Active immunization for reducing osteoarthritic, neuropathic, and cancer pain

Abstract

An immunogenic fusion protein is provided capable of conferring immunity to osteoarthritis in mammals, including canine, feline, and equine as well as methods of their use and preparation are also provided. More specifically, where an immunogenic fusion protein includes a first antigen derived from nerve growth factor, a second antigen that includes single or multiple repeat units of substance P or hemokinin-1, and a linker separating the first antigen from the second antigen, a nucleic acid encoding the immunogenic fusion protein, a vector comprising the nucleic acid; a host cell comprising the immunogenic fusion protein; a vaccine comprising the fusion protein; and a method of vaccination against osteoarthritis or osteoarthritis associated pain in dogs.

Description

ACTIVE IMMUNIZATION FOR REDUCING OSTEOARTHRITIC, NEUROPATHIC, AND CANCER PAINTECHNICAL FIELD

[0001] The present disclosure relates to the field of vaccine technology, immunotherapy and veterinary medicine and concerns the development of an immunogenic fusion protein capable of conferring immunity to osteoarthritis in mammals, including canine, feline, and equine as well as methods of their use and preparation. It further pertains to a nucleic acid molecule encoding the immunogenic fusion protein; a vector; a host cell; a vaccine; and a method of vaccination against osteoarthritis in dogs.BACKGROUND

[0002] Chronic pain is a prevalent and challenging issue in veterinary medicine, often associated with degenerative and autoimmune conditions affecting the osteoarticular system. OA is a leading cause of chronic inflammatory pain, and significantly impacts animal health and welfare. Current therapeutic approaches, including analgesics and anti-inflammatory drugs such as corticosteroids, are hindered by limited efficacy, adverse side effects, and the logistical difficulties of long-term administration. These limitations underscore the urgent need for innovative, well-tolerated, and cost-effective treatments that can provide sustained relief from chronic pain in veterinary practice.

[0003] OA is estimated to affect 20-37% of dogs over one year of age, with a higher prevalence in aging animals. The disease progresses through a complex interplay of local and systemic factors involving joint tissues, sensory innervation, and the immune system.

[0004] Osteoarthritis pain not only affects the physical mobility of animals but also significantly impairs their overall quality of life, impacting their ability to perform basic activities and interact socially. Chronic OA pain is associated with behavioral changes such as reduced activitylevels, irritability, and altered appetite, which can strain the human-animal bond. Despite advancements in understanding OA pathophysiology, effective pain management remains elusive.

[0005] In the field of veterinary medicine, previous developments have aimed at targeting neurotrophic factors and neuropeptides implicated in pain and inflammation to reduce OA pain. Among these, nerve growth factor (NGF) and the neuropeptides substance P (SP) hemokinin-1 (HK-1) are of particular relevance. These mediators participate in peripheral sensitization of nociceptive fibers, amplification of pain signaling, and neurogenic inflammation at the joint level, thereby contributing to the establishment and perpetuation of a chronic pain state that is refractory to conventional therapies.

[0006] In this context, active immunization approaches directed against nociceptive mediators have been proposed to modulate chronic pain. One example is application WO 2023139542 (WO’ 542), which describes a recombinant fusion protein, NGF-1SP, comprising nociceptive mediators, for example, neurotrophic factor (NGF) and substance P (SP) in active immunization to reduce pain and inflammation in subjects with osteoarthritis, the entire contents of which are hereby incorporated by reference herein.Despite the advances in the progress towards a suitable vaccine for the treatment and / or prevention of osteoarthritis chronic pain, there remains a need to explore vaccine strategies capable of eliciting protective immunity against multiple pain mediators with roles in neurogenic inflammation in OA, that improve its clinical applicability with efficacy, stability, and practicality of immunogenic fusion proteins of the present invention. In particular, constructions based on a single SP copy still present room for improvement in terms of overall immunogenicity and in the magnitude and duration of the antibody response against NGF and against the neurogenic mediators involved in the pathophysiology of chronic pain. Additionally, the prior art does not fully exploit the possibility of directing, in a simultaneous and effective manner, humoral responses against multiple neuropeptides, such as SP and HK-1, which act in a complementary fashion in nociceptive sensitization and neurogenic inflammation.

[0007] Accordingly, starting from the NGF-SP fusion protein described in WO’542, the objective technical problem is to provide improved vaccine constructs that exhibit increased immunogenicity and a more robust and durable antibody response against NGF and against neurogenic mediators of pain, in particular substance P (SP) and hemokinin-1 (HK-1), while maintaining suitable properties of structural stability, expression, and formulation for use as a vaccine for the treatment of chronic pain associated with osteoarthritis in mammals.BRIEF SUMMARY

[0008] In one embodiment of the present disclosure, an immunogenic fusion protein is provided comprising, a first antigen derived from nerve growth factor (NGF), and having at least 90%, at least 95%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, or and SEQ ID NO: 25; a second antigen comprising single or multiple repeat units wherein the repeat units have at least 90%, at least 95%, or at least 99% sequence identity with an a seqeunce selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, a flexible linker amino acid sequence consisting of 4-20 amino acids separating the first antigen from the second antigen, and a flexible linker between multiple repeat units (if present) of the second antigen, where the fusion protein amplifies an immune response that comprises eliciting cross reacting antibodies against either or both antigens, and wherein the fusion protein does not include rNGF-ISP (SEQ ID NO: 19).

[0009] In another embodiment of the present disclosure, the fusion protein comprises a repeat configuration of the multiple units of the second antigen.

[0010] In yet another embodiment of the present disclosure, a nucleic acid is provided that encodes the immunogenic fusion protein of the present disclosure.

[0011] In one embodiment of the present disclosure, a vector is provided that comprises the nucleic acid molecule of the present disclosure.

[0012] In yet another embodiment, a host cell expressing the immunogenic fusion protein of the present disclosure is provided or comprising the nucleic acid molecule of the present disclosure is provided.

[0013] In one embodiment of the present disclosure, a vaccine composition comprising the fusion protein of the present disclosure, or the nucleic acid molecule of the present disclosure, or the vector of the present disclosure or the host cell of the present disclosure is provided.

[0014] In another embodiment, a method of treating a condition selected from nociceptive and / or inflammatory related pain, osteoarthritis associated pain, chronic osteoarthritis related pain, and refractory osteoarthritis related pain in mammals is provided, comprising administering the vaccine composition of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Features and advantages will be apparent from the following, more particular, description of various exemplary embodiments, as illustrated in the accompanying drawings.

[0016] Fig. 1A-C show the design, production and purification, expression and solubility profiles of rNGF-3SP and rNGF-5SP immunogenic fusion proteins containing 3 and 5 SP multiple repeat units. Fig. 1A is a schematic representation of the amino acid sequences of the recombinant fusion proteins rNGF-3SP and rNG-5SP. Fig. IB is a schematic view of the workflow illustrating the production and purification for the key process steps involved in production of immunogenic fusion proteins. Fig. 1C shows an SDS-PAGE analysis of the expression and solubility profiles of rNGF-3SP and rNGF-5SP.

