US20160193316A1 - Inducing Cellular Immune Responses to Plasmodium Falciparum Using Peptide and Nucleic Acid Compositions - Google Patents

Inducing Cellular Immune Responses to Plasmodium Falciparum Using Peptide and Nucleic Acid Compositions Download PDF

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US20160193316A1
US20160193316A1 US14/980,150 US201514980150A US2016193316A1 US 20160193316 A1 US20160193316 A1 US 20160193316A1 US 201514980150 A US201514980150 A US 201514980150A US 2016193316 A1 US2016193316 A1 US 2016193316A1
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peptide
hla
seq
epitopes
peptides
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Alessandro Sette
John Sidney
Scott Southwood
Brian D. Livingston
Robert Chestnut
Denise Marie Baker
Esteban Celis
Ralph T. Kubo
Howard M. Grey
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Epimmune Inc
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Epimmune Inc
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Priority claimed from US08/821,739 external-priority patent/US20020168374A1/en
Priority claimed from US09/017,743 external-priority patent/US20020177694A1/en
Priority claimed from US09/189,702 external-priority patent/US7252829B1/en
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Priority to US14/980,150 priority Critical patent/US20160193316A1/en
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Definitions

  • PF Plasmodium falciparum
  • Anti-sporozoite antibodies are by themselves, in general, not completely efficacious in clearing the infection (Egan et al., Science 236:453, 1987). However, high concentrations of antibodies directed against the repeated region of the major B cell antigen of the sporozoite/circumsporozoite protein (CSP) have been shown to prevent liver cell infection in certain experimental models (Egan et al., Science 236:453, 1987; Potocnjak, P. et al., Science 207:71, 1980).
  • CSP central sporozoite/circumsporozoite protein
  • constructs encompassing CSP-repeat B cell epitopes and the optimized helper epitope PADRETM are highly immunogenic, and can protect in vitro against sporozoite invasion in both mouse and human liver cells, and protect mice in vivo against live sporozoite challenge (Franke et al., Vaccine 17:1201-1205, 1999)
  • PF-specific CD4 + T cells also have a role in malarial immunity beyond providing help for B cell and CTL responses.
  • Experiments by Renia et al. (Renia, et al., Proc. Natl. Acad. Sci. USA 88:7963, 1991) demonstrated that HTLs directed against the Plasmodium yoelli CS protein could in fact adoptivley transfer protection against malaria.
  • CD8 + CTLs can eliminate Plasmodium berghei - or Plasmodium yoelii -infected mouse hepatocytes from in vitro culture in a major histocompatibility complex (MHC)-restricted and antigen-restricted manner (Hoffman et al., Science 244:1078-1081, 1989; Weiss et al., J. Exp. Med. 171:763-773, 1990). Further, it has also been shown that the immunity that developed in mice vaccinated with irradiated sporozoites is also dependent upon the present of CD8+ T cells.
  • MHC major histocompatibility complex
  • CSP circumsporozoite
  • HLA-Bw53 MHC class I human leukocyte antigen (HLA)-Bw53 has been associated with resistance to severe malaria in The Gambia, and CTLs to a conserved epitope restricted by the HLA-Bw53 allele have been identified on P. falciparum LSA-1 (Hill et al., Nature 352:595-600, 1991; Hill et al., Nature 340:434-439, 1992). Since HLA-Bw53 is found in 15%-40% of the population of sub-Saharan Africa but in less than 1% of Caucasians and Asians, these data suggest evolutionary selection on the basis of protection against severe malaria.
  • HLA class I molecules are expressed on the surface of almost all nucleated cells. Following intracellular processing of antigens, epitopes from the antigens are presented as a complex with the HLA class I molecules on the surface of such cells. CTL recognize the peptide-HLA class I complex, which then results in the destruction of the cell bearing the HLA-peptide complex directly by the CTL and/or via the activation of non-destructive mechanisms e.g., the production of interferon.
  • This invention applies our knowledge of the mechanisms by which antigen is recognized by T cells, for example, to develop epitope-based vaccines directed towards PF. More specifically, this application communicates our discovery of specific epitope pharmaceutical compositions and methods of use in the prevention and treatment of PF infection.
  • epitope-based vaccines Upon development of appropriate technology, the use of epitope-based vaccines has several advantages over current vaccines, particularly when compared to the use of whole antigens in vaccine compositions. There is evidence that the immune response to whole antigens is directed largely toward variable regions of the antigen, allowing for immune escape due to mutations.
  • the epitopes for inclusion in an epitope-based vaccine are selected from conserved regions of antigens of pathogenic organisms or tumor-associated antigens, which thereby reduces the likelihood of escape mutants. Furthermore, immunosuppressive epitopes that may be present in whole antigens can be avoided with the use of epitope-based vaccines.
  • An additional advantage of an epitope-based vaccine approach is the ability to combine selected epitopes (CTL and HTL), and further, to modify the composition of the epitopes, achieving, for example, enhanced immunogenicity. Accordingly, the immune response can be modulated, as appropriate, for the target disease. Similar engineering of the response is not possible with traditional approaches.
  • epitope-based immune-stimulating vaccines Another major benefit of epitope-based immune-stimulating vaccines is their safety. The possible pathological side effects caused by infectious agents or whole protein antigens, which might have their own intrinsic biological activity, is eliminated.
  • An epitope-based vaccine also provides the ability to direct and focus an immune response to multiple selected antigens from the same pathogen. Thus, patient-by-patient variability in the immune response to a particular pathogen may be alleviated by inclusion of epitopes from multiple antigens from that pathogen in a vaccine composition.
  • a “pathogen” may be an infectious agent or a tumor associated molecule.
  • a need has existed to modulate peptide binding properties, e.g., so that peptides that are able to bind to multiple HLA antigens do so with an affinity that will stimulate an immune response.
  • Identification of epitopes restricted by more than one HLA allele at an affinity that correlates with immunogenicity is important to provide thorough population coverage, and to allow the elicitation of responses of sufficient vigor to prevent or clear an infection in a diverse segment of the population. Such a response can also target a broad array of epitopes.
  • the technology disclosed herein provides for such favored immune responses.
  • epitopes for inclusion in vaccine compositions of the invention are selected by a process whereby protein sequences of known antigens are evaluated for the presence of motif or supermotif-bearing epitopes. Peptides corresponding to a motif- or supermotif-bearing epitope are then synthesized and tested for the ability to bind to the HLA molecule that recognizes the selected motif. Those peptides that bind at an intermediate or high affinity i.e., an IC 50 (or a K D value) of 500 nM or less for HLA class I molecules or an IC 50 of 1000 nM or less for HLA class II molecules, are further evaluated for their ability to induce a CTL or HTL response. Immunogenic peptide epitopes are selected for inclusion in vaccine compositions.
  • Supermotif-bearing peptides may additionally be tested for the ability to bind to multiple alleles within the HLA supertype family.
  • peptide epitopes may be analogued to modify binding affinity and/or the ability to bind to multiple alleles within an HLA supertype.
  • the invention also includes embodiments comprising methods for monitoring or evaluating an immune response to PF in patient having a known HLA-type.
  • Such methods comprise incubating a T cell sample from the patient with a peptide composition comprising an PF epitope consisting essentially of an amino acid sequence described in Tables VII to Table XX or Table XXII which binds the product of at least one HLA allele present in the patient, and detecting for the presence of a T cell that binds to the peptide.
  • a CTL peptide epitope may, for example, be used as a component of a tetrameric complex for such an analysis.
  • An alternative modality for defining the peptide epitopes in accordance with the invention is to recite the physical properties, such as length; primary structure; or charge, which are correlated with binding to a particular allele-specific HLA molecule or group of allele-specific HLA molecules.
  • a further modality for defining peptide epitopes is to recite the physical properties of an HLA binding pocket, or properties shared by several allele-specific HLA binding pockets (e.g. pocket configuration and charge distribution) and reciting that the peptide epitope fits and binds to said pocket or pockets.
  • FIG. 1 provides a graph of total frequency of genotypes as a function of the number of PF candidate epitopes bound by HLA-A and B molecules, in an average population.
  • the peptide epitopes and corresponding nucleic acid compositions of the present invention are useful for stimulating an immune response to PF by stimulating the production of CTL or HTL responses.
  • the peptide epitopes which are derived directly or indirectly from native PF protein amino acid sequences, are able to bind to HLA molecules and stimulate an immune response to PF.
  • the complete sequence of the PF proteins to be analyzed can be obtained from Genbank.
  • Peptide epitopes and analogs thereof can also be readily determined from sequence information that may subsequently be discovered for heretofore unknown variants of PF, as will be clear from the disclosure provided below.
  • peptide epitopes of the invention have been identified in a number of ways, as will be discussed below. Also discussed in greater detail is that analog peptides have been derived and the binding activity for HLA molecules modulated by modifying specific amino acid residues to create peptide analogs exhibiting altered immunogenicity. Further, the present invention provides compositions and combinations of compositions that enable epitope-based vaccines that are capable of interacting with HLA molecules encoded by various genetic alleles to provide broader population coverage than prior vaccines.
  • a “computer” or “computer system” generally includes: a processor; at least one information storage/retrieval apparatus such as, for example, a hard drive, a disk drive or a tape drive; at least one input apparatus such as, for example, a keyboard, a mouse, a touch screen, or a microphone; and display structure. Additionally, the computer may include a communication channel in communication with a network. Such a computer may include more or less than what is listed above.
  • Cross-reactive binding indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.
  • a “cryptic epitope” elicits a response by immunization with an isolated peptide, but the response is not cross-reactive in vitro when intact whole protein which comprises the epitope is used as an antigen.
  • a “dominant epitope” is an epitope that induces an immune response upon immunization with a whole native antigen (see, e.g., Sercarz, et al., Annu. Rev. Immunol. 11:729-766, 1993). Such a response is cross-reactive in vitro with an isolated peptide epitope.
  • an “epitope” is a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor (TCR) proteins and/or Major Histocompatibility Complex (MHC) receptors.
  • TCR T cell receptor
  • MHC Major Histocompatibility Complex
  • an epitope is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, TCR or HLA molecule.
  • epitope and peptide are often used interchangeably. It is to be appreciated, however, that isolated or purified protein or peptide molecules larger than and comprising an epitope of the invention are still within the bounds of the invention.
  • Human Leukocyte Antigen or “HLA” is a human class I or class II MHC protein (see, e.g., Stites, et al., I MMUNOLOGY , 8 TH E D ., Lange Publishing, Los Altos, Calif. (1994).
  • HLA supertype or family describes sets of HLA molecules grouped on the basis of shared peptide-binding specificities. HLA class I molecules that share somewhat similar binding affinity for peptides bearing certain amino acid motifs are grouped into HLA supertypes.
  • HLA superfamily, HLA supertype family, HLA family, and HLA xx-like molecules are synonyms.
  • IC 50 is the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide is observed. Given the conditions in which the assays are run (i.e., limiting HLA proteins and labeled peptide concentrations), these values approximate K D values. Assays for determining binding are described in detail, e.g., in PCT publications WO 94/20127 and WO 94/03205. It should be noted that IC 50 values can change, often dramatically, if the assay conditions are varied, and depending on the particular reagents used (e.g., HLA preparation, etc.). For example, excessive concentrations of HLA molecules will increase the apparent measured IC 50 of a given ligand.
  • binding is expressed relative to a reference peptide.
  • the IC 50 's of the peptides tested may change somewhat, the binding relative to the reference peptide will not significantly change.
  • the assessment of whether a peptide is a good, intermediate, weak, or negative binder is generally based on its IC 50 , relative to the IC 50 of a standard peptide.
  • Binding may also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al., Nature 339:392, 1989; Christnick et al., Nature 352:67, 1991; Busch et al., Immunol. 2:443, 1990; Hill et al., J. Immunol. 147:189, 1991; del Guercio et al., J. Immunol. 154:685, 1995), cell free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21:2069, 1991), immobilized purified MHC (e.g., Hill et al., J. Immunol.
  • high affinity with respect to HLA class I molecules is defined as binding with an IC 50 , or K D value, of 50 nM or less; “intermediate affinity” is binding with an IC 50 or K D value of between about 50 and about 500 nM.
  • High affinity with respect to binding to HLA class II molecules is defined as binding with an IC 50 or K D value of 100 nM or less; “intermediate affinity” is binding with an IC 50 or K D value of between about 100 and about 1000 nM.
  • nucleic or percent “identity,” in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.
  • immunogenic peptide or “peptide epitope” is a peptide that comprises an allele-specific motif or supermotif such that the peptide will bind an HLA molecule and induce a CTL and/or HTL response.
  • immunogenic peptides of the invention are capable of binding to an appropriate HLA molecule and thereafter inducing a cytotoxic T cell response, or a helper T cell response, to the antigen from which the immunogenic peptide is derived.
  • isolated or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state.
  • isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.
  • MHC Major Histocompatibility Complex
  • motif refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule.
  • Peptide motifs are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.
  • a “negative binding residue” or “deleterious residue” is an amino acid which, if present at certain positions (typically not primary anchor positions) in a peptide epitope, results in decreased binding affinity of the peptide for the peptide's corresponding HLA molecule.
  • peptide is used interchangeably with “oligopeptide” in the present specification to designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the alpha-amino and carboxyl groups of adjacent amino acids.
  • the preferred CTL-inducing peptides of the invention are 13 residues or less in length and usually consist of between about 8 and about 11 residues, preferably 9 or 10 residues.
  • the preferred HTL-inducing peptides are less than about 50 residues in length and usually consist of between about 6 and about 30 residues, more usually between about 12 and 25, and often between about 15 and 20 residues.
  • “Pharmaceutically acceptable” refers to a non-toxic, inert, and physiologically compatible composition.
  • a “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule.
  • One to three, usually two, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding grooves of an HLA molecule, with their side chains buried in specific pockets of the binding grooves themselves.
  • the primary anchor residues are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 9-residue peptide epitope in accordance with the invention.
  • the primary anchor positions for each motif and supermotif are set forth in Table 1.
  • analog peptides can be created by altering the presence or absence of particular residues in these primary anchor positions. Such analogs are used to modulate the binding affinity of a peptide comprising a particular motif or supermotif.
  • Promiscuous recognition is where a distinct peptide is recognized by the same T cell clone in the context of various HLA molecules. Promiscuous recognition or binding is synonymous with cross-reactive binding.
  • a “protective immune response” or “therapeutic immune response” refers to a CTL and/or an HTL response to an antigen derived from an infectious agent or a tumor antigen, which prevents or at least partially arrests disease symptoms or progression.
  • the immune response may also include an antibody response which has been facilitated by the stimulation of helper T cells.
  • residue refers to an amino acid or amino acid mimetic incorporated into an oligopeptide by an amide bond or amide bond mimetic.
  • a “secondary anchor residue” is an amino acid at a position other than a primary anchor position in a peptide which may influence peptide binding.
  • a secondary anchor residue occurs at a significantly higher frequency amongst bound peptides than would be expected by random distribution of amino acids at one position.
  • the secondary anchor residues are said to occur at “secondary anchor positions.”
  • a secondary anchor residue can be identified as a residue which is present at a higher frequency among high or intermediate affinity binding peptides, or a residue otherwise associated with high or intermediate affinity binding.
  • analog peptides can be created by altering the presence or absence of particular residues in these secondary anchor positions. Such analogs are used to finely modulate the binding affinity of a peptide comprising a particular motif or supermotif.
  • a “subdominant epitope” is an epitope which evokes little or no response upon immunization with whole antigens which comprise the epitope, but for which a response can be obtained by immunization with an isolated peptide, and this response (unlike the case of cryptic epitopes) is detected when whole protein is used to recall the response in vitro or in vivo.
  • a “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles.
  • a supermotif-bearing peptide is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.
  • Synthetic peptide refers to a peptide that is not naturally occurring, but is man-made using such methods as chemical synthesis or recombinant DNA technology.
  • each residue is generally represented by standard three letter or single letter designations.
  • the L -form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol
  • the D -form for those amino acids having D -forms is represented by a lower case single letter or a lower case three letter symbol.
  • Glycine has no asymmetric carbon atom and is simply referred to as “Gly” or G. Symbols for the amino acids are shown below.
  • T cells recognize antigens The mechanism by which T cells recognize antigens has been delineated during the past ten years. Based on our understanding of the immune system we have developed efficacious peptide epitope vaccine compositions that can induce a therapeutic or prophylactic immune response to PF in a broad population. For an understanding of the value and efficacy of the claimed compositions, a brief review of immunology-related technology is provided.
  • a complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993).
  • class I and class II allele-specific HLA binding motifs allows identification of regions within a protein that have the potential of binding particular HLA antigen(s).
  • the present inventors have found that the correlation of binding affinity with immunogenicity, which is disclosed herein, is an important factor to be considered when evaluating candidate peptides.
  • candidates for epitope-based vaccines have been identified.
  • additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, antigenicity, and immunogenicity.
  • HLA transgenic mice see, e.g., Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997);
  • peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice.
  • splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week.
  • Peptide-specific T cells are detected using, e.g., a 51 Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.
  • recall responses are detected by culturing PBL from subjects that have been naturally exposed to the antigen, for instance through infection, and thus have generated an immune response “naturally”, or from patients who were vaccinated against the infection.
  • PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of “memory” T cells, as compared to “naive” T cells.
  • APC antigen presenting cells
  • T cell activity is detected using assays for T cell activity including 51 Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.
  • epitope selection encompassing identification of peptides capable of binding at high or intermediate affinity to multiple HLA molecules is preferably utilized, most preferably these epitopes bind at high or intermediate affinity to two or more allele-specific HLA molecules.
  • CTL-inducing peptides of interest for vaccine compositions preferably include those that have an IC 50 or binding affinity value for class I HLA molecules of 500 nM or better (i.e., the value is ⁇ 500 nM).
  • HTL-inducing peptides preferably include those that have an IC 50 or binding affinity value for class II HLA molecules of 1000 nM or better, (i.e., the value is ⁇ 1,000 nM).
  • peptide binding is assessed by testing the capacity of a candidate peptide to bind to a purified HLA molecule in vitro. Peptides exhibiting high or intermediate affinity are then considered for further analysis. Selected peptides are tested on other members of the supertype family. In preferred embodiments, peptides that exhibit cross-reactive binding are then used in cellular screening analyses or vaccines.
  • HLA binding affinity is correlated with greater immunogenicity.
  • Greater immunogenicity can be manifested in several different ways. Immunogenicity corresponds to whether an immune response is elicited at all, and to the vigor of any particular response, as well as to the extent of a population in which a response is elicited. For example, a peptide might elicit an immune response in a diverse array of the population, yet in no instance produce a vigorous response. In accordance with these principles, close to 90% of high binding peptides have been found to be immunogenic, as contrasted with about 50% of the peptides which bind with intermediate affinity. Moreover, higher binding affinity peptides leads to more vigorous immunogenic responses. As a result, less peptide is required to elicit a similar biological effect if a high affinity binding peptide is used. Thus, in preferred embodiments of the invention, high affinity binding epitopes are particularly useful.
  • binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes on bound antigens has been determined for the first time in the art by the present inventors.
  • the correlation between binding affinity and immunogenicity was analyzed in two different experimental approaches (see, e.g., Sette, et al., J. Immunol. 153:5586-5592, 1994).
  • the immunogenicity of potential epitopes ranging in HLA binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice.
  • HBV hepatitis B virus
  • DR restriction was associated with intermediate affinity (binding affinity values in the 100-1000 nM range). In only one of 32 cases was DR restriction associated with an IC 50 of 1000 nM or greater.
  • 1000 nM can be defined as an affinity threshold associated with immunogenicity in the context of DR molecules.
  • the binding affinity of peptides for HLA molecules can be determined as described in Example 1, below.
  • Peptides of the present invention may also comprise epitopes that bind to MEW class II DR molecules.
  • This increased heterogeneity of HLA class II peptide ligands is due to the structure of the binding groove of the HLA class II molecule which, unlike its class I counterpart, is open at both ends. Crystallographic analysis of HLA class II DRB*0101-peptide complexes showed that the major energy of binding is contributed by peptide residues complexed with complementary pockets on the DRB*0101 molecules.
  • P1 may represent the N-terminal residue of a class II binding peptide epitope, but more typically is flanked towards the N-terminus by one or more residues. Other studies have also pointed to an important role for the peptide residue in the 6 th position towards the C-terminus, relative to P1, for binding to various DR molecules.
  • HLA class I and class II molecules can be classified into a relatively few supertypes, each characterized by largely overlapping peptide binding repertoires, and consensus structures of the main peptide binding pockets.
  • peptides of the present invention are identified by any one of several HLA-specific amino acid motifs (see, e.g., Tables or if the presence of the motif corresponds to the ability to bind several allele-specific HLA antigens, a supermotif.
  • the HLA molecules that bind to peptides that possess a particular amino acid supermotif are collectively referred to as an HLA “supertype.”
  • peptide epitopes bearing a respective supermotif or motif are included in Tables as designated in the description of each motif or supermotif below.
  • the IC 50 values of standard peptides used to determine binding affinities for Class I peptides are shown in Table IV.
  • the IC 50 values of standard peptides used to determine binding affinities for Class II peptides are shown in Table V.
  • the peptides used as standards for the binding assays described herein are examples of standards; alternative standard peptides can also be used when performing binding analyses.
  • peptide epitope sequences listed in each Table protein sequence data for four P. falciparum antigens were evaluated for the presence of the designated supermotif or motif. These antigens are: EXP-1, LSA-1, SSP2, and CSP. Nineteen sequences were available for CSP, 10 sequences were available for SSP, and one sequence each was available for EXP-1 and LSA-1. Peptide epitopes were additionally evaluated on the basis of their conservancy among the protein sequences for the PF antigens for which multiple sequences were available. A criterion for conservancy requires that the entire sequence of an HLA class I binding peptide be totally (i.e., 100%) conserved in 79% of the sequences available for a specific protein.
  • a criterion for conservancy requires that the entire 9-mer core region of an HLA class II binding peptide be totally conserved in 79% of the sequences available for a specific protein.
  • the percent conservancy of the selected peptide epitopes is indicated on the Tables.
  • the frequency, i.e. the number of sequences of the PF protein antigen in which the totally conserved peptide sequence was identified, is also shown.
  • the “pos” (position) column in the Tables designates the amino acid position in the PF protein that corresponds to the first amino acid residue of the epitope.
  • the “number of amino acids” indicates the number of residues in the epitope sequence.
  • HLA class I peptide epitope supermotifs and motifs delineated below are summarized in Table I.
  • the HLA class I motifs set out in Table I(a) are those most particularly relevant to the invention claimed here.
  • Primary and secondary anchor positions are summarized in Table II.
  • Allele-specific HLA molecules that comprise HLA class I supertype families are listed in Table VI.
  • peptide epitopes may be listed in both a motif and a supermotif Table. The relationship of a particular motif and respective supermotif is indicated in the description of the individual motifs.
  • the HLA-A1 supermotif is characterized by the presence in peptide ligands of a small (T or S) or hydrophobic (L, I, V, or M) primary anchor residue in position 2, and an aromatic (Y, F, or W) primary anchor residue at the C-terminal position of the epitope (see, e.g., Sette and Sidney, Immunogenetics , in press, 1999).
  • the corresponding family of HLA molecules that bind to the A1 supermotif i.e., the HLA-A1 supertype
  • is comprised of at least A*0101, A*2601, A*2602, A*2501, and A*3201 see, e.g., DiBrino, M. et al., J.
  • HLA-A2 supermotif which presence in peptide ligands corresponds to the ability to bind several different HLA-A2 and -A28 molecules.
  • the HLA-A2 supermotif comprises peptide ligands with L, I, V, M, A, T, or Q as a primary anchor residue at position 2 and L, I, V, M, A, or T as a primary anchor residue at the C-terminal position of the epitope.
  • the corresponding family of HLA molecules (i.e., the HLA-A2 supertype that binds these peptides) is comprised of at least: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*0209, A*0214, A*6802, and A*6901.
  • Other allele-specific HLA molecules predicted to be members of the A2 supertype are shown in Table VI.
  • binding to each of the individual allele-specific HLA molecules can be modulated by substitutions at the primary anchor and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • peptide epitopes that comprise an A2 supermotif are set forth on the attached Table VIII.
  • the motifs comprising the primary anchor residues V, A, T, or Q at position 2 and L, I, V, A, or T at the C-terminal position are those most particularly relevant to the invention claimed herein.
  • the HLA-A3 supermotif is characterized by the presence in peptide ligands of A, L, I, V, M, S, or, T as a primary anchor at position 2, and a positively charged residue, R or K, at the C-terminal position of the epitope, e.g., in position 9 of 9-mers (see, e.g., Sidney et al., Hum. Immunol. 45:79, 1996).
  • Exemplary members of the corresponding family of HLA molecules (the HLA-A3 supertype) that bind the A3 supermotif include at least A*0301, A*1101, A*3101, A*3301, and A*6801.
  • allele-specific HLA molecules predicted to be members of the A3 supertype are shown in Table VI.
  • peptide binding to each of the individual allele-specific HLA proteins can be modulated by substitutions of amino acids at the primary and/or secondary anchor positions of the peptide, preferably choosing respective residues specified for the supermotif.
  • the HLA-A24 supermotif is characterized by the presence in peptide ligands of an aromatic (F, W, or Y) or hydrophobic aliphatic (L, I, V, M, or T) residue as a primary anchor in position 2, and Y, F, W, L, I, or M as primary anchor at the C-terminal position of the epitope (see, e.g., Sette and Sidney, Immunogenetics , in press, 1999).
  • the corresponding family of HLA molecules that bind to the A24 supermotif i.e., the A24 supertype
  • Other allele-specific HLA molecules predicted to be members of the A24 supertype are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • Representative peptide epitopes that comprise the A24 supermotif are set forth on the attached Table X.
  • the HLA-B7 supermotif is characterized by peptides bearing proline in position 2 as a primary anchor, and a hydrophobic or aliphatic amino acid (L, I, V, M, A, F, W, or Y) as the primary anchor at the C-terminal position of the epitope.
  • the corresponding family of HLA molecules that bind the B7 supermotif is comprised of at least twenty six HLA-B proteins including: B*0702, B*0703, B*0704, B*0705, B*1508, B*3501, B*3502, B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101, B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501, B*5502, B*5601, B*5602, B*6701, and B*7801 (see, e.g., Sidney, et al., J. Immunol.
  • the HLA-B27 supermotif is characterized by the presence in peptide ligands of a positively charged (R, H, or K) residue as a primary anchor at position 2, and a hydrophobic (F, Y, L, W, M, I, A, or V) residue as a primary anchor at the C-terminal position of the epitope (see, e.g., Sidney and Sette, Immunogenetics , in press, 1999).
  • Exemplary members of the corresponding family of HLA molecules that bind to the B27 supermotif include at least B*1401, B*1402, B*1509, B*2702, B*2703, B*2704, B*2705, B*2706, B*3801, B*3901, B*3902, and B*7301.
  • Other allele-specific HLA molecules predicted to be members of the B27 supertype are shown in Table VI.
  • Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • the HLA-B44 supermotif is characterized by the presence in peptide ligands of negatively charged (D or E) residues as a primary anchor in position 2, and hydrophobic residues (F, W, Y, L, I, M, V, or A) as a primary anchor at the C-terminal position of the epitope (see, e.g., Sidney et al., Immunol. Today 17:261, 1996).
  • Exemplary members of the corresponding family of HLA molecules that bind to the B44 supermotif include at least: B*1801, B*1802, B*3701, B*4001, B*4002, B*4006, B*4402, B*4403, and B*4006.
  • Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions; preferably choosing respective residues specified for the supermotif.
  • the HLA-B58 supermotif is characterized by the presence in peptide ligands of a small aliphatic residue (A, S, or T) as a primary anchor residue at position 2, and an aromatic or hydrophobic residue (F, W, Y, L, I, V, M, or A) as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Sidney and Sette, Immunogenetics , in press, 1999 for reviews of relevant data).
  • Exemplary members of the corresponding family of HLA molecules that bind to the B58 supermotif include at least: B*1516, B*1517, B*5701, B*5702, and B*5801.
  • Allele-specific HLA molecules predicted to be members of the B58 supertype are shown in Table VI.
  • Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • the HLA-B62 supermotif is characterized by the presence in peptide ligands of the polar aliphatic residue Q or a hydrophobic aliphatic residue (L, V, M, I, or P) as a primary anchor in position 2, and a hydrophobic residue (F, W, Y, M, I, V, L, or A) as a primary anchor at the C-terminal position of the epitope (see, e.g., Sidney and Sette, Immunogenetics , in press, 1999).
  • Exemplary members of the corresponding family of HLA molecules that bind to the B62 supermotif include at least: B*1501, B*1502, B*1513, and B5201.
  • Allele-specific HLA molecules predicted to be members of the B62 supertype are shown in Table VI.
  • Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • the HLA-A1 motif is characterized by the presence in peptide ligands of T, S, or M as a primary anchor residue at position 2 and the presence of Y as a primary anchor residue at the C-terminal position of the epitope.
  • An alternative allele-specific A1 motif is characterized by a primary anchor residue at position 3 rather than position 2. This motif is characterized by the presence of D, E, A, or S as a primary anchor residue in position 3, and a Y as a primary anchor residue at the C-terminal position of the epitope (see, e.g., DiBrino et al., J.
  • Peptide binding to HLA A1 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.
  • peptide epitopes that comprise either A1 motif are set forth on the attached Table XV. Those epitopes comprising T, S, or M at position 2 and Y at the C-terminal position are also included in the listing of HLA-A1 supermotif-bearing peptide epitopes listed in Table VII, as these residues are a subset of the A1 supermotif primary anchors.
  • HLA-A2*0201 motif was determined to be characterized by the presence in peptide ligands of L or M as a primary anchor residue in position 2, and L or V as a primary anchor residue at the C-terminal position of a 9-residue peptide (see, e.g., Falk et al., Nature 351:290-296, 1991) and was further found to comprise an I at position 2 and I or A at the C-terminal position of a nine amino acid peptide (see, e.g., Hunt et al., Science 255:1261-1263, Mar. 6, 1992; Parker et al., J. Immunol. 149:3580-3587, 1992).
  • the A*0201 allele-specific motif has also been defined by the present inventors to additionally comprise V, A, T, or Q as a primary anchor residue at position 2, and M or T as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Kast et al., J. Immunol. 152:3904-3912, 1994).
  • the HLA-A*0201 motif comprises peptide ligands with L, I, V, M, A, T, or Q as primary anchor residues at position 2 and L, I, V, M, A, or T as a primary anchor residue at the C-terminal position of the epitope.
  • A*0201 motif Representative peptide epitopes that comprise an A*0201 motif are set forth on the attached Table VIII.
  • the A*0201 motifs comprising the primary anchor residues V, A, T, or Q at position 2 and L, I, V, A, or T at the C-terminal position are those most particularly relevant to the invention claimed herein
  • the HLA-A3 motif is characterized by the presence in peptide ligands of L, M, V, I, S, A, T, F, C, G, or D as a primary anchor residue at position 2, and the presence of K, Y, R, H, F, or A as a primary anchor residue at the C-terminal position of the epitope (see, e.g., DiBrino et al., Proc. Natl. Acad. Sci USA 90:1508, 1993; and Kubo et al., J. Immunol. 152:3913-3924, 1994).
  • Peptide binding to HLA-A3 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.
  • A3 motif Representative peptide epitopes that comprise the A3 motif are set forth on the attached Table XVI. Those peptide epitopes that also comprise the A3 supermotif are also listed in Table IX.
  • the A3 supermotif primary anchor residues comprise a subset of the A3- and A11-allele specific motif primary anchor residues.
  • the HLA-A11 motif is characterized by the presence in peptide ligands of V, T, M, L, I, S, A, G, N, C, D, or F as a primary anchor residue in position 2, and K, R, Y, or H as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Zhang et al., Proc. Natl. Acad. Sci USA 90:2217-2221, 1993; and Kubo et al., J. Immunol. 152:3913-3924, 1994).
  • Peptide binding to HLA-A11 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.
  • peptide epitopes that comprise the A11 motif are set forth on the attached Table XVII; peptide epitopes comprising the A3 allele-specific motif are also present in this Table because of the extensive overlap between the A3 and A11 motif primary anchor specificities. Further, those peptide epitopes that comprise the A3 supermotif are also listed in Table IX.
  • the HLA-A24 motif is characterized by the presence in peptide ligands of Y, F, W, or M as a primary anchor residue in position 2, and F, L, I, or W as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Kondo et al., J. Immunol. 155:4307-4312, 1995; and Kubo et al., J. Immunol. 152:3913-3924, 1994).
  • Peptide binding to HLA-A24 molecules can be modulated by substitutions at primary and/or secondary anchor positions; preferably choosing respective residues specified for the motif.
  • peptide epitopes that comprise the A24 motif are set forth on the attached Table XVIII. These epitopes are also listed in Table X, which sets forth HLA-A24-supermotif-bearing peptide epitopes, as the primary anchor residues characterizing the A24 allele-specific motif comprise a subset of the A24 supermotif primary anchor residues.
  • HLA DRB1*0401 HLA DRB1*0401
  • DRB1*0101 HLA DRB1*0101
  • DRB1*0701 HLA DRB1*0401
  • HLA DRB1*0101 HLA DRB1*0101
  • DRB1*0701 HLA DRB1*0701
  • Peptides that bind to these DR molecules carry a supermotif characterized by a large aromatic or hydrophobic residue (Y, F, W, L, I, V, or M) as a primary anchor residue in position 1, and a small, non-charged residue (S, T, C, A, P, V, I, L, or M) as a primary anchor residue in position 6 of a 9-mer core region. Allele-specific secondary effects and secondary anchors for each of these HLA types have also been identified (Southwood et al., supra). These are set forth in Table III. Peptide binding to HLA-DRB1*0401, DRB1*0101, and/or DRB1*0701 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • conserved 9-mer core regions i.e., sequences that are 100% conserved in at least 79% of the PF antigen protein sequences used for the analysis
  • conserved 9-mer core regions comprising the DR-1-4-7 supermotif, wherein position 1 of the supermotif is at position 1 of the nine-residue core
  • Respective exemplary peptide epitopes of 15 amino acid residues in length, each of which comprise a conserved nine residue core are also shown in section “a” of the Table.
  • Cross-reactive binding data for exemplary 15-residue supermotif-bearing peptides are shown in Table XIXb.
  • motifs characterize peptide epitopes that bind to HLA-DR3 molecules (see, e.g., Geluk et al., J. Immunol. 152:5742, 1994).
  • first motif (submotif DR3A) a large, hydrophobic residue (L, I, V, M, F, or Y) is present in anchor position 1 of a 9-mer core, and D is present as an anchor at position 4, towards the carboxyl terminus of the epitope.
  • core position 1 may or may not occupy the peptide N-terminal position.
  • the alternative DR3 submotif provides for lack of the large, hydrophobic residue at anchor position 1, and/or lack of the negatively charged or amide-like anchor residue at position 4, by the presence of a positive charge at position 6 towards the carboxyl terminus of the epitope.
  • L, I, V, M, F, Y, A, or Y is present at anchor position 1; D, N, Q, E, S, or T is present at anchor position 4; and K, R, or H is present at anchor position 6.
  • Peptide binding to HLA-DR3 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.
  • conserved 9-mer core regions i.e., those sequences that are 100% conserved in at least 79% of the PF antigen protein sequences used for the analysis
  • a nine residue sequence comprising the DR3A submotif wherein position 1 of the motif is at position 1 of the nine residue core
  • Respective exemplary peptide epitopes of 15 amino acid residues in length, each of which comprise a conserved nine residue core are also shown in Table XXa.
  • Table XXb shows binding data of exemplary DR3 submotif A-bearing peptides.
  • each of the HLA class I or class II peptide epitopes set out in the Tables herein are deemed singly to be an inventive aspect of this application. Further, it is also an inventive aspect of this application that each peptide epitope may be used in combination with any other peptide epitope.
  • Vaccines that have broad population coverage are preferred because they are more commercially viable and generally applicable to the most people. Broad population coverage can be obtained using the peptides of the invention (and nucleic acid compositions that encode such peptides) through selecting peptide epitopes that bind to HLA alleles which, when considered in total, are present in most of the population.
  • Table XXI lists the overall frequencies of the HLA class I supertypes in various ethnicities (Table XXIa) and the combined population coverage achieved by the A2-, A3-, and B7-supertypes (Table XXIb). The A2-, A3-, and B7 supertypes are each present on the average of over 40% in each of these five major ethnic groups.
  • the B44-, A1-, and A24-supertypes are present, on average, in a range from 25% to 40% in these major ethnic populations (Table XXIa). While less prevalent overall, the B27-, B58-, and B62 supertypes are each present with a frequency >25% in at least one major ethnic group (Table XXIa).
  • Table XXIb summarizes the estimated prevalence of combinations of HLA supertypes that have been identified in five major ethnic groups. The incremental coverage obtained by the inclusion of A1-, A24-, and B44-supertypes with the A2, A3, and B7 coverage and coverage obtained with all of the supertypes described herein, is shown.
  • CTL and HTL responses are not directed against all possible epitopes. Rather, they are restricted to a few “immunodominant” determinants (Zinkernagel, et al., Adv. Immunol. 27:5159, 1979; Bennink, et al., J. Exp. Med. 168:19351939, 1988; Rawle, et al., J. Immunol. 146:3977-3984, 1991).
  • dominance and subdominance is relevant to immunotherapy of both infectious diseases and cancer.
  • recruitment of subdominant epitopes can be important for successful clearance of the infection, especially if dominant CTL or HTL specificities have been inactivated by functional tolerance, suppression, mutation of viruses and other mechanisms (Franco, et al., Curr. Opin. Immunol. 7:524-531, 1995).
  • CTLs recognizing at least some of the highest binding affinity peptides might be functionally inactivated. Lower binding affinity peptides are preferentially recognized at these times, and may therefore be preferred in therapeutic or prophylactic anti-cancer vaccines.
  • TAA tumor infiltrating lymphocytes
  • CTL tumor infiltrating lymphocytes
  • T cells to dominant epitopes may have been clonally deleted, selecting subdominant epitopes may allow existing T cells to be recruited, which will then lead to a therapeutic or prophylactic response.
  • the binding of HLA molecules to subdominant epitopes is often less vigorous than to dominant ones. Accordingly, there is a need to be able to modulate the binding affinity of particular immunogenic epitopes for one or more HLA molecules, and thereby to modulate the immune response elicited by the peptide, for example to prepare analog peptides which elicit a more vigorous response. This ability would greatly enhance the usefulness of peptide epitope-based vaccines and therapeutic agents.
  • peptides with suitable cross-reactivity among all alleles of a superfamily are identified by the screening procedures described above, cross-reactivity is not always as complete as possible, and in certain cases procedures to increase cross-reactivity of peptides can be useful; moreover, such procedures can also be used to modify other properties of the peptides such as binding affinity or peptide stability. Having established the general rules that govern cross-reactivity of peptides for HLA alleles within a given motif or supermotif, modification (i.e., analoging) of the structure of peptides of particular interest in order to achieve broader (or otherwise modified) HLA binding capacity can be performed.
  • peptides which exhibit the broadest cross-reactivity patterns can be produced in accordance with the teachings herein.
  • the present concepts related to analog generation are set forth in greater detail in U.S. Ser. No. 09/226,775 filed Jan. 6, 1999, now abandoned.
  • the strategy employed utilizes the motifs or supermotifs which correlate with binding to certain HLA molecules.
  • the motifs or supermotifs are defined by having primary anchors, and in many cases secondary anchors.
  • Analog peptides can be created by substituting amino acid residues at primary anchor, secondary anchor, or at primary and secondary anchor positions.
  • analogs are made for peptides that already bear a motif or supermotif.
  • Preferred secondary anchor residues of supermotifs and motifs that have been defined for HLA class I and class II binding peptides are shown in Tables II and III, respectively.
  • residues are defined which are deleterious to binding to allele-specific HLA molecules or members of HLA supertypes that bind the respective motif or supermotif (Tables II and III). Accordingly, removal of such residues that are detrimental to binding can be performed in accordance with the present invention.
  • the incidence of cross-reactivity increased from 22% to 37% (see, e.g., Sidney, J. et al., Hu. Immunol. 45:79, 1996).
  • one strategy to improve the cross-reactivity of peptides within a given supermotif is simply to delete one or more of the deleterious residues present within a peptide and substitute a small “neutral” residue such as Ala (that may not influence T cell recognition of the peptide).
  • An enhanced likelihood of cross-reactivity is expected if, together with elimination of detrimental residues within a peptide, “preferred” residues associated with high affinity binding to an allele-specific HLA molecule or to multiple HLA molecules within a superfamily are inserted.
  • the analog peptide when used as a vaccine, actually elicits a CTL response to the native epitope in vivo (or, in the case of class II epitopes, elicits helper T cells that cross-react with the wild type peptides), the analog peptide may be used to immunize T cells in vitro from individuals of the appropriate HLA allele. Thereafter, the immunized cells' capacity to induce lysis of wild type peptide sensitized target cells is evaluated.
  • antigen presenting cells cells that have been either infected, or transfected with the appropriate genes, or, in the case of class II epitopes only, cells that have been pulsed with whole protein antigens, to establish whether endogenously produced antigen is also recognized by the relevant T cells.
  • Another embodiment of the invention is to create analogs of weak binding peptides.
  • Class I binding peptides exhibiting binding affinities of 500-5000 nM, and carrying an acceptable but suboptimal primary anchor residue at one or both positions can be “fixed” by substituting preferred anchor residues in accordance with the respective supertype.
  • the analog peptides can then be tested for crossbinding activity.
  • Another embodiment for generating effective peptide analogs involves the substitution of residues that have an adverse impact on peptide stability or solubility in, e.g., a liquid environment. This substitution may occur at any position of the peptide epitope.
  • a cysteine (C) can be substituted out in favor of ⁇ -amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity.
  • Representative analog peptides are set forth in Table XXII.
  • the Table indicates the length and sequence of the analog peptide as well as the motif or supermotif, if appropriate.
  • the information in the “Fixed Nomenclature” column indicates the residues substituted at the indicated position numbers for the respective analog.
  • a native protein sequence e.g., a tumor-associated antigen, or sequences from an infectious organism, or a donor tissue for transplantation
  • a means for computing such as an intellectual calculation or a computer
  • the information obtained from the analysis of native peptide can be used directly to evaluate the status of the native peptide or may be utilized subsequently to generate the peptide epitope.
  • Computer programs that allow the rapid screening of protein sequences for the occurrence of the subject supermotifs or motifs are encompassed by the present invention; as are programs that permit the generation of analog peptides. These programs are implemented to analyze any identified amino acid sequence or operate on an unknown sequence and simultaneously determine the sequence and identify motif-bearing epitopes thereof; analogs can be simultaneously determined as well.
  • the identified sequences will be from a pathogenic organism or a tumor-associated peptide.
  • the target molecules considered herein include, without limitation, the EXP1, LSA1, SSP2, and CSP1 proteins of PF.
  • peptides may also be selected on the basis of their conservancy.
  • a presently preferred criterion for conservancy defines that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide, be totally (i.e., 100%) conserved in at least 79% of the sequences evaluated for a specific protein. This definition of conservancy has been employed herein; although, as appreciated by those in the art, lower or higher degrees of conservancy can be employed as appropriate for a given antigenic target.
  • ⁇ G a 1i ⁇ a 2i ⁇ a 3i . . . ⁇ a ni
  • a ji is a coefficient that represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids.
  • Additional methods to identify preferred peptide sequences include the use of neural networks and molecular modeling programs (see, e.g., Milik et al., Nature Biotechnology 16:753, 1998; Altuvia et al., Hum. Immunol. 58:1, 1997; Altuvia et al, J. Mol. Biol. 249:244, 1995 ; Buus, S. Curr. Opin. Immunol. 11:209-213, 1999; Brusic, V. et al., Bioinformatics 14:121-130, 1998; Parker et al., J. Immunol.
  • a protein sequence or translated sequence may be analyzed using software developed to search for motifs, for example the “FINDPATTERNS’ program (Devereux, et al. Nucl. Acids Res. 12:387-395, 1984) or MotifSearch 1.4 software program (D. Brown, San Diego, Calif.) to identify potential peptide sequences containing appropriate HLA binding motifs.
  • the identified peptides can be scored using customized polynomial algorithms to predict their capacity to bind specific HLA class I or class II alleles.
  • Peptides in accordance with the invention can be prepared synthetically, by recombinant DNA technology or chemical synthesis, or from natural sources such as native tumors or pathogenic organisms.
  • Peptide epitopes may be synthesized individually or as polyepitopic peptides.
  • the peptide will preferably be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides may be synthetically conjugated to native fragments or particles.
  • the peptides in accordance with the invention can be a variety of lengths, and either in their neutral (uncharged) forms or in forms which are salts.
  • the peptides in accordance with the invention are either free of modifications such as glycosylation, side chain oxidation, or phosphorylation; or they contain these modifications, subject to the condition that modifications do not destroy the biological activity of the peptides as described herein.
  • the peptide epitope will be as small as possible while still maintaining substantially all of the immunologic activity of the native protein.
  • HLA class II binding peptide epitopes may be optimized to a length of about 6 to about 30 amino acids in length, preferably to between about 13 and about 20 residues.
  • the peptide epitopes are commensurate in size with endogenously processed pathogen-derived peptides or tumor cell peptides that are bound to the relevant HLA molecules.
  • peptides of other lengths can also be carried out using the techniques described herein. Moreover, it is preferred to identify native peptide regions that contain a high concentration of class I and/or class II epitopes. Such a sequence is generally selected on the basis that it contains the greatest number of epitopes per amino acid length. It is to be appreciated that epitopes can be present in a frame-shifted manner, e.g. a 10 amino acid long peptide could contain two 9 amino acid long epitopes and one 10 amino acid long epitope; upon intracellular processing, each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. This larger, preferably multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source.
  • the peptides of the invention can be prepared in a wide variety of ways.
  • the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols. (See, for example, Stewart & Young, S OLID P HASE P EPTIDE S YNTHESIS , 2 D. ED ., Pierce Chemical Co., 1984).
  • individual peptide epitopes can be joined using chemical ligation to produce larger peptides that are still within the bounds of the invention.
  • recombinant DNA technology can be employed wherein a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • These procedures are generally known in the art, as described generally in Sambrook et al., M OLECULAR C LONING, A L ABORATORY M ANUAL , Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).
  • recombinant polypeptides which comprise one or more peptide sequences of the invention can be used to present the appropriate T cell epitope.
  • nucleotide coding sequence for peptide epitopes of the preferred lengths contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al., J. Am. Chem. Soc. 103:3185 (1981). Peptide analogs can be made simply by substituting the appropriate and desired nucleic acid base(s) for those that encode the native peptide sequence; exemplary nucleic acid substitutions are those that encode an amino acid defined by the motifs/supermotifs herein.
  • the coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein.
  • the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host.
  • promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence.
  • the resulting expression vectors are transformed into suitable bacterial hosts.
  • yeast, insect or mammalian cell hosts may also be used, employing suitable vectors and control sequences.
  • HLA binding peptides Once HLA binding peptides are identified, they can be tested for the ability to elicit a T-cell response.
  • the preparation and evaluation of motif-bearing peptides are described in PCT publications WO 94/20127 and WO 94/03205. Briefly, peptides comprising epitopes from a particular antigen are synthesized and tested for their ability to bind to the appropriate HLA proteins. These assays may involve evaluating the binding of a peptide of the invention to purified HLA class I molecules in relation to the binding of a radioiodinated reference peptide. Alternatively, cells expressing empty class I molecules (i.e. lacking peptide therein) may be evaluated for peptide binding by immunofluorescent staining and flow microfluorimetry.
  • peptide binding examples include peptide-dependent class I assembly assays and/or the inhibition of CTL recognition by peptide competition. Those peptides that bind to the class I molecule, typically with an affinity of 500 nM or less, are further evaluated for their ability to serve as targets for CTLs derived from infected or immunized individuals, as well as for their capacity to induce primary in vitro or in vivo CTL responses that can give rise to CTL populations capable of reacting with selected target cells associated with a disease. Corresponding assays are used for evaluation of HLA class II binding peptides. HLA class II motif-bearing peptides that are shown to bind, typically at an affinity of 1000 nM or less, are further evaluated for the ability to stimulate HTL responses.
  • T cell responses include proliferation assays, lymphokine secretion assays, direct cytotoxicity assays, and limiting dilution assays.
  • antigen-presenting cells that have been incubated with a peptide can be assayed for the ability to induce CTL responses in responder cell populations.
  • Antigen-presenting cells can be normal cells such as peripheral blood mononuclear cells or dendritic cells.
  • mutant non-human mammalian cell lines that are deficient in their ability to load class I molecules with internally processed peptides and that have been transfected with the appropriate human class I gene, may be used to test for the capacity of the peptide to induce in vitro primary CTL responses.
  • PBMCs Peripheral blood mononuclear cells
  • the appropriate antigen-presenting cells are incubated with peptide, after which the peptide-loaded antigen-presenting cells are then incubated with the responder cell population under optimized culture conditions.
  • Positive CTL activation can be determined by assaying the culture for the presence of CTLs that kill radio-labeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed forms of the antigen from which the peptide sequence was derived.
  • HTL activation may also be assessed using such techniques known to those in the art such as T cell proliferation and secretion of lymphokines, e.g. IL-2 (see, e.g. Alexander et al., Immunity 1:751-761, 1994).
  • HLA transgenic mice can be used to determine immunogenicity of peptide epitopes.
  • transgenic mouse models including mice with human A2.1, A11 (which can additionally be used to analyze HLA-A3 epitopes), and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed.
  • HLA-DR1 and HLA-DR3 mouse models have also been developed. Additional transgenic mouse models with other HLA alleles may be generated as necessary.
  • Mice may be immunized with peptides emulsified in Incomplete Freund's Adjuvant and the resulting T cells tested for their capacity to recognize peptide-pulsed target cells and target cells transfected with appropriate genes.
  • CTL responses may be analyzed using cytotoxicity assays described above.
  • HTL responses may be analyzed using such assays as T cell proliferation or secretion of lymphokines.
  • Exemplary immunogenic peptide epitopes are set out in Table XXIII
  • HLA class I and class II binding peptides as described herein can be used, in one embodiment of the invention, as reagents to evaluate an immune response.
  • the immune response to be evaluated may be induced by using as an immunogen any agent that may result in the production of antigen-specific CTLs or HTLs that recognize and bind to the peptide epitope(s) to be employed as the reagent.
  • the peptide reagent need not be used as the immunogen.
  • Assay systems that may be used for such an analysis include relatively recent technical developments such as tetramers, staining for intracellular lymphokines and interferon release assays, or ELISPOT assays.
  • a peptide of the invention may be used in a tetramer staining assay to assess peripheral blood mononuclear cells for the presence of antigen-specific CTLs following exposure to a pathogen or immunogen.
  • the HLA-tetrameric complex is used to directly visualize antigen-specific CTLs (see, e.g., Ogg et al., Science 279:2103-2106, 1998; and Altman et al., Science 174:94-96, 1996) and determine the frequency of the antigen-specific CTL population in a sample of peripheral blood mononuclear cells.
  • a tetramer reagent using a peptide of the invention may be generated as follows: A peptide that binds to an HLA molecule is refolded in the presence of the corresponding HLA heavy chain and ⁇ 2 -microglobulin to generate a trimolecular complex. The complex is biotinylated at the carboxyl terminal end of the heavy chain at a site that was previously engineered into the protein. Tetramer formation is then induced by the addition of streptavidin. By means of fluorescently labeled streptavidin, the tetramer can be used to stain antigen-specific cells. The cells may then be identified, for example, by flow cytometry. Such an analysis may be used for diagnostic or prognostic purposes.
  • Peptides of the invention may also be used as reagents to evaluate immune recall responses.
  • patient PBMC samples from individuals infected with PF may be analyzed for the presence of antigen-specific CTLs or HTLs using specific peptides.
  • a blood sample containing mononuclear cells may be evaluated by cultivating the PBMCs and stimulating the cells with a peptide of the invention. After an appropriate cultivation period, the expanded cell population may be analyzed, for example, for CTL or for HTL activity.
  • the peptides may also be used as reagents to evaluate the efficacy of a vaccine.
  • PBMCs obtained from a patient vaccinated with an immunogen may be analyzed using, for example, either of the methods described above.
  • the patient is HLA typed, and peptide epitope reagents that recognize the allele-specific molecules present in that patient are selected for the analysis.
  • the immunogenicity of the vaccine is indicated by the presence of PF epitope-specific CTLs and/or HTLs in the PBMC sample.
  • the peptides of the invention may also be used to make antibodies, using techniques well known in the art (see, e.g. CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley/Greene, NY; and Antibodies A Laboratory Manual Harlow , Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989), which may be useful as reagents to diagnose PF infection.
  • Such antibodies include those that recognize a peptide in the context of an HLA molecule, i.e., antibodies that bind to a peptide-WIC complex.
  • Vaccines that contain an immunogenically effective amount of one or more peptides as described herein are a further embodiment of the invention.
  • vaccine compositions that contain an immunogenically effective amount of one or more peptides as described herein.
  • Such vaccine compositions can include, for example, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol.
  • lipopeptides e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995
  • PLG poly(DL-lactide-co-glycolide)
  • Toxin-targeted delivery technologies also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.
  • vaccines in accordance with the invention encompass compositions of one or more of the claimed peptide(s).
  • the peptide(s) can be individually linked to its own carrier; alternatively, the peptide(s) can exist as a homopolymer or heteropolymer of active peptide units.
  • Such a polymer has the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response.
  • the composition may be a naturally occurring region of an antigen or may be prepared, e.g., recombinantly or by chemical synthesis.
  • useful carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L -lysine, poly L -glutamic acid, influenza, hepatitis B virus core protein, and the like.
  • the vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline.
  • the vaccines also typically include an adjuvant.
  • Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P 3 CSS).
  • P 3 CSS tripalmitoyl-S-glycerylcysteinlyseryl-serine
  • the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later infection, or at least partially resistant to developing an ongoing chronic infection, or derives at least some therapeutic benefit when the antigen was tumor-associated.
  • class I peptide vaccines of the invention may be desirable to combine with vaccines which induce or facilitate neutralizing antibody responses to the target antigen of interest, particularly to surface antigens.
  • a preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention.
  • An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a PADRETM (Epimmune, San Diego, Calif.) molecule (described, for example, in U.S. Pat. No. 5,736,142).
  • PADRETM Epimmune, San Diego, Calif.
  • any of these embodiments can be administered as a nucleic acid mediated modality.
  • the vaccine compositions of the invention may also be used in combination with antiviral drugs such as interferon- ⁇ .
  • the peptides of the invention can also be expressed by viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus, for example, as a vector to express nucleotide sequences that encode the peptides of the invention.
  • the recombinant vaccinia virus Upon introduction into an acutely or chronically infected host or into a non-infected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL and/or HTL response.
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S.
  • BCG Bacillus Calmette Guerin
  • BCG vectors are described in Stover et al., Nature 351:456-460 (1991).
  • a wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.
  • Antigenic peptides are used to elicit a CTL and/or HTL response ex vivo, as well. Ex vivo administration is described, for example, in application U.S. Ser. No. 09/016,361 filed Jan. 30, 1998, now abandoned.
  • the resulting CTL or HTL cells can be used to treat chronic infections, or tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention.
  • CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide.
  • APC antigen-presenting cells
  • the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (an infected cell or a tumor cell).
  • CTL destroy
  • HTL facilitate destruction
  • Transfected dendritic cells may also be used as antigen presenting cells.
  • dendritic cells are transfected, e.g., with a minigene construct in accordance with the invention, in order to elicit immune responses. Minigenes will be discussed in greater detail in a following section.
  • Vaccine compositions may also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.
  • DNA or RNA encoding one or more of the peptides of the invention can also be administered to a patient.
  • This approach is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720; and in more detail below.
  • DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).
  • the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene.
  • Exemplary epitopes that may be utilized in a vaccine to treat or prevent PF infection are set out in Tables XXXIII and XXXIV.
  • the multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.
  • Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with PF clearance.
  • this includes 3-4 epitopes that come from at least one antigen of PF.
  • HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one PF antigen (see e.g., Rosenberg et al., Science 278:1447-1450).
  • Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC 50 of 500 nM or less, or for Class II an IC 50 of 1000 nM or less.
  • Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage.
  • a Monte Carlo analysis a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.
  • epitopes from cancer-related antigens it is often preferred to select analogs because the patient may have developed tolerance to the native epitope.
  • selecting epitopes for infectious disease-related antigens it is preferable to select either native or analoged epitopes.
  • native or analoged epitopes Of particular relevance for infectious disease vaccines (but for cancer-related vaccines as well), are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence.
  • a peptide comprising “transcendent nested epitopes” is a peptide that has both HLA class I and HLA class II epitopes in it.
  • a sequence that has the greatest number of epitopes per provided sequence Preferably, one avoids providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide.
  • a longer peptide sequence such as a sequence comprising nested epitopes, it is important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.
  • an objective is to generate the smallest peptide possible that encompasses the epitopes of interest.
  • the principles employed are similar, if not the same as those employed when selecting a peptide comprising nested epitopes.
  • the peptide encoded thereby is analyzed to determine whether any “junctional epitopes” have been created.
  • a junctional epitope is an actual binding epitope, as predicted, e.g., by motif analysis, that only exists because two discrete peptide sequences are encoded directly next to each other.
  • junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
  • Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section.
  • a preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention. The use of multi-epitope minigenes is described below and in, e.g., application U.S. Ser. No. 09/311,784, now U.S. Pat. No.
  • a multi-epitope DNA plasmid encoding nine dominant HLA-A*0201- and A11-restricted epitopes derived from the polymerase, envelope, and core proteins of HBV and human immunodeficiency virus (HIV), the PADRETM universal HTL epitope, and an endoplasmic reticulum-translocating signal sequence was engineered. Immunization of HLA transgenic mice with this plasmid construct resulted in strong CTL induction responses against the nine epitopes tested, similar to those observed with a lipopeptide of known immunogenicity in humans, and significantly greater than immunization in oil-based adjuvants.
  • the amino acid sequences of the epitopes may be reverse translated.
  • a human codon usage table can be used to guide the codon choice for each amino acid.
  • These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created.
  • additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal.
  • HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.
  • the minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.
  • Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells.
  • a promoter with a down-stream cloning site for minigene insertion a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance).
  • Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
  • introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene.
  • mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.
  • the minigene is cloned into the polylinker region downstream of the promoter.
  • This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.
  • immunostimulatory sequences appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.
  • a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used.
  • proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRETM, Epimmune, San Diego, Calif.).
  • HTL epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction.
  • immunosuppressive molecules e.g. TGF- ⁇
  • TGF- ⁇ immunosuppressive molecules
  • Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli , followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
  • Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available.
  • Cationic lipids can also be used in the formulation; in addition, glycolipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) can also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987).
  • PINC protective, interactive, non-condensing compounds
  • Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes.
  • the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays.
  • the transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection.
  • a plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • HTL epitopes are then chromium-51 ( 51 Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51 Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.
  • In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations.
  • Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product.
  • the dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (IP) for lipid-complexed DNA).
  • splenocytes are harvested and restimulated for 1 week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, 51 Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is evaluated in transgenic mice in an analogous manner.
  • nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253.
  • particles comprised solely of DNA are administered.
  • DNA can be adhered to particles, such as gold particles.
  • Vaccine compositions comprising the peptides of the present invention, or analogs thereof, which have immunostimulatory activity may be modified to provide desired attributes, such as improved serum half life, or to enhance immunogenicity.
  • the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response.
  • T helper epitopes in conjunction with CTL epitopes to enhance immunogenicity is illustrated, for example, in applications U.S. Ser. No. 08/197,484, now U.S. Pat. No. 6,419,931, and U.S. Ser. No. 08/464,234, now abandoned.
  • Particularly preferred CTL epitope/HTL epitope conjugates are linked by a spacer molecule.
  • the spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions.
  • the spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids.
  • the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues.
  • the CTL peptide may be linked to the T helper peptide without a spacer.
  • the CTL peptide epitope may be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide.
  • the amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.
  • the HTL peptide epitopes used in the invention can be modified in the same manner as CTL peptides. For instance, they may be modified to include D -amino acids or be conjugated to other molecules such as lipids, proteins, sugars and the like.
  • the T helper peptide is one that is recognized by T helper cells present in the majority of the population. This can be accomplished by selecting amino acid sequences that bind to many, most, or all of the HLA class II molecules. These are known as “loosely HLA-restricted” or “promiscuous” T helper sequences.
  • amino acid sequences that are promiscuous include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO: 3799), Plasmodium falciparum CS protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 3800), and Streptococcus 18 kD protein at positions 116 (GAVDSILGGVATYGAA; SEQ ID NO: 3801).
  • Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.
  • An alternative of a pan-DR binding epitope comprises all “L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.
  • HTL peptide epitopes can also be modified to alter their biological properties.
  • peptides comprising HTL epitopes can contain D -amino acids to increase their resistance to proteases and thus extend their serum half-life.
  • the epitope peptides of the invention can be conjugated to other molecules such as lipids, proteins or sugars, or any other synthetic compounds, to increase their biological activity.
  • the T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.
  • lipids have been identified as agents capable of priming CTL in vivo against viral antigens.
  • palmitic acid residues can be attached to the ⁇ - and ⁇ -amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide.
  • the lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant.
  • a particularly effective immunogenic comprises palmitic acid attached to ⁇ - and ⁇ -amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.
  • E. coli lipoproteins such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P 3 CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide.
  • P 3 CSS tripalmitoyl-S-glycerylcysteinlyseryl-serine
  • P 3 CSS tripalmitoyl-S-glycerylcysteinlyseryl-serine
  • additional amino acids can be added to the termini of a peptide to provide for ease of linking peptides one to another, for coupling to a carrier support or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like.
  • Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced at the C- or N-terminus of the peptide or oligopeptide, particularly class I peptides.
  • modification at the carboxyl terminus of a CTL epitope may, in some cases, alter binding characteristics of the peptide.
  • the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH 2 acylation, e.g., by alkanoyl (C 1 -C 20 ) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.
  • peptides of the present invention and pharmaceutical and vaccine compositions of the invention are useful for administration to mammals, particularly humans, to treat and/or prevent malaria.
  • Vaccine compositions containing the peptides of the invention are administered to an individual susceptible to, or otherwise at risk for, malaria or to a patient infected with PF to elicit an immune response against PF antigens and thus enhance the patient's own immune response capabilities.
  • peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective CTL and/or HTL response to the PF antigen and to cure or at least partially arrest or slow symptoms and/or complications.
  • Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
  • the vaccine compositions of the invention may also be used purely as prophylactic agents.
  • the level of expected exposure e.g., a traveler versus a resident of an area where malaria is endemic determines the magnitude of response that is desired to be achieved by the vaccination. Therefore, some vaccination regimens may employ higher doses of the vaccine compositions, or more doses may be administered.
  • the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 ⁇ g and the higher value is about 10,000; 20,000; 30,000; or 50,000 ⁇ g.
  • Dosage values for a human typically range from about 500 ⁇ g to about 50,000 ⁇ g per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 ⁇ g to about 50,000 ⁇ g of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine.
  • the immunogenicity of the vaccine may be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.
  • peptides comprising CTL and/or HTL epitopes of the invention induce immune responses when presented by HLA molecules and contacted with a CTL or HTL specific for an epitope comprised by the peptide.
  • the manner in which the peptide is contacted with the CTL or HTL is not critical to the invention.
  • the peptide can be contacted with the CTL or HTL either in vivo or in vitro. If the contacting occurs in vivo, the peptide itself can be administered to the patient, or other vehicles, e.g., DNA vectors encoding one or more peptides, viral vectors encoding the peptide(s), liposomes and the like, can be used, as described herein.
  • the immunogenic peptides of the invention are generally administered to an individual who has not been infected with PF.
  • the peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences.
  • compositions may also be used to treat individuals already infected with PF. Patients can be treated with the immunogenic peptide epitopes separately or in conjunction with other treatments, as appropriate.
  • administration should generally begin at the first diagnosis of PF infection. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. Loading doses followed by boosting doses may be required.
  • the peptide or other compositions used for prophylaxis or the treatment of PF infection can be used, e.g., in persons who are not manifesting symptoms of disease but who act as a disease vector.
  • the dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 ⁇ g and the higher value is about 10,000; 20,000; 30,000; or 50,000 ⁇ g.
  • Dosage values for a human typically range from about 500 ⁇ g to about 50,000 ⁇ g per 70 kilogram patient.
  • Boosting dosages of between about 1.0 ⁇ g to about 50000 ⁇ g of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood.
  • the peptides and compositions of the present invention may be employed in serious disease states, that is, life-threatening or potentially life threatening situations.
  • a representative dose is in the range disclosed above.
  • Administration should continue until at least clinical symptoms or laboratory tests indicate that the PF infection has been eliminated or substantially abated and for a period thereafter.
  • the dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.
  • compositions for therapeutic treatment are intended for parenteral, topical, oral, intrathecal, or local administration.
  • the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like.
  • These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered.
  • compositions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • a human unit dose form of the peptide composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, preferably an aqueous carrier, and is administered in a volume of fluid that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17 th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985).
  • the peptides of the invention may also be administered via liposomes, which serve to target the peptides to a particular tissue, such as lymphoid tissue, or to target selectively to infected cells, as well as to increase the half-life of the peptide composition.
  • Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
  • liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions.
  • Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
  • a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells.
  • a liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
  • nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.
  • the immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are 0.01%-20% by weight, preferably 1%-10%.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters such as mixed or natural glycerides may be employed.
  • the surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%.
  • the balance of the composition is ordinarily propellant.
  • a carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
  • kits can be provided in kit form together with instructions for vaccine administration.
  • the kit would include desired peptide compositions in a container, preferably in unit dosage form and instructions for administration.
  • An alternative kit would include a minigene construct with desired nucleic acids of the invention in a container, preferably in unit dosage form together with instructions for administration. Lymphokines such as IL-2 or IL-12 may also be included in the kit.
  • kit components that may also be desirable include, for example, a sterile syringe, booster dosages, and other desired excipients.
  • binding assays can be performed with peptides that are either motif-bearing or not motif-bearing.
  • Epstein-Barr virus (EBV)-transformed homozygous cell lines, fibroblasts, CIR, or 721.22 transfectants were used as sources of HLA class I molecules. These cells were maintained in vitro by culture in RPMI 1640 medium supplemented with 2 mM L-glutamine (GIBCO, Grand Island, N.Y.), 50 ⁇ M 2-ME, 100 ⁇ g/ml of streptomycin, 100 U/ml of penicillin (Irvine Scientific) and 10% heat-inactivated FCS (Irvine Scientific, Santa Ana, Calif.). Cells were grown in 225-cm 2 tissue culture flasks or, for large-scale cultures, in roller bottle apparatuses. The specific cell lines routinely used for purification of MHC class I and class II molecules are listed in Table XXIV.
  • Cell lysates were prepared and HLA molecules purified in accordance with disclosed protocols (Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, cells were lysed at a concentration of 10 8 cells/ml in 50 mM Tris-HCl, pH 8.5, containing 1% Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland), 150 mM NaCl, 5 mM EDTA, and 2 mM PMSF. Lysates were cleared of debris and nuclei by centrifugation at 15,000 ⁇ g for 30 min.
  • HLA molecules were purified from lysates by affinity chromatography. Lysates prepared as above were passed twice through two pre-columns of inactivated Sepharose CL4-B and protein A-Sepharose. Next, the lysate was passed over a column of Sepharose CL-4B beads coupled to an appropriate antibody. The antibodies used for the extraction of HLA from cell lysates are listed in Table XXV. The anti-HLA column was then washed with 10-column volumes of 10 mM Tris-HCL, pH 8.0, in 1% NP-40, PBS, 2-column volumes of PBS, and 2-column volumes of PBS containing 0.4% n-octylglucoside.
  • MHC molecules were eluted with 50 mM diethylamine in 0.15M NaCl containing 0.4% n-octylglucoside, pH 11.5. A 1/25 volume of 2.0M Tris, pH 6.8, was added to the eluate to reduce the pH to ⁇ 8.0. Eluates were then be concentrated by centrifugation in Centriprep 30 concentrators at 2000 rpm (Amicon, Beverly, Mass.). Protein content was evaluated by a BCA protein assay (Pierce Chemical Co., Rockford, Ill.) and confirmed by SDS-PAGE.
  • protease inhibitors were 1 mM PMSF, 1.3 nM 1.10 phenanthroline, 73 ⁇ M pepstatin A, 8 mM EDTA, 6 mM N-ethylmaleimide (for Class II assays), and 200 ⁇ M N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK). All assays were performed at pH 7.0 with the exception of DRB1*0301, which was performed at pH 4.5, and DRB1*1601 (DR2w21 ⁇ 1 ) and DRB4*0101 (DRw53), which were performed at pH 5.0. pH was adjusted as described elsewhere (see Sidney et al., in Current Protocols in Immunology , Margulies, Ed., John Wiley & Sons, New York, Section 18.3, 1998).
  • Radiolabeled peptides were iodinated using the chloramine-T method. Representative radiolabeled probe peptides utilized in each assay, and its assay specific IC 50 nM, are summarized in Tables IV and V. Typically, in preliminary experiments, each MHC preparation was titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays were performed using these HLA concentrations.
  • ⁇ 1 molecules are not separated from ⁇ 3 (and/or ⁇ 4 and ⁇ 5 ) molecules.
  • the ⁇ 1 specificity of the binding assay is obvious in the cases of DRB1*0101 (DR1), DRB1*0802 (DR8w2), and DRB1*0803 (DR8w3), where no ⁇ 3 is expressed.
  • Binding assays as outlined above may be used to analyze supermotif and/or motif-bearing epitopes as, for example, described in Example 2.
  • Vaccine compositions of the invention may include multiple epitopes that comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Additional experimental details that may be relevant to this example are found in Doolan, D. L. et al., Immunity 7:97, 1997. Calculation of population coverage was performed using the strategy described below.
  • a ji is a coefficient which represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids.
  • the crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains).
  • residue j occurs at position i in the peptide, it is assumed to contribute a constant amount j i to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide. This assumption is justified by studies from our laboratories that demonstrated that peptides are bound to MHC and recognized by T cells in essentially an extended conformation (data omitted herein).
  • the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.
  • the five immunogenic peptides were then tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802).
  • the peptide SSP2 14-23 which was immunogenic in primary human CTL cultures and contains the SSP2 14-22 epitope (rather than SSP2 14-22 itself), was included in the analysis.
