# Patent

## Title
Immunomodulatory methods and systems for treatment and/or prevention of atherosclerosis

## Abstract

Immunostimulatory methods and systems for treating or preventing atherosclerosis and/or a condition associated thereto in an individual.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present disclosure is an application under section 371 of International patent application PCT/US2010/056623 filed Nov. 12, 2010, which claims priority to U.S. provisional application 61/261,331 entitled “Immunomodulatory Methods and Systems for Treatment and/or Prevention of Atherosclerosis” filed on Nov. 14, 2009. Each of these disclosures are herein incorporated by reference in their entirety.


FIELD
The present disclosure relates to immunomodulatory methods and systems that are suitable for treatment or prevention of atherosclerosis and/or conditions associated thereto.
BACKGROUND
Atherosclerosis is currently viewed as a chronic lipid-related and immune-mediated inflammatory disease of the arterial walls. Many immune components have been identified that participate in atherogenesis. Pre-clinical studies have yielded promising results suggesting that immuno-modulatory therapies targeting these components can reduce atherosclerosis.
SUMMARY
Provided herein, are methods and systems for inducing immunomodulatory responses in an individual. In several embodiments, the immunomodulatory responses induced by the methods and systems of the present disclosure are associated to a therapeutic and/or preventive effect related to atherosclerosis in the individual or a condition associated thereto.
According to a first aspect, a CD8(+) T cell activated by an immunogenic fragment of ApoB100 or an immunogenically active portion thereof is described together with related methods and systems. The activated CD8(+) T cell is specific for the immunogenic fragment or the immunogenically active portions thereof.
According to a second aspect, a CD8(+) T cell activated by an immunogenic fragment of ApoB100 or an immunogenically active portion thereof is described for use as a medicament and in particular for use in treatment and/or prevention of atherosclerosis in an individual. The activated CD8(+) T cell is specific for the immunogenic fragment or the immunogenically active portions thereof. The immunogenic fragment or immunogenically active portion thereof is associated to atherosclerosis reduction in an individual.
According to a third aspect, a method and system to treat and/or prevent atherosclerosis in an individual is described. The method comprises administering to the individual an effective amount of a CD8(+) Tcell activated by an immunogenic fragment of ApoB100 or an immunogenically active portion thereof, the immunogenic fragment or immunogenically active portion thereof associated with atherosclerosis reduction, the activated CD8(+)Tcell being specific for the immunogenic fragment or the immunogenically active portion thereof. The system comprises at least two of an activated CD8(+) T cell, one or more suitable adjuvants.
According to a fourth aspect, a method and system to provide activated CD8(+)T cell is described. The method comprises contacting a CD8(+) T cell with an immunogenic fragment of ApoB100 or an immunogenically active portion thereof for a time and under condition to obtain a CD8(+)Tcell specific for the immunogenic fragment or the immunogenically active portions thereof. In some embodiments, wherein the activated CD8(+) T cell is suitable to treat and/or prevent atherosclerosis and/or a condition associated thereto. The immunogenic fragment or immunogenically active portion thereof is associated to atherosclerosis reduction. The system comprises at least two of a CD8(+) T cell, an immunogenic fragment of ApoB100, an immunogenically active portion of said immunogenic fragment, and a T cell enhancer.
According to a fifth aspect, a composition is described comprising at least one activated CD8(+)T cell herein described together with a suitable vehicle.
According to a sixth aspect, a method and system to identify a therapeutic agent for treatment and/or prevention of atherosclerosis in an individual is described. The method comprises contacting a CD 8(+) T cell with an immunogenic fragment of ApoB100 or an immunogenically active portion thereof for a time and under condition to provide an activated CD8(+) T cell, the activated CD8(+)Tcell being specific for the immunogenic fragment or the immunogenically active portion thereof. The method further comprises administering the activated CD8(+) T cell to an animal model and detecting atherosclerosis reduction in the animal model. The system comprises at least two of a CD8(+) T cell, a T cell enhancer, an animal model, reagents to detect T cell activation, and reagents to detect atherosclerosis reduction.
The T cells, compositions, methods and systems herein described can be used in connection with applications wherein reduction of plaque, attenuation of plaque growth and/or a therapeutic or preventive effect for atherosclerosis in an individual is desired.
The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.



BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and examples sections, serve to explain the principles and implementations of the disclosure.
FIG. 1 shows p210 immunization confers athero-protective effect. (A) Immunization with native p210 resulted in a significant reduction in aortic atherosclerosis when compared to PBS and cBSA/Alum group (n=9-10 each group, representative picture from each group shown). (B) P210 immunization significantly reduced macrophage infiltration and DC presence assessed by MOMA-2 (n=9-10 each group) and CD11c (n=7-12 each group) immuno-reactivity, respectively in aortic sinus plaques.
FIG. 2 shows an effect of p210 immunization on DCs. One week after primary immunization, (A) CD11c(+) or (B) CD11c(+)CD86(+) cells at the immunization sites was significantly reduced in p210/cBSA/alum group when compared to cBSA/alum group. N=10 each group. (C) One week after third immunization, p210 immunized mice had reduced CD11c(+)CD86(+) cells in lymph nodes compared to cBSA/alum group (n=5 in each group; ANOVA followed by multiple group comparison).
FIG. 3 shows IgM or IgG titer against p210 before and after p210 immunization. (A) The p210 IgG titers were low before immunization and remained low in the PBS group at euthanasia but significantly increased in cBSA/alum and p210/cBSA/alum groups, with the highest titer in the cBSA/alum group. (B) The p210 IgM titers were low before immunization and significantly increased at euthanasia with no difference among 3 groups of mice. N=5 for 6-7 week time-point and n=9 for 25 week time-point.
FIG. 4 shows activated lymphocyte population after immunization in vivo. (A) CD8(+)CD25(+) T-cell population in the lymph nodes was significantly higher in p210/cBSA/alum group when compared to that of PBS or cBSA/alum groups; (B) CD4(+)CD25(+) T-cells in the lymph nodes did not differ among the three groups. There was a significantly larger population of splenic CD8(+)CD25(+)IL-10(+) T-cells in p210/cBSA/alum group among 3 groups (C) without difference in splenic CD8(+)CD25(+)IL12(+) T-cells among 3 groups (D). Splenic CD4(+)CD25(+)IL-10(+) T-cell population significantly increased in the cBSA/alum group, but was significantly attenuated by the p210/cBSA/alum immunization (E) and (F) splenic CD4(+)CD25(+)IL12(+) T-cells did not differ among 3 groups. N=9-10 in each group for (A) and (B); n=5 in each group for (C), (D), (E) and (F).
FIG. 5 shows adoptive transfer of CD8(+) T-cells from p210 immunized donors recapitulated the athero-protective effect of p210 immunization but not by transfer of B-cells or CD4(+)CD25(+) T-cells. (A) The recipient mice of CD8(+) T-cells from p210/cBSA/alum immunized donors developed significantly smaller atherosclerotic lesions compared to the recipient mice of CD8(+) T-cells from other 2 groups (n=9-10 each group). (B) Adoptive transfer of B-cells from p210/cBSA/alum donors did not reduce atherosclerosis when compared to the recipient mice of B-cells from PBS or cBSA/alum groups (n=9 each group). Recipient mice of CD4(+)CD25(+) T-cells (n=9-13 each group) with 2 different doses (C. 1×105 cells/mouse or D. 3×105 cells/mouse) did not reproduce the athero-reducing effect of p210 immunization.
FIG. 6 shows increased cytolytic activity of CD8(+) T cells from p210 immunized mice against dendritic cells in vitro. CD8(+) T-cells from p210 immunized mice significantly had a higher cytolytic activity against when compared to those from PBS or BSA/alum groups. Experiments were repeated 4 times with CD8(+) T-cells pooled from 5 mice in each group each time. Duplicate or triplicate was done each time with total of 11 data-points in each group altogether.
FIG. 7 shows CD8(+) T-cells from p210 immunized mice containing higher level of Granzyme B when compared to those from PBS or cBSA/alum group; whereas there is no difference in perforin level
FIG. 8 shows IgG titers against KLH or TNP after p210 immunization. (A) Prior immunization with p210 did not affect the efficacy of subsequent T-cell dependent (KLH, n=3-6 each group) or (B) T-cell independent (TNP, n=4-5 each group) immunization as assessed by the IgG antibody titers when compared to mice received PBS or cBSA/alum.



