WO2013186178A1

WO2013186178A1 - Stimulation of cellular immune response against epstein-barr virus (ebv)

Info

Publication number: WO2013186178A1
Application number: PCT/EP2013/061929
Authority: WO
Inventors: Stefan Stevanovic; Christina KYZIRAKOS; Christoph GRABENBAUER; Elina BARSAUME; Stefan AUDEHM; Hans-Georg Rammensee; Thomas Feger
Original assignee: Eberhard-Karls-Universitaet Universitaetsklinikum
Priority date: 2012-06-15
Filing date: 2013-06-10
Publication date: 2013-12-19
Also published as: DE102012105193B4; DE102012105193A1

Abstract

Pharmaceutical composition for stimulating the cellular immune response against the Epstein-Barr virus (EBV), comprising at least three different peptides, each of which has different amino acid sequences selected from among the group consisting of SEQ ID Nos. 1 to 78.

Description

Stimulation of cellular immune response against Epstein-Barr virus (EBV)

The present invention relates to a pharmaceutical composition for stimulating the cellular immune response against the Epstein-Barr virus (EBV), a process for the preparation of such a pharmaceutical composition and peptides contained therein.

The Epstein-Barr virus (EBV), also called human herpes virus 4 (HHV 4), is a human pathogenes, enveloped, double-stranded DNA virus from the family of herpesviruses. The main transmission path of the virus is a droplet infection or a contact or smear infection. Transmissions may also be involved in transplantations or blood transfusions. The infection with the virus takes place mostly in childhood. From the age of 40, approximately 98% of people are infected with EBV. In most cases, chronic infections are asymptomatic. In 30% to 60% of all cases, however, the so-called Pfeiffer glandular fever or infectious mononucleosis may occur. EBV is also causally related to lymph node cancer, Burkitt's lymphoma, Hodgkin's lymphoma and other lymphomas. Especially in immunocompromised individuals there is an increased occurrence of EBV-associated diseases.

EBV plays an important role in patients undergoing organ transplantation. These are immunosuppressed to prevent the rejection of the foreign organ. This promotes the multiplication of EBV and the development of the so-called lymphoproliferative disease after transplantation (English, "post-transplant lymphoproliferative disorder", PTLD).

A targeted therapy against the Pfeiffer glandular fever does not exist so far. In most cases, there is a purely symptomatic treatment. Accordingly, the treatment methods of Hodgkin's or Burkitt's lymphoma consist of classic chemotherapy and radiation therapies. The therapy of PTLD has not been satisfactorily resolved. Often it comes here to the use of chemo and radiation therapies and various methods of immune stimulation. The adoptive transfer of polyclonal T cell lines for the treatment and prevention of PTLD in hematopoietic stem cell transplantation is described, for example, in Heslop et al. (2010), Long-term outcome of EBV-specific T-cell infusion to prevent or treat EBV-related lymphoproliferative disease in transplant recipients, Blood 1 15 (5), pp. 925-935. The polyclonal T cell lines used there were generated by the repeated stimulation of mononuclear cells of the peripheral blood (PBMC) of the donor with autologous EBV-infected lymphoblastoid cell lines (LCL). However, this is very time consuming. The generation of LCL alone can take up to 5 weeks; see. Rooney et al. (1995), Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr virus-related lymphoproliferation, Lancet 345 (8941), pp. 9-13, and Wilkie et al. (2004), Establishment and characterization of a bank of cytotoxic T lymphocytes for immunotherapy of Epstein-Barr virus-associated diseases, Journal of Immuno-therapy 27 (4), pages 309-316. Another disadvantage of this treatment method results from the polyclonality of the T cell lines. These can trigger an immunological reaction in the sense of a graft-versus-host disease.

Against this background, it is the object of the present invention to provide a novel pharmaceutical composition with which EBV-associated diseases can be prevented or treated, wherein the problems described in the prior art should be largely avoided.

This object is achieved by providing a pharmaceutical composition for stimulating the cellular immune response against the Epstein-Barr virus (EBV), which has at least three different peptides each having different amino acid sequences, wherein the amino acid sequences are each selected from the group from SEQ ID No. 1 to 78.

The inventors have surprisingly discovered that the administration of at least three different peptides having three different amino acid sequences from the group found by the inventors leads to stimulation of the cellular immune response to EBV in almost all EBV-infected individuals. The peptides provided by the inventors detect so-called "frequently recognized epitopes" (FREP) from EBV, that is epitopes of EBV, which are frequently recognized by the cellular immune system. The peptides provided by the inventors capture both MHC class I and MHC class II restricted EBV specific epitopes.

Against this background, the particular advantage of the pharmaceutical composition according to the invention is that the MHC or HLA restriction is practically overcome. In other words, the pharmaceutical composition according to the invention triggers stimulation of the cellular immune response in EBV-infected individuals, regardless of which MHC or HLA profile is present. It is therefore not necessary to perform MHC or HLA typing of the affected individual prior to administration. The treatment with the composition according to the invention is therefore particularly cost-effective.

Furthermore, it is not absolutely necessary to first remove cells of the immune system from the EBV-infected individual in order then to stimulate them ex vivo and then to administer them to the individual again, as described in the Heslop et al. (2010, supra) is the case. Rather, the pharmaceutical composition may be administered directly to the individual and thus promptly induce immune stimulation.

