WO2017060283A1

WO2017060283A1 - Specific immunodominant peptide epitopes for polyomavirus vaccine

Info

Publication number: WO2017060283A1
Application number: PCT/EP2016/073760
Authority: WO
Inventors: Hans H. HIRSCH; Céline LEBOEUF
Original assignee: Universität Basel
Priority date: 2015-10-06
Filing date: 2016-10-05
Publication date: 2017-04-13

Abstract

The invention relates to immunodominant peptide epitopes of BK, JC and MC polyomavirus; carriers, such as virus-like particles, virosomes or nanoparticles, comprising such peptide epitopes; vaccines against polyomavirus infection comprising such peptide epitopes and/or loaded carriers; and nucleic acids encoding these peptide epitopes. The invention further relates to diagnostic and therapeutic methods using these peptide epitopes in polyomavirus-associated diseases such as nephropathy or hemorrhagic cystitis of importance for transplant recipients.

Description

Specific immunodominant peptide epitopes for polyomavirus vaccine Field of the Invention The invention relates to recombinant immunodominant peptide epitopes of polyomavirus, and vaccines against polyomavirus infection comprising such epitopes.

Background Art BK polyomavirus (BKPyV) is a small non-enveloped double-stranded DNA virus and one of by now at least 12 human polyomaviruses (Rinaldo C.H., Hirsch H. H., APMIS

2013;121 :681-4). Specific antibody surveys indicate that BKPyV infects 80-95% of the human population, mostly during childhood without specific symptoms or signs. BKPyV then persists in the renourinary tract as evidenced by asymptomatic shedding into the urine. Disease manifestations arise almost exclusively in individuals with altered immune functions and appear to involve co-factors linked to specific clinical settings. Thus, polyomavirus-associated nephropathy (PyVAN) occurs in 1 -15% of kidney transplant recipients, while polyomavirus-associated hemorrhagic cystitis (PyVHC) affects 5-15% of allogeneic haematopoietic stem cell transplant patients. PyVAN and PyVHC have significant impact on morbidity, graft, and patient survival. Despite considerable virological research, randomized clinical studies are either lacking or failed to demonstrate effective antiviral approaches. In kidney transplant recipients, high-level BKPyV viruria and viremia have been identified as markers of progression to PyVAN. Hence, current management strategies recommend screening kidney transplant recipients for viremia followed by reducing immunosuppression. As BKPyV-specific T-cell responses are low and approximately 50- to 100-fold lower than those to cytomegalovirus, these assays have not readily entered clinical practice. Moreover, the risk factors of BKPyV replication and nephropathy vary in different kidney transplant studies and include steroid pulses for acute rejection, maintenance immunosuppression such as tacrolimus-mycophenolate versus cyclosporine-mycophenolate, older age of recipients, male gender, and higher number of HLA-mismatches. According to the recent OPTN/SRTR report, these risk factors are present in a substantial number of kidney transplant patients (Matas A.J. et al., Am J Transplant. 2015;15 Suppl 2:1 -34). Cellular immune responses to overlapping peptide pools encoded in the early viral gene region (EVGR), containing the partially overlapping large T and small T proteins, or in the late viral gene region (LVGR) of BKPyV DNA genome have been investigated (Binggeli S. et al., Am J Transplant. 2007;7:1 131 -9; Trydzenskaya H. et al., Transplantation

201 1 ;92:1269-77; Weist B.J. et al., Med Microbiol Immunol. 2014;203:395-408). T-cell responses to the LVGR-encoded capsid viral protein VP1 were generally more

pronounced than those to EVGR-encoded viral proteins. Moreover, interferon (IFN)-y responses were largely derived from CD4+T-cells and to a lesser extent from CD8+T-cells (Binggeli S. et al., Am J Transplant. 2007;7:1 131 -9). Since most of these studies used overlapping 15mer-peptide pools, the contribution of individual CD8+T cell-restricted epitopes to these responses are largely undefined. With few exceptions, HLA-restricted T- cell responses to BKPyV are mostly reported from HLA-A^*02 individuals (Provenzano M. et al., J Transl Med. 2006;4:47; Randhawa P.S. et al., Hum Immunol. 2006;67:298-302). To better characterize BKPyV-specific CD8+T-cell epitopes, a bioinformatics approach was chosen to predict 9mer-epitopes encoded in BKPyV EVGR and presented by 14 common HLA types in Europe and North America for experimental testing in adult healthy individuals and paediatric kidney transplant recipients.

A patent application describing HLA-A^*02-restricted immunogenic epitopes within BKPyV VP1 has been published in 2007 (US 2007/0026503). However, the present inventors have found that LTag is responsible for higher CD8-mediated responses than VP1 , and is more suitable for preventing and treating viremia and BKPyV nephropathy.

A patent application published in 2008 (WO 2008/1 16468, Dako Denmark AS, Sch0ller J et al.) lists thousands of MHC monomers and multimers and also thousands of potential protein binders, describing numerous potential uses in immune monitoring, diagnostics, prognostics, therapy and vaccines of bacterial, viral and fungal diseases, parasitic diseases, allergic diseases, transplantation related diseases, cancer, and autoimmune and inflammatory diseases, but without indicating any specific use thereof. In contrast, the present inventors, based on in vitro T-cell functional assays, demonstrate immune responses in healthy individuals and pediatric and adult kidney transplant recipients, caused by BKPyV early viral gene region epitopes predicted to be presented by different HLA molecules and described in the present patent application. Summary of the Invention

The invention relates to recombinant immunodominant peptide epitopes of a

polyomavirus, such as BK, JC or MC polyomavirus, carriers, such as virus-like particles, virosomes or nanoparticles, comprising such epitopes, vaccines against polyomavirus infection comprising such peptides and/or carriers, and methods of prophylaxis and treatment using such vaccines.

The invention further relates to nucleic acids encoding these proteins and epitopes, vectors comprising such DNA, and host cells comprising such vectors.

The invention further relates to a diagnostic method using these peptide epitopes or nucleic acids encoding these. Brief Description of the Figures

Figure 1 : In silico prediction of BKPyV EVGR immunogenic epitopes

The graph depicts 20 top-scoring 9mer-epitopes predicted in BKPyV EVGR (early viral gene region) sequence for common HLA-A and -B types in Europe and North America, according to Immune Epitope Database (X) and Syfpeithi ) algorithms.

Figure 2: BKPyV IgG serology of 42 healthy individuals

Normalized BKPyV IgG antibody levels are shown at 1 :100, 1 :200 and 1 :400 dilutions (median, box shows 25^th and 75^th percentile, whiskers 5^th and 95^th percentile). Positive serological status was defined as OD₄92n_m≥ 0.100 (dotted line) at the 1 :200 dilution.

Figure 3: Characterization of BKPyV EVGR 9mer-specific immune responses

(A) IFN-γ ELISpot assay using PBMCs directly after isolation from fresh blood (d-1 , left panel) or after 9 day-expansion with BKPyV EVGR peptides (d+9, right panel). Cells were treated with medium (NC, negative control), Staphylococcus enterotoxin B (SEB), the pooled 9mer-peptides (9mP), an overlapping 15mer-peptide pool spanning the BKPyV EGFR sequence (15mP) or with a longer peptide pool (LPP).

(B) Rechallenge of 9-day expanded T-cells with 9mer sub-pools 9msA to 9msH, and 9ms1 to 9ms9. For an explanation of the sub-pools see Table 8. Each peptide was present in two sub-pools for cross-identification. (C) 9mer-epitope identification by restimulating expanded cells with single 9mer-peptides contained in the sub-pools eliciting the highest responses (e.g. 9msA, 9ms4, 9ms5).

(D) HLA streptamer staining using PE-labelled StrepTactin without (left panel,

NC=negative control) and with (right panel) HLA-B^*0702 molecules bearing 9m27 peptide. (E) CD8+T-cell proliferation during in vitro expansion. PBMCs were stained at day 0 with CFSE dye that dilutes upon cell division (y-axis). Proliferation of CD8+T-cells (left panel) and HLA-B^*07-positive 9m27-specific T-cells (right panel) are shown.

(F) Epitope-specific degranulation of CD8+T-cells using PE-Cy7-labelled CD107a antibody upon 5h-restimulation with 9m27 (right panel) or with another BKPyV EVRG peptide as negative control (left panel). HLA-B^*07 9m27-specific T cells are shown.

(G) 9mer-specific cytotoxic activity of expanded T-cells. Autologous PHA blasts stained with ⁵¹Cr and pulsed with 9mP (■) or 9m27 (A ) were used as target cells and incubated for 4h with expanded T-cells (effector cells). Percentage of target cells lysis (y-axis) at the different effectontarget cells ratios (E:T, x-axis) is shown (See materials and methods for details).

Figure 4: Breadth and strength of BKPyV EVGR epitope-specific immune responses in healthy individuals

The 9mer-epitope responses in IFN-γ ELISpot assay (dots) found in at least 40% of healthy individuals listed in Table 7, with positive streptamer staining (solid line: median).

Figure 5: BKPyV viremia and HLA-matching in 118 pediatric kidney transplant recipients.

The percentage of viremic (left side) and non-viremic (right side) kidney transplant recipients according to HLA type is shown. For each HLA type, the percentage of patients displaying matched (blank) or mismatched (dotted) allele with their kidney donor is shown. The number of kidney transplant recipients with most common HLA-types is indicated on the left. BKPyV viremia was analyzed for single HLA-types versus the whole population (p-value "P1 ") or by comparing viremia occurrence in matched versus mismatched patients (p-value "P2") (Fisher's exact test).

Figure 6: BKPyV and JCPyV seroprevalence in viremic and non-viremic kidney transplant recipients.

Plasma samples from 98 patients harvested at the time of transplantation (TO), 6 months post-transplantation (T6) and 12 months post-transplantation (T12) were tested at 1 :200 dilution in a BKPyV (upper panel) or JCPyV (lower panel) IgG ELISA using virus-like particles. The dotted and empty boxes display viremic patients and non-viremic patients, respectively. The optical density was measured at 492nm and was normalized to an internal laboratory reference serum (nOD). The dotted line shows the cut-off for seropositivity.

Statistical analysis was performed using Kruskal-Wallis nonparametric test.

*^** p < 0.0001

Figure 7: Direct BKPyV IFN^y T-cell responses.

PBMCs from viremic (left, A) or non-viremic (right, B) kidney transplant patients were stimulated with BKPyV LTag 15mP or 9mP in a IFN-γ ELISpot assay one day after thawing. Data are expressed as Spot Forming Unit (SFU) per 10⁶ cells. Each dot represents one sample and the black bar shows the mean SFU/10⁶ cells.

Statistical analysis was performed using Student's t-test.

* p < 0.05

*^* p < 0.001

Figure 8: In vitro expanded BKPyV IFN^y T-cell responses.

PBMCs from viremic (left, A) or non-viremic (right, B) kidney transplant patients were expanded in vitro with BKPyV LTag 15mP in the presence of cytokines. After two weeks, the cells were stimulated with BKPyV LTag 15mP or 9mP in a IFN-γ ELISpot assay. Data are expressed as Spot Forming Unit (SFU) per 10⁶ cells. Each dot represents one sample and the black bar shows the mean SFU/10⁶ cells.

Statistical analysis was performed using Student's t-test.

*^** p < 0.0001

Figure 9: Diversity of BKPyV LTag-specific T-cell responses in kidney transplant recipients.