DETAILED DESCRIPTION
T cells and related methods and systems are herein described that are suitable for treatment and/or prevention of atherosclerosis or a condition associated thereto in an individual
The term “T cells” as used herein indicates T lymphocytes belonging to a group of white blood cells known as lymphocytes, and participate in humoral or cell-mediated immunity. T cells can be distinguished from other lymphocyte types, such as B cells and natural killer cells (NK cells) by the presence of special markers on their cell surface such as T cell receptors (TCR). Additional markers identifying T cell include CD1a, CD3, CD4, CD8, and additional markers possibly associated to a T cell state and/or functionality as will be understood by a skilled person.
The term “treating” or “treatment” as used herein indicates any activity that is part of a medical care for, or that deals with, a condition medically or surgically. The term “preventing” or “prevention” as used herein indicates any activity, which reduces the burden of mortality or morbidity from a condition in an individual. This takes place at primary, secondary and tertiary prevention levels, wherein: a) primary prevention avoids the development of a disease; b) secondary prevention activities are aimed at early disease treatment, thereby increasing opportunities for interventions to prevent progression of the disease and emergence of symptoms; and c) tertiary prevention reduces the negative impact of an already established disease by restoring function and reducing disease-related complications.
The term “atherosclerosis” as used herein indicates a condition in which an artery wall thickens as the result of a build-up of fatty materials such as cholesterol. It is a syndrome typically affecting arterial blood vessels, in large part due to the accumulation of oxidized lipids, macrophage, activated lymphocytes, and promoted by low-density lipoproteins (plasma proteins that carry cholesterol and triglycerides). It is commonly referred to as a hardening of the arteries. Atherosclerosis is currently viewed as a chronic lipid-related and immune-mediated inflammatory disease of the arterial walls. Many immune components have been identified that participate in atherogenesis and pre-clinical studies have yielded promising results suggesting that immuno-modulatory therapies targeting these components can reduce atherosclerosis. One of the strategies is to use apoB-100 related peptides as immunogens and several candidate peptides have been identified for such use, see references (1) (2).
The term “condition” as used herein indicates as usually the physical status of the body of an individual (as a whole or of one or more of its parts) that does not conform to a physical status of the individual (as a whole or of one or more of its parts) that is associated with a state of complete physical, mental and possibly social well-being. Conditions herein described include but are not limited to disorders and diseases wherein the term “disorder” indicates a condition of the living individual that is associated to a functional abnormality of the body or of any of its parts, and the term “disease” indicates a condition of the living individual that impairs normal functioning of the body or of any of its parts and is typically manifested by distinguishing signs and symptoms. Exemplary conditions include but are not limited to injuries, disabilities, disorders (including mental and physical disorders), syndromes, infections, deviant behaviours of the individual and atypical variations of structure and functions of the body of an individual or parts thereof.
The wording “associated to” as used herein with reference to two items indicates a relation between the two items such that the occurrence of a first item is accompanied by the occurrence of the second item, which includes but is not limited to a cause-effect relation and sign/symptoms-disease relation.
The term “individuals” as used herein indicates a single biological organism such as higher animals and in particular vertebrates such as mammals and more particularly human beings.
In an embodiment, a CD8(+) Tcell activated by an immunogenic fragment of ApoB100 is described, the activated CD8(+)Tcell specific for the immunogenic fragment.
The term “CD8(+) T cells” indicates T cells expressing the CD8 glycoprotein at their surface, wherein the CD8 (cluster of differentiation 8) glycoprotein is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). Similarly to the TCR, CD8 binds to a major histocompatibility complex (MHC) molecule, but is specific for the class I MHC protein. Exemplary CD8 T cells comprise cytotoxic memory CD8 T cells, regulatory CD8 T cells, cytotoxic effector CD8 T-cells and additional cells identifiable by a skilled person. Two isoforms of the CD8 protein have been identified, alpha and beta, each encoded by a different gene. In humans, both genes are located on chromosome 2 in position 2p12.
In particular, in some embodiments of the T cell, compositions methods and systems herein described CD8(+) T cells can be activated using one or more immunogenic fragments of ApoB100 or an immunogenically active portion thereof.
The term “activated” and activation as used herein indicate the process by which a T cells interacts with an antigen presenting cell which presents a specific antigen for a time and under condition resulting in a T cell having a preassigned immunological role (e.g. cytotoxicity) within the immune system. The term “antigen-presenting cell” (APC) indicates a cell that displays antigen complex with major histocompatibility complex (MHC) on its surface. T-cells recognize this complex using their T-cell receptor (TCR). Exemplary APCs comprise dendritic cells (DCs) which are known to play an important role in linking innate and acquired immunity, see references (3, 4), and both immune responses participate in atherogenesis, see references (5), (6).
Detection of T cells and in particular, CD8(+) T cells, can be performed by detection of markers such as CD8, alone or in combination with TCR and additional markers identifiable by a skilled person. Detection of activated CD8(+) T cells can be performed by detection of T cells markers and other markers such as CD25, CD44, CD62 and additional markers identifiable by a skilled person using process and techniques suitable for detecting surface markers.
The terms “detect” or “detection” as used herein indicates the determination of the existence, presence or fact of a molecule or cell in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate. The “detect” or “detection” as used herein can comprise determination of chemical and/or biological properties of the target, including but not limited to ability to interact, and in particular bind, other compounds, ability to activate another compound and additional properties identifiable by a skilled person upon reading of the present disclosure. The detection can be quantitative or qualitative. A detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the target or signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the target or signal. A detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the target or signal in terms of relative abundance to another target or signal, which is not quantified.
Exemplary techniques suitable for detecting T cell markers comprise use of suitable monoclonal or polyclonal antibodies or antigen-specific HLA or MHC pentamers or hexamers labeled with an appropriate molecule allowing detection as well as additional methods and techniques identifiable by a skilled person. In an exemplary approach T cell markers are identified by flow cytometric analysis as described in the Examples section.
Activated CD8(+) T cells according to the present disclosure are activated with one or more immunogenic fragment of ApoB100 or an immunogenically active portion thereof and are typically specific for the immunogenic fragment or the immunogenically active portion used for the activation.
The wording “specific” “specifically” or “specificity” as used herein with reference to the immunogenic response refers to the ability of an immunological agent to direct the immunological activity towards an antigen, together with substantially less to no immunological activity towards other antigen that may be present. As consequence, CD8 (+) T cells herein are specifically activated towards the immunogenic fragment or active portion used to activate them and not for other antigens.
Exemplary antigenic properties that can be used to identify CD8 T cell specific for the immunogenic fragments comprise humoral and/or cellular responses detectable using methods and techniques such as the ones exemplified in the Examples section as well as other methods and techniques identifiable by a skilled person. Exemplary methods and systems for detecting antigenic properties in the sense of the present disclosure comprise ELISA and in particular serum ELISA and additional methods exemplified in the Examples section. Exemplary techniques suitable for detecting T cell markers comprise use of suitable monoclonal or polyclonal antibodies or antigen-specific HLA or MHC pentamers or hexamers labeled with an appropriate molecule allowing detection as well as additional methods and techniques identifiable by a skilled person. In an exemplary approach T cell markers are identified by flow cytometric analysis as described in the Examples section.
The term “immunogenic fragment” or “antigenic fragment” as used herein indicates a portion of a polypeptide of any length capable of generating an immune response, such as an antigen. An antigen is a molecule recognized by the immune system. An antigenic fragment of ApoB100 is accordingly a portion of apoB-100 that presents antigenic properties.
The term “fragment of ApoB100” in the sense of the present disclosure comprises not only fragments of any length from ApoB100, but also peptides produced by genetic recombination or chemically synthesized comprising sequences from ApoB100. The term “immunogenic fragments” in the sense of the present disclosure further comprise also derivative of any fragment, such as mutated fragments (including fragments with replaced, added or deleted residues) oxidative derivative and/or peptide treated with MDA or copper, which maintain a detectable antigenic property of the original fragment (e.g. a specific humoral and/or cellular response).
The term “derivative” as used herein with reference to a first peptide (e.g., an immunogenic fragment), indicates a second peptide that is structurally related to the first peptide and is derivable from the first peptide by a modification that introduces a feature that is not present in the first peptide while retaining functional properties of the first peptide. Accordingly, a derivative peptide of an immunogenic fragment, or of any portion thereof, (e.g. an epitope thereof), usually differs from the original immunogenic fragment or portion thereof by modification of the amino acidic sequence that might or might not be associated with an additional function not present in the original peptide or portion thereof. A derivative peptide of an immunogenic fragment or of any portion thereof retains however one or more of the immunogenic activities that are herein described in connection with an immunogenic fragment or portion thereof. The antigenic properties can be verified with methods and systems such as the ones already described for the immunogenic fragments and additional methods and systems identifiable to a skilled person. Typically, a derivative of an immunogenic fragment comprises at least one epitope of the immunogenic fragment
The term “immunogenically active portion” in the sense of the present disclosure indicates any part of a reference antigen that can elicit specific immune response. Exemplary immunogenically active portions are epitopes typically formed by 5 or more residues within an immunogenic fragment. In some embodiments, epitopes within one or more fragments can overlap.
Immunogenic fragments can be expressed by recombinant technology, such as a fusion with an affinity or epitope tag, chemical synthesis of an oligopeptide, either free or conjugated to carrier proteins, or any other methods known in the art to express the ApoB-100 peptides.
Exemplary fragments of ApoB100 are peptides each comprising one of the sequences listed in the Sequence Listing as SEQ ID NO: 1 to SEQ ID NO: 302 described in further detail in the Examples section. Methods and systems suitable to identify an immunogenic fragment in the sense of the present are described in WO 02/080954, hereby incorporated by reference. Additional methods are exemplified in the Examples section (see e.g. Example 1).
The term “protein” or “polypeptide” or “peptide” as used herein indicates an organic polymer composed of two or more amino acid monomers and/or analogs thereof. The term “polypeptide” includes amino acid polymers of any length including full length proteins or peptides, as well as analogs and fragments thereof. A peptide of three or more amino acids is also called an oligopeptide. As used herein the term “amino acid”, “amino acidic monomer”, or “amino acid residue” refers to any of the twenty amino acids including synthetic amino acids with unnatural side chains and including both D and L optical isomers. The term “amino acid analog” refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, isotope, or with a different functional group but is otherwise identical to its natural amino acid analog.
In an embodiment, the immunogenic fragment of ApoB100 is associated to atherosclerosis reduction. In particular, in some embodiments, immunogenic fragments of ApoB100 are immunogenic fragments producing an immune response associated to atherosclerosis reduction in the individual or in an animal model. In some of those embodiments, a percentage atherosclerosis reduction is at least 20%, or at least 30% to 50% or 40% to 80%. Reference is made to Examples section wherein embodiments of the present disclosure are exemplified with reference to immunogenic fragment p210 associated with a reduction of atherosclerosis of about 57.6% (see Example 2).
In some embodiments, the immunogenic fragment associated to atherosclerosis reduction comprises at least one of peptide, each comprising p1 (SEQ ID NO: 1), p2 (SEQ ID NO: 2), p10 (SEQ ID NO: 10), p11 (SEQ ID NO:11), p25 (SEQ ID NO:25), p30-p34 (SEQ ID NOs:30-34), p40 (SEQ ID NO:40), p40 (SEQ ID NO:40), p45 (SEQ ID NO:45), p68 (SEQ ID NO:68), p74 (SEQ ID NO:74), p94 (SEQ ID NO:94), p99 (SEQ ID NO:99), p100 (SEQ ID NO:100), p102(SEQ ID NO:102), p103 (SEQ ID NO: 103), p105 (SEQ ID NO:105), p107 (SEQ ID NO:107), pill (SEQ ID NO:111), p129 (SEQ ID NO:129), p143 (SEQ ID NO:143), p148 (SEQ ID NO:148), p149 (SEQ ID NO:149), p154 (SEQ ID NO:154), p162 (SEQ ID NO:162), p169 (SEQ ID NO:169), p177 (SEQ ID NO:177), p199 (SEQ ID NO:199), p210 (SEQ ID NO:210), p222 (SEQ ID NO:222), p236 (SEQ ID NO:236), p252 (SEQ ID NO:252), or p301 (SEQ ID NO:301).
In an embodiment, the one or more immunogenic fragments associated to atherosclerosis reduction comprises one or more peptides each comprising p2 (SEQ ID NO:2), p11 (SEQ ID NO:11), p32 (SEQ ID NO:32), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), p148 (SEQ ID NO:148), p162 (SEQ ID NO:162), or p210 (SEQ ID NO:210).
In an embodiment, the one or more immunogenic fragments associated to atherosclerosis reduction comprises two peptides each comprising p143 (SEQ ID NO: 143), or p210 (SEQ ID NO:210). In an embodiment, the one or more immunogenic fragments associated to atherosclerosis reduction comprises three peptides each comprising, one of p11 (SEQ ID NO:11), p25 (SEQ ID NO: 25), or p74 (SEQ ID NO:74). In an embodiment, the one or more immunogenic fragments associated to atherosclerosis reduction comprises five peptides each comprising one of p99 (SEQ ID NO: 99), p100 (SEQ ID NO: 100), p102 (SEQ ID NO: 102), p103 (SEQ ID NO: 103), and p105 (SEQ ID NO: 105).
In an embodiment, the one or more immunogenic fragments associated to atherosclerosis reduction comprises one or more peptides each comprising p2 (SEQ ID NO: 2), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), or p210 (SEQ ID NO:210).
In an embodiment, the one or more immunogenic fragments associated to atherosclerosis reduction comprise a peptide comprising amino acids 16-35 of human apoB-100 (p2; SEQ ID NO:2).
In an embodiment the one or more immunogenic fragments associated to atherosclerosis reduction comprise a peptide comprising amino acids 661-680 of human apoB-100 (p45; SEQ ID NO:45).
In an embodiment, the one or more immunogenic fragments associated to atherosclerosis reduction comprise a peptide comprising amino acids 3136-3155 of human apoB-100 (P210; SEQ ID NO: 210).
Exemplary data showing association of the above peptides to atherosclerosis reduction are shown in International application WO 02/080954, herein incorporated by reference in its entirety (see in particular Table 1, Table 2, Table A and Table B). In particular for some of those peptides or combination thereof a percentage reduction of 64.6% (p143 and p210), 59.6% (p11, p25 and p74), 56.8% (p129, p148, and p167), p67.7 (p2), 57.9% (p210), 55.2% (p301), 47.4% (p45), 31% (p1) has been detected (see W0/02080954 incorporated herein by reference in its entirety, and in particular Table B).
In some embodiments, activated CD8(+)T cell suitable herein described, can be activated by contacting a CD8(+) T cell with one or more immunogenic fragment of ApoB100, or an immunogenically active portion thereof, for a time and under condition to obtain a CD8(+)Tcell specific for the immunogenic fragment.
In an embodiment, the contacting can be performed in vivo by administering an effective amount of immunogenic fragment or of an immunogenically active portion thereof to the individual. In some embodiments, the effective amount can be determined in animal models. In some embodiment, following the administering, activated T cell are isolated from the individual (e.g. from blood or other fluid samples) according to methods identifiable by a skilled person.
Effective amounts of an immunogenic fragment to activate T cells in vivo will depend on the individual wherein the activation is performed and will be identifiable by a skilled person. For example in an embodiment the T cell activation can be performed with an effective amount of from about 1 to about 100 μg immunogenic fragment or immunogenically active portion thereof. In an embodiment, T cell activation can be performed with an effective amount of from about 1 to about 100 mg immunogenic fragment or immunogenically active portion thereof. Additional effective amounts are identifiable by a skilled person in view of the individual where activation is performed and the desired activation.
In an embodiment, the contacting can be performed in vitro using suitable techniques such as pulsing the T cells with an appropriate amount of immunogenic fragment or active portion thereof (see e.g. (41) and (42) herein incorporated by reference in their entirety).
Effective amounts of an immunogenic fragment to activate T cells in vitro are identifiable by a skilled person. For example in an embodiment the T cell activation can be performed with an effective amount of from about 1 to about 100 μg immunogenic fragment or immunogenically active portion thereof.
In an embodiment, the contacting can be performed by contacting the CD8(+) T cell together with an enhancer of CD8(+) T cell activation
The terms “enhancer” and “enhance” as it pertains to a molecule in connection with CD8 T cell refers to the ability of a molecule to modify the immune response by promoting the activation of cells of the immune system. The choice of appropriate enhancer can allow control of activation of the immune response. Exemplary enhancers include cytokines such as IL-2. The term “cytokine” as used herein refers cell signaling molecules that act as has immunomodulating agents, and comprise proteins such as interleukins and interferons as would be identifiable to a skilled person. Selection of a suitable cytokine can result under appropriate conditions in the preferential induction of a humoral or cellular immune response.