  • the peptide Exp-1 83 which was positive in the murine CTL assays and the peptide CSP 425 and SSP2 230 , were also analyzed for cross-reactive binding. As shown in Table XXVI, all eight of these peptides were found to be A2-supertype cross-reactive binders with six of these binding to three or more A2 supertype alleles.
  • the PF protein sequences scanned above were also examined for the presence of conserved peptides with the HLA-A3 supermotif primary anchors. Further analysis using the A03 and A11 algorithms (see, e.g., Gulukota et al, J. Mol. Biol. 267:1258-1267, 1997 and Sidney et al, Human Immunol. 45:79-93, 1996) identified 203 conserved 9- or 10-mer motif-containing peptide sequences that scored high in either or both algorithms. Of these candidates, twenty five peptides were identified that bound A3 and/or A11 with binding affinities of ⁇ 500 nM.
  • HLA-A3 supertype cross-reactive binding peptides derived from conserved regions of PF proteins were identified.
  • HLA-A1 and -A24 epitopes can also be incorporated into potential vaccine constructs.
  • CTL induced in A*0201/K b transgenic mice exhibit specificity similar to CTL induced in the human system (see, e.g., Vitiello et al., J Exp. Med. 173:1007-1015, 1991; Wentworth et al., Eur. J. Immunol. 26:97-101, 1996). Accordingly, these mice were used to evaluate the immunogenicity of the fourteen conserved A*0201 motif-bearing high affinity binding peptides identified in Example 2 above.
  • mice were injected subcutaneously at the base of the tail with each peptide (50 ⁇ g/mouse) emulsified in IFA in the presence of an excess of an IA b -restricted helper peptide (140 ⁇ g/mouse) (HBV core 128-140, Sette et al., J Immunol. 153:5586-5592, 1994).
  • splenocytes were incubated in the presence of peptide-loaded syngenic LPS blasts. After six days, cultures were assayed for cytotoxic activity using peptide-pulsed targets. The data indicated that 5 of the 14 peptides were capable of inducing primary CTL responses in A*0201/K b transgenic mice. (For these studies, a peptide was considered positive if it induced CTL (L.U. 30/10 6 cells in at least two transgenic animals (Wentworth et al., Eur. Immunol. 26:97-101, 1996).
  • the fourteen peptides that bound to HLA-A*0201 with good affinity were also tested for immunogenicity with PBMCs from at least four malaria-naive human donors.
  • the induction of primary CTL responses in vitro with PBMCs from normal naive humans requires a brief treatment of the antigen-presenting cells with acidic buffer and subsequent neutralization in the presence of excess B 2 -microglobulin and exogenous peptide (Wentworth et al., supra). By ensuring that the majority of the HLA class I molecules are occupied by exogenous peptide, these steps are essential for the induction of primary CTL responses. Such responses cannot be induced using methods developed for the induction of recall CTL responses.
  • a peptide was considered positive if yielding more than 2 LU 30 /10 6 cells (lytic units 20% per 10 4 cells, where one lytic unit corresponds to the number of effector cells required to induce 30% 51 Cr release from 10,000 target cells during a 6 hr assay.) or 15% peptide-specific lysis, respectively, in at least two different primary CTL cultures.
  • the five peptides that were positive in HLA transgenic mice were also shown to induce primary CTL responses.
  • HLA-A2 cross-reactive binding peptides were tested for their ability to elicit in vitro recall responses from PBMCs of six volunteers, each of whom had an HLA-A*0201 allele, immunized with irradiated sporozoites. The results demonstrated that all of the A2-binding peptides were recognized in association with HLA-A*0201.
  • the immunogenicity of the eight supermotif-bearing peptides was also evaluated in recall responses using PBMC from volunteers bearing HLA-A3 supertype alleles who had previously been immunized with irradiated sporozoites. All the peptides were recognized in association with both A3 and A33. The fraction of individuals responding to each peptide varied for the supertype overall from 50% for one of the peptides to 100% for three of the peptides.
  • Immunogenicity was also evaluated using PBMCs of semi-immune or nonimmune individuals naturally exposed to malaria. In this population, recall CTL responses (percentage specific lysis greater than 10%) were detected for five of the eight A3-binding peptides.
  • Immunogenicity of A3 supermotif-bearing peptides can also be evaluated in transgenic mice that bear a human HLA-A11 allele using methodology analagous to that for immunogenicity studies using HLA-A2.1 transgenic mice.
  • Both peptides were found to be capable of inducing CTL responses.
  • the two peptides were recognized as CTL epitopes in the context of three of the five B7 supertype alleles.
  • HLA motifs and supermotifs are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein.
  • the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analogued, or “fixed” to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analog peptides that exhibit modulated binding affinity are set forth in this example.
  • the primary anchor residues are analogued to modulate binding activity.
  • peptide engineering strategies are implemented to further increase the cross-reactivity of the A3-supertype candidate epitopes identified above.
  • the main anchors of A3-supermotif-bearing peptides are altered, for example, to introduce a preferred V, S, or M at position 2.
  • each engineered analog is initially tested for binding to the prototype A3 supertype alleles A3 and A11; then, if binding capacity is maintained, for additional A3-supertype cross-reactivity.
  • analogs of HLA-A2 supermotif-bearing epitopes may also be generated.
  • peptides binding to A2-supertype molecules may be engineered at primary anchor residues to possess a preferred residue (L, I, V, or M) at position 2 and/or a preferred I or V as a position 9 primary anchor residue.
  • analog peptides are then tested for the ability to bind the A2 supermotif prototype allele, A*0201. Those peptides that demonstrate 500 nM binding capacity are then tested for A2-supertype cross-reactivity.
  • B7 supermotif-bearing peptides may, for example, be engineered to possess a preferred residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. ( J. Immunol. 157:3480-3490, 1996).
  • HLA supermotifs are of value in engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties.
  • binding capacity of an analog of the B7 supermotif-bearing peptide Pf SSP2 126 is analyzed.
  • the peptide may be substituted with an F at position 1, rather than and L.
  • the peptide, which binds to 3 of 5 B7 supertype portions is then analyzed for the ability to bind all five B7-supertype molecules with a good affinity.
  • results from previous binding evaluations may be analyzed to identify conserved (8-, 9-, 10-, or 11-mer) peptides which bind, minimally, 3/5 B7 supertype molecules with weak affinity (IC 50 of 500 nM-5 ⁇ M). This analysis identifies additional candidate peptides that can be analogued. These peptides are tested for enhanced binding affinity and B7-supertype cross-reactivity.
  • Peptide epitopes bearing an HLA class II supermotif or motif may also be identified as outlined below using methodology similar to that described in Examples 1-3.
  • the protein sequences from the same four PF antigens used for the identification of HLA Class I supermotif/motif sequences were analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences were selected comprising a DR-supermotif, further comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total). It was also required that the 9-mer core sequence be 100% conserved in at least 79% of the sequences analyzed.
  • PF-derived peptides identified above were tested for their binding capacity for various common HLA-DR molecules. All peptides were initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least 2 of these 3 DR molecules were then tested for binding to DR2w2 ⁇ 1, DR2w2 ⁇ 2, DR6w19, and DR9 molecules in secondary assays. Finally, peptides binding at least 2 of the 4 secondary panel DR molecules, and thus cumulatively at least 4 of 7 different DR molecules, were screened for binding to DR4w15, DR5w11, and DR8w2 molecules in tertiary assays.
  • Peptides binding at least 7 of the 10 DR molecules comprising the primary, secondary, and tertiary screening assays were considered cross-reactive DR binders.
  • the composition of these screening panels, and the phenotypic frequency of associated antigens, are shown in Table XXX.
  • HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations
  • DR3 binding capacity is an important criterion in the selection of HTL epitopes.
  • data generated previously indicated that DR3 only rarely cross-reacts with other DR alleles (Sidney et al., J. Immunol. 149:2634-2640, 1992; Geluk et al., J. Immunol. 152:5742-5748, 1994; Southwood et al., J. Immunol. 160:3363-3373, 1998).
  • This is not entirely surprising in that the DR3 peptide-binding motif appears to be distinct from the specificity of most other DR alleles.
  • DR3 binding epitopes identified in this manner that are found to induce immunological responses as in Example 6 below may then be included in vaccine compositions with DR supermotif-bearing peptide epitopes.
  • the immunogenicity of the HLA class II binding epitopes identified in Example 5 was evaluated in a study testing PBMC from either healthy volunteers previously immunized with an irradiated sporozoite vaccine, and thereby immune to malaria, or PBMC from naturally exposed individuals from the Irian Java (Indonesia) region where malaria is highly endemic. Vigorous responses were seen in volunteers vaccinated with whole irradiate sporozoites. All peptides were recognized in at least one immune individual, but not in either of the two individuals for which pre-immunization sample were available. All individuals recognized at least two, and up to nine different epitopes.
  • This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.
  • the A3-like supertype may also include A34, A66, and A*7401, these alleles were not included in overall frequency calculations.
  • confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901.
  • the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602).
  • Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups (see Table XXI). Coverage may be extended by including peptides bearing the A1 and A24 motifs. On average, A1 is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%. An analagous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.
  • candidate peptide epitopes derived from conserved regions of PF have been identified (Table XXXIII) These include eight HLA-A2 supermotif-bearing epitopes, eight HLA-A3 supermotif-bearing epitopes, and two HLA-B7 supermotif-bearing epitope, each capable of binding to multiple A2-, A3-, or B7-supertype molecules, and immunogenic in HLA transgenic mice or antigenic for human PBL.
  • four A1 motif-bearing and four A24 motif-bearing epitopes are also include candidate CTL epitopes for inclusion in a vaccine composition.
  • average population coverage (i.e., recognition of at least one PF epitope) is predicted to be, on average, greater than 95% (range of 90.6%-99.1%), in five major ethnic populations.
  • the potential redundancy of coverage afforded by these epitopes can be estimated using the game theory Monte Carlo simulation analysis, which is known in the art (see e.g., Osborne, M. J. and Rubinstein, A. “A course in game theory” MIT Press, 1994). As shown in FIG. 1 , it is estimated that 90% of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize 8 or more of the candidate epitopes described herein.
  • This example determines that CTL induced by native or analogued peptide epitopes identified and selected as described in Examples 1-6 recognize endogenously synthesized, i.e., native antigens.
  • Effector cells isolated from transgenic mice that are immunized with peptide epitopes as in Example 3, for example HLA-A2 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells.
  • effector cells Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated.
  • An additional six days later, these cell lines are tested for cytotoxic activity on 51 Cr labeled Jurkat-A2.1/K b target cells in the absence or presence of peptide, and also tested on 51 Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with PF expression vectors.
  • transgenic mouse model to be used for such an analysis depends upon the epitope(s) that is being evaluated.
  • HLA-A*0201/K b transgenic mice several other transgenic mouse models including mice with human A11, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed.
  • HLA-DR1 and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.
  • This example illustrates the induction of CTLs and HTLs in transgenic mice by use of a PF CTL/HTL peptide conjugate whereby the vaccine composition comprises peptides administered to a PF-infected patient or an individual at risk for malaria.
  • the peptide composition can comprise multiple CTL and/or HTL epitopes. This analysis demonstrates enhanced immunogenicity that can be achieved by inclusion of one or more HTL epitopes in a vaccine composition.
  • Such a peptide composition can comprise a lipidated HTL epitope conjugated to a preferred CTL epitope containing, for example, at least one CTL epitope selected from Tables VII-XVIII, or an analog of that epitope.
  • the HTL epitope is, for example, selected from Table XIX or XX.
  • Lipopeptides are prepared by coupling the appropriate fatty acid to the amino terminus of the resin bound peptide. A typical procedure is as follows: A dichloromethane solution of a four-fold excess of a pre-formed symmetrical anhydride of the appropriate fatty acid is added to the resin and the mixture is allowed to react for two hours. The resin is washed with dichloromethane and dried. The resin is then treated with trifluoroacetic acid in the presence of appropriate scavengers [e.g. 5% (v/v) water] for 60 minutes at 20° C. After evaporation of excess trifluoroacetic acid, the crude peptide is washed with diethyl ether, dissolved in methanol and precipitated by the addition of water. The peptide is collected by filtration and dried.
  • appropriate scavengers e.g. 5% (v/v) water
  • mice Immunization of transgenic mice is performed as described (Alexander et al., J. Immunol. 159:4753-4761, 1997).
  • A2/K b mice which are transgenic for the human HLA A2.1 allele and are useful for the assessment of the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearing epitopes, are primed subcutaneously (base of the tail) with 0.1 ml of peptide conjugate formulated in saline, or DMSO/saline. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide.
  • Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/K b chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991)
  • spleen cells (30 ⁇ 10 6 cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10 ⁇ 10 6 cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity.
  • Target cells 1.0 to 1.5 ⁇ 10 6
  • Peptide is added where required at a concentration of 1 ⁇ g/ml.
  • 10 4 51 Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 ⁇ l) in U-bottom 96-well plates. After a 6 hour incubation period at 37° C., a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter.
  • % 51 Cr release data is expressed as lytic units/10 6 cells.
  • One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a 6 hour 51 Cr release assay.
  • the lytic units/10 6 obtained in the absence of peptide is subtracted from the lytic units/10 6 obtained in the presence of peptide.
  • the results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using the CTL epitope as outlined in Example 3. Analyses similar to this may be performed to evaluate the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures it is found that a CTL response is induced, and concomitantly that an HTL response is induced, upon administration of such compositions.
  • the peptides in the composition may be in the form of a nucleic acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or may be single and/or polyepitopic peptides.
  • the following principles are utilized when selecting an array of epitopes for inclusion in a vaccine composition. Each of the following principles are balanced in order to make the selection.
  • Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with PF clearance.
  • this includes 3-4 epitopes that come from at least one antigen of PF.
  • it has been observed that patients who spontaneously clear PF generate an immune response to at least 3 epitopes on at least one PF antigen.
  • HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one PF antigen.
  • Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC 50 of 500 nM or less, or for Class II an IC 50 of 1000 nM or less.
  • Sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage.
  • epitopes are selected to provide at least 80% population coverage.
  • a Monte Carlo analysis a statistical evaluation known in the art and discussed herein, can be employed to assess breadth, or redundancy, of population coverage.
  • nested epitopes When selecting epitopes for PF antigens it may be preferable to select native epitopes. Therefore, of particular relevance for infectious disease vaccines, are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A peptide comprising “transcendent nested epitopes” is a peptide that has both HLA class I and HLA class II epitopes in it.
  • a sequence that has the greatest number of epitopes per provided sequence is provided.
  • a limitation on this principle is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide.
  • the sequence is screened in order to insure that it does not have pathological or other deleterious biological properties.
  • an objective is to generate the smallest peptide possible that encompasses the epitopes of interest.
  • the principles employed are similar, if not the same as those employed when selecting a peptide comprising nested epitopes. Additionally, however, upon determination of the nucleic acid sequence to be provided as a minigene, the peptide encoded thereby is analyzed to determine whether any “junctional epitopes” have been created.
  • a junctional epitope is an actual binding epitope, as predicted, e.g., by motif analysis. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that epitope, which is not present in a native PF protein sequence. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
  • Peptide epitopes for inclusion in vaccine compositions are, for example, selected from those listed in Tables XXXIII and XXXIV.
  • a vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude of an immune response that clears an acute PF infection.
  • Minigene plasmids may, of course, contain various configurations of CTL and/or HTL epitopes or epitope analogs as described herein.
  • Expression plasmids have been constructed and evaluated as described, for example, in U.S. Ser. No. 09/311,784 filed May 13, 1999, now U.S. Pat. No. 6,534,482, and in Ishioka et al., J. Immunol. 162:3915-3925, 1999.
  • a minigene expression plasmid may include multiple CTL and HTL peptide epitopes.
  • HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes.
  • Preferred epitopes are identified, for example, in Tables XXXIII and XXXIV.
  • HLA class I supermotif or motif-bearing peptide epitopes derived from multiple PF antigens, e.g., EXP-1, SSP2, CSP and LSA-1, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage.
  • HLA class II epitopes are selected from multiple PF antigens to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct.
  • the selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.
  • This example illustrates the methods to be used for construction of such a minigene-bearing expression plasmid.
  • Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.
  • the minigene DNA plasmid contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein.
  • the sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
  • Overlapping oligonucleotides for example eight oligonucleotides, averaging approximately 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified.
  • the oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence.
  • the final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR.
  • a Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.
  • the full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product for 25 additional cycles.
  • the full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.
  • HLA-A11/K b transgenic mice are immunized intramuscularly with 100 ⁇ g of naked cDNA.
  • a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.
  • Splenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a 51 Cr release assay.
  • the results indicate the magnitude of the CTL response directed against the A3-restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine. It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A3 supermotif peptide epitopes as does the polyepitopic peptide vaccine.
  • a similar analysis is also performed using other HLA-A2 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A2 and HLA-B7 motif or supermotif epitopes.
  • I-A b restricted mice are immunized intramuscularly with 100 ⁇ g of plasmid DNA.
  • a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant.
  • CD4 + T cells i.e. HTLs
  • HTLs are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene).
  • the HTL response is measured using a 3 H-thymidine incorporation proliferation assay, (see, e.g., Alexander et al., Immunity 1:751-761, 1994). the results indicate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.
  • DNA minigenes constructed as described in Example 11, may also be evaluated as a vaccine in combination with a boosting agent using a prime boost protocol.
  • the boosting agent may consist of recombinant protein (e.g., Barnett et al., Aids Res. and Human Reotroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med. 5:526-34, 1999).
  • recombinant protein e.g., Barnett et al., Aids Res. and Human Reotroviruses
  • the efficacy of the DNA minigene may be evaluated in transgenic mice.
  • A2.1/K b transgenic mice are immunized IM with 100 ⁇ g of the DNA minigene encoding the immunogenic peptides. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 10 7 pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 ⁇ g of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost.
  • splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an IFN-y ELISA. It is found that the minigene utilized in a prime-boost mode elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis is also performed using other HLA-A11 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes.
  • Vaccine compositions of the present invention are used to prevent PF infection in persons who are at risk for such infection.
  • a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) containing multiple CTL and HTL epitopes such as those selected in Examples 9 and/or 10, which are also selected to target greater than 80% of the population, is administered to individuals at risk for PF infection.
  • the composition is provided as a single lipidated polypeptide that encompasses multiple epitopes.
  • the vaccine is administered in an aqueous carrier comprised of Freunds Incomplete Adjuvant.
  • the dose of peptide for the initial immunization is from about 1 to about 50,000 ⁇ g, generally 100-5,000 ⁇ g, for a 70 kg patient.
  • the initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required.
  • the composition is found to be both safe and efficacious as a prophylaxis against PF infection.
  • polyepitopic peptide composition can be administered as a nucleic acid in accordance with methodologies known in the art and disclosed herein.
  • a native PF polyprotein sequence is screened, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes and is preferably less in length than an entire native antigen.
  • This relatively short sequence that contains multiple distinct, even overlapping, epitopes is selected and used to generate a minigene construct.
  • the construct is engineered to express the peptide, which corresponds to the native protein sequence.
  • the “relatively short” peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length.
  • the protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes.
  • epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with frame shifted overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.
  • the vaccine composition will preferably include, for example, three CTL epitopes and at least one HTL epitope from PF.
  • This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide.
  • an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.
  • the embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (absent analogs) directs the immune response to multiple peptide sequences that are actually present in native PF antigens thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions.
  • computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.
  • the PF peptide epitopes of the present invention are used in conjunction with peptide epitopes from target antigens related to one or more other diseases, to create a vaccine composition that is useful for the prevention or treatment of PF as well as the one or more other disease(s).
  • the other diseases include, but are not limited to, HIV, HCV, and HBV.
  • a polyepitopic peptide composition comprising multiple CTL and HTL epitopes that target greater than 98% of the population may be created for administration to individuals at risk for both PF and HIV infection.
  • the composition can be provided as a single polypeptide that incorporates the multiple epitopes from the various disease-associated sources, or can be administered as a composition comprising one or more discrete epitopes.
  • Peptides of the invention may be used to analyze an immune response for the presence of specific CTL or HTL populations directed to PF. Such an analysis may be performed in a manner as that described by Ogg et al., Science 279:2103-2106, 1998.
  • peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.
  • tetramers highly sensitive human leukocyte antigen tetrameric complexes
  • tetramers highly sensitive human leukocyte antigen tetrameric complexes
  • tetramers are used for a cross-sectional analysis of, for example, PF HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of infection or following immunization using an PF peptide containing an A*0201 motif.
  • Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and ⁇ 2-microglobulin are synthesized by means of a prokaryotic expression system.
  • the heavy chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site.
  • the heavy chain, ⁇ 2-microglobulin, and peptide are refolded by dilution.
  • the 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Mo.), adenosine 5′triphosphate and magnesium.
  • Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer-phycoerythrin.
  • PBMCs For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 ⁇ l of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive uninfected donors.
  • the percentage of cells stained with the tetramer is then determined by flow cytometry.
  • the results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the extent of immune response to the PF epitope, and thus the stage of infection with PF, the status of exposure to PF, or exposure to a vaccine that elicits a protective or therapeutic response.
  • the peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from infection, who are chronically infected with PF, or who have been vaccinated with a PF vaccine.
  • the class I restricted CTL response of persons who have been vaccinated may be analyzed.
  • the vaccine may be any PF vaccine.
  • PBMC are collected from vaccinated individuals and HLA typed.
  • Appropriate peptide epitopes of the invention that are preferably highly conserved and, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.
  • PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 ⁇ g/ml), and Hepes (10 mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats.
  • a synthetic peptide comprising an epitope of the invention is added at 10 ⁇ g/ml to each well and HBV core 128-140 epitope is added at 1 ⁇ g/ml to each well as a source of T cell help during the first week of stimulation.
  • a positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific 51 Cr release, based on comparison with uninfected control subjects as previously described (Rehermann, et al., Nature Med. 2:1104,1108, 1996; Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J. Clin. Invest. 98:1432-1440, 1996).
  • Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, Mass.) or established from the pool of patients as described (Guilhot, et al. J. Virol. 66:2670-2678, 1992).
  • Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 ⁇ M, and labeled with 100 ⁇ Ci of 51 Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after which they are washed four times with HBSS.
  • Cytolytic activity is determined in a standard 4-h, split well 51 Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target (E/T) ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100 ⁇ [(experimental release ⁇ spontaneous release)/maximum release ⁇ spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis, Mo.). Spontaneous release is ⁇ 25% of maximum release for all experiments.
  • the class II restricted HTL responses may also be analyzed.
  • Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5 ⁇ 10 5 cells/well and are stimulated with 10 ⁇ g/ml synthetic peptide, whole antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 10 U/ml IL-2. Two days later, 1 3 H-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for 3 H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of 3 H-thymidine incorporation in the presence of antigen divided by the 3 H-thymidine incorporation in the absence of antigen.
  • Such a vaccine regimen is includes an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptides mixture administered in an adjuvant.
  • the initial immunization may be performed using an expression vector, such as that constructed in Example 11, in the form of naked DNA administered IM (or SC or ID) in the amounts of 0.5-5, typically 100 g, at multiple sites.
  • the DNA 0.1 to 1000 mg
  • the DNA can also be administered using a gene gun.
  • a booster dose is then administered.
  • the booster can be recombinant fowlpox virus administered at a dose of 5-10 7 to 5 ⁇ 10 9 pfu.
  • Alternative recombinant virus, such as MVA, canarypox, adenovirus, and adeno-associated viruses can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered.
  • Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
  • a human clinical trial to evaluate an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study and carried out as a randomized, double-blind, placebo-controlled trial in patients are not infected with Pf.
  • Such a trial is designed, for example, as follows:
  • a total of about 27 subjects are enrolled and divided into 3 groups:
  • Group I 3 subjects are injected with placebo and 6 subjects are injected with 5 ⁇ g of peptide composition
  • Group II 3 subjects are injected with placebo and 6 subjects are injected with 50 ⁇ g peptide composition;
  • Group III 3 subjects are injected with placebo and 6 subjects are injected with 500 ⁇ g of peptide composition.
  • the endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity.
  • Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy.
  • Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
  • the vaccine is found to be both safe and efficacious.
  • a prophylactic field trial can also be conducted to evaluate a vaccine composition of the invention.
  • issues of patient compliance are also considered in the determination of vaccine efficacy.
  • Vaccines comprising peptide epitopes of the invention may be administered using dendritic cells.
  • the immunogenic peptide epitopes are used to elicit a CTL and/or HTL response ex vivo.
  • CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptides.
  • APC antigen-presenting cells
  • the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., PF-infected cells.
  • Another way of identifying motif-bearing peptides is to elute them from cells bearing defined MHC molecules.
  • EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can then be infected with a pathogenic organism, e.g., PF, HIV, etc. or transfected with nucleic acids that express the antigen of interest. Thereafter, peptides produced by endogenous antigen processing of peptides produced consequent to infection (or as a result of transfection) will bind to HLA molecules within the cell and be transported and displayed on the cell surface.
  • a pathogenic organism e.g., PF, HIV, etc.
  • nucleic acids that express the antigen of interest.
  • the peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al., J. Immunol. 152:3913, 1994). Because, as disclosed herein, the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.
  • cell lines that do not express any endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells may then be used as described, i.e., they may be infected with a pathogenic organism or transfected with nucleic acid encoding an antigen of interest to isolate peptides corresponding to the pathogen or antigen of interest that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.
  • Terminus (Primary Anchor) (Primary Anchor) (Primary Anchor) (Primary Anchor) A1 TI LVMS FWY A2 LIVM ATQ IV MATL A3 VSMA TLI RK A24 YF WIVLMT FI YWLM B7 P VILF MWYA B27 RHK FYL WMIVA B44 E D FWYLIMVA B58 ATS FWY LIVMA B62 QL IVMP FWY MIVLA MOTIFS A1 TSM Y A1 DE AS Y A2.1 LM VQIAT V LIMAT A3 LMVISATF CGD KYR HFA A11 VTMLISAGN CDF K RYH A24 YFW M FLIW A*3101 MVT ALIS R K A*3301 MVALF IST RK A*6801 AVT MSLI RK B*0702 P LMF W YAIV B*3501 P LMFWY IVA B51 P LIVF WYAM
  • Terminus (Primary Anchor) (Primary Anchor) (Primary Anchor) (Primary Anchor) A1 TI LVMS FWY A2 VQAT V LIMAT A3 VSMA TLI RK A24 YF WIVLMT FI YWLM B7 P VILF MWYA B27 RHK FYL WMIVA B58 ATS FWY LIVMA B62 QL IVMP FWY MIVLA MOTIFS A1 TSM Y A1 DE AS Y A2.1 VQAT * V LIMAT A3.2 LMVISATF CGD KYR HFA A11 VTMLISAGN CDF K RHY A24 YFW FLIW *If 2 is V, or Q, the C-term is not L Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearingif it has primary anchors at each primary anchor position for a motif or supermotifas specified in the above table.
  • SEQ STANDARD STANDARD ID BINDING ALLELE PEPTIDE SEQUENCE NO: AFFINITY (nM) A*0101 944.02 YLEPAIAKY 3575 25 A*0201 941.01 FLPSDYFPSV 3576 5.0 A*0202 941.01 FLPSDYFPSV 3577 4.3 A*0203 941.01 FLPSDYFPSV 3578 10 A*0205 941.01 FLPSDYFPSV 3579 4.3 A*0206 941.01 FLPSDYFPSV 3580 3.7 A*0207 941.01 FLPSDYFPSV 3581 23 A*6802 1072.34 YVIKVSARV 3582 8.0 A*0301 941.12 KVFPYALINK 3583 11 A*1101 940.06 AVDLYHFLK 3584 6.0 A*3101 941.12 KVFPYALINK 3585 18 A*3301 1083.02 STLPETYVVRR 3586 29 A*
  • A3-supertype peptides are tested for binding ato A*03, A*11, A*31011, A*3301, and A*6801.
  • B7-supertype peptides are tested for binding to B*0702, B*3501, B*5101, B*5301, and B*5401.
  • A1 and A24 peptides are tested for binding to A*0101, and A*2402, respectively.
  • A3-supertype peptides are tested for binding to A*03, A*11, A*31011, A*3301, and A*6801.
  • B7-supertype peptides are tested for binding to B*0702, B*3501, B*5101, B*5301, and B*5401.
  • A1 and A24 peptides are tested for binding to A*0101 and A*2402, respectively.

Abstract

This invention uses our knowledge of the mechanisms by which antigen is recognized by T cells to identify and prepare Plasmodium falciparum epitopes, and to develop epitope-based vaccines directed towards malaria. More specifically, this application communicates our discovery of pharmaceutical compositions and methods of use in the prevention and treatment of malaria. In particular, this application discloses isolated peptides comprising oligopeptides, for example the oligopeptides LLACAGLAY, FLIFFDLFLV, FMKAVCVEV, VLAGLLGNV, GLIMVLSFL, KILSVFFLA, GLLGNVSTV, VLLGGVGLVL, ILSVSSFLFV, QTNFKSLLR, LACAGLAYK, ALFFIIFNK, LLACAGLAYK, HVLSHNSYEK, FILVNLLIFH, FQDEENIGIY, PSDGKCNLY, YYIPHQSSL, FYFILVNLL, KYLVIVFLI and KYKLATSVL, or isolated peptides conjugated with T helper peptides that are used as antigens in epitope-based vaccines to prevent and/or treat malaria.

Description

    CROSS-REFERENCES TO RELATED APPLICATIONS
  • This application is a divisional application of U.S. application Ser. No. 09/390,061, filed Sep. 3, 1999, wherein U.S. application Ser. No. 09/390,061 is a continuation-in-part of U.S. application Ser. No. 09/017,743, filed Feb. 3, 1998 (abandoned); and is a continuation-in-part of U.S. application Ser. No. 08/821,739, filed Mar. 20, 1997 (abandoned); and is a continuation-in-part of U.S. application Ser. No. 08/452,843, filed May 30, 1995 (abandoned); and is a continuation-in-part of U.S. application Ser. No. 08/454,033, filed May 26, 1995 (abandoned); and is a continuation-in-part of U.S. application Ser. No. 08/344,824, filed Nov. 23, 1994 (abandoned); said Ser. No. 09/017,743 (abandoned) is a continuation-in-part of U.S. application Ser. No. 08/753,615, filed Nov. 23, 1996 (abandoned); which is a continuation-in-part of U.S. application Ser. No. 08/590,298, filed Jan. 23, 1996 (abandoned); which is a continuation-in-part of said Ser. No. 08/452,843, filed May 30, 1995 (abandoned); which is a continuation-in-part of said Ser. No. 08/344,824, filed Nov. 23, 1994 (abandoned); which is a continuation-in-part of U.S. application Ser. No. 08/278,634, filed Jul. 21, 1994 (abandoned); said Ser. No. 08/821,739 (abandoned) claims the benefit of U.S. Provisional Application No. 60/013,833, filed Mar. 21, 1996 (now inactive); and is a continuation-in-part of U.S. application Ser. No. 08/451,913, filed May 26, 1995 (abandoned).
  • This application is related to U.S. Ser. No. 09/189,702 filed Nov. 10, 1998, now U.S. Pat. No. 7,252,829, which is a CIP of U.S. Ser. No. 08/205,713 filed Mar. 4, 1994 (abandoned), which is a CIP of Ser. No. 08/159,184 filed Nov. 29, 1993 and now abandoned, which is a CIP of Ser. No. 08/073,205 filed Jun. 4, 1993 and now abandoned, which is a CIP of Ser. No. 08/027,146 filed Mar. 5, 1993 and now abandoned. The present application is also related to U.S. Ser. No. 09/226,775 (abandoned), which is a CIP of abandoned U.S. Ser. No. 08/815,396, which claims benefit of abandoned U.S. Ser. No. 60/013,113 filed Mar. 21, 1996. Furthermore, the present application is related to U.S. Ser. No. 09/017,735 (abandoned), which is a CIP of abandoned U.S. Ser. No. 08/589,108; U.S. Ser. No. 08/454,033 (abandoned); and U.S. Ser. No. 08/349,177 (abandoned). The present application is also related to U.S. Ser. No. 09/017,524 (abandoned), U.S. Ser. No. 08/821,739 (abandoned), which claims benefit of abandoned U.S. Ser. No. 60/013,833 filed Mar. 21, 1996; and U.S. Ser. No. 08/347,610 (abandoned), which is a CIP of U.S. Ser. No. 08/159,339, now U.S. Pat. No. 6,037,135, which is a CIP of abandoned U.S. Ser. No. 08/103,396, which is a CIP of abandoned U.S. Ser. No. 08/027,746, which is a CIP of abandoned U.S. Ser. No. 07/926,666. The present application is also related to U.S. Ser. No. 09/017,743 (abandoned), which is a CIP of abandoned U.S. Ser. No. 08/590,298; and U.S. Ser. No. 08/452,843 (abandoned), which is a CIP of U.S. Ser. No. 08/344,824 (abandoned), which is a CIP of abandoned U.S. Ser. No. 08/278,634. The present application is also related to PCT application PCT/US99/12066 filed May 28, 1999 which claims benefit of provisional U.S. Ser. No. 60/087,192, filed May 29, 1998 (now inactive), and U.S. Ser. No. 09/009,953 (abandoned), which is a CIP of abandoned U.S. Ser. No. 60/036,713 and abandoned U.S. Ser. No. 60/037,432. In addition, the present application is related to U.S. Ser. No. 09/098,584 (abandoned), U.S. Ser. No. 09/239,043 now U.S. Pat. No. 6,689,363, and to Provisional U.S. Patent Application 60/117,486 filed Jan. 27, 1999 (now inactive). The present application is also related to Ser. No. 09/350,401 filed Jul. 8, 1999, and U.S. Ser. No. 09/357,737 filed Jul. 19, 1999. All of the above applications are incorporated herein by reference.
    • FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
  • This invention was funded, in part, by the United States government under grants with the National Institutes of Health. The U.S. government has certain rights in this invention.
    • REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
  • The content of the electronically submitted sequence listing (Name: 2473 0510005_SeqListing_ST25.txt, Size: 962,033 bytes; and Date of Creation: Dec. 21, 2015) filed herewith was originally filed with U.S. application Ser. No. 09/390,061 and is incorporated herein by reference in its entirety.
    • INDEX I. Background of the Invention II. Summary of the Invention III. Brief Description of the Figures IV. Detailed Description of the Invention
      • A. Definitions
      • B. Stimulation of CTL and HTL responses
      • C. Binding Affinity of Peptide Epitopes for HLA Molecules
      • D. Peptide Epitope Binding Motifs and Supermotifs
        • 1. HLA-A1 supermotif
        • 2. HLA-A2 supermotif
        • 3. HLA-A3 supermotif
        • 4. HLA-A24 supermotif
        • 5. HLA-B7 supermotif
        • 6. HLA-B27 supermotif
        • 7. HLA-B44 supermotif
        • 8. HLA-B58 supermotif
        • 9. HLA-B62 supermotif
        • 10. HLA-A1 motif
        • 11. HLA-A2.1 motif
        • 12. HLA-A3 motif
        • 13. HLA-A11 motif
        • 14. HLA-A24 motif
        • 15. HLA-DR-1-4-7 supermotif
        • 16. HLA-DR3 motifs
      • E. Enhancing Population Coverage of the Vaccine
      • F. Immune Response-Stimulating Peptide Epitope Analogs
      • G. Computer Screening of Protein Sequences from Disease-Related Antigens for Supermotif- or Motif-Containing Epitopes
      • H. Preparation of Peptide Epitopes
      • I. Assays to Detect T-Cell Responses
      • J. Use of Peptide Epitopes for Evaluating Immune Responses
      • K. Vaccine Compositions
        • 1. Minigene Vaccines
        • 2. Combinations of CTL Peptides with Helper Peptides
      • L. Administration of Vaccines for Therapeutic or Prophylactic Purposes
      • M. Kits
    V. Examples VI. Claims VII. Abstract I. BACKGROUND OF THE INVENTION
  • Malaria, which is caused by infection with the parasite Plasmodium falciparum (PF), represents a major world health problem. Approximately 500 million people in the world are at risk from the disease, with approximately 200 million people actually harboring the parasites. An estimated 1 to 2 million deaths occur each year due to malaria. (Miller et al., Science 234:1349, 1986).
  • Fatal outcomes are not confined to first infections, and constant exposure is apparently a prerequisite for maintaining immunity. Naturally acquired sterile immunity is rare, if it exists at all. Accordingly, major efforts to develop an efficacious malaria vaccine have been undertaken.
  • Human volunteers injected with irradiated PF sporozoites are resistant to subsequent sporozoite challenges, which demonstrates that development of a malaria vaccine is indeed immunologically feasible. Furthermore, these immune individuals developed a vigorous response, including antibodies, and cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) components, directed against multiple antigens. Reproducing the breadth and multiplicity of this response in a vaccine, however, is a task of large proportions. The epitope approach, as described herein, may represent a solution to this challenge, in that it allows the incorporation of various antibody, CTL and HTL epitopes, from various proteins, in a single vaccine composition.
  • Anti-sporozoite antibodies are by themselves, in general, not completely efficacious in clearing the infection (Egan et al., Science 236:453, 1987). However, high concentrations of antibodies directed against the repeated region of the major B cell antigen of the sporozoite/circumsporozoite protein (CSP) have been shown to prevent liver cell infection in certain experimental models (Egan et al., Science 236:453, 1987; Potocnjak, P. et al., Science 207:71, 1980). The present inventors have shown that constructs encompassing CSP-repeat B cell epitopes and the optimized helper epitope PADRE™ (San Diego, Calif.) are highly immunogenic, and can protect in vitro against sporozoite invasion in both mouse and human liver cells, and protect mice in vivo against live sporozoite challenge (Franke et al., Vaccine 17:1201-1205, 1999)
  • PF-specific CD4+ T cells also have a role in malarial immunity beyond providing help for B cell and CTL responses. Experiments by Renia et al. (Renia, et al., Proc. Natl. Acad. Sci. USA 88:7963, 1991) demonstrated that HTLs directed against the Plasmodium yoelli CS protein could in fact adoptivley transfer protection against malaria.
  • Considerable data implicate CTLs in protection against pre-erythrocytic-stage malaria. CD8+ CTLs can eliminate Plasmodium berghei- or Plasmodium yoelii-infected mouse hepatocytes from in vitro culture in a major histocompatibility complex (MHC)-restricted and antigen-restricted manner (Hoffman et al., Science 244:1078-1081, 1989; Weiss et al., J. Exp. Med. 171:763-773, 1990). Further, it has also been shown that the immunity that developed in mice vaccinated with irradiated sporozoites is also dependent upon the present of CD8+ T cells. These T cells accumulate in inflammatory liver infiltrates subsequent to challenge. Passive transfer of circumsporozoite (CSP)-specific CTL clones as long as three hours after inoculation of sporozoites (i.e., after the parasites have left the bloodstream and infected liver cells) were capable of protecting animals against infection (Romero et al., Nature 341:323, 1989).
  • It is notable that CTL-restricted responses directed against a single antigen are insufficient to protect mice with different MHC alleles, and a combination of multiple antigens was required even to protect mice from the most common laboratory strains of Plasmodium. These data indicate that a combination of epitopes form several antigens is necessary to elicit a protective CTL response.
  • Indirect evidence that CTLs are important in protective immunity against Pf in humans has also accumulated. It has been reported that cytotoxic CD8+ T cells can be identified in humans immunized with PF sporozoites (Moreno, et al., Int. Immunol. 3:997, 1991). Further, humans immunized with irradiated sporozoites or naturally exposed to malaria can generate a CTL response to the pre-erythrocytic-stage antigens, CSP, sporozoite surface protein 2 (SSP2), liver-stage antigen-1 (LSA-1), and exported protein-1 (Exp-1) (see, e.g. Malik et al., Proc. Natl. Acad. Sci. USA 88, 3300-3304, 1991; Doolan et al., Int. Immunol. 3:511-516, 1991; Hill et al., Nature 360:434-439, 1992). Additionally, there is evidence that the polymorphism within the CSP may be the result of selection by CTLs of parasites that express variant forms (MCutchan and Water, Immunol. Lett. 25:23-26, 1990). This is based on the observation that the variation is nonsynonymous at the nucleotide level, thereby indicating selective pressure at the protein level. The polymorphism primarily maps to identified CTL and T helper epitopes (Doolan et al., Int. Immunol. 5:27-46, 1993); and CTL responses to some of the parasite variants do not cross-react (Hill et al., supra). Finally, the MHC class I human leukocyte antigen (HLA)-Bw53 has been associated with resistance to severe malaria in The Gambia, and CTLs to a conserved epitope restricted by the HLA-Bw53 allele have been identified on P. falciparum LSA-1 (Hill et al., Nature 352:595-600, 1991; Hill et al., Nature 340:434-439, 1992). Since HLA-Bw53 is found in 15%-40% of the population of sub-Saharan Africa but in less than 1% of Caucasians and Asians, these data suggest evolutionary selection on the basis of protection against severe malaria.
  • Thus, antibody, and both HLA class I and class II restricted responses directed against multiple sporozoite antigens appear to be involved in generating protective immunity to malaria. Furthermore, several important antigenic epitopes against which humoral and cellular immunity is focused have already been exactly delineated.
  • HLA class I molecules are expressed on the surface of almost all nucleated cells. Following intracellular processing of antigens, epitopes from the antigens are presented as a complex with the HLA class I molecules on the surface of such cells. CTL recognize the peptide-HLA class I complex, which then results in the destruction of the cell bearing the HLA-peptide complex directly by the CTL and/or via the activation of non-destructive mechanisms e.g., the production of interferon.
  • In view of the heterogeneous immune response observed with PF infection, induction of a multi-specific cellular immune response directed simultaneously against multiple PF epitopes appears to be important for the development of an efficacious vaccine against PF. There is a need, however, to establish vaccine embodiments that elicit immune responses that correspond to responses seen in patients that clear PF infection.
  • The information provided in this section is intended to disclose the presently understood state of the art as of the filing date of the present application. Information is included in this section which was generated subsequent to the priority date of this application. Accordingly, information in this section is not intended, in any way, to delineate the priority date for the invention.
    • II. SUMMARY OF THE INVENTION
  • This invention applies our knowledge of the mechanisms by which antigen is recognized by T cells, for example, to develop epitope-based vaccines directed towards PF. More specifically, this application communicates our discovery of specific epitope pharmaceutical compositions and methods of use in the prevention and treatment of PF infection.
  • Upon development of appropriate technology, the use of epitope-based vaccines has several advantages over current vaccines, particularly when compared to the use of whole antigens in vaccine compositions. There is evidence that the immune response to whole antigens is directed largely toward variable regions of the antigen, allowing for immune escape due to mutations. The epitopes for inclusion in an epitope-based vaccine are selected from conserved regions of antigens of pathogenic organisms or tumor-associated antigens, which thereby reduces the likelihood of escape mutants. Furthermore, immunosuppressive epitopes that may be present in whole antigens can be avoided with the use of epitope-based vaccines.
  • An additional advantage of an epitope-based vaccine approach is the ability to combine selected epitopes (CTL and HTL), and further, to modify the composition of the epitopes, achieving, for example, enhanced immunogenicity. Accordingly, the immune response can be modulated, as appropriate, for the target disease. Similar engineering of the response is not possible with traditional approaches.
  • Another major benefit of epitope-based immune-stimulating vaccines is their safety. The possible pathological side effects caused by infectious agents or whole protein antigens, which might have their own intrinsic biological activity, is eliminated.
  • An epitope-based vaccine also provides the ability to direct and focus an immune response to multiple selected antigens from the same pathogen. Thus, patient-by-patient variability in the immune response to a particular pathogen may be alleviated by inclusion of epitopes from multiple antigens from that pathogen in a vaccine composition. A “pathogen” may be an infectious agent or a tumor associated molecule.
  • One of the most formidable obstacles to the development of broadly efficacious epitope-based immunotherapeutics, however, has been the extreme polymorphism of HLA molecules. To date, effective non-genetically biased coverage of a population has been a task of considerable complexity; such coverage has required that epitopes be used that are specific for HLA molecules corresponding to each individual HLA allele; impractically large numbers of epitopes would therefore have to be used in order to cover ethnically diverse populations. Thus, there has existed a need for peptide epitopes that are bound by multiple HLA antigen molecules for use in epitope-based vaccines. The greater the number of HLA antigen molecules bound, the greater the breadth of population coverage by the vaccine.
  • Furthermore, as described herein in greater detail, a need has existed to modulate peptide binding properties, e.g., so that peptides that are able to bind to multiple HLA antigens do so with an affinity that will stimulate an immune response. Identification of epitopes restricted by more than one HLA allele at an affinity that correlates with immunogenicity is important to provide thorough population coverage, and to allow the elicitation of responses of sufficient vigor to prevent or clear an infection in a diverse segment of the population. Such a response can also target a broad array of epitopes. The technology disclosed herein provides for such favored immune responses.
  • In a preferred embodiment, epitopes for inclusion in vaccine compositions of the invention are selected by a process whereby protein sequences of known antigens are evaluated for the presence of motif or supermotif-bearing epitopes. Peptides corresponding to a motif- or supermotif-bearing epitope are then synthesized and tested for the ability to bind to the HLA molecule that recognizes the selected motif. Those peptides that bind at an intermediate or high affinity i.e., an IC50 (or a KD value) of 500 nM or less for HLA class I molecules or an IC50 of 1000 nM or less for HLA class II molecules, are further evaluated for their ability to induce a CTL or HTL response. Immunogenic peptide epitopes are selected for inclusion in vaccine compositions.
  • Supermotif-bearing peptides may additionally be tested for the ability to bind to multiple alleles within the HLA supertype family. Moreover, peptide epitopes may be analogued to modify binding affinity and/or the ability to bind to multiple alleles within an HLA supertype.
  • The invention also includes embodiments comprising methods for monitoring or evaluating an immune response to PF in patient having a known HLA-type. Such methods comprise incubating a T cell sample from the patient with a peptide composition comprising an PF epitope consisting essentially of an amino acid sequence described in Tables VII to Table XX or Table XXII which binds the product of at least one HLA allele present in the patient, and detecting for the presence of a T cell that binds to the peptide. A CTL peptide epitope may, for example, be used as a component of a tetrameric complex for such an analysis.
  • An alternative modality for defining the peptide epitopes in accordance with the invention is to recite the physical properties, such as length; primary structure; or charge, which are correlated with binding to a particular allele-specific HLA molecule or group of allele-specific HLA molecules. A further modality for defining peptide epitopes is to recite the physical properties of an HLA binding pocket, or properties shared by several allele-specific HLA binding pockets (e.g. pocket configuration and charge distribution) and reciting that the peptide epitope fits and binds to said pocket or pockets.
  • As will be apparent from the discussion below, other methods and embodiments are also contemplated. Further, novel synthetic peptides produced by any of the methods described herein are also part of the invention.
    • III. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 provides a graph of total frequency of genotypes as a function of the number of PF candidate epitopes bound by HLA-A and B molecules, in an average population.
    •  IV. DETAILED DESCRIPTION OF THE INVENTION
  • The peptide epitopes and corresponding nucleic acid compositions of the present invention are useful for stimulating an immune response to PF by stimulating the production of CTL or HTL responses. The peptide epitopes, which are derived directly or indirectly from native PF protein amino acid sequences, are able to bind to HLA molecules and stimulate an immune response to PF. The complete sequence of the PF proteins to be analyzed can be obtained from Genbank. Peptide epitopes and analogs thereof can also be readily determined from sequence information that may subsequently be discovered for heretofore unknown variants of PF, as will be clear from the disclosure provided below.
  • The peptide epitopes of the invention have been identified in a number of ways, as will be discussed below. Also discussed in greater detail is that analog peptides have been derived and the binding activity for HLA molecules modulated by modifying specific amino acid residues to create peptide analogs exhibiting altered immunogenicity. Further, the present invention provides compositions and combinations of compositions that enable epitope-based vaccines that are capable of interacting with HLA molecules encoded by various genetic alleles to provide broader population coverage than prior vaccines.
    • IV.A. DEFINITIONS
  • The invention can be better understood with reference to the following definitions, which are listed alphabetically:
  • A “computer” or “computer system” generally includes: a processor; at least one information storage/retrieval apparatus such as, for example, a hard drive, a disk drive or a tape drive; at least one input apparatus such as, for example, a keyboard, a mouse, a touch screen, or a microphone; and display structure. Additionally, the computer may include a communication channel in communication with a network. Such a computer may include more or less than what is listed above.
  • “Cross-reactive binding” indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.
  • A “cryptic epitope” elicits a response by immunization with an isolated peptide, but the response is not cross-reactive in vitro when intact whole protein which comprises the epitope is used as an antigen.
  • A “dominant epitope” is an epitope that induces an immune response upon immunization with a whole native antigen (see, e.g., Sercarz, et al., Annu. Rev. Immunol. 11:729-766, 1993). Such a response is cross-reactive in vitro with an isolated peptide epitope.
  • With regard to a particular amino acid sequence, an “epitope” is a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor (TCR) proteins and/or Major Histocompatibility Complex (MHC) receptors. In an immune system setting, in vivo or in vitro, an epitope is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, TCR or HLA molecule. Throughout this disclosure epitope and peptide are often used interchangeably. It is to be appreciated, however, that isolated or purified protein or peptide molecules larger than and comprising an epitope of the invention are still within the bounds of the invention.
  • “Human Leukocyte Antigen” or “HLA” is a human class I or class II MHC protein (see, e.g., Stites, et al., IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos, Calif. (1994).
  • An “HLA supertype or family”, as used herein, describes sets of HLA molecules grouped on the basis of shared peptide-binding specificities. HLA class I molecules that share somewhat similar binding affinity for peptides bearing certain amino acid motifs are grouped into HLA supertypes. The terms HLA superfamily, HLA supertype family, HLA family, and HLA xx-like molecules (where xx denotes a particular HLA type), are synonyms.
  • Throughout this disclosure, results are expressed in terms of “IC50's.” IC50 is the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide is observed. Given the conditions in which the assays are run (i.e., limiting HLA proteins and labeled peptide concentrations), these values approximate KD values. Assays for determining binding are described in detail, e.g., in PCT publications WO 94/20127 and WO 94/03205. It should be noted that IC50 values can change, often dramatically, if the assay conditions are varied, and depending on the particular reagents used (e.g., HLA preparation, etc.). For example, excessive concentrations of HLA molecules will increase the apparent measured IC50 of a given ligand.
  • Alternatively, binding is expressed relative to a reference peptide. Although as a particular assay becomes more, or less, sensitive, the IC50's of the peptides tested may change somewhat, the binding relative to the reference peptide will not significantly change. For example, in an assay run under conditions such that the IC50 of the reference peptide increases 10-fold, the IC50 values of the test peptides will also shift approximately 10-fold. Therefore, to avoid ambiguities, the assessment of whether a peptide is a good, intermediate, weak, or negative binder is generally based on its IC50, relative to the IC50 of a standard peptide.
  • Binding may also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al., Nature 339:392, 1989; Christnick et al., Nature 352:67, 1991; Busch et al., Immunol. 2:443, 1990; Hill et al., J. Immunol. 147:189, 1991; del Guercio et al., J. Immunol. 154:685, 1995), cell free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21:2069, 1991), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890, 1994; Marshall et al., J. Immunol. 152:4946, 1994), ELISA systems (e.g., Reay et al., EMBO J. 11:2829, 1992), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425, 1993); high flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353, 1994), and measurement of class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476, 1990; Schumacher et al., Cell 62:563, 1990; Townsend et al., Cell 62:285, 1990; Parker et al., J. Immunol. 149:1896, 1992).
  • As used herein, “high affinity” with respect to HLA class I molecules is defined as binding with an IC50, or KD value, of 50 nM or less; “intermediate affinity” is binding with an IC50 or KD value of between about 50 and about 500 nM. “High affinity” with respect to binding to HLA class II molecules is defined as binding with an IC50 or KD value of 100 nM or less; “intermediate affinity” is binding with an IC50 or KD value of between about 100 and about 1000 nM.
  • The terms “identical” or percent “identity,” in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.
  • An “immunogenic peptide” or “peptide epitope” is a peptide that comprises an allele-specific motif or supermotif such that the peptide will bind an HLA molecule and induce a CTL and/or HTL response. Thus, immunogenic peptides of the invention are capable of binding to an appropriate HLA molecule and thereafter inducing a cytotoxic T cell response, or a helper T cell response, to the antigen from which the immunogenic peptide is derived.
  • The phrases “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.
  • “Major Histocompatibility Complex” or “MHC” is a cluster of genes that plays a role in control of the cellular interactions responsible for physiologic immune responses. In humans, the MHC complex is also known as the HLA complex. For a detailed description of the MHC and HLA complexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3RD ED., Raven Press, New York, 1993.
  • The term “motif” refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.
  • A “negative binding residue” or “deleterious residue” is an amino acid which, if present at certain positions (typically not primary anchor positions) in a peptide epitope, results in decreased binding affinity of the peptide for the peptide's corresponding HLA molecule.
  • The term “peptide” is used interchangeably with “oligopeptide” in the present specification to designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the alpha-amino and carboxyl groups of adjacent amino acids. The preferred CTL-inducing peptides of the invention are 13 residues or less in length and usually consist of between about 8 and about 11 residues, preferably 9 or 10 residues. The preferred HTL-inducing peptides are less than about 50 residues in length and usually consist of between about 6 and about 30 residues, more usually between about 12 and 25, and often between about 15 and 20 residues.
  • “Pharmaceutically acceptable” refers to a non-toxic, inert, and physiologically compatible composition.
  • A “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding grooves of an HLA molecule, with their side chains buried in specific pockets of the binding grooves themselves. In one embodiment, for example, the primary anchor residues are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 9-residue peptide epitope in accordance with the invention. The primary anchor positions for each motif and supermotif are set forth in Table 1. For example, analog peptides can be created by altering the presence or absence of particular residues in these primary anchor positions. Such analogs are used to modulate the binding affinity of a peptide comprising a particular motif or supermotif.
  • “Promiscuous recognition” is where a distinct peptide is recognized by the same T cell clone in the context of various HLA molecules. Promiscuous recognition or binding is synonymous with cross-reactive binding.
  • A “protective immune response” or “therapeutic immune response” refers to a CTL and/or an HTL response to an antigen derived from an infectious agent or a tumor antigen, which prevents or at least partially arrests disease symptoms or progression. The immune response may also include an antibody response which has been facilitated by the stimulation of helper T cells.
  • The term “residue” refers to an amino acid or amino acid mimetic incorporated into an oligopeptide by an amide bond or amide bond mimetic.
  • A “secondary anchor residue” is an amino acid at a position other than a primary anchor position in a peptide which may influence peptide binding. A secondary anchor residue occurs at a significantly higher frequency amongst bound peptides than would be expected by random distribution of amino acids at one position. The secondary anchor residues are said to occur at “secondary anchor positions.” A secondary anchor residue can be identified as a residue which is present at a higher frequency among high or intermediate affinity binding peptides, or a residue otherwise associated with high or intermediate affinity binding. For example, analog peptides can be created by altering the presence or absence of particular residues in these secondary anchor positions. Such analogs are used to finely modulate the binding affinity of a peptide comprising a particular motif or supermotif.
  • A “subdominant epitope” is an epitope which evokes little or no response upon immunization with whole antigens which comprise the epitope, but for which a response can be obtained by immunization with an isolated peptide, and this response (unlike the case of cryptic epitopes) is detected when whole protein is used to recall the response in vitro or in vivo.
  • A “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. Preferably, a supermotif-bearing peptide is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.
  • “Synthetic peptide” refers to a peptide that is not naturally occurring, but is man-made using such methods as chemical synthesis or recombinant DNA technology.
  • The nomenclature used to describe peptide compounds follows the conventional practice wherein the amino group is presented to the left (the N-terminus) and the carboxyl group to the right (the C-terminus) of each amino acid residue. When amino acid residue positions are referred to in a peptide epitope they are numbered in an amino to carboxyl direction with position one being the position closest to the amino terminal end of the epitope, or the peptide or protein of which it may be a part. In the formulae representing selected specific embodiments of the present invention, the amino- and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by standard three letter or single letter designations. The L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form for those amino acids having D-forms is represented by a lower case single letter or a lower case three letter symbol. Glycine has no asymmetric carbon atom and is simply referred to as “Gly” or G. Symbols for the amino acids are shown below.
  • Single Letter Three Letter Amino
    Symbol Symbol Acids
    A Ala Alanine
    C Cys Cysteine
    D Asp Aspartic Acid
    E Glu Glutamic Acid
    F Phe Phenylalanine
    G Gly Glycine
    H His Histidine
    I Ile Isoleucine
    K Lys Lysine
    L Leu Leucine
    M Met Methionine
    N Asn Asparagine
    P Pro Proline
    Q Gln Glutamine
    R Arg Arginine
    S Ser Serine
    T Thr Threonine
    V Val Valine
    W Trp Tryptophan
    Y Tyr Tyrosine
    • IV.B. STIMULATION OF CTL AND HTL RESPONSES
  • The mechanism by which T cells recognize antigens has been delineated during the past ten years. Based on our understanding of the immune system we have developed efficacious peptide epitope vaccine compositions that can induce a therapeutic or prophylactic immune response to PF in a broad population. For an understanding of the value and efficacy of the claimed compositions, a brief review of immunology-related technology is provided.
  • A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are described herein and are set forth in Tables I, II, and III (see also, e.g., Southwood, et al., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, access via web at: http://134.2.96.221/scripts.hlaserver.d11/home.htm; Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics, in press, 1999).
  • Furthermore, x-ray crystallographic analysis of HLA-peptide complexes has revealed pockets within the peptide binding cleft of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e.g., Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991.)
  • Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that have the potential of binding particular HLA antigen(s).
  • The present inventors have found that the correlation of binding affinity with immunogenicity, which is disclosed herein, is an important factor to be considered when evaluating candidate peptides. Thus, by a combination of motif searches and HLA-peptide binding assays, candidates for epitope-based vaccines have been identified. After determining their binding affinity, additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, antigenicity, and immunogenicity.
  • Various strategies can be utilized to evaluate immunogenicity, including:
  • 1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1, 1998); This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a 51Cr-release assay involving peptide sensitized target cells.
  • 2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997); In this method, peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected using, e.g., a 51Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.
  • 3) Demonstration of recall T cell responses from immune individuals who have effectively been vaccinated, recovered from infection, and/or from chronically infected patients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). In applying this strategy, recall responses are detected by culturing PBL from subjects that have been naturally exposed to the antigen, for instance through infection, and thus have generated an immune response “naturally”, or from patients who were vaccinated against the infection. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of “memory” T cells, as compared to “naive” T cells. At the end of the culture period, T cell activity is detected using assays for T cell activity including 51Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.
  • The following describes the peptide epitopes and corresponding nucleic acids of the invention.
    • IV.C. BINDING AFFINITY OF PEPTIDE EPITOPES FOR HLA MOLECULES
  • As indicated herein, the large degree of HLA polymorphism is an important factor to be taken into account with the epitope-based approach to vaccine development. To address this factor, epitope selection encompassing identification of peptides capable of binding at high or intermediate affinity to multiple HLA molecules is preferably utilized, most preferably these epitopes bind at high or intermediate affinity to two or more allele-specific HLA molecules.
  • CTL-inducing peptides of interest for vaccine compositions preferably include those that have an IC50 or binding affinity value for class I HLA molecules of 500 nM or better (i.e., the value is ≦500 nM). HTL-inducing peptides preferably include those that have an IC50 or binding affinity value for class II HLA molecules of 1000 nM or better, (i.e., the value is ≦1,000 nM). For example, peptide binding is assessed by testing the capacity of a candidate peptide to bind to a purified HLA molecule in vitro. Peptides exhibiting high or intermediate affinity are then considered for further analysis. Selected peptides are tested on other members of the supertype family. In preferred embodiments, peptides that exhibit cross-reactive binding are then used in cellular screening analyses or vaccines.
  • As disclosed herein, higher HLA binding affinity is correlated with greater immunogenicity. Greater immunogenicity can be manifested in several different ways. Immunogenicity corresponds to whether an immune response is elicited at all, and to the vigor of any particular response, as well as to the extent of a population in which a response is elicited. For example, a peptide might elicit an immune response in a diverse array of the population, yet in no instance produce a vigorous response. In accordance with these principles, close to 90% of high binding peptides have been found to be immunogenic, as contrasted with about 50% of the peptides which bind with intermediate affinity. Moreover, higher binding affinity peptides leads to more vigorous immunogenic responses. As a result, less peptide is required to elicit a similar biological effect if a high affinity binding peptide is used. Thus, in preferred embodiments of the invention, high affinity binding epitopes are particularly useful.
  • The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes on bound antigens has been determined for the first time in the art by the present inventors. The correlation between binding affinity and immunogenicity was analyzed in two different experimental approaches (see, e.g., Sette, et al., J. Immunol. 153:5586-5592, 1994). In the first approach, the immunogenicity of potential epitopes ranging in HLA binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL from acute hepatitis patients. Pursuant to these approaches, it was determined that an affinity threshold value of approximately 500 nM (preferably 50 nM or less) determines the capacity of a peptide epitope to elicit a CTL response. These data are true for class I binding affinity measurements for naturally processed peptides and for synthesized T cell epitopes. These data also indicate the important role of determinant selection in the shaping of T cell responses (see, e.g., Schaeffer et al. Proc. Natl. Acad. Sci. USA 86:4649-4653, 1989).
  • An affinity threshold associated with immunogenicity in the context of HLA class II DR molecules has also been delineated (see, e.g., Southwood et al. J. Immunology 160:3363-3373,1998, and U.S. Ser. No. 09/009,953 filed Jan. 21, 1998, now U.S. Pat. No. 6,413,517). In order to define a biologically significant threshold of DR binding affinity, a database of the binding affinities of 32 DR-restricted epitopes for their restricting element (i.e., the HLA molecule that binds the motif) was compiled. In approximately half of the cases (15 of 32 epitopes), DR restriction was associated with high binding affinities, i.e. binding affinity values of 100 nM or less. In the other half of the cases (16 of 32), DR restriction was associated with intermediate affinity (binding affinity values in the 100-1000 nM range). In only one of 32 cases was DR restriction associated with an IC50 of 1000 nM or greater. Thus, 1000 nM can be defined as an affinity threshold associated with immunogenicity in the context of DR molecules.
  • The binding affinity of peptides for HLA molecules can be determined as described in Example 1, below.
    • IV.D. PEPTIDE EPITOPE BINDING MOTIFS AND SUPERMOTIFS
  • Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues required for allele-specific binding to HLA molecules have been identified. The presence of these residues correlates with binding affinity for HLA molecules. The identification of motifs and/or supermotifs that correlate with high and intermediate affinity binding is an important issue with respect to the identification of immunogenic peptide epitopes for the inclusion in a vaccine. Kast et al. (J. Immunol. 152:3904-3912, 1994) have shown that motif-bearing peptides account for 90% of the epitopes that bind to allele-specific HLA class I molecules. In this study all possible peptides of 9 amino acids in length and overlapping by eight amino acids (240 peptides), which cover the entire sequence of the E6 and E7 proteins of human papillomavirus type 16, were evaluated for binding to five allele-specific HLA molecules that are expressed at high frequency among different ethnic groups. This unbiased set of peptides allowed an evaluation of the predictive value of HLA class I motifs. From the set of 240 peptides, 22 peptides were identified that bound to an allele-specific HLA molecule with high or intermediate affinity. Of these 22 peptides, 20 (i.e. 91%) were motif-bearing. Thus, this study demonstrates the value of motifs for the identification of peptide epitopes for inclusion in a vaccine: application of motif-based identification techniques will identify about 90% of the potential epitopes in a target antigen protein sequence.
  • Such peptide epitopes are identified in the Tables described below.
  • Peptides of the present invention may also comprise epitopes that bind to MEW class II DR molecules. A greater degree of heterogeneity in both size and binding frame position of the motif, relative to the N and C termini of the peptide, exists for class II peptide ligands. This increased heterogeneity of HLA class II peptide ligands is due to the structure of the binding groove of the HLA class II molecule which, unlike its class I counterpart, is open at both ends. Crystallographic analysis of HLA class II DRB*0101-peptide complexes showed that the major energy of binding is contributed by peptide residues complexed with complementary pockets on the DRB*0101 molecules. An important anchor residue engages the deepest hydrophobic pocket (see, e.g., Madden, D. R. Ann. Rev. Immunol. 13:587, 1995) and is referred to as position 1 (P1). P1 may represent the N-terminal residue of a class II binding peptide epitope, but more typically is flanked towards the N-terminus by one or more residues. Other studies have also pointed to an important role for the peptide residue in the 6th position towards the C-terminus, relative to P1, for binding to various DR molecules.
  • In the past few years evidence has accumulated to demonstrate that a large fraction of HLA class I and class II molecules can be classified into a relatively few supertypes, each characterized by largely overlapping peptide binding repertoires, and consensus structures of the main peptide binding pockets. Thus, peptides of the present invention are identified by any one of several HLA-specific amino acid motifs (see, e.g., Tables or if the presence of the motif corresponds to the ability to bind several allele-specific HLA antigens, a supermotif. The HLA molecules that bind to peptides that possess a particular amino acid supermotif are collectively referred to as an HLA “supertype.”
  • The peptide motifs and supermotifs described below, and summarized in Tables I-III, provide guidance for the identification and use of peptide epitopes in accordance with the invention.
  • Examples of peptide epitopes bearing a respective supermotif or motif are included in Tables as designated in the description of each motif or supermotif below. The Tables include a binding affinity ratio listing for some of the peptide epitopes. The ratio may be converted to IC50 by using the following formula: IC50 of the standard peptide/ratio=IC50 of the test peptide (i.e., the peptide epitope). The IC50 values of standard peptides used to determine binding affinities for Class I peptides are shown in Table IV. The IC50 values of standard peptides used to determine binding affinities for Class II peptides are shown in Table V. The peptides used as standards for the binding assays described herein are examples of standards; alternative standard peptides can also be used when performing binding analyses.
  • To obtain the peptide epitope sequences listed in each Table, protein sequence data for four P. falciparum antigens were evaluated for the presence of the designated supermotif or motif. These antigens are: EXP-1, LSA-1, SSP2, and CSP. Nineteen sequences were available for CSP, 10 sequences were available for SSP, and one sequence each was available for EXP-1 and LSA-1. Peptide epitopes were additionally evaluated on the basis of their conservancy among the protein sequences for the PF antigens for which multiple sequences were available. A criterion for conservancy requires that the entire sequence of an HLA class I binding peptide be totally (i.e., 100%) conserved in 79% of the sequences available for a specific protein. Similarly, a criterion for conservancy requires that the entire 9-mer core region of an HLA class II binding peptide be totally conserved in 79% of the sequences available for a specific protein. The percent conservancy of the selected peptide epitopes is indicated on the Tables. The frequency, i.e. the number of sequences of the PF protein antigen in which the totally conserved peptide sequence was identified, is also shown. The “pos” (position) column in the Tables designates the amino acid position in the PF protein that corresponds to the first amino acid residue of the epitope. The “number of amino acids” indicates the number of residues in the epitope sequence.
    • HLA Class I Motifs Indicative of CTL Inducing Peptide Epitopes:
  • The primary anchor residues of the HLA class I peptide epitope supermotifs and motifs delineated below are summarized in Table I. The HLA class I motifs set out in Table I(a) are those most particularly relevant to the invention claimed here. Primary and secondary anchor positions are summarized in Table II. Allele-specific HLA molecules that comprise HLA class I supertype families are listed in Table VI. In some cases, peptide epitopes may be listed in both a motif and a supermotif Table. The relationship of a particular motif and respective supermotif is indicated in the description of the individual motifs.
    • IV.D.1. HLA-A1 SUPERMOTIF
  • The HLA-A1 supermotif is characterized by the presence in peptide ligands of a small (T or S) or hydrophobic (L, I, V, or M) primary anchor residue in position 2, and an aromatic (Y, F, or W) primary anchor residue at the C-terminal position of the epitope (see, e.g., Sette and Sidney, Immunogenetics, in press, 1999). The corresponding family of HLA molecules that bind to the A1 supermotif (i.e., the HLA-A1 supertype) is comprised of at least A*0101, A*2601, A*2602, A*2501, and A*3201 (see, e.g., DiBrino, M. et al., J. Immunol. 151:5930, 1993; DiBrino, M. et al., J. Immunol. 152:620, 1994; Kondo, A. et al., Immunogenetics 45:249, 1997). Other allele-specific HLA molecules predicted to be members of the A1 supertype are shown in Table VI. Peptides binding to each of the individual HLA proteins can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • Representative peptide epitopes that comprise the A1 supermotif are set forth on the attached Table VII.
    • IV.D.2. HLA-A2 Supermotif
  • Primary anchor specificities for allele-specific HLA-A2.1 molecules (see, e.g., Falk et al., Nature 351:290-296, 1991; Hunt et al., Science 255:1261-1263, 1992; Parker et al., J. Immunol. 149:3580-3587, 1992; Ruppert et al., Cell 74:929-937, 1993) and cross-reactive binding among HLA-A2 and -A28 molecules have been described. (See, e.g., Fruci et al., Human Immunol. 38:187-192, 1993; Tanigaki et al., Human Immunol. 39:155-162, 1994; Del Guercio et al., J. Immunol. 154:685-693, 1995; Kast et al., J. Immunol. 152:3904-3912, 1994 for reviews of relevant data.) These primary anchor residues define the HLA-A2 supermotif; which presence in peptide ligands corresponds to the ability to bind several different HLA-A2 and -A28 molecules. The HLA-A2 supermotif comprises peptide ligands with L, I, V, M, A, T, or Q as a primary anchor residue at position 2 and L, I, V, M, A, or T as a primary anchor residue at the C-terminal position of the epitope.
  • The corresponding family of HLA molecules (i.e., the HLA-A2 supertype that binds these peptides) is comprised of at least: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*0209, A*0214, A*6802, and A*6901. Other allele-specific HLA molecules predicted to be members of the A2 supertype are shown in Table VI. As explained in detail below, binding to each of the individual allele-specific HLA molecules can be modulated by substitutions at the primary anchor and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • Representative peptide epitopes that comprise an A2 supermotif are set forth on the attached Table VIII. The motifs comprising the primary anchor residues V, A, T, or Q at position 2 and L, I, V, A, or T at the C-terminal position are those most particularly relevant to the invention claimed herein.
