Peptide vaccines

Abstract

Embodiments of the disclosure encompass methods and compositions for treating thrombosis, including with respect to any thrombosis-related medical conditions, such as cardiovascular disease. In specific embodiments, one or more peptide vaccines are utilized in methods as immunogenic compositions, including in certain aspects as vaccines. In particular embodiments, the peptide vaccines derive from the extracellular loop of a receptor polypeptide.

Description

PEPTIDE VACCINESBACKGROUND

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 683,478, filed August 15, 2024, which is incorporated by reference herein in its entirety.

[0002] This invention was made with government support under R15HL115567 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] This application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on 14 August 2025, is named TAMU_P0007WO_Sequence_Listing.xml and is 7,001 bytes in size.I. Technical Field

[0004] This disclosure relates at least to the fields of immunology, pharmacology, cell biology, molecular biology, and medicine.II. Background

[0005] Platelet activation processes start upon injury to blood vessels that exposes the subendothelial matrix, leading the platelets to attach to them, where they become activated, and exert their hemostatic function. Excessive platelet aggregation is associated with thrombotic disorders, such as arterial thrombosis. To manage such diseases, medications that inhibit thrombosis are continuously being sought, despite potential underlying drawbacks that include hemorrhage. The present disclosure satisfies a long-felt need in the art of treating thrombosis and related medical conditions, including at least cardiovascular diseases.SUMMARY

[0006] Embodiments of the disclosure include methods and compositions for eliciting an immune reaction in an individual in need thereof. In specific embodiments, the disclosure concerns treatment or prevention of cardiac disease, a thromboembolic condition, or both. InTAMU.P0007WO / 1001354698 - 1 -particular embodiments, immunogenic compositions are encompassed that comprise specific peptides, including peptides that come from an extracellular loop of a receptor.

[0007] In specific embodiments, the compositions and methods of the disclosure treat or prevent cardiac disease and / or a thromboembolic condition. The compositions may prevent onset of disease or they may delay onset of disease, reduce severity and / or prevalence of one or more symptoms of disease, reduce duration of a medical condition, and / or hasten recovery from disease. In specific embodiments, the methods and compositions are utilized as part of routine health care, wherein in some embodiments, the methods and compositions are not utilized as part of routine health care. The individual receiving the methods and compositions of the disclosure may be doing so because of having a risk of disease that is greater than the general population. The individual may be receiving the methods and compositions of the disclosure because of being of a certain age. The individual may be of any gender. The individual may have a vocation that requires long periods of immobility, such as being required to remain in a sitting position. The individual may be pregnant. The individual may have a clotting disorder, cancer, heart condition, diabetes, high blood pressure, kidney disease, chronic inflammation, arthritis, irritable bowel syndrome, infectious disease, CNS disease, or a combination thereof, in some embodiments. The individual may be an invalid, bedridden, a paraplegic, or a quadriplegic, in some embodiments.

[0008] Embodiments of the disclosure may include methods and compositions that treat or prevent cardiac disease and / or a thromboembolic condition by utilizing one or more peptides comprising functional activity to treat or prevent cardiac disease and / or a thromboembolic condition in the individual. In specific embodiments, the sequence of the peptide is from a receptor, and in certain embodiments the peptide is part or all of an extracellular loop of a receptor.

[0009] Embodiments of the disclosure encompass methods and compositions corresponding to the human EL2 sequence (TGVRKNKTITCYDTTSDEYLRSYF; SEQ ID NO: 1; T192-F215; 24 amino acids) of the purinergic P2Y1-R, in alternative embodiments with additional [CGG] amino acids synthesized to permit directional conjugation to VLP, such as with SEQ ID NO:6: CGGTGVRKNKTITCYDTTSDEYLRSYF; 27 amino acids. Such methods may encompass methods of treating a medical condition, methods of preventing a medical condition, methods of delaying onset of a medical condition, methods of reducing severity and / or prevalence of a medical condition, methods of reducing duration of a medical condition, and / or methods of hastening recovery from a medical condition.TAMU. P0007WO / 1001354698 - 2 -

[0010] Embodiments of the disclosure encompass methods and compositions corresponding to the EL2 sequence of the human 5-HT2AR (QDDSKVFKEGSCLLADDN (SEQ ID NO:2) [Q216-N233]; 18 amino acids), optionally in alternative embodiments with additional [CGG] amino acids synthesized to permit directional conjugation to VLP. Embodiments of the disclosure encompass methods and compositions corresponding to any peptide disclosed herein or a peptide that is at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to any peptide disclosed herein. Embodiments of the disclosure encompass methods and compositions corresponding to CGGQDDSKVFKEGSCLLADDN (SEQ ID NO:5; 21 amino acids) that includes the [CGG], Any embodiment with respect to SEQ ID NO:2 encompassed herein may additionally or alternatively utilize the peptide of SEQ ID NO: 5 or be relevant to the peptide of SEQ ID NO: 5. Such methods may encompass methods of treating a medical condition, methods of preventing a medical condition, methods of delaying onset of a medical condition, methods of reducing severity and / or prevalence of a medical condition, methods of reducing duration of a medical condition, and / or methods of hastening recovery from a medical condition.

[0011] In specific embodiments, compositions and methods comprise SEQ ID NO: 1, SEQ ID NO:2, a combination thereof, or a peptide that is at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 1 or SEQ ID NO:2. In some embodiments, an immunogenic composition comprises a peptide comprising the sequence of SEQ ID NO: 1 and / or a peptide that comprises the sequence of SEQ ID NO:2, and combinations may be included. Methods may include administering a therapeutically effective amount of a peptide comprising the sequence of SEQ ID NO: 1, a peptide comprising the sequence of SEQ ID NO:2, or both. In embodiments wherein an individual receives an effective amount of a peptide comprising SEQ ID NO:1 or a peptide that is at least 85% identical to a peptide comprising SEQ ID NO:1 and the individual also receives an effective amount of a peptide comprising SEQ ID NO:2 or a peptide that is at least 85% identical to a peptide comprising SEQ ID NO:2, the peptides may or may not be administered at the same time. They may be administered successively and in any order.

[0012] Embodiments of the disclosure include compositions, comprising an isolated peptide comprising SEQ ID NO: 1; an isolated peptide comprising sequence that is at least 85% identical to SEQ ID NO: 1; an isolated peptide comprising SEQ ID NO:2; an isolated peptide comprising sequence that is at least 85% identical to SEQ ID NO:2; or any combination thereof. In specific embodiments, the peptide sequence is at least 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 1.TAMU. P0007WO / 1001354698 - 3 -In some embodiments, the peptide is at least, exactly, or no more than 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In specific aspects, the peptide is at least, exactly, or no more than 18 amino acids in length. In specific embodiments, the peptide of claim lb) has 1, 2, 3, or more amino acid substitutions compared to SEQ ID NO: 1 or compared to SEQ ID NO:2. In certain embodiments, the peptide comprises an N-terminal extension of 1, 2, 3, or more amino acids compared to SEQ ID NO: 1; an N-terminal extension of 1, 2, 3, or more amino acids compared to SEQ ID NO:2; or both. In some aspects, the peptide comprises a C-terminal extension of 1, 2, 3, or more amino acids compared to SEQ ID NO: 1; a C-terminal extension of 1, 2, 3, or more amino acids compared to SEQ ID NO:2; or both.

[0013] Any composition encompassed herein may be comprised in a pharmaceutically acceptable carrier. In some embodiments, the peptide is linked to or comprised within an agent, such as a viral particle, another peptide, a bead, a scaffold, a therapeutic agent, or a combination thereof. Any composition may comprise one or more adjuvants, one or more stabilizers, one or more preservatives, one or more antibiotics, or a combination thereof. Any composition may be an immunogenic composition or a vaccine composition.

[0014] Embodiments of the disclosure include methods of eliciting an immune response in an individual, comprising administering to the individual an effective amount of any composition encompassed herein. An individual may have cardiac disease, may be at risk for cardiac disease, may have thrombosis, or may be at risk for thrombosis. In any embodiment, the composition may be administered once or multiple times. In any embodiment, the duration of time between administrations may be about 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more years. In any embodiment, the individual is: (a) pregnant; (b) at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years old; (c) bedridden; (d) immobile because of injury, because of surgery or hospitalization, and / or because of travel; (e) has a family history of cardiac disease or thromboembolism; (f) has a blood clotting disorder, cancer, heart condition, diabetes, high blood pressure, kidney disease, chronic inflammation, arthritis, irritable bowel syndrome, or a combination thereof; (g) has estrogen-containing contraception or hormone replacement therapy; (h) is a smoker; and / or (i) is a paraplegic or quadriplegic.

[0015] Embodiments of the disclosure include methods of reducing thrombosis, or the risk of thrombosis, in an individual, comprising administering to the individual an effective amount ofTAMU. P0007WO / 1001354698 - 4 -any composition encompassed herein. The individual may have cardiac disease, may be at risk for cardiac disease, may have thrombosis, or may be at risk for thrombosis.

[0016] Embodiments of the disclosure include methods of treating or preventing thrombosis or cardiovascular disease in an individual, comprising administering to the individual an effective amount of any composition encompassed herein.

[0017] Embodiments of the disclosure include kits comprising any composition encompassed herein.

[0018] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0019] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The subject matter of the present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0021] FIG. 1A. A cartoon model depicting one embodiment of a protocol for the immunization / vaccination with EL2Vac as well as studies used to characterize its activity / effects in terms of managing disease and causing adverse events. The protocol may be applied to development of any composition encompassed herein.

[0022] FIG. IB. A cartoon model depicting potential inhibition of ADP-induced activation of the P2Y1-R by EL2Vac. In specific aspects, vaccination with the EL2Vac will trigger an immune response, and generate antibodies targeted against the EL2 of the P2Y1-R, which will prevent ADP from binding, and inhibit ADP-induced platelet activation.TAMU. P0007WO / 1001354698 - 5 -

[0023] FIG. 2. Antibody concentration in mice vaccinated with EL2Vac, EL2rVac, or VLP. Mice were immunized by VLP coupled with the EL2 sequence (CGGTGVRKNK- TITCYDTTSDEYLRSYF; SEQ ID NO:6 (T192-F215)), random EL2 (LYKDGTICTYVFYNKRRSDTETST; SEQ ID NO:3) or VLP. The EL2Ab- which was measured in the vaccinated mice using ELISA and the EL2 cognate peptide- was detectable at significant levels in the EL2Vac mice, but not the EL2rVac and VLP controls. The titers were assessed in five distinct vaccination groups, each comprising of five mice (**P < 0.001).

[0024] FIG. 3. Platelet aggregation assessment in the vaccinated mice. Platelets were obtained from mice vaccinated with 20 pg of EL2Vac or the EL2rVac and stimulated with two different agonists, (top) 10 pM ADP or (bottom) 1 pM U46619 before their aggregation response was measured in a lumi-aggregometry system. EL2Vac showed a selective inhibition to ADP -induced platelet aggregation. Each test was done in triplicate using three separate groups, with blood pooled from 6 mice per group.

[0025] FIG. 4. P-Selectin expression assessment in the vaccinated mice. Platelets were obtained from mice vaccinated with the EL2Vac or the EL2rVac and stimulated with two different agonists, 10 pM ADP or 1 pM U46619 before their alpha granule secretion / P-Sel ectin expression was measured using flow cytometry. The experiment was conducted in triplicate, each time utilizing three separate groups, and blood was collected from eight mice in each group and pooled per group. The error bars represent the SEM, NS denotes insignificance, whereas the asterisks (**) denote P< 0.001 indicating a statistically significant difference in P-Selectin expression between the comparison groups.

[0026] FIG. 5. Occlusion time in the vaccinated mice. Mice vaccinated with the EL2Vac or the EL2rVac were subjected to the FeCLs-induced carotid artery injury thrombosis model. The time for occlusion was found to be delayed in the EL2Vac, unlike EL2rVac. Each point represents a single animal; **P< 0.001.

[0027] FIG. 6. Tail bleeding time in the vaccinated mice. Mice vaccinated with the EL2Vac or the EL2rVac were subjected to the tail bleeding time assay. The bleeding time was not impacted in the EL2Vac or EL2rVac mice. Each point represents a single animal. The abbreviation NS denotes insignificance, (P> 0.05).

[0028] FIG. 7. Structural representation of the SH TAR highlighting the EL2 domain. This figure shows the detailed 3D structure of the serotonin 2A receptor as generated using AlphaFoldTAMU. P0007WO / 1001354698 - 6 -https: / / alphafbld ebi.ac.uk / , focusing on the extracellular loop 2 (EL2) sequence, which depicts amino acid Q216to N233. The highlighted regions within the structure represent “zoom-in” view of the specific amino acid residues of interest within the EL2 domain that are critical for ligand binding and receptor activity.

[0029] FIG. 8. Comparison of Antibody Levels Following Vaccination with VLP, EL2- 5HTVac, and EL2rVac Vaccines. The bar graph represents the concentration of antibodies (measured in ng / ml) in serum samples from mice vaccinated with VLP, a vaccine containing the random peptide sequence / EL2rVac, and a vaccine comprised of a peptide sequence corresponding to the EL2 of 5-HT2AR / EL2-5HTVac (30 pg). Data are presented as mean ± SEM from five separate experimental groups with five mice in each. The asterisks (***), denote a P-value < 0.001, indicating a statistically significant difference in antibody concentration between the 5-HT2AR Peptide Vaccine and the other two groups.

