WO2010003053A2

WO2010003053A2 - Hsv-2 peptides for stimulation of cytotoxic t lymphocytes

Info

Publication number: WO2010003053A2
Application number: PCT/US2009/049506
Authority: WO
Inventors: Peter J. Wettstein; Nancy D. Borson; Michael A. Strausbauch
Original assignee: Mayo Foundation For Medical Education And Research
Priority date: 2008-07-03
Filing date: 2009-07-02
Publication date: 2010-01-07
Also published as: WO2010003053A3

Abstract

Compositions containing HLA-A2 binding-peptides and adjuvants are described as well as methods of using such compositions to stimulate immune responses to herpes simplex virus-2 infection in mammals.

Description

HS V-2 Peptides for Stimulation of Cytotoxic T Lymphocytes

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority from U.S. Provisional Application Serial No. 61/078,085, filed on July 3, 2008. The disclosure of the prior application is incorporated by reference in its entirety.

TECHNICAL FIELD

This document relates to methods and materials for stimulating an immune response to herpes simplex virus 2 (HSV -2) infection, and more particularly, to using compositions containing HLA- A2 binding peptides for stimulating an immune response to HS V-2.

BACKGROUND

HSV-2 infection in the human population is a significant clinical problem that has not been successfully treated with antiviral drugs. The inability of antiviral drugs to cure HSV-2 infections is a major impetus for the development of effective preventative and therapeutic vaccines. Infection with HSV-2 stimulates strong T and B cell responses. The importance of cytotoxic T lymphocytes (CTLs) in the immune response to HSV-2 infection and the infiltration of infected ganglia by CTLs indicate that vaccinations to activate and expand HSV-2-specific CTLs should have strong therapeutic potential.

SUMMARY This document is based on the identification of HLA- A2 binding peptides from HSV-2 proteins that can be used to induce an immune response against HSV-2 infection. For example, the HLA- A2 binding peptides described herein can be used in compositions for stimulating the activation and expansion of HSV-2 specific CTLs. In one aspect, this document features an isolated HLA- A2 binding peptide. The peptide can comprise or consist essentially of an amino acid sequence selected from the group consisting of CLFQPLMFI (SEQ ID NO:7), RLRNAIREV (SEQ ID NO: 1), ALASADETL (SEQ ID NO:2), ILARLANRV (SEQ ID NO:3), MLIDLGLDL (SEQ ID NO:4), VLENLATRL (SEQ ID NO:5), RRLSDIKDI (SEQ ID NO:6), VLANFCSAL (SEQ ID NO: 10), VLVDQTGNV (SEQ ID NO: 13), FVSSSSAQV (SEQ ID NO: 14), AVAAVVPRV (SEQ ID NO: 15), ALDQACFRI (SEQ ID NO: 17), VLLLSTRDL (SEQ ID NO: 18), and RLRAWLREL (SEQ ID NO: 19). For example, the peptide can have the amino acid sequence CLFQPLMFI (SEQ ID NO:7) or VLANFCSAL (SEQ ID NO: 10). The HLA-A2 binding peptide further can include a CpG-interacting amino acid sequence. The CpG-interacting amino acid sequence can include a cysteine residue and/or at least one positively charged amino acid. For example, the CpG-interacting amino acid sequence can comprise or consist essentially of the sequence set forth in SEQ ID NO:44 (CASANA) or SEQ ID NO:45 (KCSRNR). The CpG-interacting amino acid sequence can be located at the amino terminus of the HLA-A2 binding peptide.

In another aspect, this document features a pharmaceutical composition that includes an HLA-A2 binding peptide and an adjuvant, wherein the HLA- A2 binding peptide includes the amino acid sequence CLFQPLMFI or VLANFCSAL. The adjuvant can be a CpG oligonucleotide. The CpG oligonucleotide can include a phosphorothioate linkage and/or a phosphorothioate backbone. In some embodiments, the composition includes the HLA- A2 binding peptide having the amino acid sequence CLFQPLMFI. In some embodiments, the composition includes the HLA- A2 binding peptide having the amino acid sequence CLFQPLMFI and the HLA-A2 binding peptide having the amino acid sequence VLANFCSAL. The HLA- A2 binding peptide further can include a CpG-interacting amino acid sequence. The CpG-interacting amino acid sequence can include a cysteine residue and/or at least one positively charged amino acid. For example, the CpG-interacting amino acid sequence can comprise or consist essentially of the sequence set forth in SEQ ID NO:44 (CASANA) or SEQ ID NO:45 (KCSRNR). The CpG-interacting amino acid sequence can be located at the amino terminus of the HLA-A2 binding peptide.