In an embodiment, the enhancer can be Interleukin 2 (IL2), Interleukin 15 (IL-15), TGF-beta (TGF-β), IL2-antiIL-2 antibody complex and/or additional enhancer identifiable by a skilled person upon reading of the present disclosure. Reference is made to the references Mitchell et al 2010 (38), Perret et al 2008 (39) and Kamimura et al 2007 (40), each incorporated by reference in its entirety, which describe exemplary use of enhancer in connection with T cell activation.
In particular in some embodiments, the enhancing is performed by reducing CD86 expression and/or IL12 secretion by dendritic cells in the individual.
In several embodiments treatment or prevention of atherosclerosis and/or a condition associated thereto in an individual, can be performed by increasing in the individual activated CD8(+) T cell specific for an immunogenic fragment of ApoB100 or an immunogenically active portion thereof.
In particular, in several embodiments, the increase of p210 specific CD8(+) or other activated CD8(+) T cell herein described is associated with a decrease of dendritic cells in the atherosclerotic plaques. More particularly this increase of specific CD8(+) T cell can be performed by administering to the individual an effective amount of peptide p210, comprising sequence KTTKQ SFDLS VKAQY KKNKH (SEQ ID NO: 210) or another immunogenic fragment herein described associated with atherosclerosis reduction in an individual.
In an embodiment, the increasing can be performed by administering to the individual an effective amount of an activated CD8(+) T cell.
In an embodiment the effective amount is expected to be comprised between about 500,000 to 2,000,000 cells. In embodiment the effective amount is expected to be comprised between about 750,000 to about 1,500,000 cells. In an embodiment, the effective amount is expected to be about 1,000,000 cells.
In particular, in an embodiment administration of about 1,000,000 cells is expected to result in both treatment and prevention of atherosclerosis. Administration is expected to be performed in accordance with dosages and schedule which will be identified based on the condition of the individual to be treated and the desired effect. For example in administration directed to prevention, administering an effective amount of activated CD8(+) T cell can performed by performing either a single administration, or a plurality of administrations (e.g. 3 administrations or more, in particular up to 6 administrations) of activated CD8(+) T cell herein described in intervals to obtain a desired immunization based on the condition of the individual. In particular, a plurality of administrations can be performed whenever a prolonged effect is desired.
Administration of CD8(+) T cell herein described can be performed according to methods to immunize an individual identifiable to a skilled person. In an embodiment, the administering can be performed by parenteral administration. Parenteral administration is a systemic route of administration where the substance is given by route other than the digestive tract and includes but is not limited to intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intradermal, administration, intraperitoneal administration, and intravesical infusion. In particular, in an embodiment the administering can be performed by intravenous administration.
In an embodiment, administration can be performed by administering activated CD8(+) T cell one time, typically via intravenous route, one time or multiple times, depending on the desired duration of the immunization effect.
In an embodiment, administering the CD8(+) T cells is expected to be particularly effective if performed in combination with one or more immunogenic fragments of ApoB100 or an immunogenically active portion thereof alone or together with a CD8(+) T cell enhancer such as IL 2. In particular, in some embodiments, the administering can be also performed by administering any combination of the activated CD8(+) T cells herein descried and any of the immunogenic fragments of ApoB100 herein described possibly in combination with enhancers herein described.
In an embodiment, increasing an activated CD8(+) T cell in the individual, the activated CD8(+)Tcell specific for an immunogenic fragment of ApoB100 or an immunogenically active portion thereof is expected to result in reducing plaque, dendritic cell content and/or macrophage content. According to a possible mechanism provided for guidance purpose only and not intended to be limited, a danger signal (such as oxidized LDL, heat shock protein) in the arterial wall can activate DCs or another APC, which induce effector T-cells with subsequent activation of immune-mediated inflammatory cascade and mediators leading to atherosclerotic plaque growth or destabilization see references (7), (8). Reduction of such DCs recruitment into arterial walls can be associated with decreased atherosclerosis.
Also in some embodiments the enhancing is performed by modulating dendritic cell phenotype in the individual to generate tolerogenic dendritic cells. Additionally in several embodiments, an individual presenting atherosclerosis and/or a condition associated thereto can be treated by enhancing a T regulatory cell population in the individual.
In an embodiment, CD8(+) T cell can be used in a method to identify a therapeutic agent for treatment and/or prevention of atherosclerosis or of a condition associated thereto. The method comprises contacting a CD8(+) T cell with an immunogenic fragment of ApoB100 or an immunogenically active portion thereof for a time and under condition to provide an activated CD8(+) T cell, the activated CD8(+)Tcell being specific for the immunogenic fragment or the immunogenically active portion thereof. The method further comprises administering the activated CD8(+) T cell to an animal model and detecting atherosclerosis reduction in the animal model.
The term “animal model” as used herein indicates a living, non-human animal used during the research and investigation of human disease, for the purpose of better understanding the disease without the added risk of causing harm to an actual human being during the process. The animal chosen will usually meet a determined taxonomic equivalency to humans, so as to react to disease or its treatment in a way that resembles human physiology as needed. Many drugs, treatments and cures for human diseases have been developed with the use of animal models. Animal models representing specific taxonomic groups in the research and study of developmental processes are also referred to as model organisms.
In an embodiment, detecting atherosclerosis reduction in the animal model can be performed by immunohistochemistry and histomorphometry techniques such as the ones illustrated in the Examples section. Detecting atherosclerosis reduction in the human can be performed by imaging techniques such as ultrasound determination of carotid intima thickness, MRI scan of carotid atherosclerosis, CT angiogram of coronary or peripheral arterial circulation.
Immunogenic fragments and/or immunogenically active portion thereof and CD8(+) T cell activated thereby which are associated to a reduction in atherosclerosis following administration in the animal model can be selected as therapeutic agent for treatment or prevention of atherosclerosis. In particular in some embodiments, immunogenic fragments or an immunogenic portion thereof or a CD8(+) T cell activated thereby associated to a reduction of at least about 20% atherosclerosis can be selected as a therapeutic agent. In an embodiment, the percentage reduction can of at about 30% to 50%, or of about 40% to 80%.
As disclosed herein, the CD8(+) Tcell, immunogenic fragments or immunogenically active portion thereof, and enhancers herein described can be provided as a part of systems to treat and/or prevent atherosclerosis or of a condition associated thereto or as part of a system to identify a therapeutic agent in treatment and/or prevention of atherosclerosis or of a condition associated thereto.
In some embodiments, the system comprises two or more of one or more agents suitable to increase CD8(+) T cell activated in the individual and/or one or more agents suitable to detect the increased CD8(+) T cell in the individual.
In some embodiments, the system comprises two or more of one or more agents suitable to increase the activated CD8(+) T cell specific for the immunogenic fragment of ApoB100 in the individual and one or more agents suitable to detect the reduced placque, dendritic cell content and/or macrophage content in the individual.
The systems can be provided in the form of kits of parts. In a kit of parts, the CD8(+) Tcell herein described and other reagents to perform the method herein described can be comprised in the kit independently. The CD8(+) Tcell herein described can be included in one or more compositions, and each CD8(+) Tcell herein described can be in a composition together with a suitable vehicle.
Additional components can include, any of the immunogenic fragments herein described or immunogenically active portion thereof alone or in combination, enhancers (e.g. IL2, IL15, IL2-antiIL2 antibody complex), adjuvant, (e.g. aluminum salts and in general aluminum adjuvants), reagents able to detect CD8(+) Tcell herein described, reagents able to detect reduced plaque such as labeled molecules and in particular, labeled antibodies, labels, microfluidic chip, reference standards, and additional components identifiable by a skilled person upon reading of the present disclosure.
The terms “label” and “labeled molecule” as used herein as a component of a complex or molecule referring to a molecule capable of detection, including but not limited to radioactive isotopes, fluorophores, chemiluminescent dyes, chromophores, enzymes, enzymes substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sols, ligands (such as biotin, avidin, streptavidin or haptens) and the like. The term “fluorophore” refers to a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable image. As a consequence, the wording “labeling signal” as used herein indicates the signal emitted from the label that allows detection of the label, including but not limited to radioactivity, fluorescence, chemiluminescence, production of a compound in outcome of an enzymatic reaction and the like.
In an embodiment, the system is directed to increase CD8(+) T cell in an individual (e.g. for producing the CD8(+) T cell) and the system comprises two or more of one or more agents suitable to increase CD8(+) T cell activated in the individual and/or one or more agents suitable to detect the increased CD8(+) T cell in the individual.
In an embodiment, the system is directed to reduce plaque, dendritic cell content and/or macrophage content (e.g. in an individual for therapeutic purposes or in an animal model for the purpose of biological research), and the system comprises two or more of one or more agents suitable to increase the activated CD8(+) T cell specific for the immunogenic fragment of ApoB100 or an immunogenically active portions thereof in the individual and one or more agents suitable to detect the reduced placque, dendritic cell content and/or macrophage content in the individual.
In some embodiments, detection of a CD8(+) Tcell herein described can be carried either via fluorescent based readouts, in which the labeled antibody is labeled with fluorophore, which includes, but not exhaustively, small molecular dyes, protein chromophores, quantum dots, and gold nanoparticles. Additional techniques are identifiable by a skilled person upon reading of the present disclosure and will not be further discussed in detail.
In particular, the components of the kit can be provided, with suitable instructions and other necessary reagents, in order to perform the methods here described. The kit will normally contain the compositions in separate containers. Instructions, for example written or audio instructions, on paper or electronic support such as tapes or CD-ROMs, for carrying out the assay, will usually be included in the kit. The kit can also contain, depending on the particular method used, other packaged reagents and materials (e.g. wash buffers and the like).
In some embodiments, the CD8(+) Tcell herein described can be included in compositions together with a suitable vehicle.
The term “vehicle” as used herein indicates any of various media acting usually as solvents, carriers, binders or diluents for T cell comprised in the composition as an active ingredient.
In some embodiments, where the composition is to be administered to an individual the composition can be a pharmaceutical anti-inflammatory composition, and comprises T cell and a pharmaceutically acceptable vehicle.
In particular, in some embodiments, disclosed are pharmaceutical compositions which contain at least one CD8(+) Tcell herein described as herein described, in combination with one or more compatible and pharmaceutically acceptable vehicles, and in particular with pharmaceutically acceptable diluents or excipients. In those pharmaceutical compositions the CD8(+) Tcell herein described can be administered as an active ingredient for treatment or prevention of a condition in an individual.
The term “excipient” as used herein indicates an inactive substance used as a carrier for the active ingredients of a medication. Suitable excipients for the pharmaceutical compositions herein disclosed include any substance that enhances the ability of the body of an individual to absorb a CD8(+) Tcell herein described. Suitable excipients also include any substance that can be used to bulk up formulations with CD8(+) Tcell herein described to allow for convenient and accurate dosage. In addition to their use in the single-dosage quantity, excipients can be used in the manufacturing process to aid in the handling of CD8(+) Tcell herein described. Depending on the route of administration, and form of medication, different excipients can be used. Exemplary excipients include but are not limited to antiadherents, binders, coatings disintegrants, fillers, flavors (such as sweeteners) and colors, glidants, lubricants, preservatives, sorbents.
The term “diluent” as used herein indicates a diluting agent which is issued to dilute or carry an active ingredient of a composition. Suitable diluent include any substance that can decrease the viscosity of a medicinal preparation.
In an embodiment, compositions herein described can further include an adjuvant. The term “adjuvant” as used herein indicates an agent that can stimulate the immune system and increase the response to a vaccine, without having any specific antigenic effect in itself. The word “adjuvant” comes from the Latin word adjuvare, meaning to help or aid. Typically, an immunologic adjuvant is defined as any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens.
Further advantages and characteristics of the present disclosure will become more apparent hereinafter from the following detailed disclosure by way of illustration only with reference to an experimental section.
EXAMPLES
The methods and systems herein described are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.
In particular, the following examples illustrate exemplary CD8(+) T cell and related process of activation and use to treat or prevent atherosclerosis. More particularly, activation of CD8 (+) T cell performed with the immunogenic fragment p210 is shown, as well as the use of the CD8 (+) T cell so obtained to treat and prevent atherosclerosis in mice models.
A person skilled in the art will appreciate the applicability and the necessary modifications to adapt the features described in detail in the present section, to additional immunogenic fragments, administered subcutaneously or using other routes of administration in vivo or in vitro according to embodiments of the present disclosure.
Unless otherwise indicated the following material and methods were followed in the Examples reported below.
Selection of Peptides and their Preparation for Immunization
The establishment and screening of human apoB-100 peptides has been reported (8). Based on Applicants pilot experiments and prior reports see references (9), (10) Applicants selected peptide 210 (p210, KTTKQ SFDLS VKAQY KKNKH—SEQ ID NO: 210) as a candidate immunogen. Native p210 peptide (Euro-Diagnostica AB, Sweden) was conjugated to cationic bovine serum albumin (cBSA) as carrier using a method described previously, see references (3), (4) Alum was used as adjuvant and mixed with peptide/cBSA conjugate with 1:1 ratio in volume. Peptide conjugation and mixing with alum were prepared fresh prior to each immunization.
Immunization Protocols
Male apoE (−/−) mice (Jackson Laboratories) were housed in an animal facility accredited by the American Association of Accreditation of Laboratory Animal Care and kept on a 12-hour day/night cycle with unrestricted access to water and food. The Institutional Animal Care and Use Committee of Cedars-Sinai Medical Center approved the experimental protocols. In a pilot experiment, p210 immunization using 100 μg dose conferred optimum athero-reduction compared to 25 or 50 μg dose. Hence 100 μg dose was used for all subsequent experiments. Mice, maintained on normal chow diet, received subcutaneous primary immunization in the dorsal area between scapulas at 6-7 weeks of age, followed by a booster at 9 and 12 weeks of age. One week after last booster, diet was switched to high cholesterol chow (TD 88137, Harlan-Teklad) and continued until euthanasia at the age of 25 weeks. Separate groups of mice receiving PBS or cBSA/alum at the same immunization time-points served as control. Some mice were sacrificed at 8 or 13 weeks of age to assess immune response against p210.
Tissue Harvesting and Preparation
At euthanasia the hearts were harvested and embedded in OCT compound (Tissue-Tek) for cryo-section. Whole aortas were cleaned, processed and stained with Oil Red O to assess the extent of atherosclerosis en face with computer-assisted histomorphometry see references (3), (4).
Immunohistochemistry and Histomorphometry
The sections from aortic sinus were stained with MOMA-2 (Serotec), or CD11c (eBioscience) antibody to identify macrophages or dendritic cells immunohistochemically using standard protocol. Oil-Red-O stain for plaque size was done using standard protocol. Computer-assisted morphometric analysis was performed to assess histomorphometry as described previously see references (3), (4).
Serum ELISA
Flat-bottomed 96-well polystyrene plates (MaxiSorp, Germany) were pre-coated with 100 ul (20 μg/ml) p210, KLH, TNP-KLH (Biosearch Technologies T-5060) or BSA (2 μg/ml for IgG or 10 μg/ml for IgM) respectively by incubation overnight at 4° C. to assess antibodies levels using standard protocol. The coating concentration was optimized in pilot experiments. Goat anti-mouse HRP-IgG (Pierce 31437) or IgM (Southern Biotech) were used as detecting antibodies and the bound antibodies were detected by developing in ABTS (Southern Biotech) as substrate and optical density values were recorded at 405 nm.
Flow Cytometric Analysis
Flow cytometric analysis was performed using standard protocols with antibodies listed in Table 1 below and a FACScan (Becton Dickinson) or a CyAn ADP analyzer (Beckman Coulter). For intracellular cytokine staining, Brefeldin A (3 μg/ml) was added to the cultured cells for 2 hours before cells subject to staining procedure. Cell membranes were permeabilized for staining intracellular molecules.