[0030] FIGS. 9A and 9B. Assessment of Occlusion and Tail Bleeding Times in Vaccinated Mice. FIG. 9A shows the occlusion times following FeC13-induced carotid artery thrombosis in mice immunized with either the 30 pg of EL2-5HTVac or EL2rVac. A notable delay in occlusion was observed in mice treated with EL2-5HTVac compared to those given EL2rVac, indicating a significant impact of the 5-HT2AR-targeted vaccine on thrombosis. FIG. 9B shows the tail bleeding times post-vaccination with EL2-5HTVac or EL2rVac. The duration of bleeding in both vaccinated groups did not show a significant difference, suggesting that the EL2-5HTVac vaccination does not adversely affect hemostasis (significance denoted by the asterisks **P < 0.001, NS denotes no significance, with P > 0.05).

[0031] FIGS. 10A and 10B. Platelet shape change and aggregation assessment of platelets from vaccinated mice. Platelets were obtained from mice vaccinated with 30 pg of EL2-5HTVac or the VLP and stimulated with 1 pM ADP in absence or presence of 15 pM of serotonin before their aggregation response was measured in a lumi-aggregometry system. FIG. 10A shows that EL2-5HTVac can selectively inhibit the serotonin-enhanced ADP-induced platelet aggregation. FIG. 10B shows that vaccination of C57 / BL6 mice with the EL2-5HTVac (30 pg) can result in inhibition of platelet shape change response induced by 15 pM serotonin (10B). Each test was done in triplicate using three separate groups, with blood pooled from 6 mice per group.

[0032] FIG. 11. Platelet aggregation assessment in the vaccinated mice. Platelets were obtained from mice vaccinated with 20 pg of EL2Vac or EL2rVac and stimulated with (C) 5TAMU. P0007WO / 1001354698 - 7 -pg / mL collagen or (D) 2 pg / L collagen before their aggregation response was measured in a lumi-aggregometry system. EL2Vac showed selective inhibition of low-dose collagen-induced platelet aggregation. Each test was performed in triplicate using three separate groups, with blood pooled from six mice per group each time.

[0033] FIG. 12. The effects of EL2Vac on ADP -triggered reduction in cAMP levels in platelets. cAMP levels were measured in EL2rVac and EL2Vac mice, as discussed in Methods. The experiment was conducted in triplicate, and blood was collected from five to six mice and pooled per group. *** p<0.0001; the abbreviation NS denotes insignificance (p > 0.05).

[0034] FIGS. 13A and 13B. Occlusion times in the vaccinated mice. FIG. 13A shows the occlusion times following FeCh-induced carotid artery thrombosis in mice vaccinated with EL2Vac or EL2rVac. The time for occlusion was found to be delayed in EL2Vac, unlike EL2rVac, and it persisted six months later. Mice additionally injected with the cognate EL2 peptide showed reversal of the antithrombotic effects of EL2Vac. Each point represents a single animal (n = 5 for EL2rVac and EL2Vac, and n = 6 for the EL2Vac 6-month experiment); NS denotes insignificance, ** p < 0.001. FIG. 13B shows the occlusion times following FeCh-induced carotid artery thrombosis in mice immunized with either the 30 pg of EL2-5HTVac or EL2rVac. A notable delay in occlusion was observed in mice treated with EL2-5HTVac compared to those given EL2rVac. Mice additionally injected with cognate EL2 peptide showed reversal of the antithrombotic effects of EL2-5HTVac.DETAILED DESCRIPTIONI. Examples of Definitions

[0035] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0036] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”TAMU. P0007WO / 1001354698 - 8 -

[0037] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0038] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0039] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0040] The term “peptide” means a polymer of amino acids of any length. The polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. Except where indicated otherwise, e.g., for the abbreviations for the uncommon or unnatural amino acids set forth herein, the three-letter and one-letter abbreviations, as used in the art, are used herein to represent amino acid residues. Groups or strings of amino acid abbreviations are used to represent peptides. Except where specifically indicated, peptides are indicated with the N- terminus of the left and the sequence is written from the N-terminus to the C-terminus.

[0041] The term “composition” is meant to encompass, and is not limited to, pharmaceutical compositions and nutraceutical compositions, such as an immunogenic composition or a vaccine, containing drug substance (e.g., nucleic acids or peptides). The composition may also contain one or more “excipients” that are inactive ingredients or compounds devoid of pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of a human.

[0042] The terms “immunogenic composition,” and “immunogenic peptide composition,” means a composition that can induce an immune response in a subject, unless clearly indicated otherwise.

[0043] The term “vaccine,” “vaccine composition,” or “peptide vaccine,” in some embodiments, means a composition that can generate acquired immunity against a pathogen orTAMU. P0007WO / 1001354698 - 9 -disease in a subject. In some embodiments, the acquired immunity does not prevent the disease in the subject but reduces its severity. In some embodiments, vaccines promote immune system tolerance for one or more proteins or agents.

[0044] The term “therapeutically effective amount” or “effective amount” refers to any amount that is necessary or sufficient for achieving or promoting a desired outcome. In some instances, an effective amount is a therapeutically effective amount. A therapeutically effective amount is any amount that is necessary or sufficient for promoting or achieving a desired biological response in a subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular agent being administered, the size and / or age of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular agent without necessitating undue experimentation.

[0045] The terms “subject” and “patient” and “individual” are used interchangeably herein. The terms refer to an animal, such as a mammal including a nonprimate and a primate (e.g., a monkey such as a cynomolgus monkey, a chimpanzee, and a human). The term “animal” also includes, but is not limited to, companion animals such as cats and dogs; zoo animals; wild animals; farm or sport animals such as ruminants, non-ruminants, livestock and fowl (e.g., horses, cattle, sheep, pigs, turkeys, ducks, and chickens); and laboratory animals, such as rodents (e.g., mice, rats), rabbits; and guinea pigs, as well as animals that are cloned or modified, either genetically or otherwise (e g., transgenic animals).

[0046] The terms “identity” or “percent identity” as used herein refer to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules and / or peptide molecules.

[0047] The terms “treating,” “treatment,” and “therapy” as used herein refer to the attempted reduction or amelioration of the progression, severity, and / or duration of a disorder, or the attempted amelioration of one or more symptoms thereof). In some embodiments, a subject is successfully “treated” or a disease or disorder if the patient shows total, partial, or transient alleviation or elimination of at least one symptom or measurable physical parameter associated with the disease or disorder.

[0048] As used herein, the terms “prevent,” “preventing”, “prevention”, “alleviate”, or “alleviating” refer to the prevention, inhibition, or lessening of the recurrence, onset, orTAMU. P0007WO / 1001354698 - 10 -development of a disorder or a symptom thereof in a subject resulting from the administration of a therapy (e.g , an immunogenic composition(s) or a vaccine(s)), or the administration of a combination of therapies (e.g., a combination of immunogenic composition(s), vaccine(s), and / or therapeutic agents).

[0049] The term “preventing”, as used herein, covers measures not only to prevent the occurrence of disease, such as risk factor reduction, but also to arrest its progress and reduce its consequences once established, also including to delay its onset.

[0050] As used herein, the term “treatment” or grammatical equivalents encompasses the improvement and / or reversal of the symptoms of disease. It comprises composition that causes an improvement in any parameter associated with disease when used in the methods of the present disclosure. The term “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. For example, those who may benefit from treatment with compositions and methods of the present disclosure include those already with a disease and / or disorder as well as those in which a disease and / or disorder is to be prevented (c.g., using a prophylactic treatment of the present disclosure).

[0051] The present disclosure provides the design and characterization of a novel approach for managing thrombotic disorders, which involves a peptide-based immunogenic composition or vaccine. Such an agent was modeled over the ligand binding site (EL2) of the platelet P2Y1-R (EL2Vac), in specific cases. The EL2Vac exhibits antiplatelet and antithrombotic effects, without causing adverse bleeding. This disclosure provides evidence regarding the ADP-ligand binding site and its importance in regulating platelet function and thrombogenesis. The disclosure also provides a novel vaccine designed to target the ligand binding domain of 5-HT2AR, which resides in the second extracellular loop (EL2). This vaccine, referred to as “EL2-5HTVac” provides a long-lasting and selective therapeutic approach without the complications of increased bleeding risk. The disclosure provides methods and compositions with therapeutic utility of such peptide- based vaccines or immunogenic compositions in the treatment of thrombotic disorders of any kind. A peptide vaccine may be administered to an individual who is already diseased to increase their immune response to a causal disease or condition. There exists a need for compositions, systems, and methods of peptide vaccines based on prediction of the target peptides that will be displayed to protect a host from disease, in specific aspects.TAMU. P0007WO / 1001354698 - 11 -II. Peptides of the Disclosure and Compositions Thereof

[0052] In some aspects, there is a method for treating an individual suffering or at risk to suffer from a thrombotic disease in the course of which one or more peptides according to the present disclosure is administered to said individual. Particular embodiments of the present disclosure provide compositions and methods for treating or preventing or reducing the risk of thrombosis in an individual caused by any reason. Embodiments include one or more peptides having functional activity, either directly or indirectly, to treat, prevent, or reduce the risk of thrombosis in an individual. In specific embodiments, the present disclosure relates to one or more peptides that are part of a vaccine, vaccine composition, immunogenic composition, or composition comprising at least one peptide comprising, consisting of, or consisting essentially of at least or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. 21, 22, 23, 24 or more amino acid residues of SEQ ID NO: 1 or SEQ ID NO:2, including at least or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. 21, 22, 23, 24 or more contiguous amino acid residues of SEQ ID NO: 1 or SEQ ID NO:2.

[0053] The peptides of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be at least 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%, or 100% (or any derivable range therein) similar, identical, or homologous with at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 contiguous amino acids SEQ ID NO: 1. The peptides of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be at least 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%, or 100% (or any derivable range therein) similar, identical, or homologous with at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 contiguous amino acids of SEQ ID NO:2.

[0054] In some embodiments, the peptide may comprise at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 contiguous amino acids of SEQ ID NO:1 that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,TAMU. P0007WO / 1001354698 - 12 -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%, or 100% (or any derivable range therein) similar, identical, or homologous with SEQ ID NO: 1.

[0055] In some embodiments, the peptide may comprise at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 contiguous amino acids of SEQ ID NO:2 that are at least, at most, or exactly 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%, or 100% (or any derivable range therein) similar, identical, or homologous with SEQ ID NO:2.

[0056] The protein from which the peptide sequence of SEQ ID NO:1 has been previously disclosed, = may be found in recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank® and GenPept® databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes, or regions thereof, including extracellular loop(s), may be amplified and / or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.

[0057] The peptides of the present disclosure in particular embodiments may be chemically synthesized by methods that are well known in the art. In some embodiments it is also possible to produce the peptides of the present disclosure using recombinant methods. The peptides can be produced in microorganisms such as bacteria, yeast or fungi, in eukaryotic cells such as mammalian or insect cells, or in a recombinant virus vector such as adenovirus, poxvirus, herpes virus, Simliki forest virus, baculovirus, bacteriophage, sindbis virus or Sendai virus. Suitable bacteria for producing the peptides include E. coli, B. subtilis or any other bacterium that is capable of expressing such peptides. Suitable yeast cells for expressing the peptides of the present disclosure include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida, Pichia pastoris or any other yeast capable of expressing peptides. Corresponding means and methods are well known in the art. Also methods for isolating and purifying recombinantly produced peptides are well known in the art and include, e.g., gel filtration, affinity chromatography, ion exchange chromatography, etc.

[0058] In some embodiment, including to facilitate isolation of the peptides of the present disclosure, fusion polypeptides may be made wherein the peptides are translationally fusedTAMU. P0007WO / 1001354698 - 13 -(covalently linked) to a heterologous polypeptide that enables isolation by affinity chromatography. Typical heterologous polypeptides are His-Tag (e.g. His6; 6 histidine residues), GST-Tag (Glutathione-S-transferase), etc. The fusion polypeptide facilitates not only the purification of the peptides but can also prevent the degradation of the peptides during the purification steps. If it is desired to remove the heterologous polypeptide after purification, the fusion polypeptide may comprise a cleavage site at the junction between the peptide and the heterologous polypeptide, in some aspects. The cleavage site may comprise or consist of an amino acid sequence that is cleaved with an enzyme specific for the amino acid sequence at the site (e.g., proteases).

[0059] The peptides of the present disclosure may be administered to any kind of mammal, including at least humans, dogs, cats, horses, captive primates, and so forth.

[0060] According to a particular embodiment at least one peptide (SEQ ID NO. 1, SEQ ID NO:2, or both) comprised in the composition of the present disclosure comprises at its N- and / or C-terminus at least one cysteine residue bound directly or via a spacer sequence thereto. This cysteine residue may serve as a reactive group in order to bind the peptide to another molecule or a carrier. For instance, this group may be used to bind the peptide to a carrier protein. The cysteine residue can be bound directly to the peptide(s) of the present disclosure or via a spacer sequence. In some embodiments, the spacer sequence comprises at least one, at least two, at least three, at least four, and optionally a maximum of ten, a minimum of five small non-polar amino acid residues, such as glycines.

[0061] In some embodiments, peptide(s) of the disclosure are comprised in a composition that may comprise a carrier. According to one embodiment of the present disclosure the carrier is selected from the group consisting of keyhole limpet haemocyanin (KLH), CRM (such as CRM197), tetanus toxoid (TT), diphtheria toxin (DT), protein D, or any other protein or peptide comprising helper T-cell epitopes.