[0017] Fig. 2 shows humoral IgG responses against the vaccinal antigen, NGF, and SP following immunization with rNGF-ISP, rNGF-3SP, and rNGF.

[0018] Fig. 3 shows the design of rNGF-HK-1 immunogenic fusion proteins containing a single HK-1 (rNGF-HK-1) and three repeat units (3) of HK-1 (rNGF-3HK-l)

[0019] Fig. 4 shows a cartoon representation of NGF-SP and NGF-3SP models obtained with Alphafold2.

[0020] Fig- 5 shows RMSD timelines measured over the triplicated simulations of each construct (in black curves), and rolling averages (shown in green).

[0021] Fig 6 shows the RMSF analysis of NGF-1SP and NGF-3SP. The left panel shows the initial representative structure showing the structural changes obtained after 0.5 ps trajectories for NGF-3SP.

[0022] Fig 7 shows the average and standard deviations epitope scores calculated between the five representative structures of each construct, NGF-1SP and NGF-3SP. The barplot (in blue residues) shows scores above the threshold, indicating residue candidates to form part of an epitope.

[0023] Fig 8 Superposition of the representative structures of NGF-1SP and NGF-3SP. DETAILED DESCRIPTION

[0024] Features, advantages, and embodiments of the present disclosure are set forth or apparent from a consideration of the following detailed description, drawings, and claims. Moreover, the following detailed description is exemplary and intended to provide further explanation without limiting the scope of the disclosure as claimed.

[0025] The singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0026] As used herein, “nociceptive factors or mediators” refers to molecules that directly or indirectly promote an increased peripheral sensitization to stimuli resulting in painful perception, including but not limiting to NGF, SP and HK-1. Nociceptive mediators activate primary afferent neurons directly or indirectly to enhance nociceptive signal transmission to the central nervous system. Excitation of primary afferents by peripherally originating mediators, so-called “peripheral sensitization” is a hallmark of tissue injury-related pain. For example, chronic OA pain (defined below) is associated with sensory disturbances that are mainly mediated by endogenous nociceptive factors produced in OA-damaged tissues. Such nociceptive mediators may also exhibit inflammatory activities. In OA, NGF produced by inflammatory cells induces the overexpression of SP in sensory neurons, which in turn contributes to hyperalgesia and neurogenic inflammation at peripheral sites.SP and NGF are recognized inflammatory mediators produced by immune cells with the ability to promote mast cell degranulation.

[0027] The term “adjuvant” refers to a substance that increases the intensity of the immune response after co-administration with an immunogen. An adjuvant may act as an immunopotentiator useful for enabling immunogenic composition or vaccine to induce potent and persistent immune responses, while reducing the dose and number of boosters. Adjuvant may also increase the stability of the immunogenic composition or vaccine.

[0028] The term “osteoarthritis (OA)” refers to a chronic joint disease characterized by loss of joint cartilage, thickening of the joint capsule and new bone formation around the joint (osteophytosis) and ultimately leading to pain and limb dysfunction. In canines, signs of OA are often non-specific and include: i) activity impairment, reluctance to exercise, decrease in overall activity, stiffness, lameness, inability to jump, changes in gait such as “bunny-hopping”, ii) pain on manipulation, behavioral changes such as aggression or signs of discomfort.

[0029] The term “refractory osteoarthritis” refers to a chronic condition that does not respond or only slightly responds to conventional treatment including NSAIDs, corticoids and opioids.

[0030] As used herein, “neurogenic inflammation” refers to the physiological process by which mediators are released directly from the sensory nerves to initiate an inflammatory reaction. This results in production of local inflammatory responses including erythema, swelling, temperature increase, tenderness, and pain. Fine unmyelinated afferent somatic C-fibers, which respond to low intensity mechanical and chemical stimulations, are largely responsible for the release of inflammatory mediators. When stimulated, these nerve fibers in the cutaneous nerves rapidly release active neuropeptides such as SP and HK-1 into the microenvironment, triggering a series of inflammatory responses.

[0031] The term “inflammatory pain” refers to the spontaneous hypersensitivity to pain that occurs in response to tissue damage and inflammation (e.g., postoperative pain, trauma, arthritis).Inflammatory pain is a type of nociceptive pain that results from activation and sensitization of nociceptors by inflammatory mediators. Often the pain improves when the inflammation subsides.

[0032] The term “recombinant protein” refers to a protein encoded by recombinant nucleic acid that has been cloned in an expression vector that supports expression of the gene and translation of messenger RNA. Escherichia coli (bacteria) is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm. Bacterial inclusion bodies are mesoscale protein aggregates commonly observed in recombinant bacteria, primarily formed by recombinant protein. Other expression system may include but are not limited to insect cells and yeast cells.

[0033] The term “chronic pain” refers to pain that is persistent and typically lasts longer than six months, extending beyond the expected period of tissue healing. Chronic pain is distinct from acute pain, as it is not merely a prolonged temporal continuation but represents a complex pathological state involving both peripheral and central mechanisms. In the context of sustained injury or illness, functional and structural reorganization of neuronal circuits occurs within the CNS, resulting in long-term alterations in pain perception, behavior, and sensitivity. This reorganization often involves central sensitization, maladaptive neuroplasticity, and dysregulated pain modulation pathways, contributing to the persistence of pain even after the original cause has resolved. Moreover, chronic pain is characterized by the continuous activation of pain signaling pathways in the nervous system, potentially lasting for weeks, months, or even years.

[0034] The term “chronic OA pain” is used to describe chronic pain associated with OA, a degenerative joint disease. Chronic OA pain encompasses both nociceptive and neuropathic components, arising from joint inflammation, structural joint damage, and central sensitization, significantly impacting the quality of life and mobility of affected individuals.

[0035] As used herein “antibody or immunoglobulin” refers to a protein produced by the B-cells of the immune system that can identify, bind and neutralize an antigen. In the context of the invention, an antibody is produced by the immune system and binds an endogenous protein or polypeptide, such as a nociceptive mediator. The antibody may have neutralizing properties and may be capable of suppressing or reducing the biological activity of the nociceptive mediator or the downstream pathway mediated by the nociceptive mediator such as blocking its binding to its specific receptor.

[0036] As used herein, “active immunization” refers to immunization that stimulates the immune system to produce antibodies against an antigen (self or foreign). Active immunization can be induced through vaccination. In the context of the invention, a vaccine or immunogenic composition comprises at least 2 self-antigens or endogenous polypeptides that stimulate the production of antibodies without causing any illness. Such antibodies may have neutralizing properties that will capture the self-antigens, blocking any of their function(s). Active immunization is often long-lasting and may be reactivated by repeated injection of boosters. In contrast, passive immunization occurs when antibodies directed against specific antigen are administered to a subject.