After two weeks in vitro expansion, PBMCs from viremic (panel A) and non-viremic (panel B) patients were stimulated with single BKPyV LTag-derived 9mers in a IFN-γ ELISpot assay. The number of patients (x-axis) displaying a positive response upon stimulation with the indicated peptides (y-axis) is shown. Epitopes that were not found frequently in healthy individuals are indicated by a star. Detailed Description of the Invention

The invention relates to recombinant immunodominant peptide epitopes of polyomavirus and peptides comprising these.

More specifically, the invention relates to a recombinant peptide consisting of 9 to 50 amino acids comprising

one epitope selected from the group of peptides of SEQ ID NO: 1 -3, 5-7, 9-1 1 , 13, 15-18, 20-33, 35, 37, 38, 40-43, 45-52, 54-56, 59, 61 -73, 75-97, and 121 -164, or

two, three, four, five or six optionally overlapping epitopes selected from the group of peptides of SEQ ID NO:1 to 97 and 121 to 164, or

one, two or three peptides selected from the group of peptides of SEQ ID NO:98 to 1 17 and optionally one, two or three further optionally overlapping epitopes selected from the group of peptides of SEQ ID NO:1 to 97 and 121 to 164.

In a particular embodiment, the invention relates to a recombinant peptide consisting of 15 to 50 amino acids comprising

More particularly, the invention relates to such a recombinant peptide consisting of at least 24, at least 25, at least 26, at least 27, at least 28, at least 36, or at least 37 amino acids.

In particular, the invention relates to a recombinant peptide consisting of 15 to 50 amino acids comprising two, three, four, five or six optionally overlapping epitopes selected from the group of peptides of SEQ ID NO: 1 -3, 5-7, 9-1 1 , 13, 15-18, 20-33, 35, 37, 38, 40-43, 45-52, 54-56, 59, 61 -73, 75-97, and 121 -164.

Peptides of SEQ ID NO: 1 -3, 5-7, 9-1 1 , 13, 15-18, 20-33, 35, 37, 38, 40-43, 45-52, 54-56, 59, 61 -73, and 75-97 are novel immunodominant peptide epitopes of BK polyomavirus determined by the present inventors for the first time. Table 1 : Immunogenic BKPyV LTag epitopes

Name BKPyV amino acid sequence SEQ ID NO Reference

9m19 LERAAWGNL 1

9m21 RAAWGNLPL 2

9m26 NLPLMRKAY 3

9m27 LPLMRKAYL 4 (1 )

9m28 PLMRKAYLR 5

9m29 LMRKAYLRK 6

9m32 KAYLRKCKE 7

9m33 AYLRKCKEF 8 (2)

9m145 HQFLSQAVF 9

9m148 LSQAVFSNR 10

9m150 QAVFSNRTL 1 1

9m156 RTLACFAVY 12 (2)

9m158 LACFAVYTT 13

9m159 ACFAVYTTK 14 (2)

9m165 TTKEKAQIL 15

9m166 TKEKAQILY 16

9m167 KEKAQILYK 17

9m169 KAQILYKKL 18

9m172 ILYKKLMEK 19 (2)

9m173 LYKKLMEKY 20

9m176 KLMEKYSVT 21

9m177 LMEKYSVTF 22

9m178 MEKYSVTFI 23

9m188 RHMCAGHNI 24

9m192 AGHNIIFFL 25

9m190 MCAGHNIIF 26

9m191 CAGHNIIFF 27

9m196 IIFFLTPHR 28

9m199 FLTPHRHRV 29

9m201 TPHRHRVSA 30

9m202 PHRHRVSAI 31

9m212 NFCQKLCTF 32

9m214 CQKLCTFSF 33

9m216 KLCTFSFLI 34 (3)

9m218 CTFSFLICK 35

9m222 FLICKGVNK 36 (2)

9m224 ICKGVNKEY 37

9m226 KGVNKEYLL 38

9m227 GVNKEYLLY 39 (2)

9m230 KEYLLYSAL 40

9m232 YLLYSALTR 41

9m235 YSALTRDPY 42

9m238 LTRDPYHTI 43

9m242 PYHTIEESI 44 (2)

9m282 TKCEDVFLL 45

9m283 KCEDVFLLL 46

9m286 DVFLLLGMY 47

9m289 LLLGMYLEF 48 9m291 LGMYLEFQY 49

9m293 MYLEFQYNV 50

9m355 LHMTREEML 51

9m359 REEMLTERF 52

9m362 MLTERFNHI 53 (4)

9m369 HILDKMDLI 54

9m402 KMDSVIFDF 55

9m403 MDSVIFDFL 56

9m406 VIFDFLHCI 57 (3)

9m410 FLHCIVFNV 58 (3)

9m408 FDFLHCIVF 59

9m414 IVFNVPKRR 60 (2)

9m415 VFNVPKRRY 61

9m417 NVPKRRYWL 62

9m418 VPKRRYWLF 63

9m569 RILQSGMTL 64

9m570 ILQSGMTLL 65

9m571 LQSGMTLLL 66

9m573 SGMTLLLLL 67

9m574 GMTLLLLLI 68

9m575 MTLLLLLIW 69

9m576 TLLLLLIWF 70

9m577 LLLLLIWFR 71

9m579 LLLIWFRPV 72 (3)

9m582 IWFRPVADF 73

9m436 TLAAGLLDL 74

9m440 GLLDLCGGK 75

9m442 LDLCGGKAL 76

9m446 GGKALNVNL 77

9m453 NLPMERLTF 78

9m529 TMNEYPVPK 79

9m531 NEYPVPKTL 80

9m533 YPVPKTLQA 81

9m535 VPKTLQARF 82

9m537 KTLQARFVR 83

9m539 LQARFVRQI 84

9m541 ARFVRQIDF 85

9m545 RQIDFRPKI 86

9m121 DSQHSTPPK 87

9m124 HSTPPKKKR 88

9m125 STPPKKKRK 89

9m126 TPPKKKRKV 90

9m127 PPKKKRKVE 91

9m128 PKKKRKVED 92

9m129 KKKRKVEDP 93

9m132 RKVEDPKDF 94

9m136 DPKDFPSDL 95

9m139 DFPSDLHQF 96

9m140 FPSDLHQFL 97

LP-LER LERAAWGNLPLMRKAYLRKCKEFHP 98

LP-DSQ DSQHSTPPKKKRKVEDPKDFPSDLHQFL 99 LP-HQF HQFLSQAVFSNRTLACFAVYTTKEK 100

LP-AVY AVYTTKEKAQILYKKLMEKYSVTFI 101

LP-RHM RHMCAGHNIIFFLTPHRHRVSAINN 102

LP-NFC NFCQKLCTFSFLICK 103

LP-FLI FLICKGVNKEYLLYS 104

LP-GVN GVNKEYLLYSALTRDPYHTIEESI 105

LP-VET VETKCEDVFLLLGMYLEFQYNVEEC 106

LP-LHM LHMTREEMLTERFNHILDKMDLIF 107

LP-KMD KMDSVIFDFLHCIVFNVPKRRYWLF 108

LP-TLA TLAAGLLDLCGGKALNVNLPMERLT 109

LP-TMN TMNEYPVPKTLQARFVRQIDFRPKI 1 10

LP-LLE LLEKRILQSGMTLLLLLIWFRPVADF 1 1 1

LTag, Large Tumor antigen; 9m, peptide consisting of 9 amino acids; LP, longer peptide. Epitopes for which a reference (1 ) to (4) is given, are already known in the state of the art. (1 ) Li J et al., J Gen Virol. 2006;87:2951 -60.

(2) Ramaswami B et al., Hum Immunol. 2009;70:722-8.

(3) Provenzano M et al., J Transl Med. 2006;4:47.

(4) Randhawa P.S. et al., Hum Immunol. 2006;67:298-302.

In a further particular embodiment, the invention relates to a recombinant peptide comprising two, three, four, five or six optionally overlapping peptides selected from the group of peptides of SEQ ID NO: 1 , 2, 4, 5, 8, 9, 12, 15, 21 , 29, 30, 34, 39, 40, 44, 48, 49, 53, 58, 66, 72, 74, 80, 81 and 97.

In yet another particular embodiment, the invention relates to a recombinant peptide comprising two, three, four, five or six optionally overlapping peptides selected from the group of peptides of SEQ ID NO: 1 , 2, 5, 7, 9, 15, 21 , 29, 30-32, 40, 46-51 , 62, 65, 66, 74, 80, 81 and 97.

For the purpose of vaccine design, recombinant peptides were constructed comprising epitopes targeting a wide range of different HLA types.

In a particular embodiment, the invention thus relates to recombinant peptides RP-14, RP- 21 , RP-34, RP-51 , RP-54 and RP-55 (Table 2). Table 2: Preferred recombinant peptides and targeted HLA sites

Recombinant peptides RP are not present in this particular epitope combination within corresponding polyomaviruses, and are fully synthetic. RP-14, -21 , -51 , -54 and -55 are especially useful for HLA-transgenic mice experiments, since some of the targeted HLA types match with the phenotype of the available HLA-transgenic mice (Taconic). Thus, those RPs will allow to investigate BKPyV-specific immune responses in 6 different HLA- transgenic mice (A^*01 , A^*02, A^*1 1 , A^*24, B^*07 and B^*44).

Appropriate proteasomal cleavages of RP-14, -21 , -34, -51 , -54 and -55 were predicted using PAPROC prediction server (http://www.paproc.de). Most predictions were confirmed with another proteasome prediction server, namely NetChop3.1

(http://www.cbs.dtu.dk/services/NetChop/).

Table 3: Predicted proteasomal cleavage sites

Predicted cleavage

Name BKPyV amino acid sequence a. a. Cleavage positions

7 A +++

8 Y +++

9 L ++

10 K ++

1 1 L +

16 Y +++

LPLMRKAYLKLLLGMYLEFG 19 F +++

RP-14 37

VNKEYLLYLLLIWFRPV 20 G +

21 V +++

23 K ++

25 Y +++

27 L +++

28 Y +++

32 I ++ 6 Y

8 L

9 Y

10 L

GVNKEYLLYLERAAWGNLK 17 N

RP-21 36

EYLLYSALRTLACFAVY 18 L

21 Y

23 L

27 L

30 L

7 F

8 Q

9 Y

1 1 P

LGMYLEFQYFPSDLHQFLR

RP-34 27 15 H

AAWGNLPL

16 Q

17 F

18 L

20 A

6 Y

9 F

10 G

1 1 V

LLLGMYLEFGVNKEYLLYLL

RP-51 27 13 K

LIWFRPV

15 Y

17 L

18 Y

22 I

8 N

9 L

13 R

LERAAWGNLPLMRKAYLRA

RP-54 26 15 A

AWGNLPL

16 Y

17 L

19 A

7 F

8 Q

9 Y

1 1 P

15 H

LGMYLEFQYFPSDLHQFLK

RP-55 27 16 Q

EYLLYSAL

17 F

18 L

20 E

21 Y

23 L

high cleavage rate; ++ intermediate c eavage rate; + low cleavage rate

Spacer and processing amino acids to improve cleavage, presentation and vaccine response are further considered within the peptides of the invention. For example, any 9mer-peptide listed above can be combined with L, A and D amino acids at their C- terminus, except for 9mer-peptides having L, F, W or Y at their C-terminus that will be combined with A and D only. Also, different combinations of 9mer-epitope sequences with or without spacers can be further considered for the synthesis of other immunogenic recombinant peptides.