[0062] In some embodiments, the peptide(s) is coupled or fused to a pharmaceutically acceptable carrier, such as KLH (Keyhole Limpet Hemocyanin), CRM, tetanus toxoid, albuminbinding protein, bovine serum albumin, a dendrimer, peptide linkers (or flanking regions) as well as the adjuvant substances described in Singh et al. (Singh et al., Nat. Biotech. 17, (1999): 1075- 1081 (in particular those in Table 1 of that document)), and O'Hagan et al. (O'Hagan and Valiante, Nature Reviews, Drug Discovery 2 (9); (2003): 727-735 (in particular the endogenous immuno-TAMU. P0007WO / 1001354698 - 14 -potentiating compounds and delivery systems described therein)), or mixtures thereof. The conjugation chemistry (e.g. ,via heterobifunctional compounds such as GMBS and others as described in “Bioconjugate Techniques”, Greg T. Hermanson) in this context can be selected from reactions known to the skilled man in the art.

[0063] In some embodiments, one can fuse at least one peptide of the present disclosure to a protein carrier, including by methods known in the art. Such proteins comprise a peptide as described herein together with an unrelated immunogenic protein. Preferably the immunogenic protein is capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis, hepatitis proteins and protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91 / 18926). Preferably a protein D derivative is used which comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids) and which may be lipidated. Another carrier which may be used to provide fusion proteins may be the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43; (1986):265-292). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion protein. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.

[0064] In specific aspects, the peptide(s) may be formulated with an adjuvant, such as one or more of a low soluble aluminum composition, in particular aluminum hydroxide, MF59, aluminum phosphate, calcium phosphate, cytokines (e.g. IL-2, IL-12, GM-CSF), saponins (e.g. QS21), MDP derivatives, CpG oligonucleotides, LPS, MPL, polyphosphazenes, emulsions (e.g. Freund's, SAF), liposomes, virosomes, iscoms, cochleates, PLG microparticles, poloxamer particles, virus-like particles (VLP), heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins (e.g. LTK63 and LTR72), microparticles and / or polymerized liposomes may be used. In addition or alternative to adjuvants may be utilized that are commercially available as, for example, AS01B, AS02A, AS15, AS-2 and derivatives thereof (GlaxoSmithKline, Philadelphia, PA); CWS, TDM, Leif, aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphorylTAMU. P0007WO / 1001354698 - 15 -lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7 or 12 may also be used as adjuvants.

[0065] In specific embodiments, adjuvants may be used that elicit a predominantly Thl-type response such as, for example, a combination of monophosphoryl lipid A, 3-O-deacylated monophosphoryl lipid A (3D-MPL), optionally with an aluminum salt (see, for example, Ribi et al., Immunology and Immunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., NY, (1986): 407-419; GB 2122204B; GB 2220211; and U.S. Pat. No. 4,912,094). A particular form of 3D-MPL is an emulsion having a small particle size less than 0.2 mm in diameter, and its method of manufacture is disclosed in WO 94 / 21292. Aqueous formulations comprising monophosphoryl lipid A and a surfactant have been described in WO 98 / 43670 and may be used. Exemplified particular adjuvants include AS01B (MPL and QS21 in a liposome formulation), 3D-MPL and QS21 in a liposome formulation, AS02A (MPL and QS21 and an oil-in-water emulsion), 3D-MPL and QS21 and an oil-in-water emulsion, and AS 15. MPL adjuvants are disclosed c.g. in U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094.

[0066] In some embodiments, an adjuvant may be used, such as a saponin or saponin mimetics or derivatives, including QS21 (Aquila Biopharmaceuticals Inc.), which may be used alone or in combination with other adjuvants. For example, an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94 / 00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as described in WO 96 / 33739. Other formulations may comprise an oil-in-water emulsion and tocopherol. A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95 / 17210 and may be used. Additional saponin adjuvants of use in the present disclosure include QS7 (described in WO 96 / 33739 and WO 96 / 11711) and QS17 (described in U.S. Pat. No. 5,057,540 and EP 0 362 279 B l).

[0067] Particular adjuvants also include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the SB AS series of adjuvants (c.g., SBAS-2, AS2', AS2, SBAS-4, or SBAS6, available from GlaxoSmithKline), Detox (Corixa), RC- 529 (Corixa, Hamilton, MT) and other amino-alkyl glucosaminide 4-phosphates (AGPs). Further example adjuvants include synthetic MPL and adjuvants based on Shiga toxin B subunit (see WO 2005 / 112991).TAMU. P0007WO / 1001354698 - 16 -

[0068] Any peptide composition of the present disclosure may be administered by any suitable route, including at least subcutaneously, intramuscularly, intradermally, intravenously (see e.g. “Handbook of Pharmaceutical Manufacturing Formulations”, Sarfaraz Niazi, CRC Press Inc, 2004). Depending on the route of administration, the medicament may comprise respective carriers, adjuvants, and / or excipients. Any composition that comprises a peptide of the present disclosure and a pharmaceutically acceptable carrier may be administered by any suitable mode of application, e.g., intradermally (i.d.), intraperitoneally (i.p.), intramuscularly (i.m.), intranasally, orally, subcutaneously (s.c ), etc. and in any suitable delivery device (O'Hagan et al., Nature Reviews, Drug Discovery 2 (9), (2003), 727-735). The peptides of the present disclosure may be formulated for intradermal, subcutaneous, or intramuscular administration, merely as examples. Means and methods for obtaining respective formulations are known to the person skilled in the art (see e.g., “Handbook of Pharmaceutical Manufacturing Formulations”, Sarfaraz Niazi, CRC Press Inc, 2004).

[0069] According to a particular embodiment of the present disclosure, the composition may be used in the treatment and / or prevention of thrombolytic disorders of any kind, including at least cardiovascular diseases, stroke, or peripheral vascular diseases in particular in mammals, such as in humans.

[0070] In some embodiments, the peptides of the present disclosure are administered to a mammal or an individual in an amount of 0.1 ng to 10 mg, such as 0.5 to 500 pg, including 1 to 100 pg, per administration. In a certain embodiment these amounts refer to all peptides (if more than one peptide is used in the vaccine) present in the composition. In another embodiment these amounts refer to each single fragment present in the composition. In some embodiments of a composition the peptides are present in different or equal amounts. However, the peptide of the present disclosure may alternatively be administered to a mammal or an individual in an amount of 0.1 ng to 10 mg, preferably 10 ng to 1 mg, in particular 100 ng to 500 pg / kg body weight.

[0071] The amount of peptides that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The dose of the composition may vary according to factors such as the disease state, age, sex and weight of the mammal or individual, and the ability of antibody to elicit a desired response in the individual. Dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may beTAMU. P0007WO / 1001354698 - 17 -proportionally reduced as indicated by the exigencies of the therapeutic situation. The dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances. For instance, the peptides and vaccine of the present disclosure may be administered to an individual at intervals of several days, one or two weeks or even months or years.

[0072] In a particular embodiment of the present disclosure, the composition is applied between 2 and 10, such as between 2 and 7, even including up to 5 and in some cases up to 4 times. This number of immunizations may lead to a basic immunization. In a particular embodiment, the time interval between the subsequent vaccinations is chosen to be between 2 weeks and 5 years, such as between 1 month and up to 3 years, including between 2 months and 1.5 years. An exemplified vaccination schedule may comprise 3 to 4 initial vaccinations over a period of 6 to 8 weeks and up to 6 months. Thereafter the administration may be repeated every two to ten years. The repeated administration of the composition may maximize the final effect of a therapeutic administration.

[0073] In some embodiments, the composition of the present disclosure is provided in an amount of 0.5 to 500 pg, such as 1 to 100 pg and alternatively from 0.1 ng to 10 mg, including 10 ng to 1 mg, in particular 100 ng to 100 pg, or, alternatively, c.g. 100 fmol to 10 umol, such as 10 pmol to 1 umol, in particular 100 pmol to 100 nmol. In some aspects, the composition may also comprise auxiliary substances, e.g., buffers, stabilizers, etc.

[0074] It is contemplated that in some compositions of the disclosure, there is between about 0.001 mg and about 10 mg of total peptide per ml.

[0075] In some embodiments, the peptide(s) of this disclosure are administered in a composition. In some embodiments, the amino acid sequences of this disclosure are administered in a pharmaceutical composition that includes a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is in the form of a spray, aerosol, gel, solution, emulsion, lipid nanoparticle, nanoparticle, or suspension. In some embodiments, the composition or pharmaceutical composition is in the form of a cationic nanoemulsion, one example of which is described by Brito et al., (2014) that is incorporated herein by reference.

[0076] In some embodiments, the one or more peptides of this disclosure are administered in a composition. In some embodiments, the one or more peptides of this disclosure are administered in a pharmaceutical composition that includes a pharmaceutically acceptable carrier. In someTAMU. P0007WO / 1001354698 - 18 -embodiments, the composition or pharmaceutical composition is comprised of the third peptide set, as described in this disclosure. In some embodiments, the pharmaceutical composition is in the form of a spray, aerosol, gel, solution, emulsion, lipid nanoparticle, nanoparticle, or suspension. In some embodiments, the composition pharmaceutical composition is in the form of a cationic nanoemulsion, one example of which is described by Brito et al., (2014) that is incorporated herein by reference. The composition may be administered to a subject with a pharmaceutically acceptable carrier, i.e., as a pharmaceutical composition. Typically, in some embodiments, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation, which in some embodiments can render the formulation isotonic.

[0077] In certain embodiments, the peptides are provided as an immunogenic composition comprising any one of the peptides described herein and a pharmaceutically acceptable carrier. In certain embodiments, the immunogenic composition further comprises an adjuvant. In certain embodiments, the peptides are conjugated with other molecules to increase their effectiveness as is known by those practiced in the art. For example, peptides can be coupled to antibodies that recognize cell surface proteins on antigen presenting cells to enhance vaccine effectiveness. One such method for increasing the effectiveness of peptide delivery is described in Woodham, et al., (2018).

[0078] In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of saline, Ringer's solution, dextrose solution, and a combination thereof. Other suitable pharmaceutically acceptable carriers known in the art are contemplated. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 2005, Mack Publishing Co. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. The formulation may also comprise a lyophilized powder. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of peptides being administered.

[0079] The phrase pharmaceutically acceptable carrier as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting theTAMU. P0007WO / 1001354698 - 19 -subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier is acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as butylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. The term carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being comingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency. The composition may also include additional agents such as an isotonicity agent, a preservative, a surfactant, and, a divalent cation, preferably, zinc.

[0080] The composition can also include an excipient, or an agent for stabilization of a peptide composition, such as a buffer, a reducing agent, a bulk protein, amino acids (such as e.g., glycine or praline) or a carbohydrate. Bulk proteins useful in formulating peptide compositions include albumin. Typical carbohydrates useful in formulating peptides include but are not limited to sucrose, mannitol, lactose, trehalose, or glucose.

[0081] Surfactants may also be used to prevent soluble and insoluble aggregation and / or precipitation of peptides or proteins included in the composition. Suitable surfactants include but are not limited to sorbitan trioleate, soya lecithin, and oleic acid. In certain cases, solution aerosols are preferred using solvents such as ethanol. Thus, formulations including peptides can also include a surfactant that can reduce or prevent surface-induced aggregation of peptides by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001% and 4% by weight of theTAMU. P0007WO / 1001354698 - 20 -formulation. In some embodiments, surfactants used with the present disclosure are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20. Additional agents known in the art can also be included in the composition.

[0082] In some embodiments, the compositions and dosage forms further comprise one or more compounds that reduce the rate by which an active ingredient will decay, or the composition will change in character. So called stabilizers or preservatives may include, but are not limited to, amino acids, antioxidants, pH buffers, or salt buffers. Nonlimiting examples of antioxidants include butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, butylated hydroxy anisole and cysteine. Nonlimiting examples of preservatives include parabens, such as methyl or propyl p-hydroxybenzoate and benzalkonium chloride. Additional nonlimiting examples of amino acids include glycine or proline.

[0083] The present disclosure also teaches the stabilization (preventing or minimizing thermally or mechanically induced soluble or insoluble aggregation and / or precipitation of an inhibitor protein) of liquid solutions containing peptides at neutral pH or less than neutral pH by the use of amino acids including proline or glycine, with or without divalent cations resulting in clear or nearly clear solutions that are stable at room temperature or preferred for pharmaceutical administration.

[0084] In one embodiment, the composition is of single unit or multiple unit dosage forms. Compositions of single unit or multiple unit dosage forms of the invention comprise a prophylactically or therapeutically effective amount of one or more compositions (e.g., a compound of the invention, or other prophylactic or therapeutic agent), typically, one or more vehicles, carriers, or excipients, stabilizing agents, and / or preservatives. Preferably, the vehicles, carriers, excipients, stabilizing agents and preservatives are pharmaceutically acceptable.

[0085] In some embodiments, the compositions and dosage forms comprise anhydrous compositions and dosage forms. Anhydrous compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Compositions and dosage forms that comprise lactose and at least one active ingredient that comprise a primary or secondary amine are preferably anhydrous if substantial contact with moisture and / or humidity during manufacturing, packaging, and / or storage is expected. An anhydrous composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are preferably packaged using materialsTAMU. P0007WO / 1001354698 - 21 -known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0086] Suitable vehicles are well known to those skilled in the art of pharmacy, and nonlimiting examples of suitable vehicles include glucose, sucrose, starch, lactose, gelatin, rice, silica gel, glycerol, talc, sodium chloride, dried skim milk, propylene glycol, water, sodium stearate, ethanol, and similar substances well known in the art. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles. Whether a particular vehicle is suitable for incorporation into a composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient and the specific active ingredients in the dosage form. Pharmaceutical vehicles can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.

