enhancing the stimulation of the immunogen by inhibiting the regulator. Conversely, an agonist mixture of activators of receptors on regulatory T cell subsets (i.e. chemokines in VTL3) led to the inhibition of the immunogen's (gD) effect, eliminating the induction of the immune response.

[0251] The antibody effect can prevent early infection and can be stimulated by appropriate antigen presentation, for example, presentation of gD2 as used in the above examples. The VTL3 immunoregulatory formulation was effective in completely blocking the induction of neutralizing antibodies. This was also consistent with the efficacy of VTL3 in blocking any induced immunity from early acute or recurrent infection as shown herein.

[0252] VTL1 is a human-compatible molecule, and therefore its effect in a human setting, rather than a guinea pig as used herein, is likely to further improve outcomes. Comparison of the affinity for the binding domain in VTL1 with that of human chemokines showed, for example, a 10-100-fold increase in interaction even when ex vivo human cells were used as a source of chemokine receptors (Catusse et al. 2007). In contrast to the pronounced inhibitory effect of VTL3, VTL1 effectively induced neutralizing antibodies together with the known immunogen gD2. Thus, the protective or inhibitory effect in the human system is likely to be higher for VTL1 and for VTL3 as a mixture of human chemokines.

[0253] As discussed above, treatment with VTL3 results in blocking immune responses and associated activities against the known immunogen gD2. This warrants further investigation in clinical settings as a prophylactic and therapeutic treatment to prevent immune stimulation and other runaway immune responses, such as in autoimmune or inflammatory diseases. In particular, VTL3 induces and induces inhibition of immune responses against a known, clinically effective immunogen (i.e., gD2) by regulatory T cells, as shown in the examples above.

[0254] Examples of uses for VTL3 include the treatment of pathogenic conditions involving excessive immune responses.For example, in rheumatoid arthritis, immune cells bearing CCR5 can be recruited to the site of disease in the joints.Therefore, blocking this recruitment of CCR5+ activated immune cells by inducing regulatory T cells can be used as a beneficial treatment.Furthermore, in combination with known immunogens such as allergens, undesirable immune responses, such as anaphylaxis, can be prevented by desensitization of individuals by immunizing them with allergens together with VTL3 formulations to induce regulatory responses and inhibit pathogenic responses.Allergens such as cat hair immunogens that can be targeted, as well as common immunogens related to hay fever or inducing asthma attacks, are well described.

[0255] Another example for use of VTL3 may be in the treatment of runaway immune responses to infectious diseases. For example, in chronic obstructive pulmonary disease, there is an immune response to viral immunogens such as rhinoviruses that result in airway obstruction. In this case, known immunogens from rhinoviruses can be selected for immunization with VTL3 formulations to prevent disease. Furthermore, in infections such as SARS-CoV2, severe late-stage disease is characterized by high levels of antibodies, including autoantibodies, runaway immune responses and cytokine storms, which can be treated with immunomodulators such as VTL3 to promote a controlled response. This can be administered as an aerosol, for example using an inhaler, to prevent local runaway immune responses in the lungs, or it can be administered intramuscularly, as shown here to prevent a systemic response.

[0256] Sequence Listing Description SEQ ID NO:1 is the amino acid sequence of the CCL5 protein precursor. MKVSAAALAVILIATALCAPASASPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMS

[0257] SEQ ID NO:2 is the amino acid sequence of the CCL5 mature protein. SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMS

[0258] SEQ ID NO:3 is the nucleotide sequence of the cDNA encoding the CCL5 mature protein.Free text: mature CCL5 cDNA. tccccatatt cctcggacac cacaccctgc tgctttgcct acattgcccg cccactgccc cgtgcccaca tcaaggagta tttctacacc agtggcaagt gctccaaccc agcagtcgtc tttgtcaccc gaaagaaccg ccaagtgtgt gccaacccag agaagaaatg ggttcgggag tacatcaact ctttggagat gagctag