The pharmaceutical composition may comprise, in addition to the peptides, a pharmaceutically acceptable carrier. Such carriers are well known in the art. They include, for example, binders, disintegrants, fillers, lubricants and buffers, salts and other substances suitable for the formulation of medicaments; see. Rowe et al. (2006), Handbook of Pharmaceutical Excipients, 5th Edition, Pharmaceutical Press, or Bauer et al. (1999), Textbook of Pharmaceutical Technology, 6th Edition, Wissenschaftliche Verlagsgesellschaft Stuttgart mbH.

The inventors have recognized that already three peptides are sufficient to capture a majority of EBV-infected individuals. This is especially true in the case of MHC class II peptides. These are not very strong in their ability to bind restrictive and can sometimes bind across alleles. The inventors were able to generate MHC class II peptides, which led to recognition rates of more than 75% among randomly selected blood donors. When using at least three MHC class II peptides according to the invention, recognition rates of approximately 100% are achieved.

The pharmaceutical composition requires in addition to the peptides of the invention no further active ingredients. However, it may contain other agents or potentiators. In the case of a vaccine or vaccine, for example, so-called adjuvants are preferred in order to non-specifically increase the immune response. Such adjuvants include aluminum hydrochloride, MF59, AS03, monophosphoryl lipid A, etc.

The problem underlying the invention is hereby completely solved.

According to a preferred development of the invention, the pharmaceutical composition has at least five different peptides according to the invention.

This measure has the advantage that the entire population of EBV-infected individuals is covered with even greater reliability and a targeted immune stimulation is achieved in these. This also applies, and in particular in the case of five selected MHC class I peptides. Thus, each human has an individual set of two HLA-A alleles and HLA-B alleles, for example HLA-A2, HLA-A3, HLA-B7 and HLA-B44. The different alleles are represented differently in the population. MHC class I epitopes are very restrictive in their binding ability, thus binding eg only to HLA-A2. If one calculates the theoretical population coverage of a vaccine within the German population consisting of peptides that bind to the most common HLA alleles, then it would be found that more than 95% of the population would be covered from a quantity of five peptides: Number of peptides Population coverage (%) Restrictions of the peptides used

1 49,8736 A2

2 69.4191 A2 + A3

3 82.1916 A2 + A3 + B7

4 91, 3564 A2 + A3 + B7 + B44

5 96.2364 A2 + A3 + B7 + B44 + B8

6 99.1 164 A2 + A3 + B7 + B44 + B8 + B35

Table 1: Theoretical population coverage with increasing number of peptides at a recognition rate of the peptides of 100%; Calculation according to Schipper et al. (1996), Minimal phenotype panels. A method for achieving maximum population coverage with a minimum of HLA antigens, human immunology 51 (2), pages 95-98; Allele frequencies of the German population from www.allelefrequencies.net

According to a preferred development, the pharmaceutical composition has at least eight different peptides.

This measure has the advantage that the composition according to the invention with even greater safety in any EBV-infected individual triggers a cellular immune response.

It is understood that the pharmaceutical composition according to the invention at least 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 may have different peptides, wherein the Amino acid sequences of the various peptides are each selected from the group consisting of SEQ ID Nos. 1 to 73.

According to a preferred development, the pharmaceutical composition comprises at least three different MHC class II peptides each having different amino acid sequences and at least five different MHC class I peptides each having different amino acid sequences, wherein the amino acid sequences of the MHC class Il peptides are each selected from the group consisting of SEQ ID Nos. 1 to 38, and wherein the amino acid sequences of the MHC class I peptides are each selected from the group consisting of: SEQ ID Nos. 39 to 78. This measure has the advantage that the MHC class I equipment and at the same time the MHC class II equipment of a human individual are detected with very high reliability, so that an optimal stimulation of the cellular immune response is ensured.

According to a preferred embodiment, the pharmaceutical composition according to the invention for the treatment and / or prophylaxis of an EBV-associated disease is formed.

In the EBV-associated disease may be Pfeiffer glandular fever (infectious mononucleosis), lymph node cancer (Hodgkin's disease), Burkitt's lymphoma, or preferably the lymphoproliferative disorder after transplantation (PTLD) act.

According to the invention, there is thus provided in a particularly advantageous manner such a pharmaceutical composition with which such EBV-associated diseases can be purposefully treated or prevented, for which hitherto no satisfactory therapeutic options are available.

Against this background, another object of the present invention relates to the use of at least three, preferably at least five, more preferably at least eight different peptides, each with different amino acid sequences, for stimulating the cellular immune response against Epstein-Barr virus (EBV), wherein the amino acid sequences are each selected from the group consisting of SEQ ID Nos. 1 to 78.

The features, advantages and developments of the pharmaceutical composition according to the invention apply equally to the use according to the invention.

Another object of the present invention relates to a process for the preparation of a pharmaceutical composition, the following steps (1) providing at least three, preferably at least five, more preferably at least eight different peptides, (2) formulation of the peptides into a pharmaceutically acceptable carrier, wherein the at least three, preferably at least five, more preferably at least eight different peptides each have different Amino acid sequences each selected from the group consisting of SEQ ID Nos. 1 to 78.

The features, advantages and developments of the pharmaceutical composition according to the invention apply equally to the manufacturing method according to the invention.

Another object of the present invention relates to a peptide for stimulating the cellular immune response against Epstein-Barr virus (EBV) or the use of a peptide for stimulating the cellular immune response against Epstein-Barr virus (EBV), wherein the peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 78.