[0087] The disclosure also provides that a composition can be packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity. In one embodiment, the composition can be supplied as a dry sterilized lyophilized powder in a delivery device suitable for administration to the lower airways of a patient. The compositions can, if desired, be presented in a pack or dispenser device that can contain one or more unit dosage forms containing the active ingredient. The pack can for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.

[0088] Methods of preparing these peptide formulations or compositions include the step of bringing into association a peptide compound of the present disclosure with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0089] Formulations of the disclosure suitable for administration may be in the form of powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouthwashes and the like, each containing a predetermined amount of a compound of the present invention (e.g., peptides) as an active ingredient.TAMU. P0007WO / 1001354698 - 22 -

[0090] A liquid composition herein can be used as such with a delivery device, or they can be used for the preparation of pharmaceutically acceptable formulations comprising peptides that are prepared for example by the method of spray drying. The methods of spray freeze-drying peptides / proteins for pharmaceutical administration disclosed in Maa et al., Curr. Pharm. Biotechnol., 2001, 1, 283-302, are incorporated herein. In another embodiment, the liquid solutions herein are freeze spray dried and the spray-dried product is collected as a dispersible peptide- containing powder that is therapeutically effective when administered to an individual.

[0091] The compounds and compositions of the present disclosure can be employed in combination therapies for thrombosis or cardiovascular disease, that is, the compounds and compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures (e.g., peptide vaccine or peptide immunogenic composition can be used in combination therapy with another treatment, such as anticoagulants, antiplatelet agents, statins, heart failure medications, antihypertensive agents, and so forth.). The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and / or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, the compound of the present invention may be administered concurrently with another therapeutic or prophylactic).

[0092] The disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the compositions of the disclosure. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0093] The current disclosure provides for dosage forms comprising nucleic acid sequences or peptides suitable for treating cardiovascular or other diseases. The dosage forms can be formulated, e.g., as sprays, aerosols, nanoparticles, liposomes, or other forms known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences; Remington: The Science and Practice of Pharmacy supra; Pharmaceutical Dosage Forms and Drug Delivery Systems by Howard C., Ansel et al., Lippincott Williams & Wilkins; 7th edition (Oct. 1, 1999).TAMU. P0007WO / 1001354698 - 23 -

[0094] Generally, a dosage form used in the acute treatment of a disease may contain larger amounts of one or more of the active ingredients it comprises than a dosage form used in the chronic treatment of the same disease. In addition, the prophylactically and therapeutically effective dosage form may vary among different conditions. For example, a therapeutically effective dosage form may contain peptides that has an appropriate immunogenic action when intending to treat cancer or other disease. On the other hand, a different effective dosage may contain nucleic acid sequences or peptides that has an appropriate immunogenic action when intending to use the peptides of the present disclosure as a prophylactic (e.g., an immunogenic composition or vaccine) against cancer or another disease / condition. These and other ways in which specific dosage forms encompassed by this invention will vary from one another and will be readily apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 2005, Mack Publishing Co.; Remington: The Science and Practice of Pharmacy by Gennaro, Lippincott Williams & Wilkins; 20th edition (2003); Pharmaceutical Dosage Forms and Drug Delivery Systems by Howard C. Ansel et al., Lippincott Williams & Wilkins; 7th edition (Oct. 1, 1999); and Encyclopedia of Pharmaceutical Technology, edited by Swarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988, which are incorporated herein by reference in their entirety.

[0095] The pH of a composition or dosage form may also be adjusted to improve delivery and / or stability of one or more active ingredients. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Compounds such as stearates can also be added to compositions or dosage forms to alter advantageously the hydrophilicity or lipophilicity of one or more active ingredients to improve delivery. In this regard, stearates can also serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery enhancing or penetration-enhancing agent. Different salts, hydrates, or solvates of the active ingredients can be used to adjust further the properties of the resulting composition.

[0096] Compositions can be formulated with appropriate carriers and adjuvants using techniques to yield compositions suitable for immunization. The compositions can include an adjuvant, such as, for example but not limited to, alum, poly IC, MF-59, squalene-based adjuvants, or liposomal based adjuvants suitable for immunization.Expression SystemsTAMU. P0007WO / 1001354698 - 24 -

[0097] In certain aspects, the disclosure provides culturing a cell line that expresses any one of the peptides of the disclosure in a culture medium comprising any of the peptides described herein.

[0098] Various expression systems for producing recombinant proteins / peptides are known in the art, and include, prokaryotic (e.g., bacteria), plant, insect, yeast, and mammalian expression systems. Suitable cell lines, can be transformed, transduced, or transfected with nucleic acids containing coding sequences for the peptides of the invention in order to produce the molecule of interest. Expression vectors containing such a nucleic acid sequence, which can be linked to at least one regulatory sequence in a manner that allows expression of the nucleotide sequence in a host cell, can be introduced via methods known in the art. Practitioners in the art understand that designing an expression vector can depend on factors, such as the choice of host cell to be transfected and / or the type and / or amount of desired protein to be expressed. Enhancer regions, which are those sequences found upstream or downstream of the promoter region in non-coding DNA regions, are also known in the art to be important in optimizing expression. If needed, origins of replication from viral sources can be employed, such as if a prokaryotic host is utilized for introduction of plasmid DNA. However, in eukaryotic organisms, chromosome integration is a common mechanism for DNA replication. For stable transfection of mammalian cells, a small fraction of cells can integrate introduced DNA into their genomes. The expression vector and transfection method utilized can be factors that contribute to a successful integration event. For stable amplification and expression of a desired protein, a vector containing DNA encoding a protein of interest is stably integrated into the genome of eukaryotic cells (for example mammalian cells), resulting in the stable expression of transfected genes. A gene that encodes a selectable marker (for example, resistance to antibiotics or drugs) can be introduced into host cells along with the gene of interest in order to identify and select clones that stably express a gene encoding a protein of interest. Cells containing the gene of interest can be identified by drug selection wherein cells that have incorporated the selectable marker gene will survive in the presence of the drug. Cells that have not incorporated the gene for the selectable marker die. Surviving cells can then be screened for the production of the desired protein molecule.

[0099] A host cell strain, which modulates the expression of the inserted sequences, or modifies and processes the nucleic acid in a specific fashion desired also may be chosen. Such modifications (for example, glycosylation and other post-translational modifications) andTAMU. P0007WO / 1001354698 - 25 -processing (for example, cleavage) of peptide / protein products may be important for the function of the peptide / protein. Different host cell strains have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. As such, appropriate host systems or cell lines can be chosen to ensure the correct modification and processing of the target protein expressed. Thus, eukaryotic host cells possessing the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.

[0100] Various culturing parameters can be used with respect to the host cell being cultured. Appropriate culture conditions for mammalian cells are well known in the art (Cleveland W L, et al., J Immunol Methods, 1983, 56(2): 221-234) or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. (Oxford University Press: New York, 1992)). Cell culturing conditions can vary according to the type of host cell selected. Commercially available medium can be utilized.

[0101] Peptides of the disclosure can be purified from any human or non-human cell which expresses the peptide, including those which have been transfected with expression constructs that express peptides of the invention. For protein recovery, isolation and / or purification, the cell culture medium or cell lysate is centrifuged to remove particulate cells and cell debris. The desired peptide molecule is isolated or purified away from contaminating soluble proteins and peptides by suitable purification techniques. Non-limiting purification methods for proteins include: size exclusion chromatography; affinity chromatography; ion exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on a resin, such as silica, or cation exchange resin, e.g., DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, e.g., Sephadex G-75, Sepharose; protein A sepharose chromatography for removal of immunoglobulin contaminants; and the like. Other additives, such as protease inhibitors (e.g., PMSF or proteinase K) can be used to inhibit proteolytic degradation during purification. Purification procedures that can select for carbohydrates can also be used, e.g., ion-exchange soft gel chromatography, or HPLC using cation- or anion exchange resins, in which the more acidic fraction(s) is / are collected.Variant PolypeptidesTAMU. P0007WO / 1001354698 - 26 -

[0102] The following is a discussion of changing the amino acids of a peptide to generate an equivalent, or even improved, second-generation variant peptide, for certain embodiments. For example, certain amino acids may be substituted for other amino acids in peptide sequence with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s functional activity, certain amino acid substitutions can be made in a peptide sequence thereof (and in its corresponding DNA coding sequence) and nevertheless produce a peptide with similar or desirable properties. It is thus contemplated by the inventors that various changes may be made in the peptides without appreciable loss of their biological utility or activity.

[0103] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.

[0104] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more non-contiguous or contiguous amino acids of the peptide, as compared to SEQ ID NO: 1. A variation in a polypeptide of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more non-contiguous or contiguous amino acids of the peptide, as compared to SEQ ID NO:2. A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein. A variant can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.

[0105] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region. The additionalTAMU. P0007WO / 1001354698 - 27 -residues on either terminus (or both termini) may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids, for example.

[0106] Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein. The deleted residues on either terminus or both termini may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids, for example.

[0107] Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0108] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties. “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes of alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0109] Alternatively, substitutions may be “non-conservative”, such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for aTAMU. P0007WO / 1001354698 - 28 -nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.

[0110] One skilled in the art can determine suitable variants of polypeptides as set forth herein using well-known techniques. One skilled in the art may identify suitable areas of the peptide molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. The skilled artisan will also be able to identify amino acid residues and portions of the peptide molecules that are conserved among similar proteins or polypeptides. In further embodiments, areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without significantly altering the biological activity or without adversely affecting the protein or polypeptide structure.[0U1] In making such changes, the hydropathy index of amino acids may be considered. The hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (—0.4); threonine (—0.7); serine (—0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathy amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157: 105-131 (1982)). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and others. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a similar biological activity. In making changes based upon the hydropathy index, in certain embodiments, the substitution of amino acids whose hydropathy indices are within ±2 is included. In some aspects of the present disclosure, those that are within ±1 are included, and in other aspects of the present disclosure, those within ±0.5 are included.

[0112] It also is understood in the art that the substitution of like amino acids can be effectively made based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states thatTAMU. P0007WO / 1001354698 - 29 -the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen binding, that is, as a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (_0.5); histidine (—0.5); cysteine (—1.0); methionine (—1.3); valine (—1.5); leucine (—1.8); isoleucine (-1.8); tyrosine (—2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 are included, in other embodiments, those which are within ±1 are included, and in still other embodiments, those within ±0.5 are included. In some instances, one may also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as “epitopic core regions.” It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0113] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides or proteins that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0114] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using standard assays for binding and / or activity, thus yielding information gathered from such routine experiments, which may allow one skilled in theTAMU. P0007WO / 1001354698 - 30 -art to determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations. Various tools available to determine secondary structure can be found on the world wide web at expasy.org / proteomics / protein structure.

[0115] In some embodiments of the disclosure, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinities, and / or (5) confer or modify other physicochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular contacts. In such embodiments, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the native antibody).III. Methods of Treatment and Prevention

[0116] In one embodiment, the subject matter disclosed herein relates to a therapeutic or preventive medical treatment started after following diagnosis of thrombosis, including cardiovascular disease, in order to prevent the disease from worsening, or curing the disease. In one embodiment, the subject matter disclosed herein relates to prophylaxis of subjects who are believed to be at risk for thrombosis or have previously been diagnosed with thrombosis (or another disease). In one embodiment, said subjects can be administered one or more compositions of the disclosure. The disclosure contemplates using any of the nucleic acid sequences or peptides produced by the systems and methods described herein. In one embodiment, the compositions described herein can be administered subcutaneously via syringe or any other suitable method know in the art.

[0117] In some embodiments, the composition(s) of the present disclosure are provided to an individual as a means of preventative, routine health care, including as a standard vaccination. Such an individual may or may not have a history of thrombosis or cardiovascular disease. Such an individual may be of a certain age, such as 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 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,TAMU. P0007WO / 1001354698 - 31 -84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 or more years of age. Such an individual may or may not be at risk of thrombosis or cardiovascular disease compared to the general population.

[0118] In some embodiments, the peptides prepared using methods of the disclosure can be used as an immunogenic composition or as a vaccine to promote an immune response against an antigen. In some embodiments, the disclosure provides compositions and methods for induction of immune response, for example induction of antibodies against one or more peptides encompassed herein. In some embodiments, the antibodies are broadly neutralizing antibodies. In some embodiments, the disclosure provides compositions and methods for induction of immune response, for example induction of a T cell response to antigens.