[0259] SEQ ID NO:4 is the amino acid sequence of the CCL17 protein precursor. MAPLKMLALVTLLLLGASLQHIHAARGTNVGRECCLEYFKGAIPLRKLKTWYQTSEDCSRDAIVFVTVQGRAICSDPNNKRVKNAVKYLQSLERS

[0260] SEQ ID NO:5 is the amino acid sequence of the CCL17 mature protein. ARGTNVGRECCLEYFKGAIPLRKLKTWYQTSEDCSRDAIVFVTVQGRAICSDPNNKRVKNAVKYLQSLERS

[0261] SEQ ID NO:6 is the nucleotide sequence of the cDNA encoding the CCL17 mature protein. Free text: mature CCL17 cDNA. gccccactga agatgctggc cctggtcacc ctcctcctgg gggcttctct gcagcacatc cacgcagctc gagggaccaa tgtgggccgg gagtgctgcc tggagtactt caagggagcc attcccctta gaaagctgaa gacgtggtac cagacatctg aggactgctc cagggatgcc atcgtttttg taactgtgca gggcagggcc atctgttcgg accccaacaa caagagagtg aagaatgcag ttaaatacct gcaaagcctt gagaggtctt ga

[0262] SEQ ID NO:7 is the amino acid sequence of the CCL20 protein precursor. MCCTKSLLLAALMSVLLLHLCGESEAASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVCANPKQTWVKYIVRLLSKKVKNM

[0263] SEQ ID NO:8 is the amino acid sequence of the CCL20 mature protein. ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVCANPKQTWVKYIVRLLSKKVKNM

[0264] SEQ ID NO:9 is the nucleotide sequence of the cDNA encoding the CCL20 mature protein. Free text: mature CCL20 cDNA. gcaagcaact ttgactgctg tcttggatac acagaccgta ttcttcatcc taaatttatt gtgggcttca cacggcagct ggccaatgaa ggctgtgaca tcaatgctat catctttcac acaaagaaaa agttgtctgt gtgcgcaaat ccaaaacaga cttgggtgaa atatattgtg cgtctcctca gtaaaaaagt caagaacatg taa

[0265] SEQ ID NO: 10 is the amino acid sequence of Met-CCL5. Free text: Met-CCL5. MSPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMS

[0266] SEQ ID NO: 11 is the nucleotide sequence of the cDNA encoding Met-CCL5. Free text: Met-CCL5 cDNA. atgtccccat attcctcgga caccacacc tgctgctttg cctacattgc ccgcccactg ccccgtgccc acatcaagga gtatttctac accagtggca agtgctccaa cccagcagtc gtctttgtca cccgaaagaa ccgccaagtg tgtgccaacc cagagaagaa atgggttcgg gagtacatca actctttgga gatgagctag

[0267] SEQ ID NO: 12 is the amino acid sequence of Met-CCL17. Free text: Met-CCL17. MARGTNVGRECCLEYFKGAIPLRKLKTWYQTSEDCSRDAIVFVTVQGRAICSDPNNKRVKNAVKYLQSLERS

[0268] SEQ ID NO: 13 is the nucleotide sequence of the cDNA encoding Met-CCL17. Free text: Met-CCL17 cDNA. atggctcgag ggaccaatgt gggccgggag tgctgcctgg agtacttcaa gggagccatt ccccttagaa agctgaagac gtggtaccag acatctgagg actgctccag ggatgccatc gtttttgtaa ctgtgcaggg cagggccatc tgttcggacc ccaacaacaa gagagtgaag aatgcagtta aatacctgca aagccttgag aggtcttga

[0269] SEQ ID NO: 14 is the amino acid sequence of Met-CCL20. Free text: Met-CCL20. MASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVCANPKQTWVKYIVRLLSKKVKNM