    • IV.D.3. HLA-A3 Supermotif
  • The HLA-A3 supermotif is characterized by the presence in peptide ligands of A, L, I, V, M, S, or, T as a primary anchor at position 2, and a positively charged residue, R or K, at the C-terminal position of the epitope, e.g., in position 9 of 9-mers (see, e.g., Sidney et al., Hum. Immunol. 45:79, 1996). Exemplary members of the corresponding family of HLA molecules (the HLA-A3 supertype) that bind the A3 supermotif include at least A*0301, A*1101, A*3101, A*3301, and A*6801. Other allele-specific HLA molecules predicted to be members of the A3 supertype are shown in Table VI. As explained in detail below, peptide binding to each of the individual allele-specific HLA proteins can be modulated by substitutions of amino acids at the primary and/or secondary anchor positions of the peptide, preferably choosing respective residues specified for the supermotif.
  • Representative peptide epitopes that comprise the A3 supermotif are set forth on the attached Table IX.
    • IV.D.4. HLA-A24 Supermotif
  • The HLA-A24 supermotif is characterized by the presence in peptide ligands of an aromatic (F, W, or Y) or hydrophobic aliphatic (L, I, V, M, or T) residue as a primary anchor in position 2, and Y, F, W, L, I, or M as primary anchor at the C-terminal position of the epitope (see, e.g., Sette and Sidney, Immunogenetics, in press, 1999). The corresponding family of HLA molecules that bind to the A24 supermotif (i.e., the A24 supertype) includes at least A*2402, A*3001, and A*2301. Other allele-specific HLA molecules predicted to be members of the A24 supertype are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • Representative peptide epitopes that comprise the A24 supermotif are set forth on the attached Table X.
    • IV.D.5. HLA-B7 Supermotif
  • The HLA-B7 supermotif is characterized by peptides bearing proline in position 2 as a primary anchor, and a hydrophobic or aliphatic amino acid (L, I, V, M, A, F, W, or Y) as the primary anchor at the C-terminal position of the epitope. The corresponding family of HLA molecules that bind the B7 supermotif (i.e., the HLA-B7 supertype) is comprised of at least twenty six HLA-B proteins including: B*0702, B*0703, B*0704, B*0705, B*1508, B*3501, B*3502, B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101, B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501, B*5502, B*5601, B*5602, B*6701, and B*7801 (see, e.g., Sidney, et al., J. Immunol. 154:247, 1995; Barber, et al., Curr. Biol. 5:179, 1995; Hill, et al., Nature 360:434, 1992; Rammensee, et al., Immunogenetics 41:178, 1995 for reviews of relevant data). Other allele-specific HLA molecules predicted to be members of the B7 supertype are shown in Table VI. As explained in detail below, peptide binding to each of the individual allele-specific HLA proteins can be modulated by substitutions at the primary and/or secondary anchor positions of the peptide, preferably choosing respective residues specified for the supermotif.
  • Representative peptide epitopes that comprise the B7 supermotif are set forth on the attached Table XI.
    • IV.D.6. HLA-B27 Supermotif
  • The HLA-B27 supermotif is characterized by the presence in peptide ligands of a positively charged (R, H, or K) residue as a primary anchor at position 2, and a hydrophobic (F, Y, L, W, M, I, A, or V) residue as a primary anchor at the C-terminal position of the epitope (see, e.g., Sidney and Sette, Immunogenetics, in press, 1999). Exemplary members of the corresponding family of HLA molecules that bind to the B27 supermotif (i.e., the B27 supertype) include at least B*1401, B*1402, B*1509, B*2702, B*2703, B*2704, B*2705, B*2706, B*3801, B*3901, B*3902, and B*7301. Other allele-specific HLA molecules predicted to be members of the B27 supertype are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • Representative peptide epitopes that comprise the B27 supermotif are set forth on the attached Table XII.
    • IV.D.7. HLA-B44 Supermotif
  • The HLA-B44 supermotif is characterized by the presence in peptide ligands of negatively charged (D or E) residues as a primary anchor in position 2, and hydrophobic residues (F, W, Y, L, I, M, V, or A) as a primary anchor at the C-terminal position of the epitope (see, e.g., Sidney et al., Immunol. Today 17:261, 1996). Exemplary members of the corresponding family of HLA molecules that bind to the B44 supermotif (i.e., the B44 supertype) include at least: B*1801, B*1802, B*3701, B*4001, B*4002, B*4006, B*4402, B*4403, and B*4006. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions; preferably choosing respective residues specified for the supermotif.
    • IV.D.8. HLA-B58 Supermotif
  • The HLA-B58 supermotif is characterized by the presence in peptide ligands of a small aliphatic residue (A, S, or T) as a primary anchor residue at position 2, and an aromatic or hydrophobic residue (F, W, Y, L, I, V, M, or A) as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Sidney and Sette, Immunogenetics, in press, 1999 for reviews of relevant data). Exemplary members of the corresponding family of HLA molecules that bind to the B58 supermotif (i.e., the B58 supertype) include at least: B*1516, B*1517, B*5701, B*5702, and B*5801. Other allele-specific HLA molecules predicted to be members of the B58 supertype are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • Representative peptide epitopes that comprise the B58 supermotif are set forth on the attached Table XIII.
    • IV.D.9. HLA-B62 Supermotif
  • The HLA-B62 supermotif is characterized by the presence in peptide ligands of the polar aliphatic residue Q or a hydrophobic aliphatic residue (L, V, M, I, or P) as a primary anchor in position 2, and a hydrophobic residue (F, W, Y, M, I, V, L, or A) as a primary anchor at the C-terminal position of the epitope (see, e.g., Sidney and Sette, Immunogenetics, in press, 1999). Exemplary members of the corresponding family of HLA molecules that bind to the B62 supermotif (i.e., the B62 supertype) include at least: B*1501, B*1502, B*1513, and B5201. Other allele-specific HLA molecules predicted to be members of the B62 supertype are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • Representative peptide epitopes that comprise the B62 supermotif are set forth on the attached Table XIV.
    • IV.D.10. HLA-A1 Motif
  • The HLA-A1 motif is characterized by the presence in peptide ligands of T, S, or M as a primary anchor residue at position 2 and the presence of Y as a primary anchor residue at the C-terminal position of the epitope. An alternative allele-specific A1 motif is characterized by a primary anchor residue at position 3 rather than position 2. This motif is characterized by the presence of D, E, A, or S as a primary anchor residue in position 3, and a Y as a primary anchor residue at the C-terminal position of the epitope (see, e.g., DiBrino et al., J. Immunol., 152:620, 1994; Kondo et al., Immunogenetics 45:249, 1997; and Kubo et al., J. Immunol. 152:3913, 1994 for reviews of relevant data). Peptide binding to HLA A1 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.
  • Representative peptide epitopes that comprise either A1 motif are set forth on the attached Table XV. Those epitopes comprising T, S, or M at position 2 and Y at the C-terminal position are also included in the listing of HLA-A1 supermotif-bearing peptide epitopes listed in Table VII, as these residues are a subset of the A1 supermotif primary anchors.
    • IV.D.11. HLA-A*0201 Motif
  • An HLA-A2*0201 motif was determined to be characterized by the presence in peptide ligands of L or M as a primary anchor residue in position 2, and L or V as a primary anchor residue at the C-terminal position of a 9-residue peptide (see, e.g., Falk et al., Nature 351:290-296, 1991) and was further found to comprise an I at position 2 and I or A at the C-terminal position of a nine amino acid peptide (see, e.g., Hunt et al., Science 255:1261-1263, Mar. 6, 1992; Parker et al., J. Immunol. 149:3580-3587, 1992). The A*0201 allele-specific motif has also been defined by the present inventors to additionally comprise V, A, T, or Q as a primary anchor residue at position 2, and M or T as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Kast et al., J. Immunol. 152:3904-3912, 1994). Thus, the HLA-A*0201 motif comprises peptide ligands with L, I, V, M, A, T, or Q as primary anchor residues at position 2 and L, I, V, M, A, or T as a primary anchor residue at the C-terminal position of the epitope. The preferred and tolerated residues that characterize the primary anchor positions of the HLA-A*0201 motif are identical to the residues describing the A2 supermotif. (For reviews of relevant data, see, e.g., Del Guercio et al., J. Immunol. 154:685-693, 1995; Ruppert et al., Cell 74:929-937, 1993; Sidney et al., Immunol. Today 17:261-266, 1996; Sette and Sidney, Curr. Opin. in Immunol. 10:478-482, 1998). Secondary anchor residues that characterize the A*0201 motif have additionally been defined (see, e.g., Ruppert et al., Cell 74:929-937, 1993). These are shown in Table II. Peptide binding to HLA-A*0201 molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.
  • Representative peptide epitopes that comprise an A*0201 motif are set forth on the attached Table VIII. The A*0201 motifs comprising the primary anchor residues V, A, T, or Q at position 2 and L, I, V, A, or T at the C-terminal position are those most particularly relevant to the invention claimed herein
    • IV.D.12. HLA-A3 Motif
  • The HLA-A3 motif is characterized by the presence in peptide ligands of L, M, V, I, S, A, T, F, C, G, or D as a primary anchor residue at position 2, and the presence of K, Y, R, H, F, or A as a primary anchor residue at the C-terminal position of the epitope (see, e.g., DiBrino et al., Proc. Natl. Acad. Sci USA 90:1508, 1993; and Kubo et al., J. Immunol. 152:3913-3924, 1994). Peptide binding to HLA-A3 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.
  • Representative peptide epitopes that comprise the A3 motif are set forth on the attached Table XVI. Those peptide epitopes that also comprise the A3 supermotif are also listed in Table IX. The A3 supermotif primary anchor residues comprise a subset of the A3- and A11-allele specific motif primary anchor residues.
    • IV.D.13. HLA-A11 Motif
  • The HLA-A11 motif is characterized by the presence in peptide ligands of V, T, M, L, I, S, A, G, N, C, D, or F as a primary anchor residue in position 2, and K, R, Y, or H as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Zhang et al., Proc. Natl. Acad. Sci USA 90:2217-2221, 1993; and Kubo et al., J. Immunol. 152:3913-3924, 1994). Peptide binding to HLA-A11 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.
  • Representative peptide epitopes that comprise the A11 motif are set forth on the attached Table XVII; peptide epitopes comprising the A3 allele-specific motif are also present in this Table because of the extensive overlap between the A3 and A11 motif primary anchor specificities. Further, those peptide epitopes that comprise the A3 supermotif are also listed in Table IX.
    • IV.D.14. HLA-A24 Motif
  • The HLA-A24 motif is characterized by the presence in peptide ligands of Y, F, W, or M as a primary anchor residue in position 2, and F, L, I, or W as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Kondo et al., J. Immunol. 155:4307-4312, 1995; and Kubo et al., J. Immunol. 152:3913-3924, 1994). Peptide binding to HLA-A24 molecules can be modulated by substitutions at primary and/or secondary anchor positions; preferably choosing respective residues specified for the motif.
  • Representative peptide epitopes that comprise the A24 motif are set forth on the attached Table XVIII. These epitopes are also listed in Table X, which sets forth HLA-A24-supermotif-bearing peptide epitopes, as the primary anchor residues characterizing the A24 allele-specific motif comprise a subset of the A24 supermotif primary anchor residues.
    • Motifs Indicative of Class II HTL Inducing Peptide Epitopes
  • The primary and secondary anchor residues of the HLA class II peptide epitope supermotifs and motifs delineated below are summarized in Table III.
    • IV.D.15. HLA DR-1-4-7 Supermotif
  • Motifs have also been identified for peptides that bind to three common HLA class II allele-specific HLA molecules: HLA DRB1*0401, DRB1*0101, and DRB1*0701 (see, e.g., the review by Southwood et al. J. Immunology 160:3363-3373,1998). Collectively, the common residues from these motifs delineate the HLA DR-1-4-7 supermotif. Peptides that bind to these DR molecules carry a supermotif characterized by a large aromatic or hydrophobic residue (Y, F, W, L, I, V, or M) as a primary anchor residue in position 1, and a small, non-charged residue (S, T, C, A, P, V, I, L, or M) as a primary anchor residue in position 6 of a 9-mer core region. Allele-specific secondary effects and secondary anchors for each of these HLA types have also been identified (Southwood et al., supra). These are set forth in Table III. Peptide binding to HLA-DRB1*0401, DRB1*0101, and/or DRB1*0701 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • Conserved 9-mer core regions (i.e., sequences that are 100% conserved in at least 79% of the PF antigen protein sequences used for the analysis), comprising the DR-1-4-7 supermotif, wherein position 1 of the supermotif is at position 1 of the nine-residue core, are set forth in Table XIXa. Respective exemplary peptide epitopes of 15 amino acid residues in length, each of which comprise a conserved nine residue core, are also shown in section “a” of the Table. Cross-reactive binding data for exemplary 15-residue supermotif-bearing peptides are shown in Table XIXb.
    • IV.D.16. HLA DR3 Motifs
  • Two alternative motifs (i.e., submotifs) characterize peptide epitopes that bind to HLA-DR3 molecules (see, e.g., Geluk et al., J. Immunol. 152:5742, 1994). In the first motif (submotif DR3A) a large, hydrophobic residue (L, I, V, M, F, or Y) is present in anchor position 1 of a 9-mer core, and D is present as an anchor at position 4, towards the carboxyl terminus of the epitope. As in other class II motifs, core position 1 may or may not occupy the peptide N-terminal position.
  • The alternative DR3 submotif provides for lack of the large, hydrophobic residue at anchor position 1, and/or lack of the negatively charged or amide-like anchor residue at position 4, by the presence of a positive charge at position 6 towards the carboxyl terminus of the epitope. Thus, for the alternative allele-specific DR3 motif (submotif DR3B): L, I, V, M, F, Y, A, or Y is present at anchor position 1; D, N, Q, E, S, or T is present at anchor position 4; and K, R, or H is present at anchor position 6. Peptide binding to HLA-DR3 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.
  • Conserved 9-mer core regions (i.e., those sequences that are 100% conserved in at least 79% of the PF antigen protein sequences used for the analysis) corresponding to a nine residue sequence comprising the DR3A submotif (wherein position 1 of the motif is at position 1 of the nine residue core) are set forth in Table XXa. Respective exemplary peptide epitopes of 15 amino acid residues in length, each of which comprise a conserved nine residue core, are also shown in Table XXa. Table XXb shows binding data of exemplary DR3 submotif A-bearing peptides.
  • Conserved 9-mer core regions (i.e., those that are 100% conserved in at least 79% conserved in the PF antigen protein sequences used for the analysis) comprising the DR3B submotif and respective exemplary 15-mer peptides comprising the DR3 submotif-B epitope are set forth in Table XXc. Table XXd shows binding data of exemplary DR3 submotif B-bearing peptides.
  • Each of the HLA class I or class II peptide epitopes set out in the Tables herein are deemed singly to be an inventive aspect of this application. Further, it is also an inventive aspect of this application that each peptide epitope may be used in combination with any other peptide epitope.
    • IV.E. ENHANCING POPULATION COVERAGE OF THE VACCINE
  • Vaccines that have broad population coverage are preferred because they are more commercially viable and generally applicable to the most people. Broad population coverage can be obtained using the peptides of the invention (and nucleic acid compositions that encode such peptides) through selecting peptide epitopes that bind to HLA alleles which, when considered in total, are present in most of the population. Table XXI lists the overall frequencies of the HLA class I supertypes in various ethnicities (Table XXIa) and the combined population coverage achieved by the A2-, A3-, and B7-supertypes (Table XXIb). The A2-, A3-, and B7 supertypes are each present on the average of over 40% in each of these five major ethnic groups. Coverage in excess of 80% is achieved with a combination of these supermotifs. These results suggest that effective and non-ethnically biased population coverage is achieved upon use of a limited number of cross-reactive peptides. Although the population coverage reached with these three main peptide specificities is high, coverage can be expanded to reach 95% population coverage and above, and more easily achieve truly multispecific responses upon use of additional supermotif or allele-specific motif bearing peptides.
  • The B44-, A1-, and A24-supertypes are present, on average, in a range from 25% to 40% in these major ethnic populations (Table XXIa). While less prevalent overall, the B27-, B58-, and B62 supertypes are each present with a frequency >25% in at least one major ethnic group (Table XXIa). Table XXIb summarizes the estimated prevalence of combinations of HLA supertypes that have been identified in five major ethnic groups. The incremental coverage obtained by the inclusion of A1-, A24-, and B44-supertypes with the A2, A3, and B7 coverage and coverage obtained with all of the supertypes described herein, is shown.
  • The data presented herein, together with the previous definition of the A2-, A3-, and B7-supertypes, indicates that all antigens, with the possible exception of A29, B8, and B46, can be classified into a total of nine HLA supertypes. By including epitopes from the six most frequent supertypes, an average population coverage of 99% is obtained for five major ethnic groups.
    • IV.F. IMMUNE RESPONSE-STIMULATING PEPTIDE ANALOGS
  • In general, CTL and HTL responses are not directed against all possible epitopes. Rather, they are restricted to a few “immunodominant” determinants (Zinkernagel, et al., Adv. Immunol. 27:5159, 1979; Bennink, et al., J. Exp. Med. 168:19351939, 1988; Rawle, et al., J. Immunol. 146:3977-3984, 1991). It has been recognized that immunodominance (Benacerraf, et al., Science 175:273-279, 1972) could be explained by either the ability of a given epitope to selectively bind a particular HLA protein (determinant selection theory) (Vitiello, et al., J. Immunol. 131:1635, 1983); Rosenthal, et al., Nature 267:156-158, 1977), or to be selectively recognized by the existing TCR (T cell receptor) specificities (repertoire theory) (Klein, J., IMMUNOLOGY, THE SCIENCE OF SELFNONSELF DISCRIMINATION, John Wiley & Sons, New York, pp. 270-310, 1982). It has been demonstrated that additional factors, mostly linked to processing events, can also play a key role in dictating, beyond strict immunogenicity, which of the many potential determinants will be presented as immunodominant (Sercarz, et al., Annu. Rev. Immunol. 11:729-766, 1993).
  • The concept of dominance and subdominance is relevant to immunotherapy of both infectious diseases and cancer. For example, in the course of chronic infectious disease, recruitment of subdominant epitopes can be important for successful clearance of the infection, especially if dominant CTL or HTL specificities have been inactivated by functional tolerance, suppression, mutation of viruses and other mechanisms (Franco, et al., Curr. Opin. Immunol. 7:524-531, 1995). In the case of cancer and tumor antigens, CTLs recognizing at least some of the highest binding affinity peptides might be functionally inactivated. Lower binding affinity peptides are preferentially recognized at these times, and may therefore be preferred in therapeutic or prophylactic anti-cancer vaccines.
  • In particular, it has been noted that a significant number of epitopes derived from known non-viral tumor associated antigens (TAA) bind HLA class I with intermediate affinity (IC50 in the 50-500 nM range). For example, it has been found that 8 of 15 known TAA peptides recognized by tumor infiltrating lymphocytes (TIL) or CTL bound in the 50-500 nM range. (These data are in contrast with estimates that 90% of known viral antigens were bound by HLA class I molecules with IC50 of 50 nM or less, while only approximately 10% bound in the 50-500 nM range (Sette, et al., J. Immunol., 153:558-5592, 1994). In the cancer setting this phenomenon is probably due to elimination or functional inhibition of the CTL recognizing several of the highest binding peptides, presumably because of T cell tolerization events.
  • Without intending to be bound by theory, it is believed that because T cells to dominant epitopes may have been clonally deleted, selecting subdominant epitopes may allow existing T cells to be recruited, which will then lead to a therapeutic or prophylactic response. However, the binding of HLA molecules to subdominant epitopes is often less vigorous than to dominant ones. Accordingly, there is a need to be able to modulate the binding affinity of particular immunogenic epitopes for one or more HLA molecules, and thereby to modulate the immune response elicited by the peptide, for example to prepare analog peptides which elicit a more vigorous response. This ability would greatly enhance the usefulness of peptide epitope-based vaccines and therapeutic agents.
  • Although peptides with suitable cross-reactivity among all alleles of a superfamily are identified by the screening procedures described above, cross-reactivity is not always as complete as possible, and in certain cases procedures to increase cross-reactivity of peptides can be useful; moreover, such procedures can also be used to modify other properties of the peptides such as binding affinity or peptide stability. Having established the general rules that govern cross-reactivity of peptides for HLA alleles within a given motif or supermotif, modification (i.e., analoging) of the structure of peptides of particular interest in order to achieve broader (or otherwise modified) HLA binding capacity can be performed. More specifically, peptides which exhibit the broadest cross-reactivity patterns, can be produced in accordance with the teachings herein. The present concepts related to analog generation are set forth in greater detail in U.S. Ser. No. 09/226,775 filed Jan. 6, 1999, now abandoned.
  • In brief, the strategy employed utilizes the motifs or supermotifs which correlate with binding to certain HLA molecules. The motifs or supermotifs are defined by having primary anchors, and in many cases secondary anchors. Analog peptides can be created by substituting amino acid residues at primary anchor, secondary anchor, or at primary and secondary anchor positions. Generally, analogs are made for peptides that already bear a motif or supermotif. Preferred secondary anchor residues of supermotifs and motifs that have been defined for HLA class I and class II binding peptides are shown in Tables II and III, respectively.
  • For a number of the motifs or supermotifs in accordance with the invention, residues are defined which are deleterious to binding to allele-specific HLA molecules or members of HLA supertypes that bind the respective motif or supermotif (Tables II and III). Accordingly, removal of such residues that are detrimental to binding can be performed in accordance with the present invention. For example, in the case of the A3 supertype, when all peptides that have such deleterious residues are removed from the population of peptides used for the analysis, the incidence of cross-reactivity increased from 22% to 37% (see, e.g., Sidney, J. et al., Hu. Immunol. 45:79, 1996). Thus, one strategy to improve the cross-reactivity of peptides within a given supermotif is simply to delete one or more of the deleterious residues present within a peptide and substitute a small “neutral” residue such as Ala (that may not influence T cell recognition of the peptide). An enhanced likelihood of cross-reactivity is expected if, together with elimination of detrimental residues within a peptide, “preferred” residues associated with high affinity binding to an allele-specific HLA molecule or to multiple HLA molecules within a superfamily are inserted.
  • To ensure that an analog peptide, when used as a vaccine, actually elicits a CTL response to the native epitope in vivo (or, in the case of class II epitopes, elicits helper T cells that cross-react with the wild type peptides), the analog peptide may be used to immunize T cells in vitro from individuals of the appropriate HLA allele. Thereafter, the immunized cells' capacity to induce lysis of wild type peptide sensitized target cells is evaluated. It will be desirable to use as antigen presenting cells, cells that have been either infected, or transfected with the appropriate genes, or, in the case of class II epitopes only, cells that have been pulsed with whole protein antigens, to establish whether endogenously produced antigen is also recognized by the relevant T cells.
  • Another embodiment of the invention is to create analogs of weak binding peptides. Class I binding peptides exhibiting binding affinities of 500-5000 nM, and carrying an acceptable but suboptimal primary anchor residue at one or both positions can be “fixed” by substituting preferred anchor residues in accordance with the respective supertype. The analog peptides can then be tested for crossbinding activity.
  • Another embodiment for generating effective peptide analogs involves the substitution of residues that have an adverse impact on peptide stability or solubility in, e.g., a liquid environment. This substitution may occur at any position of the peptide epitope. For example, a cysteine (C) can be substituted out in favor of α-amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substituting α-amino butyric acid for C not only alleviates this problem, but actually improves binding and crossbinding capability in certain instances (see, e.g., the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, 1999). Substitution of cysteine with α-amino butyric acid may occur at any residue of a peptide epitope, i.e. at either anchor or non-anchor positions.
  • Representative analog peptides are set forth in Table XXII. The Table indicates the length and sequence of the analog peptide as well as the motif or supermotif, if appropriate. The information in the “Fixed Nomenclature” column indicates the residues substituted at the indicated position numbers for the respective analog.
    • IV.G. COMPUTER SCREENING OF PROTEIN SEQUENCES FROM DISEASE-RELATED ANTIGENS FOR SUPERMOTIF- OR MOTIF-BEARING PEPTIDES
  • In order to identify supermotif- or motif-bearing epitopes in a target antigen, a native protein sequence, e.g., a tumor-associated antigen, or sequences from an infectious organism, or a donor tissue for transplantation, is screened using a means for computing, such as an intellectual calculation or a computer, to determine the presence of a supermotif or motif within the sequence. The information obtained from the analysis of native peptide can be used directly to evaluate the status of the native peptide or may be utilized subsequently to generate the peptide epitope.
  • Computer programs that allow the rapid screening of protein sequences for the occurrence of the subject supermotifs or motifs are encompassed by the present invention; as are programs that permit the generation of analog peptides. These programs are implemented to analyze any identified amino acid sequence or operate on an unknown sequence and simultaneously determine the sequence and identify motif-bearing epitopes thereof; analogs can be simultaneously determined as well. Generally, the identified sequences will be from a pathogenic organism or a tumor-associated peptide. For example, the target molecules considered herein include, without limitation, the EXP1, LSA1, SSP2, and CSP1 proteins of PF.
  • In cases where the sequence of multiple variants of the same target protein are available, peptides may also be selected on the basis of their conservancy. A presently preferred criterion for conservancy defines that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide, be totally (i.e., 100%) conserved in at least 79% of the sequences evaluated for a specific protein. This definition of conservancy has been employed herein; although, as appreciated by those in the art, lower or higher degrees of conservancy can be employed as appropriate for a given antigenic target.
  • It is important that the selection criteria utilized for prediction of peptide binding are as accurate as possible, to correlate most efficiently with actual binding. Prediction of peptides that bind, for example, to HLA-A*0201, on the basis of the presence of the appropriate primary anchors, is positive at about a 30% rate (see, e.g., Ruppert, J. et al. Cell 74:929, 1993). However, by extensively analyzing peptide-HLA binding data disclosed herein, data in related patent applications, and data in the art, the present inventors have developed a number of allele-specific polynomial algorithms that dramatically increase the predictive value over identification on the basis of the presence of primary anchor residues alone. These algorithms take into account not only the presence or absence of primary anchors, but also consider the positive or deleterious presence of secondary anchor residues (to account for the impact of different amino acids at different positions). The algorithms are essentially based on the premise that the overall affinity (or AG) of peptide-HLA interactions can be approximated as a linear polynomial function of the type:

  • ΔG=a 1i ×a 2i ×a 3i . . . ×a ni
  • where aji is a coefficient that represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. An important assumption of this method is that the effects at each position are essentially independent of each other. This assumption is justified by studies that demonstrated that peptides are bound to HLA molecules and recognized by T cells in essentially an extended conformation. Derivation of specific algorithm coefficients has been described, for example, in Gulukota, K. et al., J. Mol. Biol. 267:1258, 1997.
  • Additional methods to identify preferred peptide sequences, which also make use of specific motifs, include the use of neural networks and molecular modeling programs (see, e.g., Milik et al., Nature Biotechnology 16:753, 1998; Altuvia et al., Hum. Immunol. 58:1, 1997; Altuvia et al, J. Mol. Biol. 249:244, 1995; Buus, S. Curr. Opin. Immunol. 11:209-213, 1999; Brusic, V. et al., Bioinformatics 14:121-130, 1998; Parker et al., J. Immunol. 152:163, 1993; Meister et al., Vaccine 13:581, 1995; Hammer et al., J. Exp. Med. 180:2353, 1994; Sturniolo et al., Nature Biotechnol. 17:555 1999).
  • For example, it has been shown that in sets of A*0201 motif-bearing peptides containing at least one preferred secondary anchor residue while avoiding the presence of any deleterious secondary anchor residues, 69% of the peptides will bind A*0201 with an IC50 less than 500 nM (Ruppert, J. et al. Cell 74:929, 1993). These algorithms are also flexible in that cut-off scores may be adjusted to select sets of peptides with greater or lower predicted binding properties, as desired.
  • In utilizing computer screening to identify peptide epitopes, a protein sequence or translated sequence may be analyzed using software developed to search for motifs, for example the “FINDPATTERNS’ program (Devereux, et al. Nucl. Acids Res. 12:387-395, 1984) or MotifSearch 1.4 software program (D. Brown, San Diego, Calif.) to identify potential peptide sequences containing appropriate HLA binding motifs. The identified peptides can be scored using customized polynomial algorithms to predict their capacity to bind specific HLA class I or class II alleles. As appreciated by one of ordinary skill in the art, a large array of computer programming software and hardware options are available in the relevant art which can be employed to implement the motifs of the invention in order to evaluate (e.g., without limitation, to identify epitopes, identify epitope concentration per peptide length, or to generate analogs) known or unknown peptide sequences.
  • In accordance with the procedures described above, PF peptide epitopes and analogs thereof that are able to bind HLA supertype groups or allele-specific HLA molecules have been identified (Tables VII-XX; Table XXII).
    • IV.H. PREPARATION OF PEPTIDE EPITOPES
  • Peptides in accordance with the invention can be prepared synthetically, by recombinant DNA technology or chemical synthesis, or from natural sources such as native tumors or pathogenic organisms. Peptide epitopes may be synthesized individually or as polyepitopic peptides. Although the peptide will preferably be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides may be synthetically conjugated to native fragments or particles.
  • The peptides in accordance with the invention can be a variety of lengths, and either in their neutral (uncharged) forms or in forms which are salts. The peptides in accordance with the invention are either free of modifications such as glycosylation, side chain oxidation, or phosphorylation; or they contain these modifications, subject to the condition that modifications do not destroy the biological activity of the peptides as described herein.
  • Desirably, the peptide epitope will be as small as possible while still maintaining substantially all of the immunologic activity of the native protein. When possible, it may be desirable to optimize HLA class I binding peptide epitopes of the invention to a length of about 8 to about 13 amino acid residues, preferably 9 to 10. HLA class II binding peptide epitopes may be optimized to a length of about 6 to about 30 amino acids in length, preferably to between about 13 and about 20 residues. Preferably, the peptide epitopes are commensurate in size with endogenously processed pathogen-derived peptides or tumor cell peptides that are bound to the relevant HLA molecules.
  • The identification and preparation of peptides of other lengths can also be carried out using the techniques described herein. Moreover, it is preferred to identify native peptide regions that contain a high concentration of class I and/or class II epitopes. Such a sequence is generally selected on the basis that it contains the greatest number of epitopes per amino acid length. It is to be appreciated that epitopes can be present in a frame-shifted manner, e.g. a 10 amino acid long peptide could contain two 9 amino acid long epitopes and one 10 amino acid long epitope; upon intracellular processing, each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. This larger, preferably multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source.
  • The peptides of the invention can be prepared in a wide variety of ways. For the preferred relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. (See, for example, Stewart & Young, SOLID PHASE PEPTIDE SYNTHESIS, 2D. ED., Pierce Chemical Co., 1984). Further, individual peptide epitopes can be joined using chemical ligation to produce larger peptides that are still within the bounds of the invention.
  • Alternatively, recombinant DNA technology can be employed wherein a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression. These procedures are generally known in the art, as described generally in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Thus, recombinant polypeptides which comprise one or more peptide sequences of the invention can be used to present the appropriate T cell epitope.
  • The nucleotide coding sequence for peptide epitopes of the preferred lengths contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al., J. Am. Chem. Soc. 103:3185 (1981). Peptide analogs can be made simply by substituting the appropriate and desired nucleic acid base(s) for those that encode the native peptide sequence; exemplary nucleic acid substitutions are those that encode an amino acid defined by the motifs/supermotifs herein. The coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such vectors and suitable host systems are now available. For expression of the fusion proteins, the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host. For example, promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vectors are transformed into suitable bacterial hosts. Of course, yeast, insect or mammalian cell hosts may also be used, employing suitable vectors and control sequences.
    • IV.I. ASSAYS TO DETECT T-CELL RESPONSES
  • Once HLA binding peptides are identified, they can be tested for the ability to elicit a T-cell response. The preparation and evaluation of motif-bearing peptides are described in PCT publications WO 94/20127 and WO 94/03205. Briefly, peptides comprising epitopes from a particular antigen are synthesized and tested for their ability to bind to the appropriate HLA proteins. These assays may involve evaluating the binding of a peptide of the invention to purified HLA class I molecules in relation to the binding of a radioiodinated reference peptide. Alternatively, cells expressing empty class I molecules (i.e. lacking peptide therein) may be evaluated for peptide binding by immunofluorescent staining and flow microfluorimetry. Other assays that may be used to evaluate peptide binding include peptide-dependent class I assembly assays and/or the inhibition of CTL recognition by peptide competition. Those peptides that bind to the class I molecule, typically with an affinity of 500 nM or less, are further evaluated for their ability to serve as targets for CTLs derived from infected or immunized individuals, as well as for their capacity to induce primary in vitro or in vivo CTL responses that can give rise to CTL populations capable of reacting with selected target cells associated with a disease. Corresponding assays are used for evaluation of HLA class II binding peptides. HLA class II motif-bearing peptides that are shown to bind, typically at an affinity of 1000 nM or less, are further evaluated for the ability to stimulate HTL responses.
  • Conventional assays utilized to detect T cell responses include proliferation assays, lymphokine secretion assays, direct cytotoxicity assays, and limiting dilution assays. For example, antigen-presenting cells that have been incubated with a peptide can be assayed for the ability to induce CTL responses in responder cell populations. Antigen-presenting cells can be normal cells such as peripheral blood mononuclear cells or dendritic cells. Alternatively, mutant non-human mammalian cell lines that are deficient in their ability to load class I molecules with internally processed peptides and that have been transfected with the appropriate human class I gene, may be used to test for the capacity of the peptide to induce in vitro primary CTL responses.
  • Peripheral blood mononuclear cells (PBMCs) may be used as the responder cell source of CTL precursors. The appropriate antigen-presenting cells are incubated with peptide, after which the peptide-loaded antigen-presenting cells are then incubated with the responder cell population under optimized culture conditions. Positive CTL activation can be determined by assaying the culture for the presence of CTLs that kill radio-labeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed forms of the antigen from which the peptide sequence was derived.
  • More recently, a method has been devised which allows direct quantification of antigen-specific CTLs by staining with Fluorescein-labelled HLA tetrameric complexes (Altman, J. D. et al., Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman, J. D. et al., Science 274:94, 1996). Other relatively recent technical developments include staining for intracellular lymphokines, and interferon release assays or ELISPOT assays. Tetramer staining, intracellular lymphokine staining and ELISPOT assays all appear to be at least 10-fold more sensitive than more conventional assays (Lalvani, A. et al., J. Exp. Med. 186:859, 1997; Dunbar, P. R. et al., Curr. Biol. 8:413, 1998; Murali-Krishna, K. et al., Immunity 8:177, 1998).
  • HTL activation may also be assessed using such techniques known to those in the art such as T cell proliferation and secretion of lymphokines, e.g. IL-2 (see, e.g. Alexander et al., Immunity 1:751-761, 1994).
  • Alternatively, immunization of HLA transgenic mice can be used to determine immunogenicity of peptide epitopes. Several transgenic mouse models including mice with human A2.1, A11 (which can additionally be used to analyze HLA-A3 epitopes), and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed. Additional transgenic mouse models with other HLA alleles may be generated as necessary. Mice may be immunized with peptides emulsified in Incomplete Freund's Adjuvant and the resulting T cells tested for their capacity to recognize peptide-pulsed target cells and target cells transfected with appropriate genes. CTL responses may be analyzed using cytotoxicity assays described above. Similarly, HTL responses may be analyzed using such assays as T cell proliferation or secretion of lymphokines. Exemplary immunogenic peptide epitopes are set out in Table XXIII
    • IV.J. USE OF PEPTIDE EPITOPES AS DIAGNOSTIC AGENTS AND FOR EVALUATING IMMUNE RESPONSES
  • HLA class I and class II binding peptides as described herein can be used, in one embodiment of the invention, as reagents to evaluate an immune response. The immune response to be evaluated may be induced by using as an immunogen any agent that may result in the production of antigen-specific CTLs or HTLs that recognize and bind to the peptide epitope(s) to be employed as the reagent. The peptide reagent need not be used as the immunogen. Assay systems that may be used for such an analysis include relatively recent technical developments such as tetramers, staining for intracellular lymphokines and interferon release assays, or ELISPOT assays.
  • For example, a peptide of the invention may be used in a tetramer staining assay to assess peripheral blood mononuclear cells for the presence of antigen-specific CTLs following exposure to a pathogen or immunogen. The HLA-tetrameric complex is used to directly visualize antigen-specific CTLs (see, e.g., Ogg et al., Science 279:2103-2106, 1998; and Altman et al., Science 174:94-96, 1996) and determine the frequency of the antigen-specific CTL population in a sample of peripheral blood mononuclear cells. A tetramer reagent using a peptide of the invention may be generated as follows: A peptide that binds to an HLA molecule is refolded in the presence of the corresponding HLA heavy chain and β2-microglobulin to generate a trimolecular complex. The complex is biotinylated at the carboxyl terminal end of the heavy chain at a site that was previously engineered into the protein. Tetramer formation is then induced by the addition of streptavidin. By means of fluorescently labeled streptavidin, the tetramer can be used to stain antigen-specific cells. The cells may then be identified, for example, by flow cytometry. Such an analysis may be used for diagnostic or prognostic purposes.
  • Peptides of the invention may also be used as reagents to evaluate immune recall responses. (see, e.g., Bertoni et al., J. Clin. Invest. 100:503-513, 1997 and Penna et al., J. Exp. Med. 174:1565-1570, 1991.) For example, patient PBMC samples from individuals infected with PF may be analyzed for the presence of antigen-specific CTLs or HTLs using specific peptides. A blood sample containing mononuclear cells may be evaluated by cultivating the PBMCs and stimulating the cells with a peptide of the invention. After an appropriate cultivation period, the expanded cell population may be analyzed, for example, for CTL or for HTL activity.
  • The peptides may also be used as reagents to evaluate the efficacy of a vaccine. PBMCs obtained from a patient vaccinated with an immunogen may be analyzed using, for example, either of the methods described above. The patient is HLA typed, and peptide epitope reagents that recognize the allele-specific molecules present in that patient are selected for the analysis. The immunogenicity of the vaccine is indicated by the presence of PF epitope-specific CTLs and/or HTLs in the PBMC sample.
  • The peptides of the invention may also be used to make antibodies, using techniques well known in the art (see, e.g. CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY; and Antibodies A Laboratory Manual Harlow, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989), which may be useful as reagents to diagnose PF infection. Such antibodies include those that recognize a peptide in the context of an HLA molecule, i.e., antibodies that bind to a peptide-WIC complex.
    • IV.K. VACCINE COMPOSITIONS
  • Vaccines that contain an immunogenically effective amount of one or more peptides as described herein are a further embodiment of the invention. Once appropriately immunogenic epitopes have been defined, they can be sorted and delivered by various means, herein referred to as “vaccine” compositions. Such vaccine compositions can include, for example, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32, 1996), viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.
  • Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the claimed peptide(s). The peptide(s) can be individually linked to its own carrier; alternatively, the peptide(s) can exist as a homopolymer or heteropolymer of active peptide units. Such a polymer has the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response. The composition may be a naturally occurring region of an antigen or may be prepared, e.g., recombinantly or by chemical synthesis.
  • Furthermore, useful carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS).
  • As disclosed in greater detail herein, upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later infection, or at least partially resistant to developing an ongoing chronic infection, or derives at least some therapeutic benefit when the antigen was tumor-associated.
  • In some instances it may be desirable to combine the class I peptide vaccines of the invention with vaccines which induce or facilitate neutralizing antibody responses to the target antigen of interest, particularly to surface antigens. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a PADRE™ (Epimmune, San Diego, Calif.) molecule (described, for example, in U.S. Pat. No. 5,736,142). Furthermore, any of these embodiments can be administered as a nucleic acid mediated modality.
  • The vaccine compositions of the invention may also be used in combination with antiviral drugs such as interferon-α.
  • For therapeutic or prophylactic immunization purposes, the peptides of the invention can also be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus, for example, as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a non-infected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL and/or HTL response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.
  • Antigenic peptides are used to elicit a CTL and/or HTL response ex vivo, as well. Ex vivo administration is described, for example, in application U.S. Ser. No. 09/016,361 filed Jan. 30, 1998, now abandoned. The resulting CTL or HTL cells, can be used to treat chronic infections, or tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen (infectious or tumor-associated antigen) are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 14 weeks), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (an infected cell or a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells. Alternatively, dendritic cells are transfected, e.g., with a minigene construct in accordance with the invention, in order to elicit immune responses. Minigenes will be discussed in greater detail in a following section.
  • Vaccine compositions may also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.
  • DNA or RNA encoding one or more of the peptides of the invention can also be administered to a patient. This approach is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720; and in more detail below. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).
  • Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. Exemplary epitopes that may be utilized in a vaccine to treat or prevent PF infection are set out in Tables XXXIII and XXXIV. The multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.
  • It is preferred that each of the following principles are balanced in order to make the selection.
  • 1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with PF clearance. For HLA Class I this includes 3-4 epitopes that come from at least one antigen of PF. For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one PF antigen (see e.g., Rosenberg et al., Science 278:1447-1450).
  • 2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, or for Class II an IC50 of 1000 nM or less.
  • 3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.
  • 4.) When selecting epitopes from cancer-related antigens it is often preferred to select analogs because the patient may have developed tolerance to the native epitope. When selecting epitopes for infectious disease-related antigens it is preferable to select either native or analoged epitopes. Of particular relevance for infectious disease vaccines (but for cancer-related vaccines as well), are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A peptide comprising “transcendent nested epitopes” is a peptide that has both HLA class I and HLA class II epitopes in it.
  • When providing nested epitopes, it is preferable to provide a sequence that has the greatest number of epitopes per provided sequence. Preferably, one avoids providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a longer peptide sequence, such as a sequence comprising nested epitopes, it is important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.
  • 5.) When creating a minigene, as disclosed in greater detail in the following section, an objective is to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same as those employed when selecting a peptide comprising nested epitopes. Furthermore, upon determination of the nucleic acid sequence to be provided as a minigene, the peptide encoded thereby is analyzed to determine whether any “junctional epitopes” have been created. A junctional epitope is an actual binding epitope, as predicted, e.g., by motif analysis, that only exists because two discrete peptide sequences are encoded directly next to each other. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
    • IV.K.1. Minigene Vaccines
  • A growing body of experimental evidence demonstrates that a number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention. The use of multi-epitope minigenes is described below and in, e.g., application U.S. Ser. No. 09/311,784, now U.S. Pat. No. 6,534,482; Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding nine dominant HLA-A*0201- and A11-restricted epitopes derived from the polymerase, envelope, and core proteins of HBV and human immunodeficiency virus (HIV), the PADRE™ universal HTL epitope, and an endoplasmic reticulum-translocating signal sequence was engineered. Immunization of HLA transgenic mice with this plasmid construct resulted in strong CTL induction responses against the nine epitopes tested, similar to those observed with a lipopeptide of known immunogenicity in humans, and significantly greater than immunization in oil-based adjuvants. Moreover, the immunogenicity of DNA-encoded epitopes in vivo correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these data show that the minigene served to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes. A similar approach may be used to develop minigenes encoding PF epitopes.
  • For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.
  • The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.
  • Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
  • Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.
  • Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.
  • In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.
  • In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego, Calif.). HTL epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.
  • Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
  • Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids can also be used in the formulation; in addition, glycolipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) can also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987).
  • Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (51Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.
  • In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (IP) for lipid-complexed DNA).
  • Twenty-one days after immunization, splenocytes are harvested and restimulated for 1 week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, 51Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is evaluated in transgenic mice in an analogous manner.
  • Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.
  • IV.K.2. Combinations of CTL Peptides with Helper Peptides
  • Vaccine compositions comprising the peptides of the present invention, or analogs thereof, which have immunostimulatory activity may be modified to provide desired attributes, such as improved serum half life, or to enhance immunogenicity.
  • For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. The use of T helper epitopes in conjunction with CTL epitopes to enhance immunogenicity is illustrated, for example, in applications U.S. Ser. No. 08/197,484, now U.S. Pat. No. 6,419,931, and U.S. Ser. No. 08/464,234, now abandoned.
  • Particularly preferred CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively, the CTL peptide may be linked to the T helper peptide without a spacer.
  • The CTL peptide epitope may be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated. The HTL peptide epitopes used in the invention can be modified in the same manner as CTL peptides. For instance, they may be modified to include D-amino acids or be conjugated to other molecules such as lipids, proteins, sugars and the like.
  • In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in the majority of the population. This can be accomplished by selecting amino acid sequences that bind to many, most, or all of the HLA class II molecules. These are known as “loosely HLA-restricted” or “promiscuous” T helper sequences. Examples of amino acid sequences that are promiscuous include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO: 3799), Plasmodium falciparum CS protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 3800), and Streptococcus 18 kD protein at positions 116 (GAVDSILGGVATYGAA; SEQ ID NO: 3801). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.
  • Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego, Calif.) are designed to most preferrably bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVWANTLKAAa (SEQ ID NO: 3802), where “X” is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all “L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.
  • HTL peptide epitopes can also be modified to alter their biological properties. For example, peptides comprising HTL epitopes can contain D-amino acids to increase their resistance to proteases and thus extend their serum half-life. Also, the epitope peptides of the invention can be conjugated to other molecules such as lipids, proteins or sugars, or any other synthetic compounds, to increase their biological activity. Specifically, the T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.
  • In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes cytotoxic T cells. Lipids have been identified as agents capable of priming CTL in vivo against viral antigens. For example, palmitic acid residues can be attached to the ε- and α-amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic comprises palmitic acid attached to ε- and α-amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.
  • As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide. (See, e.g., Deres, et al., Nature 342:561, 1989). Peptides of the invention can be coupled to P3CSS, for example, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P3CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses to infection.
  • As noted herein, additional amino acids can be added to the termini of a peptide to provide for ease of linking peptides one to another, for coupling to a carrier support or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced at the C- or N-terminus of the peptide or oligopeptide, particularly class I peptides. However, it is to be noted that modification at the carboxyl terminus of a CTL epitope may, in some cases, alter binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH2 acylation, e.g., by alkanoyl (C1-C20) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.
    • IV.L. ADMINISTRATION OF VACCINES FOR THERAPEUTIC OR PROPHYLACTIC PURPOSES
  • The peptides of the present invention and pharmaceutical and vaccine compositions of the invention are useful for administration to mammals, particularly humans, to treat and/or prevent malaria. Vaccine compositions containing the peptides of the invention are administered to an individual susceptible to, or otherwise at risk for, malaria or to a patient infected with PF to elicit an immune response against PF antigens and thus enhance the patient's own immune response capabilities. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective CTL and/or HTL response to the PF antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
  • The vaccine compositions of the invention may also be used purely as prophylactic agents. The level of expected exposure (e.g., a traveler versus a resident of an area where malaria is endemic) determines the magnitude of response that is desired to be achieved by the vaccination. Therefore, some vaccination regimens may employ higher doses of the vaccine compositions, or more doses may be administered.
  • Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine may be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.
  • As noted above, peptides comprising CTL and/or HTL epitopes of the invention induce immune responses when presented by HLA molecules and contacted with a CTL or HTL specific for an epitope comprised by the peptide. The manner in which the peptide is contacted with the CTL or HTL is not critical to the invention. For instance, the peptide can be contacted with the CTL or HTL either in vivo or in vitro. If the contacting occurs in vivo, the peptide itself can be administered to the patient, or other vehicles, e.g., DNA vectors encoding one or more peptides, viral vectors encoding the peptide(s), liposomes and the like, can be used, as described herein.
  • For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual who has not been infected with PF. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences.
  • The pharmaceutical compositions may also be used to treat individuals already infected with PF. Patients can be treated with the immunogenic peptide epitopes separately or in conjunction with other treatments, as appropriate.
  • For therapeutic use, administration should generally begin at the first diagnosis of PF infection. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. Loading doses followed by boosting doses may be required.
  • The peptide or other compositions used for prophylaxis or the treatment of PF infection can be used, e.g., in persons who are not manifesting symptoms of disease but who act as a disease vector. In this context, it is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to effectively stimulate a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.
  • The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50000 μg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. The peptides and compositions of the present invention may be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.
  • Thus, for treatment of a chronically infected individual, a representative dose is in the range disclosed above. Initial doses followed by boosting doses at established intervals, e.g., from four weeks to six months, may be required, possibly for a prolonged period of time to effectively immunize an individual. Administration should continue until at least clinical symptoms or laboratory tests indicate that the PF infection has been eliminated or substantially abated and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.
  • The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, intrathecal, or local administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • A human unit dose form of the peptide composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, preferably an aqueous carrier, and is administered in a volume of fluid that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985).
  • The peptides of the invention may also be administered via liposomes, which serve to target the peptides to a particular tissue, such as lymphoid tissue, or to target selectively to infected cells, as well as to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
  • For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
  • For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.
  • For aerosol administration, the immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are 0.01%-20% by weight, preferably 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
    • IV.M. KITS
  • The peptide and nucleic acid compositions of this invention can be provided in kit form together with instructions for vaccine administration. Typically the kit would include desired peptide compositions in a container, preferably in unit dosage form and instructions for administration. An alternative kit would include a minigene construct with desired nucleic acids of the invention in a container, preferably in unit dosage form together with instructions for administration. Lymphokines such as IL-2 or IL-12 may also be included in the kit. Other kit components that may also be desirable include, for example, a sterile syringe, booster dosages, and other desired excipients.
  • The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield alternative embodiments in accordance with the invention.
    • V. EXAMPLES
  • The following examples illustrate identification, selection, and use of immunogenic Class I and Class II peptide epitopes for inclusion in vaccine compositions.
    • Example 1 HLA Class I and Class II Binding Assays
  • The following example of peptide binding to HLA molecules demonstrates quantification of binding affinities of HLA class I and class II peptides. Binding assays can be performed with peptides that are either motif-bearing or not motif-bearing.
  • Epstein-Barr virus (EBV)-transformed homozygous cell lines, fibroblasts, CIR, or 721.22 transfectants were used as sources of HLA class I molecules. These cells were maintained in vitro by culture in RPMI 1640 medium supplemented with 2 mM L-glutamine (GIBCO, Grand Island, N.Y.), 50 μM 2-ME, 100 μg/ml of streptomycin, 100 U/ml of penicillin (Irvine Scientific) and 10% heat-inactivated FCS (Irvine Scientific, Santa Ana, Calif.). Cells were grown in 225-cm2 tissue culture flasks or, for large-scale cultures, in roller bottle apparatuses. The specific cell lines routinely used for purification of MHC class I and class II molecules are listed in Table XXIV.
  • Cell lysates were prepared and HLA molecules purified in accordance with disclosed protocols (Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, cells were lysed at a concentration of 108 cells/ml in 50 mM Tris-HCl, pH 8.5, containing 1% Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland), 150 mM NaCl, 5 mM EDTA, and 2 mM PMSF. Lysates were cleared of debris and nuclei by centrifugation at 15,000×g for 30 min.
  • HLA molecules were purified from lysates by affinity chromatography. Lysates prepared as above were passed twice through two pre-columns of inactivated Sepharose CL4-B and protein A-Sepharose. Next, the lysate was passed over a column of Sepharose CL-4B beads coupled to an appropriate antibody. The antibodies used for the extraction of HLA from cell lysates are listed in Table XXV. The anti-HLA column was then washed with 10-column volumes of 10 mM Tris-HCL, pH 8.0, in 1% NP-40, PBS, 2-column volumes of PBS, and 2-column volumes of PBS containing 0.4% n-octylglucoside. Finally, MHC molecules were eluted with 50 mM diethylamine in 0.15M NaCl containing 0.4% n-octylglucoside, pH 11.5. A 1/25 volume of 2.0M Tris, pH 6.8, was added to the eluate to reduce the pH to ˜8.0. Eluates were then be concentrated by centrifugation in Centriprep 30 concentrators at 2000 rpm (Amicon, Beverly, Mass.). Protein content was evaluated by a BCA protein assay (Pierce Chemical Co., Rockford, Ill.) and confirmed by SDS-PAGE.
  • A detailed description of the protocol utilized to measure the binding of peptides to Class I and Class II MEW has been published (Sette et al., Mol. Immunol. 31:813, 1994; Sidney et al., in Current Protocols in Immunology, Margulies, Ed., John Wiley & Sons, New York, Section 18.3, 1998). Briefly, purified MHC molecules (5 to 500 nM) were incubated with various unlabeled peptide inhibitors and 1-10 nM 125I-radiolabeled probe peptides for 48 h in PBS containing 0.05% Nonidet P-40 (NP40) (or 20% w/v digitonin for H-2 IA assays) in the presence of a protease inhibitor cocktail. The final concentrations of protease inhibitors (each from CalBioChem, La Jolla, Calif.) were 1 mM PMSF, 1.3 nM 1.10 phenanthroline, 73 μM pepstatin A, 8 mM EDTA, 6 mM N-ethylmaleimide (for Class II assays), and 200 μM N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK). All assays were performed at pH 7.0 with the exception of DRB1*0301, which was performed at pH 4.5, and DRB1*1601 (DR2w21β1) and DRB4*0101 (DRw53), which were performed at pH 5.0. pH was adjusted as described elsewhere (see Sidney et al., in Current Protocols in Immunology, Margulies, Ed., John Wiley & Sons, New York, Section 18.3, 1998).
  • Following incubation, MHC-peptide complexes were separated from free peptide by gel filtration on 7.8 mm×15 cm TSK200 columns (TosoHaas 16215, Montgomeryville, Pa.), eluted at 1.2 mls/min with PBS pH 6.5 containing 0.5% NP40 and 0.1% NaN3. Because the large size of the radiolabeled peptide used for the DRB1*1501 (DR2w2β1) assay makes separation of bound from unbound peaks more difficult under these conditions, all DRB1*1501 (DR2w2β1) assays were performed using a 7.8 mm×30 cm TSK2000 column eluted at 0.6 mls/min. The eluate from the TSK columns was passed through a Beckman 170 radioisotope detector, and radioactivity was plotted and integrated using a Hewlett-Packard 3396A integrator, and the fraction of peptide bound was determined.
  • Radiolabeled peptides were iodinated using the chloramine-T method. Representative radiolabeled probe peptides utilized in each assay, and its assay specific IC50 nM, are summarized in Tables IV and V. Typically, in preliminary experiments, each MHC preparation was titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays were performed using these HLA concentrations.
  • Since under these conditions [label]<[HLA] and IC50≧[HLA], the measured IC50 values are reasonable approximations of the true KD values. Peptide inhibitors are typically tested at concentrations ranging from 120 μg/ml to 1.2 ng/ml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the IC50 of a positive control for inhibition by the IC50 for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide). For database purposes, and inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into IC50 nM values by dividing the IC50 nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation has proven to be the most accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC.
  • Because the antibody used for HLA-DR purification (LB3.1) is α-chain specific, β1 molecules are not separated from β3 (and/or β4 and β5) molecules. The β1 specificity of the binding assay is obvious in the cases of DRB1*0101 (DR1), DRB1*0802 (DR8w2), and DRB1*0803 (DR8w3), where no β3 is expressed. It has also been demonstrated for DRB1*0301 (DR3) and DRB3*0101 (DR52a), DRB1*0401 (DR4w4), DRB1*0404 (DR4w14), DRB1*0405 (DR4w15), DRB1*1101 (DR5), DRB1*1201 (DR5w12), DRB1*1302 (DR6w19) and DRB1*0701 (DR7). The problem of β chain specificity for DRB1*1501 (DR2w2β1), DRB5*0101 (DR2w2β2), DRB1*1601 (DR2w21β1), DRB5*0201 (DR51Dw21), and DRB4*0101 (DRw53) assays is circumvented by the use of fibroblasts. Development and validation of assays with regard to DRβ molecule specificity have been described previously (see, e.g., Southwood et al., J. Immunol. 160:3363-3373, 1998).
  • Binding assays as outlined above may be used to analyze supermotif and/or motif-bearing epitopes as, for example, described in Example 2.
    • Example 2 Identification of Conserved HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes
  • Vaccine compositions of the invention may include multiple epitopes that comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Additional experimental details that may be relevant to this example are found in Doolan, D. L. et al., Immunity 7:97, 1997. Calculation of population coverage was performed using the strategy described below.
  • Computer Searches and Algorithms for Identification of Supermotif and/or Motif-Bearing Epitopes
  • Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs were performed as follows. All translated PF protein sequences were analyzed using a text string search software program, e.g., MotifSearch 1.4 (D. Brown, San Diego) to identify potential peptide sequences containing appropriate HLA binding motifs;
  • alternative programs are readily produced in accordance with information in the art in view of the motif/supermotif disclosure herein. Furthermore, such calculations can be made mentally. Identified A2-, A3-, and DR-supermotif sequences were scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms take into account both extended and refined motifs (that is, to account for the impact of different amino acids at different positions), and are essentially based on the premise that the overall affinity (or AG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:

  • “ΔG”=a 1i ×a 2i ×a 3i ×a ni
  • where aji is a coefficient which represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue j occurs at position i in the peptide, it is assumed to contribute a constant amount ji to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide. This assumption is justified by studies from our laboratories that demonstrated that peptides are bound to MHC and recognized by T cells in essentially an extended conformation (data omitted herein).
  • The method of derivation of specific algorithm coefficients has been described in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate of ji. For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.
    • Selection of HLA-A2 Supertype Cross-Reactive Peptides
  • Complete protein sequences from PF antigens were aligned, then scanned, utilizing motif identification software, to identify conserved 9- and 10-mer sequences containing the HLA-A*0201-motif main anchor specificity. Following conservancy determination and algorithm analysis to take into account the influence of secondary anchors, 53 peptides containing the HLA-A*0201 of potential interest were identified and tested for their capacity to bind to purified HLA-A*0201 molecules in vitro. Fifteen peptides bound A*0201 with IC50 values ≦500 nM.
  • Fourteen of these peptides were subsequently tested for immunogenicity as described below. Of these, 5 scored positive both in primary in vitro CTL responses and in HLA transgenic mice.
  • The five immunogenic peptides were then tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). The peptide SSP214-23, which was immunogenic in primary human CTL cultures and contains the SSP214-22 epitope (rather than SSP214-22 itself), was included in the analysis. In addition, the peptide Exp-183, which was positive in the murine CTL assays and the peptide CSP425 and SSP2230, were also analyzed for cross-reactive binding. As shown in Table XXVI, all eight of these peptides were found to be A2-supertype cross-reactive binders with six of these binding to three or more A2 supertype alleles.
    • Selection of HLA-A3 Supermotif-Bearing Epitopes
  • The PF protein sequences scanned above were also examined for the presence of conserved peptides with the HLA-A3 supermotif primary anchors. Further analysis using the A03 and A11 algorithms (see, e.g., Gulukota et al, J. Mol. Biol. 267:1258-1267, 1997 and Sidney et al, Human Immunol. 45:79-93, 1996) identified 203 conserved 9- or 10-mer motif-containing peptide sequences that scored high in either or both algorithms. Of these candidates, twenty five peptides were identified that bound A3 and/or A11 with binding affinities of ≦500 nM. These peptides were then tested for binding cross-reactivity to the other common A3-supertype alleles (A*3101, A*3301, and A*6801). Seven of them bound at least three of the five HLA-A3-supertype molecules tested. An eighth peptide, LSA-111 was also considered for further study because it bound strongly to two of the A3 supertype alleles and weakly to the other two A3 supertype alleles. (Table XXVII)
  • In summary, eight HLA-A3 supertype cross-reactive binding peptides derived from conserved regions of PF proteins were identified.
    • Selection of HLA-B7 Supermotif Bearing Epitopes
  • When the same PF target antigen protein sequences were also analyzed for the presence of conserved 9- or 10-mer peptides with the HLA-B7-supermotif, 26 sequences were identified. Of these 26, 24 corresponding peptides were synthesized and tested for binding to HLA-B*0702, the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Four of the peptides bound B*0702 with IC50 of ≦500 nM. These four peptides were then tested for binding to other common B7-supertype molecules (B*3501, B*51, B*5301, and B*5401). As shown in Table XXVIII, one peptide was capable of to four of the five B7 supertype alleles; another was found to bind three of the five alles.
    • Selection of A1 and A24 Motif-Bearing Epitopes
  • To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into potential vaccine constructs.
  • An analysis of the protein sequence data from the PF target antigens utilized above identified 40 A1- and 81 A24-motif-containing conserved sequences. Testing for binding to the appropriate HLA molecule (i.e., A1 or A24) was performed on a subset of those peptides. Four A1-motif peptides and four A24-motif peptides, shown in Table Table XXIX, were found to have binding capacities of 500 nM or less for the appropriate allele-specific HLA molecule.
    • Example 3 Confirmation of Immunogenicity Evaluation of A*0201 Immunogenicity
  • It has been shown that CTL induced in A*0201/Kb transgenic mice exhibit specificity similar to CTL induced in the human system (see, e.g., Vitiello et al., J Exp. Med. 173:1007-1015, 1991; Wentworth et al., Eur. J. Immunol. 26:97-101, 1996). Accordingly, these mice were used to evaluate the immunogenicity of the fourteen conserved A*0201 motif-bearing high affinity binding peptides identified in Example 2 above.
  • CTL induction in transgenic mice following peptide immunization has been described (Vitiello et al., J. Exp. Med. 173:1007-1015, 1991; Alexander et al.; J. Immunol. 159:4753-4761, 1997). In these studies, mice were injected subcutaneously at the base of the tail with each peptide (50 μg/mouse) emulsified in IFA in the presence of an excess of an IAb-restricted helper peptide (140 μg/mouse) (HBV core 128-140, Sette et al., J Immunol. 153:5586-5592, 1994). Eleven days after injection, splenocytes were incubated in the presence of peptide-loaded syngenic LPS blasts. After six days, cultures were assayed for cytotoxic activity using peptide-pulsed targets. The data indicated that 5 of the 14 peptides were capable of inducing primary CTL responses in A*0201/Kb transgenic mice. (For these studies, a peptide was considered positive if it induced CTL (L.U. 30/106 cells in at least two transgenic animals (Wentworth et al., Eur. Immunol. 26:97-101, 1996).
  • The fourteen peptides that bound to HLA-A*0201 with good affinity were also tested for immunogenicity with PBMCs from at least four malaria-naive human donors. The induction of primary CTL responses in vitro with PBMCs from normal naive humans requires a brief treatment of the antigen-presenting cells with acidic buffer and subsequent neutralization in the presence of excess B2-microglobulin and exogenous peptide (Wentworth et al., supra). By ensuring that the majority of the HLA class I molecules are occupied by exogenous peptide, these steps are essential for the induction of primary CTL responses. Such responses cannot be induced using methods developed for the induction of recall CTL responses. A peptide was considered positive if yielding more than 2 LU30/106 cells (lytic units 20% per 104 cells, where one lytic unit corresponds to the number of effector cells required to induce 30% 51Cr release from 10,000 target cells during a 6 hr assay.) or 15% peptide-specific lysis, respectively, in at least two different primary CTL cultures. The five peptides that were positive in HLA transgenic mice were also shown to induce primary CTL responses.
  • The HLA-A2 cross-reactive binding peptides were tested for their ability to elicit in vitro recall responses from PBMCs of six volunteers, each of whom had an HLA-A*0201 allele, immunized with irradiated sporozoites. The results demonstrated that all of the A2-binding peptides were recognized in association with HLA-A*0201.
  • In addition to investigating whether the peptides could be recognized as CTL epitopes, the ability of the peptides to induce specific cytokine responses was also measured. In particular, induction of interferon-γ and TNF-α were measured, both of which have been implicated in protective immunity against malaria. PBMC from irradiated sporozoite-immunized volunteers and PBMC from naturally exposed individuals were tested. The results indicate that significant peptide-induced cytokine responses were observed for all of the A2 supermotif-bearing peptides. (See Doolan et al., Immunity 7:97-112, 1997.)
    • Evaluation of A*03/A11 Immunogenicity
  • The immunogenicity of the eight supermotif-bearing peptides was also evaluated in recall responses using PBMC from volunteers bearing HLA-A3 supertype alleles who had previously been immunized with irradiated sporozoites. All the peptides were recognized in association with both A3 and A33. The fraction of individuals responding to each peptide varied for the supertype overall from 50% for one of the peptides to 100% for three of the peptides.
  • Immunogenicity was also evaluated using PBMCs of semi-immune or nonimmune individuals naturally exposed to malaria. In this population, recall CTL responses (percentage specific lysis greater than 10%) were detected for five of the eight A3-binding peptides.
  • Immunogenicity of A3 supermotif-bearing peptides can also be evaluated in transgenic mice that bear a human HLA-A11 allele using methodology analagous to that for immunogenicity studies using HLA-A2.1 transgenic mice.
    • Evaluation of B7 Immunogenicity
  • Immunogenicity of two B7 supermotif-bearing peptides, SSP2539 and the HLA-B-restricted peptide Pfs1677 was also examined in individuals who had been exposed to PF, either through immunization or natural exposure, as described for the evaluation of A2- and A3-supermotif-bearing peptides.
  • Both peptides were found to be capable of inducing CTL responses. The two peptides were recognized as CTL epitopes in the context of three of the five B7 supertype alleles.
    • Example 4 Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs
  • HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analogued, or “fixed” to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analog peptides that exhibit modulated binding affinity are set forth in this example.
    • Analoging at Primary Anchor Residues
  • The primary anchor residues are analogued to modulate binding activity. For example, peptide engineering strategies are implemented to further increase the cross-reactivity of the A3-supertype candidate epitopes identified above. On the basis of the data disclosed, e.g., in related and U.S. Ser. No. 09/226,775, now abandoned, the main anchors of A3-supermotif-bearing peptides are altered, for example, to introduce a preferred V, S, or M at position 2.
  • To analyze the cross-reactivity of the analog peptides, each engineered analog is initially tested for binding to the prototype A3 supertype alleles A3 and A11; then, if binding capacity is maintained, for additional A3-supertype cross-reactivity.
  • Similarly, analogs of HLA-A2 supermotif-bearing epitopes may also be generated. For example, peptides binding to A2-supertype molecules may be engineered at primary anchor residues to possess a preferred residue (L, I, V, or M) at position 2 and/or a preferred I or V as a position 9 primary anchor residue.
  • The analog peptides are then tested for the ability to bind the A2 supermotif prototype allele, A*0201. Those peptides that demonstrate 500 nM binding capacity are then tested for A2-supertype cross-reactivity.
  • Similarly to the A2- and A3-motif bearing peptides, peptide binding to B7-supertype alleles may be improved, where possible, to achieve increased cross-reactive binding. B7 supermotif-bearing peptides may, for example, be engineered to possess a preferred residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490, 1996).
    • Analoging at Secondary Anchor Residues
  • Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties. For example, the binding capacity of an analog of the B7 supermotif-bearing peptide Pf SSP2126, representing a discreet single amino acid substitution at position one, is analyzed. The peptide may be substituted with an F at position 1, rather than and L. The peptide, which binds to 3 of 5 B7 supertype alles, is then analyzed for the ability to bind all five B7-supertype molecules with a good affinity.
  • Because so few B7-supertype cross-reactive epitopes were identified in the initial binding screen, results from previous binding evaluations may be analyzed to identify conserved (8-, 9-, 10-, or 11-mer) peptides which bind, minimally, 3/5 B7 supertype molecules with weak affinity (IC50 of 500 nM-5 μM). This analysis identifies additional candidate peptides that can be analogued. These peptides are tested for enhanced binding affinity and B7-supertype cross-reactivity.
  • Engineered analogs with sufficiently improved binding capacity or cross-reactivity are tested as described in Example 2 for the ability of the peptide to induce CTL responses using PBMC from individuals who had previously been exposed to Pf antigens. Immunogenicity may also be studied in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization.
  • In conclusion, these data demonstrate that by the use of even single amino acid substitutions, it is possible to increase the binding affinity and/or cross-reactivity of peptide ligands for HLA supertype molecules.
    • Example 5 Identification of Conserved PF-Derived Sequences with HLA-DR Binding Motifs
  • Peptide epitopes bearing an HLA class II supermotif or motif may also be identified as outlined below using methodology similar to that described in Examples 1-3.
    • Selection of HLA-DR-Supermotif-Bearing Epitopes
  • To identify PF-derived, HLA class II HTL epitopes, the protein sequences from the same four PF antigens used for the identification of HLA Class I supermotif/motif sequences were analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences were selected comprising a DR-supermotif, further comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total). It was also required that the 9-mer core sequence be 100% conserved in at least 79% of the sequences analyzed.
  • The conserved, PF-derived peptides identified above were tested for their binding capacity for various common HLA-DR molecules. All peptides were initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least 2 of these 3 DR molecules were then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, and DR9 molecules in secondary assays. Finally, peptides binding at least 2 of the 4 secondary panel DR molecules, and thus cumulatively at least 4 of 7 different DR molecules, were screened for binding to DR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides binding at least 7 of the 10 DR molecules comprising the primary, secondary, and tertiary screening assays were considered cross-reactive DR binders. The composition of these screening panels, and the phenotypic frequency of associated antigens, are shown in Table XXX.
  • In conclusion, 8 cross-reactive DR-binding peptides derived from 6 independent regions were identified that bind 7 or more HLA DR alleles. Five other peptides were also identified that bound between 4 and 6 DR alleles (Table XXXI).
    • Selection of Conserved DR3 Motif Peptides
  • Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is an important criterion in the selection of HTL epitopes. However, data generated previously indicated that DR3 only rarely cross-reacts with other DR alleles (Sidney et al., J. Immunol. 149:2634-2640, 1992; Geluk et al., J. Immunol. 152:5742-5748, 1994; Southwood et al., J. Immunol. 160:3363-3373, 1998). This is not entirely surprising in that the DR3 peptide-binding motif appears to be distinct from the specificity of most other DR alleles.
  • To efficiently identify peptides that bind DR3, target proteins were analyzed for conserved sequences carrying one of the two DR3 specific binding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). Peptides containing a DR3 motif were then synthesized and tested for their DR3 binding capacity. Three peptides were found to bind DR3 with an affinity of 1 μM or less (Table XXXI), and thereby qualify as HLA class II high affinity binders. On of these peptides was also identified above as a cross-reactive DR binding peptide.
  • DR3 binding epitopes identified in this manner that are found to induce immunological responses as in Example 6 below may then be included in vaccine compositions with DR supermotif-bearing peptide epitopes.
    • Example 6 Immunogenicity of PF-Derived HTL Epitopes
  • The immunogenicity of the HLA class II binding epitopes identified in Example 5 was evaluated in a study testing PBMC from either healthy volunteers previously immunized with an irradiated sporozoite vaccine, and thereby immune to malaria, or PBMC from naturally exposed individuals from the Irian Java (Indonesia) region where malaria is highly endemic. Vigorous responses were seen in volunteers vaccinated with whole irradiate sporozoites. All peptides were recognized in at least one immune individual, but not in either of the two individuals for which pre-immunization sample were available. All individuals recognized at least two, and up to nine different epitopes.
