Method for antigen-specific stimulation of t-lymphocytes with synthetic peptide libraries

Abstract

The invention relates to a method for the antigen-specific stimulation of T-lymphocytes with synthetic peptide libraries, comprising the following steps: a) dividing the whole amino acid sequence of the antigen into protein fragments with partial amino acid sequences: b) synthesising a peptide library containing these protein fragments: c) incubating a suspension containing CD8- and/or CD4- T-lymphocytes with all of the protein fragments of the peptide library in a single culture batch. The method can be used for immunostimulation of T-lymphocytes of mammals, especially humans, as well as for diagnosis purposes, in order to determine whether a mammal, especially a human being, has previously responded to a specific protein with its/his/her immune system, and the strength of this response.

Description

[0001] The invention relates to a method for antigen-specific stimulation of T lymphocytes with synthetic peptide libraries, comprising the following steps:

[0002] (a) subdividing the total amino acid sequence of the antigen into protein fragments with partial amino acid sequences;

[0003] (b) synthesizing a peptide library containing these protein fragments;

[0004] (c) incubating a suspension containing CD8+ and/or CD4+ T lymphocytes with all the protein fragments of the peptide library in a single culture run.

[0005] The method can be employed for both the immunostimulation of T lymphocytes of mammals, especially humans, and for diagnostics in order to establish whether a mammal, especially a human, has previously responded to a specific protein with its immune system, and if so, how strong such response is.

BACKGROUND OF THE INVENTION

[0006] The immune response of CD8+ T lymphocytes to protein antigens can be detected only with a great expenditure using known methods. It depends on the presentation of the epitopes derived from these antigens on MHC class I molecules on cells and can be measured through measuring a cytotoxic response induced by exposure. This experimental set-up is usual and takes one to several weeks in which the CD8+ T lymphocytes must be stimulated with the antigen in a suitable cell culture and are then incubated in a cytotoxicity test with suitable target cells which have been loaded with peptides from this antigen or transfected with the antigen or parts thereof. The induction of a response of the CD8+ T lymphocytes is measured from the degree of destruction of target cells, which requires suitable controls and includes a great experimental and time expenditure.

[0007] The detection of the immune response of CD4+ T lymphocytes to protein antigens is somewhat less complicated. The response of CD4+ T lymphocytes to protein antigens depends on the presentation of the epitopes derived from these antigens on MHC class II molecules on cells and can be measured through the proliferation of such cells in the presence of the antigen or upon exposure to this antigen, e.g., through the incorporation of tritiated thymidine. This experimental set-up is usual and takes several days up to a week or longer. The presence of a CD4+ T lymphocyte response to protein antigens can further be measured in a known method in which a suspension containing CD4+ T lymphocytes is incubated with the corresponding protein followed by detecting the CD4+ T lymphocyte induction through the presence of intracellular cytokines by flow cytometry.

[0008] The presence of a CD8+ T lymphocyte or CD4+ T lymphocyte response to individual epitopes can further be measured in a known method in which a suspension containing CD8+ and/or CD4+ T lymphocytes is incubated with peptides from this protein followed by detecting the CD8+ or CD4+ T lymphocyte induction through the presence of intracellular cytokines by flow cytometry, making use of the fact that peptides can be charged directly from outside onto the MHC class I or MHC class II molecules on cells, circumventing intracellular processing. In this method, it can be achieved by a suitable grouping of peptides that stimulating peptides can be identified and thus epitopes can be determined. The grouping used in this way distributes all possible epitopes to several, and mostly a large number of, runs so that it can be established whether individual peptides from this protein can induce a T lymphocyte response and it can be established which of the peptides occurring in the individual groups have led to such stimulation (this is described in F. Kern et al., Journal of Virology, October 1999, p. 8179-8184, and in WO 99/36568).

[0009] However, this grouping allows neither to determine systematically in a single measurement with a corresponding control whether a T lymphocyte response against the protein is present at all, nor to tell how strong the response (the proportion of the reactive lymphocytes in percent of the total CD8+ or CD4+ T lymphocytes) to this protein is all in all. To do this, the usual grouping in this method for the identification of epitopes would require several stimulation and measuring runs, depending on the number of peptides used. The application described in the literature aims at the precise identification of epitopes and therefore uses groups of peptides whose size is chosen in such a way that as few as possible individual peptides must be tested to establish the stimulating activity of a peptide group. However, the smaller the group size is chosen, the more groups have to be tested. Therefore, as the most favorable variant, a number of groups is chosen in this method which is twice the square root of the next square number exceeding the number of the peptides (unless the number of peptides is itself a square number).

[0010] This may be exemplified by the pp65 protein of the human cytomegalovirus. 138 peptides were synthesized which cover the amino acid sequence of the whole protein (561 amino acids) to the full length thereof, neighboring peptides overlapping by 9 amino acids each. 138 is not a square number. The next higher square number from 138 is 144 (12.times.12). Thus, the peptides were distributed to 2.times.12, i.e. 24, groups in such a way that each peptide occurs in exactly two different groups. By combining the groups with positive results (stimulation), the stimulating peptide can be concluded directly (when only two groups show a positive result), or it can be narrowed down to a small number of candidate peptides which can be retested individually, if more then two groups of peptides have resulted in positive stimulation results. The principle of this grouping has been described in some detail by F. Kern et al., Journal of Virology, October 1999, p. 8179-8184. A possibility for telling by one single run with a corresponding negative control whether a protein has a stimulating effect on CD8+ T lymphocytes, i.e., whether the amino acid sequence of this protein contains epitopes which are recognized by CD8+ T lymphocytes, has not been described to date.

DESCRIPTION OF THE INVENTION

[0011] The object of the invention is to provide a possibility for how to employ protein antigens of known sequence for the immunostimulation of CD8+ and CD4+ T lymphocytes, wherein cellular antigen processing is not necessary and individual antigenic determinants (epitopes) need not be identified. It has now been found that a sufficient immunostimulation can be achieved by incubation with T lymphocytes of a special peptide library of individual fragments of the antigen with some overlapping of the fragments. The stimulation can be detected by flow cytometry. Thus, it can be established whether an organism (human or animal) has built up a T lymphocyte response against the immunizing antigen after an exposure which has occurred (also well-aimed immunization). This T lymphocyte reactivity can be examined in terms of its time course. A further object of the invention is to provide a method by which protein antigens whose amino acid sequences are known can be identified as T-lymphocyte-stimulating protein antigens within a short time and with comparably few expenditure. This further provides a possibility for examining prior to the selection of a protein for the identification of epitopes whether T-lymphocyte-stimulating antigenic determinants are at all present in this protein.

[0012] Thus, the present invention relates to

[0013] (1) a method for the antigen-specific stimulation of T lymphocytes with synthetic peptide libraries, comprising the following steps:

[0014] (a) subdividing the total amino acid sequence of the antigen into protein fragments with partial amino acid sequences, wherein said protein fragments have a minimum length of 9 amino acid residues (also briefly referred to as "AA" in the following), and wherein adjacent or neighboring protein fragments are overlapping with their partial amino acid sequence;

[0015] (b) synthesizing a peptide library containing the protein fragments defined in (a);

[0016] (c) incubating a suspension containing CD8+ and/or CD4+ T lymphocytes with all the protein fragments of the peptide library obtained in (b) in a single culture run;

[0017] (2) in a preferred embodiment of method (1), it is adapted for in-vivo and in-vitro immunostimulation of T lymphocytes of mammals, especially humans;

[0018] (3) stimulated T lymphocytes obtainable by the method as defined above under (2);

[0019] (4) use of a peptide library as defined above under (1) for the preparation of a medicament for in-vivo immunostimulation of T lymphocytes of mammals; and

[0020] (5) a composition for in-vitro and in-vivo immunostimulation of T lymphocytes of mammals, comprising one or more peptide libraries as defined above under (1).

DESCRIPTION OF FIGURES

[0021] FIG. 1: Peptides for whole pool HCMV IE-1 (laboratory strain AD169). The sequence of the starting protein, VIE1-HCMVA 55 kDa immediate-early protein 1 (IE1), human cytomegalovirus (strain AD169), is known from Swiss-Prot P13202 and depicted in SEQ ID NO:1.

[0022] FIG. 2: Peptides for whole pool HCMV pp65 (laboratory strain AD169). The sequence of the starting protein, PP65-HCMVA 65 kDa lower matrix phosphoprotein (pp65), human cytomegalovirus (strain AD169), is known from Swiss-Prot P06725 and depicted in SEQ ID NO:2.

[0023] FIG. 3: Detection of intracellular interferon-gamma in CD8+ T lymphocytes upon stimulation with the peptide libraries described. The marker CD69 was used as an activation marker in addition to interferon-gamma. The representation has been limited to CD3+/CD8+ events, stating the average fluorescence intensity.