[0119] In some embodiments, the peptides prepared using methods of the disclosure can be combined with additional therapeutic components. In some embodiments, the combination can be encoded in one or more nucleic acids that encode the peptides produced with the methods described herein and additional therapeutic components (e.g., peptides or proteins) that are known in the art. In some embodiments, the combination is created by adding the peptides or proteins that encode the additional therapeutic components of the peptides that result from the methods described here for combined formulation and packaging. An example of the combination of components is the creation of vaccines that contain components of tumor cell associated proteins, such as MICA or MICB (Badrinath et al., 2022). In some embodiments, peptide components to invoke an adaptative immune response can be added to such combined vaccines (e.g., MICA or MICB) by using one or more nucleic acids to encode the components and packaging the nucleic acids in a mRNA-LNP or DNA formulation, or separately formulating different components as mRNA-LNP or DNA and then combining them for packaging or immediately before administration to a person. In some embodiments, cancer or other vaccines that encode one or more protein fragments to produce an antibody response can be combined with a peptide vaccine using the methods described herein to produce a cellular immune response.Examples

[0120] The following examples are included to demonstrate particular embodiments of the subject matter of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventorsTAMU. P0007WO / 1001354698 - 32 -to function well in the practice of the subject matter of the disclosure, and thus can be considered to constitute particular modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the subject matter of the disclosure.EXAMPLE 1DESIGN AND PHARMACOLOGICAL CHARACTERIZATION OF A NOVEL ANTITHROMBOTIC P2Y1 RECEPTOR-BASED VACCINE

[0121] This disclosure provides the development of a novel peptide-based vaccine that targets the purinergic ADP P2Yi receptor (abbreviated EL2Vac), and its pharmacological characterization. Thus, EL2Vac was designed and developed that is rationalized over the ligand binding domain of the P2Yi receptor, which resides in its second extracellular loop (EL2). The vaccine’s ability to trigger an immune response (antibody production) in the immunized mice, to modulate platelet function, as well as its antithrombotic activity and any effects on hemostasis, was characterized by employing a thrombosis model and the tail bleeding time assay. Results showed significant levels of antibody production in mice treated with EL2Vac, in comparison with the random peptide vaccine control (EL2rVac). Moreover, there was significant inhibition of the ADP-induced aggregation response in platelets from the EL2Vac mice, relative to those from the control. This inhibition was selective for ADP, as when using another agonist, namely that for the thromboxane A2 receptor (TPR), U46619, no detectable effects on aggregation were found. As for its capacity to protect against thrombosis, the data showed a significant delay in the occlusion time of the EL2Vac mice in contrast with the control vaccine. Interestingly, the E12Vac did not appear to prolong the tail bleeding time, supporting the notion that it is devoid of bleeding diathesis. The disclosure documents the design and evaluation of a novel EL2Vac that selectively targets the P2Yi receptor and protects from thrombus formation without impairing hemostasis. Thus, the EL2Vac provides a clinical option to treat thromboembolic disorders.TAMU. P0007WO / 1001354698 - 33 -

[0122] Hemostasis is the process of maintaining the blood vessels' integrity after vascular injury by forming a clot at the site of the wound. This can be attained- in part- through interactions involving endothelial cells, platelets, and the coagulation cascade. Platelets are anucleate discoid shape cells that do not interact with the vessel wall unless the subendothelial matrix gets exposed, allowing them to attach and get activated through the surface receptors and adhesion molecules. Thus, the primary mechanism for hemostasis is initiated through platelet adhesion, activation, and aggregation to the uncovered subendothelial matrix upon injury. Furthermore, platelets can act as a promoter for the coagulation cascade through their procoagulant surface, which is known as secondary hemostasis. One important platelet surface receptor family is the G-protein coupled receptors (GPCRs), which is essential for platelet activation and aggregation. Among the various platelets activators, adenosine 5 ’-diphosphate (ADP) is secreted from the dense granules, and is responsible fortheir activation via interaction and subsequent stimulation of two separate GPCRs: the purinergic P2Yi and P2Y12 receptors.

[0123] The P2Y1-R and P2Y12-R are responsible for total platelet aggregation triggered by ADP. The activation of P2Y12-R leads to intensification and maintaining the aggregation response as a result of inhibiting the Gi-dependent adenylyl cyclase and decreasing the cyclic adenosine monophosphate (cAMP). Moreover, P2Y1-R activation is thought to be essential for platelet shape alteration, but also plays a pivotal role in ADP -induced aggregation through Gq-dependent phospholipase C (PLC) stimulation that results in the making of inositol triphosphate (IP3) and, consequentially, the release of intracellular calcium. Regardless of its vital role in hemostasis, excessive aggregation can lead to serious conditions that include arterial thrombosis. Subsequently, P2Y12 antagonist had been extensively used to prevent the propagation of platelet aggregation (e.g., clopidogrel) due to its prominent role in platelets aggregation in comparison to P2Yi, but such a strategy has also showed a high risk of bleeding. It is noteworthy that no P2Yi- R antagonist has been approved for clinical use up to date. However, Fabre et al. demonstrated a vital role of P2Y1-R in platelet aggregation, using P2Y i-R deficient mice that were found to exhibit impaired ADP -induced platelet aggregation and delayed thrombosis, which were supported by a separate report by Lenain et al. To this end, antagonism of P2Yi receptor has garnered some interest, in light of the transgenic and pharmacological approaches clearly documenting it as a mediator for thrombotic disease (shown in Table 1). Nevertheless, those antagonists had not beenTAMU. P0007WO / 1001354698 - 34 -clinically approved- due to various limitations. Noteworthy, the latter antagonists had been designed based on the structure of ADP and ATP regardless of the P2Y1-R binding domain.

[0124] Table 1 P2Y1-R antagonists: pharmacological action and limitations.P2Y1-R antagonist Pharmacological action LimitationsComplete inhibition ofA2P5P and A3P5P Not selective platelet aggregationInhibition of plateletTransient actionMRS2179 function and delay in requiring high doses thrombosis formationEnhanced inhibition ofPotential forADP-induced aggregation, irreversible inhibitionMRS2500 antithrombotic activity, when used with higher targeting ability than clopidogrelMRS2179Unknown in vivoInhibited ADP / P2Yi effectiveness, shortDi -aryl urea receptor-mediated platelet biological half-life, aggregation in vitro toxicityBenzofuran-substituted Inhibited P-selectin Poor potency, oral urea derivatives expression bioavailability issues

[0125] In pursuit of P2Y1-R inhibitors, an antibody targeting the ligand binding domain, namely second extracellular loop (EL2) of the receptor protein, was developed. The results demonstrated that this antibody, abbreviated EL2Ab, effectively selectively inhibited ADP- induced platelet function and protected against thrombogenesis.

[0126] Vaccines have long been established as a therapeutic approach for a host of diseases / infections, and more recently, for managing HIV, HPV, and cancers. This is especially the case for peptide-based vaccines because of their proven safety profile, low cost, ease of manufacturing, and, most importantly, better patient compliance. In this connection, peptideTAMU. P0007WO / 1001354698 - 35 -vaccines use a 20-30 amino acid sequence to produce an immunogenic peptide representing a particular epitope, which would be sufficient to develop an appropriate cellular and humoral response. FIG. 1A depicts an embodiment of a vaccination protocol / strategy and the dosing timeline, as well as various studies that may be conducted to characterize the biological activity of an immunogenic composition, at least in terms of efficacy and potential side effects.

[0127] Based on the aforementioned considerations, the present disclosure provides design and characterization of a peptide-based vaccine modeled over the EL2 domain of the P2Y1-R (EL2Vac), relative to the random EL2 peptide vaccine control (EL2rVac), for its antithrombotic activity (FIG. IB).EXAMPLE 2QUANTIFICATION OF ANTIBODY PRODUCTION IN VACCINATED MICE

[0128] To confirm that vaccination with EL2Vac efficiently induced an immune response; serum samples were collected from mice vaccinated with the EL2 peptide (EL2Vac), or the random EL2 peptide (EL2rVac), and virus like particles (VLP) controls, and the concentration of the antibody produced was determined using ELISA utilizing the EL2 cognate peptide. The EL2Vac resulted in the production of significant levels of antibody, unlike EL2rVac and VLP (FIG. IB). Thus, this result indicates that EL2Vac was successful in generating antibodies against EL2 (EL2Ab) that would presumably interact with the EL2 region of P2Y i-R and exert a biological effect.EXAMPLE 3PLATELET COUNTS IN VACCINATED MICE

[0129] Since previous studies showed an altered platelet count due to vaccine-induced immune-response, it was sought to determine whether EL2Vac would produce any effects. The results indicated no significant difference in the platelet counts between the EL2Vac mice, in comparison with the EL2rVac and VLP groups, suggesting that EL2Ab production and interaction with the P2Y1-R does not affect the platelet life span (Table 2).TAMU. P0007WO / 1001354698 - 36 -

[0130] Table 2. Platelets counts for the reference (Basal), Vehicle, EL2Vac, and VLP. All counts had been determined individually for each mouse (5 mice per group) and in a triplicate manner, while data in the table represents the mean ± SEM for each group.EXAMPLE 4THE EL2VAC INHIBITS ADP-INDUCED PLATELET AGGREGATION

[0131] Previous work had demonstrated the ability of a custom-made antibody against the EL2 domain of P2Y1-R (EL2Ab) to block ADP -induced platelet aggregation. In the present disclosure it was assessed whether the EL2Vac would exhibit a similar activity. Indeed, EL2Vac was found to exert significant inhibitory effects on platelet aggregation induced by 10 pM ADP (FIG. 2), unlike the EL2rVac that was without any effects. To evaluate its specificity, the capacity of EL2Vac to inhibit non-ADP mediated platelet aggregation was examined. As one might expect, the EL2Vac had no apparent effects on the platelet aggregation induced by 1 pM of the TPR agonist, U46619 (FIG. 3), or a high dose 5 pg / mL of collagen (FIG. 11). Of note, low-dose collagen-triggered platelet activation is thought to involve P2Y1-R. Interestingly, and consistent with this notion, when a low dose 2 pg / mL of collagen was utilized, significant inhibition was observed (FIG. 11). This data supports the selectivity of the EL2Vac for the P2Y1-R.

[0132] P -selectin expression was examined on the surface of activated platelets as a measure of secretion of alpha granules in the context of our antithrombotic vaccine. The EL2Vac exertedTAMU. P0007WO / 1001354698 - 37 -significant inhibition of / ’-selectin expression following activation by ADP, whereas the EL2rVac control was without any effects (FIG. 4). Moreover, neither the EL2Vac, nor the EL2rVac exhibited any activity against U46619 (FIG. 4).EXAMPLE 5THE EL2VAC PROLONGS THE OCCLUSION TIME WITHOUT AFFECTING THE TAIL BLEEDING TIME

[0133] In the next series of studies, thrombogenesis was investigated following vaccinations using the FeC13-induced thrombosis model. The data, as shown in FIG. 5, revealed that the EL2Vac, but not EL2rVac, significantly prolonged the time needed for occlusion of the carotid artery. These data show the ability of the developed vaccine to prevent thrombus formation and support its clinical use for managing thrombotic disorders.

[0134] The capacity of the vaccination-based approach to modulate hemostasis was evaluated by using the tail bleeding time assay. Interestingly, the EL2Vac did not exert any detectable effects, much like the control EL2rVac (FIG. 6). Taken together, these findings indicate that EL2Vac exerts thromboprotective activity without modulating hemostasis / increasing the risk of bleeding.EXAMPLE 6SIGNIFICANCE OF CERTAIN EMBODIMENTS OF EXAMPLES 1-5

[0135] The primary hemostatic pathway for any sustained injuries in blood vessels involves platelet activation, which will result in the initiation of subsequent events, including aggregation, and secretion. Thus, injury-induced exposure of the subendothelial tissues, enables platelets to adhere and then promote the formation of a plug to prevent further hemorrhage. At the same time, attachment of platelets to the exposed subendothelial matrix will trigger a series of intraplatelet signaling and activation cascades, including release of ADP from the granules, which will subsequently interact with its GPCRs on the platelets surface, specifically, P2Y1 -R and P2Y12- R. ADP will initiate the conformational changes of the GP Ilb-IIIa to facilitate fibrinogen binding and, thus, aggregation of the platelets. Despite the significant role of platelet aggregation in maintaining hemostasis of blood vessels, their unregulated and / or hyperactivation is one of the causes of arterial thrombosis.TAMU. P0007WO / 1001354698 - 38 -

[0136] With regard to the ADP GPCRs, signaling downstream of P2Y12-R has been found to play a more prominent role in platelet activation, than that of the P2Y1-R; however, that appears to come at the expense of a significant underlying bleeding phenotype upon pharmacological targeting / antagonizing the P2Y12-R. Consequently, in particular embodiments antagonism of the P2Y1-R leads to a less potent and safer alternative. Thus far, because of the limitations of the designed P2Y1-R antagonists none are yet to receive approval by the FDA. This is attributed to the fact / drawback that their design is based on the structure of ADP and / or ATP, neglecting the ligand binding domain on the P2Y1-R. To this end, given research that the P2Y1-R ligand binding domain for ADP is located on the EL2 region of the receptor protein, the inventors developed and showed that an antibody against this region (EL2Ab; passive immunization) is able to selectively inhibit ADP -induced platelet function, and protect from thrombogenesis, without adverse bleeding. In light of limitations of antibody-based therapies, a peptide-based vaccine modeled over EL2 was sought. This vaccine would be expected to initiate an immune response (active immunization) in the host and produce the “EL2Ab”, all while maintaining the same pharmacological activity and safety profde. Indeed, ELISA-based antibody quantification revealed robust levels of the EL2Ab in mice injected with the EL2Vac, whereas it was not detectable in the control mice (EL2rVac or VLP). This indicates that the EL2 peptide-based vaccine elicits an immune response, unlike the random peptide one, or VLP.