[0270] SEQ ID NO: 15 is the nucleotide sequence of the cDNA encoding Met-CCL20. Free text: Met-CCL20 cDNA. atggcaagca actttgactg ctgtcttgga tacacagacc gtattcttca tcctaaattt attgtgggct tcacacggca gctggccaat gaaggctgtg acatcaatgc tatcatcttt cacacaaaga aaaagttgtc tgtgtgcgca aatccaaaac agacttgggt gaaatatatt gtgcgtctcc tcagtaaaaa agtcaagaac atgtaa

[0271] SEQ ID NO: 16 is the nucleotide sequence of VTL101 iciU83A-N cDNA. Free text: VTL101. gtcgaaatgt ccattcggct ttttattggt tttttttata cggcatatat tggtatggct atcggattta tatgtagttc ccccgatgcg gagctgtttt ccgaaaaatc acgtatttcg tcttctgtct tgttaggatg tttgttgtgt tgcatggatt ggtccgctgc cgtacccgtc tggtttggag cagggctcga tgtgtga

[0272] SEQ ID NO: 17 is the nucleotide sequence of the VTL1016 variant of VTL101 iciU83A-N mutated in the polyT tract with its own Kozak sequence. Free text: VTL1 variant VTL1016 or VIT1. gtcgaaatgt ccattcggct ttttattggt ttcttttata cggcatatat tggtatggct atcggattta tatgtagttc ccccgatgcg gagctgtttt ccgaaaaatc acgtatttcg tcttctgtct tgttaggatg tttgttgtgt tgcatggatt ggtccgctgc cgtacccgtc tggtttggag cagggctcga tgtgtga

[0273] SEQ ID NO: 18 is the nucleotide sequence of the VTL1017 variant of VTL101 iciU83A-N, human codon optimized, no polyT and no TACC, manually adjusted. Free text: VTL1 variant VTL1017. gtcgaaatgt ccatccgcct tttcattggc ttcttttaca cagcatacat cgggatggct ataggcttca tttgctcctc tccagacgcg gagctgtttt cagagaaaag ccggatatct agtagcgtgc tgctcggatg tctgctctgt tgcatggact ggtccgctgc cgtcccagtg tggttcggcg ctggactgga tgtgtga

[0274] SEQ ID NO: 19 is the amino acid sequence encoded by human endogenous chromosomally integrated human beta-herpesvirus 6A VTL1 and its VTL1016 and VTL1017 variants, showing an additional C-terminal extension of 8 amino acids. MSIRLFIGFFYTAYIGMAIGFICSSPDAELFSEKSRISSSVLLGCLLCCMDWSAAVPVWFGAGLD

[0275] SEQ ID NO:20 is the amino acid sequence encoded by VTL101, VTL1016 and VTL1017, cleaved after the signal sequence to give the mature secreted product. FICSSPDAELFSEKSRISSSVLLGCLLCCMDWSAAVPVWFGAGLDV

[0276] SEQ ID NO: 21 is the nucleotide sequence of VTL1018 variant of VTL101 iciU83A-N, which has been mutated to disrupt the polyT tract and retains its own Kozak sequence. Free text: VTL1 variant VTL1018. gtcgaaatgt ccattcggct ttttattggt ttcttttata cggcatatat tggtatggct atcggattta tatgtagttc ccccgatgcg gagctgtttt ccgaaaaatc acgtatttcg tcttctgtct tgttaggatg tttgttgtgt tgcatggatt ggtccgctgc cgtacctggg aaaacagagc cttttagaaa actttttgat gcaatcatga ttaaaaagct aaaaagttgt tctgctgctt acccgtctgg tttggagcag ggctcgatgt gtgatatggc agatgcatcg ccgacaagtc ttgaattagg attgtcgaaa ttagacaaag aatcatga