DETAILED DESCRIPTION OF THE INVENTION

[0024] "Antigens" in the method according to the invention are those antigens which have a peptide basic structure (i.e., proteins, parts of proteins or polypeptides etc.). The antigen in step (a) of the above defined method is an antigen (i.e., protein, part of a protein, or polypeptide) to which a T lymphocyte stimulation is desired, or on which it is to be tested whether such a stimulation has already occurred.

[0025] "Proteins or peptides" in the present invention have a sequence of at least nine AAs as an essential feature.

[0026] A "peptide library" within the meaning of the application is a complex mixture of peptides which in their entirety cover the complete sequence of a protein antigen or partial antigen, which is in such a way that successive peptides are overlapping along this sequence.

[0027] Therefore, in the method according to embodiment (1) of the invention, it may be necessary to determine the total amino acid sequence of the antigen prior to the above mentioned step (a), especially when the amino acid sequence of the antigen is not known.

[0028] It does not matter how the sequence of the antigen has been established. Thus, for a new protein, the sequence can be analyzed for the first time, or for a known protein, it may be read from a data base. It is only important that the amino acid sequence of the protein or partial protein has been determined.

[0029] In a preferred embodiment of method (1) according to the invention, the protein fragments have a minimum length of 15 AAs and/or a maximum length of 35 AAs, preferably 25 AAs. It is further preferred that an overlap of 8 AAs, preferably 11 AAs, is present between neighboring protein fragments. In addition, the synthetic protein fragments may be extended by a maximum of 7 natural or artificial AAs and/or a protective group at either or both of their N terminus and C terminus. These extensions of natural or artificial AAs are non-overlapping sequences.

[0030] Suitable protective groups on the N terminus of the protein fragments are alkyl, aryl, alkylaryl, aralkyl, alkylcarbonyl or arylcarbonyl, having from 1 to 10 carbon atoms, an acyl group having from 1 to 7 carbon atoms, etc. Preferred protective groups for the N terminus are the naphthoyl, naphthylacetyl, naphthylpropionyl and benzoyl groups. Suitable protective groups for the C terminus of the protein fragments are alkoxy or aryloxy groups having from 1 to 10 carbon atoms or an amino group. Further protective groups are described in Houben-Weyl (1974), Georg Thieme Verlag, 4th Edition. The description of the protective groups in the above reference is included herein by reference.

[0031] Further, it is preferred that the concentration of the individual protein fragments of the peptide library is at least 1 ng/ml, preferably from about 0.1 to about 10 .mu.g/ml in the culture run (final concentration). Particularly preferred is a concentration of about 1 .mu.g/ml of culture broth.

[0032] In addition, it is preferred that the incubation solution (i.e., the culture broth) further contains one or more compounds having costimulatory properties, such as costimulatory antibodies (e.g. anti-CD28 or anti-CD49d) or other molecules having costimulatory properties. (e.g., stimulatory CTLA4-Ig). These compounds are preferably contained in the culture broth in final concentration of from 0.1 to 10 .mu.g/ml.

[0033] A particularly preferred embodiment of the method (1) according to the invention for the antigen-specific stimulation of T lymphocytes with synthetic peptide libraries comprises the following steps:

[0034] (a.sub.1) determining the total amino acid sequence of the antigen, which is a protein or part of a protein;

[0035] (a.sub.2) subdividing the total amino acid sequence in protein fragments having partial amino acid sequences, wherein the protein fragments have a minimum length of 9 (preferably 15) AAs, optionally have a maximum length of 25 AAs, and wherein adjacent or neighboring protein fragments are over-lapping with their partial amino acid sequence, an overlap of 8 AAs, especially an overlap of 11 AAs, being preferred;

[0036] (b) synthesizing a peptide library containing the protein fragments defined in (a2), optionally extended by a maximum of 7 natural or artificial amino acids and/or a protective group at either or both of the N terminus and C terminus;

[0037] (c) incubating a suspension containing CD8+ and/or CD4+ T lymphocytes with all the protein fragments of the peptide library in a single culture run.

[0038] Preferred is the use of the method according to the invention for identifying stimulating or non-stimulating mixtures of all protein fragments in a single culture run, wherein the following steps are added:

[0039] (d) identifying (preferably flow-cytometric identifying) of

[0040] (i) at least one T-cell cytokine which was induced by the protein fragment or fragments and synthesized in the T lymphocytes, wherein said cytokine or cytokines are intracellular or bound to the cell membrane; and/or

[0041] (ii) at least one activation marker which was induced by the protein fragment or fragments and synthesized in the T lymphocytes, wherein said activation marker or markers are intracellular or bound to the cell membrane.

[0042] The method (1) according to the invention is also suitable for establishing whether T-lymphocyte-stimulating antigenic determinants are present in an antigen.

[0043] The method according to the invention is further suitable for diagnostics, especially to establish whether a mammal, especially a human, has previously responded to a specific protein with its immune system, and how strong such response is.

[0044] According to the preferred embodiment (2) of the invention, the method is suitable for immunostimulation of T lymphocytes of mammals, especially humans, for both in-vitro and in-vivo applications. This method may further include the expanding of the stimulated T lymphocytes.

[0045] The above mentioned embodiments of the method according to the invention may also be designed to employ several different synthetic peptide libraries (from different antigens) together in one culture run or in separated culture runs.

[0046] Suspensions containing T lymphocytes within the meaning of this application are characterized by containing cells which can present MHC-bound peptides. Thus, the presenting cells may also be T lymphocytes in addition to the antigen-presenting cells.

[0047] An advantage of the method according to the invention is the fact that the identification of at least one T-cell cytokine or activation marker is effected on the level of the individual cell. Thus, it is possible to exactly determine the phenotype of the responding cells. Cytokines and surface markers are described in some detail in Abul K. Abbas et al. (1997), Cellular and Molecular Immunology, Philadelphia, 3rd Edition, ISBN 0-7216-4024-9.

[0048] It is known that protein fragments binding to MHC class I molecules (MHC=major histocompatibility complex) usually have a length of 9 amino acids, while protein fragments binding to MHC class II molecules are somewhat longer and more variable in length.

[0049] An advantage of methods (1) and (2) according to the invention is the fact that, despite of the short incubation time, the protein fragments are taken up by the MHC molecules present on the cell surface sufficiently to enable an unambiguous identification of a T-cell stimulation after six hours, for example.

[0050] In the method according to the invention, the suspension containing T lymphocytes can be derived from whole blood, peripheral white blood cells (PWBC), splenocytes, thymocytes, bone marrow, cerebrospinal fluid, lymph node cells, etc.

[0051] In the method according to the invention, it is particularly advantageous that processing of the T lymphocytes is not required. Thus, the T lymphocytes need not be enriched, and further, the removal or destruction of other cells is not necessary. Thus, the method according to the invention can be practiced more simply in a routine manner.

[0052] Preferred is a method according to the invention for antigen-specific stimulation of T lymphocytes with synthetic peptide libraries in which the suspension containing the T lymphocytes is derived from patients to be treated, from other donors or from animals. If the suspension containing T lymphocytes is derived from a patient, the identification can be used, for example, for establishing to which protein of a virus a CD8+ or CD4+ T lymphocyte response can be induced. The peptide library employed for examining this reactivity can then be selectively employed for the stimulation of further T lymphocytes of the same or other patients. The cells thus induced and stimulated for proliferation can be expanded in vivo or ex vivo and subsequently retransfused to the patient.

[0053] The method according to the invention can also be used in veterinary medicine. It is possible to use a wide variety of animal species and also constellations of animal patients and donors as the source of the suspension containing T lymphocytes.

[0054] Advantageous is a method according to the invention for the antigen-specific stimulation of T lymphocytes with synthetic peptide libraries in which the antigens, which are proteins or partial proteins, are derived from microorganisms, macroorganisms, cells, cell cultures and/or tissues from donors or patients. Microorganisms include, for example, viruses, bacteria, fungi, protozoans, parasites. Macroorganisms include, for example, all multicellular eukaryotes. This source is just important for influencing allergies. Included are animals and plants. There may be used cells, cell cultures or even whole tissues consisting of one or more layers or cell types.

[0055] Preferred is a method according to the invention for antigen-specific stimulation of T lymphocytes with synthetic peptide libraries in which the stimulation is detected by means of a flow cytometer. What is essential is the principle that markers present in the cell or on its surface, such as cytokines or surface markers, will contact with a specific detector, for example, an antibody, the detector being loaded with a fluorescent dye. Upon excitation by laser light of this fluorescent dye on the cells focused in a liquid stream, the flow cytometer records the emitted scattered light and fluorescence signals, which enables the simultaneous or later analysis of the cells. Such techniques are described in some detail in Howard M. Shapiro (1995), Practical Flow Cytometry, New York, 3rd Edition, ISBN 0-471-30376-3. The detection of the intracellular cytokines is described in ILL. Picker et al. (1995), Blood, Vol. 86, p. 1408.