TABLE 1





Antigen
Clone
Type
Supplier







CD4
GK1.5
FITC-Rat IgG2b, κ
BD Pharmingen


CD8b.2
53-5.8
FITC-Rat IgG1, κ
BD Pharmingen


CD25
PC61.5
PE-Rat IgG1, λ
eBioscience


IL-10
JES5-16E3
Percp-Cy5.5-Rat
eBioscience




IgG2a, κ


IL-12
Clone C17.8
Percp-Cy5.5-Rat
eBioscience




IgG2b, κ


CD11c
HL3
FITC-Hamster IgG1, λ
BD Pharmingen


CD86
GL1
PE-Rat IgG2a, κ
BD Pharmingen


TGF-Beta
1D11
APC-Mouse IgG1
R&D system


Granzyme B
16G6
Alexa-Fluo 647 Rat
eBioscience




IgG2b, κ


Perforin
eBioOMAK-D
FITC-Rat IgG2a, κ
eBioscience









Adoptive Transfer Experiment
Male apoE (−/−) mice on regular chow received subcutaneous immunization as described in previous paragraph and were sacrificed at 13 weeks of age as donors. Splenocytes from the same treatment group were pooled before cell isolation. Donor CD8(+) T-cells, CD4(+)CD25(+) T-cells or B-cells were isolated using Dynabeads FlowComp (Invitrogen) according to the manufacturer's protocols. CD4(+) T-cells were negatively selected from the splenocytes followed by positive selection of CD4(+)CD25(+) cells. B cells were negatively isolated whereas CD8(+) T-cells were positively isolated first and released from beads. The purity of pooled CD8(+) T-cells, CD4(+)CD25(+) T-cells and B-cells was 90%, 80% and 70%, respectively. The isolated CD8(+) T-cells (1×106 cells/mouse), CD4(+)CD25(+) T-cells (1×105 or 3×105 cells/mouse) or B-cells (2×107 cells/mouse) were then adoptively transferred to naïve male apoE (−/−) recipient mice at 6-7 weeks of age via tail vein injection. In the published literatures of vascular biology, the number of adoptively transferred lymphocytes varied greatly. For B-cells transfer, the number of 2×107 cells/mouse was chosen based on two prior reports, see references (11), (12). For CD4(+)CD25(+) T-cells transfer, the number of cells transferred ranged from 5×104 cells/mouse to 1×106 cells/mouse in the published literature see references (13), (14), (15). Hence Applicants chose 2 intermediate doses for our experiment. As to CD8(+) T-cells, 1×106 cells was chosen based on a report from the field of autoimmune disease, see reference (16). Applicants did not adoptively transferred CD4(+) T-cells because naïve or antigen-primed CD4(+) T-cells are known to be pro-atherogenic, see references. (17), (18). Recipient mice were fed normal chow until 13 weeks of age when chow was switched to high cholesterol diet until euthanasia at 25 weeks of age. Aortas were harvested to assess the extent of atherosclerosis.
KLH or Trinitrophenyl-Lipopolysaccharide (TNP-LPS) Immunization
Applicants also tested if p210 immunization affected the efficacy of subsequent immunization with other antigens. KLH was chosen as a prototypical T-cell dependent and TNP as a T-cell independent antigen. Male C57/BL6 mice on regular chow received subcutaneous immunization with p210 conjugate or adjuvant control as described in previous paragraphs for apoE (−/−) mice. At 13 and 15 weeks of age mice were subcutaneously immunized with 100 μg KLH (with alum as adjuvant) at injection sites away from p210 sites or injected intraperitoneally with 100 μg TNP-LPS (Sigma). KLH or TNP immunization was done in separate groups of mice. Blood was collected via retro-orbital puncture at euthanasia (16 weeks of age).
In Vitro Generation of BM-Derived Dendritic Cells (BMDCs)
The method for generating BMDC with GM-CSF was adapted from previous publication with modification, see reference (19). Briefly, bone marrow cells from femurs and tibiae of male apoE−/− mice were plated into 10 cm culture plates (Falcon) with 20 ml complete RPMI-1640 containing 10 ng/ml GM-CSF (R&D Systems) and 10 ng/ml IL-4 (Invitrogen). Cells were washed and fed on day 3 and day 5 by removing the old medium followed by replenishing with fresh culture medium with GM-CSF and IL-4. On day 8, the immature DC appeared as non-adherent cells under the microscope and harvested by vigorous pipetting and subcultured into new culture plates with 2×105 DCs in 1.5 ml medium.
In Vitro CD8+ T-Cells Isolation and Co-Culture with Dendritic Cells
Donor mice [male apoE (−/−) mice] for CD8(+) T-cells were immunized with PBS, cBSA/Alum, or cBSA/Alum/P210 according to the schedule described in earlier paragraphs and splenocytes were harvested at 13 weeks of age. CD8(+) T-cells were negatively isolated using a CD8 selection Dynabeads kit (Invitrogen) per manufacturer's protocol. The selected CD8(+) T-cells were then co-culture with DCs in a CD8:DC ratio of 3:1. A series of pilot studies has been performed to determine the optimal CD8:DC ratio for this assay. After co-culture for 4 hours, cells were collected and processed for flow cytometric determination of CD11c and 7-AAD by LSR II flow cytometer (BD Biosciences) and data was analyzed with Summit V4.3 software. Dendritic cell death without CD8(+) T-cells in the co-culture was used as baseline and percentage of specific lysis of cells was calculated using a method described previously, see reference (20).
Statistics
Data are presented as mean±Std. Number of animals in each group is listed in text or description of the figures. Data were analyzed by ANOVA followed by Newman-Keuls multiple group comparison, or by t-test when appropriate. P<0.05 was considered as statistically significant and horizontal bars in each figure indicated statistically significant difference between groups.
Example 1
Immunogenic Fragments of ApoB-100
Specific immunogenic epitopes by focusing on the single protein found in LDL, apolipoprotein B-100 (apo B) were characterized. A peptide library comprised of 302 peptides, 20 amino acid residues in length, covering the complete 4563 amino acid sequence of human apo B was produced. The peptides were produced with a 5 amino acid overlap to cover all sequences at break points. Peptides were numbered 1-302 starting at the N-terminal of apo B as indicated in Table 2 below.