[0037] The term “immune activation” refers to the enhancement or improvement of immunogenicity. In one embodiment, immune activation is afforded by the multiple repeat configurations of the fusion proteins of the present disclosure. In another embodiment, the term “immune activation,” refers to the enhancement or improvement of immunogenicity. In certain embodiments, immune activation is facilitated through the unique structural and functional properties of the multiple repeat configurations present within the fusion proteins disclosed herein. These configurations are specifically designed to potentiate immune responses by optimizing antigen presentation and interaction with immune effector mechanisms.

[0038] Fusion Proteins

[0039] In one embodiment, the present disclosure improves on the fusion protein of WO’542.

[0040] The inventors have found surprisingly that an immunogenic fusion protein of the present disclosure, based on for example, a NGF polypeptide as a first antigen, produced a greater immune response against critical mediators of SP and Hemokinin- 1 (HK-1), when the mediators were presented as multiple epitopes of a second antigen in the fusion protein.

[0041] In one embodiment, the inventors found that the immunogenic fusion protein of the present disclosure afforded long term antibody production and minimized repeat administration and / or frequency of dosing to aging pets, for example, canine, feline, or equine.

[0042] In another embodiment the present disclosure provides for enhancing substance P (SP)-like epitopes by increasing the number of SP sequences in the immunogenic fusion protein to significantly improve its immunogenicity.

[0043] In another embodiment, the observed enhancement in immunogenicity was non-linear and did not directly correlate with the increase in peptide chain length of SP sequences.

[0044] In another embodiment, a second antigen SP is provided having multiple repeat units, for example, 3 or 5 repeat units.

[0045] In one embodiment, a second antigen SP is provided having 3 repeat units.

[0046] In yet another embodiment of the present disclosure, SEQ ID NO: 1 is provided showing the sequence for rNGF fused with 3 repeat units of SP (rNGF-3SP).

[0047] In one embodiment, a second antigen SP is provided having 5 repeat units.

[0048] In one embodiment, SEQ ID NO: 4 is provided showing the sequence for rNGF fused with 5 repeat units of SP (rNGF-5SP).

[0049] In one embodiment, the second antigen is Hemokinin-1 (HK-1).

[0050] In one embodiment, the present disclosure provides for enhancing HK-l-like epitopes by increasing the number of HK-1 sequences to significantly improve its immunogenicity.

[0051] In another embodiment, the observed enhancement in immunogenicity for HK-1 sequences was predicted on the basis of behavior for SP sequences, was non-linear and did not directly correlate with the increase in peptide chain length of HK-1 sequences.

[0052] In another embodiment, a second antigen HK-1 is provided having single or multiple repeat units, for example, 3 or 5 repeat units.

[0053] In one embodiment, a second antigen HK-1 is provided having a single unit.

[0054] In yet another embodiment of the present disclosure, SEQ ID NO: 7 is provided showing the sequence for rNGF fused with a single unit of HK-1 (rNGF-HKl).

[0055] In one embodiment, a second antigen HK-1 is provided having 3 repeat units.

[0056] In yet another embodiment of the present disclosure, SEQ ID NO: 10 is provided showing the sequence for rNGF fused with 3 repeat units of HK-1 (rNGF-3HKl).

[0057] In one embodiment, a second antigen HK-1 is provided having 5 repeat units.

[0058] In yet another embodiment of the present disclosure, SEQ ID NO: 13 is provided showing the sequence for rNGF fused with 5 repeat units of HK-1 (rNGF-5HKl).

[0059] In one embodiment, the immunogenic fusion protein comprises a first antigen, rNGF polypeptide and a second antigen of one or more SP or HK-1 peptides, for example three or five SP or HK-1 peptides, each connected by a flexible amino acid linker, enhancing the immunogenicity of the recombinant fusion protein. The linker is of a length such that the polypeptides are linked without substantial interference. In some embodiments, a linker may be chosen to maintain structural flexibility while ensuring non-native conformations of the recombinant fusion protein. In other embodiments, a linker may also provide additional beneficial properties to the protein, such as increased protein expression in expression systems, improved biophysical properties such as stability and solubility, improved protein purification and detection and / or increased enzymatic activity.

[0060] In another embodiment, exemplary linkers may have the formula (Gly-Ser)n with, optionally, some Glu or Lys residues dispersed throughout to increase solubility, where n can be an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0061] In one embodiment, an exemplary linker may be a flexible linker with formula (GS)n or (Gly-Ser)n.

[0062] In one embodiment, n is 1 or 2.

[0063] In another embodiment, n is 2.

[0064] In yet another embodiment of the present disclosure, either second antigen, for example, SP or HK-1 is fused to the NGF antigen by a flexible amino acid linker, for example, GSGS, as shown in SEQ ID NOS: 1, 4, 7, 10 or 13.

[0065] In one embodiment, second antigens, SP or HK-1 are in a multiple repeat configuration and each repeat unit is separated by a linker, for example, GSGS as shown in SEQ ID NOS: 1, 4, 10 or 13.

[0066] In one embodiment of the present disclosure, the first antigen comprises at least 90% amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NO: 16., SEQ ID NO: 23, SEQ ID NO: 24, or and SEQ ID NO: 25.

[0067] In another embodiment of the present disclosure, the first antigen comprises at least 95% amino acid sequence identity with SEQ ID NO: 16.

[0068] In another embodiment of the present disclosure, the first antigen comprises at least 99% amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NO: 16., SEQ ID NO: 23, SEQ ID NO: 24, or and SEQ ID NO: 25.

[0069] In one embodiment of the present disclosure, SEQ ID NO: 16 refers to canine or Canus NGF.

[0070] In another embodiment of the present disclosure, examples of the first antigen are described in WO ’542, which are hereby incorporated by reference.

[0071] In one embodiment of the present disclosure, the second antigen comprises at least 90%, at least 95%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

[0072] In another embodiment of the present disclosure, the second antigen comprises at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 18.

[0073] In one embodiment of the present disclosure, immunogenic fusion proteins of any of the preceding sequences can be adapted to target specific mammalian species, including dogs, cats,horses, and humans. This versatility allows for tailored applications across a range of pain and inflammatory conditions in companion animals, and potentially humans.

[0074] In another embodiment, while the fusion proteins exemplified herein are related to canine proteins (NGF, SP and HK-1), the present disclosure is equally applicable to other species by incorporating the relevant proteins from that species to construct analogous recombinant fusion proteins. Methods for production and use are identical to those for canine proteins and fusion proteins and the fusion proteins are constructed in a similar manner. For example, completely analogous recombinant fusion can be prepared from the following species shown in Table 1.

[0075] Table 1. Proteins in related mammalian species that can be used to construct analogous immunogenic fusion proteins

[0076] In another embodiment of the present disclosure, the target specific mammalian species is dog.

[0077] Nucleic Acids

[0078] In one embodiment of the present disclosure, nucleic acid DNA molecules encoding the preceding amino acid sequences are shown in SEQ ID NOS: 2, 5, 8, 11, and 14.

[0079] In another embodiment, the nucleic acid DNA molecule of the present disclosure comprises SEQ ID NO: 2.

[0080] In another embodiment, the nucleic acid DNA molecule of the present disclosure comprises SEQ ID NO: 5.