Another object of the present invention relates to a peptide for stimulating the cellular immune response against the Epstein-Barr virus (EBV) or the use of a peptide for stimulating the cellular immune response to the Epstein-Barr virus (EBV), wherein the peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4, 6, 8, 10 to 14, 16, 18, to 20, 22, 24 to 38, 48, 49, 54 , 56, 61, 62, 66, 68, 69.

Another object of the present invention relates to a nucleic acid molecule, which may be DNA or RNA or a genetic vector or plasmid encoding the peptide of the invention.

The invention also relates to a host, for example. A microorganism such as a bacterium containing the nucleic acid molecule of the invention. The features, advantages and developments mentioned for the pharmaceutical composition according to the invention apply correspondingly to the peptide according to the invention and to its use and to the nucleic acid molecule according to the invention.

Another object of the present invention relates to a method for stimulating the cellular immune response of a living organism against EBV, which comprises the administration of the pharmaceutical composition according to the invention and / or of the peptide or peptides according to the invention in the living being.

A further subject of the invention is a method for the diagnostic examination of a living being for an infection with EBV, which comprises the following steps: (1) administration of the pharmaceutical composition according to the invention and / or the peptide according to the invention and / or the peptides according to the invention into the animal , (2) measuring the immune response of the livestock, (3) correlating an immune reaction of the livestock with a positive diagnosis of EBV infection.

The immune reaction of the living organism can be measured, for example, via the secretion of cytokine, with IFN-γ being a particularly suitable marker which can be determined, for example, in the context of the so-called ELISpot assay.

The features and advantages of the pharmaceutical composition according to the invention apply correspondingly to the stimulation method according to the invention and the diagnostic method according to the invention.

It is understood that the features mentioned above and those yet to be explained not only in the combination specified, but also in other combinations or alone, without departing from the scope of the present invention. The invention will now be explained in more detail with reference to embodiments, from which further features and advantages. The embodiments are merely illustrative and do not limit the scope of the present invention. Reference is made to the attached figures.

The figures show the following:

FIG. 1 A) Amplification of peptide-specific reactions in vitro leads to detectable reactions. Spot-forming units of PBMCs from eight healthy donors, measured by IFN-γ ELISpot after stimulation with 10 μg ml of the peptide BLLF1 ₁₆₇ ex vivo and after in vitro amplification. The background (stimulation with the control peptide FlnA) was subtracted.

B) Example of result of IFN-γ ELISpot screening. The PBMCs of a healthy blood donor were tested for specific memory cells for ten different epitope candidates after amplification in vitro. Spot values of ^ 10, which were at least three times higher than the spot values of the control peptides, were defined as positive reactions. The positive reactions are marked with an asterisk. FlnA: control peptide; 0: medium control, PHA: positive control phytohemagglutinin.

Fig. 2 A) PBMCs from 16 randomly selected blood donors respond to the

Peptide mix 1 by IFN-γ production after amplification in vitro. The cells were amplified and stimulated with 10 μg ml of the peptide mix (2 g / ml per peptide) and 2 and 10 μg ml of FlnA (control peptide). Spot values of ^ 10, which were at least three times higher than the spot values of the control peptides, were defined as positive reactions. FlnA: control peptide, 0: medium control, PHA: positive control phytohemagglutinin. B) Stimulation with peptide mix 1 induces a multifunctional CD4 ⁺ T cell response in seropositive but not seronegative donors. PBMCs from five seropositive and two seronegative donors were stimulated with 10 μg ml of peptide mix 1 after preamplification and stained intracellularly for IFN-γ, TNF, granzyme B, CD154 and IL2. Gating strategy: live lymphocytes were selected and selected in CD4 ⁺ and CD8 ⁺ T cells. Duplets were excluded.

C) The responsive cells of the seropositive individuals are highly functional in comparison to the responsive cells of the seronegative individuals. The arcs shown visualize the various functions that are simultaneously expressed by the CD4 ⁺ cells of five seropositive and two seronegative individuals. In all seropositive individuals, one-sixth to about one-third of the cells (white to mid-gray) express at least three functions. In the seronegative individuals, the reacting cells mainly show a function (black parts).

Fig. 3 Reaction of 16 blood donors to the peptide mix 2.

FIG. 4 Reactions of 16 blood donors to the peptide mix 3.

FIG. 5 Reactions of 4 blood donors to the peptide mixtures 4 to 13.

embodiments

1 . material and methods

1 .1 Selection of candidate epitopes

The candidate epitopes were analyzed using the database SYFPEITHI, University of Tübingen, Interfaculty Institute of Cell Biology, Germany (www.syfpeithi.de). The protein sequences were added to the database

Taken from SwissProt (www.uniprot.org). For each antigen, 2% of the possible number of peptides were selected for screening. peptide synthesis

The peptides were synthesized as described in Meyer et al. (2009), Identification of natural MHC class II presented phosphopeptides and tumor-derived MHC class I phospholigands, J. Proteome Res. 8 (7), pp. 3666-3674. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

Buffy Coats or Leukapheresis products were provided by the Institute for Clinical and Experimental Transfusion Medicine, University of Tübingen, Germany. The PBMCs were isolated by standard gradient separation with Ficoll (PAA) and cryopreserved in fetal calf serum (PAA) with 10% DMSO (Merck) at -80 ° C until use. Pre-amplification of specific reactions