TABLE 2







Exemplary immunogenic fragments












Apolipoprotein



Peptide
Sequence
B aa
SEQ ID NO





P1:
EEEML ENVSL VCPKD ATRFK
aa 1-20
SEQ ID NO: 1


 


P2:
ATRFK HLRKY TYNYE AESSS
aa 16-35
SEQ ID NO: 2


 


P3:
AESSS GVPGT ADSRS ATRIN
aa 31-50
SEQ ID NO: 3


 


P4:
ATRIN CKVEL EVPQL CSFIL
aa 46-65
SEQ ID NO: 4


 


P5:
CSFIL KTSQC TLKEV YGFNP
aa 61-80
SEQ ID NO: 5


 


P6:
YGFNP EGKAL LKKTK NSEEF
aa 76-95
SEQ ID NO: 6


 


P7:
NSEEF AAAMS RYELK LAIPE
aa 91-110
SEQ ID NO: 7


 


P8:
LAIPE GKQVF LYPEK DEPTY
aa 106-125
SEQ ID NO: 8


 


P9:
DEPTY ILNIK RGIIS ALLVP
aa 121-140
SEQ ID NO: 9


 


P10:
ALLVP PETEE AKQVL FLDTV
aa 136-155
SEQ ID NO: 10


 


P11:
FLDTV YGNCS THFTV KTRKG
aa 151-170
SEQ ID NO: 11


 


P12:
KTRKG NVATE ISTER DLGQC
aa 166-185
SEQ ID NO: 12


 


P13:
DLGQC DRFKP IRTGI SPLAL
aa 181-200
SEQ ID NO: 13


 


P14:
SPLAL IKGMT RPLST LISSS
aa 196-215
SEQ ID NO: 14


 


P15:
LISSS QSCQY TLDAK RKHVA
aa 211-230
SEQ ID NO: 15


 


P16:
RKHVA EAICK EQHLF LPFSY
aa 226-245
SEQ ID NO: 16


 


P17:
LPFSY NNKYG MVAQV TQTLK
aa 241-260
SEQ ID NO: 17


 


P18:
TQTLK LEDTP KINSR FFGEG
aa 256-275
SEQ ID NO: 18


 


P19:
FFGEG TKKMG LAFES TKSTS
aa 271-290
SEQ ID NO: 19


 


P20:
TKSTS PPKQA EAVLK TLQEL
aa 286-305
SEQ ID NO: 20


 


P21:
TLQEL KKLTI SEQNI QRANL
aa 301-320
SEQ ID NO: 21


 


P22:
QRANL FNKLV TELRG LSDEA
aa 316-335
SEQ ID NO: 22


 


P23:
LSDEA VTSLL PQLIE VSSPI
aa 331-350
SEQ ID NO: 23


 


P24:
VSSPI TLQAL VQCGQ PQCST
aa 346-365
SEQ ID NO: 24


 


P25:
PQCST HILQW LKRVH ANPLL
aa 361-380
SEQ ID NO: 25


 


P26:
ANPLL IDVVT YLVAL IPEPS
aa 376-395
SEQ ID NO: 26


 


P27:
IPEPS AQQLR EIFNM ARDQR
aa 391-410
SEQ ID NO: 27


 


P28:
ARDQR SRATL YALSH AVNNY
aa 406-425
SEQ ID NO: 28


 


P29:
AVNNY HKTNP TGTQE LLDIA
aa 421-440
SEQ ID NO: 29


 


P30:
LLDIA NYLME QIQDD CTGDE
aa 436-455
SEQ ID NO: 30


 


P31:
CTGDE DYTYL ILRVI GNMGQ
aa 451-470
SEQ ID NO: 31


 


P32:
GNMGQ TMEQL TPELK SSILK
aa 466-485
SEQ ID NO: 32


 


P33:
SSILK CVQST KPSLM IQKAA
aa 481-500
SEQ ID NO: 33


 


P34:
IQKAA IQALR KMEPK DKDQE
aa 496-515
SEQ ID NO: 34


 


P35:
DKDQE VLLQT FLDDA SPGDK
aa 511-530
SEQ ID NO: 35


 


P36:
SPGDK RLAAY LMLMR SPSQA
aa 526-545
SEQ ID NO: 36


 


P37:
SPSQA DINKI VQILP WEQNE
aa 541-560
SEQ ID NO: 37


 


P38:
WEQNE QVKNF VASHI ANILN
aa 556-575
SEQ ID NO: 38


 


P39:
ANILN SEELD IQDLK KLVKE
aa 571-590
SEQ ID NO: 39


 


P40:
KLVKE ALKES QLPTV MDFRK
aa 586-605
SEQ ID NO: 40


 


P41:
MDFRK FSRNY QLYKS VSLPS
aa 601-620
SEQ ID NO: 41


 


P42:
VSLPS LDPAS AKIEG NLIFD
aa 616-635
SEQ ID NO: 42


 


P43:
NLIFD PNNYL PKESM LKTTL
aa 631-650
SEQ ID NO: 43


 


P44:
LKTTL TAFGF ASADL IEIGL
aa 646-665
SEQ ID NO: 44


 


P45:
IEIGL EGKGF EPTLE ALFGK
aa 661-680
SEQ ID NO: 45


 


P46:
ALFGK QGFFP DSVNK ALYWV
aa 676-695
SEQ ID NO: 46


 


P47:
ALYWV NGQVP DGVSK VLVDH
aa 691-710
SEQ ID NO: 47


 


P48:
VLVDH FGYTK DDKHE QDMVN
aa 706-725
SEQ ID NO: 48


 


P49:
QDMVN GIMLS VEKLI KDLKS
aa 721-740
SEQ ID NO: 49


 


P50:
KDLKS KEVPE ARAYL RILGE
aa 736-755
SEQ ID NO: 50


 


P51:
RILGE ELGFA SLHDL QLLGK
aa 751-770
SEQ ID NO: 51


 


P52:
QLLGK LLLMG ARTLQ GIPQM
aa 766-785
SEQ ID NO: 52


 


P53:
GIPQM IGEVI RKGSK NDFFL
aa 781-800
SEQ ID NO: 53


 


P54:
NDFFL HYIFM ENAFE LPTGA
aa 796-815
SEQ ID NO: 54


 


P55:
LPTGA GLQLQ ISSSG VIAPG
aa 811-830
SEQ ID NO: 55


 


P56:
VIAPG AKAGV KLEVA NMQAE
aa 826-845
SEQ ID NO: 56


 


P57:
NMQAE LVAKP SVSVE FVTNM
aa 841-860
SEQ ID NO: 57


 


P58:
FVTNM GIIIP DFARS GVQMN
aa 856-875
SEQ ID NO: 58


 


P59:
GVQMN TNFFH ESGLE AHVAL
aa 871-890
SEQ ID NO: 59


 


P60:
AHVAL KAGKL KFIIP SPKRP
aa 886-905
SEQ ID NO: 60


 


P61:
SPKRP VKLLS GGNTL HLVST
aa 901-920
SEQ ID NO: 61


 


P62:
HLVST TKTEV IPPLI ENRQS
aa 916-935
SEQ ID NO: 62


 


P63:
ENRQS WSVCK QVFPG LNYCT
aa 931-950
SEQ ID NO: 63


 


P64:
LNYCT SGAYS NASST DSASY
aa 946-965
SEQ ID NO: 64


 


P65:
DSASY YPLTG DTRLE LELRP
aa 961-980
SEQ ID NO: 65


 


P66:
LELRP TGEIE QYSVS ATYEL
aa 976-995
SEQ ID NO: 66


 


P67:
ATYEL QREDR ALVDT LKFVT
aa 991-1010
SEQ ID NO: 67


 


P68:
LKFVT QAEGA KQTEA TMTFK
aa 1006-1025
SEQ ID NO: 68


 


P69:
TMTFK YNRQS MTLSS EVQIP
aa 1021-1040
SEQ ID NO: 69


 


P70:
EVQIP DFDVD LGTIL RVNDE
aa 1036-1055
SEQ ID NO: 70


 


P71:
RVNDE STEGK TSYRL TLDIQ
aa 1051-1070
SEQ ID NO: 71


 


P72:
TLDIQ NKKIT EVALM GHLSC
aa 1066-1085
SEQ ID NO: 72


 


P73:
GHLSC DTKEE RKIKG VISIP
aa 1081-1100
SEQ ID NO: 73


 


P74:
VISIP RLQAE ARSEI LAHWS
aa 1096-1115
SEQ ID NO: 74


 


P75:
LAHWS PAKLL LQMDS SATAY
aa 1111-1130
SEQ ID NO: 75


 


P76:
SATAY GSTVS KRVAW HYDEE
aa 1126-1145
SEQ ID NO: 76


 


P77:
HYDEE KIEFE WNTGT NVDTK
aa 1141-1160
SEQ ID NO: 77


 


P78:
NVDTK KMTSN FPVDL SDYPK
aa 1156-1175
SEQ ID NO: 78


 


P79:
SDYPK SLHMY ANRLL DHRVP
aa 1171-1190
SEQ ID NO: 79


 


P80:
DHRVP ETDMT FRHVG SKLIV
aa 1186-1205
SEQ ID NO: 80


 


P81:
SKLIV AMSSW LQKAS GSLPY
aa 1201-1220
SEQ ID NO: 81


 


P82:
GSLPY TQTLQ DHLNS LKEFN
aa 1216-1235
SEQ ID NO: 82


 


P83:
LKEFN LQNMG LPDFH IPENL
aa 1231-1250
SEQ ID NO: 83


 


P84:
IPENL FLKSD GRVKY TLNKN
aa 1246-1260
SEQ ID NO: 84


 


P85:
TLNKN SLKIE IPLPF GGKSS
aa 1261-1280
SEQ ID NO: 85


 


P86:
GGKSS RDLKM LETVR TPALH
aa 1276-1295
SEQ ID NO: 86


 


P87:
TPALH FKSVG FHLPS REFQV
aa 1291-1310
SEQ ID NO: 87


 


P88:
REFQV PTFTI PKLYQ LQVPL
aa 1306-1325
SEQ ID NO: 88


 


P89:
LQVPL LGVLD LSTNV YSNLY
aa 1321-1340
SEQ ID NO: 89


 


P90:
YSNLY NWSAS YSGGN TSTDH
aa 1336-1355
SEQ ID NO: 90


 


P91:
TSTDH FSLRA RYHMK ADSVV
aa 1351-1370
SEQ ID NO: 91


 


P92:
ADSVV DLLSY NVQGS GETTY
aa 1366-1385
SEQ ID NO: 92


 


P93:
GETTY DHKNT FTLSC DGSLR
aa 1381-1400
SEQ ID NO: 93


 


P94:
DGSLR HKFLD SNIKF SHVEK
aa 1396-1415
SEQ ID NO: 94


 