[0056] The advantage of this method according to the invention for antigen-specific stimulation of T lymphocytes with synthetic peptide libraries is that a reagent for the immunostimulation of T lymphocytes can be made available within a very short period of time and, as compared to the conventional method, with very low expenditure. It is further advantageous that individual stimulating epitopes need not to be identified.

[0057] In a single run (one tube or one well or one flask, etc.), the T lymphocytes of a donor/patient (CD8 and/or CD4) can be stimulated simultaneously with all possible antigenic determinants of the protein (or proteins when several peptide libraries are used) without needing to be specifically known. For example, the T lymphocytes of a patient who has undergone a bone marrow transplantation could be incubated with HLA-identical dendritic cells which were previously incubated with such a mixture of peptides, and thus these T lymphocytes could be stimulated with all epitopes relevant (i.e., binding) to the particular HLA type without these epitopes needing to be known or becoming known by the method. The only critical point is that they stimulate T lymphocytes and belong to the selected protein or proteins. These cells could be retransferred to the patient within the scope of an adoptive immunotherapy.

[0058] A preferred source of the T lymphocytes to be stimulated are those (human or animal) donors which have previously build up an immunological primary response to the antigen or in which such an immune response to the antigen has been induced by exposure. This may have occurred, for example, within the scope of an infection or also within the scope of an immunization. This situation also prevails in an auto-immune response.

[0059] Another advantage is that the MHC type of the donor need not be known. A further advantage is that the stimulation of both CD8+ and CD4+ T lymphocytes can be examined simultaneously and in a single run.

[0060] The stimulated T lymphocytes according to embodiment (3) of the invention are preferably obtained by in-vitro stimulation. The stimulated lymphocytes are capable of being transfused into a patient.

[0061] The medicament according to embodiment (4) of the invention may contain further immunoreactive compounds, such as the above defined compounds having co-stimulating properties, in addition to usual additives and auxiliaries. The medicament may also contain several of the above defined peptide libraries.

[0062] The composition according to embodiment (5) can be a pharmaceutical composition, i.e., for the in-vivo treatment of humans and animals, or a diagnostic composition or a so-called kit, i.e., primary for in-vitro application, wherein the peptide library is respectively adapted to the antigen to be stimulated. As to further components of the composition, the same applies as has been set forth above with respect to embodiment (4).

[0063] The present invention is further illustrated by the following non-limiting Example.

EXAMPLE

[0064] Mononuclear cells were prepared from the peripheral blood of two patients obtained by venous puncture. The patients possessed antibodies against the human cytomegalovirus (HCMV). The cells prepared by standard methods were incubated for six hours under optimized conditions with peptide libraries for the HCMV proteins 65 kD lower matrix phosphoprotein (pp65) and 55 kDa immediate-early protein 1 (IE1). This is done according to the method described in Kern et al., Eur. J. Immunol. 30: 1676-1682 (2000), which comprises the following steps:

[0065] 1. Resuspension of PBMC (2.5.times.10.sup.6/ml in RPMI 1640 with 2 mM glutamine added) after Ficoll preparation (standard protocol).

[0066] 2. 400 .mu.l of this suspension was mixed in an incubation vessel (sterile tube from Falcon No. 2054, 5 ml) with 100 .mu.l of peptide solution (containing 10 .mu.g of each individual peptide in RPMI 1640 with 2 mM glutamine added).

[0067] 3. Incubation at 37.degree. C. under an H.sub.2O-saturated atmosphere with 5% CO.sub.2 (standard incubator).

[0068] 4. After 2 hours, there was added 500 .mu.l of RPMI 1640 to which 20% fetal calf serum (v/v) and additionally glutamine (2 mM) and 10 .mu.g of Brefeldin A (BFA, final concentration in the mix was 10 .mu.g/ml) had been added. The final concentration of fetal calf serum in the mix is 10% (v/v). The final concentration of each individual peptide is 1 .mu.g/ml. BFA serves to retain synthetic cytokines in the cells, which is of advantage for the detection of the intracellular cytokines. The final volume of the mix is 1 ml.

[0069] 5. After further incubation for 4 h under the same conditions (i.e., a total incubation time of 6 h), the incubation was stopped by adding ice-cold PBS buffer solution.

[0070] 6. This was followed by centrifugation (8 min, 400 g), decantation and further processing of the samples according to a standard protocol, including detachment from the tube wall using 2 mM EDTA/PBS solution, fixation, permeabilization and staining with monoclonal antibodies.

[0071] 7. Analysis on a flow cytometer (e.g., a four-color fluorescence flow cytometer of the type FacsCalibur (Becton Dickinson)).

[0072] The sequences of IE-1 and pp65 have been deposited in the SWISS-PROT data base, European Bioinformatics Institute, under the Nos. P13202 (see also SEQ ID NO: 1) and P06725 (see also SEQ ID NO: 2). In addition, the sequences of both proteins are described in M. S. Chee, A. T. Bankier, S. Becks et al., Curr. Top. Microbiol. Immunol. 154: 125-169 (1990).

[0073] The peptide library which represents the 55 kD immediate-early protein 1 consisted of peptides of 15 amino acids length each with 9 overlaps between successive peptides (see FIG. 1), and the peptide library which represents the 65 kD lower matrix phosphoprotein consisted of peptides of 15 amino acids length each with 11 overlaps between successive peptides (see FIG. 2).

[0074] In two different individuals (coluumns "I)" and "II)" in FIG. 3), incubation with the peptide libraries resulted in the production of IFN-gamma in T cells, which was detected by measurement on a flow cytometer on the level of the individual cells (J. L. Picker et al. (1995), Blood, Vol. 86, p. 1408-1419), or else in no detectable stimulation. Individual I exhibited a CD8+ T lymphocyte response to IE-1, but not to pp65, whereas individual II exhibited a CD8+ T lymphocyte response to both proteins. Incubation with an irrelevant peptide did not produce this effect (control).