A composition described above further can include a different peptide that stimulates a CTL response against HSV-2. For example, the different peptide can be selected from the group consisting of GAPRSSSSGGAPLRAGVGSGS (SEQ ID NO:35); APAWSRRTL (SEQ ID NO:36); AIDYVHCKGIIHRDI (SEQ ID NO:37); AVPLLSAGGAAPPHP (SEQ ID NO:38); ELYYGPVSPADPESP (SEQ ID NO:39); PMRARPRGEVRFLHY (SEQ ID NO:40); RPRGEVRFLHYDEAG (SEQ ID NO:41); VAGFNKRVFCAAVGR (SEQ ID NO:42); and STAPEVGTYTPLRYAC (SEQ ID NO:43). This document also features a method for inducing an immune response to an HSV-2 infection in a subject. The method includes administering to the subject an amount of a pharmaceutical composition effective to induce the immune response, the pharmaceutical composition including an HLA- A2 binding peptide and an adjuvant, wherein the HLA-A2 binding peptide comprises or consists essentially of the amino acid sequence CLFQPLMFI (SEQ ID NO:7) or VLANFCSAL (SEQ ID NO: 10). The adjuvant can be a CpG oligonucleotide. The CpG oligonucleotide can include a phosphorothioate linkage and/or a phosphorothioate backbone. In some embodiments, the composition includes the HLA- A2 binding peptide having the amino acid sequence CLFQPLMFI. In some embodiments, the composition includes the HLA- A2 binding peptide having the amino acid sequence CLFQPLMFI and the HLA-A2 binding peptide having the amino acid sequence VLANFCSAL. Such compositions further can include a different peptide that stimulates a CTL response against HSV-2, e.g., a peptide selected from the group consisting of GAPRSSSSGGAPLRAGVGSGS (SEQ ID NO:35); APAWSRRTL (SEQ ID

NO:36); AIDYVHCKGIIHRDI (SEQ ID NO:37); AVPLL S AGG AAPPHP (SEQ ID NO:38); ELYYGPVSPADPESP (SEQ ID NO:39); PMRARPRGEVRFLHY (SEQ ID NO:40); RPRGEVRFLHYDEAG (SEQ ID NO:41); VAGFNKRVFCAAVGR (SEQ ID NO:42); and STAPEVGTYTPLRYAC (SEQ ID NO:43). The HLA-A2 binding peptide further can include a CpG-interacting amino acid sequence. The CpG- interacting amino acid sequence can include a cysteine residue and/or at least one positively charged amino acid. For example, the CpG-interacting amino acid sequence can comprise or consist essentially of the sequence set forth in SEQ ID NO:44 (CASANA) or SEQ ID NO:45 (KCSRNR). The CpG-interacting amino acid sequence can be located at the amino terminus of the HLA-A2 binding peptide.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph depicting the stabilization of HLA- A2 expression by T2 cells by the addition of exogenous HSV-2 peptides. Test peptides were as follows: (1) peptides #1-30 derive from HSV-2 proteins, (2) an influenza peptide with high binding affinity, and (3) an irrelevant peptide that does not bind to HLA-A2 molecules. T2 cells were labeled with peptides and incubated at 37°C for extending time intervals after which the cells were stained with fluorochrome-labeled anti-HLA- A2 antibody for analysis by flow cytometry. Values on the y-axes are median fluorescent intensities .

FIG. 2 is a graph depicting that the combination of HSV-2 #7 peptide + CpG primes a strong CTL responses. The values on the y-axis are numbers of spots/400,000 responders.

FIG. 3 is a graph depicting that the combination of HSV-2 #10 peptide + CpG primes detectable CTL responses. The values on the y-axis are numbers of spots/400,000 responders.

FIG. 4 is a graph depicting booster vaccinations with #7 peptide + CpG increases frequencies of specific CTLs. The values on the y-axis are numbers of spots/400,000 responders. ' * ' signifies that numbers of spots were too numerous to accurately count, and the cited numbers are minimal estimates.

FIG. 5 is a graph depicting booster vaccinations with #10 peptide + CpG increase frequencies of specific CTLs. All- hemizygotes were primed with lOOμg peptide + lOOμg CpG; three mice received second vaccinations with 50μg peptide + 50μg CpG. Spleens were harvested at 1 Id for enrichment of CD8⁺ CTLs for analysis in an IFNγ Elispot assay. The values on the y-axis are numbers of spots/400,000 responders. FIG. 6 contains two graphs depicting the number of spots per 200,000 CTLs when various immunizing peptides (SEQ ID NO:7; CASANA-CLFQPLMFI (SEQ ID NO:31); KCSRNR-CLFQPLMFI (SEQ ID NO:32); CLFQPLMFI- ANASAC (SEQ ID NO:33); or CLFQPLMFI-RNRSCK (SEQ ID NO:34) are combined with CpG adjuvant for in vivo priming. In the top panel, 100 nM CLFQPLMFI was used to pulse the A2/A2 stimulators. In the bottom panel, 10 nM CLFQPLMFI was used to pulse the A2/A2 stimulators.