P95:
SHVEK LGNNP VSKGL LIFDA
aa 1411-1430
SEQ ID NO: 95


 


P96:
LIFDA SSSWG PQMSA SVHLD
aa 1426-1445
SEQ ID NO: 96


 


P97:
SVHLD SKKKQ HLFVK EVKID
aa 1441-1460
SEQ ID NO: 97


 


P98:
EVKID GQFRV SSFYA KGTYG
aa 1456-1475
SEQ ID NO: 98


 


P99:
KGTYG LSCQR DPNTG RLNGE
aa 1471-1490
SEQ ID NO: 99


 


P100:
RLNGE SNLRF NSSYL QGTNQ
aa 1486-1505
SEQ ID NO: 100


 


P101:
QGTNQ ITGRY EDGTL SLTST
aa 1501-1520
SEQ ID NO: 101


 


P102:
SLTST SDLQS GIIKN TASLK
aa 1516-1535
SEQ ID NO: 102


 


P103:
TASLK YENYE LTLKS DTNGK
aa 1531-1550
SEQ ID NO: 103


 


P104:
DTNGK YKNFA TSNKM DMTFS
aa 1546-1565
SEQ ID NO: 104


 


P105:
DMTFS KQNAL LRSEY QADYE
aa 1561-1580
SEQ ID NO: 105


 


P106:
QADYE SLRFF SLLSG SLNSH
aa 1576-1595
SEQ ID NO: 106


 


P107:
SLNSH GLELN ADILG TDKIN
aa 1591-1610
SEQ ID NO: 107


 


P108:
TDKIN SGAHK ATLRI GQDGI
aa 1606-1625
SEQ ID NO: 108


 


P109:
GQDGI STSAT TNLKC SLLVL
aa 1621-1640
SEQ ID NO: 109


 


P110:
SLLVL ENELN AELGL SGASM
aa 1636-1655
SEQ ID NO: 110


 


P111:
SGASM KLTTN GRFRE HNAKF
aa 1651-1670
SEQ ID NO: 111


 


P112:
HNAKF SLDGK AALTE LSLGS
aa 1666-1685
SEQ ID NO: 112


 


P113:
LSLGS AYQAM ILGVD SKNIF
aa 1681-1700
SEQ ID NO: 113


 


P114:
SKNIF NFKVS QEGLK LSNDM
aa 1696-1715
SEQ ID NO: 114


 


P115:
LSNDM MGSYA EMKFD HTNSL
aa 1711-1730
SEQ ID NO: 115


 


P116:
HTNSL NIAGL SLDFS SKLDN
aa 1726-1745
SEQ ID NO: 116


 


P117:
SKLDN IYSSD KFYKQ TVNLQ
aa 1741-1760
SEQ ID NO: 117


 


P118:
TVNLQ LQPYS LVTTL NSDLK
aa 1756-1775
SEQ ID NO: 118


 


P119:
NSDLK YNALD LTNNG KLRLE
aa 1771-1790
SEQ ID NO: 119


 


P120:
KLRLE PLKLH VAGNL KGAYQ
aa 1786-1805
SEQ ID NO: 120


 


P121:
KGAYQ NNEIK HIYAI SSAAL
aa 1801-1820
SEQ ID NO: 121


 


P122:
SSAAL SASYK ADTVA KVQGV
aa 1816-1835
SEQ ID NO: 122


 


P123:
KVQGV EFSHR LNTDI AGLAS
aa 1831-1850
SEQ ID NO: 123


 


P124:
AGLAS AIDMS TNYNS DSLHF
aa 1846-1865
SEQ ID NO: 124


 


P125:
DSLHF SNVFR SVMAP FTMTI
aa 1861-1880
SEQ ID NO: 125


 


P126:
FTMTI DAHTN GNGKL ALWGE
aa 1876-1895
SEQ ID NO: 126


 


P127:
ALWGE HTGQL YSKFL LKAEP
aa 1891-1910
SEQ ID NO: 127


 


P128:
LKAEP LAFTF SHDYK GSTSH
aa 1906-1925
SEQ ID NO: 128


 


P129:
GSTSH HLVSR KSISA ALEHK
aa 1921-1940
SEQ ID NO: 129


 


P130:
ALEHK VSALL TPAEQ TGTWK
aa 1936-1955
SEQ ID NO: 130


 


P131:
TGTWK LKTQF NNNEY SQDLD
aa 1951-1970
SEQ ID NO: 131


 


P132:
SQDLD AYNTK DKIGV ELTGR
aa 1966-1985
SEQ ID NO: 132


 


P133:
ELTGR TLADL TLLDS PIKVP
aa 1981-2000
SEQ ID NO: 133


 


P134:
PIKVP LLLSE PINII DALEM
aa 1996-2015
SEQ ID NO: 134


 


P135:
DALEM RDAVE KPQEF TIVAF
aa 2011-2030
SEQ ID NO: 135


 


P136:
TIVAF VKYDK NQDVH SINLP
aa 2026-2045
SEQ ID NO: 136


 


P137:
SINLP FFETL QEYFE RNRQT
aa 2041-2060
SEQ ID NO: 137


 


P138:
RNRQT IIVVV ENVQR NLKHI
aa 2056-2075
SEQ ID NO: 138


 


P139:
NLKHI NIDQF VRKYR AALGK
aa 2071-2090
SEQ ID NO: 139


 


P140:
AALGK LPQQA NDYLN SFNWE
aa 2086-2105
SEQ ID NO: 140


 


P141:
SFNWE RQVSH AKEKL TALTK
aa 2101-2120
SEQ ID NO: 141


 


P142:
TALTK KYRIT ENDIQ IALDD
aa 2116-2135
SEQ ID NO: 142


 


P143:
IALDD AKINF NEKLS QLQTY
aa 2131-2150
SEQ ID NO: 143


 


P144:
QLQTY MIQFD QYIKD SYDLH
aa 2146-2165
SEQ ID NO: 144


 


P145:
SYDLH DLKIA IANII DEIIE
aa 2161-2180
SEQ ID NO: 145


 


P146:
DEIIE KLKSL DEHYH IRVNL
aa 2176-2195
SEQ ID NO: 146


 


P147:
IRVNL VKTIH DLHLF IENID
aa 2191-2210
SEQ ID NO: 147


 


P148:
IENID FNKSG SSTAS WIQNV
aa 2206-2225
SEQ ID NO: 148


 


P149:
WIQNV DTKYQ IRIQI QEKLQ
aa 2221-2240
SEQ ID NO: 149


 


P150:
QEKLQ QLKRH IQNID IQHLA
aa 2236-2255
SEQ ID NO: 150


 


P151:
IQHLA GKLKQ HIEAI DVRVL
aa 2251-2270
SEQ ID NO: 151


 


P152:
DVRVL LDQLG TTISF ERIND
aa 2266-2285
SEQ ID NO: 152


 


P153:
ERIND VLEHV KHFVI NLIGD
aa 2281-2300
SEQ ID NO: 153


 


P154:
NLIGD FEVAE KINAF RAKVH
aa 2296-2315
SEQ ID NO: 154


 


P155:
RAKVH ELIER YEVDQ QIQVL
aa 2311-2330
SEQ ID NO: 155


 


P156:
QIQVL MDKLV ELTHQ YKLKE
aa 2326-2345
SEQ ID NO: 156


 


P157:
YKLKE TIQKL SNVLQ QVKIK
aa 2341-2360
SEQ ID NO: 157


 


P158:
QVKIK DYFEK LVGFI DDAVK
aa 2356-2375
SEQ ID NO: 158


 


P159:
DDAVK KLNEL SFKTF IEDVN
aa 2371-2390
SEQ ID NO: 159


 


P160:
IEDVN KFLDM LIKKL KSFDY
aa 2386-2405
SEQ ID NO: 160


 


P161:
KSFDY HQFVD ETNDK IREVT
aa 2401-2420
SEQ ID NO: 161


 


P162:
IREVT QRLNG EIQAL ELPQK
aa 2416-2435
SEQ ID NO: 162


 


P163:
ELPQK AEALK LFLEE TKATV
aa 2431-2450
SEQ ID NO: 163


 


P164:
TKATV AVYLE SLQDT KITLI
aa 2446-2465
SEQ ID NO: 164


 


P165:
KITLI INWLQ EALSS ASLAH
aa 2461-2480
SEQ ID NO: 165


 


P166:
ASLAH MKAKF RETLE DTRDR
aa 2476-2495
SEQ ID NO: 166


 


P167:
DTRDR MYQMD IQQEL QRYLS
aa 2491-2510
SEQ ID NO: 167


 


P168:
QRYLS LVGQV YSTLV TYISD
aa 2506-2515
SEQ ID NO: 168


 


P169:
TYISD WWTLA AKNLT DFAEQ
aa 2521-2540
SEQ ID NO: 169


 


P170:
DFAEQ YSIQD WAKRM KALVE
aa 2536-2555
SEQ ID NO: 170


 


P171:
KALVE QGFTV PEIKT ILGTM
aa 2551-2570
SEQ ID NO: 171


 


P172:
ILGTM PAFEV SLQAL QKATF
aa 2566-2585
SEQ ID NO: 172


 


P173:
QKATF QTPDF IVPLT DLRIP
aa 2581-2600
SEQ ID NO: 173


 


P174:
DLRIP SVQIN FKDLK NIKIP
aa 2596-2615
SEQ ID NO: 174


 


P175:
NIKIP SRFST PEFTI LNTFH
aa 2611-2630
SEQ ID NO: 175


 


P176:
LNTFH IPSFT IDFVE MKVKI
aa 2626-2645
SEQ ID NO: 176


 


P177:
MKVKI IRTID QMQNS ELQWP
aa 2641-2660
SEQ ID NO: 177


 


P178:
ELQWP VPDIY LRDLK VEDIP
aa 2656-2675
SEQ ID NO: 178


 


P179:
VEDIP LARIT LPDFR LPEIA
aa 2671-2690
SEQ ID NO: 179


 


P180:
LPEIA IPEFI IPTLN LNDFQ
aa 2686-2705
SEQ ID NO: 180


 


P181:
LNDFQ VPDLH IPEFQ LPHIS
aa 2701-2720
SEQ ID NO: 181


 


P182:
LPHIS HTIEV PTFGK LYSIL
aa 2716-2735
SEQ ID NO: 182


 


P183:
LYSIL KIQSP LFTLD ANADI
aa 2731-2750
SEQ ID NO: 183


 


P184:
ANADI GNGTT SANEA GIAAS
aa 2746-2765
SEQ ID NO: 184


 


P185:
GIAAS ITAKG ESKLE VLNFD
aa 2761-2780
SEQ ID NO: 185


 


P186:
VLNFD FQANA QLSNP KINPL
aa 2776-2795
SEQ ID NO: 186


 


P187:
KINPL ALKES VKFSS KYLRT
aa 2791-2810
SEQ ID NO: 187


 


P188:
KYLRT EHGSE MLFFG NAIEG
aa 2806-2825
SEQ ID NO: 188


 


P189:
NAIEG KSNTV ASLHT EKNTL
aa 2821-2840
SEQ ID NO: 189


 


P190:
EKNTL ELSNG VIVKI NNQLT
aa 2836-2855
SEQ ID NO: 190


 


P191:
NNQLT LDSNT KYFHK LNIPK
aa 2851-2870
SEQ ID NO: 191


 


P192:
LNIPK LDFSS QADLR NEIKT
aa 2866-2885
SEQ ID NO: 192


 


P193:
NEIKT LLKAG HIAWT SSGKG
aa 2881-2900
SEQ ID NO: 193


 


P194:
SSGKG SWKWA CPRFS DEGTH
aa 2896-2915
SEQ ID NO: 194


 