[0277] SEQ ID NO: 22 is the nucleotide sequence of the VTL1019 variant of VTL101 iciU83A-N that has been mutated to disrupt splicing through the direct repeat TACC and splice donor / acceptor sites and retains the Kozak consensus site. Free text: VTL1 variant VTL1019. gtcgaaatgt ccattcggct ttttattggt tttttttata cggcatatat tggtatggct atcggattta tatgtagttc ccccgatgcg gagctgtttt ccgaaaaatc acgtatttcg tcttctgtct tgttaggatg tttgttgtgt tgcatggatt ggtccgctgc cgtgccaggg aaaacagagc cttttagaaa actttttgat gcaatcatga ttaaaaagct aaaaagttgt tctgctgctt atccatctgg tttggagcag ggctcgatgt gtgatatggc agatgcatcg ccgacaagtc ttgaattagg attgtcgaaa ttagacaaag aatcatga

[0278] SEQ ID NO: 23 is the nucleotide sequence of the VTL1020 variant of VTL101 iciU83A-N, which has been mutated to disrupt the poly-T region and splicing and retains its own Kozak sequence. Free text: VTL1 variant VTL1020. gtcgaaatgt ccattcggct ttttattggt ttcttttata cggcatatat tggtatggct atcggattta tatgtagttc ccccgatgcg gagctgtttt ccgaaaaatc acgtatttcg tcttctgtct tgttaggatg tttgttgtgt tgcatggatt ggtccgctgc cgtgccaggg aaaacagagc cttttagaaa actttttgat gcaatcatga ttaaaaagct aaaaagttgt tctgctgctt atccatctgg tttggagcag ggctcgatgt gtgatatggc agatgcatcg ccgacaagtc ttgaattagg attgtcgaaa ttagacaaag aatcatga

[0279] SEQ ID NO: 24 is the nucleotide sequence of the VTL1021 variant of VTL101 iciU83A-N that has been maximized for human codon usage and manually adjusted to disrupt splicing as underlined and to retain the Kozak sequence. Free text: VTL1 variant VTL1021. gtcgaaatga gcatcagact gttcatcggc ttcttctaca ccgcctacat cggcatggcc atcggcttca tctgcagcag ccccgacgcc gagctgttca gcgagaagag cagaatcagc agcagcgtgc tgctgggctg cctgctgtgc tgcatggact ggagcgccgc cgtgccaggc aagaccgagc ccttcagaaa gctgttcgac gccatcatga tcaagaagct gaagagctgc agcgccgcct atcctagcgg cctggagcag ggcagcatgt gcgacatggc cgacgccagc cccaccagcc tggagctggg cctgagcaag ctggacaagg agagctga

[0280] SEQ ID NO:25 is the amino acid sequence encoded by VTL1018, VTL1019, VTL1020 and VTL201. Free text: VTL1 agonist protein. MSIRLFIGFFYTAYIGMAIGFICSSPDAELFSEKSRISSSVLLGCLLCCMDWSAAVPGKTEPFRKLFDAIMIKKLKSCSAAYPSGLEQGSMCDMADASPTSLELGLSKLDKES

[0281] SEQ ID NO:26 is the amino acid sequence encoded by VTL1018, VTL1019, VTL1020 and VTL201 from which the signal sequence is cleaved to give the mature secreted product.Free text: VTL1 agonist mature protein. FICSSPDAELFSEKSRISSSVLLGCLLCCMDWSAAVPGKTEPFRKLFDAIMIKKLKSCSAAYPSGLEQGSMCDMADASPTSLELGLSKLDKES

[0282] SEQ ID NO:27 is a nucleotide sequence encoding the CCL5 protein precursor including the signal sequence. ATGAAGGTCTCCGCGGCAGCCCTCGCTGTCATCCTCATTGCTACTGCCCTCTGCGCTCCTGCATCTGCCTCCCCATATTCCTCGGACACCACACCCTGCTGCTTTGCCTACATTGCCCGCCCACTGCCCCGTGCCCAC ATCAAGGAGTATTTCTACACCAGTGGCAAGTGCTCCAACCCAGCAGTCGTCTTTGTCACCCGAAAGAACCGCCAAGTGTGTGCCAACCCAGAGAAGAAATGGGTTCGGGAGTACATCAACTCTTTGGAGATGAGCTAG