[0137] The next set of studies investigated the pharmacological activity of EL2Vac and its safety in the context of bleeding. The results showed selective inhibition of ADP-induced platelet aggregation by the EL2Vac, whereas the EL2rVac had no effects (FIG. 6). This indicates that the vaccine-generated EL2Ab interacts with (the ADP binding site) and blocks the P2Y1-R. Furthermore, based on the connection between the P2Y1-R and thrombotic disorders, the EL2Vac in specific aspects exhibits antithrombotic activity. Indeed, when the EL2Vac mice were subjected to a widely used thrombosis model, the time of occlusion was significantly delayed in contrast to those vaccinated with EL2rVac (FIG. 5). These data provide further evidence of the integral role the P2Y1-R has in the genesis of thrombosis, and that a vaccine based on its ligand binding domain could exhibit thrombo-protective effects. Interestingly, and with regard to its safety, the EL2Vac had no apparent effects on the bleeding time, which indicates that normal hemostasis can be maintained and is consistent with the notion that P2Y1-R may present a “safer” therapeutic target. It is important to note that findings with the EL2Vac appear to be “similar” to those observed withTAMU. P0007WO / 1001354698 - 39 -the TPR vaccine, as well as those with the custom P2Y1-R antibody (EL2Ab), especially in terms of lack of detectable effects on bleeding. Additionally, thrombocytopenia, which had been associated with some of the antibody / vaccine-based therapeutic approaches, was also evaluated in the present study. There were no changes in the platelets counts as a result of the EL2Vac, which might be expected as similar results were found with cocaine and angiotension I (Ang I) vaccines, which further supports the safety of the EL2Vac. In specific aspects, these findings indicate that the EL2Vac is devoid of thrombocytopenia.

[0138] Additional characterization of the antiplatelet activity of EL2Vac, namely its effects on alpha granule secretion, revealed that it significantly and selectively reduced p-selectin expression in response to activation by ADP / P2Y1-R but not TPR. These findings indicate that-as expected- the EL2Ab generated due to the EL2Vac selectively binds to the P2Y1-R, and not to TPR, thereby inhibiting ADP -induced platelet activation.EXAMPLE 7EXAMPLES OF MATERIALS AND METHODS

[0139] Provided are examples of materials and methods for Examples 1-6.

[0140] Materials and Animals

[0141] Disposables related to platelet aggregation were purchased from Chrono-Log (Havertown, PA). The thromboxane A2 receptor agonist U46619 was obtained from Cayman Chemical (Ann Arbor, MI). ADP, VLP, as well as complete and incomplete Freund’s adjuvants were purchased from Sigma Aldrich (St Louis, MO). The anti-P-selectin antibody was obtained from Cell Signaling Technology, Inc (Danvers, MA). The BD FACSTM lysing solution was purchased from BD Biosciences (Franklin Lakes, NJ).

[0142] C57BL / 6J mice, mixed gender aged 8-10 weeks, were purchased from Jackson Laboratories (Bar Harbor, ME) and kept under normal conditions (temperature of 24°C, 12 light cycles) while divided into groups of 1-4 mice at each group with ad libitum access to water and food. Any experiment performed on the animals was approved prior to their use by the Institutional Animal Care and Use Committee.

[0143] Methods

[0144] EL2 and EL2Ab Peptide Based Vaccination ProtocolTAMU. P0007WO / 1001354698 - 40 -

[0145] Mice were vaccinated using a peptide linked to VLP, corresponding to the human EL2 sequence (CGGTGVRKNKTITCYDTTSDEYLRSYF; SEQ ID NO:6; T192-F215) of the purinergic P2Y1-R. Control mice were immunized with a random version of EL2 (CGGLYKDGTICTYVFYNKRRSDTETST; SEQ ID NO:4). In each case, an additional sequence of amino acids, specifically CGG was synthesized to allow directional conjugation to VLP. Of note, VLP alone was also utilized as a control in the initial experiments to evaluate the immune response triggered by the vaccinations. The vaccines (20 pg) and VLP dissolved in Freund's complete adjuvant were injected into the mice intraperitoneally. The mice received three additional boosts of peptides (using Freund's incomplete adjuvant) on days 14, 28, and 42. Unless stated otherwise, the described analyses were conducted two weeks after completing the immunization regimen (day 56).

[0146] Quantification of Antibody Production in the Vaccinated Mice

[0147] ELISAs were conducted to assess antibody production in the vaccinated mice, namely EL2Vac, EL2rVac, and VLP. Nunc-Immuno MicroWell 96-well plates were coated with cognate EL2 peptide at 12.5 pg per well and incubated at room temperature for 18-24 hours. Next, the plates were washed (3X) with modified Tyrode's buffer, before being treated with 5% BSA in modified Tyrode's buffer for 1 hour to prevent nonspecific binding. The plates were washed for another time (3X) with modified Tyrode's buffer, and the EL2 peptide was added to the wells in triplicate using serial dilutions. The antibodies presumably generated by the vaccination (EL2Ab) were incubated for 1 hour, washed, and those attached to the immobilized peptide were detected by incubating the wells with goat anti-mouse IgG (heavy and light chains) labeled with horseradish peroxidase for 1 hour. The wells were washed one final time before introducing the horseradish peroxidase substrate solution. Following a 10-minute incubation in darkness, the reaction was stopped using 2 N H2SO4, and the antibody’s presence was evaluated by obtaining the absorbance at 490 nm, as previously described.

[0148] Effect of the Peptide Based Vaccines on Platelet Aggregation

[0149] Blood was obtained from mice that had received the EL2Vac and EL2rVac vaccines.Platelets were obtained through centrifugation. Platelet aggregation was carried out using a Model 700, 2 channel whole blood / optical Lumi-Aggregometry System (Chronolog), at 37 °C with stirring (1000 rpm). The platelet counts were adjusted following Manolopoulos P. et al. and Gearing K. et al. protocols, before being activated using 10 pM ADP, or 1 pM U46619, and theirTAMU. P0007WO / 1001354698 - 41 -aggregation response measured. Each experiment was replicated a minimum of 3 times, and the blood used for these experiments was pooled from no less than three distinct sets of 6 to 8 vaccinated mice groups.

[0150] Effect of the Peptide Based Vaccines on Surface Expression of P-Sel ectin

[0151] Flow cytometry experiments were executed on platelets obtained from vaccinated mice (EL2Vac and EL2rVac), as per our previously established protocol. In summary, platelets (at a concentration of 2.0 x 108) were activated with 10 pM ADP or 1 pM U46619, for 3 minutes, and the reactions stopped by fixing the platelets with 2% formaldehyde for 30 minutes at 20-25 °C. Subsequently, the platelets were incubated with anti-P-selectin antibody at room temperature in darkness for 30 minutes. Finally, platelets were diluted 2.5-fold using HEPES / Tyrode's buffer (pH 7.4), samples transferred to fluorescence-activated cell sorting tubes, and fluorescence intensities were assessed using a flow cytometer (BD Accuri C6). The collected data were subjected to analysis using the CFlow Plus software.

[0152] Effect of the Peptide Based Vaccines on FeC13-induced Thrombosis

[0153] The flow measurements of the carotid artery FeC13-induced thrombosis in the vaccinated mice (EL2Vac and EL2rVac) were performed using a Perivascular Transit-time Flowmeter (Transonic Systems). In brief, mice that had been vaccinated were anesthetized using isoflurane, before the left carotid artery was exposed, and cleaned. The initial blood flow in the carotid artery was measured using a Transonic microflow probe (0.5 mm; Transonic Systems Inc, Ithaca, NY). Once the blood flow stabilized, a filter paper disc (diameter of 1 mm) immersed in 7.5% FeC13 was applied to the artery for 3 minutes, and the blood flow was continually monitored for 45 minutes or until the blood flow reached a stable occlusion state (with no blood flow for 2 mins). Time taken for the vessel to occlude was determined by calculating the time difference between achieving stable occlusion and the removal of the FeC13 filter paper. For statistical analysis purposes, an occlusion time of 45 minutes was set as the threshold.

[0154] Effect of the Peptide Based Vaccines on the Tail Bleeding

[0155] The process of hemostasis in the vaccinated mice (EL2Vac and EL2rVac) was evaluated using the tail transection method, in order to assess bleeding as a potential adverse event. In brief, the mice were anesthetized using isoflurane and positioned on a homeothermic blanket at 37°C. The tails of the vaccinated mice were then transected at a point 5 mm away from the tip, and immediately immersed in 37°C saline solution. The duration until stoppage of blood flow / bleedingTAMU. P0007WO / 1001354698 - 42 -was recorded. If needed, measurements were concluded at the 10-minute mark to prevent excessive blood loss.

[0156] Statistical Analysis

[0157] Every experiment was repeated a minimum of 3 times. Data analysis was conducted using GraphPad PRISM (Version 7.0) statistical software (San Diego, CA). For assessing differences in mean occlusion and bleeding times, the Mann- Whitney test was employed. The flow cytometry and ELISA data were analyzed using student t-test. The data are expressed as the mean ± SEM. Results were considered statistically significant when the P-value was less than 0.05 (using a 2-tailed P value) unless otherwise specified.EXAMPLE 8MANAGING THROMBUS FORMATION WITH EL2-5HTVAC: A SELECTIVE VACCINATION-BASED APPROACH TARGETING THE PLATELET SEROTONIN 5-HT2AR

[0158] Cardiovascular diseases (CVDs) are the leading global cause of death, largely attributable to thrombotic events that can result in conditions such as myocardial infarction and stroke. The serotonin 2A receptor (5-HT2AR) has been identified as a key mediator in platelet aggregation and thrombogenesis, making it a promising target for anti -thrombotic therapies. Current 5-HT2AR antagonists, however, have been limited by non- selectivity and adverse effects. The following Examples provide a novel vaccine designed to target the ligand binding domain of 5-HT2AR, which resides in the second extracellular loop (EL2). This vaccine, referred to as “EL2- 5HTVac” is expected to provide a long-lasting and selective therapeutic approach without the complications of increased bleeding risk. In the following Examples, it is demonstrated that EL2- 5HTVac induces a robust immune response with a significant elevation in EL2-specific antibodies, in comparison with the controls. Furthermore, vaccinated mice exhibited prolonged occlusion times in a FeC13-induced carotid artery thrombosis model, without extending tail bleeding times, indicating a favorable safety profile. The EL2-5HTVac effectively inhibited the serotonin-induced platelet shape change. Additionally, it also blocked serotonin-enhanced ADP-induced platelet aggregation, indicating an ability to prevent serotonin-facilitated amplification of platelet activation. These findings indicate that EL2-5HTVac offers a dual advantage of thrombo-TAMU. P0007WO / 1001354698 - 43 -protection and maintenance of hemostasis, in specific embodiments overcoming limitations of existing anti -thrombotic strategies. One can characterize the long-term efficacy and safety of EL2- 5HTVac, as well as the feasibility of a vaccination approach, in larger animal models for clinical application.

[0159] Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality worldwide, posing challenges for healthcare systems as well as medical research. At the heart of many CVDs is thrombosis, a process that, while essential for hemostasis, can lead to acute pathological conditions such as myocardial infarction and stroke when dysregulated. Despite decades of progress and advancements in drug discovery, all current therapies are associated with bleeding adverse events. Thus, the search for therapeutic agents that can modulate thrombotic activity without increasing bleeding risk has long been and remains a priority in cardiovascular medicine. In this context, the serotonin 2A receptor (5-HT2AR), which plays a pivotal role in platelet aggregation and thrombogenesis, is considered a promising target for novel antithrombotic drugs as well as other medical conditions. Serotonin (5HT) itself, though a weak inducer of platelet activation, significantly amplifies aggregation initiated by other agonists, thus enhancing thrombus formation. Its receptor- the 5-hydroxytryptamine 2A receptor (5-HT2AR) which specifically couples to the Gq protein- is implicated in the secondary amplification of platelet aggregation within the growing thrombus; rather than in the primary formation and stabilization of a hemostatic plug, delineating it from other thrombotic mediators like thromboxane A2 (TXA2). It is noteworthy that there is clinical and experimental evidence suggesting that targeting 5-HT2AR can dissociate anti-thrombotic effects from undesirable prolongation of bleeding time, further highlighting its clinical potential. Nonetheless, and despite this knowledge, the development of effective 5-HT2AR antagonists (Table 3) has been hampered by issues such as significant toxicity / adverse side effects, as well as lack of receptor selectivity. This is evidenced, at least in part, by the limited clinical utility of compounds like ketanserin and sarpogrel ate, and the experimental antagonist AR246686. A pivotal limitation in the development of 5-HT2AR- targeted therapies has been the empirical nature of antagonist design, which underscores a critical gap in understanding the receptor's ligand-binding domains. To this end, research efforts were undertaken in order to elucidate these domains, and particularly identified the second extracellular loop (EL2) of 5-HT2AR as a key region for ligand binding and receptor activation. ThisTAMU. P0007WO / 1001354698 - 44 -extracellular segment was found to contain amino acid coordination sites, namely S219, L229, A230, and N233, which are either shared or unshared between a number of ligands. Additionally, an antibody raised against this sequence inhibits both ligand binding and platelet aggregation, making it the first functional antibody against the platelet 5-HT2AR. Nevertheless, the contributions of the EL2 site to in vivo 5-HT2AR-dependent platelet activation (e.g., hemostasis / bleeding time) and the genesis of thrombosis remain to be defined. Building on these findings, this disclosure provides a novel approach to 5-HT2AR modulation through the development of a vaccine targeting the EL2 domain. This strategy represents a significant departure from conventional antagonist-based therapies, providing a targeted, long-lasting, and compliance-friendly solution to modulate thrombotic activity, all without adverse bleeding activity. Taken together, by focusing exclusively on platelet 5-HT2ARS, this approach aims to selectively modulate thrombosis risk in individuals with CVD, thereby addressing a critical unmet therapy need, and setting the stage for a new frontier in anti -thrombotic drugs.