DETAILED DESCRIPTION

In general, this document provides methods and materials for inducing immune responses to HSV-2 in mammals (e.g., humans) using HLA-A2 binding peptides. As used herein, "HLA- A2 binding peptides" are fragments of HSV proteins that have the ability to bind HLA-A2. For example, HLA-A2 binding peptides can be from HSV-2 proteins involved in reactivation and early viral replication, such as the ICP22, ICP27, ICPO, ICP4, or VP16 proteins. The amino acid sequence of the ICPO, ICP4, VP 16, ICP22, and ICP27 proteins is provided in GenBank (Accession Nos. NP_044528, NP_044544, and NP_044518, NP_044531, and NP_044525 respectively). HLA- A2 binding peptides typically range from 6 to 100 amino acids (e.g., 6-75, 6-60, 6-50, 6-25, 6-20, 6-15, 7-18, 7-15, 7-12, 8-10, or 9-12 amino acids) in length. For example, an HLA-A2 binding peptide can be 7, 8, 9, 10, 11, or 12 amino acids in length. Peptides can be synthesized on a solid phase and used directly as "crude peptide" or purified to >95% purity (e.g., by high performance liquid chromatography (HPLC)).

Binding capacities of HLA- A2 binding peptides can be assessed, for example, using T2 cells, an HLA- A2 -positive cell line that requires exogenous peptides for stable class I expression. Stabilized class I expression can be examined by incubating the cells with a test peptide then staining the cells with fluorochrome-labeled anti- HLA-A2 antibodies. Flow cytometry can be used to estimate off-rates of peptide:HLA-A2 binding.

Non-limiting examples of HLA-A2 binding peptides include the following: RLRNAIREV (SEQ ID NO:1), ALASADETL (SEQ ID NO:2), ILARLANRV (SEQ ID NO:3), MLIDLGLDL (SEQ ID NO:4), VLENLATRL (SEQ ID NO:5), RRLSDIKDI (SEQ ID NO:6), CLFQPLMFI (SEQ ID NO:7), VLANFCSAL (SEQ ID NO: 10), VLVDQTGNV (SEQ ID NO: 13), FVSSSSAQV (SEQ ID NO: 14), AVAAVVPRV (SEQ ID NO: 15), ALDQACFRI (SEQ ID NO: 17), VLLLSTRDL (SEQ ID NO:18), and RLRAWLREL (SEQ ID NO:19). Peptides having the sequence CLFQPLMFI (SEQ ID NO:7) or VLANFCSAL (SEQ ID NO: 10) are particularly useful as such peptides can stimulate an in vivo cytotoxic T lymphocyte (CTL) response. CTLs recognize processed peptides that are presented by cell- surface molecules encoded by the class I genes of the major histocompatibility complex (HLA in humans). As such, HLA-A2 binding peptides can be used to stimulate the activation and expansion of HSV-2-specifϊc CTLs in a subject. The level of activated CTLs can be monitored by a variety of methods known in the art, including, but not limited to, ELISPOT assays.

Compositions Containing HLA- A2 binding peptides

An HLA- A2 binding peptide that stimulates a CTL response can be combined with an adjuvant to form a pharmaceutical composition that can be used to elicit an immune response against HSV-2 when administered to a mammal (e.g., a human). A composition described herein can contain a mixture of peptides. For example, the mixture can contain fragments from different naturally-occurring proteins, or different fragments from the same naturally-occurring protein. Fragments from the same naturally-occurring proteins can contain overlapping or nonoverlapping amino acid sequences. The use of various protein fragments can provide a collection of peptides with varying stability and varying CTL activation capabilities. For example, in some embodiments, a composition can include an HLA- A2 binding peptide having the sequence CLFQPLMFI (SEQ ID NO:7) or an HLA- A2 binding peptide having the sequence VLANFCSAL (SEQ ID NO: 10). In some embodiments, a composition can include an HLA- A2 binding peptide having the sequence CLFQPLMFI and an HLA- A2 binding peptide having the sequence VLANFCSAL. In some embodiments, the composition also contains another peptide from an HSV protein that stimulates a CTL response. For example, a peptide having the sequence GAPRSSSSGGAPLRAGVGSGS (SEQ ID NO:35); APAWSRRTL (SEQ ID NO:36); AIDYVHCKGIIHRDI (SEQ ID NO:37); AVPLL S AGG AAPPHP (SEQ ID NO:38); ELYYGPVSPADPESP (SEQ ID NO:39); PMRARPRGEVRFLHY (SEQ ID NO:40); RPRGEVRFLHYDEAG (SEQ ID NO:41); VAGFNKRVFCAAVGR (SEQ ID NO:42); or STAPEVGTYTPLRYAC (SEQ ID NO:43) can be included in a composition.