  • In the case of Irian Java population, PBMC from over 100 different individuals were screened for reactivity. Proliferation and secretion of various lymphokines has been measured. The results demonstrate that also in this semi-immune chronically exposed population, all peptides are recognized, with the percentage of individuals yielding positive responses ranging from 7% to 29% for IFN-γ, 36% to 51% for TNF-α and 12% to 2% for proliferative responses (Table XXII.
  • In conclusion, the immunogenicity of class II epitopes derived from conserved regions of the PF genome has been demonstrated.
    • Example 7 Calculation of Phenotypic Frequencies of HLA-Supertypes in Various Ethnic Backgrounds to Determine Breadth of Population Coverage
  • This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.
  • In order to analyze population coverage, gene frequencies of HLA alleles were determined. Gene frequencies for each HLA allele were calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol. 45:79-93, 1996). To obtain overall phenotypic frequencies, cumulative gene frequencies were calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=1−(1−Cgf)2].
  • Where frequency data was not available at the level of DNA typing, correspondence to the serologically defined antigen frequencies was assumed. To obtain total potential supertype population coverage no linkage disequilibrium was assumed, and only alleles confirmed to belong to each of the supertypes were included (minimal estimates). Estimates of total potential coverage achieved by inter-loci combinations were made by adding to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered (e.g., total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3, A11, A31, A*3301, and A*6801. Although the A3-like supertype may also include A34, A66, and A*7401, these alleles were not included in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602).
  • Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups (see Table XXI). Coverage may be extended by including peptides bearing the A1 and A24 motifs. On average, A1 is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%. An analagous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.
    • Summary of Candidate HLA Class I and Class II Epitopes
  • In summary, on the basis of the data presented in the above examples, candidate peptide epitopes derived from conserved regions of PF have been identified (Table XXXIII) These include eight HLA-A2 supermotif-bearing epitopes, eight HLA-A3 supermotif-bearing epitopes, and two HLA-B7 supermotif-bearing epitope, each capable of binding to multiple A2-, A3-, or B7-supertype molecules, and immunogenic in HLA transgenic mice or antigenic for human PBL. In addition four A1 motif-bearing and four A24 motif-bearing epitopes are also include candidate CTL epitopes for inclusion in a vaccine composition.
  • With these 26 CTL epitopes (as disclosed herein and from the art), average population coverage, (i.e., recognition of at least one PF epitope), is predicted to be, on average, greater than 95% (range of 90.6%-99.1%), in five major ethnic populations. The potential redundancy of coverage afforded by these epitopes can be estimated using the game theory Monte Carlo simulation analysis, which is known in the art (see e.g., Osborne, M. J. and Rubinstein, A. “A course in game theory” MIT Press, 1994). As shown in FIG. 1, it is estimated that 90% of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize 8 or more of the candidate epitopes described herein.
  • A list of PF-derived HTL epitopes that would be preferred for use in the design of minigene constructs or other vaccine formulations is summarized in Table XXXIV. As shown, 13 different peptide-binding regions have been identified which bind multiple HLA-DR molecules or bind HLA-DR3.
  • It is estimated that each of 10 common DR molecules recognizing the DR supermotif, and DR3, are covered by a minimum of 2 epitopes. Correspondingly, the total estimated population coverage represented by this panel of epitopes is, on average, in excess of 94% in each of the 5 major ethnic populations (Table XXXV).
    • Example 8 Recognition of Generation of Endogenous Processed Antigens after Priming
  • This example determines that CTL induced by native or analogued peptide epitopes identified and selected as described in Examples 1-6 recognize endogenously synthesized, i.e., native antigens.
  • Effector cells isolated from transgenic mice that are immunized with peptide epitopes as in Example 3, for example HLA-A2 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on 51Cr labeled Jurkat-A2.1/Kb target cells in the absence or presence of peptide, and also tested on 51Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with PF expression vectors.
  • The result will demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized PF antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that is being evaluated. In addition to HLA-A*0201/Kb transgenic mice, several other transgenic mouse models including mice with human A11, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.
    • Example 9 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice
  • This example illustrates the induction of CTLs and HTLs in transgenic mice by use of a PF CTL/HTL peptide conjugate whereby the vaccine composition comprises peptides administered to a PF-infected patient or an individual at risk for malaria. The peptide composition can comprise multiple CTL and/or HTL epitopes. This analysis demonstrates enhanced immunogenicity that can be achieved by inclusion of one or more HTL epitopes in a vaccine composition. Such a peptide composition can comprise a lipidated HTL epitope conjugated to a preferred CTL epitope containing, for example, at least one CTL epitope selected from Tables VII-XVIII, or an analog of that epitope. The HTL epitope is, for example, selected from Table XIX or XX.
  • Lipopeptide preparation: Lipopeptides are prepared by coupling the appropriate fatty acid to the amino terminus of the resin bound peptide. A typical procedure is as follows: A dichloromethane solution of a four-fold excess of a pre-formed symmetrical anhydride of the appropriate fatty acid is added to the resin and the mixture is allowed to react for two hours. The resin is washed with dichloromethane and dried. The resin is then treated with trifluoroacetic acid in the presence of appropriate scavengers [e.g. 5% (v/v) water] for 60 minutes at 20° C. After evaporation of excess trifluoroacetic acid, the crude peptide is washed with diethyl ether, dissolved in methanol and precipitated by the addition of water. The peptide is collected by filtration and dried.
  • Immunization procedures: Immunization of transgenic mice is performed as described (Alexander et al., J. Immunol. 159:4753-4761, 1997). For example, A2/Kb mice, which are transgenic for the human HLA A2.1 allele and are useful for the assessment of the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearing epitopes, are primed subcutaneously (base of the tail) with 0.1 ml of peptide conjugate formulated in saline, or DMSO/saline. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide.
  • Cell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/Kb chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991)
  • In vitro CTL activation: One week after priming, spleen cells (30×106 cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10×106 cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity.
  • Assay for cytotoxic activity: Target cells (1.0 to 1.5×106) are incubated at 37° C. in the presence of 200 μl of 51Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 104 51Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96-well plates. After a 6 hour incubation period at 37° C., a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release=100×(experimental release−spontaneous release)/(maximum release−spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % 51Cr release data is expressed as lytic units/106 cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a 6 hour 51Cr release assay. To obtain specific lytic units/106, the lytic units/106 obtained in the absence of peptide is subtracted from the lytic units/106 obtained in the presence of peptide. For example, if 30% 51Cr release is obtained at the effector (E): target (T) ratio of 50:1 (i.e., 5×105 effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5×104 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [(1/50,000)−(1/500,000)]×106=18 LU.
  • The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using the CTL epitope as outlined in Example 3. Analyses similar to this may be performed to evaluate the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures it is found that a CTL response is induced, and concomitantly that an HTL response is induced, upon administration of such compositions.
    • Example 10 Selection of CTL and HTL Epitopes for Inclusion in a PF-Specific Vaccine
  • This example illustrates the procedure for the selection of peptide epitopes for vaccine compositions of the invention. The peptides in the composition may be in the form of a nucleic acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or may be single and/or polyepitopic peptides.
  • The following principles are utilized when selecting an array of epitopes for inclusion in a vaccine composition. Each of the following principles are balanced in order to make the selection.
  • 1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with PF clearance. For HLA Class I this includes 3-4 epitopes that come from at least one antigen of PF. In other words, it has been observed that patients who spontaneously clear PF generate an immune response to at least 3 epitopes on at least one PF antigen. For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one PF antigen.
  • 2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, or for Class II an IC50 of 1000 nM or less.
  • 3.) Sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. For example, epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art and discussed herein, can be employed to assess breadth, or redundancy, of population coverage.
  • 4.) When selecting epitopes for PF antigens it may be preferable to select native epitopes. Therefore, of particular relevance for infectious disease vaccines, are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A peptide comprising “transcendent nested epitopes” is a peptide that has both HLA class I and HLA class II epitopes in it.
  • When providing nested epitopes, a sequence that has the greatest number of epitopes per provided sequence is provided. A limitation on this principle is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a longer peptide sequence, such as a sequence comprising nested epitopes, the sequence is screened in order to insure that it does not have pathological or other deleterious biological properties.
  • 5.) When creating a minigene, as disclosed in greater detail in Example 11, an objective is to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same as those employed when selecting a peptide comprising nested epitopes. Additionally, however, upon determination of the nucleic acid sequence to be provided as a minigene, the peptide encoded thereby is analyzed to determine whether any “junctional epitopes” have been created. A junctional epitope is an actual binding epitope, as predicted, e.g., by motif analysis. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that epitope, which is not present in a native PF protein sequence. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
  • Peptide epitopes for inclusion in vaccine compositions are, for example, selected from those listed in Tables XXXIII and XXXIV. A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude of an immune response that clears an acute PF infection.
    • Example 11 Construction of Minigene Multi-Epitope DNA Plasmids
  • This example describes the design and construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of CTL and/or HTL epitopes or epitope analogs as described herein. Expression plasmids have been constructed and evaluated as described, for example, in U.S. Ser. No. 09/311,784 filed May 13, 1999, now U.S. Pat. No. 6,534,482, and in Ishioka et al., J. Immunol. 162:3915-3925, 1999.
  • A minigene expression plasmid may include multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. Preferred epitopes are identified, for example, in Tables XXXIII and XXXIV. HLA class I supermotif or motif-bearing peptide epitopes derived from multiple PF antigens, e.g., EXP-1, SSP2, CSP and LSA-1, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from multiple PF antigens to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.
  • This example illustrates the methods to be used for construction of such a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.
  • The minigene DNA plasmid contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
  • Overlapping oligonucleotides, for example eight oligonucleotides, averaging approximately 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.
  • For the first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended: Oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM (NH4)2SO4, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product for 25 additional cycles. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.
    • Example 12 The Plasmid Construct and the Degree to which it Induces Immunogenicity
  • The degree to which the plasmid construct prepared using the methodology outlined in Example 11 is able to induce immunogenicity is evaluated through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analysed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in U.S. Ser. No. 09/311,784 filed May 13, 1999, now U.S. Pat. No. 6,534,482, and Alexander et al., Immunity 1:751-761, 1994. To assess the capacity of the pMin minigene construct to induce CTLs in vivo, HLA-A11/Kb transgenic mice, for example, are immunized intramuscularly with 100 μg of naked cDNA. As a means of comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.
  • Splenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a 51Cr release assay. The results indicate the magnitude of the CTL response directed against the A3-restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine. It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A3 supermotif peptide epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA-A2 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A2 and HLA-B7 motif or supermotif epitopes.
  • To assess the capacity of a class II epitope encoding minigene to induce HTLs in vivo, I-Ab restricted mice, for example, are immunized intramuscularly with 100 μg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant.
  • CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a 3H-thymidine incorporation proliferation assay, (see, e.g., Alexander et al., Immunity 1:751-761, 1994). the results indicate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.
  • DNA minigenes, constructed as described in Example 11, may also be evaluated as a vaccine in combination with a boosting agent using a prime boost protocol. The boosting agent may consist of recombinant protein (e.g., Barnett et al., Aids Res. and Human Reotroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med. 5:526-34, 1999).
  • For example, the efficacy of the DNA minigene may be evaluated in transgenic mice. In this example, A2.1/Kb transgenic mice are immunized IM with 100 μg of the DNA minigene encoding the immunogenic peptides. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 107 pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an IFN-y ELISA. It is found that the minigene utilized in a prime-boost mode elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis is also performed using other HLA-A11 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes.
    • Example 13 Peptide Composition for Prophylactic Uses
  • Vaccine compositions of the present invention are used to prevent PF infection in persons who are at risk for such infection. For example, a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) containing multiple CTL and HTL epitopes such as those selected in Examples 9 and/or 10, which are also selected to target greater than 80% of the population, is administered to individuals at risk for PF infection. The composition is provided as a single lipidated polypeptide that encompasses multiple epitopes. The vaccine is administered in an aqueous carrier comprised of Freunds Incomplete Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against PF infection.
  • Alternatively, the polyepitopic peptide composition can be administered as a nucleic acid in accordance with methodologies known in the art and disclosed herein.
    • Example 14 Polyepitopic Vaccine Compositions Derived from Native PF Sequences
  • A native PF polyprotein sequence is screened, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes and is preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct, even overlapping, epitopes is selected and used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The “relatively short” peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with frame shifted overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.
  • The vaccine composition will preferably include, for example, three CTL epitopes and at least one HTL epitope from PF. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.
  • The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (absent analogs) directs the immune response to multiple peptide sequences that are actually present in native PF antigens thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions.
  • Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.
    • Example 15 Polyepitopic Vaccine Compositions Directed to Multiple Diseases
  • The PF peptide epitopes of the present invention are used in conjunction with peptide epitopes from target antigens related to one or more other diseases, to create a vaccine composition that is useful for the prevention or treatment of PF as well as the one or more other disease(s). Examples of the other diseases include, but are not limited to, HIV, HCV, and HBV.
  • For example, a polyepitopic peptide composition comprising multiple CTL and HTL epitopes that target greater than 98% of the population may be created for administration to individuals at risk for both PF and HIV infection. The composition can be provided as a single polypeptide that incorporates the multiple epitopes from the various disease-associated sources, or can be administered as a composition comprising one or more discrete epitopes.
    • Example 16 Use of Peptides to Evaluate an Immune Response
  • Peptides of the invention may be used to analyze an immune response for the presence of specific CTL or HTL populations directed to PF. Such an analysis may be performed in a manner as that described by Ogg et al., Science 279:2103-2106, 1998. In the following example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.
  • In this example highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) are used for a cross-sectional analysis of, for example, PF HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of infection or following immunization using an PF peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and β2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, β2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Mo.), adenosine 5′triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer-phycoerythrin.
  • For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive uninfected donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the extent of immune response to the PF epitope, and thus the stage of infection with PF, the status of exposure to PF, or exposure to a vaccine that elicits a protective or therapeutic response.
    • Example 17 Use of Peptide Epitopes to Evaluate Recall Responses
  • The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from infection, who are chronically infected with PF, or who have been vaccinated with a PF vaccine.
  • For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any PF vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that are preferably highly conserved and, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.
  • PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.
  • In the microculture format, 4×105 PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 ml of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rIL-2 and 105 irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific 51Cr release, based on comparison with uninfected control subjects as previously described (Rehermann, et al., Nature Med. 2:1104,1108, 1996; Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J. Clin. Invest. 98:1432-1440, 1996).
  • Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, Mass.) or established from the pool of patients as described (Guilhot, et al. J. Virol. 66:2670-2678, 1992).
  • Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 μM, and labeled with 100 μCi of 51Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after which they are washed four times with HBSS.
  • Cytolytic activity is determined in a standard 4-h, split well 51Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target (E/T) ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100×[(experimental release−spontaneous release)/maximum release−spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis, Mo.). Spontaneous release is <25% of maximum release for all experiments.
  • The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to PF or a PF vaccine.
  • The class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5×105 cells/well and are stimulated with 10 μg/ml synthetic peptide, whole antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 10 U/ml IL-2. Two days later, 1 3H-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for 3H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of 3H-thymidine incorporation in the presence of antigen divided by the 3H-thymidine incorporation in the absence of antigen.
    • Example 18 Induction of CTL Responses Using a Prime Boost Protocol
  • A prime boost protocol similar in its underlying principle to that used to evaluated the efficacy of a DNA vaccine in transgenic mice, which was described in Example 12, may also be used for the administration of the vaccine to humans. Such a vaccine regimen is includes an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptides mixture administered in an adjuvant.
  • For example, the initial immunization may be performed using an expression vector, such as that constructed in Example 11, in the form of naked DNA administered IM (or SC or ID) in the amounts of 0.5-5, typically 100 g, at multiple sites. The DNA (0.1 to 1000 mg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5×109 pfu. Alternative recombinant virus, such as MVA, canarypox, adenovirus, and adeno-associated viruses can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples will be obtained before immunization as well as at intervals following administration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
  • Analysis of the results will indicate that a magnitude of sufficient response to achieve protective immunity against Pf is generated.
    • Example 19 Induction of Specific CTL Response in Humans
  • A human clinical trial to evaluate an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study and carried out as a randomized, double-blind, placebo-controlled trial in patients are not infected with Pf. Such a trial is designed, for example, as follows:
  • A total of about 27 subjects are enrolled and divided into 3 groups:
  • Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;
  • Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition;
  • Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.
  • After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.
  • The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.
  • Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.
  • Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
  • The vaccine is found to be both safe and efficacious.
  • A prophylactic field trial can also be conducted to evaluate a vaccine composition of the invention. In such a trial, issues of patient compliance are also considered in the determination of vaccine efficacy.
    • Example 20 Administration of Vaccine Compositions Using Dendritic Cells
  • Vaccines comprising peptide epitopes of the invention may be administered using dendritic cells. In this example, the immunogenic peptide epitopes are used to elicit a CTL and/or HTL response ex vivo.
  • Ex vivo CTL or HTL responses to a particular antigen (infectious or tumor-associated antigen) are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptides. After an appropriate incubation time (typically about 14 weeks), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., PF-infected cells.
    • Example 21 Alternative Method of Identifying Motif-Bearing Peptides
  • Another way of identifying motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing, have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can then be infected with a pathogenic organism, e.g., PF, HIV, etc. or transfected with nucleic acids that express the antigen of interest. Thereafter, peptides produced by endogenous antigen processing of peptides produced consequent to infection (or as a result of transfection) will bind to HLA molecules within the cell and be transported and displayed on the cell surface.
  • The peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al., J. Immunol. 152:3913, 1994). Because, as disclosed herein, the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.
  • Alternatively, cell lines that do not express any endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells may then be used as described, i.e., they may be infected with a pathogenic organism or transfected with nucleic acid encoding an antigen of interest to isolate peptides corresponding to the pathogen or antigen of interest that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.
  • As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than infection or transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.
  • The above examples are provided to illustrate the invention but not to limit its scope. For example, the human terminology for the Major Histocompatibility Complex, namely HLA, is used throughout this document. It is to be appreciated that these principles can be extended to other species as well. Thus, other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, patents, and patent application cited herein are hereby incorporated by reference for all purposes.
  • TABLE I
    SUPERMOTIFS POSITION POSITION POSITION
    2  3  C Terminus 
    (Primary Anchor) (Primary Anchor) (Primary Anchor)
    A1 TI LVMS FWY
    A2 LIVM ATQ IV MATL
    A3 VSMA TLI RK
    A24 YF WIVLMT FI YWLM
    B7 P VILF MWYA
    B27 RHK FYL WMIVA
    B44 ED FWYLIMVA
    B58 ATS FWY LIVMA
    B62 QL IVMP FWYMIVLA
    MOTIFS
    A1 TSM Y
    A1 DE AS Y
    A2.1 LM VQIAT V LIMAT
    A3 LMVISATF CGD KYR HFA
    A11 VTMLISAGN CDF K RYH
    A24 YFW M FLIW
    A*3101 MVT ALIS R K
    A*3301 MVALF IST RK
    A*6801 AVT MSLI RK
    B*0702 P LMFWYAIV
    B*3501 P LMFWY IVA
    B51 P LIVF WYAM
    B*5301 P IMFWY ALV
    B*5401 P ATIV LMFWY
    Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearingif it has primary anchors at each primary anchor position for a motif or superrnotifas specified in the above table.
  • TABLE Ia
    SUPERMOTIFS POSITION POSITION POSITION
    2  3  C Terminus 
    (Primary Anchor) (Primary Anchor) (Primary Anchor)
    A1 TI LVMS FWY
    A2 VQAT V LIMAT
    A3 VSMA TLI RK
    A24 YF WIVLMT FI YWLM
    B7 P VILF MWYA
    B27 RHK FYL WMIVA
    B58 ATS FWY LIVMA
    B62 QL IVMP FWY MIVLA
    MOTIFS
    A1 TSM Y
    A1 DE AS Y
    A2.1 VQAT* V LIMAT
    A3.2 LMVISATF CGD KYR HFA
    A11 VTMLISAGN CDF K RHY
    A24 YFW FLIW
    *If 2 is V, or Q, the C-term is not L
    Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearingif it has primary anchors at each primary anchor position for a motif or supermotifas specified in the above table.
  • TABLE 11
    Position
    SUPERMOTIFS 1 2 3 4 5
    A1 1° Anchor
    TILVMS
    A2 1° Anchor
    LIVMATQ
    A3 preferred 1° Anchor  YFW (4/5)
    VSMATLI RK
    deleterious DE (3/5);  DE (4/5)
    P (5/5)
    A24 1° Anchor
    YFWIVLMT
    B7 preferred FWY (5/5) 1° Anchor FWY (4/5)
    LIVM (3/5) P
    deleterious DE (3/5); 
    P(5/5);
    G(4/5); 
    A(3/5);
    QN (3/5)
    B27 1° Anchor
    RHK
    B44 1° Anchor
    ED
    B58 1° Anchor
    ATS
    B62 1° Anchor
    QLIVMP
    Position
    SUPERMOTIFS 6 7 8 C-terminus
    A1 1° Anchor
    FWY
    A2 1° Anchor
    LIVMAT
    A3 YFW (3/5)  YFW (4/5) P (4/5) 1° Anchor
    RK
    A24 1° Anchor
    FIYWLM
    B7 FWY (3/5)  1° Anchor
    VILFMWYA
    /5) QN (4/5) DE (4/5)
    B27 1° Anchor
    FYLWMIVA
    B44 1° Anchor
    FWYLIMVA
    B58 1° Anchor
    FWYLIVMA
    B62 1° Anchor
    FWYMIVLA
    Position
    MOTIFS 1 2 3 4 5
    A1 preferred GFYW 1° Anchor DEA YFW
    9-mer STM
    deleterious DE RHKLIVM A G
    P
    A1 preferred GRHK ASTCLIV 1° Anchor GSTC
    9-mer M DEAS
    deleterious A RHKDEPY DE PQN
    FW
    Position
    MOTIFS 6 7 8 C-terminus
    A1 P DEQN YFW 1° Anchor
    9-mer Y
    A
    A1 ASTC LIVM DE 1° Anchor
    9-mer Y
    RHK PG GP
    Position
    1 2 3 4
    A1 peferred YFW 1° Anchor DEAQN A
    10- STM
    mer deleterious GP RHKGLIV DE
    M
    A1 preferred YFW STCLIVM 1° Anchor A
    l0- DEAS
    mer deleterious RHK RHKDEPY
    FW
    A2.1 preferred YFW 1° Anchor YFW STC
    9-mer LMIVQAT
    deleterious DEP DERKH
    A2.1 preferred AYFW 1° Anchor LVIM G
    l0- LMIVQAT
    mer deleterious DEP DE RKHA
    A3 preferred RHK 1° Anchor YFW PRHKYFW
    LMVISAT
    FCGD
    deleterious DEP DE
    A11 preferred A 1° Anchor YFW YFW
    VTLMISA
    GNCDF
    deleterious DEP
    A24 preferred YFWRHK 1° Anchor STC
    9-mer YFWM
    deleterious DEG DE G
    A24 preferred 1° Anchor P
    10- YFWM
    mer deleterious GDE QN
    A3101 preferred RHK 1° Anchor YFW P
    MVTALIS
    deleterious DEP DE
    A3301 preferred 1° Anchor YFW
    MVALFIS
    T
    deleterious GP DE
    A6801 preferred YFWSTC 1° Anchor
    AVTMSLI
    deleterious GP DEG
    B0702 preferred RHKFWY 1° Anchor RHK
    P
    deleterious DEQNP DEP DE
    B3501 preferred FWYLIVM 1° Anchor FWY
    P
    deleterious AGP
    B51 preferred LIVMFWY 1° Anchor FWY STC
    P
    deleterious  AGPDERHKSTC
    B5301 preferred LIVMFWY 1° Anchor FWY STC
    P
    deleterious  AGPQN
    B5401 preferred FWY 1° Anchor FWYLIVM
    P
    deleterious  GPQNDE GDESTC
    Position
    9
    or C-
    6 7 8 terminus
    A1 peferred PASTC GDE P
    10-
    mer deleterious QNA RHKYFW RHK A
    A1 preferred PG G YFW
    l0-
    mer deleterious G PRHK QN
    A2.1 preferred A P 1° Anchor
    9-mer VLIMAT
    deleterious RKH DERKH
    A2.1 preferred G FYWL
    l0- VIM
    mer deleterious RKH DERK RKH
    A3 preferred A YFW P 1° Anchor
    KYRHFA
    deleterious
    A11 preferred YFW YFW P 1° Anchor
    KRYH
    deleterious A G
    A24 preferred YFW YFW 1° Anchor
    9-mer FLIW
    deleterious DERHK G AQN
    A24 preferred P
    10-
    mer deleterious DE A QN DEA
    A3101 preferred YFW YFW AP 1° Anchor
    RK
    deleterious DE DE DE
    A3301 preferred AYFW 1° Anchor
    RK
    deleterious
    A6801 preferred YFW P 1° Anchor
    RK
    deleterious A
    B0702 preferred RHK RHK PA 1° Anchor
    LMFWYAIV
    deleterious GDE QN DE
    B3501 preferred FWY 1° Anchor
    LMFWYIVA
    deleterious G
    B51 preferred G FWY 1° Anchor
    LIVFWYAM
    deleterious  G DEQN GDE
    B5301 preferred LIVMFWY FWY 1° Anchor
    IMFWYALV
    deleterious  G RHKQN DE
    B5401 preferred ALIVM FWYAP 1° Anchor
    ATIVLMFW
    Y
    deleterious  DE QNDGE DE
    Italicized residues indicate less preferred or ″tolerated″ residues. The information in Table II is specific for 9-meRs unless otherwise specified.
  • TABLE III
    POSITION
    SEQ ID 1° 1°
    NO: MOTIFS anchor 1 2 3 4 5 anchor 6 7 8 9
    DR4 preferred FMYLIYW M T I VSTCPALIM MH MEI
    deleterious W R WDE
    DR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM
    deleterious C CH FD CWD GDE D
    3841 DR7 preferred MFLIVWY M W A IVMSACTPL M IV
    3842 deleterious C G GRD N G
    DR Supermotif MFLIVWY VMSTACPLI
    DR3 MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1° anchor 6
    motif a LIVMFY D
    preferred
    motif b LIVMFAY DNQEST KRH
    preferred
    Italicized residues indicate less preferred or ″tolerated″ residues.
  • TABLE IV
    HLA Class I Standard Peptide Binding Affinity.
    SEQ STANDARD
    STANDARD ID BINDING
    ALLELE PEPTIDE SEQUENCE NO: AFFINITY (nM)
    A*0101  944.02 YLEPAIAKY 3575 25
    A*0201  941.01 FLPSDYFPSV 3576  5.0
    A*0202  941.01 FLPSDYFPSV 3577  4.3
    A*0203  941.01 FLPSDYFPSV 3578 10
    A*0205  941.01 FLPSDYFPSV 3579  4.3
    A*0206  941.01 FLPSDYFPSV 3580  3.7
    A*0207  941.01 FLPSDYFPSV 3581 23
    A*6802 1072.34 YVIKVSARV 3582  8.0
    A*0301  941.12 KVFPYALINK 3583 11
    A*1101  940.06 AVDLYHFLK 3584  6.0
    A*3101  941.12 KVFPYALINK 3585 18
    A*3301 1083.02 STLPETYVVRR 3586 29
    A*6801  941.12 KVFPYALINK 3587  8.0
    A*2402  979.02 AYIDNYNKF 3588 12
    B*0702 1075.23 APRTLVYLL 3589  5.5
    B*3501 1021.05 FPFKYAAAF 3590  7.2
    B51 1021.05 FPFKYAAAF 3591  5.5
    B*5301 1021.05 FPFKYAAAF 3592  9.3
    B*5401 1021.05 FPFKYAAAF 3593 10
  • TABLE V
    HLA Class II Standard Peptide Binding Affinity.
    Binding
    Standard Affinity
    Allele Nomenclature Peptide SEQ ID Sequence (nM)
    DRB1*0101 DR1  515.01 3594 PKYVKQNTLKLAT    5.0
    DRB1*0301 DR3  829.02 3595 YKTIAFDEEARR  300
    DRB1*0401 DR4w4  515.01 3596 PKYVKQNTLKLAT   45
    DRB1*0404 DR4w14  717.01 3597 YARFQSQTTLKQKT   50
    DRB1*0405 DR4w15  717.01 3598 YARFQSQTTLKQKT   38
    DRB1*0701 DR7  553.01 3599 QYIKANSKFIGITE   25
    DRB1*0802 DR8w2  553.01 3600 QYIKANSKFIGITE   49
    DRB1*0803 DR8w3  553.01 3601 QYIKANSKFIGITE 1600
    DRB1*0901 DR9  553.01 3602 QYIKANSKFIGITE   75
    DRB1*1101 DR5w11  553.01 3603 QYIKANSKFIGITE   20
    DRB1*1201 DR5w12 1200.05 3604 EALIHQLKINPYVLS  298
    DRB1*1302 DR6w19  650.22 3605 QYIKANAKFIGITE    3.5
    DRB1*1501 DR2w2β1  507.02 3606 GRTQDENPVVHFFK    9.1
    NIVTPRTPPP
    DRB3*0101 DR52a  511 3607 NGQIGNDPNRDIL  470
    DRB4*0101 DRw53  717.01 3608 YARFQSQTTLKQKT   58
    DRB5*0101 DR2w2β2  553.01 3609 QYIKANSKFIGITE   20

    The “Nomenclature” column lists the allelic designations used in Tables XIX and XX.
  • Table VI
    HLA- Allelle-specific HLA-supertype members
    supertype Verifieda Predictedb
    A1 A*0101, A*2501, A*2601, A*2602, A*3201 A*0102, A*2604, A*3601, A*4301, A*8001
    A2 A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*0208, A*0210, A*0211, A*0212, A*0213
    A*0209, A*0214, A*6802, A*6901
    A3 A*0301, A*1101, A*3101, A*3301, A*6801 A*0302, A*1102, A*2603, A*3302, A*3303, A*3401,
    A*3402, A*6601, A*6602, A*7401
    A24 A*2301, A*2402, A*3001 A*2403, A*2404, A*3002, A*3003
    B7 B*0702, B*0703, B*0704, B*0705, B*1508, B*3501, B*3502, B*3503, B*1511, B*4201, B*5901
    B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101, B*5102,
    B*5103, B*5104, B*5105, B*5301, B*5401, B*5501, B*5502, B*5601,
    B*5602, B*6701, B*7801
    B27 B*1401, B*1402, B*1509, B*2702, B*2703, B*2704, B*2705, B*2701, B*2707, B*2708, B*3802, B*3903, B*3904,
    B*2706, B*3801, B*3901, B*3902, B*7301 B*3905, B*4801, B*4802, B*1510, B*1518, B*1503
    B44 B*1801, B*1802, B*3701, B*4402, B*4403, B*4404, B*4001, B*4101, B*4501, B*4701, B*4901, B*5001
    B*4002, B*4006
    B58 B*5701, B*5702, B*5801, B*5802, B*1516, B*1517
    B62 B*1501, B*1502, B*1513, B*5201 B*1301, B*1302, B*1504, B*1505, B*1506, B*1507,
    B*1515, B*1520, B*1521, B*1512, B*1514, B*1510
    aVerified alleles include alleles whose specificity has been determined by pool sequencing analysis, peptide binding assays, or by analysis of the sequences of CTL epitopes.
    bPredicted alleles are alleles whose specificity is predicted on the basis of B and F pocket structure to overlap with the supertype specificity.
  • TABLE VII
    Malaria A01 Super Motif Peptides With Binding Data
    No. of Sequence Conservancy
    Protein Sequence Position Amino Acids Frequency (%) A*010I Seq. Id.
    CSP AILSVSSF 6 8 19 100 1
    CSP AILSVSSFLF 6 10 19 100 2
    CSP ALFQEYQCY 18 9 19 100 3
    CSP EMNYYGKQENW 52 11 19 100 4
    CSP FLFVEALF 13 8 19 100 5
    CSP FLFVEALFQEY 13 11 19 100 6
    CSP FVEALFQEY 15 9 19 100 3.4000 7
    CSP GLIMVLSF 421 8 19 100 8
    CSP GLIMVLSFLF 421 10 19 100 9
    CSP ILSVSSFLF 7 9 19 100 10
    CSP IMVLSFLF 423 8 19 100 11
    CSP KIQNSLSTEW 357 10 19 79 12
    CSP KLAILSVSSF 4 10 19 100 13
    CSP KMEKCSSVF 405 9 19 100 14
    CSP LIMVLSFLF 422 9 19 100 15
    CSP LSVSSFLF 8 8 19 100 16
    CSP NLYNELEMNY 46 10 19 100 17
    CSP NLYNELEMNYY 46 11 19 100 18
    CSP NTRVLNELNY 31 10 19 100 0.0096 19
    CSP PSDKHIEQY 346 9 19 79 20
    CSP RVLELNY 33 8 19 100 21
    CSP SIGLIMVLSF 419 10 19 100 22
    CSP SSFLFVEALF 11 10 19 100 23
    CSP SSIGLIMVLSF 418 11 19 100 24
    CSP VSSFLFVEALF 10 11 19 100 25
    CSP EVNKRKSKY 66 9 1 100 26
    EXP FLALFFIIF 8 9 1 100 27
    EXP ILSVFFLALF 3 10 1 100 28
    EXP ILSVFFLALFF 3 11 1 100 29
    EXP KILSVFFLALF 2 11 1 100 30
    EXP LLGGVGLVLY 92 10 1 100 31
    EXP LSVFFLALF 4 9 1 100 32
    EXP LSVFFLALFF 4 10 1 100 33
    EXP LVEVNKRKSKY 64 11 1 100 34
    EXP NTEKGRHPF 102 9 1 100 35
    EXP SVFFLALF 5 8 1 100 36
    EXP SVFFLALFF 5 9 1 100 37
    EXP VLLGGVGLVLY 91 11 1 100 38
    LSA DLDEFKPIVQY 1781 11 1 100 39
    LSA DVLQEDLY 1646 8 1 100 40
    LSA DVNDFQISKY 1751 10 1 100 41
    LSA ELPSENERGY 1662 10 1 100 42
    LSA ELPSENERGYY 1662 11 1 100 43
    LSA ELSEDITTKY 1897 9 1 100 44
    LSA ELSEDITKYF 1897 10 1 100 45
    LSA ETVNISDVNDF 1745 11 1 100 46
    LSA FIKSLFHIF 1877 9 1 100 47
    LSA FILVNLLIF 11 9 1 100 48
    LSA HILYISFY 3 8 1 100 49
    LSA HILYISFYF 3 9 1 100 50
    LSA HVLSHNSY 59 8 1 100 51
    LSA IINDDDDKKKY 127 11 1 100 52
    LSA ILVNLLIF 12 8 1 100 53
    LSA ILYISFYF 4 8 1 100 54
    LSA KIKKGKKY 1834 8 1 100 55
    LSA KSLYDEHIKKY 1854 11 1 100 56
    LSA KTKNNENNKF 68 10 1 100 57
    ISA KTKNNENNKFF 68 11 1 100 58
    LSA LSEDITKY 1898 8 1 100 59
    LSA LSEDITKYF 1898 9 1 100 60
    LSA NISDVNDF 1748 8 1 100 61
    LSA NLGVSENIF 103 9 1 100 62
    ISA NVKNVSQTNF 88 10 1 100 63
    ISA PIVQYDNF 1787 8 1 100 64
    LSA PSENERGY 1664 8 1 100 65
    LSA PSENERGYY 1664 9 1 100 0.0790 66
    LSA QVNKEKEKF 1869 9 1 100 67
    LSA SLYDEHIKKY 1855 10 1 100 68
    LSA TVNISDVNDF 1746 10 1 100 69
    SSP2 ALLACAGLAY 509 10 10 100 70
    SSP2 ASCGVWDEW 242 9 10 100 71
    SSP2 ATPYAGEPAPF 526 11 8 80 72
    SSP2 CSGSIRRHNW 55 10 10 100 73
    SSP2 DLDEPEQF 546 8 10 100 74
    SSP2 EVCNDEVDLY 41 10 8 80 75
    SSP2 EVEKTASCGVW 237 11 10 100 76
    SSP2 FLIFFDLF 14 8 10 100 77
    SSP2 FVVPGAATPY 520 10 8 80 78
    SSP2 GIGQGINVAF 189 10 10 100 79
    SSP2 GINVAFNRF 193 9 10 100 80
    SSP2 GSIRRHNW 57 8 10 100 81
    SSP2 IVFLIFFDLF 12 10 10 100 82
    SSP2 KTASCGVW 240 8 10 100 83
    SSP2 KTASCGVWDEW 240 11 10 100 84
    SSP2 LLACAGLAY 510 9 10 100 85
    SSP2 LLACAGLAYKF 510 11 10 100 86
    SSP2 LLSTNLPY 121 8 9 90 87
    SSP2 LVIVFLIF 10 8 10 100 88
    SSP2 LVIVFLIFF 10 9 10 100 89
    SSP2 NIVDEIKY 31 8 10 100 90
    SSP2 NLYADSAW 213 8 10 100 91
    SSP2 NVKNVIGPF 222 9 10 100 92
    SSP2 NVKYLVIVF 6 9 10 100 93
    SSP2 PSDGKCNLY 207 9 10 100 0.5400 94
    SSP2 RLPEENEW 554 8 10 100 95
    SSP2 SLLSTNLPY 120 9 9 90 96
    SSP2 VIVFLIFF 11 8 10 100 97
    SSP2 VIVFLIFFDLF 11 11 10 100 98
    SSP2 VVPGAATPY 521 9 8 80 99
    SSP2 YLVIVFLIF 9 9 10 100 100
    SSP2 YLVIVFLIFF 9 10 10 100 101
  • TABLE VIII 
    Malaria A02 Motif Peptides With Binding Information
    No. of Sequence Conservancy
    Protein Sequence Position Amino Acids Frequency (%) A*0201
    CSP HIEQYLKKI 350 9 15 79
    CSP KIQNSLST 361 8 15 79
    CSP YLKKIQNSL 358 9 15 79
    CSP YLKKIQNSLST 358 11 15 79
    CSP NANANNAV 335 8 16 84
    CSP NVDENANANNA 331 11 16 84
    CSP ELNYDNAGI 37 9 18 95
    CSP ELNYDNAGINL 37 11 18 95
    CSP GINLYNEL 44 8 18 95
    CSP GINLYNELEM 44 10 18 95
    CSP NAGINLYNEL 42 10 18 95
    CSP SLSTEWSPCSV 365 11 18 95
    CSP AILSVSSFL 6 9 19 100 0.0220
    CSP AILSVSSFLFV 6 11 19 100
    CSP DIEKKICKM 402 9 19 100
    CSP GIQVRIKPGSA 380 11 19 100
    CSP GLIMVLSFL 425 9 19 100 0.0630
    CSP GLIMVLSFLFL 425 11 19 100
    CSP ILSVSSFL 7 8 19 100
    CSP ILSVSSFLFV 7 10 19 100 0.0300
    CSP IMVLSFLFL 427 9 19 100 0.0007
    CSP IQVRIKPGSA 381 10 19 100
    CSP KICKMEKCSSV 406 11 19 100
    CSP KLAILSVSSFL 4 11 19 100
    CSP KLRICPICHKKL 104 10 19 100 0.0001
    CSP KMEKCSSV 409 8 19 100
    CSP KMEKCSSVFNV 409 11 19 100
    CSP KQENWYSL 58 8 19 100
    CSP LAILSVSSFL 5 10 19 100
    CSP LIMVLSFL 426 8 19 100
    CSP LIMVLSFLFL 426 10 19 100 0.0019
    CSP MMRKLAIL 1 8 19 100
    CSP MMRKLAILSV 1 10 19 100 0.0012
    CSP MVLSFLFL 428 8 19 100
    CSP NVDPNANPNA 300 10 19 100
    CSP NANPNVDPNA 196 10 19 100
    CSP NLYNELEM 46 8 19 100
    CSP NMPNDPNRNV 323 10 19 100 0.0007
    CSP NQGNGQGHNM 315 10 19 100
    CSP NTRVLNEL 31 8 19 100
    CSP NVDENANA 331 8 19 100
    CSP NVDPNANPNA 200 10 19 100
    CSP NVDPNANPNV 128 10 19 100
    CSP NVVNSSIGL 418 9 19 100
    CSP NVVNSSIGLI 418 10 19 100
    CSP NVVNSSIGLIM 418 11 19 100
    CSP QVRIKPGSA 382 9 19 100
    CSP RVLNELNYDNA 33 11 19 100
    CSP SIGLIMVL 423 8 19 100
    CSP SIGLIMVLSFL 423 11 19 100
    CSP SLKKNSRSL 64 9 19 100 0.0001
    CSP STEWSPCSV 367 9 19 100
    CSP STEWSPCSVT 367 10 19 100
    CSP SVFNVVNSSI 415 10 19 100 0.0005
    CSP SVSSFLFV 9 8 19 100
    CSP SVSSFLFVEA 9 10 19 100
    CSP SVSSFLFVEAL 9 11 19 100
    CSP SVTCQNGI 374 8 19 100
    CSP SVTCQNGIQV 374 10 19 100
    CSP VLNELNYDNA 34 10 19 100
    CSP VTCGNGIQV 375 9 19 100 0.0011
    CSP VTCGNGIQVRI 375 11 19 100
    CSP VVNSSIGL 419 8 19 100
    CSP VVNSSIGLI 419 9 19 100
    CSP VVNSSIGL1M 419 10 19 100
    CSP VVNSSIGLIMV 419 11 19 100
    CSP YQCYGSSSNT 23 10 19 100
    EXP ATSVLAGL 77 8 1 100
    Exp ATSVLAGLL 77 9 1 100
    EXP DMIKKEEEL 56 9 1 100
    EXP DNMUCEEELV 56 10 1 100
    EXP DVHDLISDM 49 9 1 100
    EXP DVHDLISDMI 49 10 1 100
    EXP EQPQGDDNINIL 147 10 1 100
    EXP EQPQGDDNNLV 147 11 1 100
    EXP EVNKRKSKYKL 66 11 1 100
    EXP FIIFNICESL 13 9 1 100
    EXP FIIFNKESLA 13 10 1 100
    EXP FLALFFII 8 8 1 100
    EXP GLLGNVST 83 8 1 100
    EXP GLLGNVSTV 83 9 1 100 0.0160
    EXP GLLGNVSTVL 83 10 1 100 0.0380
    EXP GLLGNVSTVLL 83 11 1 100
    EXP GVGLVLYNT 95 9 1 100
    EXP IIFNKESL 14 8 1 100
    EXP IIFNKESLA 14 9 1 100
    EXP ILSVFFLA 3 8 1 100
    EXP ILSVFFLAL 3 9 1 100 0.0058
    EXP KIGSSDPA 111 8 1 100
    EXP KIGSSDPADNA 111 11 1 100
    EXP KILSVFFL 2 8 1 100
    EXP KILSVFFLA 2 9 1 100 0.8500
    EXP KILSVFFLAL 2 10 1 100
    EXP KLATSVLA 75 8 1 100
    EXP KLATSVLAGL 75 10 1 100 0.0047
    EXP KLATSVLAGLL 75 11 1 100
    EXP KTNKGTGSGV 24 10 1 100
    EXP LABCTNKGT 21 9 1 100
    EXP LAGLLGNV 81 8 1 100
    EXP LAGLLGNVST 81 10 1 100
    EXP LAGLLGNVSTV 81 11 1 100
    EXP LATSVLAGL 76 9 1 100
    EXP LATSVLAGLL 76 10 1 100
    EXP LIDVHDLI 47 8 1 100
    EXP LIDVHDLISDM 47 11 1 100
    EXP LLGGVCLV 92 8 1 100
    EXP LLGGVCLVL 92 9 1 100 0.0038
    EXP LLGNVSTV 84 8 1 100
    EXP LLGNVSTVL 84 9 1 100 0.0350
    EXP LLGNVSTVLL 84 10 1 100 0.0059
    EXP MIKKEEEL 37 8 1 100
    EXP MIKKEEELV 57 9 1 100
    EXP MIKKEEELVEV 37 11 1 100
    EXP NADPQVTA 134 8 1 100
    EXP NADPQVTAQDV 134 11 1 100
    EXP NTEKGRHPFKI 102 11 1 100
    EXP NVSTVLLGGV 87 10 1 100
    EXP PADNANPDA 117 9 1 100
    EXP PLIDVHDL 46 8 1 100
    EXP PLIDVHDLI 46 9 1 100
    EXP PQGDDNNL 149 8 1 100
    EXP PQGDDNNLV 149 9 1 100
    EXP PQVTAQDV 137 8 1 100
    EXP PQVTAQDVT 137 9 1 100
    EXP QVTAQDVT 138 8 1 100
    EXP SLAEKTNKGT 20 10 1 100
    EXP STVLLGGV 89 8 1 100
    EXP STVLLGQVGL 89 10 1 100
    EXP STVLLGGVGLV 89 11 1 100
    EXP SVFFLALFFI 5 10 1 100 0.0017
    EXP SVPFLALFFH 5 11 1 100
    EXP SVLACLLGNV 79 10 1 100 0.0022
    EXP TVLLGGVGL 90 9 1 100
    EXP TVLLGGVCLV 90 10 1 100
    EXP TVLLGGVGLVL 90 11 1 100
    EXP VLAGLLGNV 80 9 1 100 0.0210
    EXP VLAGLLGNVST 80 11 1 100
    EXP VLLGGVCL 91 8 1 100
    EXP VLLGOVGLV 91 9 1 100 0.0290
    EXP VLLGGVCLVL 91 10 1 100 0.0290
    LSA DIQNHILET 1138 9 1 100
    LSA DIQNHTLETV 1738 10 1 100
    LSA DITKYFMKL 1901 9 1 100
    LSA DUDEFKPI 1781 8 1 100
    LSA DLDEFKPIV 1781 9 1 100 0.0001
    LSA DLEEKAAKET 148 10 1 100
    LSA DLEEKAAKETL 148 11 1 100
    LSA DLEQDRLA 1388 8 1 100
    LSA DLEQERLA 1609 8 1 100
    LSA DLEQERRA 1575 8 1 100
    LSA DLEMADT 1626 9 1 100
    LSA DUERTXASKET 1184 11 1 100
    LSA DLYGRLEI 1651 8 1 100
    LSA DLYGRLEIPA 1651 10 1 100
    LSA DLYGRLEIPAI 1651 11 1 100
    LSA DVLAEDLYGRL 1646 11 1 100
    LSA EILQIVDEL 1890 9 1 100
    LSA EISAEYDDSL 1763 10 1 100
    LSA EISAEYDDSLI 1763 11 1 100
    LSA EISIIEKT 1692 8 1 100
    LSA ELSEDITKYFM 1897 11 1 100
    LSA ELTMSNVKNV 83 10 1 100
    LSA EQDRLWEKL 1390 10 1 100
    LSA EQERLAKEKL 1611 10 1 100
    LSA EQERLANEKL 1526 10 1 100
    LSA EQERRAKEKL 1577 10 1 100
    LSA EQKEDKSA 1730 8 1 100
    LSA EQKEDKSADI 1730 10 1 100
    LSA EQQRDLEQERL 1605 11 1 100
    LSA EQQRDLEQRKA 1622 11 1 100
    LSA EQQSDLEQDRL 1384 11 1 100
    LSA EQQSDLEQERL 1588 11 1 100
    LSA EQQSDLERT 1180 9 1 100
    LSA EQQSDLERTKA 1180 11 1 100
    LSA EQQSDSEQERL 517 11 1 100
    LSA EQRKADTKKNL 1628 11 1 100
    LSA ETLQEQQSDL 1193 10 1 100
    LSA ETLWQQSDL 156 10 1 100
    LSA ETVNISDV 1745 8 1 100
    LSA FIKSLFHI 1877 8 1 100
    LSA FILVNLLI 11 8 1 100
    LSA FILVNLLIFIT 11 11 1 100
    LSA FQDEENIGI 1794 9 1 100
    LSA FQISKYEDE1 1755 10 1 100
    LSA GIOCSSEEL 1822 9 1 100
    LSA GIYKELEDL 1801 9 1 100
    LSA GIYKELEDLI 1801 10 1 100
    LSA GQDENRQEDL 140 10 1 100
    LSA GQQSDIEQISRL 1129 11 1 100
    LSA GVSENTFL 105 8 1 100
    LSA HIFDGDNEI 1883 9 1 100
    LSA HIFDGDNEIL 1883 10 1 100
    LSA HIKKYKNDKQV 1860 11 1 100
    LSA HILYISFYFI 3 10 1 100 0.0033
    LSA HILYISFYFIL 3 11 1 100
    LSA HLEEKKDGSI 1718 10 1 100
    LSA HTLETVNI 1742 8 1 100
    LSA HTLETVNISDV 1742 11 1 100
    LSA HVLSHNSYEKT 59 11 1 100
    LSA IIDONRESI 1695 10 1 100
    LSA IIEKTNRESIT 1695 11 1 100
    LSA IIKNSEKDEI 25 10 1 100
    LSA IIKNSEKDEII 25 11 1 100
    LSA ILQIVDEL 1891 8 1 100
    LSA ILVNLLIFHI 12 10 1 100 0.0076
    LSA ILYISFYFI 4 9 1 100 0.0023
    LSA ILYISFYFIL 4 10 1 100 0.0035
    LSA ILYISFYFILV 4 11 1 100
    LSA IQNHTLET 1739 8 1 100
    LSA IQNHTLETV 1739 9 1 100
    LSA IQNHTLETVN1 1739 11 1 100
    LSA IKYFMKL 1902 8 1 100
    LSA ITTNVEGRRDI 1704 11 1 100
    LSA IVDELSEDI 1894 9 1 100
    LSA IVDELSEDIT 1894 10 1 100
    LSA KADTICKNI 1631 8 1 100
    LSA KIIKNSEKDEI 24 11 1 100
    LSA KIKKGKKYEICT 1834 11 1 100
    LSA KLNKEGKL 116 8 1 100
    LSA KLNKEGKLI 116 9 1 100
    LSA KLQEQQRDL 1619 9 1 100
    LSA KLQGQQSDL 1585 9 1 100 0.0019
    LSA KLQGQQSDL 1126 9 1 100
    LSA KQVNKEKEKFI 1868 11 1 100
    LSA KTNRESIT 1698 8 1 100
    LSA KTINIRESITT 1698 9 1 100
    LSA KTNRESITNV 1698 11 1 100
    LSA LAEDLYGRL 1648 9 1 100
    LSA LAEDLYGRLEI 1648 11 1 100
    LSA LIDEEEDDEDL 1772 11 1 100
    LSA LIEICNENL 1809 8 1 100
    LSA LIEKNENLDDL 1809 11 1 100
    LSA LIFHJNGKI 17 9 1 100
    LSA LIFHINGKII 17 10 1 100 0.0002
    LSA LLIFHINGKI 16 10 1 100
    LSA LLIFHINGKII 16 11 1 100
    LSA LLRNLGVSENI 100 11 1 100
    LSA LQEQQRDL 1620 8 1 100
    LSA LQEQQSDL 1586 8 1 100
    LSA LQEQQSDLERT 1178 11 1 100
    LSA LQOQQSDL 1127 8 1 100
    LSA LQIVDELSEDI 1892 11 1 100
    LSA LTMSNVKNV 84 9 1 100 0.0010
    LSA LVNLLIFHI 13 9 1 100 0.0006
    LSA N1FLKENKL 109 9 1 100
    LSA NIGIYKEL 1799 8 1 100
    LSA NIGIYKELEDL 1799 11 1 100
    LSA NISDVNDFQI 1748 10 1 100
    LSA NLDDLDEGI 1815 9 1 100
    LSA NLERKKEHGDY 1637 11 1 100
    LSA NLGVSEN1 103 8 1 100
    LSA NLOVSENIFL 103 10 1 100
    LSA NLLIFHINGKI 15 11 1 100
    LSA NVEGRRDI 1707 8 1 100
    LSA NVKNVSQT 88 8 1 100
    LSA NVSQTNFKSL 91 10 1 100
    LSA NVSQTNFKSLL 91 11 1 100
    LSA QISKYEDEI 1756 9 1 100
    LSA QISKYEDEISA 1756 11 1 100
    LSA QIVDELSEDI 1893 10 1 100
    LSA QIVDELSEDIT 1893 11 1 100
    LSA QQRDLEQERL 1606 10 1 100
    LSA QQRDLEQERLA 1606 11 1 100
    LSA QQRDLEQERRA 1538 11 1 100
    LSA QQRDLEQRKA 1623 10 1 100
    LSA QQSDLEQDRL 1385 10 1 100
    LSA QQSDLEQDRLA 1385 11 1 100
    LSA QQSDLEQERL 1589 10 1 100
    LSA QQSDLEQDRLA 1589 11 1 100
    LSA QQSDLEQERRA 1572 11 1 100
    LSA QQSDLERT 1181 8 1 100
    LSA QQ6DLERTKA 1181 10 1 100
    LSA QQSDSEQERL 518 10 1 100
    LSA QQSDSEQERLA 518 11 1 100
    LSA QINFKSLL 94 8 1 100
    LSA QTNFKSLLRNL 94 11 1 100
    LSA QVNKEKEKFI 1869 10 1 100
    LSA RLEIPA1EL 1655 9 1 100
    LSA RQEDLEEKA 145 9 1 100
    LSA RQEDLEEKAA 145 10 1 100
    LSA RTKASKET 1187 8 1 100
    LSA RTKASKETL 1187 9 1 100
    LSA SADIQNHT 1736 8 1 100
    LSA SADIQNHTL 1736 9 1 100
    LSA SADIONITTLET 1736 11 1 100
    LSA SAEYDDSL 1765 8 1 100
    LSA SAEYDDSLI 1765 9 1 100
    LSA SIIEKTNRESI I694 11 1 100
    LSA SLLRNLGV 99 8 1 100
    LSA SQTNFKSL 93 8 1 100
    LSA SQTNFKSLL 93 9 1 100
    LSA TLETVRISOV 1743 10 1 100
    LSA TLQEQQSDL 1194 9 1 100
    LSA TLQGQQSDL 157 9 1 100
    LSA TMSNVKNV 85 8 1 100
    LSA TMSNVICNVSQT 85 11 1 100
    LSA TTNVEGRRDI 1705 10 1 100
    LSA VLAEDLYGRL 1647 10 1 100
    LSA VLSHNSYEKT 60 10 1 100
    LSA YIPHQSSL 1672 8 1 100
    LSA YISFYFIL 6 8 1 100
    LSA YISFYFILV 6 9 1 100 0.0016
    LSA YISFYFILVNL 6 11 1 100
    SSP2 AATPYAGEPA 525 10 8 80
    SSP2 ATPYAGEPA 526 9 8 80
    SSP2 EILHEGCTSEL 267 11 8 80
    SSP2 EVCNDEVDL 41 9 8 80
    SSP2 EVCNDEVDLVL 41 11 8 80
    SSP2 EVDLYLLM 46 8 8 80
    SSP2 FVVPGAATPYA 520 11 8 80
    SSP2 GAATPYAGEPA 524 11 8 80
    SSP2 ILHEGCTSEL 268 10 8 80
    SSP2 LLSTNLPYGRT 121 11 8 80
    SSP2 NLPYGRTNL 125 9 8 80
    SSP2 SIRRHNWVNHA 58 11 8 80
    SSP2 STNLPYGRT 123 9 8 80
    SSP2 STNLPYQRTNI 123 11 8 80
    SSP2 VVPGAATPYA 521 10 8 80
    SSP2 WVNHAVPL 64 8 8 80
    SSP2 WVNHAVPLA 64 9 8 80 0.0008
    SSP2 WVNHAVPLAM 64 10 8 80
    SSP2 YAGEPAPFDET 529 11 8 80
    SSP2 ALLQVRKHL 136 9 9 90 0.0010
    SSP2 DALLQVRKHL 135 10 9 90
    SSP2 DAWNKEKALI 106 11 9 90
    SSP2 DQPRPRGDNFA 302 11 9 90
    SSP2 EIKYREEV 35 8 9 90
    SSP2 IQDSLKESRKL 168 11 9 90
    SSP2 IVDEIKYREEV 32 11 9 90
    SSP2 LLQVRKHL 137 8 9 90
    SSP2 LQVRKHLNDRI 138 11 9 90
    SSP2 QVRKHLNDRI 139 10 9 90 0.0001
    SSP2 SLICESRKL 171 8 9 90
    SSP2 ALLACAGL 509 8 10 100
    SSP2 ALLACAGLA 509 9 10 100 0.0006
    SSP2 AMICLIQQL 72 8 10 100
    SSP2 AMICLIQQLNL 72 10 10 100 0.0006
    SSP2 AVCVEVEKT 233 9 10 100
    SSP2 AVCVEVEKTA 233 10 10 100
    SSP2 AVFGIGQGI 186 9 10 100 0.0001
    SSP2 AVFGIGQGINV 186 11 10 100
    SSP2 AVPLAMKL 68 8 10 100
    SSP2 AVPLAMKLI 68 9 10 100 0.0001
    SSP2 CAGLAYKFV 513 9 10 100
    SSP2 CAGLAYKFVV 513 10 10 100 0.0015
    SSP2 CVEVEKTA 235 8 10 100
    SSP2 DASKNKEKA 106 9 10 100
    SSP2 DASKNKD(AL 106 10 10 100
    SSP2 DLDEPEQFRL 546 10 10 100 0.0001
    SSP2 DLFLVNGRDV 19 10 10 100
    SSP2 DVQNNIVDEI 27 10 10 100
    SSP2 EIIRLHSDA 99 9 10 100
    SSP2 ELHEGCT 267 8 10 100
    SSP2 ETLGEEDKDL 538 10 10 100
    SSP2 EVEKTASCGV 237 10 10 100
    SSP2 FLIFFDLFL 14 9 10 100 1.2000
    SSP2 FLIFFDLFLV 14 10 10 100 0.8000
    SSP2 FLVNGRDV 21 8 10 100
    SSP2 FMKAVCVEV 230 9 10 100 0.0290
    SSP2 FVVPQAAT 520 8 10 100
    SSP2 GIAGGLAL 503 8 10 100
    SSP2 GIAGGLALL 503 9 10 100 0.0022
    SSP2 GIAGGLALLA 503 10 10 100
    SSP2 GIGQGTNV 189 8 10 100
    SSP2 GIGQGINVA 189 9 10 100
    SSP2 GINVAFNRFL 193 10 10 100
    SSP2 GINVAFNRFLV 193 11 10 100
    SSP2 GIPDSIQDSL 163 10 10 100
    SSP2 GLALLACA 507 8 10 100
    SSP2 GLALLACAGL 507 10 10 100 0.0170
    SSP2 GLALLACAGLA 507 11 10 100
    SSP2 GLAYKFVV 515 8 10 100
    SSP2 GLAYKFVVPGA 515 11 10 100
    SSP2 GTRSRKREI 260 9 10 100
    SSP2 GTRSRKREIL 260 10 10 100
    SSP2 GVKIAVFGI 182 9 10 100
    SSP2 GVWDEWSPCSV 245 11 10 100
    SSP2 HAVPLAMKL 67 9 10 100
    SSP2 HAVPLAMKLI 67 10 10 100
    SSP2 HLGNVKYL 3 8 10 100
    SSP2 HLGNVKYLV 3 9 10 100 0.0017
    SSP2 HLGNVKYLVI 3 10 10 100
    SSP2 HLGNVKYLVIV 3 11 10 100
    SSP2 HLNDRINRENA 143 11 10 100
    SSP2 HVPNSEDRET 445 10 10 100
    SSP2 LAGGIAGGL 500 9 10 100
    SSP2 IAGGIAGGLA 500 10 10 100
    SSP2 IAGGIAGGLAL 500 11 10 100
    SSP2 LAGGLALL 504 8 10 100
    SSP2 LAGGLALLA 504 9 10 100 0.0001
    SSP2 IACTGLALLACA 504 11 10 100
    SSP2 IAVFGIGQGI 185 10 10 100
    SSP2 IIRLHSDA 100 8 10 100
    SSP2 ILTDGIPDSI 159 10 10 100
    SSP2 IVFLIFFDL 12 9 10 100 0.0024
    SSP2 IVFLIFFDLFL 12 11 10 100
    SSP2 KAVCVEVEKT 232 10 10 100
    SSP2 KAVCVEVEKTA 232 11 10 100
    SSP2 KIAGGIAGGL 499 10 10 100
    SSP2 KIAGGIAGGLA 499 11 10 100
    SSP2 KIAVFGIGQI 184 11 10 100
    SSP2 KLIQQLNL 74 8 10 100
    SSP2 LACAGLAYKFV 511 11 10 100
    SSP2 LALLACAGL 508 9 10 100
    SSP2 LALLACAGLA 508 10 10 100
    SSP2 LAMKLIQQL 71 9 10 100
    SSP2 LAMKLIQQLNL 71 11 10 100
    SSP2 LAYKFVVPGA 516 10 10 100
    SSP2 LAYICFVVPGAA 516 11 10 100
    SSP2 LIFFDLFL I5 8 10 100
    SSP2 LIFFDLFLV 15 9 10 100 0.0890
    SSP2 LLACAGLA 510 8 10 100
    SSP2 LLMDCSGSI 51 9 10 100 0.0460
    SSP2 LMDCSGSI 52 8 10 100
    SSP2 LTDGIPDSI 160 9 10 100
    SSP2 LVIVFLIFFDL 10 11 10 100
    SSP2 LVNGRDVQNNI 22 11 10 100
    SSP2 LVVILTDGI 156 9 10 100
    SSP2 NANQLVVI 152 8 10 100
    SSP2 NANQLVVIL 152 9 10 100
    SSP2 NANQLVVILT 152 10 10 100
    SSP2 NIPEDSEKEV 366 10 10 100
    SSP2 NLYADSAWENV 213 11 10 100
    SSP2 NQLVVILT 154 8 10 100
    SSP2 NQLVVILTDGI 154 11 10 100
    SSP2 NVAFNRFL 195 8 10 100
    SSP2 NVAFNRFLV 195 9 10 100 0.0001
    SSP2 NVIGPFMKA 225 9 I0 100 0.0002
    SSP2 NVIGPFMKAV 225 10 10 100 0.0008
    SSP2 NVKNVIGPFM 222 10 10 100
    SSP2 NVKYLVIV 6 8 10 100
    SSP2 NVKYLVIVFL 6 10 10 100
    SSP2 NVKYLVIVFLI 6 11 10 100
    SSP2 PAPFDETL 533 8 10 100
    SSP2 PLANIKLIQQL 70 10 10 100
    SSP2 QLVVILIDGI 155 10 10 100 0.0002
    SSP2 RINRENANQL 147 10 10 100
    SSP2 RINRENANQLV 147 11 10 100
    SSP2 SAWENVKNV 218 9 10 100 0.0019
    SSP2 SAWENVKNVI 218 10 10 100
    SSP2 SIRRHNWV 58 8 10 100
    SSP2 SQDNNGNRHV 437 10 10 100
    SSP2 SVTCGKGT 254 8 10 100
    SSP2 TLGEEDKDL 539 9 10 100 0.0001
    SSP2 VAFNRFLV 196 8 10 100
    SSP2 VIGPFMKA 226 8 10 100
    SSP2 VIGPFMKAV 226 9 10 100 0.0004
    SSP2 VIGPFMKAVCV 226 11 10 100
    SSP2 VILTDGIPDSI 158 11 10 100
    SSP2 VIVFLIFFDL 11 10 10 100 0.0038
    SSP2 VQNNIVDEI 28 9 10 100
    SSP2 VVILTDGI 157 8 10 100
    SSP2 YADSAWENV 215 9 10 100
    SSP2 YLLMDCSGSI 50 10 10 100 0.1700
    SSP2 YLVIVFLI 9 8 10 100
    Protein A*0202 A*0203 A*0206 A*6802 Seq. Id
    CSP 102
    CSP 103
    CSP 104
    CSP 105
    CSP 106
    CSP 107
    CSP 108
    CSP 109
    CSP 110
    CSP 111
    CSP 112
    CSP 113
    CSP 114
    CSP 115
    CSP 116
    CSP 117
    CSP 118
    CSP 119
    CSP 120
    CSP 121
    CSP 122
    CSP 123
    CSP 124
    CSP 125
    CSP 126
    CSP 127
    CSP 128
    CSP 129
    CSP 130
    CSP 131
    CSP 132
    CSP 133
    CSP 134
    CSP 135
    CSP 136
    CSP 137
    CSP 138
    CSP 139
    CSP 140
    CSP 141
    CSP 142
    CSP 143
    CSP 144
    CSP 145
    CSP 146
    CSP 147
    CSP 148
    CSP 149
    CSP 150
    CSP 151
    CSP 152
    CSP 153
    CSP 154
    CSP 155
    CSP 156
    CSP 157
    CSP 158
    CSP 159
    CSP 160
    CSP 161
    CSP 162
    CSP 163
    CSP 164
    CSP 165
    CSP 166
    CSP 167
    CSP 168
    EXP 169
    EXP 170
    EXP 171
    EXP 172
    EXP 173
    EXP 174
    EXP 175
    EXP 176
    EXP 177
    EXP 178
    EXP 179
    EXP 180
    EXP 181
    EXP 182
    EXP 183
    EXP 184
    EXP 185
    EXP 186
    EXP 187
    EXP 188
    EXP 189
    EXP 190
    EXP 191
    EXP 192
    EXP 193
    EXP 194
    EXP 195
    EXP 196
    EXP 197
    EXP 198
    EXP 199
    EXP 200
    EXP 201
    EXP 202
    EXP 203
    EXP 204
    EXP 205
    EXP 206
    EXP 207
    EXP 208
    EXP 209
    EXP 210
    EXP 211
    EXP 212
    EXP 213
    EXP 214
    EXP 215
    EXP 216
    EXP 217
    EXP 218
    EXP 219
    EXP 220
    EXP 221
    EXP 222
    EXP 223
    EXP 224
    EXP 225
    EXP 226
    EXP 227
    EXP 228
    EXP 229
    EXP 230
    EXP 231
    EXP 232
    EXP 233
    EXP 234
    DT 235
    EXP 236
    EXP 237
    EXP 238
    EXP 239
    EXP 240
    EXP 241
    LSA 242
    LSA 243
    LSA 244
    LSA 245
    LSA 246
    LSA 247
    LSA 248
    LSA 249
    LSA 250
    LSA 251
    LSA 252
    LSA 253
    LSA 255
    LSA 256
    LSA 257
    LSA 258
    LSA 259
    LSA 260
    LSA 261
    LSA 262
    LSA 263
    LSA 264
    LSA 265
    LSA 266
    LSA 267
    LSA 268
    LSA 269
    LSA 270
    LSA 271
    LSA 272
    LSA 273
    LSA 274
    LSA 275
    LSA 276
    LSA 277
    LSA 278
    LSA 279
    LSA 280
    LSA 281
    LSA 282
    LSA 283
    LSA 284
    LSA 285
    LSA 286
    LSA 287
    LSA 288
    LSA 289
    LSA 290
    LSA 291
    LSA 292
    LSA 293
    LSA 294
    LSA 295
    LSA 296
    LSA 297
    LSA 298
    LSA 299
    LSA 300
    LSA 301
    LSA 302
    LSA 303
    LSA 304
    LSA 305
    LSA 306
    LSA 307
    LSA 308
    LSA 309
    LSA 310
    LSA 311
    LSA 312
    LSA 313
    LSA 314
    LSA 315
    LSA 316
    LSA 317
    LSA 318
    LSA 319
    LSA 320
    LSA 321
    LSA 322
    LSA 323
    LSA 324
    LSA 325
    LSA 326
    LSA 327
    LSA 328
    LSA 329
    LSA 330
    LSA 331
    LSA 332
    LSA 333
    LSA 334
    LSA 335
    LSA 336
    LSA 337
    LSA 338
    LSA 339
    LSA 340
    LSA 341
    LSA 342
    LSA 343
    LSA 344
    LSA 345
    LSA 346
    LSA 347
    LSA 348
    LSA 349
    LSA 350
    LSA 351
    LSA 352
    LSA 353
    LSA 354
    LSA 355
    LSA 356
    LSA 357
    LSA 258
    LSA 359
    LSA 360
    LSA 361
    LSA 362
    LSA 363
    LSA 364
    LSA 365
    LSA 366
    LSA 367
    LSA 368
    LSA 369
    LSA 370
    LSA 371
    LSA 372
    LSA 373
    LSA 374
    LSA 375
    LSA 376
    LSA 377
    LSA 378
    LSA 379
    LSA 380
    LSA 381
    LSA 382
    LSA 383
    LSA 384
    LSA 385
    LSA 386
    LSA 387
    LSA 388
    LSA 389
    LSA 390
    LSA 391
    LSA 392
    LSA 393
    LSA 394
    LSA 395
    LSA 396
    LSA 397
    LSA 398
    LSA 399
    LSA 400
    LSA 401
    LSA 402
    LSA 403
    LSA 404
    LSA 405
    SSP2 406
    SSP2 407
    SSP2 408
    SSP2 409
    SSP2 410
    SSP2 411
    SSP2 412
    SSP2 413
    SSP2 414
    SSP2 415
    SSP2 416
    SSP2 417
    SSP2 418
    SSP2 419
    SSP2 420
    SSP2 421
    SSP2 422
    SSP2 423
    SSP2 424
    SSP2 425
    SSP2 426
    SSP2 427
    SSP2 428
    SSP2 429
    SSP2 430
    SSP2 431
    SSP2 432
    SSP2 433
    SSP2 434
    SSP2 435
    SSP2 436
    SSP2 437
    SSP2 438
    SSP2 439
    SSP2 440
    SSP2 441
    SSP2 442
    SSP2 443
    SSP2 444
    SSP2 445
    SSP2 446
    SSP2 447
    SSP2 448
    SSP2 449
    SSP2 450
    SSP2 451
    SSP2 452
    SSP2 453
    SSP2 454
    SSP2 455
    SSP2 456
    SSP2 457
    SSP2 458
    SSP2 459
    SSP2 460
    SSP2 461
    SSP2 462
    SSP2 463
    SSP2 464
    SSP2 465
    SSP2 466
    SSP2 467
    SSP2 468
    SSP2 469
    SSP2 470
    SSP2 471
    SSP2 472
    SSP2 473
    SSP2 474
    SSP2 475
    SSP2 476
    SSP2 477
    SSP2 478
    SSP2 479
    SSP2 480
    SSP2 481
    SSP2 482
    SSP2 483
    SSP2 484
    SSP2 485
    SSP2 486
    SSP2 487
    SSP2 488
    SSP2 489
    SSP2 490
    SSP2 491
    SSP2 492
    SSP2 493
    SSP2 494
    SSP2 495
    SSP2 496
    SSP2 497
    SSP2 498
    SSP2 499
    SSP2 500
    SSP2 501
    SSP2 502
    SSP2 503
    SSP2 504
    SSP2 505
    SSP2 506
    SSP2 507
    SSP2 508
    SSP2 509
    SSP2 510
    SSP2 511
    SSP2 512
    SSP2 513
    SSP2 514
    SSP2 515
    SSP2 516
    SSP2 517
    SSP2 518
    SSP2 519
    SSP2 520
    SSP2 521
    SSP2 522
    SSP2 523
    SSP2 524
    SSP2 525
    SSP2 526
    SSP2 327
    SSP2 528
    SSP2 529
    SSP2 530
    SSP2 531
    SSP2 532
    SSP2 533
    SSP2 534
    SSP2 535
    SSP2 536
    SSP2 337
    SSP2 538
    SSP2 339
    SSP2 540
    SSP2 541
    SSP2 542
    SSP2 543
    SSP2 544
    SSP2 545
    SSP2 546
    SSP2 547
    SSP2 548
    SSP2 549
    SSP2 550
    SSP2 551
    SSP2 552
    SSP2 553
    SSP2 554
    SSP2 555
    SSP2 556
    SSP2 557
  • TABLE IX
    Malaria A03 Super Motif Peptides With Binding Data
    No. of Sequence Conservancy
    Protein Sequence Position Amino Acids Frequency (%) A*301
    CSP DIEKKICK 402 8 19 100
    CSP DIEKKICKMEK 402 11 19 100
    CSP ELEMNYYGK 50 9 19 100 0.0001
    CSP KLRKPKHK 104 8 19 100
    CSP KLRKPKHKK 104 9 19 100 0.1300
    CSP KLRKPKHKKLK 104 11 19 100
    CSP NANANNAVK 335 9 16 84 0.0001
    CSP NANPNANPNK 304 10 19 100 0.0005
    CSP NMPNDPNR 323 8 19 100
    CSP SVTCGNGIQVR 374 11 19 100
    CSP VTCGNGIQVR 375 10 19 100 0.0005
    CSP YSLKKNSR 63 8 19 100
    EXP ALFFIIFNK 10 9 1 100 1.1000
    EXP DLISDMIK 52 8 1 100
    EXP DLISDMIKK 52 9 1 100 0.0001
    EXP DVHDLISDMIK 49 11 1 100
    EXP ELVEVNKR 63 8 1 100
    EXP ELVEVNKRK 63 9 1 100 0.0001
    EXP ELVEVNKRKSK 63 11 1 100
    EXP ESLAEKTNK 19 9 1 100 0.0001
    EXP EVNKRKSK 66 8 1 100
    EXP EVNKRKSKYK 66 10 1 100 0.0005
    EXP FLALFFIIFNK 8 11 1 100
    EXP GLVLYNTEK 97 9 1 100 0.0069
    EXP GLVLYNTEKGR 97 11 1 100
    EXP GSGVSSKK 30 8 1 100
    EXP GSGVSSKKK 30 9 1 100 0.0003
    EXP GSGVSSKKKNK 30 11 1 100
    EXP GTGSGVSSK 28 9 1 100 0.0039
    EXP GTGSGVSSKK 28 10 1 100 0.0071
    EXP GTGSGVSSKKK 28 11 1 100
    EXP GVGLVLYNTEK 95 11 1 100
    EXP GVSSKKKNK 32 9 1 100 0.0001
    EXP GVSSKKKNKK 32 10 1 100 0.0011
    EXP IIFNKESLAEK 14 11 1 100
    EXP LALFFTIIFNK 9 10 1 100 0.014
    EXP LISDMIKK 53 8 1 100
    EXP LVEVNKRK 64 8 1 100
    EXP LVEVNKRKSK 64 10 1 100 0.0005
    EXP LVLYNTEK 98 8 1 100
    EXP LVLYNTEKGR 98 10 1 100 0.0005
    EXP NTEKGRHPFK 102 10 1 100 0.0047
    EXP SLAEKTNK 20 8 1 100
    EXP SSKKKNKK 34 8 1 100
    EXP VLYNTEKGRS 99 9 1 100 0.0110
    EXP VSSKKKNK 33 8 1 100
    EXP VSSKKKNKK 33 9 1 100 0.0001
    LSA AIELPSENER 1660 10 1 100 0.0001
    LSA DIHKGHLEEK 1713 10 1 100 0.0004
    LSA DIHKGHLEEKK 1713 11 1 100
    LSA DITKYFMK 1901 8 1 100
    LSA DLDEGIEK 1818 8 1 100
    LSA DLEEKAAK 148 8 1 100
    LSA DLEQDRLAK 1388 9 1 100 0.0001
    LSA DLEQDRLAKEK 1388 11 1 100
    LSA DLEQERLAK 1609 9 1 100 0.0001
    LSA DLEQERLAKEK 1609 11 1 100
    LSA DLEQERLANEK 1524 11 1 100
    LSA DLEQERRAK 1575 9 1 100 0.0001
    LSA DLEQERRAIUEK 1575 11 1 100
    LSA DLEQRKADTK 1626 10 1 100 0.0001
    LSA DLEQRKADTKK 1626 11 1 100
    LSA DLERTICASK 1184 9 1 100 0.0001
    LSA DSEQERLAK 521 9 1 100 0.0001
    LSA CGEQERLAKEK 521 11 1 100
    LSA DSKEISIIEK 1689 10 1 100 0.0001
    LSA DTKKNLER 1633 8 1 100
    LSA DTKKNLERK 1633 9 1 100 0.0001
    LSA DTKKKNLERKK 1633 10 1 100 0.0001
    LSA DVLAEDLYGR 1646 10 1 100 0.0001
    LSA DVNDFQISK 1751 9 1 100 0.0001
    LSA EIIKSNLR 33 8 1 100
    LSA EISIIEKTNR 1692 10 1 100 0.0001
    LSA ELEDLIEK 1805 8 1 100
    LSA ELPSENER 1662 8 1 100
    LSA ELSEDITK 1897 8 1 100
    LSA ELSEEKIK 1829 8 1 100
    LSA ELSEEKIKK 1829 9 1 100 0.0002
    LSA ELSEEKIKKGK 1829 11 1 100
    LSA ELTMSNVK 83 8 1 100
    LSA ESITTNVEGR 1702 10 1 100 0.0001
    LSA ESITTNVEGRR 1702 11 1 100
    LSA FLKENKLNK 111 9 1 100 0.0260
    LSA GSIKPEQK 1725 8 1 100
    LSA GSIKPEQKEDK 1725 11 1 100
    LSA GSSNSRNR 42 8 1 100
    LSA GVSENIFLK 105 9 1 100 0.2700
    LSA HIINDDDDK 126 9 1 100 0.0002
    LSA HIINDDDDKK 126 10 1 100 0.0001
    LSA HIINDDDDKKK 126 11 1 100
    LSA HIKKYKNDK 1860 9 1 100 0.0002
    LSA HINGKIIK 20 8 1 100
    LSA HLEEKICDGSIK 1718 11 1 100
    LSA HVLSHNSYEK 59 10 1 100 0.0170
    LSA QNDDDDK 127 8 1 100
    LSA IINDDDDKK 127 9 1 100 0.0002
    LSA IINDDDDIUCK 127 10 1 100 0.0001
    LSA ISDVNDFQISK 1749 11 1 100
    LSA ISIIEKTNR 1693 9 1 100 0.0001
    LSA ITTNVEGR 1704 8 1 100
    LSA ITINVEGRR 1704 9 1 100 0.0002
    LSA IVDELSEDMC 1894 11 1 100
    LSA ICADIKKNLER 1631 10 1 100 0.0001
    LSA KADTKKNLERK 1631 11 1 100
    LSA KIIKNSEK 24 8 1 100
    LSA ICQUCGKKYEK 1834 10 1 100 0.0081
    LSA KLQEQQSDLER 1177 11 1 100
    LSA KSLYDEHIK 1854 9 1 100 0.0005
    LSA KSLYDEHIKIC 1854 10 1 100 0.0094
    LSA KSSEELSEEK 1825 10 1 100 0.0001
    LSA KTKDNNFK 1843 8 1 100
    LSA KTKNNENNK 68 9 1 100 0.0028
    LSA LAEDLYGR 1648 8 1 100
    LSA LAKEKLQEQQR 1615 11 1 100
    LSA LANEKLQEQQR 1530 11 1 100
    LSA LIFHINGK 17 8 1 100
    LSA LIGHINKKIIK 17 11 1 100
    LSA LLIFHINKGK 16 9 1 100 0.0260
    LSA LSEDITKYFMK 1898 11 1 100
    LSA LSEEKIKK 1830 8 1 100
    LSA LSEEKIKKGK 1830 10 1 100 0.0004
    LSA LSEEKIKKGKK 1830 11 1 100
    LSA LSHNSYEK 61 8 1 100
    LSA LSHNSYEKTK 61 10 1 100 0.0004
    LSA NIFLKENK 109 8 1 100
    LSA NKFLKENKLNK 109 11 1 100
    LSA LNDDLDEGIEK 1815 11 1 100
    LSA NLGVSENTFLK 103 11 1 100
    LSA NLLIGHINGK 15 10 1 100 0.0049
    LSA NLRSGSSNSR 38 10 1 100 0.0004
    LSA NSEKDETIK 28 9 1 100 0.0002
    LSA NSRNRINEEK 45 10 1 100 0.0004
    LSA NVEGRRDIHK 1707 10 1 100 0.0004
    LSA NVKNVSQTNFK 88 11 1 100
    LSA NVSQTNFK 91 8 1 100
    LSA PAIELPSENER 1659 11 1 100
    LSA QSDLEQDR 1386 8 1 100
    LSA QSDLEQDRLAK 1386 11 1 100
    LSA QSKLEQER 1590 8 1 100
    LSA QSDLEQERLAK 1590 11 1 100
    LSA QSKLEQERR 1573 9 1 100 0.0002
    LSA QSDLEQERRAK 1573 11 1 100
    LSA QSDLERTK 1182 8 1 100
    LSA QSDLERTKASK 1182 11 1 100
    LSA QSDSEQER 519 8 1 100
    LSA QSDSEQERLAK 519 11 1 100
    LSA QSSLPQDNR 1676 9 1 100 0.0002
    LSA QTNFKSLLR 94 9 1 100 0.0320
    LSA QVNKEKEK 1869 8 1 100
    LSA QVNKEKEKFIK 1869 11 1 100
    LSA RINEEKHEK 49 9 1 100 0.0033
    LSA RINEEKHEKK 49 10 1 100 0.0024
    LSA RSGSSNSR 40 8 1 100
    LSA RSGSSNSRNR 40 10 1 100 0.0011
    LSA SIIEKTNR 1694 8 1 100
    LSA SIKPEQKEDK 1726 10 1 100 0.0002
    LSA SITTNVEGR 1703 9 1 100 0.0002
    LSA SITTNVEGRR 1703 10 1 100 0.0002
    LSA SLPQDNRDNSR 1678 11 1 100
    LSA SLYDEHIK 1855 8 1 100
    LSA SLYDEHIKK 1855 9 1 100 0.0460
    LSA SLYDEHIKKYK 1855 11 1 100
    LSA SSEELSEEK 1826 9 1 100 0.0002
    LSA SSEELSEEKIK 1826 11 1 100
    LSA SSLPQDNR 1677 8 1 100
    LSA TTNVEGRR 1705 8 1 100
    LSA VLAEDLYGR 1647 9 1 100 0.0013
    LSA VLSHNSYEK 60 9 1 100 0.0280
    LSA VLSHNSYEKTK 60 11 1 100
    LSA VSENTIFLK 106 8 1 100
    LSA VSENIFLKENK 106 11 1 100
    LSA VSQTNFKSLLR 92 11 1 100
    LSA YIKGQDENR 137 9 1 100 0.0025
    SSP2 ALLACAGLAYK 509 11 10 100
    SSP2 AVCVEVEK 233 8 10 100
    SSP2 CSVTCGKGTR 253 10 10 100 0.0002
    SSP2 DALLQVRK 135 8 9 90
    SSP2 DASKNKEK 106 8 10 100
    SSP2 DIPKKPENK 392 9 10 100 0.0004
    SSP2 DLDEPEQFR 546 9 10 100 0.0002
    SSP2 DLFLVNGR 19 8 10 100
    SSP2 DSAWENVK 217 8 10 100
    SSP2 DSIQDSLK 166 8 10 100
    SSP2 DSIQDSLKESR 166 11 10 100
    SSP2 DSLKESRK 170 8 9 90
    SSP2 DVPKNPEDDR 378 10 10 100 0.0002
    SSP2 DVQNNIVDEIK 27 11 10 100
    SSP2 EIIRLHSDASK 99 11 10 100
    SSP2 ELQEQCEEER 276 10 8 80 0.0002
    SSP2 ETLGEEDK 538 8 10 100
    SSP2 EVPSDVPK 374 8 10 100
    SSP2 FLVGCHPSDCK 201 11 10 100
    SSP2 FMKAVCVEVEK 230 11 10 100
    SSP2 GINVAFNR 193 8 10 100
    SSP2 GIPDSIQDSLK 163 11 10 100
    SSP2 HAVPLAMK 67 8 10 100
    SSP2 HLNDRINR 143 8 10 100
    SSP2 HSDASKNK 104 8 10 100
    SSP2 HSDASKNKEK 104 10 10 100 0.0004
    SSP2 HVPNSEDR 445 8 10 100
    SSP2 HVPNSEDRETR 445 11 9 90
    SSP2 IIRLHSDASK 100 10 10 100 0.0230
    SSP2 1VDEIKYR 32 8 9 90
    SSP2 KAVCVEVEK 232 9 10 100 0.0004
    SSP2 KVLDNERK 421 8 8 80
    SSP2 LACAGLAYK 511 9 10 100 0.0240
    SSP2 LLACAGLAYK 510 10 10 100 0.9500
    SSP2 LLMDCSGSIR 51 10 10 100 0.0004
    SSP2 LLMDCSGSIRR 51 11 10 100
    SSP2 LLQVRKHLNDR 137 11 9 90
    SSP2 LLSTNLPYGR 121 10 8 80 0.0017
    SSP2 LMDCSGSIR 52 9 10 100 0.0004
    SSP2 LMDCSGSIRR 52 10 10 100 0.0015
    SSP2 LSTNLPYGR 122 9 8 80 0.0004
    SSP2 LVGCHPSDGK 202 10 10 100 0.0004
    SSP2 NIPEDSEK 366 8 10 100
    SSP2 NIVDEIKYR 31 9 9 90 0.0005
    SSP2 NLPNDKSDR 406 9 10 100 0.0005
    SSP2 NSEDRETFT 448 8 9 90
    SSP2 NVIGPFMK 225 8 10 100
    SSP2 NVICNVIGPFMK 222 11 10 100
    SSP2 PSPNPEEGK 328 9 10 100 0.0005
    SSP2 QSQDNNGNR 436 9 10 100 0.0005
    SSP2 QVRKHLNDR 139 9 9 90 0.0005
    SSP2 RLHSDASK 102 8 10 100
    SSP2 RLHSDASKNK 102 10 10 100 0.0240
    SSP2 SIQDSLKESR 167 10 10 100 0.0004
    SSP2 SIQDSLKESRK 167 11 9 90
    SSP2 SLLSTNLPYGR 120 11 8 80
    SSP2 STNLPYGR 123 8 8 80
    SSP2 SVTCGKGTR 254 9 10 100 0.0005
    SSP2 SVTCGKGTRSR 254 11 10 100
    SSP2 VTCGKGTR 255 8 10 100
    SSP2 VPCGKGTISR 255 10 10 100 0.0004
    SSP2 VTCGKGTRSRK 255 11 10 100
    SSP2 WSPCSVTCGK 250 10 10 100 0.0004
    SSP2 WVNHAVPLAMK 64 11 8 80
    SSP2 YADSAWENVK 215 10 10 100 0.0004
    SSP2 YLLMDCSGSIR 50 11 10 100
    Protein A*1101 A*3101 A*3301 A*6801 Seq id.