P195:
DEGTH ESQIS FTIEG PLTSF
aa 2911-2930
SEQ ID NO: 195


 


P196:
PLTSF GLSNK INSKH LRVNQ
aa 2926-2945
SEQ ID NO: 196


 


P197:
LRVNQ NLVYE SGSLN FSKLE
aa 2941-2960
SEQ ID NO: 197


 


P198:
FSKLE IQSQV DSQHV GHSVL
aa 2956-2975
SEQ ID NO: 198


 


P199:
GHSVL TAKGM ALFGE GKAEF
aa 2971-2990
SEQ ID NO: 199


 


P200:
GKAEF TGRHD AHLNG KVIGT
aa 2986-3005
SEQ ID NO: 200


 


P201:
KVIGT LKNSL FFSAQ PFEIT
aa 3001-3020
SEQ ID NO: 201


 


P202:
PFEIT ASTNN EGNLK VRFPL
aa 3016-3035
SEQ ID NO: 202


 


P203:
VRFPL RLTGK IDFLN NYALF
aa 3031-3050
SEQ ID NO: 203


 


P204:
NYALF LSPSA QQASW QVSAR
aa 3046-3065
SEQ ID NO: 204


 


P205:
QVSAR FNQYK YNQNF SAGNN
aa 3061-3080
SEQ ID NO: 205


 


P206:
SAGNN ENIME AHVGI NGEAN
aa 3076-3095
SEQ ID NO: 206


 


P207:
NGEAN LDFLN IPLTI PEMRL
aa 3091-3110
SEQ ID NO: 207


 


P208:
PEMRL PYTII TTPPL KDFSL
aa 3106-3125
SEQ ID NO: 208


 


P209:
KDFSL WEKTG LKEFL KTTKQ
aa 3121-3140
SEQ ID NO: 209


 


P210:
KTTKQ SFDLS VKAQY KKNKH
aa 3136-3155
SEQ ID NO: 210


 


P211:
KKNKH RHSIT NPLAV LCEFI
aa 3151-3170
SEQ ID NO: 211


 


P212:
LCEFI SQSIK SFDRH FEKNR
aa 3166-3185
SEQ ID NO: 212


 


P213:
FEKNR NNALD FVTKS YNETK
aa 3181-3200
SEQ ID NO: 213


 


P214:
YNETK IKFDK YKAEK SHDEL
aa 3196-3215
SEQ ID NO: 214


 


P215:
SHDEL PRTFQ IPGYT VPVVN
aa 3211-3230
SEQ ID NO: 215


 


P216:
VPVVN VEVSP FTIEM SAFGY
aa 3226-3245
SEQ ID NO: 216


 


P217:
SAFGY VFPKA VSMPS FSILG
aa 3241-3260
SEQ ID NO: 217


 


P218:
FSILG SDVRV PSYTL ILPSL
aa 3256-3275
SEQ ID NO: 218


 


P219:
ILPSL ELPVL HVPRN LKLSL
aa 3271-3290
SEQ ID NO: 219


 


P220:
LKLSL PHFKE LCTIS HIFIP
aa 3286-3305
SEQ ID NO: 220


 


P221:
HIFIP AMGNI TYDFS FKSSV
aa 3301-3320
SEQ ID NO: 221


 


P222:
FKSSV ITLNT NAELF NQSDI
aa 3316-3335
SEQ ID NO: 222


 


P223:
NQSDI VAHLL SSSSS VIDAL
aa 3331-3350
SEQ ID NO: 223


 


P224:
VIDAL QYKLE GTTRL TRKRG
aa 3346-3365
SEQ ID NO: 224


 


P225:
TRKRG LKLAT ALSLS NKFVE
aa 3361-3380
SEQ ID NO: 225


 


P226:
NKFVE GSHNS TVSLT TKNME
aa 3376-3395
SEQ ID NO: 226


 


P227:
TKNME VSVAK TTKAE IPILR
aa 3391-3410
SEQ ID NO: 227


 


P228:
IPILR MNFKQ ELNGN TKSKP
aa 3406-3425
SEQ ID NO: 228


 


P229:
TKSKP TVSSS MEFKY DFNSS
aa 3421-3440
SEQ ID NO: 229


 


P230:
DFNSS MLYST AKGAV DHKLS
aa 3436-3455
SEQ ID NO: 230


 


P231:
DHKLS LESLT SYFSI ESSTK
aa 3451-3470
SEQ ID NO: 231


 


P232:
ESSTK GDVKG SVLSR EYSGT
aa 3466-3485
SEQ ID NO: 232


 


P233:
EYSGT IASEA NTYLN SKSTR
aa 3481-3500
SEQ ID NO: 233


 


P234:
SKSTR SSVKL QGTSK IDDIW
aa 3496-3515
SEQ ID NO: 234


 


P235:
IDDIW NLEVK ENFAG EATLQ
aa 3511-3530
SEQ ID NO: 235


 


P236:
EATLQ RIYSL WEHST KNHLQ
aa 3526-3545
SEQ ID NO: 236


 


P237:
KNHLQ LEGLF FTNGE HTSKA
aa 3541-3560
SEQ ID NO: 237


 


P238:
HTSKA TLELS PWQMS ALVQV
aa 3556-3575
SEQ ID NO: 238


 


P239:
ALVQV HASQP SSFHD FPDLG
aa 3571-3590
SEQ ID NO: 239


 


P240:
FPDLG QEVAL NANTK NQKIR
aa 3586-3605
SEQ ID NO: 240


 


P241:
NQKIR WKNEV RIHSG SFQSQ
aa 3601-3620
SEQ ID NO: 241


 


P242:
SFQSQ VELSN DQEKA HLDIA
aa 3616-3635
SEQ ID NO: 242


 


P243:
HLDIA GSLEG HLRFL KNIIL
aa 3631-3650
SEQ ID NO: 243


 


P244:
KNIIL PVYDK SLWDF LKLDV
aa 3646-3665
SEQ ID NO: 244


 


P245:
LKLDV TTSIG RRQHL RVSTA
aa 3661-3680
SEQ ID NO: 245


 


P246:
RVSTA FVYTK NPNGY SFSIP
aa 3676-3695
SEQ ID NO: 246


 


P247:
SFSIP VKVLA DKFIT PGLKL
aa 3691-3710
SEQ ID NO: 247


 


P248:
PGLKL NDLNS VLVMP TFHVP
aa 3706-3725
SEQ ID NO: 248


 


P249:
TFHVP FTDLQ VPSCK LDFRE
aa 3721-3740
SEQ ID NO: 249


 


P250:
LDFRE IQIYK KLRTS SFALN
aa 3736-3755
SEQ ID NO: 250


 


P251:
SFALN LPTLP EVKFP EVDVL
aa 3751-3770
SEQ ID NO: 251


 


P252:
EVDVL TKYSQ PEDSL IPFFE
aa 3766-3785
SEQ ID NO: 252


 


P253:
IPFFE ITVPE SQLTV SQFTL
aa 3781-3800
SEQ ID NO: 253


 


P254:
SQFTL PKSVS DGIAA LDLNA
aa 3796-3815
SEQ ID NO: 254


 


P255:
LDLNA VANKI ADFEL PTIIV
aa 3811-3830
SEQ ID NO: 255


 


P256:
PTIIV PEQTI EIPSI KFSVP
aa 3826-3845
SEQ ID NO: 256


 


P257:
KFSVP AGIVI PSFQA LTARF
aa 3841-3860
SEQ ID NO: 257


 


P258:
LTARF EVDSP VYNAT WSASL
aa 3856-3875
SEQ ID NO: 258


 


P259:
WSASL KNKAD YVETV LDSTC
aa 3871-3890
SEQ ID NO: 259


 


P260:
LDSTC SSTVQ FLEYE LNVLG
aa 3886-3905
SEQ ID NO: 260


 


P261:
LNVLG THKIE DGTLA SKTKG
aa 3901-3920
SEQ ID NO: 261


 


P262:
SKTKG TLAHR DFSAE YEEDG
aa 3916-3935
SEQ ID NO: 262


 


P263:
YEEDG KFEGL QEWEG KAHLN
aa 3931-3950
SEQ ID NO: 263


 


P264:
KAHLN IKSPA FTDLH LRYQK
aa 3946-3965
SEQ ID NO: 264


 


P265:
LRYQK DKKGI STSAA SPAVG
aa 3961-3980
SEQ ID NO: 265


 


P266:
SPAVG TVGMD MDEDD DFSKW
aa 3976-3995
SEQ ID NO: 266


 


P267:
DFSKW NFYYS PQSSP DKKLT
aa 3991-4010
SEQ ID NO: 267


 


P268:
DKKLT IFKTE LRVRE SDEET
aa 4006-4025
SEQ ID NO: 268


 


P269:
SDEET QIKVN WEEEA ASGLL
aa 4021-4040
SEQ ID NO: 269


 


P270:
ASGLL TSLKD NVPKA TGVLY
aa 4036-4055
SEQ ID NO: 270


 


P271:
TGVLY DYVNK YHWEH TGLTL
aa 4051-4070
SEQ ID NO: 271


 


P272:
TGLTL REVSS KLRRN LQNNA
aa 4066-4085
SEQ ID NO: 272


 


P273:
LQNNA EWVYQ GAIRQ IDDID
aa 4081-4100
SEQ ID NO: 273


 


P274:
IDDID VRFQK AASGT TGTYQ
aa 4096-4115
SEQ ID NO: 274


 


P275:
TGTYQ EWKDK AQNLY QELLT
aa 4111-4130
SEQ ID NO: 275


 


P276:
QELLT QEGQA SFQGL KDNVF
aa 4126-4145
SEQ ID NO: 276


 


P277:
KDNVF DGLVR VTQKF HMKVK
aa 4141-4160
SEQ ID NO: 277


 


P278:
HMKVK HLIDS LIDFL NFPRF
aa 4156-4175
SEQ ID NO: 278


 


P279:
NFPRF QFPGK PGIYT REELC
aa 4171-4190
SEQ ID NO: 279


 


P280:
REELC TMFIR EVGTV LSQVY
aa 4186-4205
SEQ ID NO: 280


 


P281:
LSQVY SKVHN GSEIL FSYFQ
aa 4201-4220
SEQ ID NO: 281


 


P282:
FSYFQ DLVIT LPFEL RKHKL
aa 4216-4235
SEQ ID NO: 282


 


P283:
RKHKL IDVIS MYREL LKDLS
aa 4231-4250
SEQ ID NO: 283


 


P284:
LKDLS KEAQE VFKAI QSLKT
aa 4246-4265
SEQ ID NO: 284


 


P285:
QSLKT TEVLR NLQDL LQFIF
aa 4261-4280
SEQ ID NO: 285


 


P286:
LQFIF QLIED NIKQL KEMKF
aa 4276-4295
SEQ ID NO: 286


 


P287:
KEMKF TYLIN YIQDE INTIF
aa 4291-4310
SEQ ID NO: 287


 


P288:
INTIF NDYIP YVFKL LKENL
aa 4306-4325
SEQ ID NO: 288


 


P289:
LKENL CLNLH KFNEF IQNEL
aa 4321-4340
SEQ ID NO: 289


 


P290:
IQNEL QEASQ ELQQI HQYIM
aa 4336-4355
SEQ ID NO: 290


 


P291:
HQYIM ALREE YFDPS IVGWT
aa 4351-4370
SEQ ID NO: 291


 


P292:
IVGWT VKYYE LEEKI VSLIK
aa 4366-4385
SEQ ID NO: 292


 


P293:
VSLIK NLLVA LKDFH SEYIV
aa 4381-4400
SEQ ID NO: 293


 


P294:
SEYIV SASNF TSQLS SQVEQ
aa 4396-4415
SEQ ID NO: 294


 


P295:
SQVEQ FLHRN IQEYL SILTD
aa 4411-4430
SEQ ID NO: 295


 


P296:
SILTD PDGKG KEKIA ELSAT
aa 4426-4445
SEQ ID NO: 296


 


P297:
ELSAT AQEII KSQAI ATKKI
aa 4441-4460
SEQ ID NO: 297


 


P298:
TKKII SDYHQ QFRYK LQDFS
aa 4457-4476
SEQ ID NO: 298


 


P299:
LQDFS DQLSD YYEKF IAESK
aa 4472-4491
SEQ ID NO: 299


 


P300:
IAESK RLIDL SIQNY HTFLI
aa 4487-4506
SEQ ID NO: 300


 


P301:
HTFLI YITEL LKKLQ STTVM
aa 4502-4521
SEQ ID NO: 301


 


P302:
STTVM NPYMK LAPGE LTIIL
aa 4517-4536
SEQ ID NO: 302









The full length sequence of ApoB100 can be found in various publications such as reference (43) (see in particular FIG. 1) herein incorporated by reference in its entirety.
Example 2
Athero-Protective Effects of p210 Immunization
Immunization with p210 reduced aortic atherosclerosis by 57% and 50% compared to PBS and cBSA/Alum group, respectively (FIG. 1A) without affecting circulating cholesterol levels or body weight (Table 3).