An "adjuvant" is an immunological compound that can enhance an immune response against a particular antigen such as a polypeptide. For example, an adjuvant can be a CpG molecule. Typically, a CpG molecule is an oligonucleotide that contains at least one unmethylated cytosine-guanine dinucleotide. CpG molecules can be about 15-25 nucleotides in length (e.g., about 18-20 nucleotides in length). The oligonucleotide can include at least one CpG consensus motif of RRCpGYY, where R is purine and Y is pyrimidine. The CpG molecules can include at least one phosphorothioate linkage in the backbone structure. The side chains of a phosphorothioate backbone contain at least one or more sulfur atoms in place of oxygen, and a phosphorothioate backbone can yield a longer half life, increased level of activity, and subtle changes in the specificity of activity (Kreig, Annu. Rev. Immunol. 20:709, 2002) compared to an oligonucleotide backbone that does not include a sulfur atom. The backbone of a CpG molecule also can include (or can alternatively include) one or more phosphorodithioate, methylphosphonate, or 2'-O- methylribonucleotides linkages.

Any CpG molecule can be used in the compositions and methods described herein. See WO 96/025555. For example, the CpG molecule 1826 (5'-TCC A TG ACG TTC CTG ACG TT-3'; SEQ ID NO:46), specific for mouse TLR (Davis et al. J₁ Immunol. 160:870, 1998) can be used as described. See also CpG molecules described in Lingnau et al. (Vaccine 20:3498-3508, 2002), for example.

In embodiments in which a CpG molecule is used as adjuvant, an HLA- A2 binding peptide described herein further can include a CpG interacting amino acid sequence, which may complex with and concentrate the adjuvant activity of CpG motifs for activation of localized antigen presenting cells (APCs). The CpG- interacting amino acid sequence can be located at any position in the HLA- A2 binding peptide, such as at, or near, the N- or C-terminus, or in about the middle of the peptide. A CpG interacting amino acid sequence typically includes no more than about 20 (e.g., 15, 12, 10, 8, or 6) amino acid residues. In particular, a CpG interacting amino acid sequence includes at least one cysteine residue and in some embodiments, one, two, three or more positively charged amino acids (e.g., arginine or lysine residues). An excess of positively charged residues may block T cell activation and expansion in a short term scenario, but may be effective in stimulating T cell activation over a long term period (e.g., longer than 30, 40, 50, 75, 100, or 150 days, or longer). This inhibition may be caused by a concentrated precipitation of CpG that sequesters both the polypeptide and CpG in an inactive complex. See, for example, U.S. Patent Publication No. 2008-0146488-A1 and U.S. Application No. 11/513,628.

In some embodiments, the CpG-interacting amino acid sequence contains a cysteine residue and no positively charged amino acids. The cysteine residue can facilitate the interaction of the HLA-A2 binding peptide with the CpG molecule to create a complex with enhanced immunogenicity. The CpG-interacting amino acid sequence can therefore include the sequence XCX, where C is cysteine, and X is any amino acid. While not wishing to be bound by theory, the cysteine residues of the CpG-interacting amino acid sequence can interact with, e.g., covalently link, the CpG- interacting amino acid sequence (and so also the HLA- A2 binding peptide to which it is attached) to a CpG molecule. See, for example, U.S. Patent Publication No. 2008- 0146488-A1 and U.S. Application No. 11/513,628.

In embodiments in which positively charged amino acids are included in a CpG interacting amino acid sequence, the positively charged amino acid residues can flank the Cys residue. In other embodiments, the positively charged amino acids are spaced at regular intervals throughout the CpG interacting amino acid sequence, such as at every other, every third, or every fourth amino acid position. It is not necessary that the placement of the positive amino acid residues has perfect periodicity. For example, the CpG interacting amino acid sequence includes a B-X or X-B sequence, or a B-X-B sequence, where B is a positively charged amino acid residue (e.g., arginine, lysine, or histidine) and X is any amino acid residue. In one embodiment, the CpG-interacting amino acid sequence can be B-X-B-X-B, B-X-X-B-X-B, B-X-X- B-X-X-B, and the like. The CpG-interacting amino acid sequence can be, for example, CASANA (SEQ ID NO:44) or KCSRNR (SEQ ID NO:45). See, for example, U.S. Patent Publication No. 2008-0146488-A1 and U.S. Application No. 11/513,628. A HLA-A2 binding peptide having the following sequence can be particularly useful: CASANACLFQPLMFI (SEQ ID NO:31).