The most preferred epitope is 9m27 (SEQ ID NO:4) because it is highly immunodominant in HLA-B^*07 and -B^*08 individuals. Therefore the preferred long peptides contain 9m27. A particularly preferred long peptide is the peptide of SEQ ID NO: 98 comprising 9m27 (SEQ ID NO:4), another published epitope (9m33, SEQ ID NO: 8) and 4 other newly identifed epitopes giving responses in a broad range of HLA types:

LP-LER: LERAAWGNLPLMRKAYLRKCKEFHP (SEQ ID NO:98)

A likewise preferred long peptide is the peptide of SEQ ID NO: 106 comprising an immunogenic area going from position 283 to 293 within EGVR, giving responses in a broad range of HLA types:

LP-VET VETKCEDVFLLLGMYLEFQYNVEEC (SEQ ID NO:106)

The most preferred recombinant peptide of SEQ ID NO 1 12 contains the best epitopes of each HLA type:

RP-14 LPLMRKAYLKLLLGMYLEFGVNKEYLLYLLLIWFRPV (SEQ ID NO:1 12)

This 37 amino acid peptide targets the following HLA: A^*01 , A^*02, A^*03, A^*1 1 , A^*24, B^*07, B^*08

Some epitopes induced IFN-γ ELISpot responses in more than 50% of tested healthy individuals. Proteins comprising such epitopes of SEQ ID NO: 1 , 2, 12, 39, 40, 48, 49, 72 and 97 are particularly preferred.

Proteins comprising the epitope of SEQ ID NO: 4 are particularly preferred. Also preferred are proteins comprising the epitope of SEQ ID NO: 39, or of SEQ ID NO: 72, or of SEQ ID NO: 34, or of SEQ ID NO: 48, or of SEQ ID NO: 2, or of SEQ ID NO: 12, or of SEQ ID NO: 97, or of SEQ ID NO: 19, or of SEQ ID NO: 49, or of SEQ ID NO: 44, or of SEQ ID NO: 47, or of SEQ ID NO: 59, or of SEQ ID NO: 1 , or of SEQ ID NO: 25, or of SEQ ID NO: 21 , or of SEQ ID NO: 15, or of SEQ ID NO: 32, or of SEQ ID NO: 40. BKPyV LTag (SEQ ID NO: 1 18) is a non-structural protein playing a key role in viral replication. BKPyV VP1 is a structural protein expressed at later time points and involved in capsid formation. It has been strongly suggested that LTag is involved in tumor formation as it contains some tumor suppressor-binding domains allowing interfering with p53 and retinoblastoma protein functions.

Since LTag is produced early in the viral life cycle, immune responses against LTag are thought to be responsible for the maintenance of viral latency. In kidney transplant recipients, BKPyV VP1 elicits higher immune responses than BKPyV LTag in vitro (Binggeli S et al., Am J Transplant. 2007;7:1 131 -9). However, LTag is responsible for higher CD8-mediated responses compared to VP1.

LTag is highly conserved among human polyoma viruses, e.g. BKPyV, JCPyV and MCPyV. BKPyV LTag and JCPyV LTag (SEQ ID NO: 1 19) share more than 80% sequence homology. Therefore it is reasonable to assume that a vaccine directed to BKPyV will be effective against other human polyoma viruses.

BKPyV and JCPyV LTag sequences have been aligned, and it was found that epitopes 9m165 (SEQ ID NO:15), 9m176 (SEQ ID NO:21 ), 9m199 (SEQ ID NO:29), 9m201 (SEQ ID NO:30), 9m216 (SEQ ID NO:34), 9m571 (SEQ ID NO:66), 9m579 (SEQ ID NO:72) are identical. Epitopes 9m145 (SEQ ID NO:9), 9m227 (SEQ ID NO:39), 9m230 (SEQ ID NO:40), and 9m436 (SEQ ID NO:74) differ only in one amino acid. Long peptides LP-AVY (SEQ ID NO:101 ) and LP-LLE (SEQ ID NO:1 1 1 ) are likewise identical, and LP-NFC (SEQ ID NO:103) and LP-FLI (SEQ ID NO:104) differ only by one amino acid. RP-14 (SEQ ID NO:1 12) is expected to give a cross-reactive response to JCPyV because the epitopes contained in this RP have (not identical but) highly conserved sequences.

The invention further relates to those closely related immunogenic epitope peptide sequences of JC polyomavirus of SEQ ID NO: 121 to 139, and peptides of 10 to 50 amino acids comprising these. Table 4: Immunogenic JCPyV LTag epitopes

BKPyV and MCPyV LTag (SEQ ID NO:120) sequences have been aligned, and it was found that epitope 9m222 (SEQ ID NO:36) is identical. Epitopes 9m570 (SEQ ID NO:65) and 9m571 (SEQ ID NO:66) differ only by one amino acid.

The invention further relates to immunogenic epitope peptide sequences of MC polyomavirus of SEQ ID NO: 140 to 164, and peptides of 10 to 50 amino acids comprising these. Table 5: Immunogenic MCPyV LTag epitopes

The invention further relates to a carrier comprising a recombinant peptide as described in the preceding paragraphs. Carriers considered are recombinant virus-like particles, virosomes, virosomes that are surrounded by a lipid envelope, liposomes consisting of one or several phospholipid bilayers that can encapsulate antigens, immunostimulating complexes (ISCOMs) made of cholesterol, phospholipids and saponin that are able to form a complex of micelles wherein antigens can be contained, polymeric nanoparticles that are made of polymers like dextran or chitosan, or non-degradable nanoparticles made of gold or carbon that can accommodate antigens

The carrier according to the invention may further comprise other B- and/or T-cell epitopes, proteins selected from the group consisting of additional foreign antigenic sequences, cytokines, CpG motifs, g-CMSF, CD19, and CD40 ligand, and/or fluorescent proteins, proteins useful for purification purposes of the particles or for attaching a label, and/or proteinaceous structures required for transport processes. Furthermore the invention relates to a nucleic acid encoding the proteins as defined above, to a vector comprising such nucleic acid, for example a baculovirus vector, and a host cell comprising such a vector, and to methods of manufacturing virus-like particles according to the invention using a baculovirus vector.

In particular a DNA of the invention encodes the amino acid sequences of SEQ ID NO:. Preferred DNA is listed in the following table: Table 6: Preferred DNA of epitopes, long peptides and preferred recombinant peptides

SE

BKPyV amino acid

Name Nucleotide sequence Q ID sequence

NO

9m19 LERAAWGNL ctggaacgcgcggcgtggggcaacctg 165

9m21 RAAWGNLPL cgcgcggcgtggggcaacctgccgctg 166

9m26 NLPLMRKAY aacctgccgctgatgcgcaaagcgtat 167

9m27 LPLMRKAYL ctgccgctgatgcgcaaagcgtatctg 168

9m28 PLMRKAYLR ccgctgatgcgcaaagcgtatctgcgc 169

9m29 LMRKAYLRK ctgatgcgcaaagcgtatctgcgcaaa 170

9m32 KAYLRKCKE aaagcgtatctgcgcaaatgcaaagaa 171

9m33 AYLRKCKEF gcgtatctgcgcaaatgcaaagaattt 172

9m145 HQFLSQAVF catcagtttctgagccaggcggtgttt 173

9m148 LSQAVFSNR ctgagccaggcggtgtttagcaaccgc 174

9m150 QAVFSNRTL caggcggtgtttagcaaccgcaccctg 175

9m156 RTLACFAVY cgcaccctggcgtgctttgcggtgtat 176

9m158 LACFAVYTT ctggcgtgctttgcggtgtataccacc 177

9m159 ACFAVYTTK gcgtgctttgcggtgtataccaccaaa 178

9m165 TTKEKAQIL accaccaaagaaaaagcgcagattctg 179

9m166 TKEKAQILY accaaagaaaaagcgcagattctgtat 180

9m167 KEKAQILYK aaagaaaaagcgcagattctgtataaa 181

9m169 KAQILYKKL aaagcgcagattctgtataaaaaactg 182

9m172 ILYKKLMEK attctgtataaaaaactgatggaaaaa 183

9m173 LYKKLMEKY ctgtataaaaaactgatggaaaaatat 184

9m176 KLMEKYSVT aaactgatggaaaaatatagcgtgacc 185

9m177 LMEKYSVTF ctgatggaaaaatatagcgtgaccttt 186

9m178 MEKYSVTFI atggaaaaatatagcgtgacctttatt 187

9m188 RHMCAGHNI cgccatatgtgcgcgggccataacatt 188

9m192 AGHNIIFFL gcgggccataacattattttttttctg 189

9m190 MCAGHNIIF atgtgcgcgggccataacattattttt 190

9m191 CAGHNIIFF tgcgcgggccataacattatttttttt 191

9m196 IIFFLTPHR attattttttttctgaccccgcatcgc 192

9m199 FLTPHRHRV tttctgaccccgcatcgccatcgcgtg 193

9m201 TPHRHRVSA accccgcatcgccatcgcgtgagcgcg 194

9m202 PHRHRVSAI ccgcatcgccatcgcgtgagcgcgatt 195 m212 NFCQKLCTF aacttttgccagaaactgtgcaccttt 196m214 CQKLCTFSF tgccagaaactgtgcacctttagcttt 197m216 KLCTFSFLI a aa ctg tg caccttta g ctttctg att 198m218 CTFSFLICK tgcacctttagctttctgatttgcaaa 199m222 FLICKGVNK tttctgatttgcaaaggcgtgaacaaa 200m224 ICKGVNKEY atttgcaaaggcgtgaacaaagaatat 201m226 KGVNKEYLL aaaggcgtgaacaaagaatatctgctg 202m227 GVNKEYLLY ggcgtgaacaaagaatatctgctgtat 203m230 KEYLLYSAL aaagaatatctgctgtatagcgcgctg 204m232 YLLYSALTR tatctgctgtatagcgcgctgacccgc 205m235 YSALTRDPY tatagcgcgctgacccgcgatccgtat 206m238 LTRDPYHTI ctgacccgcgatccgtatcataccatt 207m242 PYHTIEESI ccgtatcataccattgaagaaagcatt 208m282 TKCEDVFLL accaaatgcgaagatgtgtttctgctg 209m283 KCEDVFLLL aaatgcgaagatgtgtttctgctgctg 210m286 DVFLLLGMY gatgtgtttctgctgctgggcatgtat 21 1m289 LLLGMYLEF ctgctgctgggcatgtatctggaattt 212m291 LGMYLEFQY ctgggcatgtatctggaatttcagtat 213m293 MYLEFQYNV atgtatctggaatttcagtataacgtg 214m355 LHMTREEML ctgcatatgacccgcgaagaaatgctg 215m359 REEMLTERF cgcgaagaaatgctgaccgaacgcttt 216m362 MLTERFNHI atgctgaccgaacgctttaaccatatt 217m369 HILDKMDLI catattctggataaaatggatctgatt 218m402 KMDSVIFDF aaaatggatagcgtgatttttgatttt 219m403 MDSVIFDFL atggatagcgtgatttttgattttctg 220m406 VIFDFLHCI gtgatttttgattttctgcattgcatt 221m410 FLHCIVFNV tttctgcattgcattgtgtttaacgtg 222m408 FDFLHCIVF tttgattttctgcattgcattgtgttt 223m414 IVFNVPKRR attgtgtttaacgtgccgaaacgccgc 224m415 VFNVPKRRY gtgtttaacgtgccgaaacgccgctat 225m417 NVPKRRYWL aacgtgccgaaacgccgctattggctg 226m418 VPKRRYWLF gtgccgaaacgccgctattggctgttt 227m569 RILQSGMTL cgcattctgcagagcggcatgaccctg 228m570 ILQSGMTLL attctgcagagcggcatgaccctgctg 229m571 LQSGMTLLL ctgcagagcggcatgaccctgctgctg 230m573 SGMTLLLLL agcggcatgaccctgctgctgctgctg 231m574 GMTLLLLLI ggcatgaccctgctgctgctgctgatt 232m575 MTLLLLLIW atgaccctgctgctgctgctgatttgg 233m576 TLLLLLIWF accctgctgctgctgctgatttggttt 234m577 LLLLLIWFR ctgctgctgctgctgatttggtttcgc 235m579 LLLIWFRPV ctgctgctgatttggtttcgcccggtg 236m582 IWFRPVADF atttggtttcgcccggtggcggatttt 237m436 TLAAGLLDL accctggcggcgggcctgctggatctg 238m440 GLLDLCGGK ggcctgctggatctgtgcggcggcaaa 239m442 LDLCGGKAL ctggatctgtgcggcggcaaagcgctg 240m446 GGKALNVNL ggcggcaaagcgctgaacgtgaacctg 241m453 NLPMERLTF aacctgccgatggaacgcctgaccttt 242m529 TMNEYPVPK accatgaacgaatatccggtgccgaaa 243m531 NEYPVPKTL aacgaatatccggtgccgaaaaccctg 244m533 YPVPKTLQA tatccggtgccgaaaaccctgcaggcg 245m535 VPKTLQARF gtgccgaaaaccctgcaggcgcgcttt 246 9m537 KTLQARFVR aaaaccctgcaggcgcgctttgtgcgc 247