[0283] SEQ ID NO:28 is a nucleotide sequence encoding the CCL17 protein precursor including the signal sequence. ATGGCCCCACTGAAGATGCTGGCCCTGGTCACCCTCCTCCTGGGGGCTTCTCTGCAGCACATCCACGCAGCTCGAGGGACCAATGTGGGCCGGGAGTGCTGCCTGGAGTACTTCAAGGGAGCCATTCCCCTTAGAAAGCTGA AGACGTGGTACCAGACATCTGAGGACTGCTCCAGGGATGCCATCGTTTTTGTAACTGTGCAGGGCAGGGCCATCTGTTCGGACCCCAACAAGAGAGTGAAGAATGCAGTTAAATACCTGCAAAGCCTTGAGAGGTCTTGA

[0284] SEQ ID NO:29 is a nucleotide sequence encoding the CCL20 protein precursor including the signal sequence. ATGTGCTGTACCAAGAGTTTGCTCCTGGCTGCTTTGATGTCAGTGCTGCTACTCCACCTCTGCGGCGAATCAGAAGCAGCAAGCAACTTTGACTGCTGTCTTGGATACACAGACCGTATTCTTCATCCTAAATTTATTGTGGGCT TCACACGGCAGCTGGCCAATGAAGGCTGTGACATCAATGCTATCATCTTTCACACAAAGAAAAAAGTTGTCTGTGTGCGCAAATCCAAAACAGACTTGGGTGAAATATATTGTGCGTCTCCTCAGTAAAAAAGTCAAGAACATGTAA Reference materials Awasthi S, Hook LM, Shaw CE, Pahar B, Stagray JA, Liu D, Veazey RS, Friedman HM. “An HSV-2 Trivalent Vaccine Is Immunogenic in Rhesus Macaques and Highly Efficacious in Guinea Pigs”. PLoS Pathog. (2017) Jan 19;13(1):e1006141. doi: 10.1371 / journal.ppat.1006141. eCollection 2017 Jan.PMID: 28103319. Baldridge JR, Crane RT. Monophosphoryl lipid A (MPL) formulations for the next generation of vaccines. Methods 1999;19:103-7. Bar-On, Y. M., Y. Goldberg, M. Mandel, O. Bodenheimer, L. Freedman, N. Kalkstein, B. Mizrahi, S. Alroy-Preis, N. Ash, R. Milo and A. 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Claims

1. A chemokine mixture suitable for regulating an immune response, comprising a first chemokine, or an agonist or antagonist mutant thereof, that is suitable for binding to CCR4, and a second chemokine, or an agonist or antagonist mutant thereof, that is suitable for binding to CCR5 and / or CCR6.

2. the first chemokine, or an agonist or antagonist variant thereof, comprises CCL17, or an agonist or antagonist variant thereof; 2. The chemokine mixture of claim 1, wherein the agonist or antagonist variant thereof preferably comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 5 or SEQ ID NO:

12.

3. the second chemokine, or agonist or antagonist variant thereof, comprises CCL5 or CCL20, or agonist or antagonist variant thereof; 3. The chemokine mixture of claim 1 or 2, wherein the agonist or antagonist variant thereof preferably comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, or SEQ ID NO:

26.

4. the chemokine mixture comprises a third chemokine, or an agonist or antagonist variant thereof, which third chemokine, or an agonist or antagonist variant thereof, is suitable for binding to a different one of CCR5 and CCR6 than the second chemokine, or an agonist or antagonist variant thereof; Preferably, the third chemokine, or agonist or antagonist variant thereof, comprises CCL20, or an agonist or antagonist variant thereof; More preferably, the agonist or antagonist variant comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:8 or SEQ ID NO:

14.