Frozen PBMCs were thawed on day 0 and incubated at high density (0.5-1 ^* 10 ⁷ cells / ml) in medium (Iscove's modified Dulbecco's medium (Lonza) containing 5% heat-inactivated human serum, 50 μM β-mercaptoethanol (Roth), 1 x penicillin / streptomycin (PAA) and 25 ug ml gentamicin sulfate (Lonza)). On day 1, the peptides were added together with medium at 2-10 μg ml each. For epitope screening, the PBMCs were probed with a pool of peptides including the control peptide (Gag_H IV 296-313 or

FLNA_HUMAN 1669-1683). On days 2, 5 and 7, IL-2 (Proleukin S, Novartis) was added together with medium 20 U / ml. The PBMCs were used on day 12 for ELISpot, intracellular cytokine stains or IFN-γ secretion assays (Miltenyi Biotec). IFN-y ELISpot assays

2-5 ^* 10 ⁵ PBMCsA wells were stimulated in 96-well antibody plates (1D1k, MabTech) (MSHAN4B, Millipore) with 10 pg / ml peptide or 10mg / ml PHA-P (Sigma) as positive control. After 24 to 26 hours of incubation, the wells were washed repeatedly and the bound IFN-γ was detected with biotinylated secondary anti-IFN-γ antibody (7-B6-1, MabTech). The bound antibody was coupled to ExtrAvidin alkaline phosphatase (Sigma) and stained with NBT / BCIP (Sigma). The developed plates were evaluated using an automated ELISpot reader (CTL).

Each peptide was analyzed by two- or three-fold measurements. The responses to the individual peptides were rated positive when the mean number of spots per well was at least 10 and the levels were over 3 times the mean of the negative control spots. Intracellular cytokine staining

10 ⁵ - 10 ⁶ PBMCs were stimulated overnight with 2-10 μg ml of the specific peptide (s) in TCM + 10 μg ml Brefeldin A (Sigma). PMA (Sigma, 150 ng / ml) and ionomycin (Sigma, 1 μΜ final concentration) were used as a positive control. After incubation, the cells were first stained with live / dead aqua (invitro) to exclude dead cells. Subsequently, cells were stained extracellularly with various antibodies: Anti-CD3-Pacific Blue (Biolegend), Anti-CD3-FITC (proprietary preparation), Anti-CD4-APC-Cy7 (BD Biosciences), Anti-CD4-FITC (proprietary preparation), Anti -CD8-PerCP (Biolegend), anti-CD8-FITC (self-preparation) and anti-CD8-PE-Cy7 (Beckman Coulter). This was followed by permeabilization with Cytofix / Cytoperm (BD Biosciences) and an intracellular staining with anti-TNF-PacificBlue (Biolegend); Anti-CD154-FITC, anti-IFN-γ-γ-Cy7 / PE / FITC (all BD Biosciences), anti-IL-2-ΡΕ (BD Biosciences), anti-GranzymeB-AlexaFluor647 (BD Biosciences). The cells were used analysis of a FACSCanto II Cytometer (BD Biosciences) and FlowJo (TreeStar). Results Amplification of specific T cells results in a more efficient rate of epitope detection.

Using the syfpeithi database, the inventors determined over one hundred epitope candidates of EBV antigens from the latent and lytic phases of the virus. These were screened on T memory cells present in healthy blood donors to identify frequently recognized EBV epitopes ("frequently recognized epitopes", freps).

Unlike CD4 ⁺ T cells, which are specific for cytomegalovirus (CMV), the incidence of CD4 ⁺ T cells specific for epitopes of EBV is less than 1 in 10,000 PBMCs present in the ELISpot react to an epitope, usually very low. To overcome this limitation and not to overlook any reaction, preexisting specific T cells were amplified in vitro in a 12 day culture prior to reading. This resulted in an amplification of the reaction to a detectable level.

As shown in Figure 1A, eight healthy donors were found that responded to the epitope BLLF1 ₁₆ 7 (SEQ ID NO: 17) after amplification in vitro. None of these reactions were directly detectable ex vivo in an ELISpot with 0.5 ^* 10 ⁶ cells per well. Amplification of specific PBMCs that produced IFN-γ varied from about 10 to about 900 fold, with a mean of 325.7 (± standard deviation 285.4). Identification of novel EBV epitopes and verified CD4 ⁺ T-cell epitopes

Each epitope candidate was screened in a first round against PBMCs from healthy blood donors by an IFN-γ ELISpot. Spot values of ^ 10, which were at least three times higher than the spot values of the control peptides, were defined as positive reactions. Peptides that elicited positive IFN-γ responses were further tested, resulting in at least 15 tested donors per peptide. An exemplary ELISpot result of a tested donor with respect to a reaction against 10 different epitope candidates of the antigen BLLF1 is shown in Figure 1B.

With this screening approach, the epitopes could be found in the following Table 2:

_ll E _{i il} MHC _t o epass ---

15

HLA antigen position sequence detection rate reference SEQ selected at random ID NO

healthy

Individuals (%)

BLLF1 268-282 PRPVSRFLGNNSILY 75 - 1

BLLF1 269-281 RPVSRFLGNNSIL - -. 2

EBNA2 276-295 PRSPTVFYNIPPMPLPPSQL 75 Long et al. 3

2005, Khanna et al. 1997

EBNA2 277-29 ₄ RSPTVFYNIPPMPLPPSQ - - 4

EBNA1 ₄ 81-500 LLAEGLRALLARSHVERTTDE 68.8 Kruger et al. 5

2003

EBNA1 ₄ 82- ₄ 99 AEGLRALLARSHVERTTD - - 6

BXLF2 126-140 LEKQLFYYIGTMLPNTRPHS 68.75 Long et al. 7

2011

BXLF2 127-139 EKQLFYYIGTMLPNTRPH - - 8

EBNA1 521-540 RRGTALAIPQCRLTPLSRLP 62.5 Demachi- 9

Okamura et al.