9m539 LQARFVRQI ctgcaggcgcgctttgtgcgccagatt 248

9m541 ARFVRQIDF gcgcgctttgtgcgccagattgatttt 249

9m545 RQIDFRPKI cgccagattgattttcgcccgaaaatt 250

9m121 DSQHSTPPK gatagccagcatagcaccccgccgaaa 251

9m124 HSTPPKKKR catagcaccccgccgaaaaaaaaacgc 252

9m125 STPPKKKRK agcaccccgccgaaaaaaaaacgcaaa 253

9m126 TPPKKKRKV accccgccgaaaaaaaaacgcaaagtg 254

9m127 PPKKKRKVE ccgccgaaaaaaaaacgcaaagtggaa 255

9m128 PKKKRKVED ccgaaaaaaaaacgcaaagtggaagat 256

9m129 KKKRKVEDP aaaaaaaaacgcaaagtggaagatccg 257

9m132 RKVEDPKDF cgcaaagtggaagatccgaaagatttt 258

9m136 DPKDFPSDL gatccgaaagattttccgagcgatctg 259

9m139 DFPSDLHQF gattttccgagcgatctgcatcagttt 260

9m140 FPSDLHQFL tttccgagcgatctgcatcagtttctg 261

LERAAWGNLPLMRK ctggaacgcgcggcgtggggcaacctgccgctgatgcgcaa

LP-LER 262

AYLRKCKEFHP agcgtatctgcgcaaatgcaaagaatttcatccg

DSQHSTPPKKKRKV gatagccagcatagcaccccgccgaaaaaaaaacgcaaa

LP-DSQ 263

EDPKDFPSDLHQFL gtggaagatccgaaagattttccgagcgatctgcatcagtttctg

HQFLSQAVFSNRTL catcagtttctgagccaggcggtgtttagcaaccgcaccctggc

LP-HQF 264

ACFAVYTTKEK gtgctttgcggtgtataccaccaaagaaaaa

AVYTTKEKAQI LYKK gcggtgtataccaccaaagaaaaagcgcagattctgtataaa

LP-AVY 265

LMEKYSVTFI aaactgatggaaaaatatagcgtgacctttatt

RHMCAGHNIIFFLTP cgccatatgtgcgcgggccataacattattttttttctgaccccgc

LP-RHM 266

HRHRVSAINN atcgccatcgcgtgagcgcgattaacaac

LP-NFC NFCQKLCTFSFLICK aacttttgccagaaactgtgcacctttagctttctgatttgcaaa 267

LP-FLI FLICKGVNKEYLLYS tttctgatttgcaaaggcgtgaacaaagaatatctgctgtatagc 268

GVN KEYLLYSALTRD ggcgtgaacaaagaatatctgctgtatagcgcgctgacccgc

LP-GVN 269

PYHTIEESI gatccgtatcataccattgaagaaagcatt

VETKCEDVFLLLGMY gtggaaaccaaatgcgaagatgtgtttctgctgctgggcatgta

LP-VET 270

LEFQYNVEEC tctggaatttcagtataacgtggaagaatgc

LHMTREEMLTERFN ctgcatatgacccgcgaagaaatgctgaccgaacgctttaac

LP-LHM 271

HILDKMDLIF catattctggataaaatggatctgattttt

KMDSVIFDFLHCIVF aaaatggatagcgtgatttttgattttctgcattgcattgtgtttaac

LP-KMD 272

NVPKRRYWLF gtgccgaaacgccgctattggctgttt

TLAAGLLDLCGGKAL accctggcggcgggcctgctggatctgtgcggcggcaaagc

LP-TLA 273

NVNLPMERLT gctgaacgtgaacctgccgatggaacgcctgacc

TMNEYPVPKTLQAR accatgaacgaatatccggtgccgaaaaccctgcaggcgcg

LP-TMN 274

FVRQIDFRPKI ctttgtgcgccagattgattttcgcccgaaaatt

LLEKRILQSGMTLLLL ctgctggaaaaacgcattctgcagagcggcatgaccctgctg

LP-LLE 275

LIWFRPVADF ctgctgctgatttggtttcgcccggtggcggatttt

LPLMRKAYLKLLLGM ctgccgctgatgcgcaaagcgtatctgaaactgctgctgggca

RP-14 YLEFGVNKEYLLYLL tgtatctggaatttggcgtgaacaaagaatatctgctgtatctgct 276

LIWFRPV gctgatttggtttcgcccggtg

GVN KEYLLYLERAA gtgaacaaagaatatctgctgtatctggaacgcgcggcgtgg

RP-21 WGNLKEYLLYSALR ggcaacctgaaagaatatctgctgtatagcgcgctgcgcacc 277

TLACFAVY ctggcgtgctttgcggtgtat

LGMYLEFQYFPSDL ctgggcatgtatctggaatttcagtattttccgagcgatctgcatc

RP-34 278

HQFLRAAWGNLPL agtttctgcgcgcggcgtggggcaacctgccgctg

LLLGMYLEFGVNKEY ctgctgctgggcatgtatctggaatttggcgtgaacaaagaata

RP-51 LLYLLLIWFRPV tctgctgtatctgctgctgatttggtttcgcccggtg 279 LERAAWGNLPLMRK ctggaacgcgcggcgtggggcaacctgccgctgatgcgcaa

RP-54 280

AYLRAAWGNLPL agcgtatctgcgcgcggcgtggggcaacctgccgctg

LGMYLEFQYFPSDL ctgggcatgtatctggaatttcagtattttccgagcgatctgcatc

RP-55 281

HQFLKEYLLYSAL agtttctgaaagaatatctgctgtatagcgcgctg

JC9m19 LDRSAWGNI ctggatcgcagcgcgtggggcaacatt 282

JC9m21 RSAWGNIPV cgcagcgcgtggggcaacattccggtg 283

JC9m27 IPVMRKAYL attccggtgatgcgcaaagcgtatctg 284

JC9m28 PVMRKAYLK ccggtgatgcgcaaagcgtatctgaaa 285

JC9m33 AYLKKCKEL gcgtatctgaaaaaatgcaaagaactg 286

JC9m145 HAFLSQAVF catgcgtttctgagccaggcggtgttt 287

JC9m156 RTVASFAVY cgcaccgtggcgagctttgcggtgtat 288

JC9m165 TTKEKAQIL accaccaaagaaaaagcgcagattctg 289

JC9m176 KLMEKYSVT aaactgatggaaaaatatagcgtgacc 290

JC9m199 FLTPHRHRV tttctgaccccgcatcgccatcgcgtg 291

JC9m201 TPHRHRVSA accccgcatcgccatcgcgtgagcgcg 292

JC9m216 KLCTFSFLI a aa ctg tg caccttta g ctttctg att 293

JC9m227 GVNKEYLFY ggcgtgaacaaagaatatctgttttat 294

JC9m230 KEYLFYSAL aaagaatatctgttttatagcgcgctg 295

JC9m242 PYAVVEESI ccgtatgcggtggtggaagaaagcatt 296

JC9m289 LLMGMYLDF ctgctgatgggcatgtatctggatttt 297

JC9m291 MGMYLDFQE atgggcatgtatctggattttcaggaa 298

JC9m362 MLVERFNFL atgctggtggaacgctttaactttctg 299

JC9m410 FLKCIVLNI tttctgaaatgcattgtgctgaacatt 300

JC9m571 LQSGMTLLL ctgcagagcggcatgaccctgctgctg 301

JC9m579 LLLIWFRPV ctgctgctgatttggtttcgcccggtg 302

JC9m436 TLAAALLDL accctggcggcggcgctgctggatctg 303

JC9m531 NEYSVPRTL aacgaatatagcgtgccgcgcaccctg 304

JC9m533 YSVPRTLQA tatagcgtgccgcgcaccctgcaggcg 305

JC9m140 FPVDLHAFL tttccggtggatctgcatgcgtttctg 306

MC9m19 IAPNCYGNI attgcgccgaactgctatggcaacatt 307

MC9m21 PNCYGNIPL ccgaactgctatggcaacattccgctg 308

MC9m27 IPLMKAAFK attccgctgatgaaagcggcgtttaaa 309

MC9m28 PLMKAAFKR ccgctgatgaaagcggcgtttaaacgc 310

MC9m33 AFKRSCLKH gcgtttaaacgcagctgcctgaaacat 31 1

MC9m145 SDYLSHAVY agcgattatctgagccatgcggtgtat 312

MC9m156 KTVSCFAIY aaaaccgtgagctgctttgcgatttat 313

MC9m165 TTSDKAIEL accaccagcgataaagcgattgaactg 314

MC9m176 KIEKFKVDF aaaattgaaaaatttaaagtggatttt 315

MC9m199 FITLSKHRV tttattaccctgagcaaacatcgcgtg 316

MC9m201 TLSKHRVSA accctgagcaaacatcgcgtgagcgcg 317

MC9m216 TFCTISFLI a ccttttg caeca tta gctttctg att 318

MC9m227 GVNKMPEMY ggcgtgaacaaaatgccggaaatgtat 319

MC9m230 KMPEMYNNL aaaatgccggaaatgtataacaacctg 320

MC9m242 PYKLLQENK ccgtataaactgctgcaggaaaacaaa 321

MC9m289 IILAHYLDF attattctggcgcattatctggatttt 322

MC9m291 LAHYLDFAK ctggcgcattatctggattttgcgaaa 323

MC9m362 MLCKKFKKH atgctgtgcaaaaaatttaaaaaacat 324

MC9m410 IIQLLTENI attattcagctgctgaccgaaaacatt 325

MC9m571 LQSGTTLLL ctgcagagcggcaccaccctgctgctg 326

MC9m579 LCLIWCLPD ctgtgcctgatttggtgcctgccggat 327

MC9m436 SFAAALIDL agctttgcggcggcgctgattgatctg 328 MC9m531 NDYFIPKTL aacgattattttattccgaaaaccctg 329

MC9m533 YFIPKTLIA tattttattccgaaaaccctgattgcg 330

MC9m140 FPIDLSDYL tttccgattgatctgagcgattatctg 331

An example of a vector comprising a DNA of the invention is a baculovirus, for example a baculovirus of the Bac-to-Bac™ system marketed by Invitrogen. Host cells are those particularly suitable for the preferred vectors. For a baculovirus vector, the corresponding host cell is an Sf9 insect cell.