Sequence CWU 1

1

260 1 491 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 1 Met Glu Ser Ser Ala Lys Arg Lys Met Asp Pro Asp Asn Pro Asp Glu 1 5 10 15 Gly Pro Ser Ser Lys Val Pro Arg Pro Glu Thr Pro Val Thr Lys Ala 20 25 30 Thr Thr Phe Leu Gln Thr Met Leu Arg Lys Glu Val Asn Ser Gln Leu 35 40 45 Ser Leu Gly Asp Pro Leu Phe Pro Glu Leu Ala Glu Glu Ser Leu Lys 50 55 60 Thr Phe Glu Gln Val Thr Glu Asp Cys Asn Glu Asn Pro Glu Lys Asp 65 70 75 80 Val Leu Ala Glu Leu Val Lys Gln Ile Lys Val Arg Val Asp Met Val 85 90 95 Arg His Arg Ile Lys Glu His Met Leu Lys Lys Tyr Thr Gln Thr Glu 100 105 110 Glu Lys Phe Thr Gly Ala Phe Asn Met Met Gly Gly Cys Leu Gln Asn 115 120 125 Ala Leu Asp Ile Leu Asp Lys Val His Glu Pro Phe Glu Glu Met Lys 130 135 140 Cys Ile Gly Leu Thr Met Gln Ser Met Tyr Glu Asn Tyr Ile Val Pro 145 150 155 160 Glu Asp Lys Arg Glu Met Trp Met Ala Cys Ile Lys Glu Leu His Asp 165 170 175 Val Ser Lys Gly Ala Ala Asn Lys Leu Gly Gly Ala Leu Gln Ala Lys 180 185 190 Ala Arg Ala Lys Lys Asp Glu Leu Arg Arg Lys Met Met Tyr Met Cys 195 200 205 Tyr Arg Asn Ile Glu Phe Phe Thr Lys Asn Ser Ala Phe Pro Lys Thr 210 215 220 Thr Asn Gly Cys Ser Gln Ala Met Ala Ala Leu Gln Asn Leu Pro Gln 225 230 235 240 Cys Ser Pro Asp Glu Ile Met Ala Tyr Ala Gln Lys Ile Phe Lys Ile 245 250 255 Leu Asp Glu Glu Arg Asp Lys Val Leu Thr His Ile Asp His Ile Phe 260 265 270 Met Asp Ile Leu Thr Thr Cys Val Glu Thr Met Cys Asn Glu Tyr Lys 275 280 285 Val Thr Ser Asp Ala Cys Met Met Thr Met Tyr Gly Gly Ile Ser Leu 290 295 300 Leu Ser Glu Phe Cys Arg Val Leu Cys Cys Tyr Val Leu Glu Glu Thr 305 310 315 320 Ser Val Met Leu Ala Lys Arg Pro Leu Ile Thr Lys Pro Glu Val Ile 325 330 335 Ser Val Met Lys Arg Arg Ile Glu Glu Ile Cys Met Lys Val Phe Ala 340 345 350 Gln Tyr Ile Leu Gly Ala Asp Pro Leu Arg Val Cys Ser Pro Ser Val 355 360 365 Asp Asp Leu Arg Ala Ile Ala Glu Glu Ser Asp Glu Glu Glu Ala Ile 370 375 380 Val Ala Tyr Thr Leu Ala Thr Ala Gly Val Ser Ser Ser Asp Ser Leu 385 390 395 400 Val Ser Pro Pro Glu Ser Pro Val Pro Ala Thr Ile Pro Leu Ser Ser 405 410 415 Val Ile Val Ala Glu Asn Ser Asp Gln Glu Glu Ser Glu Gln Ser Asp 420 425 430 Glu Glu Glu Glu Glu Gly Ala Gln Glu Glu Arg Glu Asp Thr Val Ser 435 440 445 Val Lys Ser Glu Pro Val Ser Glu Ile Glu Glu Val Ala Pro Glu Glu 450 455 460 Glu Glu Asp Gly Ala Glu Glu Pro Thr Ala Ser Gly Gly Lys Ser Thr 465 470 475 480 His Pro Met Val Thr Arg Ser Lys Ala Asp Gln 485 490 2 561 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 2 Met Glu Ser Arg Gly Arg Arg Cys Pro Glu Met Ile Ser Val Leu Gly 1 5 10 15 Pro Ile Ser Gly His Val Leu Lys Ala Val Phe Ser Arg Gly Asp Thr 20 25 30 Pro Val Leu Pro His Glu Thr Arg Leu Leu Gln Thr Gly Ile His Val 35 40 45 Arg Val Ser Gln Pro Ser Leu Ile Leu Val Ser Gln Tyr Thr Pro Asp 50 55 60 Ser Thr Pro Cys His Arg Gly Asp Asn Gln Leu Gln Val Gln His Thr 65 70 75 80 Tyr Phe Thr Gly Ser Glu Val Glu Asn Val Ser Val Asn Val His Asn 85 90 95 Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln Glu Pro Met Ser Ile Tyr 100 105 110 Val Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn Val 115 120 125 His His Tyr Pro Ser Ala Ala Glu Arg Lys His Arg His Leu Pro Val 130 135 140 Ala Asp Ala Val Ile His Ala Ser Gly Lys Gln Met Trp Gln Ala Arg 145 150 155 160 Leu Thr Val Ser Gly Leu Ala Trp Thr Arg Gln Gln Asn Gln Trp Lys 165 170 175 Glu Pro Asp Val Tyr Tyr Thr Ser Ala Phe Val Phe Pro Thr Lys Asp 180 185 190 Val Ala Leu Arg His Val Val Cys Ala His Glu Leu Val Cys Ser Met 195 200 205 Glu Asn Thr Arg Ala Thr Lys Met Gln Val Ile Gly Asp Gln Tyr Val 210 215 220 Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val Pro Ser Gly Lys Leu 225 230 235 240 Phe Met His Val Thr Leu Gly Ser Asp Val Glu Glu Asp Leu Thr Met 245 250 255 Thr Arg Asn Pro Gln Pro Phe Met Arg Pro His Glu Arg Asn Gly Phe 260 265 270 Thr Val Leu Cys Pro Lys Asn Met Ile Ile Lys Pro Gly Lys Ile Ser 275 280 285 His Ile Met Leu Asp Val Ala Phe Thr Ser His Glu His Phe Gly Leu 290 295 300 Leu Cys Pro Lys Ser Ile Pro Gly Leu Ser Ile Ser Gly Asn Leu Leu 305 310 315 320 Met Asn Gly Gln Gln Ile Phe Leu Glu Val Gln Ala Ile Arg Glu Thr 325 330 335 Val Glu Leu Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe Phe Asp 340 345 350 Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr Ser Glu His Pro Thr 355 360 365 Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Glu Tyr Arg His Thr 370 375 380 Trp Asp Arg His Asp Glu Gly Ala Ala Gln Gly Asp Asp Asp Val Trp 385 390 395 400 Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu Val Thr Thr Glu Arg Lys 405 410 415 Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Gly Ala Ser Thr Ser 420 425 430 Ala Gly Arg Lys Arg Lys Ser Ala Ser Ser Ala Thr Ala Cys Thr Ser 435 440 445 Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu Ser Thr Val Ala Pro 450 455 460 Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile His Asn Pro Ala 465 470 475 480 Val Phe Thr Trp Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg Asn Leu 485 490 495 Val Pro Met Val Ala Thr Val Gln Gly Gln Asn Leu Lys Tyr Gln Glu 500 505 510 Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Phe Ala Glu Leu Glu 515 520 525 Gly Val Trp Gln Pro Ala Ala Gln Pro Lys Arg Arg Arg His Arg Gln 530 535 540 Asp Ala Leu Pro Gly Pro Cys Ile Ala Ser Thr Pro Lys Lys His Arg 545 550 555 560 Gly 3 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 3 Met Glu Ser Ser Ala Lys Arg Lys Met Asp Pro Asp Asn Pro Asp 1 5 10 15 4 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 4 Gln Thr Met Leu Arg Lys Glu Val Asn Ser Gln Leu Ser Leu Gly 1 5 10 15 5 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 5 Ser Asp Ser Leu Val Ser Pro Pro Glu Ser Pro Val Pro Ala Thr 1 5 10 15 6 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 6 Val Ser Pro Pro Glu Ser Pro Val Pro Ala Thr Ile Pro Leu Ser 1 5 10 15 7 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 7 Glu Ser Pro Val Pro Ala Thr Ile Pro Leu Ser Ser Val Ile Val 1 5 10 15 8 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 8 Pro Ala Thr Ile Pro Leu Ser Ser Val Ile Val Ala Glu Asn Ser 1 5 10 15 9 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 9 Pro Leu Ser Ser Val Ile Val Ala Glu Asn Ser Asp Gln Glu Glu 1 5 10 15 10 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 10 Val Ile Val Ala Glu Asn Ser Asp Gln Glu Glu Ser Glu Gln Ser 1 5 10 15 11 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 11 Glu Asn Ser Asp Gln Glu Glu Ser Glu Gln Ser Asp Glu Glu Glu 1 5 10 15 12 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 12 Gln Glu Glu Ser Glu Gln Ser Asp Glu Glu Glu Glu Glu Gly Ala 1 5 10 15 13 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 13 Glu Gln Ser Asp Glu Glu Glu Glu Glu Gly Ala Gln Glu Glu Arg 1 5 10 15 14 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 14 Glu Glu Glu Glu Glu Gly Ala Gln Glu Glu Arg Glu Asp Thr Val 1 5 10 15 15 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 15 Arg Lys Glu Val Asn Ser Gln Leu Ser Leu Gly Asp Pro Leu Phe 1 5 10 15 16 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 16 Glu Gly Ala Gln Glu Glu Arg Glu Asp Thr Val Ser Val Lys Ser 1 5 10 15 17 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 17 Asn Ser Gln Leu Ser Leu Gly Asp Pro Leu Phe Pro Glu Leu Ala 1 5 10 15 18 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 18 Ser Leu Gly Asp Pro Leu Phe Pro Glu Leu Ala Glu Glu Ser Leu 1 5 10 15 19 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 19 Pro Leu Phe Pro Glu Leu Ala Glu Glu Ser Leu Lys Thr Phe Glu 1 5 10 15 20 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 20 Glu Leu Ala Glu Glu Ser Leu Lys Thr Phe Glu Gln Val Thr Glu 1 5 10 15 21 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 21 Glu Ser Leu Lys Thr Phe Glu Gln Val Thr Glu Asp Cys Asn Glu 1 5 10 15 22 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 22 Thr Phe Glu Gln Val Thr Glu Asp Cys Asn Glu Asn Pro Glu Lys 1 5 10 15 23 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 23 Val Thr Glu Asp Cys Asn Glu Asn Pro Glu Lys Asp Val Leu Ala 1 5 10 15 24 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 24 Cys Asn Glu Asn Pro Glu Lys Asp Val Leu Ala Glu Leu Val Lys 1 5 10 15 25 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 25 Ala Lys Arg Lys Met Asp Pro Asp Asn Pro Asp Glu Gly Pro Ser 1 5 10 15 26 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 26 Pro Glu Lys Asp Val Leu Ala Glu Leu Val Lys Gln Ile Lys Val 1 5 10 15 27 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 27 Val Leu Ala Glu Leu Val Lys Gln Ile Lys Val Arg Val Asp Met 1 5 10 15 28 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 28 Leu Val Lys Gln Ile Lys Val Arg Val Asp Met Val Arg His Arg 1 5 10 15 29 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 29 Ile Lys Val Arg Val Asp Met Val Arg His Arg Ile Lys Glu His 1 5 10 15 30 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 30 Val Asp Met Val Arg His Arg Ile Lys Glu His Met Leu Lys Lys 1 5 10 15 31 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 31 Arg His Arg Ile Lys Glu His Met Leu Lys Lys Tyr Thr Gln Thr 1 5 10 15 32 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 32 Lys Glu His Met Leu Lys Lys Tyr Thr Gln Thr Glu Glu Lys Phe 1 5 10 15 33 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 33 Leu Lys Lys Tyr Thr Gln Thr Glu Glu Lys Phe Thr Gly Ala Phe 1 5 10 15 34 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 34 Thr Gln Thr Glu Glu Lys Phe Thr Gly Ala Phe Asn Met Met Gly 1 5 10 15 35 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 35 Glu Lys Phe Thr Gly Ala Phe Asn Met Met Gly Gly Cys Leu Gln 1 5 10 15 36 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 36 Met Asp Pro Asp Asn Pro Asp Glu Gly Pro Ser Ser Lys Val Pro 1 5 10 15 37 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 37 Gly Ala Phe Asn Met Met Gly Gly Cys Leu Gln Asn Ala Leu Asp 1 5 10 15 38 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 38 Met Met Gly Gly Cys Leu Gln Asn Ala Leu Asp Ile Leu Asp Lys 1 5 10 15 39 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 39 Cys Leu Gln Asn Ala Leu Asp Ile Leu Asp Lys Val His Glu Pro 1 5 10 15 40 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 40 Ala Leu Asp Ile Leu Asp Lys Val His Glu Pro Phe Glu Glu Met 1 5 10 15 41 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 41 Leu Asp Lys Val His Glu Pro Phe Glu Glu Met Lys Cys Ile Gly 1 5 10 15 42 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 42 His Glu Pro Phe Glu Glu Met Lys Cys Ile Gly Leu Thr Met Gln 1 5 10 15 43 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 43 Glu Glu Met Lys Cys Ile Gly Leu Thr Met Gln Ser Met Tyr Glu 1 5 10 15 44 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 44 Cys Ile Gly Leu Thr Met Gln Ser Met Tyr Glu Asn Tyr Ile Val 1 5 10 15 45 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 45 Thr Met Gln Ser Met Tyr Glu Asn Tyr Ile Val Pro Glu Asp Lys 1 5 10 15 46 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 46 Met Tyr Glu Asn Tyr Ile Val Pro Glu Asp Lys Arg Glu Met Trp 1 5 10 15 47 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 47 Asn Pro Asp Glu Gly Pro Ser Ser Lys Val Pro Arg Pro Glu Thr 1 5 10 15 48 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 48 Tyr Ile Val Pro Glu Asp Lys Arg Glu Met Trp Met Ala Cys Ile 1 5 10 15 49 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 49 Glu Asp Lys Arg Glu Met Trp Met Ala Cys Ile Lys Glu Leu His 1 5 10 15 50 15 PRT

Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 50 Glu Met Trp Met Ala Cys Ile Lys Glu Leu His Asp Val Ser Lys 1 5 10 15 51 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 51 Ala Cys Ile Lys Glu Leu His Asp Val Ser Lys Gly Ala Ala Asn 1 5 10 15 52 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 52 Glu Leu His Asp Val Ser Lys Gly Ala Ala Asn Lys Leu Gly Gly 1 5 10 15 53 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 53 Val Ser Lys Gly Ala Ala Asn Lys Leu Gly Gly Ala Leu Gln Ala 1 5 10 15 54 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 54 Ala Ala Asn Lys Leu Gly Gly Ala Leu Gln Ala Lys Ala Arg Ala 1 5 10 15 55 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 55 Leu Gly Gly Ala Leu Gln Ala Lys Ala Arg Ala Lys Lys Asp Glu 1 5 10 15 56 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 56 Leu Gln Ala Lys Ala Arg Ala Lys Lys Asp Glu Leu Arg Arg Lys 1 5 10 15 57 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 57 Ala Arg Ala Lys Lys Asp Glu Leu Arg Arg Lys Met Met Tyr Met 1 5 10 15 58 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 58 Gly Pro Ser Ser Lys Val Pro Arg Pro Glu Thr Pro Val Thr Lys 1 5 10 15 59 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 59 Lys Asp Glu Leu Arg Arg Lys Met Met Tyr Met Cys Tyr Arg Asn 1 5 10 15 60 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 60 Arg Arg Lys Met Met Tyr Met Cys Tyr Arg Asn Ile Glu Phe Phe 1 5 10 15 61 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 61 Met Tyr Met Cys Tyr Arg Asn Ile Glu Phe Phe Thr Lys Asn Ser 1 5 10 15 62 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 62 Tyr Arg Asn Ile Glu Phe Phe Thr Lys Asn Ser Ala Phe Pro Lys 1 5 10 15 63 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 63 Glu Phe Phe Thr Lys Asn Ser Ala Phe Pro Lys Thr Thr Asn Gly 1 5 10 15 64 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 64 Lys Asn Ser Ala Phe Pro Lys Thr Thr Asn Gly Cys Ser Gln Ala 1 5 10 15 65 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 65 Phe Pro Lys Thr Thr Asn Gly Cys Ser Gln Ala Met Ala Ala Leu 1 5 10 15 66 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 66 Thr Asn Gly Cys Ser Gln Ala Met Ala Ala Leu Gln Asn Leu Pro 1 5 10 15 67 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 67 Ser Gln Ala Met Ala Ala Leu Gln Asn Leu Pro Gln Cys Ser Pro 1 5 10 15 68 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 68 Ala Ala Leu Gln Asn Leu Pro Gln Cys Ser Pro Asp Glu Ile Met 1 5 10 15 69 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 69 Lys Val Pro Arg Pro Glu Thr Pro Val Thr Lys Ala Thr Thr Phe 1 5 10 15 70 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 70 Asn Leu Pro Gln Cys Ser Pro Asp Glu Ile Met Ala Tyr Ala Gln 1 5 10 15 71 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 71 Cys Ser Pro Asp Glu Ile Met Ala Tyr Ala Gln Lys Ile Phe Lys 1 5 10 15 72 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 72 Glu Ile Met Ala Tyr Ala Gln Lys Ile Phe Lys Ile Leu Asp Glu 1 5 10 15 73 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 73 Tyr Ala Gln Lys Ile Phe Lys Ile Leu Asp Glu Glu Arg Asp Lys 1 5 10 15 74 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 74 Ile Phe Lys Ile Leu Asp Glu Glu Arg Asp Lys Val Leu Thr His 1 5 10 15 75 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 75 Leu Asp Glu Glu Arg Asp Lys Val Leu Thr His Ile Asp His Ile 1 5 10 15 76 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 76 Arg Asp Lys Val Leu Thr His Ile Asp His Ile Phe Met Asp Ile 1 5 10 15 77 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 77 Leu Thr His Ile Asp His Ile Phe Met Asp Ile Leu Thr Thr Cys 1 5 10 15 78 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 78 Asp His Ile Phe Met Asp Ile Leu Thr Thr Cys Val Glu Thr Met 1 5 10 15 79 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 79 Met Asp Ile Leu Thr Thr Cys Val Glu Thr Met Cys Asn Glu Tyr 1 5 10 15 80 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 80 Pro Glu Thr Pro Val Thr Lys Ala Thr Thr Phe Leu Gln Thr Met 1 5 10 15 81 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 81 Thr Thr Cys Val Glu Thr Met Cys Asn Glu Tyr Lys Val Thr Ser 1 5 10 15 82 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 82 Glu Thr Met Cys Asn Glu Tyr Lys Val Thr Ser Asp Ala Cys Met 1 5 10 15 83 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 83 Asn Glu Tyr Lys Val Thr Ser Asp Ala Cys Met Met Thr Met Tyr 1 5 10 15 84 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 84 Val Thr Ser Asp Ala Cys Met Met Thr Met Tyr Gly Gly Ile Ser 1 5 10 15 85 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 85 Ala Cys Met Met Thr Met Tyr Gly Gly Ile Ser Leu Leu Ser Glu 1 5 10 15 86 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 86 Thr Met Tyr Gly Gly Ile Ser Leu Leu Ser Glu Phe Cys Arg Val 1 5 10 15 87 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 87 Gly Ile Ser Leu Leu Ser Glu Phe Cys Arg Val Leu Cys Cys Tyr 1 5 10 15 88 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 88 Leu Ser Glu Phe Cys Arg Val Leu Cys Cys Tyr Val Leu Glu Glu 1 5 10 15 89 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 89 Cys Arg Val Leu Cys Cys Tyr Val Leu Glu Glu Thr Ser Val Met 1 5 10 15 90 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 90 Cys Cys Tyr Val Leu Glu Glu Thr Ser Val Met Leu Ala Lys Arg 1 5 10 15 91 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 91 Val Thr Lys Ala Thr Thr Phe Leu Gln Thr Met Leu Arg Lys Glu 1 5 10 15 92 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 92 Leu Glu Glu Thr Ser Val Met Leu Ala Lys Arg Pro Leu Ile Thr 1 5 10 15 93 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 93 Ser Val Met Leu Ala Lys Arg Pro Leu Ile Thr Lys Pro Glu Val 1 5 10 15 94 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 94 Ala Lys Arg Pro Leu Ile Thr Lys Pro Glu Val Ile Ser Val Met 1 5 10 15 95 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 95 Leu Ile Thr Lys Pro Glu Val Ile Ser Val Met Lys Arg Arg Ile 1 5 10 15 96 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 96 Pro Glu Val Ile Ser Val Met Lys Arg Arg Ile Glu Glu Ile Cys 1 5 10 15 97 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 97 Ser Val Met Lys Arg Arg Ile Glu Glu Ile Cys Met Lys Val Phe 1 5 10 15 98 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 98 Arg Arg Ile Glu Glu Ile Cys Met Lys Val Phe Ala Gln Tyr Ile 1 5 10 15 99 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 99 Glu Ile Cys Met Lys Val Phe Ala Gln Tyr Ile Leu Gly Ala Asp 1 5 10 15 100 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 100 Lys Val Phe Ala Gln Tyr Ile Leu Gly Ala Asp Pro Leu Arg Val 1 5 10 15 101 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 101 Gln Tyr Ile Leu Gly Ala Asp Pro Leu Arg Val Cys Ser Pro Ser 1 5 10 15 102 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 102 Thr Thr Phe Leu Gln Thr Met Leu Arg Lys Glu Val Asn Ser Gln 1 5 10 15 103 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 103 Gly Ala Asp Pro Leu Arg Val Cys Ser Pro Ser Val Asp Asp Leu 1 5 10 15 104 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 104 Leu Arg Val Cys Ser Pro Ser Val Asp Asp Leu Arg Ala Ile Ala 1 5 10 15 105 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 105 Ser Pro Ser Val Asp Asp Leu Arg Ala Ile Ala Glu Glu Ser Asp 1 5 10 15 106 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 106 Asp Asp Leu Arg Ala Ile Ala Glu Glu Ser Asp Glu Glu Glu Ala 1 5 10 15 107 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 107 Ala Ile Ala Glu Glu Ser Asp Glu Glu Glu Ala Ile Val Ala Tyr 1 5 10 15 108 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 108 Glu Ser Asp Glu Glu Glu Ala Ile Val Ala Tyr Thr Leu Ala Thr 1 5 10 15 109 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 109 Glu Glu Ala Ile Val Ala Tyr Thr Leu Ala Thr Ala Gly Val Ser 1 5 10 15 110 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 110 Val Ala Tyr Thr Leu Ala Thr Ala Gly Val Ser Ser Ser Asp Ser 1 5 10 15 111 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 111 Leu Ala Thr Ala Gly Val Ser Ser Ser Asp Ser Leu Val Ser Pro 1 5 10 15 112 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 112 Gly Val Ser Ser Ser Asp Ser Leu Val Ser Pro Pro Glu Ser Pro 1 5 10 15 113 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 113 Glu Glu Arg Glu Asp Thr Val Ser Val Lys Ser Glu Pro Val Ser 1 5 10 15 114 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 114 Asp Thr Val Ser Val Lys Ser Glu Pro Val Ser Glu Ile Glu Glu 1 5 10 15 115 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 115 Val Lys Ser Glu Pro Val Ser Glu Ile Glu Glu Val Ala Pro Glu 1 5 10 15 116 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 116 Pro Val Ser Glu Ile Glu Glu Val Ala Pro Glu Glu Glu Glu Asp 1 5 10 15 117 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 117 Ile Glu Glu Val Ala Pro Glu Glu Glu Glu Asp Gly Ala Glu Glu 1 5 10 15 118 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 118 Ala Pro Glu Glu Glu Glu Asp Gly Ala Glu Glu Pro Thr Ala Ser 1 5 10 15 119 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 119 Glu Glu Asp Gly Ala Glu Glu Pro Thr Ala Ser Gly Gly Lys Ser 1 5 10 15 120 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 120 Ala Glu Glu Pro Thr Ala Ser Gly Gly Lys Ser Thr His Pro Met 1 5 10 15 121 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 121 Thr Ala Ser Gly Gly Lys Ser Thr His Pro Met Val Thr Arg Ser 1 5 10 15 122 15 PRT Artificial Sequence Description of Artificial Sequence HCMV IE-1 Fragment 122 Gly Lys Ser Thr His Pro Met Val Thr Arg Ser Lys Ala Asp Gln 1 5 10 15 123 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 123 Met Glu Ser Arg Gly Arg Arg Cys Pro Glu Met Ile Ser Val Leu 1 5 10 15 124 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 124 His Glu Thr Arg Leu Leu Gln Thr Gly Ile His Val Arg Val Ser 1 5 10 15 125 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 125 Asp Asp Val Trp Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu Val 1 5 10 15 126 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 126 Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu Val Thr Thr Glu Arg 1 5 10 15 127 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 127 Asp Ser Asp Glu Glu Leu Val Thr Thr Glu Arg Lys Thr Pro Arg 1 5 10 15 128 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 128 Glu Leu Val Thr Thr Glu Arg Lys Thr Pro Arg Val Thr Gly Gly 1 5 10 15 129 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 129 Thr Glu Arg Lys Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala 1 5 10 15 130 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 130 Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Gly Ala Ser Thr 1 5 10 15 131 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 131 Thr Gly Gly Gly Ala Met Ala Gly Ala Ser Thr Ser Ala Gly Arg 1 5 10 15 132 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 132 Ala Met Ala Gly Ala Ser Thr Ser Ala Gly Arg Lys Arg Lys Ser 1 5 10 15 133 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 133 Ala Ser Thr Ser Ala Gly Arg Lys Arg Lys Ser Ala Ser Ser Ala