[0160] Table 3. 5 -HT2 A Receptor AntagonistsN. Name of the Structural Class Property DescriptionAntagonist1 Pirenperone Piperidines 5-HT2A, Pirenperone is a5-HT2C research compound known for its action as a serotonin receptor antagonist.2 Ketanserin Phthalimides 5-HT2A Ketanserin is commonly used for its antihypertensive properties byTAMU. P0007WO / 1001354698 - 45 -blocking 5-HT2A receptors.3 Ritanserin Azathiophenes 5-HT2A, Ritanserin is5-HT2B, studied for its5-HT2C potential in treating schizophrenia and anxiety by targeting serotonin receptors.4 Mianserin Azapines 5-HT2A, Mianserin is an5-HT2C antidepressant that acts as a 5- HT2A and 5- HT2C antagonist.5 Methysergide Ergolines 5-HT1, Methysergide is5-HT2A, used to prevent5-HT2C, migraine5-HT5, headaches,5-HT6, acting on5-HT7 multiple serotonin receptors.6 LY 314228, Arylaminoguanidines 5-HT2A LY compoundsLY 320954 are investigational drugs studied for their 5-TAMU. P0007WO / 1001354698 - 46 -HT2A receptor antagonism.7 ICI-169369 Phenylquinolines 5-HT2 ICI-169369 is a compound known for its selective antagonism towards serotonin receptors.8 MDL-100907 Piperidines 5-HT2A MDL-100907 is a highly selective 5- HT2A receptor antagonist, explored for treating psychiatric disorders.9 AT-1015 Piperidines 5-HT2A AT-1015 is another selective antagonist of the 5-HT2A receptor, with potential clinical applications.10 R-102444 Pyrrolidines 5-HT2A R- 102444 represents a pyrrolidineTAMU. P0007WO / 1001354698 - 47 -class compound with action as a 5-HT2A receptor antagonist.> 11 DV-7028 Pyridotriazines 5-HT2A DV-7028 is a pyridotri azine derivative investigated for its antagonistic properties on serotonin receptors.12 MCI-9042 (o-amino- 5-HT2A Sarpogrelate is(Sarpogrelate) alkoxy )phenyl- used clinically, ethyl ethylbenzene especially inAsia, to treat peripheral arterial disease by blocking 5- HT2A receptors.

[0161] ICI-169369: 2-(2-Dimethylaminoethylthio)-3-phenylquinoline, MDL-100907: R-(+)- a-(2, 3 -dimethoxyphenyl) 1 - [2-(4-fluorophenyl ethyl)] -4-piperidine-methanol , AT -1015: N- [2- [4- (5H-dibenzo(a,d)cycloheptene-5-ylidene)piperidino]ethyl]-l -formyl -4-piperidinecarboxamide monohydrate, R-102444: (2R,4R)-4-lauroyloxy-2-[2-[2-[2-(3- methoxy)phenyl]ethyl]phenoxy]ethyl-l-methyl pyrrolidine hydrochloride, DV-7028: 3-[2-[4-(4- fhiorobenzoyl) piperidin- 1 -yl] ethyl]-6,7,8,9-tetrahydro-2H-pyrido[l,2-a]-l,3,5-triazine 2,4(3H)- dione maleate, MCI-9042: (R,S)-l-[2-[2-(3-methoxyphenyl)ethyl] phenoxy]-3-(dimethylamino)- 2-propyl hydrogen succinate chloride.TAMU. P0007WO / 1001354698 - 48 -EXAMPLE 9EXAMPLES OF METHODS AND MATERIALS

[0162] Provided are examples of methods and materials for Examples 8 and 10-13.

[0163] Materials and Animals

[0164] Materials for platelet aggregation studies were sourced from Chrono-Log corporation (Havertown, PA). ADP and virus-like particles (VLP), along with Freund’s adjuvants, were obtained from Sigma Aldrich (St. Louis, MO). Cell Signaling Technology, Inc (Danvers, MA) supplied the anti-P-selectin antibody. BD Biosciences (Franklin Lakes, NJ) provided the BD FACS™ lysing solution. The C57BL / 6J mice (mixed gender, 8-10 weeks old) were acquired from Jackson Laboratories (Bar Harbor, ME) and maintained in standard conditions (24°C, 12-hour light / dark cycles), housed in groups of 1-4, with unrestricted access to food and water. All animal experiments were pre-approved by the Institutional Animal Care and Use Committee.

[0165] Vaccination Protocol Using EL2 and EL2Ab Peptides

[0166] Mice were immunized (vaccinated) with a VLP-conjugated peptide corresponding to the EL2 sequence of the human 5-HT2AR (CGG QDDSKVFKEGSCLLADDN (SEQ ID NO:5) [Q216-N233]) (see FIG. 7), with additional [CGG] amino acids synthesized to permit directional conjugation to VLP. The control mice received a scrambled version of EL2 (CGG GESCVKFDANQLDDKLSD; SEQ ID NO:7) for comparison, again with the additional [CGG] amino acids. Each vaccine, containing 30 pg of peptide and VLP in Freund's complete adjuvant, was administered intraperitoneally. Subsequent boosts were given on days 14, 28, and 42 with the peptides in Freund's incomplete adjuvant. Immunization efficacy was evaluated two weeks postfinal vaccination (day 56).

[0167] Antibody Quantification in Vaccinated Mice

[0168] ELISA assays were performed to evaluate the “production” of antibodies in mice vaccinated with EL2-5HTVac, EL2rVac, and VLP. For this purpose, Nunc-Immuno MicroWell 96-well plates were coated with the EL2 sequence peptide, with each well receiving 12.5 pg, and left to incubate at room temperature for 18-24 hours. Following this incubation, the plates were subjected to three washes with modified Tyrode's buffer. To block nonspecific binding sites, 5% BSA in modified Tyrode's buffer was applied to the plates for 1 hour. After another series of three washes with the buffer, serial dilutions of the EL2 peptide were added to the wells in triplicate.TAMU. P0007WO / 1001354698 - 49 -The EL2 -recognizing antibodies (EL2Ab), which are anticipated to be produced as a result of the vaccination process, were then added and allowed to incubate for 1 hour. After washing away unbound antibodies, the plates were incubated with goat anti-mouse IgG conjugated to horseradish peroxidase for another hour. A final wash was conducted before the addition of the horseradish peroxidase substrate solution. The substrate was incubated in the dark for 10 minutes, after which the reaction was halted with 2N H2SO4. The detection of antibodies was accomplished by measuring the absorbance at 490 nm, following previously established methods.

[0169] Investigating Thrombus Formation via the FeC13-Induced Carotid Artery Occlusion

[0170] Blood flow assessments for FeCE-induced carotid artery thrombosis in mice immunized with EL2-5HTVac and EL2rVac were carried out with a Perivascular Flowmeter (Transonic Systems). Initially, vaccinated mice were anesthetized with isoflurane, then the left carotid artery was surgically exposed and cleaned. Baseline blood flow measurements were taken using a 0.5 mm Transonic microflow probe (Transonic Systems Inc, Ithaca, NY). Following stabilization of blood flow, a 1 mm diameter filter paper disc soaked in 7.5% FeCE was placed on the artery for 3 minutes. Blood flow was continuously monitored over the next 45 minutes or until a stable occlusion was observed (indicated by the absence of blood flow for 2 minutes). The occlusion time was calculated based on the interval between the application of the FeCE disc and the moment stable occlusion was achieved. For the purposes of statistical analysis, the maximum occlusion time considered was 45 minutes.

[0171] Tail Bleeding to Evaluate Hemostasis

[0172] The tail transection method used to assess hemostasis in vaccinated mice, and the time to bleeding cessation was recorded for each mouse. Measurements were capped at 10 minutes to limit blood loss.

[0173] Assessing the Vaccines ’ Impact on Platelet Aggregation

[0174] Blood samples were collected from mice vaccinated with either EL2-5HTVac or EL2rVac. These samples were then centrifuged to isolate platelets and count was adjusted as described before. Aggregation of platelets was assessed using a Model 700, two-channel whole blood / optical Lumi-Aggregometry System (Chrono-log), operating at a temperature of 37 °C and a stirring speed of 1200 rpm. Activation of the platelets was achieved using 1 pM ADP, 15 pM serotonin, or a combination of 1 pM ADP and 15 pM serotonin before their aggregation responsesTAMU. P0007WO / 1001354698 - 50 -were recorded. To ensure reliability, each test was conducted at least three times, utilizing blood pooled from at least three separate groups of 6 to 8 mice that had been vaccinated.

[0175] Assessing the effect of the Peptide-Based Vaccines on Surface Expression ofP-Selectin

[0176] Flow cytometry experiments were executed on platelets obtained from vaccinated

[0177] mice (EL2Vac and EL2rVac), as per our previously established protocol

[0031] . In summary, platelets (at a concentration of 2.0 x 108) were activated with 10 pM ADP or 1 pMU46619 for 3 min, and the reactions were stopped by fixing the platelets with 2% formaldehyde for 30 min at 20-25 ° C. Subsequently, the platelets were incubated with anti-P-selectin antibody at room temperature in the dark for 30 min. Finally, platelets were diluted 2.5-fold using HEPES / Tyrode’s buffer (pH 7.4), samples transferred to fluorescence-activated cell sorting tubes, and fluorescence intensities were assessed using a flow cytometer (BD Accuri C6, BD Biosciences, San Jose, CA, USA). The collected data were subjected to analysis using the CFlow Plus software (Version 1.0.34.1).

[0178] Assessing the effect of the Peptide-Based Vaccines on FeCh-Induced Thrombosis

[0179] The flow measurements of the carotid artery FeCE-induced thrombosis in the vaccinated mice (EL2Vac and EL2rVac) were performed using a Perivascular Transit-time Flowmeter (Transonic Systems, Ithaca, NY, USA). In brief, mice that had been vaccinated were anesthetized using isoflurane before the left carotid artery was exposed and cleaned. The initial blood flow in the carotid artery was measured using a Transonic microflowprobe (0.5 mm; Transonic Systems Inc., Ithaca, NY, USA). Once the blood flow stabilized, a fdter paper disc (diameter of 1 mm) immersed in 7.5% FeCk was applied to the artery for 3 min, and the blood flow was continually monitored for 45 min or until the blood flow reached a stable occlusion state (with no blood flow for 2 min). The time taken for the vessel to occlude was determined by calculating the time difference between achieving stable occlusion and removing the FeCh fdter paper. For statistical analysis, an occlusion time of 45 min was set as the threshold

[0057] ,

[0180] Assessing the effect of the Peptide-Based Vaccines on the Tail Bleeding

[0181] The process of hemostasis in the vaccinated mice (EL2Vac and EL2rVac) was evaluated using the tail transection method, in order to assess bleeding as a potential adverse event. In brief

[0058] , the mice were anesthetized using isoflurane and positioned on a homeothermic blanket at 37 ° C. The tails of the vaccinated mice were then transected at a point 5 mm away from the tip, and immediately immersed in 37 C saline solution. The duration until stoppage of bloodTAMU. P0007WO / 1001354698 - 51 -flow / bleeding was recorded. Tf needed, measurements were concluded at the 10 min mark to prevent excessive blood loss.

[0182] Effect of the Peptide-Based Vaccine on cAMP Levels.

[0183] The cAMP assay was conducted as we described before

[0031] , Mouse PRP (500 pL) samples were collected from the EL2rVac and EL2Vac mice. Next, platelets are treated with0.5 pM forskolin before the addition of 10 pM ADP, and incubated at room temperature for 1 min. Next, the phosphodiesterase inhibitor RO20-1724 (100 pM) was added, and platelets were spun down, and the pellet was snap frozen in liquid nitrogen and stored at - 70 ° C. Upon use, the pellet is resuspended in sodium acetate buffer (50 mM; pH 4.0), sonicated, boiled for 4 min, centrifuged, and the supernatant transferred to a new tube. The concentration of cAMP in the supernatant is measured according to standard protocols

[0059] , whereas the standard curve samples were prepared by adding known concentrations of cAMP to the supernatant from the EL2rVac platelets.