The invention further relates to a vaccine against polyomavirus infection comprising a a carrier loaded with peptides of the invention as described above, and optionally further viscosity-regulating compounds, stabilizing compounds and/or an adjuvant increasing the immunogenicity, as it is known in the state of the art. Such a vaccine may comprise an adjuvant selected from the group consisting of aluminium hydroxide, alum, AS01 , AS02, AS03, AS04, MF59, MPL, QS21 , ISCOMs, IC31 , unmethylated CpG, AD VAX, and

Freund's reagent. Cytokines such as GM-CSF, IL-2, IL-7, IL-12 or type-l IFNs could be used as additional adjuvants.

The invention further relates to methods of prophylaxis of polyomavirus infection and treatment of against polyomavirus infection using such vaccines. Polyomavirus infections considered are (1 ) BKPyV reactivation in kidney transplant recipients causing nephropathy and/or graft loss, (2) BKPyV reactivation in hematopoietic stem cells recipients that can cause hemorrhagic cystitis, (3) JCPyV reactivation that is responsible for progressive multifocal leukoencephalopathy in some immunocompromised patients such as AIDS individuals, natalizumab-treated multiple sclerosis patients and transplant recipients, and (4) Merkel cell carcinoma.

In one embodiment the vaccines comprising VLPs of this invention are used in a method of prophylaxis. A method for vaccinating a human uses a vaccine of the present invention comprising VLPs in the range of 1 μg to 100 mg/dose, in particular 10 μg to 10 mg/dose. An average human of 70 kg is assumed to receive at least a single vaccination. Preferably a dosage regimen comprising 3 doses applied at 0, 8 and 24 weeks, optionally followed by a second vaccination round 12-24 months after the last immunization is chosen. Preferred routes of administration are subcutaneous and intramuscular administration, but intradermal and intranasal are also suitable administrations.

The vaccines of this invention are likewise used in a method of therapeutic treatment. A method for vaccinating a human for treatment purposes uses a vaccine of the present invention comprising VLPs in the range of 1 μg to 100 mg/dose, in particular 10 μg to 10 mg/dose. An average human of 70 kg is assumed to receive at least a single vaccination. Preferably a dosage regimen comprising 3 doses applied at 0, 8 and 24 weeks, optionally followed by a second vaccination round 12-24 months after the last immunization is chosen. The vaccines of the present invention comprises VLPs in the range of 1 μg to 100 mg/dose, in particular 5 μg to 10 mg/dose for adults and half this dose for children.

The method of treatment of the invention is particularly important for immunocompromised individuals, and especially for solid organ, bone marrow and/or stem cell transplant recipients. For these patients a fast and effective CD8+ and CD4+ T-cell response is crucial. To address this topic the vaccine of the present invention is in the range of 1 μg to 100 mg/dose. An average human of 70 kg is assumed to receive at least once a vaccination. Preferably a dosage regimen comprising 3 doses applied at 0, 2 and 4 weeks before transplantation, optionally followed by a second vaccination round 1 , 4, 8 weeks after the transplantation is chosen.

The invention further relates to a diagnostic method using these peptide epitopes or nucleic acids encoding these. The polyoma EVGR epitopes herein described can be used in a diagnostic assay for detecting BKPyV-specific immune responses in patients at risk for BKPyV disease. This includes patients undergoing kidney transplantation or other immunocompromised patients. Furthermore, such an assay can be used to identify patients regaining BKPyV- specific T-cell control and aid in the tailoring of immunosuppression reduction, which currently is the only recommended treatment option for patients with BKPyV viremia and nephropathy. An assay according to the invention relies on a standardized collection of patient's blood either in specialized tubes coated with the BKPyV 9mer-peptides to stimulate CD8+T-cells, or uses an ELISpot format in microtiter plates. The immunogenic 9mer peptides induce IFN-γ production and the amount of released IFN-γ is subsequently measured by ELISA, or the number of IFN-v-secreting cells is determined by ELISpot, or the number of IFN-γ producing cells is enumerated by flow-cytometry, or are captured by magnetic beads coated with monoclonal antibodies or streptamers.

The lack of polyomavirus-specific immunity allows identifying patients at risk of polyomavirus replication and disease, which helps for individualized therapy, allows following recovery of individual patients, and/or allows selecting and enriching BKPyV- specific T-cells for adoptive cell therapy.

In particular the invention relates to an immunological test of BKPyV-specific T-cell response using the BKVyP peptide epitopes and measure cytokine production, such as production of interferon-gamma, tumor necrosis factor alpha, interleukin-2, interleukin-4, interleukin-33, interleukin-15, interleukin-17, and the like.

Furthermore the invention relates to capture and adoptive T-cell transfer of peptide responsive T-cells for immunotherapy of patients with disease or at risk of disease.

The rationale for the invention as described is based on the following results:

Bioinformatic prediction of HLA-A and HLA-B-binding BKPyV 9mer-epitopes

Syfpeithy and IEBD programs were used to predict 20 top-scoring 9mer-epitopes encoded in the BKPyV early viral gene region (EVGR) for each of 14 HLA-A and -B types prevalent in Europe and North America. The predictions for each HLA-type were visualized relative to the BKPyV EVGR sequence (Figure 1 ). Although each HLA-type appeared to have its own unique 9mer pattern, there were clearly sequence stretches, where the predicted epitopes appeared to cluster locally as well as across several HLA-types by both algorithms (Figure 1 ). To focus on immunodominant epitopes, a total of 73 predicted 9mer-epitopes including epitopes identified in previous studies were selected from different prominent clusters present across most HLA-A and -B types for chemical synthesis and experimental testing. In addition, 1 1 longer peptide stretches were selected for domains where several predicted 9mers overlapped. Twenty-four additional 9mer predictions outside of these clusters were synthesized resulting in a total number of 97 9mer-epitope candidates. Experimental testing of predicted BKPyV 9mer-epitopes in healthy individuals

PBMCs were obtained from 42 healthy individuals (HI, median age was 46 years old; see Table 7) being BKPyV-lgG seropositive as defined by the normalized OD₄92n_m of >0.1 at 200-fold dilution (Figure 2), which was previously shown to be very high sensitive and specific (Kardas P et al., J Clin Virology 2015;71 :28-33). Because of the low BKPyV- specific T-cell frequency in PBMCs, an in vitro expansion protocol was adopted (Binggeli S et al., Am J Transplant. 2007;7:1 131 -9) and PBMCs were stimulated using BKPyV EVGR 15mP or with a pool of longer peptides (LPP). IFN-γ ELISpot assays were performed before and after expansion using 15mP, LPP, 9mP and 9msP sub-pools for single epitope cross-identification.

15mP is a pool of 180 overlapping 15mer-peptides spanning EVGR sequence. 9mP is a pool of 9mer-peptides corresponding to the BKPyV EVGR 9mer-epitopes predicted by two computer algorithms (Syfpeithy and Immune Epitope Data Base (IEDB)). LPP is a pool of long peptides (15-27aa) covering those BKPyV EVGR 9mer-epitopes. 9msP are sub- pools containing 8-10 9mer-peptides, each one being contained in two sub-pools in order to be cross-identified (Table 8).

Table 7: BKPyV EVGR 9mer responses in IFN^y ELISpot assay

HLA-A HLA-B

*01 *02 *03 *11 *24 *32 *07 *08 *35 *39 *40 *44 *51

42 7 17 16 4 11 6 15 4 8 6 5 8 5

19 ++ +++ +++ +++ +++ +++ +++ +++ ++

21 ++ +++ ++ ++ ++ +++ ++ ++ ++ +++ ++

26 ++ ++ +++ ++ ++ +++ ++ ++ ++ ++

27 ++ ++ +++ +++ +++ ++ ++++ +++ + ++ ++ +++ ++

28 +++ ++ +++ ++++ ++ +++ +++ + ++ +++ ++++ ++

29 ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++

32 ++ + +++ ++ ++ + +++ +++ ++ ++++ ++

33 ++ +++ ++ ++ ++ ++ +++ ++ ++ +++ ++

145 ++ ++ +++ ++++ ++ +++ ++ ++ ++

148 ++ + + ++ ++ +++ ++ + ++ + ++

150 ++ + ++ + ++ + + ++ ++

156 ++ + ++ + ++++ ++ ++ ++ +++ + ++++

158 ++ + ++ ++ ++ ++ ++ ++ ++ + +++

159 ++ + + ++ ++ ++ ++ ++ + +++

165 ++ + + ++ +++ + + ++ +++ +++

166 + + + ++ + + + ++

167 ++ ++ + + +++ + + ++ ++ ++ ++

169 ++ ++ ++ ++ + ++

172 ++ + + ++ ++ ++ ++ ++ ++ + ++ ++ + ++ ++ ++ ++ ++ ++ ++ +++ ++ ++ + ++

++ + ++ ++ ++++ ++ +++ + + ++++

++ + +++ +++ ++ ++ ++ +++ ++ ++ + +++

++ + ++ + ++ ++ +++ ++ + +++

++ + + ++ ++ ++ ++ + ++ +++

++ + + ++ + ++ + ++

+ + + ++ ++ ++ ++ ++

++ ++ + + ++ ++ + ++ ++

++ + ++ ++ ++ ++ ++ ++

++ + ++ ++ ++ ++ + ++ ++ ++ ++ ++

+++ ++ +++ +++ ++++ +++ +++ ++ ++ +++ ++ +++

++ ++ +++ +++ ++ ++ +++ ++ ++ ++ ++ +++

++ + ++ ++ ++ ++ ++ + ++ +++ +++

+ ++ ++ ++ ++ + + ++

+ + + + + + ++

++ ++ ++ ++ ++ ++ ++ ++

++ + +++ ++ + +++

++ + +++ ++ ++ +++ ++ ++ ++

++ + ++ ++ ++ ++ + +++ + ++

++ ++ ++++ ++ +++ ++++ + +++ ++ ++ ++

++ + ++ ++++ ++ ++ ++ ++++ + ++ ++ ++

++ ++ +++ ++ ++ +++ ++ +++ + ++

++ ++ +++ ++ ++ ++ + ++ ++

++ + ++ ++ ++ ++ ++ ++ ++ ++ ++

+ + + + ++ + + ++ +

+ + + + ++ + ++

++ + ++ ++ ++ ++ ++ ++ ++++ ++ ++

++ +++ ++ ++ ++ ++ ++ ++ +++ ++++ +++ ++ ++ ++

+++ ++ +++ +++ ++++ ++ +++ ++ +++ +++ ++ +++

+++ ++ +++ +++ ++ ++ +++ ++ ++++ ++++ + ++

+++ ++ +++ +++ ++ ++ +++ ++ ++++ ++++ ++ ++

++ + ++ ++++ + ++ ++ +++ ++

++ + ++ ++ ++ ++ ++ +++ + ++

++ + ++ + ++ + ++

++ ++ ++ ++ + ++ ++ ++ ++ ++

+ + + + + ++ + ++

++ ++ + ++ ++ + +++ ++ ++ ++

++ + ++ ++ ++ ++ ++ + ++

++ + ++ ++ + ++ ++ + ++

++ * ++ + ++ ++ + ++

+ + + ++ + ++ + ++

++ + ++ ++ + ++ ++ ++ ++

++ ++ + ++ ++++ ++ ++ ++ +++ ++

+ + +

++ + ++ ++ + ++ ++ + ++

++ ++ + ++++ + +++ ++ +++ +++ ++ ++

++ ++ + + ++ +++ + ++

++ ++ ++ +++ +++ ++ ++

++ + ++ ++ ++ + ++

++ + ++ + ++ ++

++ + ++ + ++ ++ ++ + ++ 577 ++ + ++ ++ ++ ++ + ++

579 ++ * + + ++ + + ++ +++ ++

582 ++ + + + + + ++ ++ ++

Column 1 displays the position of the 9-mer epitopes within BKPyV LTag protein.