In some embodiments, a composition contains an HLA- A2 binding peptide and CpG molecules in an oil emulsion, such as Incomplete Freund's Adjuvant. The HLA-A2 binding peptide further can include a CpG interacting amino acid sequence as discussed above. Optionally, a composition can include MPL-AF (monophosphoryl Lipid A adjuvant (MPL) mixed with dipalmitoyl phosphatidyl choline (DPPC)). Compositions can contain a molar ratio of CpG:peptide that will effectively deliver the maximum amount of peptide to incite a strong immune response while simultaneously minimizing the dose of CpG to avoid a toxic response. For example the molar ratio of CpG:peptide in the pharmaceutical composition can be, e.g., about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, or 9: 1.

CpG:peptide complexes can be pre-formed before adding to a solution for administration to a human. For example, the complexes can be formed by first resuspending the CpG molecule in an aqueous buffer, such as phosphate buffered saline (PBS), and then adding the HLA- A2 binding peptide, and incubating the solution for a time sufficient to allow complexes to form. Alternatively, the CpG molecule and HLA- A2 binding peptide can be added together in a solvent such as dimethylsulfoxide (DMSO), the complexes allowed to form, and the solution diluted in an aqueous buffer (e.g., PBS). The choice of conditions for complexing may be dependent upon the relative hydrophobicity of the peptides. Complexing of CpG with relatively hydrophobic peptides may proceed more efficiently in organic solvents such as DMSO due to the insolubility of such peptides in aqueous solutions. A pharmaceutical composition described herein can be used as a vaccine to prevent HSV-2 infection, ameliorate one or more symptoms of HSV-2 infection, or decrease severity or duration of HSV-2 infection. A pharmaceutical composition described herein can be provided in solution, such as in an aqueous or organic solution (e.g., an oil emulsion), or can be provided in lyophilized form. The compositions described herein can be administered on multiple occasions and at varying concentrations. Toxicity and therapeutic efficacy of the compositions disclosed herein (e.g., pharmaceutical compositions) can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Peptides or other compounds that exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (that is, the concentration of the peptide(s) and CpG molecules which achieves a half-maximal inhibition of symptoms, e.g., CTL-activation) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. The amount of peptide and adjuvant (e.g., CpG molecule) in a vaccine dose is selected as an amount that induces an immunoprotective response without significant, adverse side effects in a subject. Such amount can vary depending on the formulation. Generally it is expected that each dose will comprise less than about 500 μg (e.g., less than about 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 20 10, 5 or 1 μg) each of peptide and CpG molecule. The dose can, optionally, comprise an equal molar ratio of the two components. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of CTL responses, antibody titres, and other responses in subjects. Following an initial vaccination, subjects may receive a boost in about 4 weeks.

Generally, administration of a pharmaceutical composition facilitates an intended purpose for both prophylaxis and treatment without undesirable side effects, such as toxicity, irritation or allergic response. Although individual needs may vary, the determination of optimal ranges for effective amounts of formulations is within the skill of the art. Human doses can readily be extrapolated from animal studies (Katocs et ah, Chapter 27 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990). Generally, the dosage required to provide an effective amount of a formulation will vary depending on several factors, including the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy, if required, and the nature and scope of the desired effect(s) (Nies et ah, Chapter 3, In: Goodman & Gilman's The Pharmacological Basis of Therapeutics. 9th Ed., Hardman et al, eds., McGraw-Hill, New York, N.Y., 1996).

Various screening methods are also provided herein. One such screening method can be used to identify HSV-2 peptides that activate CTLs. The CTL- activating peptides can be used in the production of the pharmaceutical compositions described herein. The methods can include, for example, combining a test peptide with a CpG molecule, administering the mixture to a mammal, such as a mouse or a rat, harvesting CTLs from the mammal, and determining whether or not the level of cytotoxic T lymphocytes {e.g., CD8⁺ CTLs) in the mammal is increased. The method further can include administering a peptide to a mammal in a formulation with an adjuvant such as a CpG molecule. By determining whether the peptide/CpG molecule composition can activate CTLs in the mammal, it can be determined that the test amino acid sequence can function in vivo as part of an effective immunotherapeutic reagent.