1 5 10 15 134 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 134 Ala Gly Arg Lys Arg Lys Ser Ala Ser Ser Ala Thr Ala Cys Thr 1 5 10 15 135 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 135 Leu Leu Gln Thr Gly Ile His Val Arg Val Ser Gln Pro Ser Leu 1 5 10 15 136 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 136 Arg Lys Ser Ala Ser Ser Ala Thr Ala Cys Thr Ser Gly Val Met 1 5 10 15 137 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 137 Ser Ser Ala Thr Ala Cys Thr Ser Gly Val Met Thr Arg Gly Arg 1 5 10 15 138 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 138 Gly Ile His Val Arg Val Ser Gln Pro Ser Leu Ile Leu Val Ser 1 5 10 15 139 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 139 Arg Val Ser Gln Pro Ser Leu Ile Leu Val Ser Gln Tyr Thr Pro 1 5 10 15 140 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 140 Pro Ser Leu Ile Leu Val Ser Gln Tyr Thr Pro Asp Ser Thr Pro 1 5 10 15 141 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 141 Leu Val Ser Gln Tyr Thr Pro Asp Ser Thr Pro Cys His Arg Gly 1 5 10 15 142 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 142 Tyr Thr Pro Asp Ser Thr Pro Cys His Arg Gly Asp Asn Gln Leu 1 5 10 15 143 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 143 Ser Thr Pro Cys His Arg Gly Asp Asn Gln Leu Gln Val Gln His 1 5 10 15 144 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 144 His Arg Gly Asp Asn Gln Leu Gln Val Gln His Thr Tyr Phe Thr 1 5 10 15 145 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 145 Asn Gln Leu Gln Val Gln His Thr Tyr Phe Thr Gly Ser Glu Val 1 5 10 15 146 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 146 Gly Arg Arg Cys Pro Glu Met Ile Ser Val Leu Gly Pro Ile Ser 1 5 10 15 147 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 147 Val Gln His Thr Tyr Phe Thr Gly Ser Glu Val Glu Asn Val Ser 1 5 10 15 148 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 148 Tyr Phe Thr Gly Ser Glu Val Glu Asn Val Ser Val Asn Val His 1 5 10 15 149 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 149 Ser Glu Val Glu Asn Val Ser Val Asn Val His Asn Pro Thr Gly 1 5 10 15 150 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 150 Asn Val Ser Val Asn Val His Asn Pro Thr Gly Arg Ser Ile Cys 1 5 10 15 151 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 151 Asn Val His Asn Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln Glu 1 5 10 15 152 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 152 Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln Glu Pro Met Ser Ile 1 5 10 15 153 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 153 Ser Ile Cys Pro Ser Gln Glu Pro Met Ser Ile Tyr Val Tyr Ala 1 5 10 15 154 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 154 Ser Gln Glu Pro Met Ser Ile Tyr Val Tyr Ala Leu Pro Leu Lys 1 5 10 15 155 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 155 Met Ser Ile Tyr Val Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile 1 5 10 15 156 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 156 Val Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn 1 5 10 15 157 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 157 Pro Glu Met Ile Ser Val Leu Gly Pro Ile Ser Gly His Val Leu 1 5 10 15 158 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 158 Pro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn Val His His Tyr 1 5 10 15 159 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 159 Leu Asn Ile Pro Ser Ile Asn Val His His Tyr Pro Ser Ala Ala 1 5 10 15 160 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 160 Ser Ile Asn Val His His Tyr Pro Ser Ala Ala Glu Arg Lys His 1 5 10 15 161 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 161 His His Tyr Pro Ser Ala Ala Glu Arg Lys His Arg His Leu Pro 1 5 10 15 162 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 162 Ser Ala Ala Glu Arg Lys His Arg His Leu Pro Val Ala Asp Ala 1 5 10 15 163 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 163 Arg Lys His Arg His Leu Pro Val Ala Asp Ala Val Ile His Ala 1 5 10 15 164 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 164 His Leu Pro Val Ala Asp Ala Val Ile His Ala Ser Gly Lys Gln 1 5 10 15 165 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 165 Ala Asp Ala Val Ile His Ala Ser Gly Lys Gln Met Trp Gln Ala 1 5 10 15 166 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 166 Ile His Ala Ser Gly Lys Gln Met Trp Gln Ala Arg Leu Thr Val 1 5 10 15 167 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 167 Gly Lys Gln Met Trp Gln Ala Arg Leu Thr Val Ser Gly Leu Ala 1 5 10 15 168 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 168 Ser Val Leu Gly Pro Ile Ser Gly His Val Leu Lys Ala Val Phe 1 5 10 15 169 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 169 Trp Gln Ala Arg Leu Thr Val Ser Gly Leu Ala Trp Thr Arg Gln 1 5 10 15 170 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 170 Leu Thr Val Ser Gly Leu Ala Trp Thr Arg Gln Gln Asn Gln Trp 1 5 10 15 171 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 171 Gly Leu Ala Trp Thr Arg Gln Gln Asn Gln Trp Lys Glu Pro Asp 1 5 10 15 172 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 172 Thr Arg Gln Gln Asn Gln Trp Lys Glu Pro Asp Val Tyr Tyr Thr 1 5 10 15 173 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 173 Asn Gln Trp Lys Glu Pro Asp Val Tyr Tyr Thr Ser Ala Phe Val 1 5 10 15 174 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 174 Glu Pro Asp Val Tyr Tyr Thr Ser Ala Phe Val Phe Pro Thr Lys 1 5 10 15 175 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 175 Tyr Tyr Thr Ser Ala Phe Val Phe Pro Thr Lys Asp Val Ala Leu 1 5 10 15 176 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 176 Ala Phe Val Phe Pro Thr Lys Asp Val Ala Leu Arg His Val Val 1 5 10 15 177 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 177 Pro Thr Lys Asp Val Ala Leu Arg His Val Val Cys Ala His Glu 1 5 10 15 178 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 178 Val Ala Leu Arg His Val Val Cys Ala His Glu Leu Val Cys Ser 1 5 10 15 179 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 179 Pro Ile Ser Gly His Val Leu Lys Ala Val Phe Ser Arg Gly Asp 1 5 10 15 180 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 180 His Val Val Cys Ala His Glu Leu Val Cys Ser Met Glu Asn Thr 1 5 10 15 181 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 181 Ala His Glu Leu Val Cys Ser Met Glu Asn Thr Arg Ala Thr Lys 1 5 10 15 182 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 182 Val Cys Ser Met Glu Asn Thr Arg Ala Thr Lys Met Gln Val Ile 1 5 10 15 183 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 183 Glu Asn Thr Arg Ala Thr Lys Met Gln Val Ile Gly Asp Gln Tyr 1 5 10 15 184 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 184 Ala Thr Lys Met Gln Val Ile Gly Asp Gln Tyr Val Lys Val Tyr 1 5 10 15 185 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 185 Gln Val Ile Gly Asp Gln Tyr Val Lys Val Tyr Leu Glu Ser Phe 1 5 10 15 186 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 186 Asp Gln Tyr Val Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val 1 5 10 15 187 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 187 Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val Pro Ser Gly Lys 1 5 10 15 188 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 188 Glu Ser Phe Cys Glu Asp Val Pro Ser Gly Lys Leu Phe Met His 1 5 10 15 189 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 189 Glu Asp Val Pro Ser Gly Lys Leu Phe Met His Val Thr Leu Gly 1 5 10 15 190 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 190 His Val Leu Lys Ala Val Phe Ser Arg Gly Asp Thr Pro Val Leu 1 5 10 15 191 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 191 Ser Gly Lys Leu Phe Met His Val Thr Leu Gly Ser Asp Val Glu 1 5 10 15 192 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 192 Phe Met His Val Thr Leu Gly Ser Asp Val Glu Glu Asp Leu Thr 1 5 10 15 193 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 193 Thr Leu Gly Ser Asp Val Glu Glu Asp Leu Thr Met Thr Arg Asn 1 5 10 15 194 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 194 Asp Val Glu Glu Asp Leu Thr Met Thr Arg Asn Pro Gln Pro Phe 1 5 10 15 195 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 195 Asp Leu Thr Met Thr Arg Asn Pro Gln Pro Phe Met Arg Pro His 1 5 10 15 196 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 196 Thr Arg Asn Pro Gln Pro Phe Met Arg Pro His Glu Arg Asn Gly 1 5 10 15 197 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 197 Gln Pro Phe Met Arg Pro His Glu Arg Asn Gly Phe Thr Val Leu 1 5 10 15 198 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 198 Arg Pro His Glu Arg Asn Gly Phe Thr Val Leu Cys Pro Lys Asn 1 5 10 15 199 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 199 Arg Asn Gly Phe Thr Val Leu Cys Pro Lys Asn Met Ile Ile Lys 1 5 10 15 200 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 200 Thr Val Leu Cys Pro Lys Asn Met Ile Ile Lys Pro Gly Lys Ile 1 5 10 15 201 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 201 Ala Val Phe Ser Arg Gly Asp Thr Pro Val Leu Pro His Glu Thr 1 5 10 15 202 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 202 Pro Lys Asn Met Ile Ile Lys Pro Gly Lys Ile Ser His Ile Met 1 5 10 15 203 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 203 Ile Ile Lys Pro Gly Lys Ile Ser His Ile Met Leu Asp Val Ala 1 5 10 15 204 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 204 Gly Lys Ile Ser His Ile Met Leu Asp Val Ala Phe Thr Ser His 1 5 10 15 205 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 205 His Ile Met Leu Asp Val Ala Phe Thr Ser His Glu His Phe Gly 1 5 10 15 206 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 206 Asp Val Ala Phe Thr Ser His Glu His Phe Gly Leu Leu Cys Pro 1 5 10 15 207 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 207 Thr Ser His Glu His Phe Gly Leu Leu Cys Pro Lys Ser Ile Pro 1 5 10 15 208 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 208 His Phe Gly Leu Leu Cys Pro Lys Ser Ile Pro Gly Leu Ser Ile 1 5 10 15 209 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 209 Leu Cys Pro Lys Ser Ile Pro Gly Leu Ser Ile Ser Gly Asn Leu 1 5 10 15 210 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 210 Ser Ile Pro Gly Leu Ser Ile Ser Gly Asn Leu Leu Met Asn Gly 1 5 10 15 211 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 211 Leu Ser Ile Ser Gly Asn Leu Leu Met Asn Gly Gln Gln Ile Phe 1 5 10 15 212 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 212 Arg Gly Asp Thr Pro Val Leu Pro His Glu Thr Arg Leu Leu Gln 1 5 10 15 213 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 213 Gly Asn Leu Leu Met Asn Gly Gln Gln Ile Phe Leu Glu Val Gln 1 5 10 15 214 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 214 Met Asn Gly Gln Gln Ile Phe Leu Glu Val Gln Ala Ile Arg Glu 1 5 10 15 215 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 215 Gln Ile Phe Leu Glu Val Gln Ala Ile Arg Glu Thr Val Glu Leu 1 5 10 15 216 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 216 Glu Val Gln Ala Ile Arg Glu Thr Val Glu Leu Arg Gln Tyr Asp 1 5 10 15 217 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65