    CSP 558
    CSP 559
    CSP 0.0003 560
    CSP 561
    CSP 0.0037 562
    CSP 563
    CSP 0.0002 0.0006 0.0096 0.0210 564
    CSP 0.0021 0.0009 0.0009 0.0054 565
    CSP 566
    CSP 567
    CSP 0.0340 568
    CSP 569
    EXP 1.2000 570
    EXP 571
    EXP 0.0003 572
    EXP 573
    EXP 574
    EXP 0.0002 575
    EXP 576
    EXP 0.0002 0.0004 0.0110 0.0260 577
    EXP 578
    EXP 0.0002 579
    EXP 580
    EXP 0.0055 581
    EXP 582
    EXP 583
    EXP 0.0065 0.0004 0.0010 0.0002 584
    EXP 585
    EXP 0.0180 586
    EXP 0.0340 587
    EXP 588
    EXP 589
    EXP 0.0002 590
    EXP 0.0002 591
    EXP 592
    EXP 0.0530 0.0072 0.0076 0.0039 593
    EXP 594
    EXP 595
    EXP 0.0002 596
    EXP 597
    EXP 0.0002 598
    EXP 0.0080 599
    EXP 600
    EXP 601
    EXP 0.0007 0.0039 0.0055 0.0022 602
    EXP 603
    EXP 0.0002 0.0004 0.0010 0.0002 604
    LSA 0.0002 0.0009 0.0008 0.0029 605
    LSA 0.0002 0.0009 0.0055 0.0046 606
    LSA 607
    LSA 608
    LSA 609
    LSA 610
    LSA 0.0002 611
    LSA 612
    LSA 0.0002 613
    LSA 614
    LSA 615
    LSA 0.0002 616
    LSA 617
    LSA 0.0002 618
    LSA 619
    LSA 0.0002 620
    LSA 0.0002 0.0004 0.0010 0.0002 621
    LSA 622
    LSA 0.0002 623
    LSA 624
    LSA 0.0002 625
    LSA 0.0002 626
    LSA 0.0002 627
    LSA 0.0018 628
    LSA 629
    LSA 0.0002 630
    LSA 631
    LSA 632
    LSA 633
    LSA 634
    LSA 0.0002 635
    LSA 636
    LSA 637
    LSA 0.0002 638
    LSA 639
    LSA 0.0005 640
    LSA 641
    LSA 642
    LSA 643
    LSA 0.6600 644
    LSA 0.0002 645
    LSA 0.0002 0.0009 0.0009 0.0003 646
    LSA 647
    LSA 0.0002 648
    LSA 649
    LSA 650
    LSA 0.0140 651
    LSA 652
    LSA 0.0002 653
    LSA 0.0002 654
    LSA 655
    LSA 0.0008 0.0320 0.0150 0.0054 656
    LSA 657
    LSA 0.0007 0.0025 0.0043 0.3200 658
    LSA 659
    LSA 0.0002 0.0086 0.0011 0.0003 660
    LSA 661
    LSA 662
    LSA 0.0007 0.0042 0.0009 0.0003 663
    LSA 664
    LSA 0.0340 0.0004 0.0010 0.0002 665
    LSA 0.0490 666
    LSA 0.0009 667
    LSA 668
    LSA 0.0038 669
    LSA 670
    LSA 671
    LSA 672
    LSA 673
    LSA 674
    LSA 0.0100 675
    LSA 676
    LSA 677
    LSA 0.0002 678
    LSA 679
    LSA 680
    LSA 0.0002 681
    LSA 682
    LSA 683
    LSA 684
    LSA 685
    LSA 0.0008 686
    LSA 0.0002 687
    LSA 0.0002 0.0004 0.0010 0.0002 688
    LSA 0.0002 689
    LSA 0.0002 690
    LSA 691
    LSA 692
    LSA 692
    LSA 694
    LSA 695
    LSA 696
    LSA 697
    LSA 0.0002 0.0006 0.0005 0.0005 698
    LSA 699
    LSA 700
    LSA 701
    LSA 702
    LSA 703
    LSA 0.0013 0.0150 0.014 0.0480 704
    LSA 0.0440 0.0820 0.0180 0.1300 705
    LSA 706
    LSA 707
    LSA 0.0370 708
    LSA 0.0018 0.0009 0.0009 0.0003 709
    LSA 710
    LSA 0.0002 711
    LSA 712
    LSA 0.0002 0.0009 0.0009 0.0003 713
    LSA 0.0027 714
    LSA 0.0002 715
    LSA 716
    LSA 717
    LSA 0.4100 718
    LSA 719
    LSA 0.0017 0.0004 0.0010 0.0002 720
    LSA 721
    LSA 722
    LSA 723
    LSA 0.0004 0.0083 0.0220 0.0032 724
    LSA 0.0280 725
    LSA 726
    LSA 727
    LSA 728
    LSA 729
    LSA 0.0002 730
    SSP2 731
    SSP2 732
    SSP2 0.0002 733
    SSP2 734
    SSP2 735
    SSP2 0.0002 736
    SSP2 0.0002 0.0004 0.0170 0.0002 737
    SSP2 738
    SSP2 739
    SSP2 740
    SSP2 741
    SSP2 742
    SSP2 0.0002 743
    SSP2 744
    SSP2 745
    SSP2 0.0002 746
    SSP2 747
    SSP2 748
    SSP2 749
    SSP2 750
    SSP2 751
    SSP2 752
    SSP2 753
    SSP2 754
    SSP2 755
    SSP2 0.0002 756
    SSP2 757
    SSP2 758
    SSP2 0.0002 0.0009 0.0009 0.0013 759
    SSP2 760
    SSP2 0.0076 0.0009 0.0005 0.0029 761
    SSP2 762
    SSP2 0.0290 0.0150 0.3200 0.1100 763
    SSP2 0.0870 764
    SSP2 0.0005 765
    SSP2 766
    SSP2 767
    SSP2 0.0025 768
    SSP2 0.0002 0.0370 0.0430 0.0010 769
    SSP2 0.0002 770
    SSP2 0.0100 0.2900 0.0760 0.2700 771
    SSP2 0.0002 772
    SSP2 773
    SSP2 0.0002 774
    SSP2 0.0002 775
    SSP2 776
    SSP2 777
    SSP2 778
    SSP2 0.0002 0.0004 0.0010 0.0002 779
    SSP2 0.0002 0.0020 0.0093 0.0018 780
    SSP2 0.0002 0.0041 0.0570 0.0002 781
    SSP2 782
    SSP2 0.0002 783
    SSP2 0.0009 784
    SSP2 785
    SSP2 786
    SSP2 787
    SSP2 0.0009 0.0031 0.0039 0.0310 788
    SSP2 789
    SSP2 790
    SSP2 0.0017 791
    SSP2 792
    SSP2 0.0002 793
    SSP2 794
    SSP2 0.0002 0.0009 0.0009 0.0077 795
    SSP2 796
  • TABLE X
    Malaria A24 Super Motif Peptides With Binding Information
    No. of Sequence Conservancy
    Protein Sequence Position Amino Acids Frequency (%) A*201 Seq Id.
    CSP AILSVSSF 6 8 18 95 797
    CSP AILSVSSFL 6 9 19 100 798
    CSP AILSVSSFLF 6 10 19 100 799
    CSP ALFQEYQCY 18 9 19 100 800
    CSP CYGSSSNTRVL 25 11 19 100 801
    CSP DIEKKICKM 402 9 19 100 802
    CSP DYENDIEKKI 398 10 18 95 803
    CSP ELNYDNAGI 37 9 18 95 804
    CSP ELNYDNAGINL 37 11 18 95 805
    CSP EMNYYGKQENW 52 11 19 100 806
    CSP FLFVEALF 13 8 19 100 807
    CSP FLFVEALFQEY 13 11 19 100 808
    CSP FVEALFQEY 15 9 19 100 809
    CSP GINLYNEL 44 8 18 95 810
    CSP GINLYNELEM 44 10 18 95 811
    CSP GLIMVLSF 425 8 19 100 812
    CSP GLIMVLSFL 425 9 19 100 813
    CSP GLIMVLSFLF 425 10 19 100 814
    CSP GLIMVLSFLFL 425 11 19 100 815
    CSP HIEQYLKKI 350 9 15 79 816
    CSP ILSVSSFL 7 8 19 100 817
    CSP ILSVSSFLF 7 9 19 100 818
    CSP IMVLSFLF 427 8 19 100 819
    CSP IMVLSFLFL 427 9 19 100 0.0008 820
    CSP KIQNSLSTEW 361 10 15 79 821
    CSP KLAILSVSSF 4 10 19 100 822
    CSP KLAILSVSSFL 4 11 19 100 823
    CSP KLRKPICHICKL 104 10 19 100 824
    CSP KMEKCSSVF 409 9 19 100 825
    CSP LFQEYQCY 19 8 19 100 826
    CSP LFVEALFQEY 14 10 19 100 827
    CSP LIMVLSFL 426 8 19 100 828
    CSP LIMVLSFLF 426 9 19 100 829
    CSP LIMVLSFLFL 426 10 19 100 830
    CSP LYNELEMNY 47 9 19 100 831
    CSP LYNELEMNYY 47 10 19 100 832
    CSP MMRKLAIL 1 8 19 100 833
    CSP MVLSFLFL 428 8 19 100 834
    CSP NLYNELEM 46 8 19 100 835
    CSP NLYNELEIINY 46 10 19 100 836
    CSP NLYNELINNYY 46 11 19 100 837
    CSP NTRVLNEL 31 8 19 100 838
    CSP NTRYLNELNY 31 10 19 100 839
    CSP NVVNSSIGL 418 9 19 100 840
    CSP NVVNSSIGLI 418 10 19 100 841
    CSP NVVNSSIGLIM 418 11 19 100 842
    CSP NYDNAGINL 39 9 18 95 0.0004 843
    CSP NYDNAGINLY 39 10 18 95 844
    CSP NYYGKQENW 54 9 19 100 845
    CSP NYYGKQENWY 54 10 19 100 846
    CSP RVLNELNY 33 8 19 100 847
    CSP SFLFVEAL 12 8 19 100 848
    CSP SFLFVEALF 12 9 19 100 849
    CSP SIGLIMVL 423 8 19 100 850
    CSP SIGLIMVLSF 423 10 19 100 851
    CSP SIGLIMVLSFL 423 11 19 100 852
    CSP SLKKNSRSL 64 9 19 100 853
    CSP SVFNVVNSSI 415 10 19 100 854
    CSP SVSSFLFVEAL 9 11 19 100 855
    CSP SVTCGNGI 374 8 19 100 856
    CSP VFNVVNSSI 416 9 19 100 857
    CSP VFNVVNSSIGL 416 11 19 100 858
    CSP VTCGNGIQVRI 375 11 19 100 859
    CSP VVNSSIGL 419 8 19 100 860
    CSP VVNSSIGLI 419 9 19 100 861
    CSP VVNSSIGL1M 419 10 19 100 862
    CSP WYSLKKNSRSL 62 11 19 100 863
    CSP YLKKIQNSL 358 9 15 79 864
    CSP YYGKQENW 55 8 19 100 865
    CSP YYGKQENWY 55 9 19 100 866
    CSP YYGKQENWYSL 55 11 19 100 867
    EXP ATSVLAGL 77 8 1 100 868
    EXP ATSVLAGLL 77 9 1 100 869
    EXP DMIKKEEEL 56 9 1 100 870
    EXP DVHDLISDM 49 9 1 100 871
    EXP DVHDLISDMI 49 10 1 100 872
    EXP EVNKRKSKY 66 9 1 100 873
    EXP EVNKRICSKYKL 66 11 1 100 874
    EXP FFIIFNKESL 12 10 1 100 875
    EXP FFLALFFI 7 8 1 100 876
    EXP FFLALFFII 7 9 1 100 877
    EXP FFLALFFIIF 7 10 1 100 878
    EXP FITFNKESL 13 9 1 100 879
    EXP FLALFFII 8 8 1 100 880
    EXP FLALFFIIF 8 9 1 100 881
    EXP GLLGNVSTVL 83 10 1 100 882
    EXP GLLGNVSTVLL 83 11 1 100 883
    EXP IIFNKESL 14 8 1 100 884
    EXP ILSVFFLAL 3 9 1 100 885
    EXP ILSVFFLALF 3 10 1 100 886
    EXP ILSVFFLALFF 3 11 1 100 887
    EXP KILSVFFL 2 8 1 100 888
    EXP KILSVFFLAL 2 10 1 100 889
    EXP KILSVFFLALF 2 11 1 100 890
    EXP KLATSVLAGL 75 10 1 100 891
    EXP KLATSVLAGLL 75 11 1 100 892
    EXP KYKLATSVL 73 9 1 100 0.0960 893
    EXP LFFIIFNICESL 11 11 1 100 894
    EXP LIDVHDLI 47 8 1 100 895
    EXP LIDVHDLISDM 47 11 1 100 896
    EXP LLGGVGLVL 92 9 1 100 897
    EXP LLGGVGLVLY 92 10 1 100 898
    EXP LLGNVSTVL 84 9 1 100 899
    EXP LLGNVSTVLL 84 10 1 100 900
    EXP LVEVNICRICSKY 64 11 1 100 901
    EXP LYNTEKGRHPF 100 11 1 100 902
    EXP MIKKEEEL 57 8 1 100 903
    EXP NTEKGRHPF 102 9 1 100 904
    EXP NTEKGRHPFKI 102 11 1 100 905
    EXP PLIDVHDL 46 8 1 100 906
    EXP PLIDVHDLI 46 9 1 100 907
    EXP STVLLGGVGL 89 10 1 100 908
    EXP SVFFLALF 5 8 1 100 909
    EXP SVFFLALFF 5 9 1 100 910
    EXP SVFFLALFFI 5 10 1 100 911
    EXP SVFFLALFFII 5 11 1 100 912
    EXP TVLLGGVGL 90 9 1 100 913
    EXP TVLLGGVQLVL 90 11 1 100 914
    EXP VFFLALFF 6 8 1 100 915
    EXP VFFLALFFI 6 9 1 100 916
    EXP VFFLALFFII 6 10 1 100 917
    EXP VFFLALFFIIF 6 11 1 100 918
    EXP VLLCrGVGL 91 8 1 100 919
    EXP VLLGGVGLVL 91 10 1 100 920
    EXP VLLGGVGLVLY 91 11 1 100 921
    LSA DFQISKYEDEI 1754 11 1 100 922
    LSA DITKYFMKI 1901 9 1 100 923
    LSA DLDEFKPI 1781 8 1 100 924
    LSA DLDEFKFIVQY 1781 11 1 100 925
    LSA DLEEXAAKETL 148 11 1 100 926
    LSA DLIEXNENL 1808 9 1 100 927
    LSA DLYGRLEI 1651 8 1 100 928
    LSA DLYGRLEIPAI 1651 11 1 100 929
    LSA DVLAEDLY 1646 8 1 100 930
    LSA DVLAEDLYGRL 1646 11 1 100 931
    LSA DVNDFQISKY 1751 10 1 100 932
    LSA EFXPIVQY 1784 8 1 100 933
    LSA EFKPIVQYDNF 1784 11 1 100 934
    LSA ERQIVDEL 1890 9 1 100 935
    LSA EISAEYDDSL 1763 10 1 100 936
    LSA EISAEYDDSLI 1763 11 1 100 937
    LSA ELPSENERGY 1662 10 1 100 938
    LSA ELPSENERGYY 1662 11 1 100 939
    LSA ELSEDTIKY 1897 9 1 100 940
    LSA ELSEDTIKYF 1897 10 1 100 941
    LSA ELSEDIDCYFM 1897 11 1 100 942
    LSA ETLQBQQSDL 1193 10 1 100 943
    LSA ETLQGQQSDL 156 10 1 100 944
    LSA ETVNISDVNDF 1745 11 1 100 945
    LSA FFDKDKEL 77 8 1 100 946
    LSA FFDKDKELTM 77 10 1 100 947
    LSA FIKSLFHI 1877 8 1 100 948
    LSA FIKSLFH1F 1877 9 1 100 949
    LSA FILVNLLI 11 8 1 100 950
    LSA FILVNLLIF 11 9 1 100 951
    LSA FILVNLLIFHI 11 11 1 100 952
    LSA FYF1LVNL 9 8 1 100 953
    LSA FYFILVNLL 9 9 1 100 7.5000 954
    LSA FYFILVNLLI 9 10 1 100 955
    LSA FYFILVNLLIF 9 11 1 100 956
    LSA GIEKSSEEL 1822 9 1 100 957
    LSA GIYKELEDL 1801 9 1 100 958
    LSA GIYKELEDL1 1801 10 1 100 959
    LSA GVSEN1FL 105 8 1 100 960
    LSA GYYIPHQSSL 1670 10 1 100 0.0074 961
    LSA HIFDGDNEI 1883 9 1 100 962
    LSA HIFDGDNEIL 1883 10 1 100 963
    LSA HILYISFY 3 8 1 100 964
    LSA HILYISFYF 3 9 1 100 965
    LSA NILYISFYFI 3 10 1 100 966
    LSA LIILYISFYFIL 3 11 1 100 967
    LSA HLEEKKDOS1 1718 10 1 100 968
    LSA FITLETVNI 1742 8 1 100 969
    LSA HVLSHNSY 59 8 1 100 970
    LSA IFDGDNEI 1884 8 1 100 971
    LSA IFDGDNEIL 1884 9 1 100 972
    LSA IFDODNEILQI 1884 11 1 100 973
    LSA IFHINGKI 18 8 1 100 974
    LSA IFHINGKII 18 9 1 100 975
    LSA IFLKENKL 110 8 1 100 976
    LSA IIEKTNRESI 1695 10 1 100 977
    LSA IIKNSEKDEI 25 10 1 100 978
    LSA IIKNSEKDEII 25 11 1 100 979
    LSA IINDDDDYKKY 127 11 1 100 980
    LSA ILQIVDEL 1891 8 1 100 981
    LSA ILVNLLIF 12 8 1 100 982
    LSA ILVNLLIFHI 12 10 1 100 983
    LSA ILYISFYF 4 8 1 100 984
    LSA ILYISFYFI 4 9 1 100 985
    LSA ILYISFYFIL 4 10 1 100 986
    LSA IIKYFMKL 1902 8 1 100 987
    LSA ITINVEGRRDI 1704 11 1 100 988
    LSA IVDELSEDI 1894 9 1 100 989
    LSA IYKELEDL 1802 8 1 100 990
    LSA IYKELEDLI 1802 9 1 100 991
    LSA KFFDKDKEL 76 9 1 100 992
    LSA KFFDKDKELTM 76 11 1 100 993
    LSA KFIKSLFHI 1876 9 1 100 994
    LSA KFIKSLFHIF 1876 10 1 100 995
    LSA KIIKNSEKDEI 24 11 1 100 996
    LSA KIKKGKKY 1834 8 1 100 997
    LSA KLNKEGKL 116 8 1 100 998
    LSA KLNKEGKLI 116 9 1 100 999
    LSA KLQEQQRDL 1619 9 1 100 1000
    LSA KLQEQQSDL 1585 9 1 100 1001
    LSA KLQGQQSDL 1126 9 1 100 1002
    LSA KTKNNENNKF 68 10 1 100 1003
    LSA KTKNNENNKFF 68 11 1 100 1004
    LSA KYEDEISAEY 1759 10 1 100 1005
    LSA KYEKTKDNNF 1140 10 1 100 0.0004 1006
    LSA LFHTFDODNEI 1881 11 1 100 1007
    LSA LIDEEEDDEDL 1772 11 1 100 1008
    LSA LISCNENL 1809 8 1 100 1009
    LSA LIEKNENLDDL 1809 11 1 100 1010
    LSA LIFHINGKI 17 9 1 100 1011
    LSA LIFHINGKII 17 10 1 100 1012
    LSA LLIFHINGKI 16 10 1 100 1013
    LSA LLIFHINGKII 16 11 1 100 1014
    LSA LLRNLGVSENI 100 11 1 100 1015
    LSA LVNLLIFHI 13 9 1 100 1016
    LSA LYDEHIKKY 1856 9 1 100 1017
    LSA LYGRLEIPAI 1652 10 1 100 1018
    LSA LYISFYFI 5 8 1 100 1019
    LSA LYISFYFIL 5 9 1 100 0.0088 1020
    LSA NFIONDKSL 1848 9 1 100 1021
    LSA NFKPNDKSLY 1848 10 1 100 1022
    LSA NFKSLLRNL 96 9 1 100 1023
    LSA NFQDEENI 1793 8 1 100 1024
    LSA NFQDEENIGI 1793 10 1 100 1025
    LSA NFQDEENIG1Y 1793 11 1 100 1026
    LSA NIFLKENKL 109 9 1 100 1027
    LSA MGIYKEL 1799 8 1 100 1028
    LSA MGIYKELEDL 1799 11 1 100 1029
    LSA MSDVNDF 1748 8 1 loo 1030
    LSA NISDVNDFQI 1748 10 1 100 1031
    LSA NLDDLDEGI 1815 9 1 100 1032
    LSA NLGVSENI 103 8 1 100 1033
    LSA NLGVSENIF 103 9 1 100 1034
    LSA NLGVSEN1FL 103 10 1 100 1035
    LSA NLLIFHINGKI 15 11 1 100 1036
    LSA NVEGRRDI 1707 8 1 100 1037
    LSA NVKNVSQTNF 88 10 1 100 1038
    LSA NVSQTNFKSL 91 10 1 100 1039
    LSA NVSQTNFKSLL 91 11 1 100 1040
    LSA PIVQYDNF 1787 8 1 100 1041
    LSA QISKYEDEI 1756 9 1 100 1042
    LSA QtVDELSEDI 1893 10 1 100 1043
    LSA QTNFKSLL 94 8 1 100 1044
    LSA QTNFKSLLFINL 94 11 1 100 1045
    LSA QVNKEKEKF 1869 9 1 100 1046
    LSA QVNKEKEKF1 1869 10 1 100 1047
    LSA QYDNFQDEENI 1790 11 1 100 1048
    LSA RLEIPAIEL 1655 9 1 100 1049
    LSA RIKASKETL 1187 9 1 100 1050
    LSA SFYFILVNL 8 9 1 100 1051
    LSA SFYFILVNLL 8 10 1 100 1052
    LSA SFYFILVNLLI 8 11 1 100 1053
    LSA SIIEKTNRESI 1694 11 1 100 1054
    LSA SLYDEHIKKY 1855 10 1 100 1055
    LSA TLQEQQSDL 1194 9 1 100 1056
    LSA TLQGQQSDL 157 9 1 100 1057
    LSA TTNVEGRRDI 1705 10 1 100 1058
    LSA TVNISDVNDF 1746 10 1 100 1059
    LSA VLAEDLYGRL 1647 10 1 100 1060
    LSA YFILVNLL 10 8 1 100 1061
    LSA YFILVNLLI 10 9 1 100 1062
    LSA YFILVNLLIF 10 10 1 100 1063
    LSA YIPHQSSL 1672 8 1 100 1064
    LSA YISFYFIL 6 8 1 100 1065
    LSA YISFYFILVNL 6 11 1 100 1066
    LSA YYIPHQSSL 1671 9 1 100 4.3000 1067
    SSP2 ALLACAGL 509 8 10 100 1068
    SSP2 ALLACAGLAY 509 10 10 100 1069
    SSP2 ALLQVRKHL 136 9 9 90 1070
    SSP2 AMKLIQQL 72 8 10 100 1071
    SSP2 AMKLIQQLNL 72 10 10 100 0.0006 1072
    SSP2 ATPYAGEPAPF 526 11 8 80 1073
    SSP2 AVFGIGQI 186 9 10 100 1074
    SSP2 AVPLAMKL 68 8 10 100 1075
    SSP2 AVPLAMKLI 68 9 10 100 1076
    SSP2 AWENVKNVI 219 9 10 100 1077
    SSP2 DLDEPEQF 546 8 10 100 1078
    SSP2 DLDEPEQFRL 546 10 10 100 1079
    SSP2 DVQNNIVDEI 27 10 10 100 1080
    SSP2 EILHEGCTSEL 267 11 8 80 1081
    SSP2 ETLGEEDKDL 538 10 10 100 1082
    SSP2 EVCNDEVDL 41 9 8 80 1083
    SSP2 EVCNDEVDLY 41 10 8 80 1084
    SSP2 EVCNDEVDLYL 41 11 8 80 1085
    SSP2 EVDLYLLM 46 8 8 80 1086
    SSP2 EVEKTASCGVW 237 11 10 100 1087
    SSP2 FLIFFDLF 14 8 10 100 1088
    SSP2 FLIFFDLFL 14 9 10 100 1089
    SSP2 FVVPGAATPY 520 10 8 80 1090
    SSP2 GIAGGLAL 503 8 10 100 1091
    SSP2 GIAGGLALL 503 9 10 100 1092
    SSP2 GIGQGINVAF 189 10 10 100 1093
    SSP2 GINVAFNRF 193 9 10 100 1094
    SSP2 GNVAFNRFL 193 10 10 100 1095
    SSP2 GIPDSIQDSL 163 10 10 100 1096
    SSP2 GLALLACAGL 507 10 10 100 1097
    SSP2 GTRSRXREI 260 9 10 100 1098
    SSP2 GTRSRKREIL 260 10 10 100 1099
    SSP2 GVKIAVFGI 182 9 10 100 1100
    SSP2 HLGNVKYL 3 8 10 100 1101
    SSP2 HLGNVKYLVI 3 10 10 100 1102
    SSP2 ILHEOCTSEL 268 10 8 80 1103
    SSP2 ILTDGIPDSI 159 10 10 100 1104
    SSP2 IVFLWFDL 12 9 10 100 1105
    SSP2 IVFLIFFDLF 12 10 10 100 1106
    SSP2 IVFLIFFDLFL 12 11 10 100 1107
    SSP2 KFVVPGAATPY 519 11 8 80 1108
    SSP2 KIAGGIAGOL 499 10 10 100 1109
    SSP2 KIAVFGIGQGI 184 11 10 100 1110
    SSP2 KLIQQLNL 74 8 10 100 1111
    SSP2 KTASCGVW 240 8 10 100 1112
    SSP2 KTASCGVWDEW 240 11 10 100 1113
    SSP2 KYKIAGGI 497 8 9 90 1114
    SSP2 KYLVIVFL 8 8 10 100 1115
    SSP2 KYLVIVFLI 8 9 10 100 4.6000 1116
    SSP2 KYLVIVFLIF 8 10 10 100 0.0003 1117
    SSP2 KYLVIVFLIFF 8 11 10 100 1118
    SSP2 LIFFDLFL 15 8 10 100 1119
    SSP2 LLACAGLAY 510 9 10 100 1120
    SSP2 LLACAGLAYKF 510 11 10 100 1121
    SSP2 LLMDCSGSI 51 9 10 100 1122
    SSP2 LLQVRKHL 137 8 9 90 1123
    SSP2 LLSTNLPY 121 8 9 90 1124
    SSP2 LMDCSGSI 52 8 10 100 1125
    SSP2 LIDGIPDSI 160 9 10 100 1126
    SSP2 LVTVFLIF 10 8 10 100 1127
    SSP2 LVIVFLIFF 10 9 10 100 1128
    SSP2 LVIVFLIFFDL 10 11 10 100 1129
    SSP2 LVNGRDVQNNI 22 11 10 100 1130
    SSP2 LVVILTDQI 156 9 10 100 1131
    SSP2 LYLLMDCSGSI 49 11 9 90 1132
    SSP2 NIVDEIKY 31 8 10 100 1133
    SSP2 NLPYGRTNL 125 9 8 80 1134
    SSP2 NLYADSAW 213 8 10 100 1135
    SSP2 NVAFNRFL 195 8 10 100 1136
    SSP2 NVKNVIGPF 222 9 10 100 1137
    SSP2 NVKNVIGPFM 222 10 10 100 1138
    SSP2 NVKYLVIVF 6 9 10 100 1139
    SSP2 NVKYLVIIVFL 6 10 10 100 1140
    SSP2 NVKYLVIVFLI 6 11 10 100 1141
    SSP2 NWVNHAVPL 63 9 8 80 1142
    SSP2 NWVNHAVPLAM 63 11 8 80 1143
    SSP2 PLAMKLIQQL 70 10 10 100 1144
    SSP2 PYAQEPAPF 528 9 8 80 0.0370 1145
    SSP2 QFRLPEENEW 552 10 10 100 1146
    SSP2 QLVVILIDGI 155 10 10 100 1147
    SSP2 QVRKHLNDFTI 139 10 9 90 1148
    SSP2 RINRENANQL 147 10 10 100 1149
    SSP2 RLPEENEW 554 8 10 100 1150
    SSP2 SLKESRKL 171 8 9 90 1151
    SSP2 SLLSTNLPY 120 9 9 90 1152
    SSP2 STNLPYGRTNL 123 11 8 80 1153
    SSP2 TLGEEDKDL 539 9 10 100 1154
    SSP2 VFGIGQGI 187 8 10 100 1155
    SSP2 VFLIFFDL 13 8 10 100 1156
    SSP2 VFLIFFDLF 13 9 10 100 1157
    SSP2 VFLIFFDLFL 13 10 10 100 1158
    SSP2 VILTDGIPDSI 158 11 10 100 1159
    SSP2 VIVFLIFF 11 8 10 100 1160
    SSP2 VIVFLIFFDL 11 10 10 100 1161
    SSP2 VIVFLIFFDLF 11 11 10 100 1162
    SSP2 VVILTDGI 157 8 10 100 1163
    SSP2 VVPGAATPY 521 9 8 80 1164
    SSP2 WVNHAVPL 64 8 8 80 1165
    SSP2 WVNHAVPLAM 64 10 8 80 1166
    SSP2 YLLMDCSGSI 50 10 10 100 1167
    SSP2 YLVIVFLI 9 8 10 100 1168
    SSP2 YLVIVFLIF 9 9 10 100 1169
    SSP2 YLVIVFLIFF 9 10 10 100 1170
  • TABLE XI
    Malaria B07 Super Motif Peptides With Binding Information
    No. of Sequence Conservancy
    Protein Sequence Position Amino Acids Frequency (%) B*0702 Seq. Id.
    CSP EPSDKHIEQY 345 10 15 79 171
    CSP EPSDKHIEQYL 345 11 15 79 172
    CSP DPNANPNA 202 8 19 100 173
    CSP DPNANPNV 130 8 19 100 174
    CSP DPNRNVDBIA 327 10 19 100 0.0002 175
    CSP MPNDPNRNV 324 9 19 100 0.0001 176
    CSP NPDPNANPNV 120 10 19 100 0.0001 177
    CSP NPNANPNA 302 8 19 100 0.0001 178
    CSP NPNVDPNA 198 8 19 100 0.0001 179
    CSP QPGDGNPDPNA 115 11 19 100 180
    CSP SPCSVTCGNGI 371 11 19 100 181
    EXP DPADNANPDA 116 10 1 100 0.0002 182
    EXP DPQVTAQDV 136 9 1 100 0.0001 183
    EXP EPLIDVHDL 45 9 1 100 0.0001 184
    EXP EPLIDVHDLI 45 10 1 100 0.0002 185
    EXP EPNADPQV 132 8 1 100 0.0001 186
    EXP EPNADPQVTA 132 10 1 100 0.0002 187
    EXP HPFKIGSSDPA 108 11 1 100 188
    EXP QPQGDDNNL 148 9 1 100 0.0001 189
    EXP QPQGDDNNLV 148 10 1 100 0.0002 190
    LSA KPEQKEDKSA 1728 10 1 100 0.0002 191
    LSA KPIVQYDNF 1786 9 1 100 0.0001 192
    LSA KPNDKSLY 1850 8 1 100 0.0004 193
    LSA LPSENERGY 1663 9 1 100 0,0001 194
    LSA LPSENERGYY 1663 10 1 100 0.0001 195
    LSA LPSENERGYYI 1663 11 1 100 196
    SSP2 EPAPFDETL 532. 9 10 100 0.0001 197
    SSP2 GPFMKAVCV 228 9 10 100 0.0023 198
    SSP2 GFFMKAVCVEV 228 11 10 100 199
    SSP2 HPSDGKCNL 206 9 10 100 0.0220 200
    SSP2 HPSDGKCNLY 206 10 10 100 0.0001 201
    SSP2 HPSDGKCNLYA 206 11 10 100 202
    SSP2 IPDSIQDSL 164 9 10 100 0.0022 203
    SSP2 IPEDSEKEV 367 9 10 100 0.0001 204
    SSP2 LPYGRTNL 126 8 8 80 0.1100 205
    SSP2 NPEDDREENF 382 10 10 100 0.0001 206
    SSP2 QPRPRGDNF 303 9 9 90 0.0160 207
    SSP2 QPRPRGDNFA 303 10 9 90 0.0009 208
    SSP2 QPRPRGDNFAV 303 11 9 90 209
    SSP2 RPRGDNFA 305 8 9 90 0.0110 210
    SSP2 RPRGDNFAV 305 9 9 90 0.4800 211
    SSP2 TPYAGEPA 527 8 8 80 212
    SSP2 TPYAGEPAPF 527 10 8 80 0.0990 213
    SSP2 VPGAATPY 522 8 8 80 214
    SSP2 VPGAAIPYA 522 9 8 80 0.0001 215
    SSP2 VPLAMKLI 69 8 10 100 0.0001 216
    SSP2 VPLAMKLIQQL 69 11 10 100 217
  • TABLE XII
    Malaria B27 Super Motif Peptides
    No. of Sequence Conservancy Seq.
    Protein Sequence Position Amino Acids Frequency (%) Id
    CSP CKMEKCSSVF 408 10 19 100 1218
    CSP DKHIEQYL 348 8 15 79 1219
    CSP DKHIEQYLKKI 348 11 15 79 1220
    CSP EKLRKPKHKKL 103 11 19 100 1221
    CSP GKQENWYSL 57 9 19 100 1222
    CSP KHIEQYLKKI 349 10 15 79 1223
    CSP KKIQNSLSTEW 360 11 15 79 1224
    CSP LKKIQNSL 359 8 15 79 1225
    CSP LKICNSRSL 65 8 19 100 1226
    CSP LRKPKHKKL 105 9 19 100 1227
    CSP RKLAILSVSSF 3 11 19 100 1228
    CSP RKPKHKKL 106 8 19 100 1229
    CSP TRVLNELNY 32 9 19 100 1230
    EXP EKGRHPFKI 104 9 1 100 1231
    EXP KKGSGEPL 40 8 1 100 1232
    EXP KKGSGEPLI 40 9 1 100 1233
    EXP KKNKKGSGEPL 37 11 1 100 1234
    EXP KRKSKYKL 69 8 1 100 1235
    EXP MKILSVFF 1 8 1 100 1236
    EXP MKILSVFFL 1 9 1 100 1237
    EXP MK1LSVFFLAL 1 11 1 100 1238
    EXP NKKGSGEPL 39 9 1 100 1239
    EXP NKKGSGEPLI 39 10 1 100 1240
    EXP NKRKSKYKL 68 9 1 100 1241
    EXP SKYKLATSVL 72 10 1 100 1242
    EXP VHDLISDM 50 8 1 100 1243
    EXP VHDLISDMI 50 9 1 100 1244
    EXP YKLATSVL 74 8 1 100 1245
    EXP YKLATSVLAGL 74 11 1 100 1246
    LSA DKDKELIM 79 8 1 100 1247
    LSA DKQVNKEKEICF 1867 11 1 100 1248
    LSA DKSADIQNHIL 1734 11 1 100 1249
    LSA DKSLYDEHI 1853 9 1 100 1250
    LSA DRLAKEKL 1392 8 1 100 1251
    LSA EHGDVLAEDL 1643 10 1 100 1252
    LSA EHODVIAEDLY 1643 11 1 100 1253
    LSA EKAAKEIL 151 8 1 100 1254
    LSA EKDEIIKSNL 30 10 1 100 1255
    LSA EKEKFIKSL 873 9 1 100 1256
    LSA EKEXFIKSLF 873 10 1 100 1257
    LSA EKFIKSLF 875 8 1 100 1258
    LSA EKFIKSLFHI 875 10 1 100 1259
    LSA EKFIKSLFHIF 875 11 1 100 1260
    LSA EXHECKHVL 53 9 1 100 1261
    LSA EKIKKGKKY 833 9 1 100 1262
    LSA DUCHVLSHNSY 56 11 1 100 1263
    LSA EKLQEQQRDL 618 10 1 100 1264
    LSA EKLQEQQSDL 584 10 1 100 1265
    LSA SCLQGQQSDL 125 10 1 100 1266
    LSA EISNENLDDL 811 9 1 100 1267
    LSA EKTICDNNF 842 8 1 100 1268
    LSA EKTKNNENNKF 67 11 1 100 1269
    LSA EXINRESI 697 8 1 100 1270
    LSA ERKKEHGDVL 639 10 1 100 1271
    LSA ERLAICEKL 613 8 1 100 1272
    LSA ERLANEKL 528 8 1 100 1273
    LSA ERRAKEKL 1579 8 1 100 1274
    LSA ERTKASKETL 1186 10 1 100 1275
    LSA FHIFDGDNEI 1882 10 1 100 1276
    LSA FHTFDGDNEIL 1882 11 1 100 1277
    LSA FHINGKII 19 8 1 100 1278
    LSA FKPIVQYDNF 1785 10 1 100 1279
    LSA FKPNDKSL 1849 8 1 100 1280
    LSA FIQNDKSLY 1849 9 1 100 1281
    LSA FKSLLRNL 97 8 1 100 1282
    LSA GHLEEKKDGSI 1717 11 1 100 1283
    LSA GKLIEHII 121 8 1 100 1284
    LSA GRLEIPAI 1654 8 1 100 1285
    LSA GRLEIPAIEL 1654 10 1 100 1286
    LSA GRRDIHKGHL 1710 10 1 100 1287
    LSA IKNSEKDEI 26 9 1 100 1288
    LSA IKNSEKDEII 26 10 1 100 1289
    LSA IKSLFHIF 1878 8 1 100 1290
    LSA KHEKKHVL 54 8 1 100 1291
    LSA KHILYISF 2 8 1 100 1292
    LSA KHILYISFY 2 9 1 100 1293
    LSA KHILYISFYF 2 10 1 100 1294
    LSA KHILYISFYFI 2 11 1 100 1295
    LSA KHVLSHNSY 58 9 1 100 1296
    LSA KKEHGDVL 1641 8 1 100 1297
    LSA KKHVLSHNSY 57 10 1 100 1298
    LSA KKYEKIKDNNF 1839 11 1 100 1299
    LSA LRNLGVSENI 101 10 1 100 1300
    LSA LRNLGVSENIF 101 11 1 100 1301
    LSA MKHILYISF 1 9 1 100 1302
    LSA MKHILYISFY 1 10 1 100 1303
    LSA MKHILYISFYF 1 11 1 100 1304
    LSA NHTLETVNi 1741 9 1 100 1305
    LSA NKEGKLIEH1 118 10 1 100 1306
    LSA NKEGKLIEHII 118 11 1 100 1307
    LSA NICEKEKSI 1871 8 1 100 1308
    LSA NKEKEKFRSL 1871 11 1 100 1309
    LSA NKFFDKDKEL 75 10 1 100 1310
    LSA NKLNKEGKL 115 9 1 100 1311
    LSA NKLNKEGKU 115 10 1 100 1312
    LSA NRGNSRDSKEI 1683 11 1 100 1313
    LSA QKEDILSADI 1731 9 1 100 1314
    LSA QRDLEQERL 1607 9 1 100 1315
    LSA QRKADTKKNL 1629 10 1 100 1316
    LSA RKADTKKNL 1630 9 1 100 1317
    LSA RKKEHGDVL 1640 9 1 100 1318
    LSA RRDIHKGHL 1711 9 1 100 1319
    LSA SKYEDEISAEY 1758 11 1 100 1320
    LSA SRDSKEISI 1687 9 1 100 1321
    LSA SRDSKEISII 1687 10 1 100 1322
    LSA TKASKEIL 1188 8 1 100 1323
    LSA TICNNENNKF 69 9 1 100 1324
    LSA TKNNENNKFF 69 10 1 100 1325
    LSA VKNVSQTNF 89 9 1 100 1326
    LSA YKELEDLI 1803 8 1 100 1327
    SSP2 CHPSDGKCNL 205 10 10 100 1328
    SSP2 CHPSDGKCNLY 205 11 10 100 1329
    SSP2 DKDLDEPEQF 544 10 10 100 1330
    SSP2 DREENFDI 386 8 10 100 1331
    SSP2 DRGVKIAVF 180 9 9 90 1332
    SSP2 DRGVKIAVEGI 180 11 9 90 1333
    SSP2 DRINRENANQL 146 11 10 100 1334
    SSP2 EKTASCGVW 239 9 10 100 1335
    SSP2 FRLPEENEW 333 9 10 100 1336
    SSP2 GKCNLYADSAW 210 11 10 100 1337
    SSP2 GKGTRSRKREI 258 11 10 100 1338
    SSP2 GRDVQNNI 25 8 10 100 1339
    SSP2 GRNNENRSY 458 9 10 100 1340
    SSP2 KHDNQNNL 400 8 10 100 1341
    SSP2 LHEGCTSEL 269 9 8 80 1342
    SSP2 MKLIQQLNL 73 9 10 100 1343
    SSP2 NHAVPLAM 66 8 8 80 1344
    SSP2 NHAVPLAMKL 66 10 8 80 1345
    SSP2 NHAVPLAMKLI 66 11 8 80 1346
    SSP2 NHLGNVKY 2 8 10 100 1347
    SSP2 NHLGNVKYL 2 9 10 100 1348
    SSP2 NHLGNVKYLVI 2 11 10 100 1349
    SSP2 NKEKALII 110 8 9 90 1350
    SSP2 NKEKALIII 110 9 9 90 1351
    SSP2 NKHDNQNNL 399 9 10 100 1352
    SSP2 NKYKIAGGI 496 9 9 90 1353
    SSP2 NRENANQL 149 8 10 100 1354
    SSP2 NRENANQLVVI 149 11 10 100 1355
    SSP2 PHGRNNENRSY 456 11 10 100 1356
    SSP2 PRPRGDNF 304 8 9 90 1357
    SSP2 RHNWVNHAVPL 61 11 8 80 1358
    SSP2 RKHLNDRI 141 8 10 100 1359
    SSP2 SKNKEKAL 108 8 10 100 1360
    SSP2 SKNKSCALI 108 9 9 90 1361
    SSP2 SKNKEKALII 108 10 9 90 1362
    SSP2 SKNKEKALIII 108 11 9 90 1363
    SSP2 TRSRKREI 261 8 10 100 1364
    SSP2 TRSRKREIL 261 9 10 100 1365
    SSP2 VKIAVFGI 183 8 10 100 1366
    SSP2 VKNVIGPF 223 8 10 100 1367
    SSP2 VKNVIGPFM 223 9 10 100 1368
    SSP2 VKYLVIVF 7 8 10 100 1369
    SSP2 VKYLVIVFL 7 9 10 100 1370
    SSP2 VKYLVIVFLI 7 10 10 100 1371
    SSP2 VKYLVIVFLIF 7 11 10 100 1372
    SSP2 VRKHLNDRI 140 9 10 100 1373
    SSP2 YKIAGGIAGGL 498 11 10 100 1374
  • TABLE XIII
    Malaria B58 Super Motif Peptides
    No. of Sequence Conservancy Seq.
    Protein Sequence Position Amino Acids Frequency (%) Id
    CSP CSSVFNVV 413 8 19 100 1375
    CSP CSVTCGNGI 373 9 19 100 1376
    CSP CSVTCGNGIQV 373 11 19 100 1377
    CSP EALFQEYQCY 17 10 19 100 1378
    CSP GSSSNTRV 27 8 19 100 1379
    CSP GSSSNTRVL 27 9 19 100 1380
    CSP LAILSVSSF 5 9 19 100 1381
    CSP LAILSVSSFL 5 10 19 100 1382
    CSP LAILSVSSFLF 5 11 19 100 1383
    CSP LSTEWSPCSV 366 10 18 95 1384
    CSP LSVSSFLF 8 8 19 100 1385
    CSP LSVSSFLFV 8 9 19 100 1386
    CSP NAGINLYNEL 42 10 18 95 1387
    CSP NANANNAV 335 8 16 84 1388
    CSP NSSIGLIM 421 8 19 100 1389
    CSP NSSIGLIMV 421 9 19 100 1390
    CSP NSSIGLIMVL 421 10 19 100 1391
    CSP NTRVLNEL 31 8 19 100 1392
    CSP NTRVLNELNY 31 10 19 100 1393
    CSP PSDKHIEQY 346 9 15 79 1394
    CSP PSDKHIEQYL 346 10 15 100 1395
    CSP SSFLFVEAL 11 9 19 100 1396
    CSP SSFLFVEALF 11 10 19 100 1397
    CSP SSIGLIMV 422 8 19 100 1398
    CSP SSIGLIMVL 422 9 19 100 1399
    CSP SSIGLIMVLSF 422 11 19 100 1400
    CSP SSNTRVLNEL 29 10 19 100 1401
    CSP SSSNTRVL 28 8 19 100 1402
    CSP SSSNTRVLNEL 28 11 19 100 1403
    CSP SSVFNVVNSSI 414 11 19 100 1404
    CSP STEWSPCSV 367 9 19 100 1405
    CSP VSSFLFVEAL 10 10 19 100 1406
    CSP VSSFLFVEALF 10 11 19 100 1407
    CSP VTCGNGIQV 375 9 19 100 1408
    CSP VTCGNGIQVRI 375 11 19 100 1409
    CSP YSLKKNSRSL 63 10 19 100 1410
    EXP ATSVLAGL 77 8 1 100 1411
    EXP ATSVLAGLL 77 9 1 100 1412
    EXP GSGEPLIDV 42 9 1 100 1413
    EXP ISDMIKKEEEL 54 11 1 100 1414
    EXP KSKYKLATSV 71 10 1 100 1415
    EXP KSKYKLATSVL 71 11 1 100 1416
    EXP KTNKGTGSGV 24 10 1 100 1417
    EXP LAGLLGNV 81 8 1 100 1418
    EXP LAGLLGNVSTV 81 11 1 100 1419
    EXP LALFFIIF 9 8 1 100 1420
    EXP LATSVLAGL 76 9 1 100 1421
    EXP LATSVLAGLL 76 10 1 100 1422
    EXP LSVFFLAL 4 8 1 100 1423
    EXP LSVFFLALF 4 9 1 100 1424
    EXP LSVFFLALFF 4 10 1 100 1425
    EXP LSVFFLALFFI 4 11 1 100 1426
    EXP NADPQVTAQDV 134 11 1 100 1427
    EXP NTEKGRHPF 102 9 1 100 1428
    EXP NTEKGRHPFKI 102 11 1 100 1429
    EXP STVLLGGV 89 8 1 100 1430
    EXP STVLIGGVGL 89 10 1 100 1431
    EXP STVLLGGVGLV 89 11 1 100 1432
    EXP TSVLAGLL 78 8 1 100 1433
    EXP TSVLAGLLGNV 78 11 1 100 1434
    EXP VSTVLLGGV 88 9 1 100 1435
    EXP VSTVLLGGVGL 88 11 1 100 1436
    LSA DSKEISII 1689 8 1 100 1437
    LSA ETLQEQQSDL 1193 10 1 100 1438
    LSA ETLQGQQSDL 156 10 1 100 1439
    LSA ETVNISDV 1745 8 1 100 1440
    LSA EIVNISDVNDF 1745 11 1 100 1441
    LSA GSSNSRNRI 42 9 1 100 1442
    LSA HTLETVNI 1742 8 1 100 1443
    LSA HTLETVNISDV 1742 11 1 100 1444
    LSA ISAEYDDSL 1764 9 1 100 1445
    LSA ISAEYDDSLI 1764 10 1 100 1446
    LSA ISDVNDFQI 1749 9 1 100 1447
    LSA ISFYFILV 7 8 1 100 1448
    LSA ISFYFILVNL 7 10 1 100 1449
    LSA ISFYFILVNLL 7 11 1 100 1450
    LSA ISKYEDEI 1757 8 1 100 1451
    LSA ITKYFMKL 1902 8 1 100 1452
    LSA ITTNVEGRRDI 1704 11 1 100 1453
    LSA KADTKKNL 1631 8 1 100 1454
    LSA KSADIQNHTL 1735 10 1 100 1455
    LSA KSLLRNLGV 98 9 1 100 1456
    LSA KSLYDEHI 1854 8 1 100 1457
    LSA KSLYDEHIKKY 1854 11 1 100 1458
    LSA KSSEELSEEKI 1825 11 1 100 1459
    LSA KTKNNENNKF 68 10 1 100 1460
    LSA KTKNNENNKFF 68 11 1 100 1461
    LSA KTNRESITTNV 1698 11 1 100 1462
    LSA LAEDLYGRL 1648 9 1 100 1463
    LSA LAEDLYGRLEI 1648 11 1 100 1464
    LSA LSEDITKY 1898 8 1 100 1465
    LSA LSEDITKYF 1898 9 1 100 1466
    LSA LSEDITKYFM 1898 10 1 100 1467
    LSA LTMSNVKNV 84 9 1 100 1468
    LSA NSEKDEII 28 8 1 100 1469
    LSA NSRDSKEI 1686 8 1 100 1470
    LSA NSRDSKEISI 1686 10 1 100 1471
    LSA NSRDSKEISII 1686 11 1 100 1472
    LSA PSENERGY 1664 8 1 100 1473
    LSA PSENERGYY 1664 9 1 100 1474
    LSA PSENERGYYI 1664 10 1 100 1475
    LSA QSDLEQDRL 1386 9 1 100 1476
    LSA QSDLEQERL 1590 9 1 100 1477
    LSA QSDSEQERL 519 9 1 100 1478
    LSA QTNFKSLL 94 8 1 100 1479
    LSA QTNFKSLLRNL 94 11 1 100 1480
    LSA RSGSSNSRNRI 40 11 1 100 1481
    LSA RTKASKETL 1187 9 1 100 1482
    LSA SADIQNHTL 1736 9 1 100 1483
    LSA SAEYDDSL 1765 8 1 100 1484
    LSA SAEYDDSLI 1765 9 1 100 1485
    LSA SSEELSEEKI 1826 10 1 100 1486
    LSA SSNSRNRI 43 8 1 100 487
    LSA TTNVEGRRDI 1705 10 1 100 488
    LSA VSQTNFKSL 92 9 1 100 489
    LSA VSQTNFKSLL 92 10 1 100 490
    SSP2 ASCGVWDEW 242 9 10 100 491
    SSP2 ASKNKEKAL 107 9 10 100 492
    SSP2 ASKNKEKALI 107 10 9 90 493
    SSP2 ASKNKEKALII 107 11 9 90 494
    SSP2 AIPYAGEPAPF 526 11 8 80 495
    SSP2 CAGLAYKF 513 8 10 100 496
    SSP2 CAGLAYKFV 513 9 10 100 497
    SSP2 CAGLAYKFVV 513 10 10 100 498
    SSP2 CSGSIRRHNW 55 10 10 100 499
    SSP2 CSGSIRRHNWV 55 11 10 100 500
    SSP2 DALLQVRKHL 135 10 9 90 501
    SSP2 DASKNKEKAL 106 10 10 100 502
    SSP2 DASKNKEKALI 106 11 9 90 503
    SSP2 DSAWENVKNV 217 10 10 100 504
    SSP2 DSAWENVKNVI 217 11 10 100 505
    SSP2 DSEKEVPSDV 370 10 10 100 506
    SSP2 DSLKESRKL 170 9 9 90 507
    SSP2 ETLGEEDKDL 538 10 10 100 508
    SSP2 GSIRRHNW 57 8 10 100 509
    SSP2 GSIRRHNWV 57 9 10 100 510
    SSP2 GTRSRKREI 260 9 10 100 511
    SSP2 GTRSRKREIL 260 10 10 100 512
    SSP2 HAVPLAMKL 67 9 10 100 513
    SSP2 HAVPLAMKLI 67 10 10 100 514
    SSP2 IAGGIAGGL 500 9 10 100 515
    SSP2 IAGGIAGGLAL 500 11 10 100 516
    SSP2 IAGGLALL 504 8 10 100 517
    SSP2 IAVFGIGQGI 185 10 10 100 518
    SSP2 KTASCGVW 240 8 10 100 519
    SSP2 KTASCGVWDEW 240 11 10 100 520
    SSP2 LACAGLAY 511 8 10 100 521
    SSP2 LACAGLAYKF 511 10 10 100 522
    SSP2 LACAGLAYKFV 511 11 10 100 523
    SSP2 LALLACAGL 508 9 10 100 524
    SSP2 LALLACAGLAY 508 11 10 100 525
    SSP2 LAMKLIQQL 71 9 10 100 526
    SSP2 LAMKLIQQLNL 71 11 10 100 527
    SSP2 LTDGIPDSI 160 9 10 100 528
    SSP2 NANQLVVI 152 8 10 100 529
    SSP2 NANQLVVIL 152 9 10 100 530
    SSP2 PAPFDETL 533 8 10 100 531
    SSP2 PSCGKCNL 207 8 10 100 532
    SSP2 PSDGKCNLY 207 9 10 100 533
    SSP2 QSQDNNGNRHV 436 11 10 100 534
    SSP2 RSRKREIL 262 8 10 100 535
    SSP2 SAWENVKNV 218 9 10 100 536
    SSP2 SAWENVKNVI 218 10 10 100 537
    SSP2 STNLPYGRTNL 123 11 8 80 538
    SSP2 TASCGVWDEW 241 10 10 100 539
    SSP2 VAFNRFLV 196 8 10 100 540
    SSP2 YADSAWENV 215 9 10 100 541
    SSP2 YAGEPAPF 529 8 8 80 1542
  • TABLE XIV
    Malaria B62 Super Motif Peptides
    No. of Sequence Conservancy Seq.
    Protein Sequence Position Amino Acids Frequency (%) Id.