[0081] In another embodiment, the nucleic acid DNA molecule of the present disclosure comprises SEQ ID NO: 8.

[0082] In another embodiment, the nucleic acid DNA molecule of the present disclosure comprises SEQ ID NO: 11.

[0083] In another embodiment, the nucleic acid DNA molecule of the present disclosure comprises SEQ ID NO: 14.

[0084] Vectors

[0085] The polynucleotide sequence according to one embodiment disclosed in the present application may be present in a vector in which the polynucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the polynucleotide sequence by a suitable host cell.

[0086] The expression vectors may contain a signal sequence or a leader sequence for membrane targeting or secretion, as well as regulatory sequences such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, an enhancer and the like. The promoter may be a constitutive or an inducible promoter. Further, the expression vector may include one or more selectable marker genes for selecting the host cell containing the expression vector and may further include a polynucleotide sequence that enables the vector to replicate in the host cell in question.

[0087] The expression vector constructed according to an embodiment may be the vector where the polynucleotide encoding the recombinant fusion protein is inserted within the multiple cloning sites (MCS) of a pT7 vector.

[0088] Known methods may be used to construct vectors including the polynucleotide sequence according to one embodiment disclosed in the present application and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination.

[0089] In one embodiment, the present disclosure provides a recombinant vector comprising the nucleic acid molecule or plasmid DNA encoding the immunogenic fusion protein of any of the preceding embodiments.

[0090] In another embodiment of the present disclosure, plasmid DNA molecules encoding the preceding amino acid sequences are shown in SEQ ID NOS: 3, 6, 9, 12, and 15.

[0091] In one embodiment, the vector of the present disclosure comprises SEQ ID NO: 3.

[0092] In one embodiment, the vector of the present disclosure comprises SEQ ID NO: 6.

[0093] In one embodiment, the vector of the present disclosure comprises SEQ ID NO: 9.

[0094] In one embodiment, the vector of the present disclosure comprises SEQ ID NO: 12.

[0095] In one embodiment, the vector of the present disclosure comprises SEQ ID NO: 15.

[0096] Host Cells

[0097] In an embodiment, the recombinant expression vector having the nucleic acid sequence encoding for at least one recombinant fusion protein may be inserted in a host cell and recombined with the host cell genome or refers to any nucleic acid including a nucleotide sequence competent to replicate spontaneously as an episome. Such a vector may include a linear nucleic acid, a plasmid, a phagemid, a cosmid, an RNA vector, a viral vector, etc.

[0098] In an embodiment, the vector may be genetically engineered to incorporate the nucleic acid sequence encoding the recombinant fusion protein in an orientation either N-terminal and / or C-terminal to a nucleic acid sequence encoding a peptide, a polypeptide, a protein domain, or a full-length protein of interest, and in the correct reading frame so that the recombinant fusion protein including NGF and SP or NGF and HK-1 may be expressed. Expression vectors may be selected from those readily available for use in prokaryotic or eukaryotic expression systems.

[0099] Standard recombinant nucleic acid methods may be used to express a genetically engineered recombinant fusion protein. The nucleic acid sequence encoding the recombinant fusion protein according to one embodiment disclosed in the present application may be cloned into a nucleic acid expression vector, e.g., with appropriate signal and processing sequences and regulatory sequences for transcription and translation, and the protein may be synthesized using automated organic synthetic methods.

[0100] In order to obtain high level expression of a cloned gene or nucleic acid, for example, a cDNA encoding the recombinant fusion protein according to one embodiment disclosed in the present application, the recombinant fusion protein sequence may be typically subcloned into an expression vector that includes a strong promoter for directing transcription, a transcription / translation terminator, and in the case of a nucleic acid encoding a protein, a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art. Bacterial expression systems for expression of the recombinant fusion protein are available in, e.g., E. coli. Bacillus sp., and Salmonella. Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. The eukaryotic expression vector may be preferably an adenoviral vector, an adeno-associated vector, or a retroviral vector.

[0101] The recombinant fusion protein may be introduced into an appropriate host cell, e.g., a bacterial cell, a yeast cell, an insect cell, or a tissue culture cell. The recombinant protein may also be introduced into embryonic stem cells in order to generate a transgenic organism. Large numbers of suitable vectors and promoters are known to those skilled in the art and are commercially available for generating the recombinant protein.

[0102] It is understood that the host cell refers to a eukaryotic or prokaryotic cell into which one or more DNAs or vectors are introduced and refers not only to the particular subject cell but also to the progeny or potential progeny thereof. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0103] The host cells may be preferably bacterial cells, and as the bacterial cells, there are, in principle, no limitations. They may be eubacteria (gram-positive or gram-negative) or archaebacteria, as long as they allow genetic manipulation for insertion of a gene of interest, preferably for sitespecific integration, and they may be cultured on a manufacturing scale. Preferably, the host cells may have the property to allow cultivation to high cell densities.

[0104] Examples of bacterial host cells that may be used in the preparation of the recombinant fusion protein are E. coli. Bacillus sublilis. Pseudomonas fluorescens as well as various Corynebacterium and Lactococcus lactis strains. Preferably, the host cells are Escherichia coli cells.

[0105] In one embodiment, the present disclosure provides a host cell expressing the immunogenic fusion protein according to any of the preceding embodiments or comprising the nucleic acid DNA molecules of the present disclosure in any preceding embodiment.

[0106] In another embodiment, the present disclosure provides a host cell where the fusion protein of any of the preceding embodiments is produced in a prokaryotic or eukaryotic expression system, for example, an inclusion body.

[0107] In one embodiment, present disclosure provides fusion proteins in any preceding embodiment, that fold correctly into mature proteins and present their epitopes effectively to the mammalian immune system.

[0108] In another embodiment, protein folding of the fusion proteins in any preceding embodiment depends on specific sets of structural conformations that make the protein immunogenic but devoid of intrinsic functional NGF-, SP- or HK-l-like biological activity.

[0109] In yet another embodiment, the present disclosure provides fusion proteins that adopt homogeneity in protein conformations for forming proper epitopes necessary for effective immunization, increasing consistency and potency of the immune response against NGF, SP and HK-1, improving the therapeutic efficacy of the vaccine of the present disclosure.

[0110] In another embodiment, the present disclosure provides fusion proteins in any preceding embodiment, that contain predictable disulfide bonds during the expression and purification process. Mature NGF contains 6 cysteine residues in each monomer, that form specific disulfide bonds, which are critical for maintaining its biologically active conformation. The expression of fusion proteins of the present disclosure in E. Coli inclusion bodies can be modeled to properly form folded protein structures, to improve batch-to-batch consistency of the vaccine, and improve uniform efficacy across different production runs.

[0111] Vaccines

[0112] In one embodiment, the present disclosure relates to active immunization, peptide vaccines and immunogenic compositions for treating osteoarthritis, and chronic pain associated with osteoarthritis, mediated by neuroimmune factors such as nerve growth factor, and the neuropeptide substance P (SP) responsible for neurogenic inflammation and pain. Preferably, the vaccines and immunogenic compositions comprise the fusions protein of the present invention.