[0184] Statistical Analysis

[0185] Analyses were performed using GraphPad PRISM software, employing Mann- Whitney tests for occlusion and bleeding times, and t-tests for ELISA and flow cytometry data. Significance was determined at P < 0.05, with data presented as mean ± SEM.EXAMPLE 10ASSESSMENT OF ANTIBODY PRODUCTION FOLLOWING VACCINATION

[0186] To evaluate the effectiveness of the EL2-5HTVac vaccination in eliciting an immune response, serum samples were obtained from mice immunized with the EL2 peptide (EL2- 5HTVac), the scrambled / random EL2 peptide (EL2rVac), and from those receiving the VLP vaccine control. The presence and quantity of antibodies generated were measured using an ELISA that employed the EL2 specific peptide. The findings revealed that vaccination with EL2-5HTVac (30 pg of the EL2 peptide) led to a significant increase in antibody levels, in contrast to the responses observed with EL2rVac and VLP controls (FIG. 8). This outcome indicates that the EL2-5HTVac vaccination successfully induced the production of EL2-targeting antibodies (EL2Ab), which are likely to bind to the EL2 region of the 5HTA2R protein and produce a biological response.TAMU. P0007WO / 1001354698 - 52 -EXAMPLE 11EL2-5HTVAC EXTENDS OCCLUSION TIME WITHOUT IMPACTING TAIL BLEEDING DURATION

[0187] Subsequent studies focused on thrombogenesis were carried out utilizing the FeCE- induced thrombosis model to assess the impact of the vaccines. The results shown in FIG. 9A demonstrated that EL2-5HTVac significantly extended the occlusion time in the carotid artery, unlike EL2rVac, indicating the vaccine's efficacy in inhibiting thrombus development. This evidence highlights the therapeutic potential of the EL2-5HTVac in the treatment of thrombotic conditions. Next, to explore the vaccine's effect on hemostasis, a tail bleeding time assay was conducted. Remarkably, EL2-5HTVac showed no significant alterations in bleeding time, similar to the EL2rVac control (FIG. 9B). These outcomes indicate that EL2-5HTVac offers thromboprotective benefits without affecting hemostatic processes or increasing bleeding risk.EXAMPLE 12THE EL2-5HTVAC INHIBITS SEROTONIN-INDUCED SHAPE CHANGE, AND SEROTONIN-ENHANCED ADP-INDUCED PLATELET AGGREGATION

[0188] Previous work had demonstrated the ability of a custom-made antibody against the EL2 domain of 5HTA2-R (EL2Ab; passive immunization) to block serotonin-enhanced ADP-induced platelet aggregation. In the present disclosure, it was assessed whether the EL2-5HTVac (active immunization) would exhibit a similar activity. Indeed, EL2-5HTVac was found to exert significant inhibitory effects on platelet aggregation induced by 1 pM ADP in the presence of 15 pM serotonin (FIG. 10A), unlike the VLP that was without any effects. This data indicates that the EL2-5HTVac is selective for the 5HTA2R. Moreover, and consistent with this notion, immunizing mice with the EL2-5HTVac did result in significant blockade of the platelet shape change induced by 15 pM serotonin (FIG. 10B), which is the response typically seen with serotonin.EXAMPLE 13EL2VAC DOES NOT INHIBIT THE P2Y12-RTAMU. P0007WO / 1001354698 - 53 -

[0189] To address whether EL2Vac interacts / interferes with P2Y12-R mediated platelet activation, the capacity of EL2Vac to inhibit ADP-mediated reduction in cAMP in the presence of forskolin was assessed. The results showed that neither EL2rVac nor EL2Vac exerted any effects on the ability of ADP to lower the cAMP levels that are elevated using forskolin (FIG. 12).EXAMPLE 14COGNATE EL2 PEPTIDE REVERSES THE ANTITHROMBOTIC EFFECTS OF EL2VAC AND EL2-5HTVAC

[0190] As described in Examples 5 and 11, EL2Vac and EL2-5HTVac significantly prolonged the time needed for occlusion of the carotid artery (FIG. 5 and FIG. 9A) in the FeCE-induced thrombosis model. Subsequent studies showed that cognate EL2 peptides can reverse the antithrombotic effects of the vaccines to occlusion times similar to EL2rVac controls (FIGS. BABB.EXAMPLE 15SIGNIFICANCE OF CERTAIN EMBODIMENTS OF EXAMPLES 8-14

[0191] To address the substantial morbidity and mortality associated with thrombotic events, as highlighted by the National Blood Clot Alliance with an estimated 274 deaths daily from blood clots, the development of safer and more targeted therapeutic interventions remains a critical goal. The 5HT2A platelet signaling pathway's pivotal role in the pathogenesis of thrombosis and other cardiovascular disorders underscores the need for innovative treatments that circumvent the limitations of current antiplatelet and anticoagulant therapies, which, while effective, often present a trade-off between efficacy and safety due to serious bleeding risks. The empirical design of previous 5HT2AR antagonists has been a significant barrier, leading to a lack of selectivity and undesirable side effects.

[0192] The development of vaccines targeting G protein-coupled receptors (GPCRs) and their ligands represents a novel immunological-derived therapeutic strategy to neutralize the actions of endogenous agonists in disease modulation, particularly in cancer therapy. Notably, vaccines have been developed and tested against GnRH and human Chorionic Gonadotropin (CG) hormones for prostate cancer, whereas the G17DT, an anti -gastrin vaccine, aimed to treat gastricTAMU. P0007WO / 1001354698 - 54 -adenocarcinoma. These efforts underline the significant potential of vaccine-based immunotherapies in targeting GPCRs for cancer treatment, with ongoing research to attempt to expand their application and efficacy in clinical settings. In the present disclosure, the inventors employed this vaccine-centered approach in an attempt to manage thrombus formation.

[0193] The results from the current study underscore the potential of 5HT2AR-based vaccine that is developed against its ligand binding pocket, namely the EL2 domain (referred to as EL2- 5HTVac). In some aspects, the EL2-5HTVac would induce a targeted immune response against the 5-HT2AR, which may have important implications for the management of thrombotic diseases. The significant elevation of antibodies specific to the EL2 sequence of the 5-HT2AR, as compared to the scrambled sequence and VLP control, is consistent with previous studies demonstrating the effectiveness of peptide-based vaccines in generating a specific immune response. The ability to induce such selective antibodies could be instrumental in developing more precise therapeutic interventions that minimize off-target effects, while also improving patient outcomes. The prolonged occlusion time observed in mice vaccinated with EL2-5HTVac without affecting the tail bleeding time provides an encouraging indication of the usefulness of targeting EL2 domain. This dissociation between antithrombotic efficacy and hemostatic safety profiles parallels the therapeutic window seen with certain anticoagulants or antiplatelets (e.g., reversible PARI antagonists and PAR4 inhibitors) that are foreseen as less risky for bleeding complication. However, unlike traditional anticoagulants and antithrombotic, the vaccine approach would also potentially offer a more compliance-prone treatment option for thrombotic-based cardiovascular diseases.

[0194] The inhibitory effect of EL2-5HTVac on ADP-induced platelet aggregation in the presence of serotonin reinforces the notion that EL2-specific antibodies can interfere with the 5- HT2AR function. This finding corroborates the mechanism by which the inhibition of serotonin- facilitated amplification of ADP-induced platelet aggregation via targeting 5-HT2AR. Indeed, the ability of the EL2-5HTVac to block serotonin-induced platelet shape change indicates specificity for 5-HT2AR, and suggests that the antibodies might be preventing the conformational changes necessary for platelet activation and aggregation, in particular embodiments.

[0195] Collectively, the results provide a strong rationale for the continued development of EL2-5HTVac as a novel therapeutic approach for thrombosis. The inventors have recently shown that a similar but passive immunization-based approach utilizing an antibody (EL2Ab) for 5-TAMU. P0007WO / 1001354698 - 55 -HT2ARs was effective as thrombo-protective. These approaches do have an advantage over classical antagonists, because of the precision and selectivity of using antibodies. Nevertheless, a vaccination approach would be more durable, as antibody treatments often require ongoing administration, while vaccines typically aim for long-term immunity after a limited number of doses. In this connection, the inventors have previously shown that a vaccine targeting the platelet thromboxane A2 receptor (TPR) is indeed a promising approach, which exhibited selective TPR- dependent antiplatelet effect and did not interfere with hemostasis. Furthermore, the TPR-based vaccine was also shown to inhibit some of the inflammatory aspects of thrombosis, in part by inhibiting platelet - leukocyte aggregates.

[0196] In conclusion, the EL2-5HTVac represents a promising new therapeutic candidate in the field of thrombosis research and related disorders, with the potential to overcome some of the limitations associated with current antithrombotic therapies.* * *

[0197] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.TAMU. P0007WO / 1001354698 - 56 -

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A composition, comprising a) an isolated peptide comprising SEQ ID NO: 1; b) an isolated peptide comprising sequence that is at least 85% identical to SEQ ID NO: 1 ; c) an isolated peptide comprising SEQ ID NO:2; d) an isolated peptide comprising sequence that is at least 85% identical to SEQ ID NO:2; or e) any combination thereof.

2. The composition of claim 1(b), wherein the peptide sequence is at least 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 1; and / or, wherein in 1(c) the peptide sequence is at least 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO:2.

3. The composition of claim 1(a) or 1(b), wherein the peptide is at least, exactly, or no more than 27 amino acids in length.

4. The composition of claim 1(c) or 1(d), wherein the peptide is at least, exactly, or no more than 18 amino acids in length.

5. The composition of any one of claims 1-4, wherein the peptide of claim lb) has 1, 2, 3, or more amino acid substitutions compared to SEQ ID NO: 1 and / or wherein the peptide of claim 1(c) has 1, 2, 3, or more amino acid substitutions compared to SEQ ID NO:2.

6. The composition of any one of claims 1-5, wherein the peptide of claim Id) has 1, 2, 3, or more amino acid substitutions compared to SEQ ID NO:2.

7. The composition of any one of claims 1-6, wherein(a) the peptide comprises an N-terminal extension of 1, 2, 3, or more amino acids compared to SEQ ID NO: 1;(b) the peptide comprises an N-terminal extension of 1, 2, 3, or more amino acids compared to SEQ ID NO:2; orTAMU. P0007WO / 1001354698 - 57 -(C) both.

8. The composition of any one of claims 1-7, wherein(a) the peptide comprises a C-terminal extension of 1, 2, 3, or more amino acids compared to SEQ ID NO: 1;(b) the peptide comprises a C-terminal extension of 1, 2, 3, or more amino acids compared to SEQ ID NO:2; or(c) both.

9. The composition of any one of claims 1-8, wherein the composition is comprised in a pharmaceutically acceptable carrier.

10. The composition of any one of claims 1-9, wherein the peptide is linked to or comprised within an agent.

11. The composition of claim 10, wherein the agent comprises a viral particle, another peptide, a bead, a scaffold, a therapeutic agent, or a combination thereof.

12. The composition of any one of claims 1-11, further comprising one or more adjuvants, one or more stabilizers, one or more preservatives, one or more antibiotics, or a combination thereof.

13. An immunogenic composition, comprising the composition of any one of claims 1-12.

14. A vaccine composition, comprising the composition of any one of claims 1-12.

15. A method of eliciting an immune response in an individual, comprising administering to the individual an effective amount of the composition of any one of claims 1-14.

16. The method of claim 15, wherein the individual has cardiac disease, is at risk for cardiac disease, has thrombosis, or is at risk for thrombosis.

17. The method of claim 15 or 16, wherein there is a single administration of the composition.TAMU. P0007WO / 1001354698 - 58 -18. The method of claim 15 or 16, wherein there are multiple administrations of the composition.

19. The method of claim 18, wherein the duration of time between administrations is about 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more years.

20. The method of any one of claims 15-19, wherein the individual is:(a) pregnant;(b) at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years old;(c) bedridden;(d) immobile because of injury, because of surgery or hospitalization, and / or because of travel;(e) has a family history of cardiac disease or thromboembolism;(f) has a blood clotting disorder, cancer, heart condition, diabetes, high blood pressure, kidney disease, chronic inflammation, arthritis, irritable bowel syndrome, or a combination thereof;(g) has estrogen-containing contraception or hormone replacement therapy;(h) is a smoker; and / or(i) is a paraplegic or quadriplegic.

21. A method of reducing thrombosis, or the risk of thrombosis, in an individual, comprising administering to the individual an effective amount of the composition of any one of claims 1-14.

22. The method of claim 21, wherein the individual has cardiac disease or is at risk for cardiac disease.

23. The method of claim 21 or 22, wherein there are multiple administrations of the composition of any one of claims 1-14.

24. The method of claim 23, wherein the duration of time between administrations is about 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more years.

25. The method of any one of claims 21-24, wherein the individual is:TAMU. P0007WO / 1001354698 - 59 -(a) pregnant;(b) at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years old;(c) bedridden;(d) immobile because of injury, because of surgery or hospitalization, and / or because of travel;(e) has a family history of cardiac disease or thromboembolism;(f) has a blood clotting disorder, cancer, heart condition, diabetes, high blood pressure, kidney disease, chronic inflammation, arthritis, irritable bowel syndrome, or a combination thereof;(g) has estrogen-containing contraception or hormone replacement therapy;(h) is a smoker; and / or(i) is a paraplegic or quadriplegic.

26. A method of treating or preventing thrombosis or cardiovascular disease in an individual, comprising administering to the individual an effective amount of the composition of any one of claims 1-14.

27. The method of claim 26, wherein the individual has cardiac disease, is at risk for cardiac disease, or is at risk for thrombosis.

28. The method of claim 26 or 27, wherein there are multiple administrations of the composition of any one of claims 1-14.

29. The method of claim 28, wherein the duration of time between administrations is about 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more years.

30. The method of any one of claims 26-29, wherein the individual is:(a) pregnant;(b) at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years old;(c) bedridden;(d) immobile because of injury, because of surgery or hospitalization, and / or because of travel;(e) has a family history of cardiac disease or thromboembolism;(f) has a blood clotting disorder, cancer, heart condition, diabetes, high blood pressure, kidney disease, chronic inflammation, arthritis, irritable bowel syndrome, or a combination thereof;TAMU. P0007WO / 1001354698 - 60 -(g) has estrogen-containing contraception or hormone replacement therapy;(h) is a smoker; and / or(i) is a paraplegic or quadriplegic.

31. A kit, comprising the composition of any one of claims 1-14.TAMU. P0007WO / 1001354698 - 61 -

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