    CSP AILSVSSF 6 8 9 100 1543
    CSP AILSVSSFLF 6 10 9 100 1544
    CSP AILSVSSFLFV 6 11 9 100 1545
    CSP ALFQEYQCY 18 9 9 100 1546
    CSP DIEKKICKM 402 9 9 100 1547
    CSP DPNANPNV 130 8 9 100 1548
    CSP ELNYDNAGI 37 9 8 95 1549
    CSP EMNYYGKQENW 52 11 9 100 1550
    CSP EPSDKHIEQY 345 10 5 79 1551
    CSP FLFVEALF 13 8 9 100 1552
    CSP FLFVEALFQEY 13 11 9 100 1553
    CSP FVEALFQEY 1S 9 9 100 1554
    CSP GINLYNELEM 44 10 8 95 1555
    CSP GLIMVLSF 425 8 9 100 1556
    CSP GLIMVLSFLF 425 10 9 100 1557
    CSP HIEQYLKKI 350 9 5 79 1558
    CSP ILSVSSFLF 7 9 9 100 1559
    CSP ILSVSSFLFV 7 10 9 100 1560
    CSP IMVLSFLF 427 8 9 100 1561
    CSP IQNSLSTEW 362 9 5 79 1562
    CSP KICKMEKCSSV 406 11 9 100 1563
    CSP KIQNSLSTEW 361 10 5 79 1564
    CSP KLAILSVSSF 4 10 9 100 1565
    CSP KMEKCSSV 409 8 9 100 1566
    CSP KMEKCSSVF 409 9 9 100 1567
    CSP KMEKCSSVFNV 409 11 9 100 1568
    CSP LIMVLSFLF 426 9 9 100 1569
    CSP MMRKLAILSV 1 10 9 100 1570
    CSP MPNDPNRNV 324 9 9 100 1571
    CSP NLYNELEM 46 8 9 100 1572
    CSP NLYNELEMNY 46 10 9 100 1573
    CSP NLYNELEMNYY 46 11 9 100 1574
    CSP NMPNDPNRNV 323 10 9 100 1575
    CSP NPDPNANPNV 120 10 9 100 1576
    CSP NQGNGQGHNM 315 10 9 100 1577
    CSP NVDPNANPNV 128 10 9 100 1578
    CSP NVVNSSIGLI 418 10 9 100 1579
    CSP NVVNSSIGLIM 418 11 9 100 1580
    CSP RVLNELNY 33 8 9 100 1581
    CSP SIGLIMVLSF 423 10 9 100 1582
    CSP SLSTEWSPCSV 365 11 8 95 1583
    CSP SPCSVTCGNGI 371 11 9 100 1584
    CSP SVFNVVNSSI 415 10 9 100 1585
    CSP SVSSFLFV 9 8 9 100 1586
    CSP SVTCGNGI 374 8 9 100 1587
    CSP SVTCGNGIQV 374 10 9 100 1588
    CSP VVNSSIGLI 419 9 9 100 1589
    CSP VVNSSIGLIM 419 10 9 100 1590
    CSP VVNSSIGLIMV 419 11 9 100 1591
    EXP DMIKKEEELV 56 10 1 100 1592
    EXP DPQVTAQDV 136 9 1 100 1593
    EXP DVHDLISDM 49 9 1 100 1594
    EXP DVHDLISDMI 49 10 1 100 1595
    EXP EPLIDVHDLI 45 10 1 100 1596
    EXP EPNADPQV 132 8 1 100 1597
    EXP EQPQGDDNNLV 147 11 1 100 1598
    EXP EVNKRKSKY 66 9 1 100 1599
    EXP FLALFFII 8 8 1 100 1600
    EXP FLALFFIIF 8 9 1 100 1601
    EXP GLLGNVSTV 83 9 1 100 1602
    EXP ILSVFFLALF 3 10 1 100 1603
    EXP ILSVFFLALFF 3 11 1 100 1604
    EXP KILSVFFLALF 2 11 1 100 1605
    EXP LIDVHDLI 47 8 1 100 1606
    EXP LIDVHDLISDM 47 11 1 100 1607
    EXP LLGGVGLV 92 8 1 100 1608
    EXP LLGGVGLVLY 92 10 1 100 1609
    EXP LLGNVSTV 84 8 1 100 1610
    EXP LVEVNKRKSKY 64 11 1 100 1611
    EXP MIKKEEELV 57 9 1 100 1612
    EXP MIKKEEELVEV 57 11 1 100 1613
    EXP NVSTVLLGGV 87 10 1 100 1614
    EXP PLIDVHDLI 46 9 1 100 1615
    EXP PQGDDNNLV 149 9 1 100 1616
    EXP PQVTAQDV 137 8 1 100 1617
    EXP QPQGDDNNLV 148 10 1 100 1618
    EXP SVFFLALF 5 8 1 100 1619
    EXP SVFFLALFF 5 9 1 100 1620
    EXP SVFFLALFFI 5 10 1 100 1621
    EXP SVFFLALFFII 5 11 1 100 1622
    EXP SVLAGLLGNV 79 10 1 100 1623
    EXP TVLLGGVGLV 90 10 1 100 1624
    EXP VLAGLLGNV 80 9 1 100 1625
    EXP VLLGGVGLV 91 9 1 100 1626
    EXP VLLGGVGLVLY 91 11 1 100 1627
    LSA DIQNHTLETV 1738 10 1 100 1628
    LSA DLDEFKPI 1781 8 1 100 1629
    LSA DLDEFKPIV 1781 9 1 100 1630
    LSA DLDEFKPIVQY 1781 11 1 100 1631
    LSA DLYGRLEI 1651 8 1 100 1632
    LSA DLYGRLEIPAI 1651 11 1 100 1633
    LSA DVLAEDLY 1646 8 1 100 1634
    LSA DVNDFQISKY 1751 10 1 100 1635
    LSA EISAEYDDSLI 1763 11 1 100 1636
    LSA ELPSENERGY 1662 10 1 100 1637
    LSA ELPSENERGYY 1662 11 1 100 1638
    LSA ELSEDITKY 1897 9 1 100 1639
    LSA ELSEDITKYF 1897 10 1 100 1640
    LSA ELSEDRKYFM 1897 11 1 100 1641
    LSA ELTMSNVKNV 83 10 1 100 1642
    LSA EQKEDKSADI 1730 10 1 100 1643
    LSA FIKSLFHI 1877 8 1 100 1644
    LSA FIKSLFHIF 1877 9 1 100 1645
    LSA FILVNLLI 11 8 1 100 1646
    LSA FILVNLLIF 11 9 1 100 1647
    LSA FILVNLLIFFII 11 11 1 100 1648
    LSA FQDEENIGI 1794 9 1 100 1649
    LSA FQDEENIGIY 1794 10 1 100 1650
    LSA FQISKYEDE1 1755 10 1 100 1651
    LSA GIYKELEDLI 1801 10 1 100 1652
    LSA HIFDGDNEI 1883 9 1 100 1653
    LSA HIKKYKNDKQV 1860 11 1 100 1654
    LSA HILYISFY 3 8 1 100 1655
    LSA HILYISFYF 3 9 1 100 1656
    LSA HILYISFYFI 3 10 1 100 1657
    LSA HLEEKKDGSI 1718 10 1 100 1658
    LSA HVLSHNSY 59 8 1 100 1659
    LSA IIEKTNRESI 1695 10 1 100 1660
    LSA IIKNSEKDEI 25 10 1 100 1661
    LSA IIKNSEKDEII 25 11 1 100 1662
    LSA IINDDDDKKKY 127 11 1 100 1663
    LSA RILVNLLIF 12 8 1 100 1664
    LSA ILVNLLIFHI 12 10 1 100 1665
    LSA ILYYISFYF 4 8 1 100 1666
    LSA ILYISFYFI 4 9 1 100 1667
    LSA ILYISFYFILV 4 11 1 100 1668
    LSA IQNHTLETV 1739 9 1 100 1669
    LSA IQNHTLETVNI 1739 11 1 100 1670
    LSA IVDELSEDI 1894 9 1 100 1671
    LSA KIIKNSEKDEI 24 11 1 100 1672
    LSA KIKKGKKY 1834 8 1 100 1673
    LSA KLNKEGKLI 116 9 1 100 1674
    LSA KPIVQYDNF 1786 9 1 100 1675
    LSA KPNDKSLY 1850 8 1 100 1676
    LSA KQVNKEKEKF 1868 10 1 100 1677
    LSA KQVNKEKEKFI 1868 11 1 100 1678
    LSA LIFHINGKI 17 9 1 100 1679
    LSA LIFHINGKII 17 10 1 100 1680
    LSA LLIFHINGKI 16 10 1 100 1681
    LSA LLIFHINGKII 16 11 1 300 1682
    LSA LLRNLGVSENI 100 11 1 100 1683
    LSA LPSENERGY 1663 9 1 100 1684
    LSA LPSENERGYY 1663 10 1 100 1685
    LSA LPSENERGYYI 1663 11 1 100 1686
    LSA LQIVDELSEDI 1892 11 1 100 1687
    LSA LVNLLIFHI 13 9 1 100 1688
    LSA NISDVNDF 1748 8 1 100 1689
    LSA NISDVNDFQI 1748 10 1 100 1690
    LSA NLDDLDEGI 1815 9 1 100 1691
    LSA NLERKKEHGDV 1637 11 1 100 1692
    LSA NLGVSENI 103 8 1 100 1693
    LSA NLGVSENIF 103 9 1 100 1694
    LSA NLLIFHTNGKI 15 11 1 100 1695
    LSA NVEGRRDI 707 8 1 100 1696
    LSA NVKNVSQTNF 88 10 1 100 1697
    LSA PIVQYDNF 787 8 1 100 1698
    LSA QISKYEDEI 756 9 1 100 1699
    LSA QIVDELSEDI 893 10 1 100 1700
    LSA QVNKEKEKF 869 9 1 100 1701
    LSA QVINIKEKEKFI 869 10 1 100 1702
    LSA SIIEKTNRESI 694 11 1 100 1703
    LSA SLLRNLGV 99 8 1 100 1704
    LSA SLYDEHIKKY 855 10 1 100 1705
    LSA TLETVNISDV 743 10 1 100 1706
    LSA TMSNVKNV 85 8 1 100 1707
    LSA TVNISDVNDF 746 10 1 100 1708
    LSA YISFYFILV 6 9 1 100 1709
    SSP2 ALLACAGLAY 509 10 10 100 1710
    SSP2 AVFGIGQGI 186 9 10 100 1711
    SSP2 AVFGIGQGINV 186 11 10 100 1712
    SSP2 AVPLAMKLI 68 9 10 100 1713
    SSP2 DLDEPDQF 546 8 10 100 1714
    SSP2 DLFLVNGRDV 19 10 10 100 1715
    SSP2 DQPRPRGDNF 302 10 9 90 1716
    SSP2 DVQNNIVDEI 27 10 10 100 1717
    SSP2 EIKYREEV 35 8 9 90 1718
    SSP2 EQFRLPEENEW 551 11 10 100 1719
    SSP2 EVCNDEVDLY 41 10 8 80 1720
    SSP2 EVDLYLLM 46 8 8 80 1721
    SSP2 EVEKTASCGV 237 10 10 100 1722
    SSP2 EVEXTASCGVW 237 11 10 100 1723
    SSP2 FLIFFDLF 14 8 10 100 1724
    SSP2 FLIFFDLFLV 14 10 10 100 1725
    SSP2 FLVNGRDV 21 8 10 100 1726
    SSP2 FMKAVCVEV 230 9 10 100 1727
    SSP2 FVVPGAATPY 520 10 8 80 1728
    SSP2 GIGQGINV 189 8 10 100 1729
    SSP2 GIGQGINVAF 189 10 10 100 1730
    SSP2 GINVAFNRF 193 9 10 100 1731
    SSP2 GINVAFNRFLV 193 11 10 100 1732
    SSP2 GLAYKFVV 515 8 10 100 1733
    SSP2 GPFMKAVCV 228 9 10 100 1734
    SSP2 GPFMKAVCVEV 228 11 10 100 1735
    SSP2 GQGINVAF 191 8 10 100 1736
    SSP2 GQGINVAFNRF 191 11 10 100 1737
    SSP2 GVKIAVFGI 182 9 10 100 1738
    SSP2 GVWDEWSPCSV 245 11 10 100 1739
    SSP2 HLGNVKYLV 3 9 10 100 1740
    SSP2 HLGNVKYLVI 3 10 10 100 1741
    SSP2 HLGNVKYLVIV 3 11 10 100 1742
    SSP2 HPSDGKCNLY 206 10 10 100 1743
    SSP2 ILTDGIPDSI 159 10 10 100 1744
    SSP2 IPEDSEKEV 367 9 10 100 1745
    SSP2 IVDEIKYREEV 32 11 9 90 1746
    SSP2 IVFLIFFDLF 12 10 10 100 1747
    SSP2 KIAVFGIGQGI 184 11 10 100 1748
    SSP2 LIFFDLFLV 15 9 10 100 1749
    SSP2 LLACAGLAY 510 9 10 100 1750
    SSP2 LLACAGLAYKF 510 11 10 100 1751
    SSP2 LLMDCSGSI 51 9 10 100 1752
    SSP2 LLSTNLPY 121 8 9 90 1753
    SSP2 LMDCSGSI 52 8 10 100 1754
    SSP2 LQVRKHLNDRI 138 11 9 90 1755
    SSP2 LVIVFLIF 10 8 10 100 1756
    SSP2 LVIVFLIFF 10 9 10 100 1757
    SSP2 LVNGRDVQNNI 22 11 10 100 1758
    SSP2 LVVILTDGI 156 9 10 100 1759
    SSP2 NIPEDSEKEV 366 10 10 100 1760
    SSP2 NIVDEIKY 31 8 10 100 1761
    SSP2 NLYADSAW 213 8 10 100 1762
    SSP2 NLYADSAWENV 213 11 10 100 1763
    SSP2 NPEDDREENF 382 10 10 100 1764
    SSP2 NQLVVILTDGI 154 11 10 100 1765
    SSP2 NVARAFIN 195 9 10 100 1766
    SSP2 NVIGPFMKAV 225 10 10 100 1767
    SSP2 NVKNVIGPF 222 9 10 100 1768
    SSP2 NVKNVIGPFM 222 10 10 100 1769
    SSP2 NVKYLVIV 6 8 10 100 1770
    SSP2 NVKYLVIVF 6 9 10 100 1771
    SSP2 NVKYLVIVFLI 6 11 10 100 1772
    SSP2 QLVVILTDGI 155 10 10 100 1773
    SSP2 QPRPRGDNF 303 9 9 90 1774
    SSP2 QPRPRGDNFAV 303 11 9 90 1775
    SSP2 QVRKHLNDR1 139 10 9 90 1776
    SSP2 RINRENANQLV 147 11 10 100 1777
    SSP2 RLPEENEW 554 8 10 100 1778
    SSP2 RPRGDNFAV 305 9 9 90 1779
    SSP2 SIRRHNWV 58 8 10 100 1780
    SSP2 SLLSTNLPY 120 9 9 90 1781
    SSP2 SQDNNGNRHV 437 10 10 100 1782
    SSP2 TPYAGEPAPF 527 10 8 80 1783
    SSP2 VIGPFMKAV 226 9 10 100 1784
    SSP2 VIGPFMKAVCV 226 11 10 100 1785
    SSP2 VILIDGIPDSI 158 11 10 100 1786
    SSP2 VIVFLIFF 11 8 10 100 1787
    SSP2 VIVFLIFFDLF 11 11 10 100 1788
    SSP2 VPGAATPY 522 8 8 80 1789
    SSP2 VPLAMKLI 69 8 10 100 1790
    SSP2 VQNNIVDEI 28 9 10 100 1791
    SSP2 VQNNIVDE1KY 28 11 10 100 1792
    SSP2 VVILTDM 157 8 10 100 1793
    SSP2 VVPGAATPY 521 9 8 80 1794
    SSP2 WVNHAVPLAM 64 10 8 80 1795
    SSP2 YLLMDCSGS1 50 10 10 100 1796
    SSP2 YLVIVFLI 9 8 10 100 1797
    SSP2 YLV1VFLIF 9 9 10 100 1798
    SSP2 YLVIVFLIFF 9 10 10 100 1799
  • TABLE XV
    Malaria A01 Motif Peptides With Binding Information
    No. of Sequence Conservancy Seq.
    Protein Sequence Pos Amino Acids Freq. (%) A*0101 Id.
    CSP DNAGINLY 41 8 19 100 1800
    CSP EPSDKHIEQY 345 10 15 79 1801
    CSP FVEALFQEY 15 9 19 100 3.4000 1802
    CSP NTRVLNELNY 31 10 19 100 0.0096 1803
    CSP NYDNAGINLY 39 10 18 95 0.0012 1804
    CSP PSDKHIEQY 346 9 15 79 1805
    CSP VEALFQEY 16 8 19 100 1806
    CSP VEALFQEYQCY 16 11 19 100 1807
    CSP YNELEMNY 48 8 19 100 1808
    CSP YNELEMNYY 48 9 19 100 1809
    EXP LVEVNKRKSKY 64 11 1 100 1810
    LSA DDDDKKKY 130 8 1 100 1811
    LSA DEENIGIY 1796 8 1 100 1812
    LSA DLDEFKPIVQY 1781 11 1 100 1813
    LSA EDEISAEY 1761 8 1 100 1814
    LSA ELSEDIAY 1897 9 1 100 1815
    LSA FQDEENIGIY 1794 10 1 100 1.1000 1816
    LSA HGDVLAEDLY 1644 10 1 100 0.0012 1817
    LSA INDDDDKKKY 128 10 1 100 1818
    LSA KSLYDEHIKKY 1854 11 1 100 1819
    LSA KYEDEISAEY 1759 10 1 100 0.0011 1820
    LSA LDEFKPIVQY 1782 10 1 100 1821
    LSA LPSENERGY 1663 9 1 100 0.6700 1822
    LSA LPSENERGYY 1663 10 1 100 0.0011 1823
    LSA LSEDIMY 1898 8 1 100 1824
    LSA LYDEHIKKY 1856 9 1 100 0.0011 1825
    LSA NDDDDKKKY 129 9 1 100 1826
    LSA PSENERGY 1664 8 1 100 1827
    LSA PSENERGYY 1664 9 1 100 0.0790 1828
    LSA QDEENIGIY 1795 9 1 100 1829
    LSA SEEKIKKGKKY 1831 11 1 100 1830
    LSA VDELSEDITKY 1895 11 1 100 1831
    LSA VNDFQISKY 1752 9 1 100 1832
    LSA YDEHGCY 1857 8 1 100 1833
    LSA YEDEISAEY 1760 9 1 100 0.0012 1834
    SSP2 CNDEVDLY 43 8 8 80 1835
    SSP2 HPSDGKCNLY 206 10 10 100 0.0260 1836
    SSP2 LLACAGLAY 510 9 10 100 1837
    SSP2 LLSTNLPY 121 8 9 90 1838
    SSP2 PSDGKCNLY 207 9 10 100 0.5400 1839
  • TABLE XVI
    Malaria A3 Motif Peptides With Binding Information
    No. of Sequence Conservancy SEQ.
    Protein Sequence Position Amino Acids Frequency (%) A*0301 Id.
    CSP NMPNDPNR 323 8 19 100 1897
    CSP NIRVLNELNY 31 10 19 100 0.0005 1898
    CSP NVDENANA 331 8 19 100 1899
    CSP NVDENANANNA 331 11 16 84 1900
    CSP NVDPNANPNA 200 10 19 100 1901
    CSP PGDGNPDPNA 116 10 19 100 1902
    CSP PSDKHIEQY 346 9 15 79 1903
    CSP PSDICHIEQYLK 346 11 15 79 1904
    CSP QCYGSSSNTR 24 10 19 100 1905
    CSP QGHNMPNDPNR 320 11 19 100 1906
    CSP QVRIKPGSA 382 9 19 100 1907
    CSP RDGNNEDNEK 95 10 19 100 0.0005 1908
    CSP RVLNEHNY 33 8 19 100 1909
    CSP RVLNELNYDNA 33 11 19 100 1910
    CSP SDKHIEQY 347 8 15 79 1911
    CSP SDKHIEQYLK 347 10 15 79 1912
    CSP SDKHIEQYLKK 347 11 15 79 1913
    CSP SFLFVEALF 12 9 19 100 1914
    CSP StGLIMVLSF 423 10 19 100 1915
    CSP SSFLFVEA 11 8 19 100 1916
    CSP SSFLFVEALF 11 10 19 100 1917
    CSP SSIGLIMVLSF 422 11 19 100 1918
    CSP SVSSFLFVEA 9 10 19 100 1919
    CSP SVTCGNGIQVR 374 11 19 100 1920
    CSP TCGNGIQVR 376 9 19 100 1921
    CSP TCGNGIQVRIK 376 11 19 100 1922
    CSP VDENANANNA 332 10 16 84 1923
    CSP VDPNANPNA 201 9 19 100 1914
    CSP VLNEINYDNA 34 10 19 100 1925
    CSP VSSFLFVEA 10 9 19 100 1926
    CSP VSSFLFVEALF 10 11 19 100 1927
    CSP VTCGNGIQVR 375 10 19 100 0.0005 1928
    CSP YDNAGINLY 40 9 18 95 1929
    CSP YGKQENWY 56 8 19 100 1930
    CSP YGKQENWYSLK 56 11 19 100 1931
    CSP YGSSSNTR 26 8 19 100 1932
    CSP YSLKKNSR 63 8 19 100 1933
    EXP ADNANPDA 118 8 1 100 1934
    EXP ADSESNGEPNA 125 11 1 100 1935
    EXP ALFFITFNIC 10 9 1 100 1.1000 1936
    EXP DDNNLVSGPEH 152 11 1 100 1937
    EXP DLISDMIK 52 8 1 100 1938
    EXP DLISDMIKK 52 9 1 100 0.0001 1939
    EXP DSESNGEPNA 126 10 1 100 1940
    EXP DVHDLISDMIK 49 11 1 100 1941
    EXP ELYEVNKR 63 8 1 100 1942
    EXP ELVEVNKRK 63 9 1 100 0.0001 1943
    EXP ELVEVNKRKSK 63 11 1 100 1944
    EXP ESLAEKTNIC 19 9 1 100 0.0001 1945
    EXP ESNGEPNA 128 8 1 100 1946
    EXP EVNKRKSK 66 8 1 100 1947
    EXP EVNKRKSKY 66 9 1 100 0.0001 1948
    EXP EVNKRKSKYK 66 10 1 100 0.0005 1949
    EXP FFIIFNKESLA 12 11 1 100 1950
    EXP FFLALFFIIF 7 10 1 100 1951
    EXP FFIFNKESLA 13 10 1 100 1952
    EXP FLALFFIIF 8 9 1 100 1953
    EXP FLALFFIIFNK 8 11 1 100 1954
    EXP GGVGLVLY 94 8 1 100 1955
    EXP GLVLYNTEK 97 9 1 100 0.0069 1956
    EXP GLVLYNTEKGR 97 11 1 100 1957
    EXP GSGEPLIDVH 42 10 1 100 0.0005 1958
    EXP GSGVSSKK 30 8 1 100 1959
    EXP GSGVSSKKK 30 9 1 100 0.0003 1960
    EXP GSGVSSKKKNK 30 11 1 100 1961
    EXP GSSDPADNA 113 9 1 100 1962
    EXP GTGSGVSSK 28 9 1 100 0.0039 1963
    EXP GTGSGVSSKX 28 10 1 100 0.0071 1964
    EXP GTGSGVSSKKK 28 11 1 100 1965
    EXP GVGLVLYNTEK 95 11 1 100 1966
    EXP GVSSKKKNK 32 9 1 100 0.0001 1967
    EXP GVSSKKKNKK 32 10 1 100 0.0011 1968
    EXP HDLISDMIK 51 9 1 100 0.0001 1969
    EXP HDLISDMIKK 51 10 1 100 0.0009 1970
    EXP IFNKESLA 15 8 1 100 1971
    EXP IFNKESLAEK 15 10 1 100 0.0005 1972
    EXP IGSSDPADNA 112 10 1 100 1973
    EXP IIFNKESLA 14 9 1 100 1974
    EXP IIFNKESLAEK 14 11 1 100 1975
    EXP ILSVFFLA 3 8 1 100 1976
    EXP ILSVFFLALF 3 10 1 100 1977
    EXP ILSVFFLALFF 3 11 1 100 1978
    EXP KGSGEPLIDVH 41 11 1 100 1979
    EXP KGTGSGVSSK 27 10 1 100 0.0005 1980
    EXP KGTGSOVSSKK 27 11 1 100 1981
    EXP KIGSSDPA 111 8 1 100 1982
    EXP KIGSSDPADNA 111 11 1 100 1983
    EXP KILSVFFLA 2 9 1 100 0.1400 1984
    EXP KILSVFFLALF 2 11 1 100 1985
    EXP KLATSVLA 75 8 1 100 1986
    EXP LALFFIIF 9 8 1 100 1987
    EXP LALFFIIFNK 9 10 1 100 0.0140 1988
    EXP LFFIIFNK 11 8 1 100 1989
    EXP LGGVGLVLY 93 9 1 100 0.0001 1990
    EXP LISDMIKK 53 8 1 100 1991
    EXP LLGGVGLVLY 92 10 1 100 0.0034 1992
    EXP LSVFFLALF 4 9 1 100 1993
    EXP LSVFFLALFF 4 10 1 100 1994
    EXP LVEVNKRK 64 8 1 100 1995
    EXP LVEVNKRKSK 64 10 1 100 0.0005 1996
    EXP LVEVNKRKSKY 64 11 1 100 1997
    EXP LVLYNIEK 98 8 1 100 1998
    EXP LVLYNTEKGR 98 10 1 100 0.0005 1999
    EXP LVLYNTEKGRH 98 11 1 100 2000
    EXP NADPQVTA 134 8 1 100 2001
    EXP NLVSGPEH 155 8 1 100 2002
    EXP NTEKGRHPF 102 9 1 100 2003
    EXP NTEKGFHPFK 102 10 1 100 0.0047 2004
    EXP PADNANPDA 117 9 1 100 2005
    EXP PFKIGSSDPA 109 10 1 100 2006
    EXP SDPADNANPDA 115 11 1. 100 2007
    EXP SGEPLIDVH 43 9 1 100 0.0001 2008
    EXP SGVSSKKK 31 8 1 100 2009
    EXP SGVSSKKKNK 31 10 1 100 0.0005 2010
    EXP SGVSSKKKNKK 31 11 1 100 2011
    EXP SLAEKTINIK 20 8 1 100 2012
    EXP SSDPADNA 114 8 1 100 2013
    EXP SSKKKNKK 34 8 1 100 2014
    EXP SVFFLALF S 8 1 100 2015
    EXP SVFFLALFF 5 9 1 100 2016
    EXP TGSGVSSK 29 8 1 100 2017
    EXP TGSGVSSKK 29 9 1 100 0.0001 2018
    EXP TGSGVSSKKK 29 10 1 100 0.0005 2019
    EXP VFFLALFF 6 8 1 100 2020
    EXP VFFLALFFIIF 6 11 1 100 2021
    EXP VGLVLYNIEK 96 10 1 100 0.0005 2022
    EXP VLLGGVGLVLY 91 11 1 100 2023
    EXP VLYNTEKGR 99 9 1 100 0.0110 2024
    EXP VLYNIEKGRH 99 10 1 100 0.0029 2025
    EXP VSSKKKNK 33 8 1 100 2026
    EXP VSSKKKNKK 33 9 1 100 0.0001 2027
    LSA ADTKKNLER 1632 9 1 100 2028
    LSA ADTKKNLERK 1632 10 1 100 0.0001 2029
    LSA ADTKKNLERKK 1632 11 1 100 2030
    LSA AIELPSENER 1660 10 1 100 0.0001 2031
    LSA DDDDKKKY 130 8 1 100 2032
    LSA DDDDKKKYIK 130 10 1 100 0.0001 2033
    LSA DDDKKKYIK 131 9 1 100 0.0001 2034
    LSA DDEDLDEF 1778 8 1 100 2035
    LSA DDEDLDEFK 1778 9 1 100 0.0001 2036
    LSA DDKKKYIK 132 8 1 100 2037
    LSA DDIDEGIEK 1817 9 1 100 0.0001 2038
    LSA DGSIKPEQK 1724 9 1 100 0.0001 2039
    LSA DIHKGHLEEK 1713 10 1 100 0.0004 2040
    LSA DIHKGHLEEKK 1713 11 1 100 2041
    LSA DITKYFMK 1901 8 1 100 2042
    LSA DLDEFKPIVQY 1781 11 1 100 2043
    LSA DLDEGIEK 1818 8 1 100 2044
    LSA DLEEKAAK 148 8 1 100 2045
    LSA DLEQDRLA 1388 8 1 100 2046
    LSA DLEQDRLAK 1388 9 1 100 0.0001 2047
    LSA DLEQDRLAKEK 1388 11 1 100 2048
    LSA DLEQERLA 1609 8 1 100 2049
    LSA DLEQERLAK 1609 9 1 100 0.0001 2050
    LSA DLEQERLAKEK 1609 11 1 100 2051
    LSA DLEQERLANEK 1524 11 1 100 2052
    LSA DLEpERRA 1575 8 1 100 2053
    LSA DLEQERRAK 1575 9 1 100 0.0001 2054
    LSA DLEQERRAKEK 1575 11 1 100 2055
    LSA DLEQRKADTK 1626 10 1 100 0.0001 2056
    LSA DLEQRKADIKK 1626 11 1 100 2057
    LSA DLERTKASK 1184 9 1 100 0.0001 2058
    LSA DLYGRLEIPA 1651 10 1 100 2059
    LSA DSEQERLA 521 8 1 100 2060
    LSA DSEQERLAK 521 9 1 100 0.0001 2061
    LSA DSEQERLAKEK 521 11 1 100 2062
    LSA DSKEISIIEK 1689 10 1 100 0.0001 2063
    LSA DTKKNLER 1633 8 1 100 2064
    LSA DTKIMERK 1633 9 1 100 0.0001 2065
    LSA DTKKNLERKK 1633 10 1 100 0.0001 2066
    LSA DVLAEDLY 1646 8 1 100 2067
    LSA DVLAEDLYGR 1646 10 1 100 0.0001 2068
    LSA DVNDFQISK 1751 9 1 100 0.0001 2069
    LSA DVNDFQISKY 1751 10 1 100 0.0003 2070
    LSA EDDEDLDEF 1777 9 1 100 2071
    LSA EDDEDLDEFK 1777 10 1 100 0.0001 2072
    LSA EDEISAEY 1761 8 1 100 2073
    LSA EDITKYFMK 1900 9 1 100 0.0001 2074
    LSA EDKSADIQNH 1733 10 1 100 2075
    LSA EDLEEKAA 147 8 1 100 2076
    LSA EDLEEKAAK 147 9 1 100 0.0002 2077
    LSA EDLYGRLEIPA 1650 11 1 100 2078
    LSA EFKPIVQY 1784 8 1 100 2079
    LSA EFPIVQYDNF 1784 11 1 100 2080
    LSA EGRRDIHK 709 8 1 100 2081
    LSA EGRRDIHKGH 1709 10 1 100 0.0001 2082
    LSA EIIKSNLR 33 8 1 100 2083
    LSA EISIIEKTNR 1692 10 1 100 0.0001 2084
    LSA ELEDLIEK 1805 8 1 100 2085
    LSA ELPSENER 1662 8 1 100 2086
    LSA ELPSENERGY 1662 10 1 100 0.0001 2087
    LSA ELPSENERGYY 1662 11 1 100 2088
    LSA ELSEDITK 1897 8 1 100 2089
    LSA ESLSEDITKY 1897 9 1 100 0.0002 2090
    LSA ELSEDITKYF 1897 10 1 100 2091
    LSA ELSEEKIK 1829 8 1 100 2092
    LSA ELSEEKIKK 1829 9 1 100 0.0002 2093
    LSA ELSEEKIKKGK 1829 11 1 100 2094
    LSA ELTMSNVK 83 8 1 100 2095
    LSA ESITTNVEGR 1702 10 1 100 0.0001 2096
    LSA ESITTNVEGRR 1702 11 1 100 2097
    LSA ETVNISDVNDF 1745 11 1 100 2098
    LSA FIKSLFHIF 1877 9 1 100 2099
    LSA FILVNLLIF 11 9 1 100 2100
    LSA FILVNLLIFH 11 10 1 100 0.0310 2101
    LSA FLKENKLNK 111 9 1 100 0.0260 2102
    LSA GDVLAEDLY 1645 9 1 100 2103
    LSA GDVLAEDLYGR 1645 11 1 100 2104
    LSA GSIKPEQK 1725 8 1 100 2105
    LSA GSIKPEQKEDK 1725 11 1 100 2106
    LSA GSSNSRNR 42 8 1 100 2107
    LSA GVSENIFLK 105 9 1 100 0.2700 2108
    LSA HGDVLAEDLY 1644 10 1 100 0.0001 2109
    LSA HIINDDDDK 126 9 1 100 0.0002 2110
    LSA HIINDDDDKK 126 10 1 100 0.0001 2111
    LSA HIINDDDDKKK 126 11 1 100 2112
    LSA HIKKYKNDK 1860 9 1 100 0.0002 2113
    LSA HILYISFY 3 8 1 100 2114
    LSA HILYISFYF 3 9 1 100 2115
    LSA HINGKIIK 20 8 1 100 2116
    LSA HLEEKKDGSIK 1718 11 1 100 2117
    LSA HVLSHNSY 59 8 1 100 2118
    LSA HVLSHNSYEK 59 10 1 100 0.0170 2119
    LSA IFHINGKIIK 18 10 1 100 0.0001 2120
    LSA IFLKENKLNK 110 10 1 100 0.0001 2121
    LSA IINDDDDK 127 8 1 100 2122
    LSA IINDDDDKK 127 9 1 100 0.0002 2123
    LSA IINDDDDKKK 127 10 1 100 0.0001 2124
    LSA IINDDDDKKKY 127 11 1 100 2125
    LSA ILVNLLIF 12 12 8 100 2126
    LSA ILVNLLIFH 12 9 1 100 0.0150 2127
    LSA ILYISFYF 4 8 1 100 2128
    LSA ISDVNDMISK 749 11 1 100 2129
    LSA ISHEKTNR 693 9 1 100 0.0001 2130
    LSA ISKYEDEISA 757 10 1 100 2131
    LSA ITTNVEGR 704 8 1 100 2132
    LSA ITTNVEGRR 704 9 1 100 0.0002 2133
    LSA DAMESEDITK 894 11 1 100 2134
    LSA KADTKKNLER 631 10 1 100 0.0001 2135
    LSA KADTKKNLERK 631 11 1 100 2136
    LSA KDEGIKSNIR 31 10 1 100 2137
    LSA KDGSIKPEQK 723 10 1 100 0.0004 2138
    LSA KDKELTMSNVK 80 11 1 100 2139
    LSA KDNNFKPNDK 845 10 1 100 0.0001 2140
    LSA KFIKSLFH 876 8 1 100 2141
    LSA KFIKSLFHIF 876 10 1 100 2142
    LSA KGHLEEKK 716 8 1 100 2143
    LSA KGKKYEKIK 837 9 1 100 0.0002 2144
    LSA KIIKNSEK 24 8 1 100 2145
    LSA KKKKGKKY 834 8 1 100 2146
    LSA KIKKGKKYEK 834 10 1 100 0.0081 2147
    LSA KLNKEGKLIEN 116 1t 1 100 2148
    LSA KLQEQQSDLER 177 11 1 100 2149
    LSA KSADIQNH 735 8 1 100 2150
    LSA KSLYDEIHK 854 9 1 100 0.0005 2151
    LSA KSLYDMIKK 854 10 1 100 0.0094 2152
    LSA KSLYDEHIKKY 854 11 1 100 2153
    LSA KSSEELSEEK 825 10 1 100 0.0001 2154
    LSA KTKDNNFK 843 8 1 100 2155
    LSA KTKNNENNK 68 9 1 100 0.0028 2156
    LSA KTKNNENNKF 68 10 1 100 2157
    LSA KTKNNENNKFF 68 11 1 100 2158
    ISA LAEDLYGR 1648 8 1 100 2159
    LSA LAKEKLQEQQR 1615 11 1 100 2160
    LSA LANEKLQEQQR 1530 11 1 100 2161
    LSA LDDLDEGIEK 1816 10 1 100 0.0001 2162
    LSA LDEFKPIVQY 1782 10 1 100 2163
    LSA LGVSENIF 104 8 1 100 2164
    LSA LGVSENIFLK 104 10 1 100 0.0001 2165
    LSA LIFHINGK 17 8 1 100 2166
    LSA LIFHINGKIIK 17 11 1 100 2167
    LSA LLIFNINGK 16 9 1 100 0.0260 2168
    LSA LSEDITKY 1898 8 1 100 2169
    LSA LSEDITKYF 1898 9 1 100 2170
    LSA LSEDITKYDAK 1898 11 1 100 2171
    LSA LSEEKIKK 1830 8 1 100 2172
    ISA LSEEKIKKGK 1830 10 1 100 0.0004 2173
    LSA LSEEKIKKGKK 1830 11 1 100 2174
    LSA LSHNSYEK 61 8 1 100 2175
    LSA LSFINSYEKTK 61 10 1 100 0.0004 2176
    LSA LVNLLIFH 13 8 1 100 2177
    LSA NDDDDKKK 129 8 1 100 2178
    LSA NDDDDKKKY 129 9 1 100 2179
    LSA NDDDDKKKYIK 129 11 1 100 2180
    LSA NDFQISKY 1753 8 1 100 2181
    LSA NDKQVNKEK 1866 9 1 100 0.0002 2182
    LSA NDKQVNKEKEK 1866 11 1 100 2183
    LSA NDKSLYDEH 1852 9 1 100 2184
    LSA NDKSLYDEHIK 1852 11 1 100 2185
    LSA NFKPNDKSLY 1848 10 1 100 2186
    LSA NFQDEENIGIY 1793 11 1 100 2187
    ISA NGKIIKNSEK 22 10 1 100 0.0004 2188
    LSA NIFLKENK 109 8 1 100 2189
    LSA NIFLKENKLNK 109 11 1 100 2190
    LSA NISDVNDF 1748 8 1 100 2191
    LSA NLDDLDEGIEK 1815 11 1 100 2192
    LSA NLERKKEH 1637 8 1 100 2193
    LSA NLGVSENIF 103 9 1 100 2194
    LSA NLGVSENIFLK 103 11 1 100 2195
    LSA NLLIFHINGK 15 10 1 100 0.0049 2196
    LSA NLRSGSSNSR 38 10 1 100 0.0004 2197
    LSA NSEKDFIKK 28 9 1 100 0.0002 2198
    LSA NSRNRKNEEK 45 10 1 100 0.0004 2199
    LSA NSRNRINEEKH 45 11 1 100 2200
    LSA NVEGRRDIH 1707 9 1 100 0.0002 2201
    LSA NVEGRRDIHK 1707 10 1 100 0.0004 2202
    LSA NVKNVSQINF 88 10 1 100 2203
    LSA NVKNVSQINFK 88 11 1 100 2204
    LSA NVSQTNFK 91 8 1 100 2205
    LSA PAIELPSENER 659 11 1 100 2206
    LSA PIVQYDNF 787 8 1 100 2207
    LSA PSENERGY 664 8 1 100 2208
    LSA PSENERGYY 664 9 1 100 0.0001 2209
    LSA QDEENIGIY 795 9 1 100 2210
    LSA QDEENIGIYK 795 10 1 100 0.0004 2211
    LSA QDNRGNSR 681 8 1 100 2212
    LSA QDNRGNSRDSK 681 11 1 100 2213
    LSA QDRLAKEK 391 8 1 100 2214
    LSA QGWISCLEM 128 11 1 100 2215
    LSA QISKYEDEISA 756 11 1 100 2216
    LSA QSDLEQDR 386 8 1 100 2217
    LSA QSDLEQDRLA 386 10 1 100 2218
    LSA QSDLEQDRLAK 386 11 1 100 2219
    LSA QSDLEQER 590 8 1 100 2220
    LSA QSDLEQERLA 590 10 1 100 2221
    LSA QSDLEQERLAK 590 11 1 100 2222
    LSA QSDLEQERR 573 9 1 100 0.0002 2223
    LSA QSDLEQERRA 573 10 1 100 2224
    LSA QSDLEQDRLAK 573 11 1 100 2225
    LSA QSDLERTK 182 8 1 100 2226
    LSA QSDLERTKA 182 9 1 100 2227
    LSA QSDLERTKASK 182 11 1 100 2228
    LSA QSDSEQER 519 8 1 100 2229
    LSA QSDSEQERLA 519 10 1 100 2230
    LSA QSDLEQDRLAK 519 11 1 100 2231
    LSA QSSLPQDNR 1676 9 1 100 0.0002 2232
    LSA QTNFKSLLR 94 9 1 100 0.0320 2233
    ISA QVNKEKEK 1869 8 1 100 2234
    ISA QVNKEIEKF 1869 9 1 100 2235
    LSA QVNKEKEKFIK 1869 11 1 100 2236
    LSA RDIHKGHLEIEK 1712 11 1 100 2237
    ISA RDLEQERLA 1608 9 1 100 2238
    LSA RDLEQERLAK 608 10 1 100 0.0004 2239
    LSA RDLEQERR 540 8 1 100 2240
    LSA RDLEQERRA 540 9 1 100 2241
    LSA RDLEQERRAK 540 10 1 100 0.0004 2242
    LSA RDLEQRKA 625 8 1 100 2243
    ISA RDLEQRKADTK 625 11 1 100 2244
    ISA RDSKEISIIEK 688 11 1 100 2245
    ISA RGNSRDSK 684 8 1 100 2246
    LSA RINEEKHEK 49 9 1 100 0.0033 2247
    LSA RINEEKNEKK 49 10 1 100 0.0024 2248
    ISA RINEEKHEKKH 49 11 1 100 2249
    LSA RSGSSNSR 40 8 1 100 2250
    ISA RSGSSNSRNR 40 10 1 100 0.0011 2251
    LSA SDLEQDRLA 1387 9 1 100 2252
    ISA SDLBQDRLAK 1387 10 1 100 0.0002 2253
    LSA SDLEQERLA 1591 9 1 100 2254
    ISA SDLEQERLAK 1591 10 1 100 0.0002 2255
    ISA SDLEQERR 1574 8 1 100 2256
    LSA SDLEQERRA 1574 9 1 100 2257
    LSA SDLEQERRAK 1574 10 1 100 0.0002 2258
    LSA SDLERTKA 1183 8 1 100 2259
    ISA SDLERTKASK 1183 10 1 100 0.0002 2260
    LSA SDSEQERLA 520 9 1 100 2261
    ISA SDSEQERLAK 520 10 1 100 0.0002 2262
    ISA SDVNDFQISK 1750 10 1 100 0.0002 2263
    LSA SDVNDFQISKY 1750 11 1 100 2264
    LSA SGSSNSRNR 41 9 1 100 0.0002 2265
    LSA SIIEKTNR 1694 8 1 100 2266
    LSA SIKPEQKEDK 1726 10 1 100 0.0002 2267
    LSA MTTNVEGR 1703 9 1 100 0.0002 2268
    LSA MTTNVEGRR 1703 10 1 100 0.0002 2269
    LSA SLPQDNRGNSR 1678 11 1 100 2270
    LSA SLYDEHKK 1855 8 1 100 2271
    LSA SLYDEHIKK 1855 9 1 100 0.0460 2272
    LSA SLYDEHIKKY 1855 10 1 100 0.0015 2273
    ISA SLYDEHIKKYK 1855 11 1 100 2274
    LSA SSEELSEEK 1826 9 1 100 0.0002 2275
    LSA SSEEBEDUK 1826 11 1 100 2276
    LSA SSLPQDNR 1677 8 1 100 2277
    LSA TTNVEGRR 1705 8 1 100 2278
    LSA TTNVEGRRIMH 1705 11 1 100 2279
    ISA TVNISDVNDF 1746 10 1 100 2280
    ISA VDESEDITK 1895 10 1 100 0.0002 2281
    LSA VDELSEDITKY 1895 11 1 100 2282
    ISA VLAEDLYGR 1647 9 1 100 0.0013 2283
    LSA VLSHNSYEK 60 9 1 100 0.0280 2284
    LSA VLSHNSYEKTK 60 11 1 100 2285
    LSA VSENIFLK 106 8 1 100 2286
    LSA VSENIFLKENK 106 11 1 100 2287
    ISA VSQTNFKSLLR 92 11 1 100 2288
    LSA YEEHDUCY 1857 8 1 100 2289
    LSA YDEHIKKYK 1857 9 1 100 0.0005 2290
    LSA YFILVNLLIF 10 10 1 100 2291
    LSA YFILVNLLWH 10 11 1 100 2292
    LSA YGRLEIPA 1653 8 1 100 2293
    LSA YIKGQDENR 137 9 1 100 0.0025 2294
    SSP2 AATPYAGEPA 525 10 8 80 2295
    SSP2 ACAGLAYK 512 8 10 100 2296
    SSP2 ACAGLAYKF 512 9 10 100 2297
    SSP2 ADSAWENVK 216 9 10 100 0.0002 2298
    SSP2 AFNRFLVGCH 197 10 10 100 2299
    SSP2 AGGIAGGLA 501 9 10 100 2300
    SSP2 AGGLALLA 505 8 10 100 2301
    SSP2 AGGLALLACA 505 10 10 100 2302
    SSP2 ALLACAGLA 509 9 10 100 0.0002 2303
    SSP2 ALLACAGLAY 509 10 10 100 0.0630 2304
    SSP2 ALLACAGLAYK 509 11 10 100 2305
    SSP2 ALLQVRKH 136 8 9 90 2306
    SSP2 ASKNKEKA 107 8 10 100 2307
    SSP2 ATPYAGEPA 526 9 8 80 2308
    SSP2 ATPYAGEPAPF 526 11 8 80 2309
    SSP2 AVCVEVEK 233 8 10 100 2310
    SSP2 AVCVEVEKTA 233 10 10 100 2311
    SSP2 CAGLAYKF 513 8 10 100 2312
    SSP2 CGKGTRSR 257 8 10 100 2313
    SSP2 CGKGTRSRK 257 9 10 100 0.0002 2314
    SSP2 CGKGIRSRKR 257 10 10 100 0.0002 2315
    SSP2 CSGSIRRH 55 8 10 100 2316
    SSP2 CSVTCGKGTR 253 10 10 100 0.0002 2317
    SSP2 CVEVEKTA 235 8 10 100 2318
    SSP2 DALLQVRK 135 8 9 90 2319
    SSP2 DALLQVRKH 135 9 9 90 0.0004 2320
    SSP2 DASKNKEK 106 8 10 100 2321
    SSP2 DASKNKEKA 106 9 10 100 2322
    SSP2 DCSGSIRR 54 8 10 100 2323
    SSP2 DCSGSIRRH 54. 9 10 100 2324
    SSP2 DDQPRPRGDNF 301 11 9 90 2325
    SSP2 DDREENFDIPK 385 11 10 100 2326
    SSP2 CCKCNLYA 209 8 10 100 2327
    SSP2 DGKCNLYADSA 209 11 10 100 2328
    SSP2 DIPKKPENK 392 9 10 100 0.0004 2329
    SSP2 DIPICKPENKH 392 10 10 100 0.0002 2330
    SSP2 DLDEPEQF 546 8 10 100 2331
    SSP2 DLDEPEQFR 546 9 10 100 0.0002 2332
    SSP2 DLFLVNGR 19 8 10 100 2333
    SSP2 DSAWENVK 217 8 10 100 2334
    SSP2 DSIQDSLK 166 8 10 100 2335
    SSP2 DSIQDSLKESR 166 11 10 100 2336
    SSP2 DSLKESRK 170 8 9 90 2337
    SSP2 DVPKNPEDDR 378 10 10 100 0.0002 2338
    SSP2 DVQNNIVDBK 27 11 10 100 2339
    SSP2 EDDQPRPR 300 8 10 100 2340
    SSP2 EDDREENF 384 8 10 100 2341
    SSP2 EDKDLDEPEQF 543 11 10 100 2342
    SSP2 EDRETRPH 450 8 9 90 2343
    SSP2 EDRETRPHGR 450 10 9 90 2344
    SSP2 EIIRLHSDA 99 9 10 100 2345
    SSP2 EIIRLHSDASK 99 11 10 100 2346
    SSP2 ELQEQCEEER 276 10 8 80 0.0002 2347
    SSP2 ETLGEEDK 538 8 10 100 2348
    SSP2 EVCNDEVDLY 41 10 8 80 0.0002 2349
    SSP2 EVPSDVPK 374 8 10 100 2350
    SSP2 FDETLGEEDK 536 10 10 100 0.0002 2351
    SSP2 FDIPKKPENK 391 10 10 100 0.0002 2352
    SSP2 FDIPKKPENKH 391 11 10 100 2353
    SSP2 FDLFLVNGR 18 9 10 100 2354
    SSP2 FFDLFLVNGR 17 10 10 100 2335
    SSP2 FGIGQGINVA 188 10 10 100 2356
    SSP2 FGIGQGINVAF 188 11 10 100 2357
    SSP2 FLIFFDLF 14 8 10 100 2358
    SSP2 FLVGCHPSDGK 201 11 10 100 2359
    SSP2 FMKAVCVEVEK 230 11 10 100 2360
    SSP2 FVVPGAATPY 520 10 8 80 0.0002 2361
    SSP2 FVVPGAATPYA 520 11 8 80 2362
    SSP2 GAATPYAGEPA 524 11 8 80 2363
    SSP2 GCHPSDGK 204 8 10 100 2364
    SSP2 GDNFAVEK 308 8 9 90 2365
    SSP2 GGIAGGLA 502 8 10 100 2366
    SSP2 GGIAGGLALLA 502 11 10 100 2367
    SSP2 GGLALLACA 506 9 10 100 2368
    SSP2 GIAGGLALLA 503 10 10 100 2369
    SSP2 GIGQGINVA 189 9 10 100 2370
    SSP2 GIGQGINVAF 189 10 10 100 2371
    SSP2 GINVAFNR 193 8 10 100 2372
    SSP2 GINVAFNRF 193 9 10 100 2373
    SSP2 GIPDSIQDSLK 163 11 10 100 2374
    SSP2 GLALLACA 507 8 10' 100 2375
    SSP2 GLALLACAGLA 507 11 10 100 2376
    SSP2 GLAYKFVVPGA 515 11 10 100 2377
    SSP2 GSIRRHNWVNH 37 11 8 80 2378
    SSP2 GTRSRKRELH 260 11 10 100 2379
    SSP2 HAVPLAMK 67 8 10 100 2380
    SSP2 HDNQNNLPNDK 401 11 10 100 2381
    SSP2 HGRNNENR 457 8 10 100 2382
    SSP2 HGRNNENRSY 457 10 10 100 0.0004 2383
    SSP2 HLNDRINR 143 8 10 100 2384
    SSP2 HLNDRINRENA 143 11 10 100 2385
    SSP2 HSDASKNK 104 8 10 100 2386
    SSP2 HSDASKNKEK 104 10 10 100 0.0004 2387
    SSP2 HSDASKNKEKA 104 11 10 100 2388
    SSP2 HVPNSEDR 445 8 10 100 2389
    SSP2 HVPNSEDRE1R 445 11 9 90 2390
    SSP2 IAGGIAGGLA 500 10 10 100 2391
    SSP2 IAGGLALLA 504 9 10 100 0.0002 2392
    SSP2 IAGGLALLACA 504 11 10 100 2393
    SSP2 IFFDLFLVNGR 16 11 10 100 2394
    SSP2 IGQGINVA 190 8 10 100 2395
    SSP2 IGQGINVAF 190 9 10 100 2396
    SSP2 IGQGINVAFNR 190 11 10 100 2397
    SSP2 RRLHSDA 100 8 10 100 2398
    SSP2 IIRLHSDASK 100 10 10 100 0.0230 2399
    SSP2 IVDEIKYR 32 8 9 90 2400
    SSP2 IVFLIFFDLF 12 10 10 100 2401
    SSP2 KAVCVEVEK 232 9 10 100 0.0004 2402
    SSP2 KAVCVEVEKTA 232 11 10 100 2403
    SSP2 KCNLYADSA 211 9 10 100 2404
    SSP2 KDLDEPEQF 545 9 10 100 2405
    SSP2 KDLDEPEQFR 545 10 10 100 2406
    SSP2 KFVVPGAA 519 8 10 100 2407
    SSP2 KFVVPGAAIPY 519 11 8 80 2408
    SSP2 KGIRSRICR 259 8 10 100 2409
    SSP2 KIAGGIAGGLA 499 11 10 100 2410
    SSP2 KVLDNERK 421 8 8 80 2411
    SSP2 LACACLAY 511 8 10 100 2412
    SSP2 LACAGLAYK 511 9 10 100 0.0240 2413
    SSP2 LACAGLAYKF 511 10 10 100 2414
    SSP2 LALLACAGLA 508 10 10 100 2415
    SSP2 LALLACAGIAY 508 11 10 100 2416
    SSP2 LAYKFVVPGA 516 10 10 100 2417
    SSP2 LAYKFVVPGAA 516 11 10 100 2418
    SSP2 LDEPEQFR 547 8 10 100 2419
    SSP2 LGNVKYLVIVF 4 11 10 100 2420
    SSP2 LLACAGLA 510 8 10 100 2421
    SSP2 LLACAGLAY 510 9 10 100 0.0120 2422
    SSP2 LLACAGLAYK 510 10 10 100 0.9500 2423
    SSP2 LLACAGLAYKF 510 11 10 100 2424
    SSP2 LLMDCSGSIR 51 10 10 100 0.0004 2425
    SSP2 LLMDCSGSIRR 51 11 10 100 2426
    SSP2 LLQVRKHLNDR 137 11 9 90 2427
    SSP2 LLSINLPY 121 8 9 90 2428
    SSP2 LLSTNLPYGR 121 10 8 80 0.0017 2429
    SSP2 LMDCSGSIR 52 9 10 100 0.0004 2430
    SSP2 LMDCSGSIRR 52 10 10 100 0.0015 2431
    SSP2 LMDCSGSIRRH 52 11 10 100 2432
    SSP2 LSTNLPYGR 122 9 8 80 0.0004 2433
    SSP2 LVGCHPSDOK 202 10 10 100 0.0004 2434
    SSP2 LVIVFLIF 10 8 10 100 2435
    SSP2 LVIVFLIFF 10 9 10 100 2436
    SSP2 MDCSGSIR 53 8 10 100 2437
    SSP2 MDCSGSIRR 53 9 10 100 2438
    SSP2 MDCSGSIRRH 53 10 10 100 2439
    SSP2 NDRINRENA 145 9 10 100 2440
    SSP2 NFDIPKKPENK 390 11 10 100 2441
    SSP2 NIPEDSEK 366 8 10 100 2442
    SSP2 NIVDSKY 31 8 10 100 2443
    SSP2 NIVDEIKYR 31 9 9 90 0.0005 2444
    SSP2 NLPNDKSDR 406 9 10 100 0.0005 2445
    SSP2 NSEDRETR 448 8 9 90 2446
    SSP2 NSEDRETRPH 448 10 9 90 0.0004 2447
    SSP2 NVIGPFMK 225 8 10 100 2448
    SSP2 NVIGPFMKA 225 9 10 100 0.0002 2449
    SSP2 NVKNVIGPF 222 9 10 100 2450
    SSP2 NVKNVIGPFMK 222 11 10 100 2451
    SSP2 NVKYLVIVF 6 9 10 100 2452
    SSP2 PCSVTCGK 252 8 10 100 2453
    SSP2 PCSVTCGKGTR 252 11 10 100 2454
    SSP2 PDSIQDSLK 165 9 10 100 0.0005 2455
    SSP2 PFDETLGEEDK 535 11 10 100 2456
    SSP2 PGAATPYA 523 8 8 80 2457
    SSP2 PSDGKCNLY 207 9 10 100 0.0002 2458
    SSP2 PSDOKCNLYA 207 10 10 1013 2459
    SSP2 PSPNPEEGK 328 9 10 100 0.0005 2460
    SSP2 QCEEERCPPK 280 10 8 80 0.0004 2461
    SSP2 QDNNGNRH 438 8 10 100 2462
    SSP2 QDSLKESR 169 8 10 100 2463
    SSP2 QDSLKESRK 169 9 9 90 0.0005 2464
    SSP2 QGINVAFNR 192 9 10 100 0.0009 2465
    SSP2 QGINVAFNRF 192 10 10 100 2466
    SSP2 QSQDNNGNR 436 9 10 100 0.0005 2467
    SSP2 QSQDNNONRH 436 10 10 100 0.0004 2468
    SSP2 QVRICHLNDR 139 9 9 90 0.0005 2469
    SSP2 RGDNFAVEK 307 9 9 90 0.0005 2470
    SSP2 RGVKIAVF 181 8 9 90 2471
    SSP2 RLHSDASK 102 8 10 100 2472
    SSP2 RLHSDASKNK 102 10 10 100 0.0240 2473
    SSP2 RSRKREILH 262 9 10 100 0.0110 2474
    SSP2 SDASICNKEK 105 9 10 100 0.0005 2475
    SSP2 SDASICNICEKA 105 10 10 100 2476
    SSP2 SDGKCNLY 208 8 10 100 2477
    SSP2 SDGKCNLYA 208 9 10 100 2478
    SSP2 SDNKYKIA 494 8 9 90 2479
    SSP2 SDVPKNPEDDR 377 11 10 100 2480
    SSP2 SIQDSLKESR 167 10 10 100 0.0004 2481
    SSP2 SIQDSLKESRK 167 11 9 90 2482
    SSP2 SIRRHNWVNH 58 10 8 80 0.0011 2483
    SSP2 SIRRHNWVNHA 58 11 8 80 2484
    SSP2 SLLSTNLPY 120 9 9 90 0.0280 2485
    SSP2 SLLSTNLPYGR 120 11 8 80 2486
    SSP2 STNLPYGR 123 8 8 80 2487
    SSP2 SVTCGKGTR 254 9 10 100 0.0005 2488
    SSP2 SVICGKGTRSR 254 11 10 100 2489
    SSP2 TCGKGTRSR 256 9 10 100 2490
    SSP2 TCGKGTRSRK 256 10 10 100 0.0004 2491
    SSP2 TCGKGIRSRKR 256 11 10 100 2492
    SSP2 VAFNRFLVGCH 196 11 10 100 2493
    SSP2 VCNDEVDLY 42 9 8 80 2494
    SSP2 VCVEVEKTA 234 9 10 100 2495
    SSP2 VFGIGQGINVA 187 11 10 100 2496
    SSP2 VFLIFFDLF 13 9 10 100 2497
    SSP2 VGCHPSDGK 203 9 10 100 0.0005 2498
    SSP2 VIGPFMKA 226 8 10 100 2499
    SSP2 VIVFLIFF 11 8 10 100 2500
    SSP2 VIVFLIFFDLF 11 11 10 100 2501
    SSP2 VTCGKGTR 255 8 10 100 2502
    SSP2 VTCGKGTRSR 255 10 10 100 0.0004 2503
    SSP2 VTCGKGTRSRK 255 11 10 100 2504
    SSP2 VVPGAATPY 521 9 8 80 0.0005 2505
    SSP2 VVPGAATPYA 521 10 8 80 2506
    SSP2 WSPCSVTCGK 250 10 10 100 0.0004 2507
    SSP2 WVNHAVPLA 64 9 8 80 0.0002 2508
    SSP2 WVNHAVPLAMK 64 11 8 80 2509
    SSP2 YADSAWENVIC 215 10 10 100 0.0004 2510
    SSP2 YAGEPAPF 529 8 8 80 2511
    SSP2 YLLMDCSGSIR 50 11 10 100 2512
    SSP2 YLVIVFLIF 9 9 10 100 2513
    SSP2 YLVIVFLIFF 9 10 10 100 2514
  • TABLE XVII
    Malaria All Motif Peptides With Binding Information
    No. of Sequence Conservancy
    Protein Sequence Position Amino Acids Frequency (%) A*1101 Seq. Id.