[0113] In another embodiment vaccines of the present disclosure, can be administered to an individual via various suitable routes. For example, the vaccine can be administered together or separately, and / or simultaneously and / or sequentially, orally, subcutaneously, intramuscularly or transdermally. In some embodiments, the present disclosure provides a pharmaceutical composition for treating pain comprising an effective amount of divalent- or multivalent vaccine, and a pharmaceutically acceptable carrier.

[0114] In yet another embodiment, vaccines of the present disclosure have a multi-valent capability against pain pathways and perception and also the capability of producing immunotherapeutic or prophylactic benefit against more than one health hazard. The immunogenic compositions of the present disclosure induce an efficient immune response, in particular trigger the production of antibodies against nociceptive mediators. Advantageously, when administered to mammals, such as dogs, cats, horses or humans, the immunogenic compositions of the present disclosure alleviate nociceptive and / or inflammatory-associated pain and reduce the need for painkillers in chronic and / or refractory pain associated with rheumatoid pain, osteoarticular inflammation, or neuropathic lesions.

[0115] In one embodiment, the immunogenic compositions of the present disclosure include appropriate carriers and / or adjuvants to the immunogenic recombinant fusion protein to facilitate a simultaneous immune response against two or more nociceptive molecules that act in concert to mediate pain signaling or perception, in the preparation of a medicament or vaccine for the treatment of pain conditions.

[0116] In another embodiment, the vaccine compositions of any of the preceding embodiments of the present disclosure can include an acceptable carrier and / or an adjuvant selected from the group consisting of oil-in-water adjuvant, polymer and water adjuvant, water-in-oil adjuvant, aluminum hydroxide adjuvant and combinations thereof.

[0117] In yet another embodiment of the present disclosure, adjuvants can be for example, complete or incomplete Freund’s adjuvant, a polymeric adjuvant such as Montanide™ gel, aluminum salts (alum), oil emulsions, saponins, immune-stimulating complexes (ISCOMs), liposomes, microparticles, nonionic block copolymers, derivatized polysaccharides, cytokines, or bacterial derivatives.

[0118] In one embodiment, vaccine compositions of the present disclosure can include potentiators useful for enabling immunogenic compositions or vaccines to induce potent and persistent immune responses.

[0119] In yet another embodiment of the present disclosure, the vaccine compositions comprising the immunogenic fusion protein of any of the preceding embodiments is provided, or comprises the nucleic acid molecule of any of the preceding embodiments, or the vector of the present disclosure or the host cell of any of the preceding embodiments.

[0120] In one embodiment of the present disclosure, vaccine compositions are provided that reduce the relative high costs for pet owners.

[0121] In another embodiment, the present disclosure provides for the immunogenic fusion protein that improves vaccines for prevention of osteoarthritis in pets with increased clinical applicability. In another embodiment, the vaccine of the present disclosure improves clinical applicability with better efficacy, stability, and practicality when administered to pets.

[0122] Methods of Treatment and Uses

[0123] In one embodiment of the present disclosure is provided a method of treating a condition selected from nociceptive and / or inflammatory related pain, osteoarthritis associated pain, chronic osteoarthritis related pain, and refractory osteoarthritis related pain in mammals comprisingadministering the fusion protein or the vaccine composition of the present disclosure. Likewise, the present invention also describes the use of the fusion protein or the vaccine composition for treating a condition selected from nociceptive and / or inflammatory related pain, osteoarthritis associated pain, chronic osteoarthritis related pain, and refractory osteoarthritis related pain in mammals.

[0124] In another embodiment of the present disclosure, the condition is nociceptive and / or inflammatory related pain.

[0125] In one embodiment of the present disclosure, the condition is osteoarthritis associated pain.

[0126] In yet another embodiment of the present disclosure, the condition is chronic osteoarthritis related pain.

[0127] In one embodiment of the present disclosure, the condition is refractory osteoarthritis related pain.

[0128] In another embodiment of the present disclosure, the mammal being treated is a dog.

[0129] In one embodiment of the present disclosure, when administered or used in a mammal, the immunogenic fusion protein having a second antigen with multiple SP repeats configuration yields a higher neutralizing titer compared to that of a second antigen with a single SP configuration, establishing the multiple epitope construct as a therapeutic solution for treating OA in mammals.

[0130] In one embodiment of the present disclosure, critical mediators HK-1 and SP are structurally related, yet distinct in its expression profile and some functional roles. While SP is predominantly synthesized and released by sensory nerve fibers, contributing to neurogenic inflammation and pain, HK-1 is primarily expressed in hematopoietic and immune cells such as macrophages, mast cells, and T cells. Both HK-1 and SP have affinity for the neurokinin-1 receptor (NK-1R) and Mas-related G protein-coupled receptor X2 (MRGPRX2) which are expressed in different immune cells including on mast cells.EXAMPLES

[0131] Specific embodiments will now be demonstrated by reference to the following examples. It should be understood that these examples are disclosed solely by way of illustrating the invention and should not be taken in any way to limit the scope of the present invention.

[0132] Example 1: Expression of recombinant immunogenic fusion proteins (rNGF variants with 3 and 5 multiple repeat units)

[0133] The fusion recombinant fusion proteins, rNGF-3SP and rNGF-5SP, were expressed using the plasmid pT7, which was transformed into E. coli BL21 (DE3) competent cells (EC0114, Thermo Fisher) following the manufacturer’s protocol. Fig. 1A illustrates the constructs’ schematic representations and amino acid sequences, including the poly-histidine tag for affinity purification, the NGF domain, and the multiple SP repeat units separated by GSGS linkers. Transformed cells were grown overnight at 37°C with continuous agitation (220 rpm) in Luria-Bertani broth supplemented with 100 pg / mL ampicillin (Sigma, A0166). A pre-culture of 10 mL was used to inoculate 1 L of Terrific Broth, which was incubated at 37°C with agitation (220 rpm) until an optical density (OD eoo) of 2 was reached. Protein expression was induced by adding 1 mM IPTG (Euromedex, EU0008-B), followed by 4 hours of incubation under the same conditions.

[0134] Cells were harvested by centrifugation at 10,000 * g for 10 minutes at 4°C, and the pellet was resuspended in 30 mM Tris buffer (pH 8) containing 150 mM NaCl and 0.5 mg / mL lysozyme. The cell suspension was sonicated (6 x 20 seconds ON, 6 x 1 minute OFF, 50% amplitude; Branson Digital Sonifier) and centrifuged at 20,000 x g for 40 minutes at 4°C. The resulting pellet underwent successive washing steps with Washing Buffer I (50 mM Tris pH 8, 50 mM NaCl, 0.5% Triton X-100, 1.5 mM P-mercaptoethanol, 1.6 M urea) and Washing Buffer II (30 mM Tris pH 8, 150 mM NaCl), each followed by centrifugation at 20,000 x g for 20 minutes at 4°C. The pellet was then solubilized in 20 mM Tris (pH 8), 500 mM NaCl, and 8 M urea. Solubility was improved with an additional sonication step (6 x 20 seconds ON, 6 x 1 minute OFF, 50% amplitude).