Column 2 shows the epitope-specific IFN-γ responses in all healthy individuals, regardless of the HLA-type (mean)

Line 3 shows the number of healthy individuals in each HLA group

+ : 1 -14% positive responders

++ : 15-29% positive responders

+++ : 30-49% positive responders

++++ :≥ 50% positive responders The approach is illustrated for healthy individual 29: PBMCs were either stimulated directly or after expansion using the indicated peptide pools (Figure 3A). IFN-γ ELISpot results showed that the in vitro T-cell expansion protocol resulted in a significant increase of BKPyV-specific T-cells that responded to both 15mP and 9mP (Figure 3A). Re- challenge with 9mer sub-pools (Table 8) showed that the highest responses could be attributed to specific sub-pools, namely 9msA and 9ms4 (Figure 3B). The single 9mer common to both of these sub-pools is 9m127. A slightly weaker response was also observed in sub-pool 9ms5 suggesting some response to 8aa-overlapping 9m 128 as well. Rechallenge of expanded cells with single 9mer-peptides confirmed that the major response to 9msP was indeed attributable to 9m127, to a lesser extent to 9m128 (Figure 3C). Based on the peptide length of 9aa, this functional IFN-γ response should originate from CD8+T-cells. To address this directly, expanded T-cells were stained with HLA- B^*07:02 9m127 streptamer, as well as with CD8 surface marker and analyzed by flow cytometry (Figure 3D). A population of HLA-B^*0702-positive 9m127-specific CD8+T-cells (right panel) could be detected representing 3.9% of total lymphocyte population.

Table 8: Composition of sub-pools

To address the proliferation following peptide stimulation, PBMCs were stained with CFSE before expansion, and labeled for CD8 and HLA-B^*0702-positive 9m127-streptamer after expansion. CSFE dilution indicated the presence of at least 9 divisions of the CD8+T-cell population (Figure 3E, left panel). HLA-B^*0702-positive 9m127-specific CD8+T-cells showed the lowest CSFE signals indicating that these cells had divided close to once per 1 -2 days during the expansion period (Figure 3E, right panel). To correlate HLA-B^*0702-positive 9m127-specific CD8+T-cells and degranulation function, expanded T-cells were stimulated with 9m127 or another BKPyV peptide (9m259) for 5h in the presence of CD107a antibody and stained for HLA-B^*0702-positive 9m127-streptamers (Figure 3F). Only 9m127 induced degranulation of nearly the entire HLA-B^*0702-positive 9m127 CD8+T-cell population.

T-cells functionality was also investigated in a killing assay where lytic activity of expanded T-cells against autologous ⁵¹Cr-labelled PHA-blasts pulsed with the single 9m127 or with 9mP was assessed (Figure 3G). The results show that 9m127 mediates a mean specific lysis of 48% at an effector:target ratio of 20:1. This single 9m127 response was comparable to the one mediated by 9mP, in line with an immunodominant BKPyV epitope.

Thus, this experimental approach permitted to functionally identify candidate 9mer- epitopes from BKPyV recognized by CD8+T-cells in BKPyV-seropositive healthy individuals (HI), even if cells were present at a low frequency among PBMCs. In some cases, the responses induced by 15mP (15mer peptide pool) and 9mP (9mer peptide pool) did not correlate, suggesting the presence of independent populations among BKPyV-specific T-cells. 9mP and LPP (long peptide pool) responses were absent in expanded PBMCs from BKPyV- and JCPyV-seronegative healthy donors indicating that the in vitro protocol of only 2 weeks expansion did not lead to significant priming of naive T-cells, but most likely induced proliferation of memory CD8+T-cells. Healthy individuals seronegative for BKPyV, but seropositive for JCPyV did show a response to these EVGR peptide pools, suggesting that LTag cross-reactive responses from JCPyV-specific T-cells were elicited.

In total, 42 healthy individuals were analyzed and the frequency of responses is summarized according HLA-type in a heat map (Table 7). There were only two 9mer responses with frequencies of less than 15% of healthy individuals (9m316, 9m518). Many 9mer-epitope responses could be detected in up to one third of healthy individuals, but there were some epitopes with more frequent responses (40 epitopes elicited IFN-γ production in 30-49% donors; 17 epitopes induced IFN-γ ELISpot responses in more than 50% of tested individuals). The presence of response hotspots supports the notion that not all 9mer-epitopes are equally potent, and certain clustering is observed. This is illustrated by the ELISpot results for epitopes eliciting responses in at least 40% healthy individuals, and for those 9mers that had been previously reported or for which streptamer binding was demonstrated (Figure 4). The overall results indicate that BKPyV EVGR- specific 9mer T-cell responses are heterogeneous in terms of frequency and strength. For some HLA-types, positive responses were found more frequently and directed towards more epitopes comparing to others: For example, in HLA-A^*03, -A^*1 1 , -B^*07, -B^*35, - B^*39, -B^*44, -B^*51 positive subjects, more than one epitope could be identified in≥50% healthy individuals (Table 5). Single 9mer-epitopes were associated with variable responses in healthy individuals (Figure 4). Values higher than 69 SFU/10⁶ cells were regarded as strong responses in accordance with a previously defined threshold of protection from BKPyV viremia in kidney transplant recipients (Binggeli S et al., Am J Transplant. 2007;7:1 131 -9).

- 9m301 elicited specific responses in 47% of HLA-B^*07 subjects with median of 292 SFU/10⁶ cells. The same 9mer-epitope was found to be immunogenic in 35% HLA-A^*02 and 45% HLA-A^*24 donors. - 9m327 induced a median response of 304 SFU/10⁶ cells in 8 HLA-A^*03-positive healthy individuals.

- 9m330 induced fairly strong responses in 53% HLA-A^*03 positive healthy individuals with median of 593 SFU/10⁶ cells. 9m330-specific IFN-γ production could also be detected in some HLA-B^*07 and -B^*39 individuals.

- 9m389 elicited a median response of 299 SFU/10⁶ cells in 6 HLA-A^*02 subjects (35%). 9m389 also elicited IFN-γ production in 50% of HLA-A^*1 1 subjects and 18% of HLA-A^*24 subjects (Table 7). In line with the predicted clusters, some areas in EVGR appeared to be more

immunogenic than others (Table 7). The domain spanning from 9m383 to 9m393 could induce frequent responses in HLA-A^*02, A^*1 1 , -B^*35, -B^*39 subjects. The domain from 9m1 19 to 9m133 appeared to be highly immunogenic across different HLA-types. HLA restriction of BKPyV EVGR-specific T-cell responses in healthy individuals

To address HLA specificity of IFN-γ inducing 9mer-epitopes, cells were stained with MHC- streptamers (Table 9).

Table 9: HLA-A and -B specificity of BKPyV EVGR CD8+T-cell responses in healthy individuals

Some MHC-streptamers showed strong staining such as HLA-B^*07 and -B^*08 positive 9m127-streptamers that were identified in 89% and 67% healthy individuals with mean values of 0.86% and 0.27%, respectively. T-cells presenting 9m127 via HLA-A^*02 molecule could not be detected, despite high 9m127-specific IFN-γ T-cell responses among HLA-A^*02 positive healthy individuals (Figure 4A), suggesting that those responses were not HLA-A^*02 restricted or that HLA-A^*02 9m127-streptamers were not efficiently presented or binding. Conversely, HLA-A^*02-positive T-cells specific for 9m679 could be detected in 50% of tested HLA-A^*02 individuals despite a low amount of responsive donors in IFN-γ ELISpot assay (18%).

- HLA-A^*03-positive 9m327-specific T-cells could be detected in 40% HLA-A^*03 HI (9m327 elicited IFN-γ responses in HLA-A^*03, -A^* 1 1 and -B^*07 donors); - HLA-A^*24-positive 9m389-specific T-cells could be detected in 14% healthy individuals (9m389 elicited IFN-γ responses in HLA-A^*02, -A^* 1 1 and -A^*24 donors);

- HLA-B^*07-positive 9m301 -specific T-cells could be detected in the only tested HLA-B^*07 individual (9m301 elicited IFN-γ responses in HLA-A^*02, -^*A24 and -B^*07 donors);

- HLA-B^*40-positive 9m1 19-specific T-cells could be detected in 50% HLA-B 0 healthy individuals (9m1 19 elicited IFN-γ responses in HLA-B^*07, -B^*40, and -B^*44 donors).

Finally, some T-cell activating 9mers were presented by more than one HLA molecule, namely 9m121 (HLA-B^*35 and -B^*39), 9m127 (HLA-B^*07 and -B^*08) and 9m240 (HLA- B^*35 and -B^*39) (Table 9).

BKPyV EVGR-specific epitope CD8+T-cell responses in pediatric kidney transplant recipients

To extend the results from healthy individuals to the clinical setting, BKPyV-specific T-cell responses were investigated in 19 pediatric kidney transplant recipients who had been protected or recovered from BKPyV viremia. Several epitopes identified in healthy individuals could be confirmed in these 19 kidney transplant recipients (Table 10).

Table 10: BKPyV EVGR-specific T cell responses in kidney transplant recipients (KTRs)

KTRs, kidney transplant recipients; SFU, spot forming unit 9m389 was recognized in 67% HLA-A^*02 patients with a mean value of 312 SFU/10⁶ cells, but HLA-A^*02 restriction could not be confirmed. This epitope induced T-cell responses in 33%, 50%, 50% and 40% H LA- A^* 1 1 , -A^*24, -B^*07 and -B^*51 patients respectively, with HLA-specificity confirmed for HLA-A^*24 and -B^*51 molecules. 9m679 was found to be immunogenic in 8 of 9 tested HLA-A^*02 patients, and specific CD8+T- cells were detectable in 36% of HLA-A^*02 patients. 9m327 could be confirmed by ELISpot assays and MHC-streptamer staining in kidney transplant recipients positive for HLA- A^*01 , -A^*03, and -A^*1 1. 9m 127 elicited T-cell responses in 100% HLA- B^*07 and -B^*08 patients, and MHC-streptamer staining identified CD8+T-cells for one HLA-B^*07 patient.

Thus, four 9mer-epitopes elicited functional IFN-γ responses in healthy individuals and kidney transplant recipients and were HLA-specific in both cohorts: 9m127 (HLA-B^*07 specific), 9m327 (HLA-A^*03 specific), 9m389 (HLA-A^*24 specific) and 9m679 (HLA-A^*02 specific). Three 9mer responses were found in kidney transplant recipients but not in healthy individuals (9m302, 9m536 and 9m633).