Fragment 217 Ile Arg Glu Thr Val Glu Leu Arg Gln Tyr Asp Pro Val Ala Ala 1 5 10 15 218 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 218 Val Glu Leu Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe Phe 1 5 10 15 219 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 219 Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe Phe Asp Ile Asp Leu 1 5 10 15 220 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 220 Val Ala Ala Leu Phe Phe Phe Asp Ile Asp Leu Leu Leu Gln Arg 1 5 10 15 221 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 221 Phe Phe Phe Asp Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr 1 5 10 15 222 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 222 Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr Ser Glu His Pro 1 5 10 15 223 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 223 Pro Val Leu Pro His Glu Thr Arg Leu Leu Gln Thr Gly Ile His 1 5 10 15 224 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 224 Leu Gln Arg Gly Pro Gln Tyr Ser Glu His Pro Thr Phe Thr Ser 1 5 10 15 225 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 225 Pro Gln Tyr Ser Glu His Pro Thr Phe Thr Ser Gln Tyr Arg Ile 1 5 10 15 226 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 226 Glu His Pro Thr Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu 1 5 10 15 227 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 227 Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Glu Tyr Arg His 1 5 10 15 228 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 228 Tyr Arg Ile Gln Gly Lys Leu Glu Tyr Arg His Thr Trp Asp Arg 1 5 10 15 229 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 229 Gly Lys Leu Glu Tyr Arg His Thr Trp Asp Arg His Asp Glu Gly 1 5 10 15 230 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 230 Tyr Arg His Thr Trp Asp Arg His Asp Glu Gly Ala Ala Gln Gly 1 5 10 15 231 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 231 Trp Asp Arg His Asp Glu Gly Ala Ala Gln Gly Asp Asp Asp Val 1 5 10 15 232 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 232 Asp Glu Gly Ala Ala Gln Gly Asp Asp Asp Val Trp Thr Ser Gly 1 5 10 15 233 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 233 Ala Gln Gly Asp Asp Asp Val Trp Thr Ser Gly Ser Asp Ser Asp 1 5 10 15 234 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 234 Ala Cys Thr Ser Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu 1 5 10 15 235 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 235 Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu Ser Thr Val Ala 1 5 10 15 236 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 236 Arg Gly Arg Leu Lys Ala Glu Ser Thr Val Ala Pro Glu Glu Asp 1 5 10 15 237 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 237 Lys Ala Glu Ser Thr Val Ala Pro Glu Glu Asp Thr Asp Glu Asp 1 5 10 15 238 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 238 Thr Val Ala Pro Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn Glu 1 5 10 15 239 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 239 Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile His Asn Pro 1 5 10 15 240 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 240 Asp Glu Asp Ser Asp Asn Glu Ile His Asn Pro Ala Val Phe Thr 1 5 10 15 241 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 241 Asp Asn Glu Ile His Asn Pro Ala Val Phe Thr Trp Pro Pro Trp 1 5 10 15 242 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 242 His Asn Pro Ala Val Phe Thr Trp Pro Pro Trp Gln Ala Gly Ile 1 5 10 15 243 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 243 Val Phe Thr Trp Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg Asn 1 5 10 15 244 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 244 Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg Asn Leu Val Pro Met 1 5 10 15 245 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 245 Ala Gly Ile Leu Ala Arg Asn Leu Val Pro Met Val Ala Thr Val 1 5 10 15 246 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 246 Ala Arg Asn Leu Val Pro Met Val Ala Thr Val Gln Gly Gln Asn 1 5 10 15 247 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 247 Val Pro Met Val Ala Thr Val Gln Gly Gln Asn Leu Lys Tyr Gln 1 5 10 15 248 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 248 Ala Thr Val Gln Gly Gln Asn Leu Lys Tyr Gln Glu Phe Phe Trp 1 5 10 15 249 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 249 Gly Gln Asn Leu Lys Tyr Gln Glu Phe Phe Trp Asp Ala Asn Asp 1 5 10 15 250 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 250 Lys Tyr Gln Glu Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile 1 5 10 15 251 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 251 Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Phe Ala Glu Leu 1 5 10 15 252 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 252 Ala Asn Asp Ile Tyr Arg Ile Phe Ala Glu Leu Glu Gly Val Trp 1 5 10 15 253 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 253 Tyr Arg Ile Phe Ala Glu Leu Glu Gly Val Trp Gln Pro Ala Ala 1 5 10 15 254 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 254 Ala Glu Leu Glu Gly Val Trp Gln Pro Ala Ala Gln Pro Lys Arg 1 5 10 15 255 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 255 Gly Val Trp Gln Pro Ala Ala Gln Pro Lys Arg Arg Arg His Arg 1 5 10 15 256 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 256 Pro Ala Ala Gln Pro Lys Arg Arg Arg His Arg Gln Asp Ala Leu 1 5 10 15 257 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 257 Pro Lys Arg Arg Arg His Arg Gln Asp Ala Leu Pro Gly Pro Cys 1 5 10 15 258 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 258 Arg His Arg Gln Asp Ala Leu Pro Gly Pro Cys Ile Ala Ser Thr 1 5 10 15 259 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 259 Asp Ala Leu Pro Gly Pro Cys Ile Ala Ser Thr Pro Lys Lys His 1 5 10 15 260 15 PRT Artificial Sequence Description of Artificial Sequence HCMV pp65 Fragment 260 Leu Pro Gly Pro Cys Ile Ala Ser Thr Pro Lys Lys His Arg Gly 1 5 10 15