5. a) the first chemokine comprises CCL17, the second chemokine comprises CCL5 or SEQ ID NO: 26, and the chemokine mixture comprises a third chemokine comprising CCL20; or b) the first chemokine comprises Met-CCL17, the second chemokine comprises Met-CCL5 or SEQ ID NO: 20, and the chemokine mixture comprises a third chemokine comprising Met-CCL20; or c) The chemokine mixture of claim 1 or 2, wherein the first chemokine comprises CCL17, the second chemokine comprises Met-CCL5 or SEQ ID NO: 20, and the chemokine mixture comprises a third chemokine comprising CCL20.

6. A polynucleotide mixture comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises a nucleic acid sequence encoding a first chemokine, or an agonist or antagonist variant thereof, that is suitable for binding to CCR4 and, optionally, also suitable for binding to CCR8, and the second polynucleotide comprises a nucleic acid sequence encoding a second chemokine, or an agonist or antagonist variant thereof, that is suitable for binding to CCR5 and / or CCR6, and the polynucleotide mixture is suitable for modulating an immune response.

7. the first polynucleotide comprises a nucleic acid sequence encoding CCL17, or an agonist or antagonist variant thereof; the second polynucleotide comprises a nucleic acid sequence encoding CCL5, or an agonist or antagonist variant thereof, CCL20, or an agonist or antagonist variant thereof, or one of SEQ ID NO: 20 and SEQ ID NO: 26; Preferably, the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:6 and SEQ ID NO:13, and the second polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:16 to SEQ ID NO:18, and SEQ ID NO:21 to SEQ ID NO:24; The polynucleotide mixture of claim 6 , wherein preferably the first and second polynucleotides each further comprise a nucleic acid sequence encoding a signal sequence.

8. the second chemokine or functional variant thereof is suitable for binding to CCR5, the polynucleotide mixture comprises a third polynucleotide, the third polynucleotide comprises a nucleic acid sequence encoding a third chemokine or an agonist or antagonist variant thereof, the third chemokine or agonist or antagonist variant thereof is suitable for binding to CCR6, Preferably, the third chemokine, or agonist or antagonist variant thereof, comprises CCL20, or an agonist or antagonist variant thereof; Preferably, the third polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 15; The polynucleotide mixture according to claim 6 or 7, wherein the third polynucleotide preferably further comprises a nucleic acid sequence encoding a signal sequence.

9. a) the first polynucleotide comprises a nucleic acid sequence encoding CCL17, the second polynucleotide comprises a nucleic acid sequence encoding CCL5 or having an amino acid sequence having at least 70% sequence identity to one of SEQ ID NOs:21 to 24, and the polynucleotide mixture comprises a third polynucleotide, the third polynucleotide comprises a nucleic acid sequence encoding CCL20, or b) the first polynucleotide comprises a nucleic acid sequence encoding Met-CCL17, the second polynucleotide comprises a nucleic acid sequence encoding Met-CCL5 or having at least 70% sequence identity to one of SEQ ID NOs: 16 to 18, and the polynucleotide mixture comprises a third polynucleotide, the third polynucleotide comprises a nucleic acid sequence encoding Met-CCL20, or c) the first polynucleotide comprises an amino acid sequence encoding CCL17, the second polynucleotide encodes Met-CCL5 or comprises an amino acid sequence having at least 70% sequence identity to one of SEQ ID NOs: 16 to 18, and the polynucleotide mixture comprises a third polynucleotide, the third polynucleotide comprising a nucleic acid sequence encoding CCL20; 8. The polynucleotide mixture according to claim 6 or 7, wherein preferably the first, second and third polynucleotides each further comprise a nucleic acid sequence encoding a signal sequence.