BKPyV replication prevalence in pediatric kidney transplant recipients

To evaluate the potential association of BKPyV replication with specific HLA-A and -B types, a prospective cohort of 1 18 consecutive pediatric kidney transplant recipients was analyzed, of whom 38 (32%) experienced BKPyV viremia. The rate of BKPyV viremia was not equally distributed across HLA-types or mismatches (Figure 5). Although the overall sample size was too small for statistically supported conclusions, HLA-A^*01 patients seemed to have a lower rate of viremia compared to the overall population (p<0.05), and mismatching for this allele was present in about half of the kidney transplant recipients. Similarly, a trend for a lower rate of BKPyV viremia was seen among HLA-A^*26, -A^*28, and -B^*57-positive patients, without a clear association with matching. Interestingly, 60% of HLA-B^*07 non-viremic patients were matched for this particular allele, whereas all of HLA-B^*07 viremic patients were mismatched (Figure 5). This observation would be in line with the hypothesis that the absence of HLA-B^*07 might increase the risk of viremia. In contrast, there was a higher proportion of HLA-B^*35-matched patients among viremic than among the non-viremic kidney transplant recipients (p<0.05). Finally, viremia seemed to be independent of matching for HLA-A^*01 , -A^*24, -A^*29, -B^*08 or -B^*51 types, for which the proportion of matched patients within viremic and non-viremic patients was similar (Figure 5). A higher rate of high-level viruria was found in HLA-A^*31 patients (p<0.05), while a trend to a lower rate was found in HLA-B^*57 patients (p<0.05) and HLA-A^*01 patients (p=0.08). Thus, even though immunogenic properties of some 9mer-epitopes could be confirmed in kidney transplant recipients, a simple association of single mismatching with risk or matching with protection could not be derived from this pediatric cohort.

BKPyV-specific immune responses in kidney transplant recipients from the Swiss

Transplant Cohort Study (STCS)

Plasma samples were used for BKPyV and JCPyV serology using a virus-like particles (VLPs)-based ELISA. As shown in Figure 6, BKPyV-specific IgG levels increase over time in patients undergoing viral reactivation. PBMCs were thawed and tested in an "ex vivo" IFN-γ ELISpot upon stimulation with BKPyV LTag-derived peptides, namely a pool of 180 overlapping 15mers (15mP) spanning the whole BKPyV LTag sequence and a pool of 97 9mers (9mP) predicted to be immunogenic in a wide range of individuals (Cioni M, Leboeuf C et al., Am J Transplant. 2016;4:1 193-1206). In parallel, PBMCs were cultured in vitro for 2 weeks in the presence of BKPyV LTag 15mP and cytokines in order to expand BKPyV LTag-specific T-cells. After expansion, the cells were tested again by IFN- γ ELISpot ("expanded" IFN-γ ELISpot) upon stimulation with 15mP, 9mP and single 9mers allowing the identification of immunodominant BKPyV LTag epitopes. As shown in Figure 7, both viremic and non-viremic patients showed very high responses to BKPyV 9mP, showing that it is possible to detect BKPyV LTag-specific CD8 T-cell responses ex vivo. Furthermore, BKPyV LTag 15mP responses at the time of transplantation and 6 months after transplantation are significantly higher in non-viremic patients than in viremic patients. Interestingly, the described expansion protocol allows a dramatic increase of the frequency of BKPyV-specific T-cells in both viremic and non-viremic patients (Figure 8). Cellular immune responses to individual BKPyV LTag epitopes in both viremic and non- viremic patients were detected, as shown by Figure 9. A high diversity of the response was observed, since 53 and 67 epitopes were identified in viremic patients and non- viremic patients, respectively. Some epitopes were frequently detected in healthy individuals (Cioni M, Leboeuf C et al., Am J Transplant. 2016;4:1 193-1206), whereas others were not.

Six patient groups were created according to the start and duration of the BKPyV viremia episode. Group 1 contains patients with viremia starting and resolving between TO and T6; group 2 contains patients viremia starting between TO and T6 and resolving between T6 and T12; group 3 contains patients viremia starting between TO and T6 and resolving after T12; group 4 contains patients viremia starting and resolving between T6 and T12; group 5 contains patients viremia starting between T6 and T12 and resolving after T12; group 6 contains patients without any viremia episode (non-viremic patients).

Discussion

BKPyV-associated nephropathy is now widely recognized as an emerging complication in kidney transplant recipients. Insufficient BKPyV-specific T-cell control of the recipient over viral replication in donor allograft is suspected as the common denominator and key mechanism. Independent single center studies indicated that reducing

immunosuppression was associated with increasing BKPyV-specific T-cell responses and coincided with clearance of viremia and nephropathy (Ginevri F et al., Am J Transplant. 2007;7:2727-35; Binggeli S et al., Am J Transplant. 2007;7:1 131 -9; Schachtner T et al., Am J Transplant. 201 1 ;1 1 :2443-52). Alternatively, the decline of cellular immunity at one month after transplantation has been proposed to identify patients at increased risk of BKPyV viremia, but no or low IFN-γ responses in at least half of patients impeded the predictive value for individual patient (Schachtner T. et al., Am J Transplant.

2015;15:2159-69). Given these experiences and the fact that mostly CD4+T-cell responses were measured by overlapping 15mer-peptide pools, a better characterization of BKPyV epitope-specific CD8+T-cells response is needed to improve current understanding and clinical utility of BKPyV-specific cellular immunity. Since BKPyV- specific CD8+T-cell responses are more frequently directed to LTag than VP1 , the BKPyV EVGR-encoded epitopes rather than the capsid antigens are important. The following aspects emerge from the present study:

1 . Systematic in silico analysis of immunogenic epitopes predicts immunogenic hotspot clusters and gaps in BKPyV EVGR for each of the 14 major HLA class I types. Predicted areas were similarly clustered across different HLA class I types, and this observation argues for potential immunodominant domains, where the virus would be particularly susceptible to immune control and selection pressure. As these domains are present in EVGR-encoding crucial viral regulatory proteins early in the viral replication cycle, at least transient escape from cellular immune control would be particularly important for BKPyV replication (Gosert R et al., J Exp Med. 2008;205:841 -52). 2. The expansion protocol used in the present study has been devised to overcome the low frequency of BKPyV-specific T-cell responses to overlapping 15mer pools (Binggeli S et al., Transplantation. 2006(S3):94), and was successfully used in the prospective study of pediatric kidney transplant recipients (Ginevri F et al., Am J Transplant. 2007;7:2727- 35). The present results demonstrate that functional CD8+T-cell responses can be amplified and target almost selectively few 9mer-epitopes located in predicted

immunodominant clusters. CFSE dye dilution and streptamer staining supported this central observation, indicating that these cells were among the most active, dividing approximatively once per 1-2 days. The 9mer responses were functionally defined by IFN-γ secretion, but could be linked in principle to 9mer-specific cell surface expression of CD107a, a marker of granzyme and perforin degranulation, and to cytotoxic activity in ⁵¹Cr release assays.

3. Although certain 9mer-epitopes were shown to be selective for specific HLA-types by streptamer staining of CD8+T-cells, presentation by different HLA class I types could be observed. This has been demonstrated for HLA-types that belong to the cross-reacting group (CREG)-1 C e.g. HLA-A^*01 , -A^*03, and -A^*1 1 , or CREG-7C e.g. HLA-B^*07 and -B^*08 (Wade J.A. et al., Blood. 2007;109:4064-70). 4. Ten of the 9mer-epitopes identified in healthy individuals could be confirmed in an unrelated cohort of 19 pediatric kidney transplant recipients protected, or recovering, from BKPyV replication. Therefore, predicted and tested immunodominant responses could be linked to the clinically relevant situation of immunosuppressed kidney transplant recipients.

Interestingly, three additional 9mer responses were found in kidney transplant recipients that had been predicted, but were not detected in healthy individuals. This might indicate that responses in healthy individuals may be lower and hence less detectable.

Conversely, the additional responses in pediatric kidney transplant recipients might result from extensive exposure to BKPyV replication and more vigorous immune response described for children (Schmidt T et al., Am J Transplant. 2014;14:1334-45).

The fact that the major site of BKPyV replication is in donor cells of the renal allograft deserves consideration as this might affect HLA presentation and modify T-cell receptor recognition of BKPyV epitopes. Interestingly, in the present prospective cohort of 1 18 pediatric kidney transplant recipients, single HLA mismatching could not be associated with BKPyV viremia. While sample size might be one important aspect (Masutani K et al., Nephrol Dial Transplant. 2013;28:31 19-26) it could also reflect the fact that different other risk factors including maintenance immunosuppression can equally well promote progression to viremia and nephropathy, and thereby contribute to the failing balance between BKPyV replication in the graft and BKPyV-specific T-cell control in an individual patient.

Materials and Methods

Healthy individuals

Peripheral blood mononuclear cells (PBMCs) were prepared from 42 healthy individuals (HI) consisting of 34 blood donors from the Swiss Red Cross blood donation center in Basel, Switzerland and from 8 other healthy volunteers. HLA types were determined fee- for-service by the Transplantation Immunology Laboratory (Basel). Participants gave written informed consent to the protocol (IRB 267/06) approved by the local institutional review board.

Pediatric kidney transplant recipients

One-hundred-eighteen consecutive pediatric kidney transplant recipients were referred to the Genova Pediatric Kidney Transplant Program between March 2003 and November 2012. Three of them were older than 21 years old but were still included in the cohort because they were initially taken care as children for their end stage renal disease in the Nephrology Unit, IRCCS, Genova, Italy. Cryopreserved PBMCs were analysed from 19 kidney transplant recipients protected (i.e. without BKPyV viremia) or recovering from BKPyV replication. The study was approved by the local IRB (867/2014).

Adult kidney transplant recipients

Kidney transplant recipients from the Swiss Transplant Cohort Study (STCS) (Project ID FUP056) will be included and tested within a retrospective study. One third of the patients experienced at least one BKPyV viremia episode within the first 12 months posttransplantation (viremic patients). Plasma and PBMCs samples were isolated from patient's blood at different timepoints (time of transplantation, 6 months and 12 months post-transplantation) and were cryopreserved in each transplant center. Cryo-preserved samples from 98 kidney transplant recipients from the Basel transplant center were analysed and the results presented here.

BKPyV IgG ELISA

BKPyV IgG serology was performed using BKPyV VP1 -derived virus-like particles as described previously (Kardas P et al., J Clin Virology 2015;71 :28-33).

In si I i co epitope prediction

Syfpeithy database (http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm) provided information about HLA class I peptide binding affinity, while Immune Epitope Data Base (http://www.iedb.org/; IEDB 2.0) provided a multiparametric prediction based on proteasomal cleavage, TAP transport and HLA class I peptide binding. The predictions were limited to HLA-A and -B types present in more than 5% of the population within Europe or North America (http://www.allelefrequencies.net/). For each HLA allele, the 20 epitopes within BKPyV EVGR sequence displaying the best scores in both algorithms were considered.

BKPyV EVGR-derived peptides

A pool of 180 overlapping 15mer-peptides (15mP) spanning BKPyV EVGR (Dunlop strain) or a pool of 1 1 longer peptides LPm1 -1 1 (LPP) covering immunodominant clusters of predicted BKPyV 9mer-epitopes were used for in vitro T-cell expansion. Cells were re- stimulated after expansion as reported (Binggeli S et al., Am J Transplant. 2007;7:1 131 -9) using 15mP or a pool of 73 predicted 9mer-peptides (9mP). The 9mer-peptides were also resuspended in different sub-pools according to a checkerboard matrix approach, from A to H and from 1 to 9 (called 9msA to H and 9ms1 to 9). Each one of the 73 peptides was present in two sub-pools. An additional set of 24 9mer-peptides that were initially not predicted by computer algorithms and 3 longer peptides were later synthesized and used to assess "prediction gaps" in EVGR sequence. All peptides were >70% pure and resuspended in DMSO (10mg/ml; Eurogentec Deutschland GmbH, Koln, Germany).