2008

EBNA1 522-539 RGTALAIPQCRLTPLSRL - - 10

Q. BLLF1 157-171 VPYI WDNCNSTNIT 50.0 - 11

BLLF1 158-170 PYIKWDNCNSTNI - - 12

EBNA-381-395 PIFIRRLHRLLLMRA 47.5-13

3A

EBNA-382-394 IFIRRLHRLLLMR - 14

3A

EBNA1 471-490 QGGSNPKFENIAEGLRALLA 43.8 Demachi- 15

Okamura et al.

2008

EBNA1 472-489 GGSNPKFENIAEGLRALL - - 16

BLLF1 167-181 STNITAVVRAQGLDV 43.75 Wallace et al. 17

1991

BLLF1 168-180 TNITAVVRAQGLD - - 18

BMRF1 261-275 SVPILRFYRSGIIAV 42,8 - 19

BMRF1 262-274 VPILRFYRSGIIA - - 20

EBNA1 514-527 KTSLYNLRRGTALA 41, 9 Khanna et al. 21

1995

EBNA1 515-526 TSLYNLRRGTAL - - 22

EBNA1 531-550 CRLTPLSRLPFGMAPGPGPQ 37.5 Long et al. 23

2005

EBNA1 532-549 RLTPLSRLPFGMAPGPGP - - 24

BRLF1 119-133 DRFFIQAPSNRVMIP 33.3 25

BRLF1 120-132 RFFIQAPSNRVMI - - 26

EBNA-134-148 YI MYAMAI RQAI RD R 33,3 - 27

3A

EBNA-135-147 I MYAMAI RQAI RD - - 28

3A

_I E _i K _l MHCpass - -

16

EBNA-287-301 DLSYIKSFVSDALGT 33,3-29

3A

EBNA-288-300 LSYIKSFVSDALG - - 30

3A

BRLF1 223-237 DVRALIKTLPRASYS 29,2 - 31

BRLF1 22 ₄ -236 VRALI TLPRASY - - 32

BNRF1 816 - 830 ITPVLQKTGSLLIAV 28.6 - 33

BNRF1 817 - 829 TPVLQKTGSLLIA - - 3 ₄

EBNA-190-20 ₄ TPTFVHLQATLGCTG 28.0-35

3A

EBNA-191-203 PTFVHLQATLGCT - - 36

3A

EBNA-179-193 SWMYSYTDHQTTPTF 16.7-37

3A

EBNA-180-192 WMYSYTDHQTTPT - - 38

3A

healthy

Individuals (%)

A * 02 BRLF1 109-117 YVLDHLIVV> 90 Saulquin et al. 39

2000

A * 02 BMLF1 280-288 GLCTLVAML 90 Scot et al. ₄ 0

1996

A * 02 LMP2 356-36 ₄ FLYALALLL 85 Lautscham et ₄ 1 al. 2003

A * 02 LMP2 ₄ 26- ₄ 3 ₄ CLGGLLTMV 80 Lee et al. 1993 ₄ 2

A * 02 LMP2 329-337 LLWTLVVLL 65 Lee et al. 1993 ₄ 3

A * 02 LMP1 159-167 YLQQNWWTL 60 Khanna et al. ₄₄

1998

A * 02 LMP1 125-133 YLLEMLWRL 58 Khanna et al. ₄ 5

Q.