[0135] Example 2: Protein purification and characterization

[0136] Fig. IB outlines the purification workflow, highlighting key steps such as solubilization in urea, immobilized metal affinity chromatography (IMAC), and endotoxin removal. Purification was performed using immobilized metal affinity chromatography (IMAC). The solubilized protein solution was filtered (0.45 pm) and applied to a 5 mL HisTrap HP column (Cytiva, GE17-5248-02) pre-charged with Ni2+and equilibrated with binding buffer (protein solubilization buffer containing 30 mM imidazole). The column was washed with five column volumes of the binding buffer, and the protein was eluted in three 10 mL steps using an elution buffer (20 mM Tris pH 8, 500 mM NaCl, 500 mM imidazole, 8 M urea). Eluted fractions were analyzed by SDS-PAGE and dialyzed to remove imidazole.

[0137] Endotoxins were removed using Miltenyi Biotec beads (130-093-657), and endotoxin levels were quantified using the LAL Chromogenic Endotoxin Quantitation Kit (Pierce, 88282) per the manufacturer’s instructions. Protein concentration was determined by absorbance at 280 nm and the Pierce 660 Method (Pierce, 22660). Purity and yield were confirmed by SDS-PAGE densitometry, and protein identity was verified by mass spectrometry.

[0138] Fig. 1C shows the expression and purification processes for rNGF-3SP (20.7 kDa) and rNGF-5SP (24 kDa) analyzed by SDS-PAGE. The non-induced fraction (Lane 1) lacked a visible protein band at the expected molecular weight. Upon induction (Lane 2), a prominent band at 20.7 kDa appeared in the total protein fraction, indicating successful expression. Minimal protein was observed in the soluble fraction (Lane 3), while the insoluble fraction (Lane 4) retained most of the recombinant protein, suggesting inclusion body formation. Similarly for the expression of rNGF-5SP, no visible protein band was detected in the non-induced fraction (Lane 5). Induction (Lane 6) resulted in a strong band at 24 kDa in the total protein fraction. Limited protein was observed in the soluble fraction (Lane 7), while the majority of rNGF-5SP was found in the insoluble fraction (Lane 8), indicating inclusion body formation.

[0139] Following IMAC purification, highly enriched rNGF-3SP and rNGF-5SP proteins were obtained, as shown in the final lanes on the right. Both proteins displayed single, distinct bands at their respective molecular weights, confirming successful purification and high purity.

[0140] These results demonstrate that rNGF-SP variants with three and five SP multiple repeats can be efficiently expressed in E. coli, predominantly in inclusion bodies and successfully purified using IMAC. The purified proteins retained high structural integrity and purity, providing a robust platform for further immunogenicity studies.

[0141] Example 3: Comparative immunogenicity of rNGF-SP immunogenic fusion protein variants in mice

[0142] The humoral anti-SP immune response was evaluated in three groups of adult male B6 mice immunized with NGF-SP (n=7), NGF-3SP (n=7), or NGF-5SP (n=3). Each group received four subcutaneous doses (one priming and three boosters) administered biweekly. Each dose contained 1 mg / kg of antigen formulated with 3% SEPPIC Montanide GEL 01 PR adjuvant in a 100 pL volume. This adjuvant, though weak in mice, was selected to align with clinical trials in dogs with osteoarthritis (OA).

[0143] Antibody titers were measured using pre-immune (PI) and hyperimmune (HI) sera, collected one week after the third booster. Data on antibody titers and optical density values demonstrated antigen-specific responses across groups and are presented in Fig. 2.

[0144] Example 4: Antibody titer assessment by ELISA

[0145] Antibody titers were determined using ELISA. Plates were coated with 2.5 pg / mL of NGF or vaccine antigen, or 10 pg / mL of SP, in 0.05 M carbonate buffer (pH 9.6). High -binding 96-well plates (Coming, REF 3590) were incubated overnight at 37°C. Blocking was performed with 200 pL PBS containing 1% gelatin for 1 hour at 37°C. Serum samples were diluted in PBS with 0.1% Tween-20 and 0.5% gelatin, and 100 pL of each diluted sample was added per well. Following a 1-hour incubation at 37°C, plates were washed five times with PBS containing 0.1% Tween-20.

[0146] Antibody titers were determined using ELISA. Plates were coated with 2.5 pg / mL of NGF or vaccine antigen, or 10 pg / mL of SP, in 0.05 M carbonate buffer (pH 9.6). High -binding 96-well plates (Coming, REF 3590) were incubated overnight at 37°C. Blocking was performed with 200 pL PBS containing 1% gelatin for 1 hour at 37°C. Serum samples were diluted in PBS with 0.1% Tween-20 and 0.5% gelatin, and 100 pL of each diluted sample was added per well. Following a 1-hour incubation at 37°C, plates were washed five times with PBS containing 0.1% Tween-20.

[0147] Example 5: Antibody titers

[0148] Active immunization with rNGF-SP, rNGF-3SP, and rNGF-5SP resulted in antibody titers of 1:4,000, 1:22,000, and 1:25,000, respectively, against the corresponding vaccine antigens. These findings indicate that the addition of SP sequences progressively enhanced the immunogenicity of the rNGF-SP construct. Notably, the observed enhancement in immunogenicity was non-linear and did not directly correlate with the increase in peptide chain length. This suggests a complex interplay between the antigenic structure and the immune system, highlighting the nuanced role of SP sequences in optimizing the immunogenic fusion protein’s ability to elicit an immune response.

[0149] Example 6: Enhanced humoral IgG responses against NGF and SP following immunization with rNGF-3SP and rNGF-5SP

[0150] To evaluate the humoral response elicited by the different SP immunogenic fusion proteins, IgG titers against the vaccinal antigen, NGF, and SP were measured in pre-immune (P I.) and hyperimmune (HI.) sera the conclusion of the immunization protocol. All groups (rNGF-SP, rNGF-3SP, and rNGF-5SP) demonstrated a significant increase in IgG levels against the respective vaccine antigens in H.I. sera compared to P. I. sera (p < 0.01). The rNGF-3SP group exhibited a significantly higher anti-NGF response compared to rNGF-SP and rNGF-5SP (p < 0.0001), suggesting that the inclusion of SP sequences in rNGF-3SP substantially enhanced anti-NGF immunogenicity. Conversely, the rNGF-SP group elicited minimal anti-NGF reactivity, while rNGF-5SP generated a moderate response. These results highlight that the structural inclusion of SP sequences in rNGF-3SP optimized the antigen’s ability to induce a robust anti-NGF response. ForSP-specific IgGs, a significant increase was observed only in the rNGF-5SP group compared to P.I. sera (p < 0.05), indicating that SP-specific immunogenicity was predominantly enhanced in this construct. Neither rNGF-SP nor rNGF-3SP showed significant anti-SP reactivity. These findings demonstrate that the addition of SP sequences progressively enhanced the humoral immune response in a construct-dependent manner. The rNGF-3SP immunogenic fusion protein was most effective at eliciting a strong anti-NGF response, while rNGF-5SP showed superior SP-specific immunogenicity. This differential response underscores the importance of antigen design in achieving targeted immune responses and optimizing vaccine efficacy.