Articles of Manufacture

Compositions or vaccines described herein can be combined with packaging materials and sold as articles of manufacture or kits. Components and methods for producing articles of manufactures are well known. The articles of manufacture may combine one or more HLA-A2 binding peptides or pharmaceutical compositions (e.g., vaccines) described herein. In addition, the articles of manufacture may further include sterile water, pharmaceutical carriers, buffers, antibodies, indicator molecules, and/or other useful reagents for solubilizing lyophilized formulations or monitoring the immune response of a mammal. Instructions describing how an HLA- A2 binding peptide or pharmaceutical composition is effective for inducing an immune response to an HSV-2 infection can be included in such kits.

The HLA- A2 binding peptides or pharmaceutical compositions can be packaged in a variety of suitable containers. For example, a composition can be contained in a bottle, vial, or syringe, composed of a material such as glass or plastic. HLA-A2 binding peptides or pharmaceutical compositions can be provided in a prepackaged unit dose form (e.g., in ampoules, syringes, or blister packs) in quantities sufficient for a single administration or for a pre-specified number of humans. Containers can be air tight and/or waterproof, and can be labeled for use, such as for a vaccine or to stimulate a CTL response.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES Example 1 -Selection of HLA- A2 Binding Peptides

A panel of HLA-A2-binding peptides from the ICPO, ICP4, ICP22, ICP27, and VP 16 proteins involved in reactivation and early viral replication of HSV-2 were identified with Rankpep software (Dana Farber Cancer Institute) (Reche et al., Human Immunology 63, 701-709 (2002)) that predicts peptides with high binding affinity. HLA-A2 was chosen since it is the most frequent class I gene in the human population. Thirty peptides were synthesized and binding capacities were ranked with an HLA-A2 -positive cell line that requires exogenous peptides for stable class I expression. Table 1 provides the sequences of the peptides and identifies from which gene they were identified.

TABLE 1

Stabilized class I expression was assessed by labeling with fluorochrome- labeled antibodies and flow cytometry to estimate off-rates of peptide :HLA-A2 binding. The results of these assays are presented in FIG. 1. The following 14 peptides had the highest binding capacity (slowest off-rates): RLRNAIREV (SEQ ID NO:1), ALASADETL (SEQ ID NO:2), ILARLANRV (SEQ ID NO:3), MLIDLGLDL (SEQ ID NO:4), VLENLATRL (SEQ ID NO:5), RRLSDIKDI (SEQ ID NO:6), CLFQPLMFI (SEQ ID NO:7), VLANFCSAL (SEQ ID NO: 10), VLVDQTGNV (SEQ ID NO: 13), FVSSSSAQV (SEQ ID NO: 14), AVAAVVPRV (SEQ ID NO : 15), ALDQACFRI (SEQ ID NO : 17), VLLLSTRDL (SEQ ID NO : 18), and RLRAWLREL (SEQ ID NO: 19).

The priming capacities of the 14 peptides with the highest binding capacity were evaluated by combining each peptide with synthetic CpG adjuvant and priming HLA-A2 transgenic mice. Two to three mice/group were immunized subcutaneously with titrated doses of peptide + CpG according to Wettstein et al., J. Immunol. 2005, 175:3681-3689. A2/A2 homozygotes and A2/- hemizygotes were primed with two doses (100 μg peptide + lOOμg CpG; 50μg peptide + 50μg CpG) and spleens were harvested at 1Od for enrichment of CD8⁺ CTLs. Frequencies of peptide-specific CTLs were estimated in IFNγ Elispot assays. Responding CTLs were stimulated with HLA- A2 transgenic and normal B6 spleen cell stimulators that had been pulsed with titrated concentrations of target peptide. One peptide (#7 in FIG. 1; CLFQPLMFI; SEQ ID NO:7) stimulated relatively strong CTL responses (FIG. 2). Both homozygous (A2/A2) and hemizygous (A2/-) recipients responded to lOOμg doses of the #7 peptide (CLFQPLMFI). Lower level responses also were observed with the #10 peptide (VLANFCSAL; SEQ ID NO: 10) (FIG. 3). Using the standard vacccination method, the remaining 12 peptides did not prime for consistent CTL responses (data not shown). Booster vaccinations increased the frequencies of CTLs responding to the #7 and #10 peptides (FIGS. 4 and 5). All- hemizygotes were primed with 100 μg peptide + 100 μg CpG then three mice received second vaccinations with 50 μg peptide + 50 μg CpG). Spleens were harvested at Hd for enrichment of CD8⁺ CTLs for analysis in an IFNγ Elispot assay.