Claims

1. A method for the antigen-specific stimulation of T lymphocytes with synthetic peptide libraries, comprising the following steps: (a) subdividing the total amino acid sequence of the antigen into protein fragments with partial amino acid sequences, wherein said protein fragments have a minimum length of 9 amino acid residues (AAs), and wherein adjacent or neighboring protein fragments are overlapping with their partial amino acid sequence; (b) synthesizing a peptide library containing the protein fragments defined in (a); (c) incubating a suspension containing CD8+ and/or CD4+ T lymphocytes with all the protein fragments of said peptide library in a single culture run.

2. The method according to claim 1, wherein the protein fragments have a minimum length of 15 AAs and/or a maximum length of 35 AAs, preferably 25 AAs.

3. The method according to claim 1 or 2, wherein an overlap of 8 AAs, preferably 11 AAs, exists between neighboring protein fragments.

4. The method according to one or more of claims 1 to 3, wherein the synthetic protein fragments are extended by a maximum of 7 natural or artificial AAs and/or a protective group at either or both of their N terminus and C terminus.

5. The method according to one or more of claims 1 to 4, wherein the concentration of the individual protein fragments of the peptide library is at least 1 ng/ml, preferably from 0.1 to 10 .mu.g/ml, in the culture mix.

6. The method according to one or more of claims 1 to 5, wherein one or more compounds having costimulatory properties, especially costimulatory antibodies, are added to the incubation solution.

7. The method according to one or more of claims 1 to 6, wherein the total amino acid sequence of the antigen is determined prior to step (a) in the method.

8. The method according to claims 1 to 7 adapted for identifying stimulating or non-stimulating mixtures of all protein fragments in a single culture run, wherein the method preferably further comprises the following steps: (d) identifying, preferably flow-cytometric identifying, of (i) at least one T-cell cytokine which was induced by the protein fragment or fragments and synthesized in the T lymphocytes and which is intracellular or bound to the cell membrane; and/or (ii) at least one activation marker which was induced by the protein fragment or fragments and synthesized in the T lymphocytes and which is intracellular or bound to the cell membrane.

9. The method according to one or more of claims 1 to 5 which is capable of establishing whether T-lymphocyte-stimulating antigenic determinants are present in an antigen.

10. The method according to one or more of claims 1 to 7 which is adapted for in-vivo and in-vitro immunostimulation of T lymphocytes of mammals, especially humans.

11. The method according to claim 10, further comprising expansion of the stimulated T lymphocytes.

12. The method according to claim 11, further comprising transfusion of the expanded T lymphocytes obtained into a patient.

13. The method according to one or more of claims 1 to 8 which is suitable for diagnostics, especially to establish whether a mammal, especially a human, has previously responded to a specific protein with its immune system, and if so, how strong such response is.

14. The method according to one or more of claims 9 to 13 which comprises the use of several different synthetic peptide libraries, wherein the incubation of the peptide libraries with the CD8+ and/or CD4+ T lymphocyte suspension is effected together in one culture run or in separated culture runs.

15. Stimulated T lymphocytes obtainable by the method according to claim 10 or 11.

16. Use of a peptide library as defined in one or more of claims 1 to 5 for the preparation of a medicament for in-vivo immunostimulation of T lymphocytes of mammals.

17. A composition for in-vitro and in-vivo immunostimulation of T lymphocytes of mammals, comprising one or more peptide libraries as defined in one or more of claims 1 to 5.