[0151] Fig.2 graphs represent the optical density (O.D.) measurements at 450 nm by ELISA, indicating IgG antibody levels in pre-immune (P.I.) and hyperimmune (H I.) sera. Serum dilutions of 1:500 were used for the vaccinal antigen and NGF, and 1:80 for SP. A: Anti-vaccinal antigen IgGs. All groups (rNGF-SP, rNGF-3SP, rNGF-5SP) showed significant increases in IgG levels in H.I. sera compared to P.I. sera (p < 0.01). B: Anti-NGF IgGs. The rNGF-3SP group demonstrated significantly higher IgG levels compared to rNGF-SP and rNGF-5SP (p < 0.0001). C: Anti-SP IgGs. Only the rNGF-5SP group showed a significant increase in anti-SP IgG levels compared to P.I. sera (p < 0.05). Data are presented as mean ± SEM. Statistical analysis was performed using paired t-tests or oneway ANOVA, as appropriate.

[0152] Example 7: Development of rNGF-SP variants substituting SP with HK-1 tandem repeats

[0153] Development of rNGF-SP variants substituting SP with HK-1 multiple repeats was undertaken to address additional inflammatory mechanisms involved in chronic pain, where a novel immunogenic protein was engineered by replacing the SP single or multiple repeats with a single or multiple repeat of the HK-1 peptide sequence. The resulting recombinant fusion protein, rNGF-3HK1, consists of a nerve growth factor (NGF) domain linked at its carboxy-terminal end to three multiple HK-1 peptide sequences, separated by flexible GSGS linkers. The HK-1 peptide sequences were derived from multiple mammalian species to enhance cross-species efficacy. The rNGF-3HKlfusion protein was designed to induce a robust immune response, generating specific antibodies capable of neutralizing both NGF and HK-1. This dual -targeting strategy aims to mitigate neurogenic inflammation and reduce pain, which are key pathological features of osteoarthritis and chronic back pain Fig. 3 illustrates the structural design of the recombinant fusion proteins using AlphaFold. Fig.3 depicts the design of NGF-HK1 and rNGF-3HKl immunogenic fusion proteins for immunization studies. Each construct contains an NGF domain linked to HK-1 peptide sequences at the carboxyterminal region via a GSGS linker. In rNGF-3HKl, three multiple repeat units of HK-1 antigen are each separated by GSGS linker sequences. All constructs include an N-terminal HisTag for purification and a TEV protease cleavage site. The multiple HK-1 peptide repeats in rNGF-3HKl are intended to enhance immunogenicity and promote antibody generation against both NGF and HK-1.

[0154] Example 8: Comparative in silico analysis of structural and dynamic properties of rNGFSP and rNGF3SP

[0155] Combining protein structural prediction and molecular dynamics simulations, the constructs rNGFSP and rNGF3SP sharing a NGF core were studied in their structural and dynamic properties. The immunogenicity of the models was estimated using a graph-based model for B-cell epitope prediction.

[0156] Methods:

[0157] Alphafold Modelling: Two constructs were studied containing an N-terminal polyhistidine tail, a central core with the neural growth factor and a C-terminal fragment formed by one or three replicas of the substance P (NGFSP and NGF3SP, respectively). Structural models were predicted with Alphafol d21. Three of the top ranked structures were selected as starting conformations for Molecular Dynamics (MD) simulations.

[0158] Molecular Dynamics: The constructs were simulated immersed in 8 M urea and 500 mM NaCl solutions. Trajectories were held for 0.5 ps and ran by triplicate with the GROMACS MD engine employing the Amber ffl4SB force-field according to Abraham, et al. (SoftwareX 2015, 1-2, 19-25. doi.org / 10.1016 / j.softx.2015.06.00) and Maier et al. (J. Chem. Theory Comput. 2015,doi.org / 10.1021). A clustering procedure using the Gromos algorithm was performed to select representative structures from the last 0.1 ps of simulation.

[0159] Epitope Prediction: Five structures from the most populated clusters were then selected to evaluate its immunogenicity using the server Graphbepi (Clifford et al, Protein Science 2022. https: / / doi.org / 10.1002 / pro.4497), which employs an artificial neural network to predict linear and conformational B-cell epitopes. The model has been trained with an epitope data set formed by high resolution structures of antibody-antigen complexes stored at the Protein Data Bank.

[0160] Results

[0161] Alphafold structure prediction: A structural and dynamic characterization of constructs NGFSP and NGF3SP was performed employing an in silico approach. Considering that the NGF acts as a dimer in its biological active form, it was relevant to evaluate the structural stability of the monomer within the construct model by Alphafold. Given as inputs the respective NGFSP and NGF3SP sequences, different 3D models were obtained with Alphafol d2. Structures were characterized for having very high predicted local distance difference test (pLDDT) scores for the NGF fragment for both NGFSP and NGF3SP constructs (Fig. 4). This score estimates how well the prediction would agree with an experimental structure. Both the polyhistidine and SP fragments obtained low or very low pLDDT scores due to the intrinsically disordered nature of the corresponding peptides.

[0162] Construct Dynamic Behavior: The structural stability of the NGF fragment was evaluated through the 1.5 ps accumulated trajectories by calculating its root mean square deviation (RMSD). This measure corresponds to the average distance (5) of the backbone atoms between a reference structure and each conformation adopted along the trajectory. In this case the initial structure was used as coordinates’ reference and the distances were calculated for residues 37 to 137, to exclude the intrinsically disordered portions of NGF.

[0163] The timelines depicted on Fig. 5, show the high structural stability of NGF-SPxl throughout the simulations, with RMSD average values over the last 0.1 ps of (2.01 ± 0.36 A).Regarding NGF-SPx3’s stability, measurements indicated a less steady behavior, presenting RMSD values of 3.87 ± 0.39 A

[0164] Next, to evaluate which region of the NGF core presented higher structural variability, the root mean square fluctuation (RMSF) was calculated. This measure corresponds to the time average RMSD calculated over each residue of the protein. In this case an average structure obtained from the simulation is used as a reference structure, and the Cas are selected to measure distances.Fig- 6 shows how the lower stability observed for NGF3SP was due to a structural loss between residues 65 and 75, corresponding to the P -|35 hairpin. The rest of the P-sheet structure was well maintained during simulations, having RMSF values around 3 A. The RMSF plot also shows the disordered behavior of the flanking regions of the NGF’s P-sheet core. In sum, the RMSD analysis of the 0.5 ps MD simulations indicated thatNGFSP possesses high stability, while NGF3SP partially lost its structure, mostly on the P4-P5 hairpin, which may be associated with increased immunogenicity.