TABLE 3







Circulating level of cholesterol and body weight of mice from PBS,


cBSA/alum and p210/cBSA/alum group












PBS
cBSA/alum
P210/cBSA/alum
P value



(n = 10)
(n = 10)
(n = 10)
(ANOVA)















Cholesterol
1503 ± 485
1395 ± 420
1135 ± 382
0.17


(mg/dl)


Body
37.9 ± 5.4
34.8 ± 5.4
34.3 ± 6.5
0.33


weight (gm)









The aortic sinus plaques from p210/cBSA/alum group contained significantly reduced macrophage and DC immuno-reactivity assessed by MOMA-2 and CD11c immuno-staining, respectively (FIG. 1B).
Example 3
Characterization of p210-Immunization Elicited Immune Responses
Since DCs are the major cell type upstream to both cellular and humoral immune responses, Applicants determined if these cells were affected by the immunization strategy. Cells from the subcutaneous immunization sites were isolated for flow cytometric analysis one week after primary immunization. The PBS group could not be included in this analysis because mice receiving PBS injection did not develop swelling or cell accumulation at the injection site.
There were significantly fewer CD11c(+) and CD11c(+)CD86(+) cells in p210/cBSA/alum group compared to cBSA/alum group at the immunization site (FIGS. 2A and 2B). When flow cytometry was performed on LN cells 1 week after the third immunization, CD11c(+)CD86(+) cells were also significantly reduced compared with cBSA/alum group (FIG. 2C).
Applicants next assessed antibody response to define the humoral immune response against p210. Before immunization all 3 groups of mice had low levels of IgG titers against p210. At euthanasia, the IgG titer against p210 remained low in the PBS group but was significantly increased in cBSA/alum group. Immunization with p210/cBSA/alum resulted in increased p210 IgG titer compared with PBS group but was significantly reduced compared with cBSA/alum group (FIG. 3A). In contrast to p210 IgG response, there was a significant increase in p210 IgM titer in all groups (FIG. 3B), suggesting an endogenous immune response against p210.
The IL-2Rα (CD25) is a well-defined lymphocyte activation marker. Applicants therefore analyzed the expression of CD25 on CD4(+) or CD8(+) T-cells from superficial cervical and axillary lymph nodes (LN) from mice one week after primary immunization to assess the T-cell immune response. CD8(+)CD25(+) T-cell population in the lymph nodes was significantly higher in p210/cBSA/alum group when compared to that of PBS or cBSA/alum groups (FIG. 4A) whereas CD4(+)CD25(+) T-cells in the lymph nodes (FIG. 4B) did not differ among 3 groups.
There was a significantly larger population of splenic CD8(+)CD25(+)IL-10(+) T-cells in p210/cBSA/alum group when compared to PBS or cBSA/alum groups (FIG. 4C) without difference in splenic CD8(+)CD25(+)IL12(+) T-cells among 3 groups (FIG. 4D). Splenic CD4(+)CD25(+)IL-10(+) T-cell population significantly increased in the cBSA/alum group. However, this increased response was significantly attenuated by the p210/cBSA/alum immunization (FIG. 4E); whereas splenic CD4(+)CD25(+)IL12(+) T-cells did not differ among the three groups (FIG. 4F).
Example 4
Adoptive Transfer of CD8(+) T-Cells from p210 Immunized Mice to Naive Recipients Recapitulates the Athero-Protective Effect of p210 Immunization
Donor CD8(+)T-cells from PBS, cBSA/alum or p210/cBSA/alum groups were adoptively transferred to 6-7 week-old unimmunized recipient apoE (−/−) mice. At euthanasia, the recipient mice injected with CD8(+) T-cells from p210/cBSA/alum group developed significantly less atherosclerotic lesions in aorta compared to the recipient mice injected with CD8(+) T-cells from PBS or cBSA/alum groups (FIG. 5A).
This reduction of aortic lesions was coupled with decreased splenic CD11c(+) DCs (PBS group: 4.3±1.7%; cBSA/alum group: 3.4±0.3%; p210/cBSA/alum group: 1.5±0.3%; n=5 each group, p<0.05 p210/cBSA/alum group vs. PBS or cBSA/alum group by ANOVA) with no difference in circulating levels of total cholesterol among 3 groups (PBS group: 1083±296 mg/dl; cBSA/alum group: 975±401 mg/dl; p210/cBSA/alum group: 1098±379 mg/dl).
Adoptive transfer of B cells isolated from the spleens of p210 immunized donor mice did not affect atherosclerosis in recipient mice compared to mice receiving B cells from other donors (FIG. 5B).
To rule out CD4(+)CD25(+) T-cells as possible athero-protective mediators induced by sub-cutaneous p210 immunization, Applicants adoptively transferred CD4(+)CD25(+)T-cells at a dose of 1×105 cells/mouse into naïve recipient apoE−/− mice. There was no difference in lesion size among the 3 groups of CD4(+)CD25(+)T-cell recipients (FIG. 5C). Transfer of a higher number of CD4(+)CD25(+) T-cells at 3×105 cells/mouse did not reduce lesion sizes in all 3 recipient groups (FIG. 5D).
Example 5
Increased Cytolytic Activity of CD8(+) T Cells from p210 Immunized Mice Against Dendritic Cells In Vitro
Given the observation that p210 immunization reduced DCs in the immunization sites and atherosclerotic plaques and adoptive transfer of CD8(+) T-cells from p210 immunized donors rendered a decrease of splenic DCs in the recipients, Applicants hypothesized that DCs could be a potential target of CD8(+) T-cells.
To test this, Applicants co-cultured bone marrow derived DCs with CD8(+) T-cells from various immunized groups. CD8(+) T-cells from p210 immunized mice significantly increased the percentage of DC death when compared to those from PBS or BSA/alum groups (FIG. 6). This increased cytolytic function of CD8(+) T-cells was associated with increased granzyme B expression but not perforin (FIG. 7).
Example 6
Immunization with p210 does not Affect the Adaptive Immune Response to Other T-Cell Dependent or Independent Antigens
Given the observations that p210 immunization decreased CD11c(+) DCs and reduced adaptive IgG response to p210, Applicants next tested if such modulation of DCs by p210 immunization would alter the host immune response to other antigens.
Applicants first immunized mice with p210 as described in previous sections followed by two separate subcutaneous KLH immunizations or intra-peritoneal injection of TNP-LPS. Using the KLH- or TNP-IgG titer as a surrogate for the efficacy of individual immunization, Applicants found that there was no difference in KLH- or TNP-IgG titers between p210 immunized mice and the titers from mice of PBS or cBSA/alum groups (FIG. 8).
Additional examples are described in US provisional Ser. No. 61/261,331 and in particular in Appendix A of said provisional which is herein incorporated in its entirety. In summary in several embodiments, Immunostimulatory T cell, compositions, methods and systems are described for treating and/or preventing atherosclerosis and/or a condition associated thereto in an individual.
The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the T cell, compositions, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Modifications of the above-described modes for carrying out the disclosure that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents, patent applications and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference.
Further, the copy of the sequence listing submitted herewith in computer readable form forms part of the present description and is incorporated herein by reference in its entireties.
It is to be understood that the disclosures are not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “plurality” includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.
Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the products, methods and system of the present disclosure, exemplary appropriate materials and methods are described herein as examples.
A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.
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## Claims

1. A pharmaceutical composition comprising:
a quantity of T cells, wherein the T cells consist of ex vivo activated CD8(+) T cells activated by one or more immunogenic fragments of ApoB100 associated with atherosclerosis reduction,
wherein the activated CD8(+) T cell is specific for the one or more immunogenic fragments of ApoB100 and
wherein the one or more immunogenic fragments are selected from SEQ ID NO: 1 to SEQ ID NO: 302.

2. The pharmaceutical composition of claim 1, wherein the one or more immunogenic fragments is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 10, (SEQ ID NO: 11, SEQ ID NO: 25, SEQ ID NOs: 30-34, SEQ ID NO: 40, SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 94, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 129, SEQ ID NO: 143, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 154, SEQ ID NO: 162, SEQ ID NO: 169, SEQ ID NO: 177, SEQ ID NO: 199, SEQ ID NO: 210, SEQ ID NO: 222, SEQ ID NO: 236, SEQ ID NO: 252, and SEQ ID NO: 301.

3. The pharmaceutical composition of claim 1, wherein the one or more immunogenic fragments is selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 11, SEQ ID NO: 32, SEQ ID NO: 45, SEQ ID NO: 74, SEQ ID NO: 102, SEQ ID NO: 148, SEQ ID NO: 162, and SEQ ID NO: 210.

4. The pharmaceutical composition of claim 1, wherein the one or more immunogenic fragments comprise a peptide having SEQ ID NO: 143 and a peptide having SEQ ID NO: 210.

5. The pharmaceutical composition of claim 1, wherein the one or more immunogenic fragments comprise a peptide having SEQ ID NO: 11, a peptide having SEQ ID NO: 25 and a peptide having SEQ ID NO: 74.

6. The pharmaceutical composition of claim 1, wherein the immunogenic fragment of ApoB100 comprises a peptide having the sequence set forth in SEQ ID NO: 2 or an immunogenic fragment thereof.

7. The pharmaceutical composition of claim 1, wherein the immunogenic fragment of ApoB100 comprises a peptide having the sequence set forth in SEQ ID NO: 45 or an immunogenic fragment thereof.

8. The pharmaceutical composition of claim 1, wherein the immunogenic fragment of ApoB100 comprises a peptide having the sequence set forth in SEQ ID NO: 102 or an immunogenic fragment thereof.

9. The pharmaceutical composition of claim 1, wherein the immunogenic fragment of ApoB100 comprises a peptide having the sequence set forth in SEQ ID NO: 210 or an immunogenic fragment thereof.

10. The pharmaceutical composition of claim 1, wherein the quantity of CD8+ T cells is about 500,000 to about 2,000,000 activated CD8(+) T cells.

11. The pharmaceutical composition of claim 1, wherein the quantity of CD8+ T cells is about 1,500,000.

12. The pharmaceutical composition of claim 1, wherein the quantity of CD8+ T cells is about 1,000,000.