o 1998

A * 02 EBNA6 28 ₄ -293 LLDFVRFMGV 58 Kerr et al. 1996 ₄ 6

A * 03 EBNA3 ₄ 71- ₄ 79 85 RLRAEAQVK Hill et al. 1995 ₄ 7

A * 03 BALF2 2 ₄ 7-255 KVAKVAPLK ₄ 3 - ₄ 8

A * 03 EBNA3 210-219 VTFSAGTFK 50 - ₄ 9

A * 11 LMP2 3 ₄ 0-3 ₄ 9 SSCSSCPLSK 75 Lee et al. 1997 50th

A * 11 EBNA ₄ 399- ₄ 08 AVFDRKSDAK 88 Steven et al. 51

1996

A * 11 EBNA ₄ ₄ 16- ₄ 2 ₄ IVTDFSVIK 63 Gavioli et al. 52

1993

A * 2 ₄ LMP2 ₄ 19- ₄ 27 TYGPVFMCL 86 Lee et al. 1997 53

A * 26 BALF2 573-581 EVVQFMNSM 63 - 5 ₄

B * 07 EBNA3 2 ₄ 7-255 RPPIFIRRL> 90 Hill et al. 1995 55

B * 07 EAD 116-125 RPQGGSRPEF 79 Pudney et al. 56

2005

B * 08 BZLF1 190-197 RAKFKQLL> 90 Bogedain et al. 57

1995

B * 08 EBNA3 193-201 FLRGRAYGL> 90 Burrows et al. 58

1990

B * 08 EBNA3 26-3 ₄ QAKWRLQTL> 90 ** Burrows et al. 59

199 ₄ B * 08 EBNA1 518-526 YNLRRGTAL> 90 ** Voo et al. 2004 60

B * 15 BZLF1 122-130 VQTAAAWF 86 Stuber et al. 61

1995

B * 15 LMP2 163-171 LLAAVASSY 42 Landmeier et 62 al. 2009

B * 15 EBNA ₄ 831-839 GQGGSPTAM 54 Rickinson and 63

Moss 1997

B * 18 BZLF1 173-180 SELEIKRY> 90 Saulquin et al. 64

2000

B * 27 EBNA6 258-266 RRIYDLIEL Brooks et al. 65

1993

B * 35 EB2 ₄ 30- ₄ 38 NPGTLSSLL> 90 Kuzushima et 66 al. 2003

B * 35 BZLF1 5 ₄ -6 ₄ EPLPQGQLTAY> 90 Saulquin et al. 67

2000

B * 35 BALF2 31-39 YPLREVATL 88 Kuzushima et 68 al. 2003

B * 35 MTP 434-442 YPAPCISGY 88 - 69

B * 35 EBNA1 ₄ 07- ₄ 17 HPVGEADYFEY 71 Khan et al. 70

2004

B * 40 LMP2 200-208 IEDPPFNSL 87 Lee et al. 1997 71

B * 44 EBNA ₄ 657-666 VEITPYKPTW 56 Rickinson and 72

Moss 1997

B * 44 EBNA6 162-171 AEGGVGWRHW 61 Morgan et al. 73

1996

B * 53 BMRF1 286-29 ₄ LPLDLSVIL> 90 * - 74

B * 53 EBNA1 ₄ 07- ₄ 17 HPVGEADYFEY> 90 * - 75

B * 53 MTP 434-442 YPAPCISGY> 90 * - 76

B * 57 LMP1 156-164 IALYLQQNW 38 Duraiswamy et 77 al. 2003

Table 2: Frequently recognized EBV-specific peptides. In each case at least 16 healthy blood donors (exceptions: three each with asterisk, 12 each with two stars) with matching HLA restriction (MHC class I epitopes) or randomly selected blood donors (MHC class II epitopes) for their reactivity to the given peptide with the ELISpot assay.

A peptide mix is recognized by over 90% of all EBV infected people.

Next, a mix containing a few, but often recognized, peptides should be created which, when combined, are sufficient to activate EBV-specific CD4 ⁺ T memory cells from as many healthy EBV carriers as possible, regardless of their HLA type.

To achieve this, the individual recognition profile of 16 healthy blood donors was compared against 9 frequently recognized epitopes, with four of the lytic and five of the latent antigens were derived after pre-stimulation:

Table 3: Reaction of 16 blood donors to 9 different peptides; the highlighted values correspond to positive immune reactions.

This comparison involved two previously known EBNA-1-derived epitopes EBNA1514 (SEQ ID NO: 21) and EBNA141 1. The epitopes EBNA3A 381 (SEQ ID NO: 13), EBNA1 514 (SEQ ID NO: 21), BMRF1 261 (SEQ ID NO: 19), BLLF1 268 (SEQ ID NO: 1) and BLLF1 167 (SEQ ID NO: 17 ) were selected because, taken together, they induced IFN-γ production in all reacting donors (12/16):

Table 4: Peptide Mix 1

With peptide mix 1, IFN-γ producing cells could be successfully stimulated in 23 of 24 randomly selected neither HLA-typed nor EBV serum status specific healthy blood donors, as detected by ELISpot. Data from 16 donors are shown in Figure 2A after amplification in vitro. The stimulated cells expressed TNF, CD154, IFNy, IL2 and granzyme B, whereas in the PBMCs of seronegative donors L13 and CG this was not detected; see. Fig. 2B.

The cytokine expression pattern among the responding cells was significantly more multifunctional in the seropositive donors than in the seronegative donors; see. Fig. 2C. While in the seropositive individuals tested a substantial proportion of cells (0.3 to 0.7% of CD4 ⁺ ) expressed all five activation markers, such cells could not be detected in the seronegative individuals.

In a further experiment, the reactions of eight other blood donors were tested for nine different peptides according to the invention:

Table 5: Reactions of eight blood donors to nine different peptides; Emphasis: immune response / IFN-y response

A second mix of five peptides, namely EBNA3A 381 (SEQ ID NO: 13), BMRF1 261 (SEQ ID NO: 19), EBNA3A 190 (SEQ ID NO: 35), BRLF1 1 19 (SEQ ID NO: 25), EBNA3A 287 (SEQ ID NO: 29) (peptide mix 2) was recognized by a total of 29/32 individuals tested (90.6%); see. Fig. 3: 14 of 16 donors tested in this experiment).

A third mix of seven epitopes, namely EBNA3A 381 (SEQ ID NO: 13), BMRF1 261 (SEQ ID NO: 19), EBNA1 514 (SEQ ID NO: 21), BLLF1 167 (SEQ ID NO: 17), BRLF1 1 19 (SEQ ID NO: 25), EBNA2 276 (SEQ ID NO: 3) and BXLF2 126 (SEQ ID NO: 7) (peptide mix 3) was recognized by 16/16 individuals tested; see. Fig. 4.