[0165] Epitope prediction: After a RMSD based structural clusterization of the simulated systems, five representative structures were selected to estimate their immunogenicity. To achieve this, the Graphbepi server was employed. This epitope predictor model recognizes not only linear but also conformational epitopes, being an appropriate approach to evaluate the structures generated with MD simulations. Figure 4 shows the per-residue epitope score for both NGF constructs. Graphbepi defines a threshold score of 0.18 to classify residues as epitope member candidates. rNGF-SP and rNGF-3SP presented several regions predicted as epitopes, belonging to the NGF and SP fragments (Fig 7). A comparison of the SP fragments between proteins indicated a foreseen increase in residues classified as epitope members, according to their number of copies (N epitopesNGF-sPxi = 10; N_epitopesNGF-sPx3 = 38). With respect to the NGF fragment, unexpectedly, the number of residues classified as epitope members also changed between constructs (N_epitopesNGFSP = 60; N_epitopesNGF-3SP = 78).

[0166] The region composed by residues 83 to 93 of NGF3SP presented a difference of 5 residues above the threshold compared with the rNGF-SP construct. A solvent accessible surface (SAS) analysis over the five representative structures indicated that the region 83-93 of NGFSP is less exposed to the solvent compared with rNGF-3SP (<SASNGF-SPX1> = 5.63 ± 0.85 nm, <SASNGF-SPX3> = 7.49 ± 1.5 nm, p-value = 0.04). These differences are explained by the interactions observed between the shorter SP fragment and the poly-histidine tail (group 1) with the 83-93 region (group 2) of NGFSP (<N_contactSNGF-sPxi> = 23 ± 15; <N_contactSNGF-sPx3> = 0.4 ± 0.9; p-value = 0.01) (Fig.8). An interatomic contact is defined when atoms from each group are distanced within 6 A.

[0167] In conclusion, the immunogenicity of the most representative conformations of the constructs estimated with an artificial neural network detecting candidate epitope regions on the NGF and SP fragments of both constructs showed increased epitope residues in the rNGF-3SP construct. The augmented SP copies of NGF3SP not only increased the number of residues classified as epitopes on the SP fragment, but also indirectly increased the epitope residues on the NGF core when compared with rNGF-SP. A greater number of SP copies increased its solubility avoiding its interaction with the region composed by residues 83-93 within the NGF core, a region of NGF that has been previously reported to interact with the monoclonal antibody MEDI1912 (PDBid: 5JZ7). A summary of the analyzed properties is presented in Table 2.Table 2: Analyzed properties of constructs NGF-1SP and NGF-3SP. RMSD averaged over the last 0.2 ps of each simulated replica.

[0168] The embodiments illustrated and discussed in this disclosure are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this disclosure should be considered as limiting the scope of the present invention. All examples presented are representative and non-limiting. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of any claims supported by the disclosure and their equivalents, the invention may be practiced other than as specifically described.

Claims

CLAIMSWhat is claimed is:

1. An immunogenic fusion protein comprising:i. a first antigen derived from nerve growth factor (NGF),ii. a second antigen comprising single or multiple repeat units derived from SP or HK- 1; and,iii. a linker amino acid sequence consisting of 4-20 amino acids separating the first antigen from the second antigen and multiple repeat units of the second antigen, if present, wherein the fusion protein amplifies an immune response that comprises eliciting cross reacting antibodies against either or both antigens, andwherein the fusion protein is not rNGF-ISP (SEQ ID NO: 19).

2. The immunogenic fusion protein of claim 1, whereini. the first antigen derived from nerve growth factor (NGF) comprises a sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25; and ii-a. the second antigen comprising multiple repeat units derived from SP has at least 90% sequence identity with SEQ ID NO: 18, orii-b. the second antigen comprising single or multiple repeat units derived from HK-1 has at least 90% sequence identity with a sequence selected from the group consisting of the amino acids selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

3. The immunogenic fusion protein of claim 1 or 2, wherein the fusion protein comprises a single unit of the second antigen derived from HK-1.

4. The immunogenic fusion protein of claim 1 or 2, wherein the fusion protein comprises a repeat configuration of the multiple units of the second antigen.

5. The fusion protein of claim 1 or 2, wherein the configuration comprises three or five repeating units of the second antigen.

6. The fusion protein according to claim 4, wherein the linker further separates each repeat unit of the multiple units of the second antigen.

7. The fusion protein of any one of claims 1-6, wherein the linker comprises (GS)n, wherein n is an integer from 2-10.

8. The fusion protein of claim 7, wherein n is 2.

9. The fusion protein according to any of claims 1-8, comprising the amino acid sequences selected from SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 13.

10. The fusion protein of claim 9, comprising SEQ ID NO: 1.

11. The fusion protein of claim 9, comprising SEQ ID NO: 4.

12. The fusion protein of claim 9, comprising SEQ ID NO: 7.

13. The fusion protein of claim 9, comprising SEQ ID NO: 10.

14. The fusion protein of claim 9, comprising SEQ ID NO: 13.

15. The fusion protein of claim 1, wherein the first antigen comprises at least 95% amino acid sequence identity with SEQ ID NO: 16.

16. The fusion protein of claim 1, wherein the first antigen is selected from a sequence comprising at least 90%, at least 95% , at least 99% or 100% sequence identity with SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, or and SEQ ID NO: 25.

17. The fusion protein of claim 1, wherein the second antigen is selected from a sequence comprising 90%, 95% or 100% sequence identity with SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

18. The fusion protein of claim 1, wherein the second antigen comprises a sequence having 90%, 95%, or 100% amino acid sequence identity with SEQ ID NO: 18.

19. A nucleic acid molecule comprising a polynucleotide encoding the immunogenic fusion protein according to any of claims 1-18.

20. The nucleic acid according to claim 19, comprising a nucleotide sequences selected from SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 14.

21. A vector comprising the nucleic acid molecule according to claim 19, comprising a nucleotide sequences selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO: 15.

22. A host cell expressing the immunogenic fusion protein according to any of claims 1-18 or comprising the nucleic acid molecule according to claim 19.

23. The host cell of claim 26, wherein the fusion protein is produced in a prokaryotic or eukaryotic expression system as an inclusion body.

24. A vaccine composition comprising the fusion protein according to any of claims 1-18, the nucleic acid molecule according to claim 19, the vector according to claim 25, or the host cell according to claim 23.

25. A method of treating a condition selected from nociceptive and / or inflammatory related pain, osteoarthritis associated pain, chronic osteoarthritis related pain, and refractory osteoarthritis related pain in a mammal comprising administering an effective amount of the vaccine composition of claim 24 to a mammal in need thereof.

26. The method of claim 25, wherein the condition is nociceptive and / or inflammatory related pain.

27. The method of claim 25, wherein the condition is osteoarthritis associated pain.

28. The method of claim 25, wherein the condition is chronic osteoarthritis related pain.

29. The method of claim 25, wherein the condition is refractory osteoarthritis related pain.

30. The method of claim 25, wherein the mammal is selected from the group consisting of a canine, a feline, an equine, and a human.

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