10. a) the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:6, the second polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:3 and one of SEQ ID NOs:21 to 24, and the polynucleotide mixture comprises a third polynucleotide, the third polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9; or b) the first polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:13, the second polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:11 and SEQ ID NO:16 to SEQ ID NO:18, and the polynucleotide mixture comprises a third polynucleotide, the third polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:15; or c) the first polynucleotide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:6, the second polynucleotide comprises an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO:11 and SEQ ID NO:16 to SEQ ID NO:18, and the polynucleotide mixture comprises a third polynucleotide, the third polynucleotide comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9; 8. The polynucleotide mixture according to claim 6 or 7, wherein preferably the first, second and third polynucleotides each further comprise a nucleic acid sequence encoding a signal sequence.

11. A composition comprising a first polynucleotide comprising a nucleic acid sequence encoding a first chemokine, or an agonist or antagonist variant thereof, suitable for binding to CCR5, CCR4 and / or CCR6, and a second chemokine, or an agonist or antagonist variant thereof, suitable for binding to one of CCR5, CCR4 and / or CCR6 that is different from the first chemokine, or an agonist or antagonist variant thereof, wherein the composition is suitable for regulating an immune response.

12. the first polynucleotide encodes CCL5, CCL17, CCL20, or an agonist or antagonist variant thereof, or encodes an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO:20 and SEQ ID NO:26, or comprises a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 to SEQ ID NO:18, and SEQ ID NO:21 to SEQ ID NO:24; the second chemokine, or agonist or antagonist variant thereof, comprises CCL5, CCL17, CCL20, or an agonist or antagonist variant thereof, or comprises an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO:20 and SEQ ID NO:26, or the composition comprises a second polynucleotide, the second polynucleotide comprising a nucleic acid sequence having at least 70% sequence identity to one of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 to SEQ ID NO:18, and SEQ ID NO:21 to SEQ ID NO:24, which is different from the first polynucleotide; Preferably, the composition further comprises a third polynucleotide comprising a nucleic acid sequence encoding a third chemokine, or an agonist or antagonist variant thereof, wherein said third chemokine, or agonist or antagonist variant thereof, is suitable for binding to a different CCR5, CCR4 and / or CCR6 than said first and second chemokines, or agonist or antagonist variants thereof, respectively, and optionally said third chemokine, or agonist or antagonist variant thereof, comprises CCL5, CCL17, CCL20, or said third chemokine, or agonist or antagonist variant thereof, comprises an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO: 20 and SEQ ID NO: 26; 12. The composition of claim 11, wherein the first, second, and third polynucleotides each further comprise a nucleic acid sequence encoding a signal sequence.

13. The first chemokine, or agonist or antagonist variant thereof, comprises CCL5, or an agonist or antagonist variant thereof, or comprises an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO:20 and SEQ ID NO:26, the second chemokine, or agonist or antagonist variant thereof, comprises CCL17, or an agonist or antagonist variant thereof, and the composition comprises CCL20, or an agonist or antagonist variant thereof.

13. The composition of claim 11 or 12, further comprising a third chemokine, or an agonist or antagonist variant thereof, comprising the first chemokine or the agonist or antagonist variant thereof, preferably wherein the first chemokine or the agonist or antagonist variant thereof comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 20, the second chemokine or the agonist or antagonist variant thereof is Met-CCL17, and the third chemokine or the agonist or antagonist variant thereof is Met-CCL20.

14. A chemokine mixture as defined in claim 1 or 2, a polynucleotide mixture as defined in claim 6 or 7, or a composition as defined in claim 11 or 12 for use in the treatment or prevention of a disease or disorder characterised by altered levels of CCR1, 4, 5, 6 and 8 or their binding chemokines, or a disease or disorder associated with dysregulated immune response, or for use as an adjuvant, wherein the disease or disorder is preferably cancer, viral infection, Alzheimer's disease, an autoimmune disease, an inflammatory disease or condition, allergy or a disease or condition associated with an overactive immune system or runaway immune response.

15. A host cell comprising the polynucleotide mixture of claim 6 or 7.

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