In the following, the inventors tested 10 further mixtures (peptide mixtures 4 to 13) with MHC class I peptides:

Peptide mix 4: BMLF1 280 (SEQ ID NO: 40), EBNA3 471 (SEQ ID NO: 47), EBNA3 247 (SEQ ID NO: 55), EBNA4 657 (SEQ ID NO: 72): Most frequent HLAs, each best peptide. Detection: 10/14, 71, 4%

Peptide mix 5: BRLF1 109 (SEQ ID NO: 39), BMLF1 280 (SEQ ID NO: 40), EBNA3 471 (SEQ ID NO: 47), BRLF1 148 (SEQ ID NO: 78), EBNA3 247 (SEQ ID NO: 47). 55), EBNA4 657 (SEQ ID NO: 72), EBNA6 162 (SEQ ID NO: 73): Most frequent HLAs, two best peptides each. Detection 9/14, 64.3%

Peptide mix 6: BRLF1 109 (SEQ ID NO: 39), EBNA3 471 (SEQ ID NO: 47), LMP2 419 (SEQ ID NO: 53), EBNA3 247 (SEQ ID NO: 55), BZLF1 190 (SEQ ID NO: 55). 57), EBN2 430 (SEQ ID NO: 66), EBNA6 162 (SEQ ID NO: 73): 7 most abundant HLAs, each best peptide. Detection 10/14, 71, 4%

Peptide mix 7: EBNA3 471 (SEQ ID NO: 47), EBNA4 399 (SEQ ID NO: 51), LMP2 419 (SEQ ID NO: 53), BALF2 573 (SEQ ID NO: 54), EBNA3 247 (SEQ ID NO: 53). 55), BZLF1 190 (SEQ ID NO: 57), BZLF1 173 (SEQ ID NO: 64), EBNA6 258 (SEQ ID NO: 65), EB2 430 (SEQ ID NO: 66), LMP2 200 (SEQ ID NO: 65). 71), EBNA6 162 (SEQ ID NO. 73), LMP1 256 (SEQ ID NO: 77): all but A ^* 02, each best peptide. Detection 13/14, 92.8%

Peptide mix 8: BRLF1 109 (SEQ ID NO: 39), BMLF1 280 (SEQ ID NO: 40), EBNA3 471 (SEQ ID NO: 47), BRLF1 148 (SEQ ID NO: 78), EBNA4 399 (SEQ ID NO: 47). 51), EBNA4 416 (SEQ ID NO: 52), LMP2 419 (SEQ ID NO: 53), BALF2 573 (SEQ ID NO: 54). All HLA-A, up to two best peptides. Detection 1 1/14, 78.5%.

Peptide mix 9: EBNA3 247 (SEQ ID NO: 55), BZLF1 190 (SEQ ID NO: 57), EBNA3 193 (SEQ ID NO: 58), EBNA4 831 (SEQ ID NO: 63), BZLF1 173 (SEQ ID NO: 58). 64), EBNA6 258 (SEQ ID NO: 65), EB2 430 (SEQ ID NO: 66), BZLF1 54 (SEQ ID NO: 67), LMP2 200 (SEQ ID NO: 71), EBNA4 657 (SEQ ID NO: 67). 72), EBNA6 162 (SEQ ID NO: 73), LMP1 156 (SEQ ID NO: 77). All HLA-B, up to two best peptides. Detection 13/14, 92.8%.

Peptide mix 10: BRLF1 109 (SEQ ID NO: 39), EBNA3 471 (SEQ ID NO: 47), EBNA3 247 (SEQ ID NO: 55), BZLF1 190 (SEQ ID NO: 57), BZLF1 173 (SEQ ID NO: 55). 64), EBN2 430 (SEQ ID NO: 66), EBNA1 407 (SEQ ID NO: 70). Random Mix I. Detection 13/14, 92.8%.

Peptide mix 1 1: BRLF1 109 (SEQ ID NO: 39), EBNA3 471 (SEQ ID NO: 47), EBNA4 399 (SEQ ID NO: 51), LMP2 419 (SEQ ID NO: 53), BALF2 573 (SEQ ID NO: 51) 54), EBNA3 247 (SEQ ID NO: 55), BZLF1 190 (SEQ ID NO: 57), BZLF1 173 (SEQ ID NO: 64), EBNA6 258 (SEQ ID NO: 65), EB2 430 (SEQ ID NO: 54) 66), LMP2 200 (SEQ ID NO: 71), EBNA6 162 (SEQ ID NO: 73), LMP1 165 (SEQ ID NO: 77). All HLAs, each best peptide. Detection 12/14, 85.7%

Peptide mix 12: BRLF1 109 (SEQ ID NO: 39), BMLF1 280 (SEQ ID NO: 40), LMP2 356 (SEQ ID NO: 41), LMP2 426 (SEQ ID NO: 42), LMP1 125 (SEQ ID NO: 41). 45), EBNA6 284 (SEQ ID NO: 46), EBNA3 471 (SEQ ID NO: 47), BRLF1 148 (SEQ ID NO: 78), EBNA4 399 (SEQ ID NO: 51), EBNA4 416 (SEQ ID NO: 47). 52), BALF2 573 (SEQ ID NO: 54), EBNA3 247 (SEQ ID NO: 55), BZLF1 190 (SEQ ID NO: 57), EBNA3 193 (SEQ ID NO: 58), LMP2 163 (SEQ ID NO: 62), EBNA4 831 (SEQ ID NO: 63), BZLF1 173 (SEQ ID NO: 64), EBNA6 258 (SEQ ID NO: 65), EB2 430 (SEQ ID NO: 66), BZLF1 54 (SEQ ID NO: 67), BALF2 31 (SEQ ID NO: 68), EBNA1 407 (SEQ ID NO: 70), LMP2 200 (SEQ ID NO: 71), EBNA4 657 (SEQ ID NO: 72), EBNA6 162 (SEQ ID NO: 73), LMP1 156 (SEQ ID NO: 77). All peptides. Detection: 14/14, 100%