    CSP ALFQEYQCY 18 9 19 100 0.0021 2515
    CSP ANANNAVK 336 8 16 84 2516
    CSP ANPNANKNK 305 9 19 100 2517
    CSP CGNGIQVR 377 8 19 100 2518
    CSP CGNGIQVRIK 377 10 19 100 0.0002 2519
    CSP DGNNEDNEX 96 9 19 100 0.0002 2520
    CSP DGNNEDNEKLR 96 11 19 100 2521
    CSP DGNNNNGDNGR 77 11 17 89 2522
    CSP DIEKKICK 402 8 19 100 2523
    CSP DIEKKICKMEK 402 11 19 100 2524
    CSP DNAGINLY 41 8 18 95 2525
    CSP DNEKLRKPK 101 9 19 100 2526
    CSP DNEKLRKPKH 101 10 19 100 2527
    CSP DNEKLRKPKHK 101 11 19 100 2528
    CSP DNGREGKDEDK 84 11 19 100 2529
    CSP EALFQEYQCY 17 10 19 100 0.0002 2530
    CSP EDNEKLRK 100 8 19 100 2531
    CSP EDNEKLRKPK 100 10 19 100 0.0002 2532
    CSP EDNEKLRKPKH 100 11 19 100 2533
    CSP EGKDEDKR 88 8 19 100 2534
    CSP ELEMNYYGK 50 9 19 100 0.0003 2535
    CSP ENANANNAVK 334 10 16 84 2536
    CSP ENKIEKKICK 400 10 19 100 2537
    CSP ENWYSLKK 60 8 19 100 2538
    CSP ENWYSLKKNSR 60 11 19 100 2539
    CSP FLFVEALFQEY 13 11 19 100 2540
    CSP FVEALFQEY 15 9 19 100 0.0003 2541
    CSP GDNGREGK 83 8 19 100 2542
    CSP GNGIQVRIK 378 9 19 100 2543
    CSP GNNEDNEK 97 8 19 100 2544
    CSP GNNEDNEKLR 97 10 19 100 2545
    CSP GNNEDNEKLRK 97 11 19 100 2546
    CSP GNNNNGDNGR 78 10 19 100 2547
    CSP HIEQYLKK 350 8 15 79 2548
    CSP HNMPNDPNR 322 9 19 100 2549
    CSP INLYNELEMNY 45 11 18 95 2550
    CSP KLRKPKHK 104 8 19 100 2551
    CSP KLRKPKHKK 104 9 19 100 0.0037 2552
    CSP KLRKPKHKKLK 104 11 19 100 2553
    CSP KNNNNEEPSDK 343 11 19 100 2554
    CSP KNNQGNGQGH 313 10 19 100 2555
    CSP LDYENDIEK 397 9 18 95 0.0002 2556
    CSP LDYENDIEKK 397 10 18 95 0.0002 2557
    CSP LFQEYQCY 19 8 19 100 2558
    CSP LFVEALFQEY 14 10 19 100 2559
    CSP LNYDNAGINLY 38 11 18 95 2560
    CSP MNYYGKQENWY 53 11 19 100 2561
    CSP NANANNAVK 335 9 16 1984 0.0002 2562
    CSP NANPNANPNK 304 10 19 100 0.0021 2563
    CSP NDIEKKICK 401 9 19 100 0.0002 2564
    CSP NGDNGREGK 82 9 19 100 0.0002 2565
    CSP NGIQVRIK 379 8 19 100 2566
    CSP NGREGKDEDK 85 10 19 100 0.0002 2567
    CSP NGEGKDEDKR 85 11 19 100 2568
    CSP NLYNELEMNY 46 10 19 100 0.0002 2569
    CSP NLYNELEMNYY 46 11 19 100 2570
    CSP NMPNDPNR 323 8 19 100 2571
    CSP NNEDNEKIR 98 9 19 100 2572
    CSP NNEDNEXLRK 98 10 19 100 2373
    CSP NNEEPSDK 346 8 19 100 2574
    CSP NNEEPSDKH 346 9 19 100 2575
    CSP NNGDNGREGK 81 10 19 100 2576
    CSP NNNEEPSDK 345 9 19 100 2577
    CSP NNNEEPSDKH 345 10 19 100 2578
    CSP NNNGDNGR 80 8 19 100 2579
    CSP NNNGDNGREGK 80 11 19 100 2580
    CSP NNNNEEPSDK 344 10 19 100 2581
    CSP NNNNEEPSDKH 344 11 19 100 2582
    CSP NNNNGDNGR 79 9 19 100 2583
    CSP NNQGNGQGH 314 9 19 100 2584
    CSP NTRVLNELNY 31 10 19 100 0.0002 2585
    CSP PNANPNANPNK 303 11 19 100 2586
    CSP PSDKHIEQY 346 9 15 79 2587
    CSP PSDKHIEQYLK 346 11 15 79 2588
    CSP QCYGSSSNTR 24 10 19 100 2589
    CSP QGHNMPNDPNR 320 11 19 100 2590
    CSP RDGNNEDNEK 95 10 19 100 0.0002  2591
    CSP RVLNELNY 33 8 19 100 2592
    CSP SDICHIEQY 347 8 15 79 2593
    CSP SDKHIEQYLK 347 10 15 79 2594
    CSP SDKHIEQYLKK 347 11 15 79 2595
    CSP SNIRVLNELNY 30 11 19 100 2596
    CSP SVTCGNGIQVR 374 11 19 100 2597
    CSP TCGNGIQVR 376 9 19 100 2598
    CSP TCGNGIQVRDC 376 11 19 100 2599
    CSP VTCGNGIQVR 375 10 19 100 0.0340 2600
    CSP YDNAGINLY 40 9 18 95 2601
    CSP YGKQENWY 56 8 19 100 2602
    CSP YGKQENWYSLK 56 11 19 100 2603
    CSP YGSSSNTR 26 8 19 100 2604
    CSP YNELEMNY 48 8 19 100 2605
    CSP YNELEMNYY 48 9 19 100 2606
    CSP YNELEMNYYGK 48 11 19 100 2607
    CSP YSLKKNSR 63 8 19 100 2608
    EXP ALFFIIFNK 10 9 1 100 1.2000 2609
    EXP DDNNLVSGPEH 152 11 1 100 2610
    EXP DLISDMIK 52 8 1 100 2611
    EXP DLISDMIKK 52 9 1 100 0.0003 2612
    EXP DNNLVSGPEH 153 10 1 100 2613
    EXP DVHDLISDMIK 49 11 1 100 2614
    EXP ELVEVNKR 63 8 1 100 2615
    EXP ELVEVNKRK 63 9 1 100 0.0002 2616
    EXP ELVEVNKRKSK 63 11 1 100 2617
    EXP EMADCTNK 19 9 1 100 0.0002 2618
    EXP EVNKRKSK 66 8 1 100 2619
    EXP EVNKRKSKY 66 9 1 100 0.0002 2620
    EXP EVNKRKSKYK 66 10 1 100 0.0002 2621
    EXP FLALFFIIFNK 8 11 1 100 2622
    EXP FNKESLAEK 16 9 1 100 2623
    EXP GGVGLVLY 94 8 1 100 2624
    EXP GLVLYNIEK 97 9 1 100 0.0055 2625
    EXP GLVLYNTEKGR 97 11 1 100 2626
    EXP GSGEPLIDVH 42 10 1 00 0.0002 2627
    EXP GSGVSSKK 30 8 1 00 2628
    EXP GSOVSSKKK 30 9 1 00 0.0065 2629
    EXP GSGVSSKKKNK 30 11 1 00 2630
    EXP GTGSGVSSK 28 9 1 00 0.0180 2631
    EXP GTGSGVSSKK 28 10 1 00 0.0340 2632
    EXP GTGSGVSSKKK 28 11 1 00 2633
    EXP GVGLVLYNIEK 95 11 1 00 2634
    EXP GVSSKKKNK 32 9 1 00 0.0002 2635
    EXP GVSSKKKNKK 32 10 1 00 0.0002 2636
    EXP HDLISDMIK 51 9 1 00 0.0002 2637
    EXP HDLISDMIKK 51 10 1 00 0.0002 2638
    EXP IFNKESLAEK 15 10 1 00 0.0003 2639
    EXP IIFNKESLAEK 14 11 1 00 2640
    EXP KGSGEPLDVH 41 11 1 00 2641
    EXP KGTGSGVSSK 27 10 1 00 0.0009 2642
    EXP KGTGSGVSSKK 27 11 1 00 2643
    EXP LALFFIIFNK 9 10 1 00 0.0530 2644
    EXP LFFIIFNK 11 8 1 00 2645
    EXP LGGVGLVLY 93 9 1 00 0.0002 2646
    EXP LISDMIKK 53 8 1 00 2647
    EXP LLGGVGLVLY 92 10 1 00 0.0003 2648
    EXP LVEVNKRK 64 8 1 00 2649
    EXP LVEVNKRKSK 64 10 1 00 0.0002 2650
    EXP LVEVNKRKSKY 64 11 1 00 2651
    EXP LVLYNIEK 98 8 1 00 2652
    EXP LVLYNTEKGR 98 10 1 00 0.0002 2653
    EXP LVLYNTEKGRH 98 11 1 00 2654
    EXP NLVSGPEH 155 8 1 00 2655
    EXP NNLVSGPEH 154 9 1 00 2656
    EXP NTEKGRHPFK 102 10 1 00 0.0080 2657
    EXP SGEPLIDVH 43 9 1 00 0.0002 2658
    EXP SGVSSKKK 31 8 1 00 2659
    EXP SGVSSKKKNK 31 10 1 00 0.0002 2660
    EXP SGVSSKKKNKK 31 11 1 00 2661
    EXP SLAEKTNK 20 8 1 00 2662
    EXP SSKKKNKK 34 8 1 00 2663
    EXP TGSGVSSK 29 8 1 00 2664
    EXP TGSGVSSKK 29 9 1 00 0.0016 2665
    EXP TGSGVSSKKK 29 10 1 00 0.0002 2666
    EXP VGLVLYNTEK 96 10 1 00 0.0052 2667
    EXP VLLGGVGLVLY 91 11 1 00 2668
    EXP VLYNTEKGR 99 9 1 00 0.0007 2669
    EXP VLYNTEXGRH 99 10 1 00 0.0002 2670
    EXP VNKRKSKY 67 8 1 00 2671
    EXP VNKFRKSKYK 67 9 1 00 2672
    EXP VSSKKKNK 33 8 1 00 2673
    EXP VSSKKKNKK 33 9 1 00 0.0002 2674
    EXP YNTEKGRH 101 8 1 00 2675
    EXP YNTEKGRHPFK 101 11 1 00 2676
    LSA ADTKKNLER 1632 9 1 00 2677
    LSA ADTKKNLERK 1632 10 1 00 0.0003 2678
    LSA ADTKKNLERKK 1632 11 1 00 2679
    LSA AIELPSENER 1660 10 1 00 0.0002 2680
    LSA ANEKLQBQQR 1531 10 1 00 2681
    LSA DDDDKKKY 130 8 1 00 2682
    LSA DDDDKKKYIK 130 10 1 100 0.0002 2683
    LSA DDDKKKYIK 131 9 1 100 0.0002 2684
    LSA DDEDLDEFK 1778 9 1 100 0.0002 2685
    LSA DDKKKYIK 132 8 1 100 2686
    LSA DDLDEGIEK 1817 9 1 100 0.0002 2687
    LSA DGSIKPEQK 1724 9 1 100 0.0002 2688
    LSA DIHKGHLEEK 1713 10 1 100 0.0002 2689
    LSA DIHKGHLEEXK 1713 11 1 100 2690
    LSA DITKYFMK 1901 8 1 100 2691
    LSA DLDEFKPIVQY 1781 11 1 100 2692
    LSA DLDEOIEK 1818 8 1 100 2693
    LSA DLEEKAAK 148 8 1 100 2694
    LSA DLEQDRLAK 1388 9 1 100 0.0002 2695
    LSA DLEQDRLAKEK 1388 11 1 100 2696
    LSA DLEQDRLAK 1609 9 1 100 0.0002 2697
    LSA DLEQERLAKEK 1609 11 1 100 2698
    LSA DLEQERLANEK 1524 11 1 100 2699
    LSA DLEQBIRAK 1575 9 1 100 0.0002 2700
    LSA DLEQERRAKEK 1575 11 1 100 2701
    LSA DLEQRKADTK 1626 10 1 100 0.0002 2702
    LSA DLEQRKADTKK 1626 11 1 100 2703
    LSA DLERTKASK 1184 9 1 100 0.0002 2704
    LSA DNNFKPNDK 1846 9 1 100 2705
    LSA DNRGNSRDSK 1682 10 1 100 2706
    LSA DSEQERLAK 521 9 1 100 0.0002 2707
    LSA DSEQERLAKEK 521 11 1 100 2708
    LSA DSKEISIIEK 1689 10 1 100 0.0002 2709
    LSA DTKKNLER 1633 8 1 100 2710
    LSA DTKKNLERK 1633 9 1 100 0.0002 2711
    LSA DTKKNLERKK 1633 10 1 100 0.0002 2712
    LSA DVLAEDLY 1646 8 1 100 2713
    LSA DVLAEDLYGR 1646 10 1 100 0.0002 2714
    LSA DVNDFQISK 1751 9 1 100 0.0018 2715
    LSA DVNDFQISKY 1751 10 1 100 0.0002 2716
    LSA EDDEDLDEFK 1777 10 1 100 0.0002 2717
    LSA EDEISAEY 1761 8 1 !CO 2718
    LSA EDMCYFMK 1900 9 1 100 0.0003 2719
    LSA EDKSADIQNH 1733 10 1 100 2720
    LSA EDLEEKAAK 147 9 1 100 0.0002 2721
    LSA EFKPIVQY 1784 8 1 100 2722
    LSA EGRRDIHK 1709 8 1 100 2723
    LSA EGRRDIHKGH 1709 10 1 100 0.0002 2724
    LSA EIIKSNLR 33 8 1 100 2725
    LSA EISIIEKTNR 1692 10 1 100 0.0002 2726
    LSA ELEDLIEK 1805 8 1 100 2727
    LSA ELPSENER 1662 8 1 100 2728
    LSA ELPSENERGY 1662 10 1 100 0.0002 2729
    LSA ELPSENERGYY 1662 11 1 100 2730
    LSA ELSEDDK 1897 8 1 100 2731
    LSA ELSEDITKY 1897 9 1 100 0.0002 2732
    LSA ELSEEKIK 1829 8 1 100 2733
    LSA ELSEEKIKK 1829 9 1 100 0.0002 2734
    LSA ELSEEKIKKGK 1829 11 1 100 2735
    LSA ELTMSNVK 83 8 1 100 2736
    LSA ENERGYYIPH 1666 10 1 100 2737
    LSA ENIFLKENK 108 9 1 100 2738
    LSA ENKLNKEGK 114 9 1 100 2739
    LSA ENNKFFDK 73 8 1 100 2740
    LSA ENNKFFDKDK 73 10 1 100 2741
    LSA ENRQEDLEEK 143 10 1 100 2742
    LSA ESITINVEGR 1702 10 1 100 0.0002 2743
    LSA ESITTNVEGRR 1702 11 1 100 2744
    LSA FILVNLLIFH 11 10 1 100 0.0060 2745
    LSA FLKENKLNK 111 9 1 100 0.0005 2746
    LSA GDVLAEDLY 1645 9 1 100 2747
    LSA GDVLAEDLYGR 1645 11 1 100 2748
    LSA GSIKPEQK 1725 8 1 100 2749
    LSA GSIKPEQKEDK 1725 11 1 100 2750
    LSA GSSNSRNR 42 8 1 100 2751
    LSA GVSENIFLK 105 9 1 100 0.6600 2752
    LSA HGDVLAEDLY 1644 10 1 100 0.0002 2753
    LSA HIINDDDDK 126 9 1 100 0.0002 2754
    LSA HIINDDDDKK 126 10 1 100 0.0002 2755
    LSA HIINDDDDKKK 126 11 1 100 2756
    LSA HIKKYKNDK 1860 9 1 100 0.0002 2757
    LSA HILYISFY 3 8 1 100 2758
    LSA HINGKIIK 20 8 1 100 2759
    LSA HLEEKDGSIK 1718 11 1 100 2760
    LSA HNSYEKTK 63 8 1 100 2761
    LSA HVLSHNSY 59 8 1 100 2762
    LSA HVLSHNSYEK 59 10 1 100 0.0140 2763
    LSA IFHINGKIIK 18 10 1 100 0.0006 2764
    LSA IFLKENIQNK 110 10 1 100 0.0002 2765
    LSA IINDDDDK 127 8 1 100 2766
    LSA IINDDDDKK 127 9 1 100 0.0002 2767
    LSA IINDDDDKKK 127 10 1 100 0.0002 2768
    LSA IINDDDDKKKY 127 11 1 100 2769
    LSA ILVNLLIFH 12 9 1 100 0.0008 2770
    LSA INDDDDKK 128 8 1 100 2771
    LSA INDDDDKKK 128 9 1 100 2772
    LSA INDDDDKKKY 128 10 1 100 2773
    LSA INEEKHSC 50 8 1 100 2774
    LSA INEEKHEKK 50 9 1 100 2775
    LSA INEEKNEKKH 50 10 1 100 2776
    LSA INGKIIKNSEK 21 11 1 100 2777
    LSA ISDVNDFQISK 1749 11 1 100 2778
    LSA ISIIEKTNR 1693 9 1 100 0.0008 2779
    LSA ITTNVEGR 1704 8 1 100 2780
    LSA ITTNVEGRR 1704 9 1 100 0.0007 2781
    LSA IVDELSEDMC 1894 11 1 100 2782
    LSA KADTKKNLER 1631 10 1 100 0.0002 2783
    LSA KADTKKNLERK 1631 11 1 100 2784
    LSA KDEIIKSTILR 31 10 1 100 2785
    LSA KDGSIKPEQK 1723 10 1 100 0.0002 2786
    LSA KDKELIMSNVK 80 11 1 100 2787
    LSA KDNNFKPNDK 1845 10 1 100 0.0002 2788
    LSA KFIKSLFH 1876 8 1 100 2789
    LSA KGHLEEKK 1716 8 1 100 2790
    LSA KGKKYEKIK 1837 9 1 100 0.0002 2791
    LSA KIIKNSEK 24 8 1 100 2792
    LSA KIKKGKKY 1834 8 1 100 2793
    LSA KIKKGKKYEK 1834 10 1 100 0.0007 2794
    LSA KLNKEGKLIEH 116 11 1 100 2795
    LSA KLQEQQSDLER 1177 11 1 100 2796
    LSA KNDKQVNK 1865 8 1 100 2797
    LSA KNDKQVNKEK 1865 10 1 100 2798
    LSA KNLERKKEH 1636 9 1 100 2799
    LSA KNNENNKFFDK 70 11 1 100 2800
    LSA KNSEKDEIIK 27 10 1 100 2801
    LSA KNVSQTNFK 90 9 1 100 2802
    LSA KSADIQNH 1735 8 1 100 2803
    LSA KSLYDEHIIK 1854 9 1 100 0.0340 2804
    LSA KSLYDEHIKK 1854 10 1 100 0.0490 2805
    LSA KSLYDEHIIKKY 1854 11 1 100 2806
    LSA KSSEELSEEK 1825 10 1 100 0.0009 2807
    LSA KTKDNNFK 1843 8 1 100 2808
    LSA KTKNNENNK 68 9 1 100 0.0038 2809
    LSA LAEDLYGR 1648 8 1 100 2810
    LSA LAKEKLQEQQR 1615 11 1 100 2811
    LSA LANEKLQEQQR 1530 11 1 100 2812
    LSA LDDLDEGIEK 1816 10 1 100 0.0002 2813
    LSA LDEFKPIVQY 1782 10 1 100 2814
    LSA LGVSENTIFLK 104 10 1 100 0.0063 2815
    LSA LIFHINGK 17 8 1 100 2816
    LSA LIFHINGKIIK 17 11 1 100 2817
    LSA LLIFHINGK 16 9 1 100 0.0100 2818
    LSA LNKEGKLIEH 117 10 1 100 2819
    LSA LSEDITKY 1898 8 1 100 2820
    LSA LSEDITKYFMK 1898 11 1 100 2821
    LSA LSEEKIKK 1830 8 1 100 2822
    LSA LSEEKIKKGK 1830 10 1 100 0.0002 2823
    LSA LSEEKIKKGKK 1830 11 1 100 2824
    LSA LSHNSYEK 61 8 1 100 2825
    LSA LSHNSYEKTK 61 10 1 100 0.0002 2826
    LSA LVNLLIFH 13 8 1 100 2827
    LSA NDDDDKKK 129 8 1 100 2828
    LSA NDDDDKKKY 129 9 1 100 2829
    LSA NDDDDKKKYIK 129 11 1 100 2830
    LSA NDFQISKY 1753 8 1 100 2831
    LSA NDKQVNKEK 1866 9 1 100 0.0002 2832
    LSA NDKQVNKEKEK 1866 11 1 100 2833
    LSA NDKSLYDEH 1852 9 1 100 2834
    LSA NDKSLYDEHIIK 1852 11 1 100 2835
    LSA NFKPNDKSLY 1848 10 1 100 2836
    LSA NFQDEENIGIY 1793 11 1 100 2837
    LSA NGKIIKNSEK 22 10 1 100 0.0002 2838
    LSA NIFLKENK 109 8 1 100 2839
    LSA NIFLKENKLNK 109 11 1 100 2840
    LSA NLDDLDEGIEK 1815 11 1 100 2841
    LSA NLERKKEH 1637 8 1 100 2842
    LSA NLGVSENIFLK 103 11 1 100 2843
    LSA NLLIFHINGK 15 10 1 100 0.0008 2844
    LSA NLRSGSSNSR 38 10 1 100 0.0002 2845
    LSA NNENNFFDK 71 10 1 100 2846
    LSA NNFKPNDK 1847 8 1 100 2847
    LSA NNFKPNDKSLY 1847 11 1 100 2848
    LSA NNKFFDKDK 74 9 1 100 2849
    LSA NSEKDEIIK 28 9 1 100 0.0002 2850
    LSA NSRNRINEEK 45 10 1 100 0.0002 2851
    LSA NSRNRINEEKH 45 11 1 100 2852
    ISA NVEGRRDIH 1707 9 1 100 0.0002 2853
    ISA NVEGRRDIHK 1707 10 1 100 0.0002 2854
    LSA NVKNVSQTNFK 88 11 1 100 2855
    LSA NVSQTNFK 91 8 1 100 2856
    LSA PAIELPSENER 659 11 1 100 2857
    LSA PNDKSLYDEFI 851 10 1 100 2858
    LSA PSENERGY 664 8 1 100 2859
    LSA PSENERGYY 664 9 1 100 0.0002 2860
    LSA QDEENIGIY 795 9 1 100 2861
    LSA QDEENIGIYK 795 10 1 100 0.0002 2862
    LSA QDNRGNSR 681 8 1 100 2863
    LSA QDNRGNSRDSK 681 11 1 100 2864
    LSA QDRLAKEK 391 8 1 100 2865
    LSA QGQQSDLEQER 128 11 1 100 2866
    LSA QSDLEQDR 386 8 1 100 2867
    LSA QSDLEQDRLAK 386 11 1 100 2868
    ISA QSDSEQER 590 8 1 100 2869
    LSA QSDLEQERLAK 590 11 1 100 2870
    LSA QSDLEQERR 573 9 1 100 0.0002 2871
    LSA QSDLEQERRAK 573 11 1 100 2872
    LSA QSDLERTK 182 8 1 100 2873
    LSA QSDLERTKASK 182 11 1 100 2874
    LSA QSDSEQER 519 8 1 100 2875
    LSA QSDSEQERLAK 519 11 1 100 2876
    ISA QSSLPQDNR 1676 9 1 100 0.0013 2877
    ISA QTNFKSLLR 94 9 1 100 0.0440 2878
    LSA QVNKEKEK 1869 8 1 100 2879
    LSA QVNKEKEKFIK 1869 11 1 100 2880
    ISA RDIHKGHLEEK 1712 11 1 100 2881
    ISA RDLEQERLAK 1608 10 1 100 0.0002 2882
    LSA RDLEQERR 1540 8 1 100 2883
    LSA RDLEQERRAK 1540 10 1 100 0.0002 2884
    ISA RDLEQRKADTK 1625 11 1 100 2885
    ISA RDSKEISIIEK 1688 11 1 100 2886
    LSA RGNSRDSK 1684 8 1 100 2887
    LSA RINEEKHEK 49 9 1 100 0.0370 2888
    LSA RINEEKHEKK 49 10 1 100 0.0018 2889
    ISA RINEEKHEKKH 49 11 1 100 2890
    LSA RNRINEEK 47 8 1 100 2891
    LSA RNRINEEKH 47 9 1 100 2892
    LSA RNRINEEKHEK 47 11 1 100 2893
    ISA RSGSSNSR 40 8 1 100 2894
    LSA RSGSSNSRNR 40 10 1 100 0.0002 2895
    LSA SDLEQDRLAK 387 10 1 100 0.0002 2896
    ISA SDLEQERLAK 591 10 1 100 0.0002 2897
    LSA SDLEQERR 574 8 1 100 2898
    LSA SDLEQERRAK 574 10 1 100 0.0002 2899
    LSA SDLERTKASK 183 10 1 100 0.0002 2900
    ISA SDSEQERLAK 520 10 1 100 0.0002 2901
    LSA SDVNDFQISK 750 10 1 100 0.0002 2902
    ISA SDVNDFQISKY 750 11 1 100 2903
    ISA SGSSNSRNR 41 9 1 100 0.0030 2904
    ISA SIIEKTNR 694 8 1 100 2905
    ISA SIKPEQKEDK 726 10 1 100 0.0002 2906
    LSA SITTNVEGR 703 9 1 100 0.0027 2907
    LSA SITTNVEGRR 703 10 1 100 0.0002 2908
    LSA SLPQDNRGNSR 678 11 1 100 2909
    LSA SLYDEHIK 835 8 1 100 2910
    LSA SLYDEHIKK 855 9 1 100 0.4100 2911
    LSA SLYDEHRKY 855 10 1 100 0.0045 2912
    LSA SLYDEHIKKVK 835 11 1 100 2913
    LSA SNLRSGSSNSR 37 11 1 100 2914
    LSA SNSRNRINEEK 44 11 1 100 2915
    LSA SSEELSEEK 1826 9 1 100 0.0017 2916
    ISA SSEELSEEKIK 1826 11 1 100 2917
    LSA SSLPQDNR 1677 8 1 100 2918
    LSA TNFKSLLR 95 8 1 100 2919
    LSA TNVEGRRDIH 1706 10 1 100 2920
    LSA TNVEGRRDIHK 1706 11 1 100 2921
    LSA TTNVEGRR 1705 8 1 100 2922
    LSA TINVEGRRDIH 1705 11 1 100 2923
    LSA VDELSEDTK 1895 10 1 100 0.0002 2924
    LSA VDELSEDITKY 1895 11 1 100 2925
    LSA VLAEDLYGR 1647 9 1 100 0.0004 2926
    LSA VLSHNSYEK 60 9 1 100 0.0280 2927
    LSA VLSHNSYEKTK 60 11 1 100 2928
    LSA VNDFQISK 1752 8 1 100 2929
    LSA VNDFQISKY 1752 9 1 100 2930
    LSA VNKEKEKFIK 1870 10 1 100 2931
    LSA VNLLIFHINGK 14 11 1 100 2932
    LSA VSENIFLK 106 8 1 100 2933
    LSA VSEMFLKENK 106 11 1 100 2934
    LSA VSQTNFKSLLR 92 11 1 100 2935
    LSA YDEHIKKY 1857 8 1 100 2936
    LSA YDEHIKKYK 1857 9 1 100 0.0002 2937
    LSA YFILVNLLIFH 10 11 1 100 2938
    LSA YIKGQDENR 137 9 1 100 0.0002 2939
    SSP2 ACAGLAYK 512 8 10 100 2940
    SSP2 ADSAWENVK 216 9 10 100 0.0009 2941
    SSP2 AFNRFLVGCH 197 10 10 100 2942
    SSP2 ALLACAGLAY 509 10 10 100 0.0110 2943
    SSP2 ALIACAGLAYK 509 11 10 100 2944
    SSP2 ALLQVRKH 136 8 9 90 2945
    SSP2 AVCVEVEK 233 8 10 100 2946
    SSP2 CGKGTRSR 257 8 10 100 2947
    SSP2 CGKGTRSRK 257 9 10 100 0.0002 2948
    SSP2 CCKGIRSRKR 257 10 10 100 0.0002 2949
    SSP2 CNDEVDLY 43 8 8 80 2950
    SSP2 CSGSIRRH 55 8 10 100 2951
    SSP2 CSVTCGKGTR 253 10 10 100 0.0002 2952
    SSP2 DALLQVRK 135 8 9 90 2953
    SSP2 DALLQVRKH 135 9 9 90 0.0002 2954
    SSP2 DASKNIUEK 106 8 10 100 2955
    SSP2 DCSGSIRR 54 8 10 100 2956
    SSP2 DCSGSIRRH 54 9 10 100 2957
    SSP2 DDREENFDIPK 385 11 10 100 2958
    SSP2 DIPKKPENK 392 9 10 100 0.0002 2959
    SSP2 DIPKKPENKH 392 10 10 100 0.0002 2960
    SSP2 DLDEPEQFR 546 9 10 100 0.0002 2961
    SSP2 DLFLVNGR 19 8 10 100 2962
    SSP2 DNQNNLPNDK 402 10 10 100 2963
    SSP2 CGAVIENVK 217 8 10 100 2964
    SSP2 DSIQDSLK 166 8 10 100 2965
    SSP2 DSIQDSLKESR 166 11 10 100 2966
    SSP2 DSLKESRK 170 8 9 90 2967
    SSP2 DVPKNPEDDR 378 10 10 100 0.0002 2968
    SSP2 DVQNNIVDEIK 27 11 10 100 2969
    SSP2 EDDQPRPR 300 8 10 100 2970
    SSP2 EDRETRPH 450 8 9 90 2971
    SSP2 EDRETRPHGR 450 10 9 90 2972
    SSP2 EIIRLHSDASK 99 11 10 100 2973
    SSP2 ELQEQCEEER 276 10 8 80 0.0002 2974
    SSP2 ENFDIPKK 389 8 10 100 2975
    SSP2 ENRSYNRK 462 8 10 100 2976
    SSP2 ETLGEEDK 538 8 10 100 2977
    SSP2 EVCNDEVDLY 41 10 8 80 0.0002 2978
    SSP2 EVPSDVPK 374 8 10 100 2979
    SSP2 FDEILGEEDK 536 10 10 100 0.0002 2980
    SSP2 FDIPKKPENK 391 10 10 100 0.0002 2981
    SSP2 FDIPKKPENKH 391 11 10 100 2982
    SSP2 FDLFLVNGR 18 9 10 100 2983
    SSP2 FFDLFLVNGR 17 10 10 100 2984
    SSP2 FLVGCHPSDGK 201 11 10 100 2985
    SSP2 FMKAVCVEVEK 230 11 10 100 2986
    SSP2 FNRFLVGCH 198 9 10 100 2987
    SSP2 FVVPGAATPY 520 10 8 80 0.0002 2988
    SSP2 GCHPSDGK 204 8 10 100 2989
    SSP2 GDNFAVEK 308 8 9 90 2990
    SSP2 GINVAFNR 193 8 10 100 2991
    SSP2 GIPDSIQDSLK 163 11 10 100 2992
    SSP2 GNRHVPNSEDR 442 11 10 100 2993
    SSP2 GSIRRHNWVNH 57 11 8 80 2994
    SSP2 GIRSRKREILH 260 11 10 100 2995
    SSP2 HAVPLAMK 67 8 10. 100 2996
    SSP2 HDNQNNLPNDK 401 11 10 100 2997
    SSP2 HGRNNENR 457 8 10 100 2998
    SSP2 HORNNENRSY 457 10 10 100 0.0002 2999
    SSP2 HLNDRINR 143 8 10 100 3000
    SSP2 HSDASKNK 104 8 10 100 3001
    SSP2 HSDASKNKEK 104 10 10 100 0.0002 3002
    SSP2 HVPNSEDR 445 8 10 100 3003
    SSP2 HVPNSEDRETR 445 11 9 90 3004
    SSP2 IFFDLFLVNGR 16 11 10 100 3005
    SSP2 IGQGINVAFNR 190 11 10 100 3006
    SSP2 IIRLHSDASK 100 10 10 100 0.0002 3007
    SSP2 IVDEIKYR 32 8 9 90 3008
    SSP2 KAVCVEVEK 232 9 10 100 0.0076 3009
    SSP2 KDLDEPEQFR 545 10 10 100 3010
    SSP2 KFVVPGAATPY 519 11 8 80 3011
    SSP2 KGTRSRKR 259 8 10 100 3012
    SSP2 KNVIGPFMK 224 9 10 100 3013
    SSP2 KVLDNERK 421 8 8 80 3014
    SSP2 LACAGLAY 511 8 10 100 3015
    SSP2 LACAGLAYK 511 9 10 100 0.0290 3016
    SSP2 LALLACAOLAY 508 11 10 100 3017
    SSP2 LDEPEQFR 547 8 10 100 3018
    SSP2 LLACAGLAY 510 9 10 100 0.0005 3019
    SSP2 LLACAGLAYK 510 10 10 100 0.0870 3020
    SSP2 LLMDCSGSIR 51 10 10 100 0.0005 3021
    SSP2 LLMDCSGSIRR 51 11 10 100 3022
    SSP2 LLQVRKHLNDR 137 11 9 90 3023
    SSP2 LLSTNLPY 121 8 9 90 3024
    SSP2 LLSTNLPYGR 121 10 8 80 0.0025 3025
    SSP2 LMDCSGSIR 52 9 10 100 0.0002 3026
    SSP2 LMDCSGSIRR 52 10 10 100 0.0002 3027
    SSP2 LMDCSGSIRRH 52 11 10 100 3028
    SSP2 LSTNLPYGR 122 9 8 80 0.0100 3029
    SSP2 LVGCHPSDGK 202 10 10 100 0.0002 3030
    SSP2 MDCSGSIR 53 8 10 100 3031
    SSP2 MDCSGSIRR 53 9 10 100 3032
    SSP2 MDCSGSIRRH 53 10 10 100 3033
    SSP2 MNHLGNVK 1 8 10 100 3034
    SSP2 MNHLGNVKY 1 9 10 100 3035
    SSP2 NFDIPKKPENK 390 11 10 100 3036
    SSP2 NIPEDSEK 366 8 10 100 3037
    SSP2 NIVDEIKY 31 8 10 100 3038
    SSP2 NIVDEIKYR 31 9 9 90 0.0002 3039
    SSP2 NLPNDKSDR 406 9 10 100 0.0002 3040
    SSP2 NNENFtSYNR 460 9 10 100 3041
    SSP2 NNENRSYNRK 460 10 10 100 3042
    SSP2 NNIVDEIK 30 8 10 100 3043
    SSP2 NNIVDEIKY 30 9 10 100 3044
    SSP2 NNIVDEIKYR 30 10 9 90 3045
    SSP2 NNLPNDKSDR 405 10 10 100 3046
    SSP2 NSEDRETR 448 8 9 90 3047
    SSP2 NSEDRETPPH 448 10 9 90 0.0002 3048
    SSP2 NVIGPFMK 225 8 10 100 3049
    SSP2 NVKNVIGPFMK 222 11 10 100 3050
    SSP2 PCSVTCGK 252 8 10 100 3051
    SSP2 PCSVTCGKGTR 252 11 10 100 3052
    SSP2 PDSIQDSLK 165 9 10 100 0.0002 3053
    SSP2 PFDETLGEEDK 535 11 10 100 3054
    SSP2 PNIPEDSEK 365 9 10 100 3055
    SSP2 PNSEDRETR 447 9 9 90 3056
    SSP2 PNSEDREMPFI 447 11 9 90 3057
    SSP2 PSDGKCNLY 207 9 10 100 0.0002 3058
    SSP2 PSPNPEEGK 328 9 10 100 0.0002 3059
    SSP2 QCEEERCPPK 280 10 8 80 0.0002 3060
    SSP2 QDNNGNRH 438 8 10 100 3061
    SSP2 QDSLKESR 169 8 10 100 3062
    SSP2 QDSLKESRK 169 9 9 90 0.0002 3063
    SSP2 QGINVAFNR 192 9 10 100 0.0780 3064
    SSP2 QNNIVDEIK 29 9 10 100 3065
    SSP2 QNNIVDEIKY 29 10 10 100 3066
    SSP2 QNNIVDBKYR 29 11 9 90 3067
    SSP2 QNNLPNDK 404 8 10 100 3068
    SSP2 QNNLPNDKSDR 404 11 10 100 3069
    SSP2 QSQDNNGNR 436 9 10 100 0.0002 3070
    SSP2 QSQDNNGNRH 436 10 10 100 0.0002 3071
    SSP2 QVRKHLNDR 139 9 9 90 0.0002 3072
    SSP2 RGDNFAVEK 307 9 9 90 0.0240 3073
    SSP2 FILHSDASK 102 8 10 100 3074
    SSP2 RLHSDASKNK 102 10 10 100 0.0002 3075
    SSP2 RNNENRSY 459 8 10 100 3076
    SSP2 RNNENRSYNR 459 10 10 100 3077
    SSP2 RNNENRSYNRK 459 11 10 100 3078
    SSP2 RSRKREILH 262 9 10 100 0.0002 3079
    SSP2 SDASKNKEK 105 9 10 100 0.0002 3080
    SSP2 SDGKCNLY 208 8 10 100 3081
    SSP2 SDVPKNPEDDR 377 11 10 100 3082
    SSP2 SIQDSLKESR 167 10 10 100 0.0009 3083
    SSP2 SIQDSLKESRK 167 11 9 90 3084
    SSP2 SIRRHNWVNH 58 10 8 80 0.0002 3085
    SSP2 SLLSTNLPY 120 9 9 90 0.0046 3086
    SSP2 SLLSTNLPYGR 120 11 8 80 3087
    SSP2 STNLPYGR 123 8 8 80 3088
    SSP2 SVTCGKOTFt 254 9 10 100 0.0009 3089
    SSP2 SVTCGKGTRSR 254 11 10 100 3090
    SSP2 TCGKGTRSR 256 9 10 100 3091
    SSP2 TCGKGIRSRK 256 10 10 100 0.0002 3092
    SSP2 TCGKGTRSRKR 256 11 10 100 3093
    SSP2 VAFNRFLVGCH 196 11 10 100 3094
    SSP2 VCNDEVDLY 42 9 8 80 3095
    SSP2 VGCHPSDGK 203 9 10 100 0.0003 3096
    SSP2 VNHAVPLAMK 65 10 8 80 3097
    SSP2 VTOGKGIR 255 8 10 100 3098
    SSP2 VTCGKGTRSR 255 10 10 100 0.0017 3099
    SSP2 VTCGKGIRSRK 255 11 10 100 3100
    SSP2 VVPGAATPY 521 9 8 80 0.0002 3101
    SSP2 WSPCSVTCGK 250 10 10 100 0.0002 3102
    SSP2 WVNHAVPLAMK 64 11 8 80 3103
    SSP2 YADSAWENVK 215 10 10 100 0.0002 3104
    SSP2 YLLMDCSGSIR 50 11 10 100 3105
  • TABLE XVIII
    Malaria A24 Motif Peptides With Binding Information
    No. of Sequence Conservancy
    Protein Sequence Position Amino Acids Frequency (%) A*2401 Seq. Id
    CSP CYGSSSNTRVL 25 11 19 100 3106
    CSP DYENDREKKI 398 10 18 95 3107
    CSP EMNYYGKQENW 52 11 19 100 3108
    CSP IMVLSFLF 427 8 19 100 3109
    CSP IMVLSFLFL 427 9 19 100 0.0008 3110
    CSP KMEKCSSVF 409 9 19 100 3111
    CSP MMRKLAIL 1 8 19 100 3112
    CSP NYDNAGINL 39 9 18 100 0.0004 3113
    CSP NYYGKQENW 54 9 19 100 3114
    CSP SFLFVEAL 12 8 19 100 3115
    CSP SFLFVEALF 12 9 19 100 3116
    CSP VFNVVNSSI 416 9 19 100 3117
    CSP VFNVVNSSIGL 416 11 19 100 3118
    CSP WYSLKKNSRSL 62 11 19 100 3119
    CSP YYGKQENW 55 8 19 100 3120
    CSP YYGKQENWYSL 55 11 19 100 3121
    EXP DMIKKEEEL 56 9 1 100 3122
    EXP FFIIFNKESL 12 10 1 100 3123
    EXP FFLALFFI 7 8 1 100 3124
    EXP FFLALFFII 7 9 1 100 3125
    EXP FFLALFFIIF 7 10 1 100 3126
    EXP KYKLATSVL 73 9 1 100 0.0960 3127
    EXP LFFIIFNKESL 11 11 1 100 3128
    EXP LYNTEKGRHPF 100 11 1 100 3129
    EXP VFFLALFF 6 8 1 100 3130
    EXP VFFLALFFI 6 9 1 100 3131
    EXP VFFLALFFII 6 10 1 100 3132
    EXP VFFLALFFIIF 6 11 1 100 3133
    LSA DFQISKYEDEI 1754 11 1 100 3134
    LSA EFKPIVQYDNF 1784 11 1 100 3135
    LSA FFDKDKEL 77 8 1 100 3136
    LSA FYFILVNL 9 8 1 100 3137
    LSA FYFILVNLL 9 9 1 100 7.5000 3138
    LSA FYFILVNLLI 9 10 1 100 3139
    LSA FYFILVNLLIF 9 11 1 100 3140
    LSA GYYIPHQSSL 1670 10 1 100 0.0074 3141
    LSA IFDGDNEI 1884 8 1 100 3142
    LSA IFDGDNEIL 1884 9 1 100 3143
    LSA IFDGDNEILQI 1884 11 1 100 3144
    LSA IFHINGKI 18 8 1 100 3145
    LSA IFHINGKII 18 9 1 100 3146
    LSA IFLKENKL 110 8 1 100 3147
    LSA IYKELEDL 1802 8 1 100 3148
    LSA IYKELEDLI 1802 9 1 100 3149
    LSA KFFDKDKEL 76 9 1 100 3150
    LSA KFIKSLFHI 1876 9 1 100 3151
    LSA KFIKSLFHIF 1876 10 1 100 3152
    LSA KYEKTKDNNF 1840 10 1 100 0.0004 3153
    LSA LFHIFDGDNEI 1881 11 1 100 3154
    LSA LYGRLETPAI 1652 10 1 100 3155
    LSA LYISFYFI 5 8 1 100 3156
    LSA LYISFYFIL 5 9 1 100 0.0088 3157
    LSA NFKPNDKSL 1848 9 1 100 3158
    LSA NFKSLLRNL 96 9 1 100 3159
    LSA NFQCEENI 1793 8 1 100 3160
    LSA NFQDEENIGI 1793 10 1 100 3161
    LSA QYDNFQDEENI 1790 11 1 100 3162
    LSA SFYFILVNL 8 9 1 100 3163
    LSA SFYFILVNLL 8 10 1 100 3164
    LSA SFYFILVNLLI 8 11 1 100 3165
    LSA YFILVNLL 10 8 1 100 3166
    LSA YFILVNLLI 10 9 1 100 3167
    LSA YFILVNLLIF 10 10 1 100 3168
    LSA YYIPHQSSL 1671 9 1 100 4.3000 3169
    SSP2 AMKLIQQL 72 8 10 100 3170
    SSP2 AMKLIQQLNL 72 10 10 100 0.0006 3171
    SSP2 AWENVKNVI 219 9 10 100 3172
    SSP2 KYKIAGGI 497 8 9 90 3173
    SSP2 KYLVIVFL 8 8 10 100 3174
    SSP2 KYLVIVFLI 8 9 10 100 4.6000 3175
    SSP2 KYLVIVFLIF 8 10 10 100 0.0003 3176
    SSP2 KYLVIVFLIFF 8 1 1 10 100 3177
    SSP2 LMDCSGSI 52 8 10 100 3178
    SSP2 LYLLMDCSGSI 49 11 9 90 3179
    SSP2 NWVNHAVPL 63 9 8 80 3180
    SSP2 PYAGEPAPF 528 9 8 80 0.0370 3181
    SSP2 QFRLPEENEW 552 1 0 10 100 3182
    SSP2 VFGIGQGI 187 8 10 100 3183
    SSP2 VFLIFFDL 13 8 10 100 3184
    SSP2 VFLIFFDLF 13 9 10 100 3185
    SSP2 VFLIFFDLFL 13 10 10 100 3186
  • TABLE XIXa
    Core Core
    Core SeqID Core Conservancy Exemplary
    Protein Sequence Num Frequency (%) Sequence
    CSP FLFVEALFQ 3187 19 100 VSSFLFVEALFQEYQ
    CSP FNVVNSSIG 3188 19 100 SSVFNVVNSSIGLIM
    CSP FQEYQCYGS 3189 19 100 EALFQEYQCYGSSSN
    CSP IEKKICKME 3190 19 100 ENDIEKKICKMEKCS
    CSP IGLIMVLSF 3191 19 100 NSSIGLIMVLSFLFL
    CSP ILSVSSFLF 3192 19 100 KLAILSVSSFLFVEA
    CSP LAILSVSSF 3193 19 100 MRKLAILSVSSFLFV
    CSP MEKCSSVFN 3194 19 100 ICKMEKCSSVFNVVN
    CSP VVNSSIGLI 3195 19 100 VFNVVNSSIGLIMVL
    CSP YQCYGSSSN 3196 19 100 FQEYQCYGSSSNTRV
    CSP YNELEMNYY 3197 19 100 INLYNELEMNYYGKQ
    CSP YDNAGINLY 3198 18  95 ELNYDNAGINLYNEL
    CSP IQNSLSTEW 3199 15  79 LKKIQNSLSTEWSPC
    CSP WSPCSVTCG 3200 10 100 STEWSPCSVTCGNGI
    LSA FILVNLLIF 3201  1 100 SFYFILVNLLIFHIN
    LSA FYFILVNLL 3202  1 100 YISFYFILVNLLIFH
    LSA IHKGHLEEK 3203  1 100 RRDIHKGHLEEKKDG
    LSA IIKSNLRSG 3204  1 100 KDEIIKSNLRSGSSN
    LSA ILVNLLIFH 3205  1 100 FYFILVNLLIFHING
    LSA INGKIIKNS 3206  1 100 IFHINGKIIKNSEKD
    LSA IPAIELPSE 3207  1 100 RLEIPAIELPSENER
    LSA IPHQSSLPQ 3208  1 100 QYYIPHQSSLPQDNR
    LSA IQNHTLETV 3209  1 100 SADIQNHTLETVNIS
    LSA ISFYFILVN 3210  1 100 ILYISFYFILVNLLI
    LSA LDEFKPIVQ 3211  1 100 DEDLDEFKPIVQYDN
    LSA LEEKAAKET 3212  1 100 QEDLEEKAAKETLQG
    LSA LEEPAIELP 3213  1 100 YGRLEEPAIELPSEN
    LSA LEQRKADTK 3214  1 100 QRDLEQRKADTKKNL
    LSA LERTKASKE 3215  1 100 QSDLERTKASKETLQ
    LSA LETVNISDV 3216  1 100 NHTLETVNISDVNDF
    LSA LIEFIENDD 3217  1 100 EGKLIEHIINDDDDK
    LSA LKENKLNKE 3218  1 100 NIFLKENKLNKEGKL
    LSA LLIFHINGK 3219  1 100 LVNLLIFHINGKIIK
    LSA LQEQQSDLE 3220  1 100 KETLQEQQSDLEQER
    LSA LQEQQSDSE 3221  1 100 KEKLQEQQSDSEQER
    LSA LQGQQSDLE 3222  1 100 KETLQGQQSDLEQER
    LSA LRNLGVSEN 3223  1 100 KSLLRNLGVSENIFL
    LSA LRSGSSNSR 3224  1 100 KSNLRSGSSNSRNRI
    LSA LTMSNVKNV 3225  1 100 DKELTMSNVKNVSQT
    LSA LVNLLXFHI 3226  1 100 YFILVNLLIFHINGK
    LSA VLSHNSYEK 3227  1 100 KKHVLSHNSYEKTKN
    LSA VNDFQISKY 3228  1 100 ISDVNDFQISKYEDE
    LSA VNISDVNDF 3229  1 100 LETVNISDVNDFQIS
    LSA YDDSLIDEE 3230  1 100 SAEYDDSLIDEEEDD
    LSA YGRLEIPAI 3231  1 100 EDLYGRLEIPAIELP
    LSA YIPHQSSLP 3232  1 100 RGYYIPHQSSLPQDN
    EXP FIUGSSDPA 3233  1 100 RHPFKIGSSDPADNA
    EXP IDVHDLISD 3234  1 100 EPLIDVHDLISDMIK
    EXP IFNKESLAE 3235  1 100 FFIIFNKESLAEKTN
    EXP IGSSDPADN 3236  1 100 PFIUGSSDPADNANP
    EXP LALFFIIFN 3237  1 100 VFFLALFFIIFNKES
    EXP LATSVLAGL 3238  1 100 KYKLATSVLAGLLGN
    EXP LGGVGLVLY 3239  1 100 TVLLGGVGLVLYNTE
    EXP LGNVSTVLL 3240  I 100 AGLLGNVSTVLLGGV
    EXP LLGNVSTVL 3241  1 100 LAGLLGNVSTVLLGG
    EXP LSVFFLALF 3242  1 100 MKILSVFFLALFFII
    EXP LVLYNTEKG 3243  1 100 GVOLVLYNTEKGRHP
    EXP VFFLALFFI 3244  1 100 ILSVFFLALFFIIFN
    EXP VHDLISDMI 3245  1 100 LIDVHDLISDMIKKE
    EXP VLAGLLGNV 3246  1 100 ATSVLAGLLGNVSTV
    EXP VLLGGVGLV 3247  1 100 VSTVLLGGVGLVLYN
    EXP VNKRKSKYK 3248  1 100 LVEVNKRKSKYKLAT
    EXP VSTVLLGGV 3249  1 100 LGNVSTVLLGGVGLV
    EXP VTAQDVTPE 3250  1 100 DPQVTAQDVTPEQPQ
    EXP YKLATSVLA 3251  1 100 KSKYKLATSVLAGLL
    SSP2 FDLFLVNGR 3252 10 100 LIFFDLFLVNGRDVQ
    SSP2 FFDLFLVNG 3253 10 100 FLIFFDLFLVNGRDV
    SSP2 FMKAVCVEV 3254 10 100 IGPFMKAVCVEVEKT
    SSP2 FNRFLVGCH 3255 10 100 NVAFNRFLVGCHPSD
    SSP2 IAGGLALLA 3256 10 100 AGGIAGGLALLACAG
    SSP2 IAVFGIGQG 3257 10 100 GVKIAVFGIGQGINV
    SSP2 LACAGLAYK 3258 10 100 LALLACAGLAYKFVV
    SSP2 LALLACAGL 3259 10 100 AGGLALLACAGLAYK
    SSP2 LAMKLIQQL 3260 10 100 AVPLAMKLIQQLNLN
    SSP2 LAYKFVVPG 3261 10 100 CAGLAYKFVVPGAAT
    SSP2 LIFFDLFLV 3262 10 100 IVFLIFFDLFLVNGR
    SSP2 LTDGIPDSI 3263 10 100 VVILTDGIPDSIQDS
    SSP2 LVGCHPSDG 3264 10 100 NRFLVGCHPSDGKCN
    SSP2 LVIVFLIFF 3265 10 100 VKYLVTVFLIFFDLF
    SSP2 LVVILTDGI 3266 10 100 ANQLVVILTDGIPDS
    SSP2 MDCSGSIRR 3267 10 100 YLLMDCSGSIRRHNW
    SSP2 MKAVCVEVE 3268 10 100 GPFMKAVCVEVEKTA
    SSP2 VEKTASCGV 3269 10 100 CVEVEKTASCGVWDE
    SSP2 VGCHPSDGK 3270 10 100 RFLVGCHPSDGKCNL
    SSP2 VIGPFMKAV 3271 10 100 VKNVIGPFMKAVCVE
    SSP2 VIVFLIFFD 3272 10 100 KYLVIVFLIFFDLFL
    SSP2 VKYLVIVFL 3273 10 100 LGNVKYLVIVFLIFF
    SSP2 VNGRDVQNN 3274 10 100 LFLVNGRDVQNNIVD
    SSP2 WDEWSPCSV 3275 10 100 CGVWDEWSPCSVTCG
    SSP2 IAGGIAGGL 3276 10 100 KYKIAGGIAGGLALL
    SSP2 VQNNIVDEI 3277 10 100 GRDVQNNIVDEIKYR
    SSP2 YLLMDCSGS 3278 10 100 VDLYLLMDCSGSIRR
    SSP2 FVVPGAATP 3279 10 100 AYKFVVPGAATPYAG
    SSP2 YKFVVPGAA 3280 10 100 GLAYKFVVPGAATPY
    SSP2 IIRLHSDAS 3281 10 100 AKEIIRLHSDASKNK
    SSP2 IIDNNPQEP 3282 10 100 EENIIDNNPQEPSPN
    SSP2 VDLYLLMDC 3283  9  90 NDEVDLYLLMDCSGS
    SSP2 LLSTNLPYG 3284  9  90 IKSLLSTNLPYGRTN
    SSP2 LHEGCTSEL 3285  8  80 REILHEGCTSELQEQ
    SSP2 VNHAVPLAM 3286  8  80 HNWVNHAVPLAMKLI
    SSP2 VPGAATPYA 3287  8  80 KFVVPGAATPYAGEP
    SSP2 VVPGAATPY 3288  8  80 YKFVVPGAATPYAGE
    SSP2 WVNHAVPLA 3289  8  80 REINWYNHAVPLAMKL
    SSP2 LSTNLPYGR 3290  8  80 KSLLSTNLPYGRTNL
    Position  Exemplary Exemplary 
    Exemplary In PF Sequence Sequence
    Protein SeqID Num Poly-Protein Frequency Conservancy (%)
    CSP 3291 10 19 100
    CSP 3292 440 19 100
    CSP 3293 17 19 100
    CSP 3294 426 19 100
    CSP 3295 447 19 100
    CSP 3296 4 19 100
    CSP 3297 2 19 100
    CSP 3298 433 19 100
    CSP 3299 442 19 100
    CSP 3300 20 19 100
    CSP 3301 45 18 95
    CSP 3302 37 18 95
    CSP 3303 385 15 79
    CSP 3304 393 19 100
    LSA 3305 8 1 100
    LSA 3306 6 1 100
    LSA 3307 1711 1 100
    LSA 3308 31 1 100
    LSA 3309 9 1 100
    LSA 3310 18 1 100
    LSA 3311 1655 1 100
    LSA 3312 1670 1 100
    LSA 3313 1736 1 100
    LSA 3314 4 1 100
    LSA 3315 1779 1 100
    LSA 3316 146 1 100
    LSA 3317 1653 1 100
    LSA 3318 1624 1 100
    LSA 3319 1182 1 100
    LSA 3320 1741 1 100
    LSA 3321 120 1 100
    LSA 3322 109 1 100
    LSA 3323 13 1 100
    LSA 3324 1192 1 100
    LSA 3325 512 1 100
    LSA 3326 155 I 100
    LSA 3327 98 1 100
    LSA 3328 36 1 100
    LSA 3329 81 1 100
    LSA 3330 10 1 100
    LSA 3331 57 1 100
    LSA 3332 1749 1 100
    LSA 3333 1744 1 100
    LSA 3334 1765 1 100
    LSA 3335 1650 1 100
    LSA 3336 1669 1 100
    EXP 3337 107 1 100
    EXP 3338 45 1 100
    EXP 3339 12 1 100
    EXP 3340 109 1 100
    EXP 3341 6 1 100
    EXP 3342 73 1 100
    EXP 3343 90 1 100
    EXP 3344 82 1 100
    EXP 3345 81 1 100
    EXP 3346 1 1 100
    EXP 3347 95 1 100
    EXP 3348 3 1 100
    EXP 3349 47 1 100
    EXP 3350 77 1 100
    EXP 3351 88 1 100
    EXP 3352 64 1 100
    EXP 3353 85 1 100
    EXP 3574 136 1 100
    EXP 3354 71 1 100
    SSP2 3355 15 10 100
    SSP2 3356 14 10 100
    SSP2 3357 227 10 100
    SSP2 3358 195 10 100
    SSP2 3359 513 10 100
    SSP2 3360 182 10 100
    SSP2 3361 520 10 100
    SSP2 3362 517 10 100
    SSP2 3363 68 10 100
    SSP2 3364 525 10 100
    SSP2 3365 12 10 100
    SSP2 3366 157 10 100
    SSP2 3367 199 10 100
    SSP2 3368 7 10 100
    SSP2 3369 153 10 100
    SSP2 3370 50 10 100
    SSP2 3371 228 10 100
    SSP2 3372 235 10 100
    SSP2 3373 200 10 100
    SSP2 3374 223 10 100
    SSP2 3375 8 10 100
    SSP2 3376 4 10 100
    SSP2 3377 20 10 100
    SSP2 3378 244 10 100
    SSP2 3379 509 9  90
    SSP2 3380 25 9  90
    SSP2 3381 47 9  90
    SSP2 3382 529 8  80
    SSP2 3383 527 8  80
    SSP2 3384 97 6  60
    SSP2 3385 317 4  40
    SSP2 3386 44 8  80
    SSP2 3387 118 5  50
    SSP2 3388 266 8  80
    SSP2 3389 62 8  80
    SSP2 3390 531 8  80
    SSP2 3391 530 8  80
    SSP2 3392 61 8  80
    SSP2 3393 119 5  50
  • TABLE XIXb
    Malaria Super Motif Peptides With Binding Data
    Core
    Core SeqID Exemplary Exemplary
    Sequence Num Sequence SeqID Num DR1 DR2w2β1
    FLFVEALFQ 3187 VSSFLFVEALFQEYQ 3291
    FNVVNSSIG 3188 SSVFNVVNSSIGLIM 3292 0.1200 0.0290
    FQEYQCYGS 3189 EQLFQEYQCYGSSSN 3293 0.0001
    IEKKICKME 3190 ENDIEKKICKMEKCS 3294
    IGLIMVLSF 3191 NSSIGLIMVLSFLFL 3295 0.0040 0.0250
    ILSVSSFLF 3192 KLAILSVSSFLFVEA 3296
    LAILSVSSF 3193 MRKLAILSVSSFLFV 3297 0.1000 0.5000
    MEKCSSVFN 3194 ICKMEKCSSVFNVVN 3298
    VVNSSGILI 3195 VFNVVNSSIGLIMVL 3299 0.0310 0.0021
    YQCYGSSSN 3196 FQEYQCYGSSSNTRV 3300
    YNELEMNYY 3197 INLYNELEMNYYGKQ 3301
    YDNAGINLY 3198 ELNYDNAGINLYNEL 3302 0.0003
    IQNSLSTEW 3199 LKKIQNSLSTEWSPC 3303
    WSPSCSVTCG 3200 STEWSPCSVTCGNGI 3304
    FILVNLLIF 3201 SFYFILVNLLIFHIN 3305 0.0009 0.0100
    FYFILVNLL 3202 YISFYFILVNLLIGH 3306 0.0029 0.0040
    IHKGHLEEK 3203 RRDIHKGHLEEKKDG 3307
    IIKSNLRSG 3204 KDEIIKSNLRSGSSN 3308
    ILVNLLIFH 3205 FYFILVNLLIFHING 3309
    INGKIIKNS 3206 IFHINGKIIKNSEKD 3310 0.0320 0.0220
    IPAIELPSE 3207 RLEIPAIELPSENER 3311
    IPHQSSLPQ 3208 GYYIPHQSSLPQDNR 3312
    IQNHTLETV 3209 SADIQNHTLETVNIS 3313 0.0001
    ISFYFILVN 3210 ILYISFYFILVNLLI 3314
    LDEFKPIQ 3211 DEDLDEFKPIVQYDN 3315
    LEEKAAKET 3212 QEDLEEKAAKETLQG 3316 0.0001
    LEIPAIELP 3213 YGRLEIPAIELPSEN 3317
    LEQRKADTK 3214 QRDLEQRKADTKKNL 3318
    LERTKASKE 3215 QDDLERTKASKETLQ 3319
    LETVNISDV 3216 NGTLETVNISDVNDF 3320 0.0001
    LIEHIINDD 3217 EGKLIEHIINDDDDK 3321
    LKENKLNKE 3218 NIFLKEKLNKEGKL 3322
    LLIFHINGK 3219 LVNLLIFHINGKIIK 3323 0.0640 0.7100
    LQEQQSDLE 3220 KETLQEQQSDLEQER 3324
    LQEQQSESE 3221 KEKLQEQQSDSEQER 3325
    LQGQQSDLE 3222 KETLQGQQSDLEQER 3326
    LRNTLGVSEN 3223 KSLLRNLGVSENIFL 3327 0.0150 0.0088
    LRSGSSNSR 3224 KSNLRSGSSNSRNRI 3328
    LTMSNVKNV 3225 DKELTMSNVKNVSQT 3329 0.0018 0.0003
    LVNLLIFHI 3226 YFILVNLLIFHINGK 3330 0.0018 0.0004
    VLSHNSYEK 3227 KKHVLSHNSYEKTKN 3331
    VNDFQISKY 3228 ISDVNDFQISKYEDE 3332 0.0001
    VNISKVNDF 3229 LETVNISDVNDFQIS 3333
    YDDSLIDEE 3230 SAEYDDSLIDEEEDD 3334
    YGRLEIPAI 3231 EDLYGRLEIPAIELP 3335 0.0004
    YIPHQSSLP 3232 RGYYIPHQSSLPQDN 3336 0.2900 0.0004
    FKIGSSDPA 3233 RHPFKIGSSDPADNA 3337 0.0044 -0.0004
    IDVHDLISH 3234 EPLIDVHDLISDMIK 3338
    IFNKESLAE 3235 FFIIFNKESLAEKTN 3339
    IGSSDPADN 3236 PFKIGSSDPADNANP 3340
    LALFFIIFN 3237 VFFALFFIIFNKES 3341 0.0006 0.0180
    LATSVLAGL 3238 KYKLATSVLAGLLGN 3342 1.2000 0.0018
    LGGVGLVLY 3239 TVLLGGVGLVLYNTE 3343 0.4900
    LGNVSTVLL 3240 AGLLGNVSTVLLGGV 3344 0.0430 0.0240
    LLGNVSTVL 3241 LAGLLGNVSTVLLGG 3345 0.0420 0.0110
    LSVFFLALF 3242 MKILSVFFLALFFII 3346 0.0017 0.0170
    LVLYNTEKG 3243 GVGLVLYNTEKGRHP 3347
    VFFLALFFI 3244 ILSVFFLALFFIIFN 3348 0.0016 0.0036
    VHDLISDMI 3245 LIDVHDLISDMIKKE 3349 0.0130
    VLAGLLGNV 3246 ATSVLAGLLGNVSTV 3350 0.2600
    VLLGGVGLV 3247 VSTVLLGGVGLVLYN 3351 0.8800 0.0080
    VNKRKSKYK 3248 LVEVNKRKSKYKLAT 3352
    VSTVLLGGV 3249 LGNVSTVLLGGVGLV 3353 0.0140 0.0001
    VTAQDVTPE 3250 DPQVTAQDVTPEQPQ 3574
    YKLATSVLA 3251 KSKYKLATSVLAGLL 3354 1.4000 0.0073
    FDLFLVNGR 3252 LIFFDLFLVNGRDVQ 3355 0.0042
    FFDLFLVNG 3253 FLIFFDLFLVNGRDV 3356
    FMKAVCVEV 3254 IGPFMKAVCVEVEKT 3357 0.0072 0.0003
    FNRFLVGCH 3255 NVAFNRFLVGCHPSD 3358
    IAGGLALLA 3256 AGGIAGGLALLACAG 3359 0.0160
    IAVFGIGQG 3257 GVKIAVFGIGQGINV 3360
    LACAGLAYK 3258 LALLACAGLAYKFVV 3361
    LALLACAGL 3259 AGGLALLACAGLAYK 3362 0.0018
    LAMKLIQQL 3260 AVPLAMKLIQQLNTN 3363 0.0015
    LAYKFVVPG 3261 CAGLAYKFVVPGAAT 3364
    LIFFDLFLV 3262 IVFLIFFDLFLVNGR 3365 0.0006 0.0048
    LTDGIPDSI 3263 VVILTDGIPDSIQDS 3366 0.0001
    LVGCHPSDG 3264 NRFLVGCHPSDGKCN 3367
    LVTVFLIFF 3265 VKYLVTVFLIFFDLF 3368 0.0001
    LVVILTDGI 3266 ANQLVVILTDGIPDS 3379 0.0038 0.0008
    MDCSGSIRR 3267 YLLMDCSGSIRRHNW 3370
    MKAVCVEVE 3268 GPFMKAVCVEVEKTA 3371
    VEKTASCGV 3269 CVEVEKTASCGVWDE 3372 0.0004
    VGCHPSDGK 3270 RFLVGCHPSDGKCNL 3373
    VIGPFMKAV 3271 VKNVIGPFMKAVCVE 3374 0.0900 0.0430
    VIVFLIFFD 3272 KYVTVFLIFFDLFL 3375 0.0012 0.0057
    VKYLVTVFL 3273 LGNVKYLVTVFLIFF 3376 0.0006 0.0033
    VNGRDVQNN 3274 LFLVNGRDVQNNTVD 3377
    WDEWSPCSV 3275 CGVWDEWSPCSVTCG 3378 0.0001
    IAGGIAGGL 3276 KYKIAGGIAGGLALL 3389 0.0380 0.0001
    YQNNIVDEI 3277 GRDVQNNIVDEIKYR 3380 0.0001 0.0001
    YLLMDCSGS 3278 VDLYLLMDCSGSIRR 3381 0.0015
    FVVPGAATP 3279 AYKFVVPGAATPYAG 3382 0.3600 -0.0009
    YKFVVPGAA 3280 GLAYKFVVPGAATPY 3383 1.6000 0.0001
    IIRLHSDAS 3281 AKEIIRLHSDASKNK 3384
    IIDNNPQEP 3282 EENIIDNNPQEPSPN 3385
    VDLYLLMDC 3283 NDEVDLYLLMDCSGS 3386 0.0001
    LLSTNLPYG 3284 IKSLSSTNLPYGRTN 3387
    LHEGCTSEL 3285 REILHEGCTSELQEQ 3388 0.0001
    VNHAVPLAM 3286 HNWVNHAVPLAMKLI 3389 0.3500 0.0250
    VPGAATPYA 3287 KFVVPGAATPYAGEP 3390 0.0230 0.0001
    VVPGAATPY 3288 YKFVVPGAATPYAGE 3391 0.1100 0.0008
    WVNHAVPLA 3289 RHNWVNHAVPLAMKL 3392 0.1900 0.0350
    LSTNLPYGR 3290 KSLLSTNLPYGRTNL 3393 0.0012
    Core
    Sequence DR2w2β2 DR3 DR4w4 DR4w15 DR5w11 DR5w12
    FLFVEALFQ
    FNVVNSSIG 0.0050 -0.0043 0.1000 0.230 0.0170 0.0051
    FQEYQCYGS -0.0005 0.0053 -0.0009 -0.0002 0.0001
    IEKKICKME
    IGLIMVLSF 0.0024 -0.0043 0.0120 0.0035 -0.0005 0.0340
    ILSVSSFLF
    LAILSVSSF 0.0130 -0.0043 0.0078 0.0270 0.0370 0.1200
    MEKCSSVFN
    VVNSSGILI 0.0006 0.0021 0.0079 0.0056 0.0002 0.0015
    YQCYGSSSN
    YNELEMNYY
    YDNAGINLY -0.0005 0.0091 -0.0009 -0.0009 -0.0002 0.0001
    IQNSLSTEW
    WSPSCSVTCG
    FILVNLLIF -0.0020 -0.0043 0.0250 0.0038 -0.0005 0.0009
    FYFILVNLL 0.0044 -0.0008 0.0210 -0.0009 0.0011 0.0006
    IHKGHLEEK
    IIKSNLRSG
    ILVNLLIFH
    INGKIIKNS 0.0660 0.0120 -0.0007 0.0038 0.0380 0.0055
    IPAIELPSE
    IPHQSSLPQ
    IQNHTLETV -0.0005 -0.0041 -0.0007 -0.0014 -0.0002 0.0001
    ISFYFILVN
    LDEFKPIQ
    LEEKAAKET -0.0005 -0.0009 -0.0009 -0.0002 0.0001
    LEIPAIELP
    LEQRKADTK
    LERTKASKE
    LETVNISDV -0.0005 -0.0007 0.0016 -0.0002 0.0015
    LIEHIINDD
    LKENKLNKE
    LLIFHINGK 0.0070 -0.0043 0.0110 -0.0030 0.2700 0.0410
    LQEQQSDLE
    LQEQQSESE
    LQGQQSDLE
    LRNTLGVSEN 0.0006 0.0210 0.0810 0.0033
    LRSGSSNSR
    LTMSNVKNV 0.0009 0.0058 0.0023 0.0074 0.0030 0.0001
    LVNLLIFHI 0.0120 -0.008 0.0160 0.0027 0.0015 0.0006
    VLSHNSYEK
    VNDFQISKY -0.0005 -0.0007 -0.0014 -0.0002 0.0001
    VNISKVNDF
    YDDSLIDEE
    YGRLEIPAI -0.0005 -0.0007 0.0170 -0.0002 0.0002
    YIPHQSSLP 0.0029 4.1000 0.2800 0.0064
    FKIGSSDPA -0.0003 -0.0008 0.4700 0.0029 0.0056 0.0001
    IDVHDLISH
    IFNKESLAE
    IGSSDPADN
    LALFFIIFN -0.0021 -0.0043 0.0047 0.0100 -0.0005 0.0002
    LATSVLAGL 0.0700 0.0010 3.2000 0.1200 0.0210 0.0073
    LGGVGLVLY -0.005 0.0032 -0.0009 -0.0062 0.0004
    LGNVSTVLL 0.0013 0.0059 0.0065 0.0360 0.0005 0.0001
    LLGNVSTVL 0.0006 0.0078 0.0160 0.0230 0.0004 0.0003
    LSVFFLALF -0.0021 -0.0043 0.0370 -0.0047 -0.0010 0.0023
    LVLYNTEKG
    VFFLALFFI 0.0091 -0.0008 0.0130 -0.0009 0.0012 0.0008
    VHDLISDMI 0.0061 0.0100 0.0310 0.0075 0.0037 0.0001
    VLAGLLGNV -0.0005 0.0021 -0.0014 0.0008 0.0043
    VLLGGVGLV 0.0005 -0.0008 0.0067 -0.0009 0.0003 0.0011
    VNKRKSKYK
    VSTVLLGGV -0.0005 -0.0008 0.0016 -0.0014 -0.0002 0.0005
    VTAQDVTPE
    YKLATSVLA 0.8500 -0.0008 6.3000 0.8100 0.6700 0.0009
    FDLFLVNGR 0.0036
    FFDLFLVNG
    FMKAVCVEV 0.0430 -0.0008 -0.0006 0.0086 -0.0004 0.0038
    FNRFLVGCH
    IAGGLALLA 0.0013 0.0014 0.0014 -0.0002 0.0007
    IAVFGIGQG
    LACAGLAYK
    LALLACAGL 0.0013 -0.0007 -0.0014 -0.0002 0.0051
    LAMKLIQQL -0.0006 0.0023 0.0013 0.0002 0.1300
    LAYKFVVPG
    LIFFDLFLV 0.0019 -0.0008 0.0130 -0.0009 0.0019 0.0016
    LTDGIPDSI -0.0006 0.1200 -0.0014 -0.0004 0.0001
    LVGCHPSDG
    LVTVFLIFF 0.0030
    LVVILTDGI -0.0005 0.0019 0.0460 0.0062 -0.0002 0.0003
    MDCSGSIRR
    MKAVCVEVE
    VEKTASCGV -0.0009 0.0021 -0.0009 -0.0002 0.0001
    VGCHPSDGK
    VIGPFMKAV 0.0800 -0.0026 -0.0020 -0.0030 0.3420 0.0920
    VIVFLIFFD -0.0020 -0.0043 0.0680 -0.0030 -0.0009 0.0021
    VKYLVTVFL 0.0012 -0.0008 0.0120 0.0045 0.0018 0.0011
    VNGRDVQNN
    WDEWSPCSV -0.0006 -0.0007 -0.0014 -0.0002 0.0001
    IAGGIAGGL 0.0480 0.0250 0.0120 0.0017 0.2300 0.3600
    YQNNIVDEI -0.0006 0.0026 -0.0006 -0.0014 -0.0004 0.0001
    YLLMDCSGS 0.0096 0.0150 -0.0014 -0.0004 0.0001
    FVVPGAATP 0.0620 0.1600 0.0036 0.6400 0.1200
    YKFVVPGAA 0.7000 -0.0008 1.0000 0.0270 1.9000 0.3500
    IIRLHSDAS
    IIDNNPQEP
    VDLYLLMDC -0.0005 0.0028 -0.0009 -0.0002 0.0001
    LLSTNLPYG
    LHEGCTSEL -0.0005 -0.0041 -0.0009 -0.0014 -0.0002 0.0001
    VNHAVPLAM 0.1400 0.2300 3.900 0.0400 0.0074 0.6000
    VPGAATPYA 0.0010 0.0620 0.1200 0.0067 0.0010 0.0860
    VVPGAATPY 0.0053 -0.0008 0.0057 -0.0014 0.0036 0.0061
    WVNHAVPLA 0.1600 0.4000 5.0000 0.0360 0.0079 0.0240
    LSTNLPYGR 0.0120
    Core Seq Exemplary Exemplary
    Sequence Id. Sequence SeqID Num DR6w19 DR7 DR8w2 DR9 DRw53
    FLFVEALFQ 3187 VSSFLFVEALFQEYQ 3291
    FNVVNSSIG 3188 SSVFNVVNSSIGLIM 3292 0.3600 0.7600 0.0550 1.2000
     
    FQEYQCYGS 3189 EQLFQEYQCYGSSSN 3293 -0.0003 0.0005
    IEKKICKME 3190 ENDIEKKICKMEKCS 3294
    IGLIMVLSF 3191 NSSIGLIMVLSFLFL 3295 0.0009 0.0690 -0.0010 0.0042
    ILSVSSFLF 3192 KLAILSVSSFLFVEA 3296
    LAILSVSSF 3193 MRKLAILSVSSFLFV 3297 0.0930 0.0500 0.0013 0.1100
    MEKCSSVFN 3194 ICKMEKCSSVFNVVN 3298
    VVNSSGILI 3195 VFNVVNSSIGLIMVL 3299 0.2600 0.1800 0.0012 0.5000
    YQCYGSSSN 3196 FQEYQCYGSSSNTRV 3300
    YNELEMNYY 3197 INLYNELEMNYYGKQ 3301
    YDNAGINLY 3198 ELNYDNAGINLYNEL 3302 -0.0003 -0.0003
    IQNSLSTEW 3199 LKKIQNSLSTEWSPC 3303
    WSPSCSVTCG 3200 STEWSPCSVTCGNGI 3304
    FILVNLLIF 3201 SFYFILVNLLIFHIN 3305 0.0004 0.0084 -0.0007 -0.0018
    FYFILVNLL 3202 YISFYFILVNLLIGH 3306 0.0003 0.0020 0.0010 -0.0003
    IHKGHLEEK 3203 RRDIHKGHLEEKKDG 3307
    IIKSNLRSG 3204 KDEIIKSNLRSGSSN 3308
    ILVNLLIFH 3205 FYFILVNLLIFHING 3309
    INGKIIKNS 3206 IFHINGKIIKNSEKD 3310 0.0120 0.0150 0.0400 0.0093  0.0020
    IPAIELPSE 3207 RLEIPAIELPSENER 3311
    IPHQSSLPQ 3208 GYYIPHQSSLPQDNR 3312
    IQNHTLETV 3209 SADIQNHTLETVNIS 3313 -0.0003 -0.0003  0.0012
    ISFYFILVN 3210 ILYISFYFILVNLLI 3314
    LDEFKPIQ 3211 DEDLDEFKPIVQYDN 3315
    LEEKAAKET 3212 QEDLEEKAAKETLQG 3316 -0.0003 -0.0002
    LEIPAIELP 3213 YGRLEIPAIELPSEN 3317
    LEQRKADTK 3214 QRDLEQRKADTKKNL 3318
    LERTKASKE 3215 QDDLERTKASKETLQ 3319 0.0010 -0.0003 -0.0005
    LETVNISDV 3216 NGTLETVNISDVNDF 3320
    LIEHIINDD 3217 EGKLIEHIINDDDDK 3321
    LKENKLNKE 3218 NIFLKEKLNKEGKL 3322 0.0530 0.1200 0.0290 0.1800
    LLIFHINGK 3219 LVNLLIFHINGKIIK 3323
    LQEQQSDLE 3220 KETLQEQQSDLEQER 3324
    LQEQQSESE 3221 KEKLQEQQSDSEQER 3325
    LQGQQSDLE 3222 KETLQGQQSDLEQER 3326
    LRNTLGVSEN 3223 KSLLRNLGVSENIFL 3327 0.5700 0.0770 0.0021 1.6000
    LRSGSSNSR 3224 KSNLRSGSSNSRNRI 3328