Example 2 -HLA- A2 Binding Peptides containing a CpG Interacting Amino Acid Sequence

CLFQPLMFI (SEQ ID NO: 7) has a cysteine residue at the first position. Cysteine residues promote disulfide bonding to the phosphorothioate backbones of synthetic CpG oligonucleotides resulting in increased immunogenicity. The effects of adding an additional Cys residue, as well as multiple positively charged amino acids that can increase immunogenicity and deposition at the site of injections, were investigated. Four additional peptides with Cys and positively charged amino acids in amino and carboxy tails were synthesized: CASANA-CLFQPLMFI (SEQ ID NO:31), KCSRNR-CLFQPLMFI (SEQ ID NO:32), CLFQPLMFI-ANASAC (SEQ ID NO:33), and CLFQPLMFI-RNRSCK (SEQ ID NO:34). These four peptides and CLFQPLMFI (SEQ ID NO:7) were dissolved in DMSO and mixed with CpG (#1826) for incubation at 37⁰C for 30min. PBS was added to dilute the peptide + CpG mixture to a volume required for subcutaneous injection at the base of the tail. Two-three

All- hemizygous mice were immunized with each peptide + CpG mixture and spleens were harvested 14 days after injection. T cells were enriched by panning over dishes coated with goat anti-mouse immunoglobulin. CD8+ CTLs then were enriched with magnetic particles coated with anti-CD8 antibody. IFNgamma Elispot assays were performed with 2-4 x 10⁵ responder CTLs and 5 xlO⁵ stimulator spleen cells per well pulsed with titrated concentrations of CLFQPLMFI (SEQ ID NO:7). Stimulator spleen cells included AlIAl homozygous and normal B6 spleen cells. The frequencies of A2-restricted CTLs specific for CLFQPLMFI (SEQ ID NO: 7) were calculated by subtracting the frequencies of CTLs that responded to peptide-pulsed B6 stimulators from those that responded to A2/A2 stimulators.

The results of the IFNgamma Elispot assay are presented in FIG. 6 and show the addition of Cys on the amino terminus of CLFQPLMFI increased immunogenicity over either the normal or carboxy-tailed CLFQPLMFI peptides. This increased immunogenicity was most evident with A2/A2 stimulators that had been pulsed with 1OnM CLFQPLMFI. These results also demonstrate that the addition of three positively charged residues significantly reduces the immunogenicity even though these residues promote the precipitation of peptide and CpG. These results have been observed in replicate experiments.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. An isolated HLA- A2 binding peptide, said peptide comprising an amino acid sequence selected from the group consisting of CLFQPLMFI (SEQ ID NO:7), RLRNAIREV (SEQ ID NO:1), ALASADETL (SEQ ID NO:2), ILARLANRV (SEQ ID NO:3), MLIDLGLDL (SEQ ID NO:4), VLENLATRL (SEQ ID NO:5), RRLSDIKDI (SEQ ID NO:6), VLANFCSAL (SEQ ID NO: 10), VLVDQTGNV (SEQ ID NO: 13), FVSSSSAQV (SEQ ID NO: 14), AVAAVVPRV (SEQ ID NO: 15), ALDQACFRI (SEQ ID NO: 17), VLLLSTRDL (SEQ ID NO: 18), and RLRAWLREL (SEQ ID NO: 19).

2. The isolated HLA-A2 binding peptide of claim 1 , wherein said peptide has the amino acid sequence CLFQPLMFI (SEQ ID NO:7).

3. The isolated HLA- A2 binding peptide of claim 1 , wherein said peptide has the amino acid sequence VLANFCSAL (SEQ ID NO: 10).

4. The isolated HLA- A2 binding peptide of claim 2, wherein said peptide further comprises a CpG-interacting amino acid sequence.

5. The isolated HLA- A2 binding peptide of claim 4, wherein said CpG-interacting amino acid sequence comprises a cysteine residue.

6. The isolated HLA-A2 binding peptide of claim 5, wherein said CpG-interacting amino acid sequence further comprises at least one positively charged amino acid.

7. The isolated HLA- A2 binding peptide of claim 4, wherein said CpG-interacting amino acid sequence comprises the sequence set forth in SEQ ID NO:44

(CASANA).

8. The isolated HLA- A2 binding peptide of claim 4, wherein said CpG-interacting amino acid sequence consists essentially of the sequence set forth in SEQ ID

NO:44 (CASANA).

9. The isolated HLA- A2 binding peptide of claim 4, wherein said CpG-interacting amino acid sequence is at the amino terminus of said HLA- A2 binding peptide.

10. A pharmaceutical composition comprising an HLA-A2 binding peptide and an adjuvant, wherein said HLA- A2 binding peptide comprises the amino acid sequence CLFQPLMFI (SEQ ID NO:7) or VLANFCSAL (SEQ ID NO: 10).