Peptide mix 13: BMLF1 280 (SEQ ID NO: 40), EBNA4 399 (SEQ ID NO: 51), BZLF1 (190 (SEQ ID NO: 57), EBNA4 831 (SEQ ID NO: 63), BZLF1 54 (SEQ ID NO 67), LMP2 200 (SEQ ID NO: 71). Random Mix II. Detection: 8/14, 57.1%

Table 6: Reactions of 14 donors (1530-1761) to various MHC class I mixes after in vitro amplification as measured by IFN-γ ELISPOT. Indicated is the stimulation index (x times the spot count over negative control). Positive reactions (at least 3-fold over negative control, at least 10 spots) are highlighted. PHA: positive control. 1729 A24 A26 B7 B38

1710 A26 A32 B51 B61

1671 A3 A23 B18 B44

1650 A1 1 - B14 B35

1613 A2 A28 B7 B57

1571 A29 A32 B27 B44

1530 A28 A31 B7 B39

1761 A2 B13 B44

1760 A28 A31 B35

1756 A23 A30 B7 B49

1755 A25 A31 B18 B60

1753 A1 A24 B7 B62

1749 A1 A24 B8 B62

1727 A2 A31 B52 B60

Table 7: HLA typing of the donors used

FIG. 5 shows representative ELISPOT results of donors 1755-1761 on the various mix combinations 4 to 13 after in vitro amplification. Each double row corresponds to a donor, each mix was measured as a duplicate. 1 10056: HIV-specific peptide, negative control. Row 12: one well positive control PHA and one well medium per donor.

3. Conclusion

The inventors provide peptides that can be used to induce stimulation of the EBV-infected individual's EBV-immune cellular immune response. The peptides can be used in a peptide mixture that induces a cellular immune response to EBV in virtually any individual without prior MHC or HLA typing. This is demonstrated by the inventors using various peptide mixtures.

Claims

claims

1 . A pharmaceutical composition for stimulating the cellular immune response against the Epstein-Barr virus (EBV) comprising at least three different peptides each having different amino acid sequences, wherein the amino acid sequences are each selected from the group consisting of: SEQ ID Nos. 1 to 78.

2. A pharmaceutical composition according to claim 1, comprising at least five different peptides.

3. A pharmaceutical composition according to claim 1 or 2, which has at least eight different peptides.

4. Pharmaceutical composition according to claim 3, which has at least three different MHC class II peptides each having different amino acid sequences and at least five different MHC class I peptides each having different amino acid sequences, wherein the amino acid sequences of the MHC class II Peptides are each selected from the group consisting of: SEQ ID Nos. 1 to 38, and wherein the amino acid sequences of the MHC class I peptides are each selected from the group consisting of: SEQ ID Nos. 39 to 78.

5. Pharmaceutical composition according to one of claims 1 to 4 for the treatment and / or prophylaxis of an EBV-associated disease.

The pharmaceutical composition of claim 5, wherein the EBV-associated disease is a post-transplant lymphoproliferative disorder (PTLD).

7. Use of at least three different peptides each having different amino acid sequences for stimulation of the cellular immune response against the Epstein-Barr virus (EBV), wherein the amino acid sequences are each selected from the group consisting of: SEQ ID Nos. 1 to 78.

8. Use according to claim 7, wherein at least five different peptides are used.

9. Use according to claim 7 or 8, wherein at least eight different peptides are used.

10. Use according to any one of claims 7 to 9, wherein at least three different MHC class II peptides each having different amino acid sequences and at least five different MHC class I peptides each having different amino acid sequences are used, wherein the amino acid sequences of the MHC Class II peptides are each selected from the group consisting of: SEQ ID NOs: 1 to 38, and wherein the amino acid sequences of the MHC class I peptides are each selected from the group consisting of: SEQ ID Nos. 39 to 78 ,

1 1. Use according to any one of claims 7 to 10 for the treatment and / or prophylaxis of an EBV-associated disease.

12. The use of claim 11, wherein the EBV-associated disease is a post-transplant lymphoproliferative disorder (PTLD).

13. A process for the preparation of a pharmaceutical composition comprising the steps of:

(1) providing at least three, preferably at least five, more preferably at least eight different peptides, (2) formulation of the peptides into a pharmaceutically acceptable carrier, wherein the at least three, preferably at least five, more preferably at least eight different peptides each have different amino acid sequences, each selected from the group consisting of: SEQ ID Nos. 1 to 78.

14. The method of claim 13, wherein in step (1) at least three different MHC class II peptides each having different amino acid sequences and at least five different MHC class I peptides each having different amino acid sequences are provided, wherein the amino acid sequences of MHC Class II peptides are each selected from the group consisting of: SEQ ID Nos. 1 to 38, and wherein the amino acid sequences of the MHC class I peptides are each selected from the group consisting of: SEQ ID Nos. 39 to 78th

15. A peptide for stimulating the cellular immune response against the Epstein-Barr virus (EBV), which has an amino acid sequence selected from the group consisting of: SEQ ID Nos. 1 to 78.

16. A peptide for stimulating the cellular immune response against the Epstein-Barr virus (EBV), which has an amino acid sequence selected from the group consisting of: SEQ ID Nos. 1, 2, 4, 6, 8, 10 to 14 , 16, 18 to 20, 22, 24 to 38, 48, 49, 54, 56, 61, 62, 66, 68, 69.