    LTMSNVKNV 3225 DKELTMSNVKNVSQT 3329 0.0430 0.0410 0.0110 0.0710  0.0024
    LVNLLIFHI 3226 YFILVNLLIFHINGK 3330 0.0013 0.0059 0.0005 0.0040  0.0290
    VLSHNSYEK 3227 KKHVLSHNSYEKTKN 3331
    VNDFQISKY 3228 ISDVNDFQISKYEDE 3332 -0.0003 -0.0003 -0.0005
    VNISKVNDF 3229 LETVNISDVNDFQIS 3333
    YDDSLIDEE 3230 SAEYDDSLIDEEEDD 3334
    YGRLEIPAI 3231 EDLYGRLEIPAIELP 3335 -0.0003 0.0021 -0.0005
    YIPHQSSLP 3232 RGYYIPHQSSLPQDN 3336 0.0004 0.1700 0.0150 0.1500
    FKIGSSDPA 3233 RHPFKIGSSDPADNA 3337 0.0003 -0.0003 0.0380 0.0950
    IDVHDLISH 3234 EPLIDVHDLISDMIK 3338
    IFNKESLAE 3235 FFIIFNKESLAEKTN 3339
    IGSSDPADN 3236 PFKIGSSDPADNANP 3340
    LALFFIIFN 3237 VFFALFFIIFNKES 3341 -0.0002 0.0056 -0.0007 -0.0018
    LATSVLAGL 3238 KYKLATSVLAGLLGN 3342 0.0072 0.6500 0.1300 2.6000
    LGGVGLVLY 3239 TVLLGGVGLVLYNTE 3343 0.0007 -0.0002
    LGNVSTVLL 3240 AGLLGNVSTVLLGGV 3344 4.6000 0.4300 0.0012 0.5300  0.0012
    LLGNVSTVL 3241 LAGLLGNVSTVLLGG 3345 0.6400 0.3800 0.0006 0.5500
    LSVFFLALF 3242 MKILSVFFLALFFII 3346 0.0019 0.0360 0.0023 0.0060
    LVLYNTEKG 3243 GVGLVLYNTEKGRHP 3347
    VFFLALFFI 3244 ILSVFFLALFFIIFN 3348 0.0005 0.0110 0.0031 -0.0003
    VHDLISDMI 3245 LIDVHDLISDMIKKE 3349 0.0004 0.0100 0.0096 0.0430  0.0940
    VLAGLLGNV 3246 ATSVLAGLLGNVSTV 3350 -0.0003 0.0005  0.0039
    VLLGGVGLV 3247 VSTVLLGGVGLVLYN 3351 0.0002 0.0020 -0.0002 0.0120
    VNKRKSKYK 3248 LVEVNKRKSKYKLAT 3352
    VSTVLLGGV 3249 LGNVSTVLLGGVGLV 3353 0.0006 -0.0003 -0.0003 -0.0005 -0.0005
    VTAQDVTPE 3250 DPQVTAQDVTPEQPQ 3574
    YKLATSVLA 3251 KSKYKLATSVLAGLL 3354 0.0082 1.9000 1.1000 2.7000  0.0150
    FDLFLVNGR 3252 LIFFDLFLVNGRDVQ 3355 0.0470
    FFDLFLVNG 3253 FLIFFDLFLVNGRDV 3356
    FMKAVCVEV 3254 IGPFMKAVCVEVEKT 3357 0.0003 0.0019 -0.0003 0.0820  0.0700
    FNRFLVGCH 3255 NVAFNRFLVGCHPSD 3358
    IAGGLALLA 3256 AGGIAGGLALLACAG 3359 -0.0003 0.0004 -0.0005
    IAVFGIGQG 3257 GVKIAVFGIGQGINV 3360
    LACAGLAYK 3258 LALLACAGLAYKFVV 3361
    LALLACAGL 3259 AGGLALLACAGLAYK 3362 0.0009 0.0003 -0.0005
    LAMKLIQQL 3260 AVPLAMKLIQQLNTN 3363 0.0770 0.0400  0.0350
    LAYKFVVPG 3261 CAGLAYKFVVPGAAT 3364
    LIFFDLFLV 3262 IVFLIFFDLFLVNGR 3365 0.0006 0.0028 0.0007 -0.0003
    LTDGIPDSI 3263 VVILTDGIPDSIQDS 3366 -0.0003 -0.0003  0.0114
    LVGCHPSDG 3264 NRFLVGCHPSDGKCN 3367
    LVTVFLIFF 3265 VKYLVTVFLIFFDLF 3368 0.0010
    LVVILTDGI 3266 ANQLVVILTDGIPDS 3379 0.0070 0.0054 -0.0002 0.0420
    MDCSGSIRR 3267 YLLMDCSGSIRRHNW 3370
    MKAVCVEVE 3268 GPFMKAVCVEVEKTA 3371
    VEKTASCGV 3269 CVEVEKTASCGVWDE 3372 0.0095 0.0005
    VGCHPSDGK 3270 RFLVGCHPSDGKCNL 3373
    VIGPFMKAV 3271 VKNVIGPFMKAVCVE 3374 0.1100 0.0590 0.0230 0.0870
    VIVFLIFFD 3272 KYVTVFLIFFDLFL 3375 0.0034 0.0130 0.0065 -0.0018
    VKYLVTVFL 3273 LGNVKYLVTVFLIFF 3376 0.0016 0.0040 0.0050 0.0012
    VNGRDVQNN 3274 LFLVNGRDVQNNTVD 3377
    WDEWSPCSV 3275 CGVWDEWSPCSVTCG 3378 -0.0003 -0.0003 -0.0006
    IAGGIAGGL 3276 KYKIAGGIAGGLALL 3389 0.2400 0.0063 1.6000 0.2600 -0.0010
    YQNNIVDEI 3277 GRDVQNNIVDEIKYR 3380 0.0810 -0.0003 -0.0003 -0.0005  0.0850
    YLLMDCSGS 3278 VDLYLLMDCSGSIRR 3381 0.0046 0.0007 -0.0010
    FVVPGAATP 3279 AYKFVVPGAATPYAG 3382 0.1700 0.1800 0.9200 0.1300
    YKFVVPGAA 3280 GLAYKFVVPGAATPY 3383 0.4900 0.1500 2.5000 0.6000 0.0190
    IIRLHSDAS 3281 AKEIIRLHSDASKNK 3384
    IIDNNPQEP 3282 EENIIDNNPQEPSPN 3385
    VDLYLLMDC 3283 NDEVDLYLLMDCSGS 3386 -0.0003 -0.0003
    LLSTNLPYG 3284 IKSLSSTNLPYGRTN 3387
    LHEGCTSEL 3285 REILHEGCTSELQEQ 3388 -0.0003 -0.0003
    VNHAVPLAM 3286 HNWVNHAVPLAMKLI 3389 0.9400 0.3800 0.200 4.000  0.0250
    VPGAATPYA 3287 KFVVPGAATPYAGEP 3390 0.0460 0.0017 0.0064 0.2500
    VVPGAATPY 3288 YKFVVPGAATPYAGE 3391 0.0017 0.0160 0.0026 0.0200
    WVNHAVPLA 3289 RHNWVNHAVPLAMKL 3392 0.8900 0.4400 1.8000 4.6000  0.0430
    LSTNLPYGR 3290 KSLLSTNLPYGRTNL 3393 0.0005
  • TABLE XXa
    Malaria DR3a Motif Peptides
    Core Ex- Position
    Core Core  Sequence emplary in Pf Exemplary Exemplary
    Core SeqID Sequence Conser- Exemplary SeqID Poly- Sequence Conser-
    Protein Sequence Num Frequency vancy (%) Sequence Num Protein Frequency vancy (%)
    CSP LFQEYQCYG 3394 19 100 VEALFQEYQCYGSSS 3449   16 19 100
    CSP LFVEALFQE 3395 19 100 SSFLFVEALFQEYQC 3450   11 19 100
    CSP MPNDPNRNV 3396 19 100 GHNMPNDPNRNVDEN 3451  347 19 100
    CSP LYNELEMNY 3397 19 100 GINLYNELEMNYYGK 3452   44 18  95
    CSP VLNELNYDN 3398 19 100 NTRVLNELNYDNAGI 3453   31 18  95
    CSP YENDIEKKI 3399 19 100 ELDYENDIEKKICKM 3454  422 12  63
    CSP LNYDNAGIN 3400 18  95 LNELNYDNAGINLYN 3455   35 18  95
    CSP LSTEWSPCS 3401 18  95 QNSLSTEWSPCSVTC 3456  389 15  79
    CSP LDYENDIEK 3402 18  95 KDELDYENDIEKKIC 3457  420 12  63
    LSA FDGDNEILQ 3403  1 100 FHIFDGDNEILQIVD 3458 1882  1 100
    LSA FDKDKELTM 3404  1 100 NKFFDKDKELTMSNV 3459   75  1 100
    LSA FQDEENIGI 3405  1 100 YDNFQDEENIGIYKE 3460 1791  1 100
    LSA IDEEEDDED 3406  1 100 DSLIDEEEDDEDLDE 3461 1770  1 100
    LSA IINDDDDKK 3407  1 100 IEHIINDDDDKKKYI 3462  124  1 100
    LSA INDDDDKKK 3408  1 100 EHIINDDDDKKKYIK 3463  125  1 100
    LSA ISAEYDDSL 3409  1 100 EDEISAEYDDSLIDE 3464 1761  1 100
    LSA IVDELSEDI 3410  1 100 ILQIVDELSEDITKY 3465 1891  1 100
    LSA IYKELEDLI 3411  1 100 NIGIYKELEDLIEKN 3466 1799  1 100
    LSA LAEDLYGRL 3412  1 100 GDVLAEDLYGRLEIP 3467 1645  1 100
    LSA LAKEKLQEQ 3413  1 100 QERLAKEKLQEQQSD 3468 1357  1 100
    LSA LAKEKLQGQ 3414  1 100 QERLAKEKLQGQQSD 3469 1119  1 100
    LSA LANEKLQEQ 3415  1 100 QERLANEKLQEQQRD 3470 1527  1 100
    LSA LEQDRLAKE 3416  1 100 QSDLEQDRLAKEKLQ 3471 1386  1 100
    LSA LEQERLAKE 3417  1 100 QSDLEQERLAKEKLQ 3472 1590  1 100
    LSA LEQERLANE 3418  1 100 QSDLEQERLANEKLQ 3473 1522  1 100
    LSA LIDEEEDDE 3419  1 100 DDSLIDEEEDDEDLD 3474 1769  1 100
    LSA LPSENERGY 3420  1 100 AIELPSENERGYYIP 3475 1660  1 100
    LSA LSEDITKYF 3421  1 100 VDELSEDITKYFMKL 3476 1895  1 100
    LSA LSEEKIKKG 3422  1 100 SEELSEEKIKKGKKY 3477 1827  1 100
    LSA LYDEHIKKY 3423  1 100 DKSLYDEHIKKYKND 3478 1853  1 100
    LSA VLAEDLYGR 3424  1 100 HGDVLAEDLYGRLEI 3479 1644  1 100
    LSA VNKEKEKFI 3425  1 100 DKQVNKEKEKFIKSL 3480 1867  1 100
    LSA VQYDNFQDE 3426  1 100 KPIVQYDNFQDEENI 3481 1786  1 100
    LSA YEDEISAEY 3427  1 100 ISKYEDEISAEYDDS 3482 1757  1 100
    LSA YKNDKQVNK 3428  1 100 IKKYKNDKQVNKEKE 3483 1861  1 100
    PfEXP FNKESLAEK 3429  1 100 FIIFNKESLAEKTNK 3484   13  1 100
    PfEXP IKKEEELVE 3430  1 100 SDMIKKEEELVEVNK 3485   55  1 100
    PfEXP LISDMIKKE 3431  1 100 VHDLISDMIKKEEEL 3486   50  1 100
    PfEXP VTPEQPQGD 3432  1 100 AQDVTPEQPQGDDNN 3487  141  1 100
    PfEXP YNTEKGRHP 3433  1 100 LVLYNTEKGRHPFKI 3488   98  1 100
    SSP2 IFFDLFLVN 3434 10 100 VFLIFFDLFLVNGRD 3489   13 10 100
    SSP2 ILTDGIPDS 3435 10 100 LVVILTDGIPDSIQD 3490  156 10 100
    SSP2 INRENANQL 3436 10 100 NDRINRENANQLVVI 3491  145 10 100
    SSP2 LHSDASKNK 3437 10 100 IIRLHSDASKNKEKA 3492  100 10 100
    SSP2 LYADSAWEN 3438 10 100 KCNLYADSAWENVKN 3493  211 10 100
    SSP2 VCVEVEKTA 3439 10 100 MKAVCVEVEKTASCG 3494  231 10 100
    SSP2 VEVEKTASC 3440 10 100 AVCVEVEKTASCGVW 3495  233 10 100
    SSP2 VPSDVPKNP 3441 10 100 EKEVPSDVPKNPEDD 3496  384 10 100
    SSP2 VWDEWSPCS 3442 10 100 SCGVWDEWSPCSVTC 3497  243 10 100
    SSP2 LLMDCSGSI 3443 10  90 DLYLLMDCSGSIRRH 3498   48  9  90
    SSP2 ILHEGCTSE 3444 10  80 KREILHEGCTSELQE 3499  265  8  80
    SSP2 IPEDSEKEV 3445 10  80 EPNIPEDSEKEVPSD 3500  376  8  80
    SSP2 YREEVCNDE 3446  9  80 EIKYREEVCNDEVDL 3501   35  8  80
    SSP2 VCNDEVDLY 3447  8  80 REEVCNDEVDLYLLM 3502   39  8  80
    SSP2 YAGEPAPFD 3448  8  80 ATPYAGEPAPFDETL 3503  538  8  80
  • TABLE XXb
    DR3a Motif Peptides With Binding Information
    Core Exemplary
    Core SeqID Exemplary SeqID
    Sequence Num Sequence Num DR1 DR2w2β1 DR2w2β2
    LFQEYQCYG 3394 VEALFQEYQCYGSSS 3449
    LFVEALFQE 3395 SSFLFVEALFQEYQC 3450
    MPNDPNRNV 3396 GHNMPNDPNRNVDEN 3451
    LYNELEMNY 3397 GINLYNELEMNYYGK 3452
    VLNELNYDN 3398 NTRVLNELNYDNAGI 3453
    YENDIEKKI 3399 ELDYENDIEKKICKM 3454
    LNYDNAGIN 3400 LNELNYDNAGINLYN 3455
    LSTEWSPCS 3401 QNSLSTEWSPCSVTC 3456
    LDYENDIEK 3402 KDELDYENDIEKKIC 3457
    FDGDNEILQ 3403 FHIFDGDNEILQIVD 3458
    FDKDKELTM 3404 NKFFDKDKELTMSNV 3459
    FQDEENIGI 3405 YDNFQDEENIGIYKE 3460
    IDEEEDDED 3406 DSLIDEEEDDEDLDE 3461
    IINDDDDKK 3407 IEHIINDDDDKKKYI 3462
    INDDDDKKK 3408 EHIINDDDDKKKYIK 3463
    ISAEYDDSL 3409 EDEISAEYDDSLIDE 3464
    IVDELSEDI 3410 ILQIVDELSEDITKY 3465 0.0001 -0.0005
    IYKELEDLI 3411 NIGIYKELEDLIEKN 3466
    LAEDLYGRL 3412 GDVLAEDLYGRLEIP 3467
    LAKEKLQEQ 3413 QERLAKEKLOEQQSD 3468
    LAKEKLQGQ 3414 QERLAKEKLOGQQSD 3469
    LANEKLQEQ 3415 QERLANEKLOEQQRD 3470
    LEQDRLAKE 3416 QSDLEQDRLAKEKLQ 3471
    LEQERLAKE 3417 QSDLEQERLAKEKLQ 3472
    LEQERLANE 3418 QSDLEQERLANEKLQ 3473
    LIDEEEDDE 3419 DDSLIDEEEDDEDLD 3474
    LPSENERGY 3420 AIELPSENERGYYIP 3475
    LSEDITKYF 3421 VDELSEDITKYFMKL 3476
    LSEEKIKKG 3422 SEELSEEKIKKGKKY 3477
    LYDEHIKKY 3423 DKSLYDEHIKKYKND 3478 0.0001 -0.0005
    VLAEDLYGR 3424 HGDVLAEDLYGRLEI 3479
    VNKEKEKFI 3425 DKOVNKEKEKFIKSL 3480
    VQYDNFQDE 3426 KPIVQYDNFQDEENI 3481
    YEDEISAEY 3427 ISKYEDEISAEYDDS 3482 0.0001 -0.0005
    YKNDKQVNK 3428 IKKYKNDKQVNKEKE 3483
    FNKESLAEK 3429 FIIFNKESLAEKTNK 3484
    IKKEEELVE 3430 SDMIKKEEELVEVNK 3485
    LISDMIKKE 3431 VHDLISDMIKKEEEL 3486
    VTPEQPQGD 3432 AQDVTPEQPQGDDNN 3487
    YNTEKGRHP 3433 LVLYNTEKGRHPFKI 3488
    IFFDLFLVN 3434 VFLIFFDLFLVNGRD 3489
    ILTDGIPDS 3435 LVVILTDGIPDSIQD 3490 0.0002 0.0001 -0.0006
    INRENANQL 3436 NDRINRENANOLVVI 3491 0.0770 0.0015
    LHSDASKNK 3437 IIRLHSDASKNKEKA 3492
    LYADSAWEN 3438 KCNLYADSAWENVKN 3493 0.0002 0.0005 -0.0010
    VCVEVEKTA 3439 MKAVCVEVEKTASCG 3494
    VEVEKTASC 3440 AVCVEVEKTASCGVW 3495 0.0001 -0.0006
    VPSDVPKNP 3441 EKEVPSDVPKNPEDD 3496
    VWDEWSPCS 3442 SCGVWDEWSPCSVTC 3497 0.0001 -0.0005
    LLMDCSGSI 3443 DLYLLMDCSGSIRRH 3498 0.0041 0.0250
    Core
    Sequence DR3 DR4w4 DR4w15 DR5w11 DR5w12
    LFQEYQCYG 0.0082
    LFVEALFQE 0.0051
    MPNDPNRNV -0.0033
    LYNELEMNY 0.0270
    VLNELNYDN -0.0033
    YENDIEKKI
    LNYDNAGIN
    LSTEWSPCS -0.0033
    LDYENDIEK
    FDGDNEILQ 0.0640
    FDKDKELTM
    FQDEENIGI -0.0033
    IDEEEDDED
    IINDDDDKK
    INDDDDKKK
    ISAEYDDSL -0.0033
    IVDELSEDI -0.0041 0.0027 0.0017 -0.0002 0.0001
    IYKELEDLI -0.0033
    LAEDLYGRL
    LAKEKLQEQ
    LAKEKLQGQ
    LANEKLQEQ -0.0033
    LEQDRLAKE 0.0038
    LEQERLAKE -0.0033
    LEQERLANE
    LIDEEEDDE
    LPSENERGY -0.0033
    LSEDITKYF
    LSEEKIKKG -0.0033
    LYDEHIKKY -0.0041 -0.0007 -0.0014 -0.0002 0.0001
    VLAEDLYGR
    VNKEKEKFI -0.0033
    VQYDNFQDE -0.0033
    YEDEISAEY -0.0041 0.0008 -0.0014 -0.0002 0.0001
    YKNDKQVNK -0.0033
    FNKESLAEK 0.0040
    IKKEEELVE -0.0033
    LISDMIKKE
    VTPEQPQGD -0.0033
    YNTEKGRHP
    IFFDLFLVN
    ILTDGIPDS 0.1400 0.3600 -0.0014 -0.0004 0.0002
    INRENANQL 0.0092 0.0011 0.0010 -0.0004 0.0001
    LHSDASKNK -0.0033
    LYADSAWEN 0.3500 -0.0055 -0.0006
    VCVEVEKTA
    VEVEKTASC -0.0041 0.0030 -0.0014 0.0003 0.0001
    VPSDVPKNP -0.0130
    VWDEWSPCS -0.0041 -0.0009 -0.0009 -0.0002 0.0001
    LLMDCSGSI 0.0300 0.0340 0.0028 -0.0002 0.0001
    Core Exemplary
    Core SeqID Exemplary SeqID
    Sequence Num Sequence Num DR6w19 DR7 DR8w2 DR9 DRw53
    LFQEYQCYG 3394 VEALFQEYQCYQSSS 3449
    LFVEALFQE 3395 SSFLFVEALFQEYQC 3450
    MPNDPNRNV 3396 GHNMPNDPNRNVDEN 3451
    LYNELEMNY 3397 GINLYNELEMNYYGK 3452
    VLNELNYDN 3398 NTRVLNELNYDNAGI 3453
    YENDIEKKI 3399 ELDYENDIEKKICKM 3454
    LNYDNAGIN 3400 LNELNYDNAGINLYN 3455
    LSTEWSPCS 3401 QNSLSTEWSPCSVTC 3456
    LDYENDIEK 3402 KDELDYENDIEKKIC 3457
    FDGDNEILQ 3403 FHIFDGDNEILQIVD 3458
    FDKDKELTM 3404 NKFFDKDKELTMSNV 3459
    FQDEENIGI 3405 YDNFQDEENIGIYKE 3460
    IDEEEDDED 3406 DSLIDEEEDDEDLDE 3461
    IINDDDDKK 3407 IEHIINDDDDKKKYI 3462
    INDDDDKKK 3408 EHIINDDDDKKKYIK 3463
    ISAEYDDSL 3409 EDEISAEYDDSLIDE 3464
    IVDELSEDI 3410 ILQIVDELSEDITKY 3465 -0.0003 -0.0003 0.0290
    IYKELEDLI 3411 NIGIYKELEDLIEKN 3466
    LAEDLYGRL 3412 GDVLAEDLYGRLEIP 3467
    LAKEKLQEQ 3413 QERLAKEKLQEQQSD 3468
    LAKEKLQGQ 3414 QERLAKEKLQGQQSD 3469
    LANEKLQEQ 3415 QERLANEKLQEQQRD 3470
    LEQDRLAKE 3416 QSDLEQDRLAKEKLQ 3471
    LEQERLAKE 3417 QSDLEQERLAKEKLQ 3472
    LEQERLANE 3418 QSDLEQERLANEKLQ 3473
    LIDEEEDDE 3419 DDSLIDEEEDDEDLD 3474
    LPSENERGY 3420 AIELPSENERGYYIP 3475
    LSEDITKYF 3421 VDELSEDITKYFMKL 3476
    LSEEKIKKG 3422 SEELSEEKIKKGKKY 3477
    LYDEHIKKY 3423 DKSLYDEHIKKYKND 3478 -0.0003 -0.0003 0.0006
    VLAEDLYGR 3424 HGDVLAEDLYGRLEI 3479
    VNKEKEKFI 3425 DKQVNKEKEKFIKSL 3480
    VQYDNFQDE 3426 KPIVQYDNFQDEENI 3481
    YEDEISAEY 3427 ISKYEDEISAEYDDS 3482 -0.0003 -0.0003 -0.0005
    YKNDKQVNK 3428 IKKYKNDKQVNKEKE 3483
    FNKESLAEK 3429 FIIFNKESLAEKTNK 3484
    IKKEEELVE 3430 SDMIKKEEELVEVNK 3485
    LISDMIKKE 3431 VHDLISDMIKKEEEL 3486
    VTPEQPQGD 3432 AQDVTPEQPQGDDNN 3487
    YNTEKGRHP 3433 LVLYNTEKGRHPFKI 3488
    IFFDLFLVN 3434 VFLIFFDLFLVNGRD 3489
    ILTDGIPDS 3435 LVVILTDGIPDSIQD 3490 0.0002 0.0046 -0.0003 0.0014 0.0480
    INRENANQL 3436 NDRINRENANQLVVI 3491 -0.0003 -0.0003 0.0096
    LHSDASKNK 3437 IIRLHSDASKNKEKA 3492
    LYADSAWEN 3438 KCNLYADSAWENVKN 3493 0.0003 -0.0014 -0.0009
    VCVEVEKTA 3439 MKAVCVEVEKTASCG 3494
    VEVEKTASC 3440 AVCVEVEKTASCGVW 3495 0.0073 0.0006 0.0022
    VPSDVPKNP 3441 EKEVPSDVPKNPEDD 3496
    VWDEWSPCS 3442 SCGVWDEWSPCSVTC 3497 -0.0003 -0.0003
    LLMDCSGSI 3443 DLYLLMDCSGSIRRH 3498 0.0072 0.0014 0.0057
    Core Exemplary
    Core SeqID Exemplary SeqID
    Sequence Num Sequence Num DR1 DR2w2β1 DR2w2β2
    ILHEGCTSE 3444 KREILHEGCTSELQE 3499
    IPEDSEKEV 3445 EPNIPEDSEKEVPSD 3500
    YREEVCNDE 3446 EIKYREEVCNDEVDL 3501
    VCNDEVDLY 3447 REEVCNDEVDLYLLM 3502 0.0003 -0.0006 0.1300
    YAGEPAPFD 3448 ATPYAGEPAPFDETL 3503
    Core
    Sequence DR3 DR4w4 DR4w15 DR5w11 DR5w12
    ILHEGCTSE
    IPEDSEKEV -0.0130
    YREEVCNDE -0.0033
    VCNDEVDLY -0.0006 -0.0014 -0.0004 0.0001
    YAGEPAPFD -0.0130
    Core Exemplary
    Core SeqID Exemplary SeqID
    Sequence Num Sequence Num DR6w19 DR7 DR8w2 DR9 DRw53
    ILHEGCTSE 3444 KREILHEGCTSELQE 3499
    IPEDSEKEV 3445 EPNIPEDSEKEVPSD 3500
    YREEVCNDE 3446 EIKYREEVCNDEVDL 3501
    VCNDEVDLY 3447 REEVCNDEVDLYLLM 3502 -0.0003 -0.0003 -0.0010
    YAGEPAPFD 3448 ATPYAGEPAPFDETL 3503
  • TABLE XXc
    Core Core
    Core SeqID Core Conservancy Exemplary
    Protein Sequence Num Frequency (%) Sequence
    CSP LKKNSRSLG 3504 19 100 WYSLKKNSRSLGEND
    CSP ANNDVKNNN 3505 3 16 NANANNDVKNNNNEE
    LSA ADIQNHTLE 3506 1 100 DKSADIQNHTLETVN
    LSA FHINGKIIK 3507 1 100 LLIFHINGKIIKNSE
    LSA FKPNDKSLY 3508 1 100 DNNFKPNDKSLYDEH
    LSA FLKENKLNK 3509 1 100 ENIFLKENKLNKEGK
    LSA IEKTNRESI 3510 1 100 ISIIEKTNRESITTN
    LSA IKNSEKDEI 3511 1 100 GKIIKNSEKDEIIKS
    LSA IKPEQKEDK 3512 1 100 DGSIKPEQKEDKSAD
    LSA IKSNLRSGS 3513 1 100 DEIIKSNLRSGSSNS
    LSA INEEKHEKK 3514 1 100 RNRINEEKHEKKHVL
    LSA LEQERRAKE 3515 1 100 QSDLEQERRAKEKLQ
    LSA LNKEGKLIE 3516 1 100 ENKLNKEGKLIEHII
    LSA LPQDNRGNS 3517 1 100 QSSLPQDNRGNSRDS
    LSA LQEQQRDLE 3518 1 100 NEKLQEQQRDLEQER
    PfEXP AEKTNKGTG 3519 1 100 ESLAEKTNKGTGSGV
    PfEXP LYNTEKGRH 3520 1 100 GLVLYNTEKGRHPFK
    PfEXP VEVNKRKSK 3521 1 100 EELVEVNKRKSKYKL
    SSP2 AWENVKNVI 3522 10 100 ADSAWENVKNVIGPF
    SSP2 FLVNGRDVQ 3523 10 100 FDLFLVNGRDVQNNI
    SSP2 LGEEDKDLD 3524 10 100 DETLGEEDKDLDEPE
    SSP2 LDNERKQSD 3525 10 80 PKVLDNERKQSDPQS
    SSP2 VLDNERKQS 3526 10 70 PPKVLDNERKQSDPQ
    SSP2 IQDSLKESR 3527 10 60 PDSIQDSLKESRKLN
    SSP2 IVDEIKYSE 3528 9 90 QNNIVDEIKYREEVC
    SSP2 ALLQVRKHL 3529 9 60 LTDALLQVRKHLNDR
    SSP2 LKESRKLND 3530 6 50 QDSLKESRKLSDRGV
    SSP2 FSNNAKEII 3531 6 40 VNVFSNNAKEIIRLH
    SSP2 YNDTPKHPE 3532 5 50 NRKYNDTPKHPEREE
    SSP2 FSNNAREII 3533 4 20 LNIFSNNAREIIRLH
    SSP2 LKESRKLSD 3534 3 30 QDSLKESRKLSDRGV
    SSP2 YNDTPKYPE 3535 2 20 NRKYNDTPKYPEREE
    SSP2 AGSDNKYKI 3536 1 10 KKKAGSDNKYKIAGG
    SSP2 ALLEVRKHL 3537 1 10 LTDALLEVRKHLNDR
    SSP2 IVDEIKYSE 3538 1 10 QNNIVDEIKYSEEVC
    Exemplary Position  Exemplary  Exemplary 
    SeqID In PF Sequence Sequence
    Protein Num Poly-Protein Frequency Conservancy (%)
    CSP 3539 62 19 100
    CSP 3540 361 3  16
    LSA 3541 1734 1 100
    LSA 3542 16 1 100
    LSA 3543 1846 1 100
    LSA 3544 108 1 100
    LSA 3545 1693 1 100
    LSA 3546 23 1 100
    LSA 3547 1724 1 100
    LSA 3548 32 1 100
    LSA 3549 47 1 100
    LSA 3550 1573 1 100
    LSA 3551 114 1 100
    LSA 3552 1676 1 100
    LSA 3553 1532 1 100
    PfEXP 3554 19 1 100
    PfEXP 3555 97 1 100
    PfEXP 3556 62 1 100
    SSP2 3557 216 10 100
    SSP2 3558 18 10 100
    SSP2 3559 549 10 100
    SSP2 3560 435 8  80
    SSP2 3561 434 7  70
    SSP2 3562 165 6  60
    SSP2 3563 29 9  90
    SSP2 3564 133 6  60
    SSP2 3565 169 5  50
    SSP2 3566 90 4  40
    SSP2 3567 479 5  50
    SSP2 3568 90 2  20
    SSP2 3569 169 3  30
    SSP2 3570 479 2  20
    SSP2 3571 501 1  10
    SSP2 3572 133 1  10
    SSP2 3573 29 1  10
  • TABLE XXd
    Malaria DR3b Motif Peptides With Binding Information
    Core Exemplary
    Core SeqID Exemplary SeqID
    Sequence Num Sequence Num DR1 DR2w2β1 DR2w2β2
    LKKNSRSLG 3504 WYSLKKNSRSLGEND 3539
    ANNDVKNNN 3505 NANANNDVKNNNNEE 3540
    ADIQNHTLE 3506 DKSADIQNHTLETVN 3541
    FHINGKIIK 3507 LLIFHINGKIIKNSE 3542 0.5700 0.2900 0.2500
    FKPNDKSLY 3508 DNNFKPNDKSLYDEH 3543
    FLKENKLNK 3509 ENIFLKENKLNKEGK 3544
    IEKTNRESI 3510 ISIIEKTNRESITIN 3545
    IKNSEKDEI 3511 GKIIKNSEKDEIIKS 3546 0.0002 -0.0021 -0.0160
    IKPEQKEDK 3512 DGSIKPEQKEDKSAD 3547
    IKSNLRSGS 3513 DEIIKSNLRSGSSNS 3548
    INEEKHEKK 3514 RNRINEEKHEKKHVL 3549
    LEQERRAKE 3515 QSDLEQERRAKEKLQ 3550
    LNKEGKLIE 3516 ENKLNKEGKLIEHII 3551 0.0001 -0.0021
    LPQDNRGNS 3517 QSSLPQDNRGNSRDS 3552
    LQEQQRDLE 3518 NEKLQEQQRDLEQER 3553
    AEKTNKGTG 3519 ESLAEKTNKGTGSGV 3554
    LYNTEKGRH 3520 GLVLYNTEKGRHPFK 3555
    VEVNKRKSK 3521 EELVEVNKRKSKYKL 3556
    AWENVKNVI 3522 ADSAWENVKNVIGPF 3557
    FLVNGRDVQ 3523 FDLFLVNGRDVQNNI 3558
    LGEEDKDLD 3524 DETLGEEDKDLDEPE 3559
    LDNERKQSD 3525 PKVLDNERKQSDPQS 3560
    VLDNERKQS 3526 PPKVLDNERKQSDPQ 3561
    IQDSLKESR 3527 PDSIQDSLKESRKLN 3562 -0.0001 0.0040 -0.0018
    IVDEIKYRE 3528 QNNIVDEIKYREEVC 3563
    ALLQVRKHL 3529 LTDALLQVRKHLNDR 3564
    LKESRKLND 3530 QDSLKESRKLNDRGV 3565
    FSNNAKEll 3531 VNVFSNNAKEIIRLH 3566
    YNDTPKHPE 3532 NRKYNDTPKHPEREE 3567
    FSNNAREII 3533 LNIFSNNAREIIRLH 3568
    LKESRKLSD 3534 QDSLKESRKLSDRGV 3569
    YNDTPKYPE 3535 NRKYNDTPKYPEREE 3570
    AGSDNKYKI 3536 KKKAGSDNKYKIAGG 3571
    ALLEVRKHL 3537 LTDALLEVRKHLNDR 3572
    IVDEIKYSE 3538 QNNIVDEIKYREEVC 3573
    Core
    Sequence DR3 DR4w4 DR4w15 DR5w11 DR5w12
    LKKNSRSLG
    ANNDVKNNN
    ADIQNHTLE
    FHINGKIIK 0.5300 0.0060 -0.0030 0.3600 0.0230
    FKPNDKSLY 0.1700
    FLKENKLNK 0.0950
    IEKTNRESI 0.1300
    IKNSEKDEI -0.0017 0.0030 -0.0010 -0.0003
    IKPEQKEDK -0.0033
    IKSNLRSGS 0.0050
    INEEKHEKK 0.0420
    LEQERRAKE
    LNKEGKLIE -0.0140 -0.0017 -0.0047 -0.0005 -0.0003
    LPQDNRGNS -0.0033
    LQEQQRDLE
    AEKTNKGTG -0.0033
    LYNTEKGRH
    VEVNKRKSK 0.0880
    AWENVKNVI -0.0130
    FLVNGRDVQ -0.0033
    LGEEDKDLD -0.0130
    LDNERKQSD -0.0130
    VLDNERKQS -0.0130
    IQDSLKESR 0.8400 -0.0055 -0.0006
    IVDEIKYRE
    ALLQVRKHL -0.0033
    LKESRKLND
    FSNNAKEll
    YNDTPKHPE
    FSNNAREII
    LKESRKLSD
    YNDTPKYPE
    AGSDNKYKI
    ALLEVRKHL
    IVDEIKYSE
    Core Exemplary
    Core SeqID Exemplary SeqID
    Sequence Num Sequence Num DR6w19 DR7 DR8w2 DR9 DRw53
    LKKNSRSLG 3504 WYSLKKNSRSLGEND 3539
    ANNDVKNNN 3505 NANANNDVKNNNNEE 3540
    ADIQNHTLE 3506 DKSADIQNHTLETVN 3541
    FHINGKIIK 3507 LLIFHINGKIIKNSE 3542 0.0330 0.1300 0.1400 0.1500
    FKPNDKSLY 3508 DNNFKPNDKSLYDEH 3543
    FLKENKLNK 3509 ENIFLKENKLNKEGK 3544
    IEKTNRESI 3510 ISIIEKTNRESITTN 3545
    IKNSEKDEI 3511 GKIIKNSEKDEIIKS 3546 -0.0011 -0.0007
    IKPEQKEDK 3512 DGSIKPEQKEDKSAD 3547
    IKSNLRSGS 3513 DEIIKSNLRSGSSNS 3548
    INEEKHEKK 3514 RNRINEEKHEKKHVL 3549
    LEQERRAKE 3515 QSDLEQERRAKEKLQ 3550
    LNKEGKLIE 3516 ENKLNKEGKLIEHII 3551 -0.0009 -0.0007
    LPQDNRGNS 3517 QSSLPQDNRGNSRDS 3552
    LQEQQRDLE 3518 NEKLQEQQRDLEQER 3553
    AEKTNKGTG 3519 ESLAEKTNKGTGSGV 3554
    LYNTEKGRH 3520 GLVLYNTEKGRHPFK 3555
    VEVNKRKSK 3521 EELVEVNKRKSKYKL 3556
    AWENVKNVI 3522 ADSAWENVKNVIGPF 3557
    FLVNGRDVQ 3523 FDLFLVNGRDVQNNI 3558
    LGEEDKDLD 3524 DETLGEEDKDLDEPE 3559
    LDNERKQSD 3525 PKVLDNERKQSDPQS 3560
    VLDNERKQS 3526 PPKVLDNERKQSDPQ 3561
    IQDSLKESR 3527 PDSIQDSLKESRKLN 3562 -0.0002 -0.0014 0.0012
    IVDEIKYRE 3528 QNNIVDEIKYREEVC 3563
    ALLQVRKHL 3529 LTDALLQVRKHLNDR 3564
    LKESRKLND 3530 QDSLKESRKLNDRGV 3565
    FSNNAKEII 3531 VNVFSNNAKEIIRLH 3566
    YNDTPKHPE 3532 NRKYNDTPKHPEREE 3567
    FSNNAREII 3533 LNIFSNNAREIIRLH 3568
    LKESRKLND 3534 QDSLKESRKLNDRGV 3569
    YNDTPKYPE 3535 NRKYNDTPKYPEREE 3570
    AGSDNKYKI 3536 KKKAGSDNKYKIAGG 3571
    ALLEVRKHL 3537 LTDALLEVRKHLNDR 3572
    IVDEIKYSE 3538 QNNIVDEIKYREEVC 3573
  • TABLE XXI
    Population coverage with combined HLA Supertypes
    PHENOTYPIC FREQUENCY
    North
    American
    HLA-SUPERTYPES Caucasian Black Japanese Chinese Hispanic Average
    a. Individual Supertypes
    A2 45.8 39.0 42.4 45.9 43.0 43.2
    A3 37.5 42.1 45.8 52.7 43.1 44.2
    B7 38.6 52.7 48.8 35.5 47.1 44.7
    A1 47.1 16.1 21.8 14.7 26.3 25.2
    A24 23.9 38.9 58.6 40.1 38.3 40.0
    B44 43.0 21.2 42.9 39.1 39.0 37.0
    B27 28.4 26.1 13.3 13.9 35.3 23.4
    B62 12.6 4.8 36.5 25.4 11.1 18.1
    B58 10.0 25.1 1.6 9.0 5.9 10.3
    b. Combined Supertypes
    A2, A3, B7 83.0 86.1 87.5 88.4 86.3 86.2
    A2, A3, B7, A24, B44, A1 99.5 98.1 100.0 99.5 99.4 99.3
    A2, A3, B7, A24, B44, A1, 99.9 99.6 100.0 99.8 99.9 99.8
    B27, B62, B58
  • TABLE XXII 
    Fixed analogs of P. falciparum CTL epitopes
    SEQ SEQ
    Supertype ID Alleles Fixing Fixed ID
    (or allele) Peptide Sequence NO: Source bounda strategy sequence NO:
    A2 1167.21 FLIFFDLFLV 3610 Pf SSP2 14 5 V2 FLIFFDLFLV 3803
    supertype 1167.16 FMKAVCVEV 3611 Pf SSP2 230 5 V2 FVKAVCVEV 3804
    1167.08 GLIMVLSFL 3612 Pf CSP 425 4 Vc GLIMVLSFV 3805
    V2 GVIMVLSFL 3806
    V2Nc GVIMVLSFV 3807
    1167.12 VLAGLLGNV 3613 Pf EXP1 80 4 V2 VLAGLLGNV 3808
    1167.13 KILSVFFLA 3614 Pf EXP1 2 3 L2 KLLSVFFLA 3809
    V2 KVLSVFFLA 3810
    Vc KILSVFFLV 3811
    L2/Vc KLLSVFFLV 3812
    V2/Vc KVLSVFFLV 3813
    1167.10 GLLGNVSTV 3615 Pf EXP1 83 3 V2 GVLGNVSTV 3814
    1167.18 ILSVSSFLFV 3616 Pf CSP 7 2 V2 IVSVSSFLFV 3815
    1167.19 VLLGGVGLVL 3617 Pf EXP1 91 2 Vc VLLGGVGLVV 3816
    V2 VVLGGVGLVL 3817
    V2/Vc VVLGGVGLVV 3818
    A3- 1167.36 LACAGLAYK 3718 Pf SSP2 511 4 V2 LVCAGLAYK 3819
    supertype 1167.32 QTNFKSLLR 3619 Pf LSA1 94 4 V2 QVNFKSLLR 3820
    1167.43 VTCGNGIQVR 3620 Pf CSP 375 4 V2 VVCGNGIQVR 3821
    1167.24 ALFFIIFNK 3621 Pf EXP1 10 3 V2 AVFFIIFNK 3822
    1167.28 GVSENIFLK 3622 Pf LSA1 105 3 —
    1167.47 HVLSHNSYEK 3623 Pf LSA1 59 3 —
    1167.51 LLACAGLAYK 3624 Pf SSP2 510 3 V2 LVACAGLAYK 3823
    1167.46 FILVNLLIFH 3625 Pf LSA1 11 2 V2 FVLVNLLIFH 3824
    Rc FILVNLLIFR 3825
    Kc FILVNLLIFK 3826
    V2/Rc FVLVNLL1FR 3827
    V2/Kc FVLVNLLIFK 3828
    B7- 1167.61 TPYAGEPAPF 3626 Pf SSP2 539 4 Ic TPYAGEPAPI 3829
    supertype
    19.0051 LPYGRTNL 3627 Pf SSP2 126 3 Ic LPYGRTNI 3830
    A1 16.0245 FQDEENIGIY 3628 Pf LSA1 1794 1 T2 FTDEENIGIY 3831
    16.0040 FVEALFQEY 3629 Pf CSP 15 1 D3 FVEALFQEY 3832
    T2 FTEALFQEY 3833
    15.0184 LPSENERGY 3630 Pf LSA1 1663 1 D3 LPDENERGY 3834
    T2 LTSENERGY 3835
    16.0130 PSDGKCNLY 3631 Pf SSP2 207 1 T2 PTDGKCNLY 3836
    A24 1167.54 FYFILVNLL 3632 Pf LSA1 9 1 Fc FYFILVNLF 3837
    1167.53 KYKLATSVL 3633 Pf EXP1 73 1 Fc KYKLATSVF 3838
    1167.56 KYLVIVFLI 3634 Pf SSP2 8 1 Fc KYLVIVFLF 3839
    1167.55 YYIPHQSSL 3635 Pf LSA1 1671 1 Fc YYIPHQSSF 3840
    aA2-supertype peptides are tested for binding to A*0201, A*0202, A*0203, A*0206, and A*6802. A3-supertype peptides are tested for binding ato A*03, A*11, A*31011, A*3301, and A*6801. B7-supertype peptides are tested for binding to B*0702, B*3501, B*5101, B*5301, and B*5401. A1 and A24 peptides are tested for binding to A*0101, and A*2402, respectively.
  • TABLE XXIII
    Plasmodium falciparum CTL-inducing epitopes
    SEQ
    ID HLA-
    Epitope NO: Antigen Residues restriction
    GLIMVLSFL 3636 CSP 386-394 A2-supertype
    ILSVSSFLFV 3637 CSP   7-16 A2-supertype
    VLAGLLGNV 3638 Exp-1  80-88 A2-supertype
    KILSVFFLA 3639 Exp-1   2-10 A2-supertype
    GLLGNVSTV 3640 Exp-1  83-91 A2-supertype
    VLLGGVGLVL 3641 Exp-1  91-100 A2-supertype
    FLIFFDLFLV 3642 SSP2  14-23 A2-supertype
    VTCGNGIQVR 3643 CSP 336-345 A3-supertype
    ALFFIIFNK 3644 Exp-1  10-18 A3-supertype
    QTNFKSLLR 3645 LSA-1  94-102 A3-supertype
    GVSENIFLK 3646 LSA-1 105-113 A3-supertype
    HVLSHNSYEK 3647 LSA-1  59-68 A3-supertype
    FILVNLLIFH 3648 LSA-1  11-20 A3-supertype
    TPYAGELPAPF 3649 SSP2 539-548 B7-supertype
    MPLETQLAI 3650 s16  77-85 B7-supertype
    MRKLAILSVSSFLVF 3651 CSP   2-16 DR-supermotif
    MNYYGKQENWYSLICK 3652 CSP  53-67 DR-supermotif
    RHNWVNHAVPLAMKLI 3653 SSP2  61-76 DR-supermotif
    VKNVIGPFMKAVCVE 3654 SSP2 223-237 DR-supermotif
    SSVFNVVNSSIGLIM 3655 CSP 410-424 DR-supermotif
    AGLLGNVSTVSTVLLGGV 3656 EXP1  82-96 DR-supermotif
    KSKYKLATSVLAGLL 3657 EXP1  71-85 DR-supermotif
    GLAYKFVVPGAATPY 3658 SSP2 512-526 DR-supermotif
    KYKIAGGIAGGLALL 3659 SSP2 494-508 DR-supermotif
  • TABLE XXIV
    MHC-peptide binding assays: cell lines and radiolabeled ligands.
    A. Class I binding assays
    Radiolabeled peptide
    Species Antigen Allele Cell line Source Sequence SEQ ID NO:
    Human A1 A*0101 Steinlin Hu. J chain 102-110 YTAVVPLVY 3660
    A2 A*0201 JY HBVc 18-27 F6->Y FLPSDYFPSV 3661
    A2 A*0202 P815  HBVc 18-27 F6->Y FLPSDYFPSV 3662
    (transfected)
    A2 A*0203 FUN HBVc 18-27 F6->Y FLPSDYFPSV 3663
    A2 A*0206 CLA HBVc 18-27 F6->Y FLPSDYFPSV 3664
    A2 A*0207 721.221  HBVc 18-27 F6->Y FLPSDYFPSV 3665
    (transfected)
    A3 GM3107 non-natural (A3CON1) KVFPYALINK 3666
    A11 BVR non-natural (A3CON1) KVFPYALINK 3667
    A24 A*2402 KAS116 non-natural (A24CON1) AYIDNYNKF 3668
    A31 A*3101 SPACH non-natural (A3CON1) KVFPYALINK 3669
    A33 A*3301 LWAGS non-natural (A3CON1) KVFPYALINK 3670
    A28/68 A*6801 C1R HBVc 141-151 T7->Y STLPETYVVRR 3671
    A28/68 A*6802 AMAI HBV pol 646-654 C4->A FTQAGYPAL 3672
    B7 B*0702 GM3107 A2 sigal seq. 5-13  APRTLVYLL 3673
    (L7->Y)
    B8 B*0801 Steinlin HIVgp 586-593 Y1->F,  FLKDYQLL 3674
    Q5->Y
    B27 B*2705 LG2 R 60s FRYNGLIHR 3675
    B35 B*3501 C1R, BVR non-natural (B35CON2) FPFKYAAAF 3676
    B35 B*3502 TISI non-natural (B35CON2) FPFKYAAAF 3677
    B35 B*3503 EHM non-natural (B35CON2) FPFKYAAAF 3678
    B44 B*4403 PITOUT EF-1 G6->Y AEMGKYSFY 3679
    B51 KAS116 non-natural (B35CON2) FPFKYAAAF 3680
    B53 B*5301 AMAI non-natural (B35CON2) FPFKYAAAF 3681
    B54 B*5401 KT3 non-natural (B35CON2) FPFKYAAAF 3682
    Cw4 Cw*0401 CIR non-natural (C4CON1) QYDDAVYKL 3683
    Cw6 Cw*0602 721.221  non-natural (C6CON1) YRHDGGNVL 3684
    transfected
    Cw7 Cw*0702 721.221  non-natural (C6CON1) YRHDGGNVL 3685
    transfected
    Mouse Db EL4 Adenovirus ElA P7->Y SGPSNTYPEI 3686
    Kb EL4 VSV NP 52-59 RGYVFQGL 3687
    Dd P815 HIV-IIIB ENV 04->Y RGPYRAFVTI 3688
    Kd P815 non-natural (KdCON1) KFNPMKTYI 3689
    Ld P815 HBVs 28-39 IPQSLDSYWTSL 3690
    B. Class II binding assays
    Radiolabeled peptide
    Species Antigen Allele Cell line Source Sequence SEQ ID NO:
    Human DR1 DRB1*0101 LG2 HA Y307-319 YPKYVKQNTLKLAT 3691
    DR2 DRB1*1501 L466.1 MBP 88-102Y VVHFFKNIVTPRTPPY 3692
    DR2 DRB1*1601 L242.5 non-natural (760.16) YAAFAAAKTAAAFA 3693
    DR3 DRB1*0301 MAT MT 65kD Y3-13 YKTIAFDEEARR 3694
    DR4w4 DRB1*0401 Preiss non-natural (717.01) YARFQSQTTLKQKT 3695
    DR4w10 DRB1*0402 YAR non-natural (717.10) YARFQRQTTLKAAA 3696
    DR4w14 DRB1*0404 BIN 40 non-natural (717.01) YARFQSQTTLKQKT 3697
    DR4w15 DRB1*0405 KT3 non-natural (717.01) YARFQSQTTLKQKT 3698
    DR7 DRB1*0701 Pitout Tet. tox. 830-843 QYIKANSKFIGITE 3699
    DR8 DRB1*0802 OLL Tet. tox. 830-843 QYIKANSKFIGITE 3700
    DR8 DRB1*0803 LUY Tet. tox. 830-843 QYIKANSKFIGITE 3701
    DR9 DRB1*0901 HID Tet. tox. 830-843 QYIKANSKFIGITE 3702
    DR11 DRB1*1101 Sweig Tet. tox. 830-843 QYIKANSKFIGITE 3703
    DR12 DRB1*1201 Herluf unknown eluted peptide EALIHQLKINPYVLS 3704
    DR13 DRB1*1302 H0301 Tet. tox. 830-843 S->A QYIKANAKFIGITE 3705
    DR51 DRB5*0101 GM3107  Tet. tox. 830-843 QYIKANAKFIGITE 3706
    or 
    L416.3
    DR51 DRB5*0201 L255.1 HA 307-319 PKYVKQNTLKLAT 3707
    DR52 DRB3*0101 MAT Tet. tox. 830-843 NGQIGNDPNRDIL 3708
    DR53 DRB4*0101 L257.6 non-natural (717.01) YARFQSQTTLKQKT 3709
    DQ3.1 QA1*0301/ PF non-natural (ROIV) YAHAAHAAHAAHAAHAA 3710
    DQB1*03(
    Mouse IAb DB27.4 non-natural (ROIV) YAHAAHAAHAAHAAHAA 3711
    IAd A20 non-natural (ROIV) YAHAAHAAHAAHAAHAA 3712
    IAk CH-12 HEL 46-61 YNTDGSTDYGILQINSR 3713
    IAs LS102.9 non-natural (ROIV) YAHAAHAAHAAHAAHAA 3714
    IAu 91.7 non-natural (ROIV) YAHAAHAAHAAHAAHAA 3715
    IEd A20 Lambda repressor 12-26 YLEDARRKKAIYEKKK 3716
    lEk CH-12 Lambda repressor 12-26 YLEDARRKKAIYEKKK 3717
  • TABLE XXV
    Monoclonal antibodies used
    in MHC purification.
    Monoclonal antibody Specificity
    W6/32 HLA-class I
    B123.2 HLA-B and C
    IVD12 HLA-DQ
    LB3.1 HLA-DR
    M1/42 H-2 class I
    28-14-8S H-2 Db and Ld
    34-5-8S H-2 Dd
    B8-24-3 H-2 Kb
    SF1-1.1.1 H-2 Kd
    Y-3 H-2 Kb
    10.3.6 H-2 IAk
    14.4.4 H-2 IEd, IEK
    MKD6 H-2 IAd
    Y3JP H-2 IAb, IAs, IAu
  • TABLE XXVI
    P. falciparum A2-supermotif CTL epitopes
    SEQ ID A2-supertype binding capacity (IC50 nM) Alleles
    Peptide AA Sequence NO: Source A*0201 A*0202 A*0203 A*0206 A*6802 bounda
    1167.21 10 FLIFFDLFLV 3718 Pf SSP2 14 12 10 5.9 11 333 5
    1167.16  9 FMKAVCVEV 3719 Pf SSP2 230 63 307 2.9 389 143 5
    1167.12  9 VLAGLLGNV 3720 Pf EXP1 80 19 24 0.67 31 606 4
    1167.08  9 GLIMVLSFL 3721 Pf CSP 425 22 20 3.6 74 4396 4
    1167.13  9 KILSVFFLA 3722 Pf EXP1 2 5.0 172 3448 8.0 9524 3
    1167.10  9 GLLGNVSTV 3723 Pf EXP1 83 24 1194 1.2 25 21053 3
    1167.19 10 VLLGGVGLVL 3724 Pf EXP1 91 94 — 2500 420 16000 2
    1167.18 10 ILSVSSFLFV 3725 Pf CSP 7 208 3583 19 587 2105 2
    * A dash indicates IC50 nM > 30000.
  • TABLE XXVII 
    P. falciparum A3-supermotif CTL epitopes
    A3-supertype binding capacity (IC50 nm)
    SEQ ID Alleles
    Peptide AA Sequence NO: Source A*301 A-1101 A*3101 A*3301 A*6801 bounda
    1167.32  9 QTNFKSLLR 3726 Pf LSA1 94 50 14 180 617 4 4
    1167.36  9 LACAGLAYK 3727 Pf SSP2 511 423 143 5294 64 32 4
    1167.43 10 VTCGNGIQVR 3728 Pf CSP 375 6875 11 15 64 444 4
    1167.24  9 ALFFIIFNK 3729 Pf EXP1 10 9.2 2.2 720 1261 73 3
    1167.51 10 LLACAGLAYK 3730 Pf SSP2 510 22 73 692 1526 24 3
    1167.28  9 GVSENIFLK 3731 Pf LSA1 105 151 5.0 2250 8286 10 3
    1167.47 10 HVLSHNSYEK 3732 Pf LSA1 59 407 200 — — 114 3
    1167.46 10 FILVNLLIFH 3733. Pf LSA1 11 733 1333 1957 397 154 2
    * A dash indicates IC50 nM > 30000.
  • TABLE XXVIII 
    P. falciparum B7-supermotif CTL epitopes
    SEQ ID B7-supertype binding capacity (IC50 nM) Alleles
    Peptide AA Sequence NO: Source B*0702 B*3501 B*5101 B*5301 B*5401 bounda
    1167.61 10 TPYAGEPAPF 3734 Pf SSP2 539 31 14 15   158 25000 4
    19.0051  8 LPYGRTNL 3735 Pf SSP2 126 50 — 32 15500   417 3
    * A dash indicates 1050 nM > 30000.
  • TABLE XXIX
    P. falciparum HLA-A*0101 and A*2402 binding peptides
    Binding capacity
    (IC50 nM)
    Motif Peptide AA Sequence SEQ ID NO: Source A*0101 A*2401
    A1 16.0040 9 FVEALFQEY 3736 Pf CSP 15   7.4
    16.0245 10 FQDEENIGIY 3737 Pf LSA1 1794 23
    15.0184 9 LPSENERGY 3738 37
    16.0130 9 PSDGKCNLY 3739 Pf SSP2 207 46
    A24 1167.55 9 YYPHQSSL 3740 Pf LSA1 1671   2.4
    1167.54 9 FYFILVNLL 3741 Pf LSA1 9 25
    1167.56 9 KYLVIVFLI 3742 Pf SSP2 8 34
    1167.53 9 KYKLATSVL 3743 Pf EXP1 73 75
  • TABLE XXX
    HLA-DR screening panels
    Screening Representative Assay Phenotypic Frequencies
    Panel Antigen Alleles Allele Alias Cauc. Blk. Jpn. Chn. Hisp. Avg.
    Primary DR1 DRB1*0101-03 DRB1*0101 (DR1) 18.5 8.4 10.7 4.5 10.1 10.4
    DR4 DRB1*0401-12 DRB1*0401 (DR4w4) 23.6 6.1 40.4 21.9 29.8 24.4
    DR7 DRB1*0701-02 DRB1*0701 (DR7) 26.2 11.1 1.0 15.0 16.6 14.0
    Panel total 59.6 24.5 49.3 38.7 51.1 44.6
    Secondary DR2 DRB1*1501-03 DRB1*1501 (DR2w2 β1) 19.9 14.8 30.9 22.0 15.0 20.5
    DR2 DRB5*0101 DRB5*0101 (DR2w2 β2) — — — — — —
    DR9 DRB1*09011, 09012 DRB1*0901 (DR9) 3.6 4.7 24.5 19.9 6.7 11.9
    DR13 DRB1*1301-06 DRB1*1302 (DR6w19) 21.7 16.5 14.6 12.2 10.5 15.1
    Panel total 42.0 33.9 61.0 48.9 30.5 43.2
    Tertiary DR4 DRB1*0405 DRB1*0405 (DR4w15) — — — — — —
    DR8 DRB1*0801-5 DRB1*0802 (DR8w2) 5.5 10.9 25.0 10.7 23.3 15.1
    DR11 DRB1*1101-05 DRB1*1101 (DR5w11) 17.0 18.0 4.9 19.4 18.1 15.5
    Panel total 22.0 27.8 29.2 29.0 39.0 29.4
    Quartemary DR3 DRB1*0301-2 DRB1*0301 (DR3w17) 17.7 19.5 0.4 7.3 14.4 11.9
    DR12 DRB1*1201-02 DRB1*1201 (DR5w12) 2.8 5.5 13.1 17.6 5.7 8.9
    Panel total 20.2 24.4 13.5 24.2 19.7 20.4
  • TABLE XXXI
    P. falciparum derived HTL candidate epitopes
    SEQ ID Binding capacity (IC50 nM)
    Peptide Sequence NO: Source DR1 DR2w2β1 DR2w2β2 DR4w4 DR4w15
    F125.04 RHNWVNHAVPLAMKLI 3744 Pf SSP2 61 26 260 83 14 317
    1188.34 HNWVNHAVPLAMKLI 3745 Pf SSP2 62 14 364 143 12 950
    1188.16 KSKYKLATSVLAGLL 3746 Pf EXP1 71 3.6 1247 24 7.1 47
    LVNLLIFHINGKIIKNSE 3747 Pf LSA1 13
    F125.02 LVNLLIFHINGKIIKNS 3748 Pf LSA1 13 78 13 426 — 1810
    27.0402 LLIFHINGKIIKNSE 3749 Pf LSA1 16 8.8 80 7500
    1188.32 GLAYKFVVPGAATPY 3750 Pf SSP2 512 3.1 - 29 45 1407
    27.0392 SSVFNVVNSSIGLIM 3751 Pf CSP 410 42 314 2500 450 1652
    27.0417 VKNVIGPFMKAVCVE 3752 Pf SSP2 223 56 212 250 — —
    27.0388 MRKLAILSVSSFLFV 3753 Pf CSP 2 50 18 1538 5769 1407
    27.0387 MNYYGKQENWYSLKK 3754 Pf CSP 53 6.4 9100 435 21 292
    1188.38 KYKIAGGIAGGLALL 3755 Pf SSP2 494 132 - 417 3750 22353
    1188.13 AGLLGNVSTVLLGGV 3756 Pf EXP1 82 116 379 15,385 6923 1056
    27.0408 QTNFKSLLRNLGVSE 3757 Pf LSA1 94 91 8273 5405 2500 1900
    35.0171 PDSIQDSLKESRKLN 3758 Pf SSP2 165 - 2285 - -
    35.0172 KCNLYADSAWENVKN 3759 Pf SSP2 211 23425 18200 - -
    Binding capacity (IC50 nM) Alleles
    Peptide DR5w11 DR6w19 DR7 DR8w2 DR9 DR3 DR5w12 bound2
    F125.04 282 3.9 23 41 33 8751 441 11
    1188.34 2703 3.7 66 68 19 1304 497 10
    1188.16 30 427 13 45 28 — —  9
    F125.02 408 66 260 766 625 19722 11610  8
    27.0402 56 106 192 350 500 566 12957  8
    1188.32 11 7.1 167 20 125 — 851  9
    27.0392 1176 9.7 33 891 63 — —  7
    27.0417 476 32 424 2130 862 — 3239  7
    27.0388 541 38 500 — 682  6
    27.0387 351 3182 3788 538 22059  6
    1188.38 87 15 3968 31 288  6
    1188.13 — 0.76 58 — 142  5
    27.0408 51 47 7813 69 —  4
    35.0171 — — — — — 357  1
    35.0172 — 11061 — — — 857  1
    A dash (—) indicates IC50 > 20 μM.
  • TABLE XXXII
    PBMC responses of individuals from the Irian Java
    endemic malaria region.
    Percent individuals yielding positive responses (n)
    Peptide IFNγ TNFα Proliferation
    CSP.2 11% (7) 59% (39)  9% (11)
    LSA1.13 16% (9) 30% (21)  8% (10)
    CSP.53  7% (4) 53% (40) 3% (4)
    SSP2.61  7% (4) 45% (36) 7% (9)
    SSP2.223 15% (9) 42% (31) 5% (6)
    CSP.410 16% (9) 47% (33) 12% (14)
    EXP1.82  29% (17) 43% (32) 6% (7)
    EXP1.71  9% (5) 49% (36) 12% (14)
    SSP2.512 14% (8) 41% (30) 3% (4)
    SSP2.62 11% (6) 42% (31) 12% (14)
    SSP2.494  7% (4) 36% (26) 2% (3)
  • TABLE XXXIII 
    P. falciparum CTL epitopes
    Supertype SEQ ID Alleles
    (or allele) Peptide AA Sequence NO: Source bounda
    A2- 1167.08  9 GLIMVLSFL 3760 Pf CSP 425 4
    supertype 1167.10  9 GLLGNVSTV 3761 Pf EXP1 83 3
    1167.12  9 VLAGLLGNV 3762 Pf EXP1 80 4
    1167.13  9 KILSVFFLA 3763 Pf EXP1 2 3
    1167.16  9 FMKAVCVEV 3764 Pf SSP2 230 5
    1167.18 10 ILSVSSFLFV 3765 Pf CSP 7 2
    1167.19 10 VLLGGVGLVL 3766 Pf EXP1 91 2
    1167.21 10 FLIFFDLFLV 3767 Pf SSP2 14 5
    A3- 1167.24  9 ALFFIIFNK 3768 PF EXP1 10 3
    supertype 1167.28  9 GVSENIFLK 3769 Pf LSA1 105 3
    1167.32  9 QTNFKSLLR 3770 Pf LSA1 94 4
    1167.36  9 LACAGLAYK 3771 Pf SSP2 511 4
    1167.43 10 VTCGNGIQVR 3772 Pf CSP 375 4
    1167.46 10 FILVNLLIFH 3773 Pf LSA1 11 2
    1167.47 10 HVLSHNSYEK 3774 Pf LSA1 59 3
    1167.51 10 LLACAGLAYK 3775 Pf SSP2 510 3
    B7- 19.0051  8 LPYGRTNL 3776 Pf SSP2 126 3
    supertype 1167.61 10 TPYAGEPAPF 3777 Pf SSP2 539 4
    A1 15.0184  9 LPSENERGY 3778 Pf LSA1 1663 1
    16.0040  9 FVEALFQEY 3779 Pf CSP 15 1
    16.0130  9 PSDGKCNLY 3780 Pf SSP2 207 1
    16.0245 10 FQDEENIGIY 3781 Pf LSA1 1794 1
    A24 1167.53  9 KYKLATSVL 3782 Pf EXP1 73 1
    1167.54  9 FYFILVNLL 3783 Pf LSA1 9 1
    1167.55  9 YYIPHQSSL 3784 Pf LSA1 1671 1
    1167.56  9 KYLVIVFLI 3785 Pf SSP2 8 1
    aA2-supertype peptides are tested for binding to A*0201, A*0202, A*0203, A*0206, and A*6802. A3-supertype peptides are tested for binding to A*03, A*11, A*31011, A*3301, and A*6801. B7-supertype peptides are tested for binding to B*0702, B*3501, B*5101, B*5301, and B*5401. A1 and A24 peptides are tested for binding to A*0101 and A*2402, respectively.
  • TABLE XXXIV 
    P. falciparum HTL epitopes
    SEQ ID Alleles
    Motif Peptide Sequence NO: Source bounda
    DR- F125.04 RHNWVNHAVPLAMKLI 3786 Pf SSP2 61 11
    supermotif 1188.16 KSKYKLATSVLAGLL 3787 Pf EXP1 71  9
    27.0402 LLIFHINGKIIKNSE 3788 Pf LSA1 16 9(DR3)
    1188.32 GLAYKFVVPGAATPY 3789 Pf SSP2 512 9
    27.0392 SSVFNVVNSSIGLIM 3790 Pf CSP 410 7
    27.0417 VKNVIGPFMKAVCVE 3791 Pf SSP2 223 7
    27.0388 MRKLAILSVSSFLFV 3792 Pf CSP 2 6
    27.0387 MNYYGKQENWYSLKK 3793 PF CSP53 6
    1188.38 KYKIAGGIAGGLALL 3794 Pf SSP2 494 6
    1188.13 AGLLGNVSTVLLGGV 3795 Pf EXP1 82 5
    27.0408 QTNFKSLLRNLGVSE 3796 Pf LSA1 94 4
    DR3 35.0171 PDSIQDSLKESRKLN 3797 Pf SSP2 165 DR3
    35.0172 KCNLYADSAWENVKN 3798 Pf SSP2 211 DR3
    aHLA-DR supermotif peptides are screened for binding to a panel alleles representing the 10 most common HLA antigens, including DR1, DR2w2 β1, DR2w2 β2, DR4w4, DR4w15, DR5w11, DR6w19, DR7, DR8w2, and DR9. Additional alleles that are tested include DR3, DR5w12, DR52a, and DR53. DR3-motif peptides are tested for binding to DR3.
  • TABLE XXXV
    Estimated population coverage by a panel of P. falciparum derived HTL epitopes
    Representative No. of Population coverage (phenotypic frequency)
    Antigen Alleles assay epitopes2 Cauc. Blk. Jpn. Chn. Hisp. Avg.
    DR1 DRB1*0101-03 DR1 11 18.5 8.4 10.7 4.5 10.1 10.4
    DR2 DRB1*1501-03 DR2w2 β1 6 19.9 14.8 30.9 22.0 15.0 20.5
    DR2 DRB5*0101 DR2w2 β2 7 — — — — — —
    DR3 DRB1*0301-2 DR3 3 17.7 19.5 0.40 7.3 14.4 11.9
    DR4 DRB1*0401-12 DR4w4 5 23.6 6.1 40.4 21.9 29.8 24.4
    DR4 DRB1*0401-12 DR4w15 3 — — — — — —
    DR7 DRB1*0701-02 DR7 8 26.2 11.1 1.0 15.0 16.6 14.0
    DR8 DRB1*0801-5 DR8w2 8 5.5 10.9 25.0 10.7 23.3 15.1
    DR9 DRB1*09011, 09012 DR9 9 3.6 4.7 24.5 19.9 6.7 11.9
    DR11 DRB1*1101-05 DR5w11 9 17.0 18.0 4.9 19.4 18.1 15.5
    DR12 DRB1*1201-2 DR5w12 2 2.8 5.5 13.1 17.6 5.7 8.9
    DR13 DRB1*1301-06 DR6w19 10 21.7 16.5 14.6 12.2 10.5 15.1
    Total 97.0 83.9 98.8 95.5 95.6 94.7

Claims (17)

1-40. (canceled)
41. An isolated peptide less than 13 amino acids in length comprising the oligopeptide: LLACAGLAY (SEQ ID NO: 3019), FLIFFDLFLV (SEQ ID NO: 3718), FMKAVCVEV (SEQ ID NO: 3719), VLAGLLGNV (SEQ ID NO: 3720), GLIMVLSFL (SEQ ID NO: 3721), KILSVFFLA (SEQ ID NO: 3722), GLLGNVSTV (SEQ ID NO: 3723), VLLGGVGLVL (SEQ ID NO: 3724), ILSVSSFLFV (SEQ ID NO: 3725), QTNFKSLLR (SEQ ID NO: 3726), LACAGLAYK (SEQ ID NO: 3727), ALFFIIFNK (SEQ ID NO: 3729), LLACAGLAYK (SEQ ID NO: 3730), HVLSHNSYEK (SEQ ID NO: 3732), FILVNLLIFH (SEQ ID NO: 3733), FQDEENIGIY (SEQ ID NO: 3737), PSDGKCNLY (SEQ ID NO: 3739), YYIPHQSSL (SEQ ID NO: 3740), FYFILVNLL (SEQ ID NO: 3741), KYLVIVFLI (SEQ ID NO: 3742) or KYKLATSVL (SEQ ID NO: 3743).
42. The isolated peptide of claim 41, wherein the peptide is LLACAGLAY (SEQ ID NO: 3019), FLIFFDLFLV (SEQ ID NO: 3718), FMKAVCVEV (SEQ ID NO: 3719), VLAGLLGNV (SEQ ID NO: 3720), GLIMVLSFL (SEQ ID NO: 3721), KILSVFFLA (SEQ ID NO: 3722), GLLGNVSTV (SEQ ID NO: 3723), VLLGGVGLVL (SEQ ID NO: 3724), ILSVSSFLFV (SEQ ID NO: 3725), QTNFKSLLR (SEQ ID NO: 3726), LACAGLAYK (SEQ ID NO: 3727), ALFFIIFNK (SEQ ID NO: 3729), LLACAGLAYK (SEQ ID NO: 3730), HVLSHNSYEK (SEQ ID NO: 3732), FILVNLLIFH (SEQ ID NO: 3733), FQDEENIGIY (SEQ ID NO: 3737), PSDGKCNLY (SEQ ID NO: 3739), YYIPHQSSL (SEQ ID NO: 3740), FYFILVNLL (SEQ ID NO: 3741), KYLVIVFLI (SEQ ID NO: 3742) or KYKLATSVL (SEQ ID NO: 3743).
43. A conjugate of an isolated peptide less than 13 amino acids in length comprising an oligopeptide selected from a group consisting of LLACAGLAY (SEQ ID NO: 3019), FLIFFDLFLV (SEQ ID NO: 3718), FMKAVCVEV (SEQ ID NO: 3719), VLAGLLGNV (SEQ ID NO: 3720), GLIMVLSFL (SEQ ID NO: 3721), KILSVFFLA (SEQ ID NO: 3722), GLLGNVSTV (SEQ ID NO: 3723), VLLGGVGLVL (SEQ ID NO: 3724), ILSVSSFLFV (SEQ ID NO: 3725), QTNFKSLLR (SEQ ID NO: 3726), LACAGLAYK (SEQ ID NO: 3727), ALFFIIFNK (SEQ ID NO: 3729), LLACAGLAYK (SEQ ID NO: 3730), HVLSHNSYEK (SEQ ID NO: 3732), FILVNLLIFH (SEQ ID NO: 3733), FQDEENIGIY (SEQ ID NO: 3737), PSDGKCNLY (SEQ ID NO: 3739), YYIPHQSSL (SEQ ID NO: 3740), FYFILVNLL (SEQ ID NO: 3741), KYLVIVFLI (SES ID NO: 3742) and KYKLATSVL (SEQ ID NO: 3743) and a T helper peptide, wherein the T helper peptide is less than about 50 amino acids in length and wherein the T helper peptide comprises a pan-DR binding epitope.
44. The conjugate of claim 43, wherein the isolated peptide is LLACAGLAY (SEQ ID NO: 3019), FLIFFDLFLV (SEQ ID NO: 3718), FMKAVCVEV (SEQ ID NO: 3719), VLAGLLGNV (SEQ ID NO: 3720), GLIMVLSFL (SEQ ID NO: 3721), KILSVFFLA (SEQ ID NO: 3722), GLLGNVSTV (SEQ ID NO: 3723), VLLGGVGLVL (SEQ ID NO: 3724), ILSVSSFLFV (SEQ ID NO: 3725), QTNFKSLLR (SEQ ID NO: 3726), LACAGLAYK (SEQ ID NO: 3727), ALFFIIFNK (SEQ ID NO: 3729), LLACAGLAYK (SEQ ID NO: 3730), HVLSHNSYEK (SEQ ID NO: 3732), FILVNLLIFH (SEQ ID NO: 3733), FQDEENIGIY (SEQ ID NO: 3737), PSDGKCNLY (SEQ ID NO: 3739), YYIPHQSSL (SEQ ID NO: 3740), FYFILVNLL (SEQ ID NO: 3741), KYLVIVFLI (SEQ ID NO: 3742) or KYKLATSVL (SEQ ID NO: 3743).
45. The conjugate of claim 43, wherein said pan-DR binding epitope is aKXVWANTLKAAa (SEQ ID NO: 3802), wherein “X” is either cycloexylalanine, phenylalanine, or tyrosine, and “a” is either D-alanine or L-alanine.
46. The conjugate of claim 45, wherein “X” is cycloexylalanine.
47. The conjugate of claim 45, wherein “X” is phenylalanine.
48. The conjugate of claim 45, wherein “X” is tyrosine.
49. The conjugate of claim 45, wherein “a” is D-alanine.
50. The conjugate of claim 45, wherein “a” is L-alanine.
51. A composition comprising the isolated peptide of claim 41.
52. A composition comprising the isolated peptide claim 42.
53. The composition of claim 51, further comprising a carrier.
54. A composition comprising the conjugate of claim 43.
55. A composition comprising the conjugate of claim 44.
56. The composition of claim 54, further comprising a carrier.
US14/980,150 1993-03-05 2015-12-28 Inducing Cellular Immune Responses to Plasmodium Falciparum Using Peptide and Nucleic Acid Compositions Abandoned US20160193316A1 (en)

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US27863494A 1994-07-21 1994-07-21
US45191395A 1995-05-26 1995-05-26
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US1311396P 1996-03-11 1996-03-11
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US75361596A 1996-11-27 1996-11-27
US08/821,739 US20020168374A1 (en) 1992-08-07 1997-03-20 Hla binding peptides and their uses
US09/017,743 US20020177694A1 (en) 1996-01-23 1998-02-03 Hla binding peptides and their uses
US8719298P 1998-05-29 1998-05-29
US09/189,702 US7252829B1 (en) 1998-06-17 1998-11-10 HLA binding peptides and their uses
US11748699P 1999-01-27 1999-01-27
US35040199A 1999-07-08 1999-07-08
US35773799A 1999-07-19 1999-07-19
US09/390,061 US9266930B1 (en) 1993-03-05 1999-09-03 Inducing cellular immune responses to Plasmodium falciparum using peptide and nucleic acid compositions
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