11. The composition of claim 10, wherein said adjuvant is a CpG oligonucleotide.

12. The composition of claim 11, wherein said CpG oligonucleotide comprises a phosphorothioate linkage.

13. The composition of claim 11, wherein said CpG oligonucleotide comprises a phosphorothioate backbone.

14. The composition of claim 10, wherein said composition comprises said HLA-A2 binding peptide having the amino acid sequence CLFQPLMFI (SEQ ID NO: 7).

15. The composition of claim 10, wherein said composition comprises said HLA- A2 binding peptide having the amino acid sequence CLFQPLMFI (SEQ ID NO: 7) and said HLA-A2 binding peptide having the amino acid sequence VLANFCSAL (SEQ ID NO: 10).

16. The composition of claim 10 further comprising a different peptide that stimulates a CTL response against HSV-2.

17. The composition of claim 16, wherein said different peptide is selected from the group consisting of GAPRSSSSGGAPLRAGVGSGS (SEQ ID NO:35); APAWSRRTL (SEQ ID NO:36); AIDYVHCKGIIHRDI (SEQ ID NO:37); AVPLLSAGGAAPPHP (SEQ ID NO:38); ELYYGPVSPADPESP (SEQ ID NO:39); PMRARPRGEVRFLHY (SEQ ID NO:40); RPRGEVRFLHYDEAG (SEQ ID NO:41); VAGFNKRVFCAAVGR (SEQ ID NO:42); and STAPEVGTYTPLRYAC (SEQ ID NO:43).

18. The composition of claim 11, wherein said HLA- A2 binding peptide further comprises a CpG-interacting amino acid sequence.

19. The composition of claim 18, wherein said CpG-interacting amino acid sequence comprises a cysteine residue.

20. The composition of claim 19, wherein said CpG-interacting amino acid sequence further comprises at least one positively charged amino acid.

21. The composition of claim 18, wherein said CpG-interacting amino acid sequence comprises the sequence set forth in SEQ ID NO:44 (CASANA).

22. A method for inducing an immune response to an herpes simplex virus-2 infection in a subject, said method comprising administering to said subject an amount of a pharmaceutical composition effective to induce said immune response, said pharmaceutical composition comprising an HLA- A2 binding peptide and an adjuvant, wherein said HLA- A2 binding peptide comprises the amino acid sequence CLFQPLMFI (SEQ ID NO:7) or VLANFCSAL (SEQ ID NO: 10).

23. The method of claim 22, wherein said adjuvant is a CpG oligonucleotide.

24. The method of claim 23, wherein said CpG oligonucleotide comprises a phosphorothioate linkage.

25. The method of claim 23, wherein said CpG oligonucleotide comprises a phosphorothioate backbone.

26. The method of claim 22, wherein said composition comprises said HLA-A2 binding peptide having the amino acid sequence CLFQPLMFI (SEQ ID NO: 7).

27. The method of claim 22, wherein said composition comprises said HLA-A2 binding peptide having the amino acid sequence CLFQPLMFI (SEQ ID NO: 7) and said HLA-A2 binding peptide having the amino acid sequence VLANFCSAL (SEQ ID NO: 10).

28. The method of claim 22, wherein said composition further comprises a different peptide that stimulates a CTL response against HSV-2.

29. The method of claim 28, wherein said different HLA-binding peptide is selected from the group consisting of GAPRSSSSGGAPLRAGVGSGS (SEQ ID NO:35); APAWSRRTL (SEQ ID NO:36); AIDYVHCKGIIHRDI (SEQ ID NO:37); AVPLLSAGGAAPPHP (SEQ ID NO:38); ELYYGPVSPADPESP (SEQ ID NO:39); PMRARPRGEVRFLHY (SEQ ID NO:40); RPRGEVRFLHYDEAG (SEQ ID NO:41); VAGFNKRVFCAAVGR (SEQ ID NO:42); and STAPEVGTYTPLRYAC (SEQ ID NO:43).

30. The method of claim 23, wherein said HLA-A2 binding peptide further comprises a CpG-interacting amino acid sequence.

31. The method of claim 30, wherein said CpG-interacting amino acid sequence comprises a cysteine residue.

32. The method of claim 31, wherein said CpG-interacting amino acid sequence further comprises at least one positively charged amino acid.

33. The method of claim 23, wherein said CpG-interacting amino acid sequence comprises the sequence set forth in SEQ ID NO:44 (CASANA).

34. The method of claim 23, wherein said CpG interacting amino acid sequence consists essentially of the sequence set forth in SEQ ID NO:44 (CASANA).