In vitro expansion of T-cells

Freshly isolated or thawed PBMCs were stimulated with LPP or 15mP (200ng/ml) in 24 well-plate and incubated for 7-14 days at 37°C 5% C0₂ before performing phenotypic and functional assays. Recombinant human IL-2 (20U/ml, Peprotech, Rocky Hill, NJ, USA) and recombinant IL-7 (5ng/ml, Peprotech) were added once a week. ELISpot assay

PDVF multiscreen filter 96 well plates (MSIPS4W10, Millipore Bedford, MA) are coated with 10ΟμΙ of anti-IFN-γ mAb 1 -D1 K (Mabtech, Nacka, Sweden) at 10Mg/ml and incubated overnight at 4°C. After three washing steps using PBS, freshly isolated PBMCs

(2.5x10⁵/well) are seeded in presence of 2μg ml of BKPyV-specific 9mer-peptides. Cells without added peptide are used as negative control, whereas cells treated with

Staphylococcus enterotoxin B (SEB) (2 g/m\; Sigma, Saint Louis, Missouri, USA) or Phytohemagglutinin-L (PHA) (2 g/m\; Roche Diagnostics GmbH, Mannheim, Germany) served as positive control. After incubation for 20-24 hours (h) at 37°C, the plates are washed five times with PBS 0.05% Tween-20 and anti-IFNy mAb 7-B6-1 -Biotin (Mabtech) is added at 1 μg ml for 3h at RT. After washing, Streptavidin ALP (Mabtech) is added at 1 μg ml for 1 h at RT. The plates are washed five times with PBS 0.05% Tween-20 and tap water before incubation with SigmaFast BCIP/NBT (Sigma-Aldrich Chemie GmbH Buchs SG, Switzerland) for 20 minutes at room temperature in the dark. Plates are rinsed with water, dried and spots counted with an ELISpot reader (Cellular Technology Ltd Europe, Bonn, Germany). ELISpot data are averaged duplicate or triplicate wells with background wells subtracted. MHC-streptamer staining

The presence of BKPyV-specific T-cells within PBMCs is investigated using MHC- streptamers obtained from custom service (IBA GmbH, Gottingen, Germany). Peptide- loaded MHC molecules are incubated with PE- or APC-coupled Strep Tactin for 45 minutes on ice before being incubated with 2-10x10⁵ cells for 45 minutes on ice. After washing with immunostaining IS buffer (IBA), cells are incubated with CD8-PE-Cy7 antibody (BD Biosciences, San Jose, CA, USA) for 15min on ice, washed with IS buffer and acquired on a flow cytometer (FACSCanto; BD Biosciences) using the FACSDiva software. Gating is performed on live cells using forward scatter and side scatter profiles, and doublets are excluded. Data are reported as percentage of specific populations after subtracting the negative control (PE or APC-coupled Strep Tactin alone).

CSFE proliferation assay

PBMCs were resuspended at a concentration of 5x10⁶/ml in PBS containing 5μΜ carboxyfluorescein diacetate succinimidyl ester (CFSE; eBioscience, Vienna, Austria). After 15min incubation at RT on a shaker, cells were washed twice with culture medium and resuspended in fresh medium for BKPyV-specific T-cell expansion described above. Cells were stained with specific MHC-streptamers and CD8 as described above and their CFSE content was analysed by flow cytometry. CD 707a degranulation assay

Expanded T-cells were resuspended in fresh medium (2x10⁶/ml) and seeded in a 96-well plate (2x10⁵ cells per well). The BKPyV 9mer-peptide of interest was added to the cultures (^g/ml) for 5h-stimulation at 37°C. Phorbol 12-myristate 13-acetate (PMA; 100ng/ml; Sigma) and ionomycin (^g/ml; Sigma) were used as positive control, and a BKPyV 9mer- peptide of another HLA specificity was used as negative control. PE-Cy7-labelled CD107a antibody (BD Biosciences) or PE-Cy7-labelled isotype control (BD Biosciences) was added during the whole period of stimulation, whereas monensin (0.3μΙ per well; BD Biosciences) and brefeldin A (1 C^g/ml; Sigma) were added for the last 4h only. Cells were then labeled for specific MHC-streptamers and CD8 as described above and analysed by flow cytometry.

Cytotoxicity assay

Specific cytotoxic activity of expanded T-cells was assessed by ⁵¹Cr-release assay of autologous PHA-stimulated blasts obtained by culturing PBMCs in the presence of PHA (4Mg/ml) for 3-6 days. PHA-blasts were loaded for 1 h at 37°C with 200μΰί ⁵¹Cr (Sodium Chromate Hartmann Analytic, Braunschweig, Germany), then pulsed for 1 h with 2 μg ml of 9mer candidate peptides or an unrelated peptide (the melanoma related peptide MAGE-4) used as negative control. Effector T-cells were incubated with 2x10³ target cells at different effector:target (E:T) cell ratios for 4h at 37°C 5%C0₂. Then 50μΙ of the supernatant was transferred to a lumaplate (Perkin Elmer, Waltham, Massachusetts, USA) and dried. Counts per minutes (cpm) were counted in a β-counter (TopCount, Perking Elmer). Killing data are the average of duplicate wells and calculated as percentage of lysis according to following formula: (Sample cpm-Spontaneous Release cpm)/(Maximum Release cpm/Spontaneous Release cpm)X100, where Spontaneous Release corresponds to ⁵¹Cr release by target cells alone and Maximum Release corresponds to ⁵¹ Cr release by target cells mechanically lysed. Data were considered reliable when Minimum release was less than 50% of Maximum Release. Statistical analysis

Proportions of viremic or viruric kidney transplant recipients, and proportions of matched or mismatched patients populations were compared using Fisher's exact test with GraphPad Prism version 4.00 (GraphPad Software, La Jolla California USA). Differences corresponding to p<0.05 were considered statistically significant.

Assessment of BKPyV viruria and viremia in pediatric kidney transplant recipients BKPyV viruria and viremia were measured at predefined time points (1 , 3, 6, 9, 12, 18, 24 months after transplantation and yearly thereafter) by the Transplantation & Clinical Virology laboratory in Basel using a quantitative real-time polymerase chain reaction (PCR). BKPyV viruria was defined by a urine viral load of >2500 genome equivalents (GEq)/mL, high-level BKPyV viruria by >7 Iog10 GEq/mL and BKPyV viremia by >1000 GEq/mL. Based on protection and recovery from BKPyV viruria and viremia, PBMCs samples of 19 kidney transplant recipients were selected and analysed.

BKPyV IgG ELISA

BKPyV VP1 -derived virus-like particles were used as antigen to detect BKPyV IgG as described (Binggeli S. et al., Am J Transplant. 2007;7:1 131 -9). Each serum sample was serially diluted 1 :100, 1 :200 and 1 :400 and the optical density (OD) was measured at 492nm. The OD₄92nm values were normalized to the OD₄₉2n_m of an internal reference serum, sera with a normalized

OO at the 1 :200 dilution were defined as IgG positive.

Isolation of peripheral blood mononuclear cells from whole blood

PBMCs from anticoagulated blood or from buffy coat preparations were diluted 1 :2 in D- PBS w/o Ca²⁺ and Mg²⁺, and overlaid on Ficoll (Lymphoprep, Axis-Shield PoC AS, Oslo, Norway). After centrifugation (room temperature, 800g; 25 minutes (min)), PBMCs were recovered, and washed twice i.e. resuspended in D-PBS w/o Ca²⁺ and Mg², and centrifuged (RT, 300g, 10min). The cells were counted and resuspended in culture medium RPMI-1640 supplemented with 5% Human Serum AB and 2mM of L-Ala-

Glutamine (all Sigma-Aldrich Chemie GmbH Buchs SG, Switzerland) or cryopreserved in culture medium containing 10% DMSO and stored in liquid nitrogen. In vitro expansion of T-cells

Freshly isolated or thawed PBMCs were seeded at a concentration of 2x10⁶/ml in culture medium in 24 well-plate after the number of viable cells was counted using Trypan Blue exclusion. PBMCs were stimulated with LPP or 15mP (200ng/ml), and incubated for 9-14 days at 37°C 5% C0₂ before phenotypic and functional assays were carried out.

Recombinant human IL-2 (20U/ml, Peprotech, Rocky Hill, NJ, USA) and recombinant IL-7 (5ng/ml, Peprotech) were added once a week.

PBMCs obtained from cryopreserved samples from pediatric KTRs were first thawed and resuspended in pre-warmed culture medium. The number of viable cells was counted using Trypan Blue solution. The cells were resuspended at the concentration of 2x10⁶/ml in culture medium, seeded in 24 well-plate and incubated with 200ng/ml 15mP at 37°C 5%C0₂. Recombinant IL-2 (20U/ml) and recombinant IL-7 (5ng/ml) were added at day 3, before performing phenotypical and functional assays at day 7. Determination of IFN-y

The protocol is adapted from QuantiFERON-TB Gold Plus™ (Qiagen).

At least three tubes have to be used in that assay, a negative control tube, a tube coated with the BKPyV antigen and a positive control tube containing a mitogen.

Each QTF-Plus™ blood collection tube is filled up with 1 ml of patient blood and shaked 10 times so the entire inner surface of the tube is coated with blood. After 16-24 hours incubation at 37°C, the tubes are centrifugated for 15 minutes at 2000 to 3000g and plasma is harvested. The plasma samples (150μΙ) can be run directly or stored for up to 28 days at 2-8°C or at -20°C for extended periods. The plasma samples are loaded into a QFT-Plus™ ELISA plate coated with anti-human IFN-γ monoclonal antibody, and mixed with an anti-human IFN-γ HRP. After 2 hours incubation at room temperature, the wells are washed 6 times and Enzyme Substrate is added for 30 minutes at room temperature in the dark. The reaction is stopped with Enzyme Stopping Solution and the optical density is read at 450nm with a microplate reader.

A standard curve allows calculating the IFN-γ concentration (Ul/ml) for each of the tested plasma samples, using the OD values of each sample.

Mouse experiments

HLA-transgenic mice from Taconic will be used for the in vivo proof-of-concept of the use of BKPyV-derived long peptides as a vaccine as follows: Three intraperitonal injection of BKPyV LTag-derived long peptides, in combination with an adjuvant compound, are performed at one week-intervals. Blood samples are collected before each immunization, and splenocytes are harvested 7 days after the last immunization. Cellular immune responses to BKPyV induced by vaccination are assessed ex vivo by stimulating splenocytes with different BKPyV LTag peptides in an IFN-γ ELISpot assay. Additionally, the presence of BKPyV-specific T-cells are investigated by cell surface staining with MHC multimers and FACS analysis. In vitro expansion of splenocytes with BKPyV-derived peptides are considered as well before doing functional and phenotypical analysis.

Five mice per group will be included in order to ensure robust statistics. Mice injected with adjuvant alone will serve as negative controls.

Claims

1 . Recombinant peptide consisting of 15 to 50 amino acids comprising

2. Recombinant peptide according to claim 1 consisting of 24 to 50 amino acids.

3. Recombinant peptide according to claim 1 or 2 comprising two, three, four, five or six optionally overlapping epitopes selected from the group of peptides of SEQ ID NO: 1 -3, 5- 7, 9-1 1 , 13, 15-18, 20-33, 35, 37, 38, 40-43, 45-52, 54-56, 59, 61 -73, 75-97, and 121 -164.

4. Recombinant peptide according to claim 1 or 2 comprising two, three, four, five or six optionally overlapping epitopes selected from the group of peptides of SEQ ID NO: 1 , 2, 4,

5. 8, 9, 12, 15, 21 , 29, 30, 34, 39, 40, 44, 48, 49, 53, 58, 66, 72, 74, 80, 81 and 97. 5. Recombinant peptide according to claim 1 or 2 comprising two, three, four, five or six optionally overlapping epitopes selected from the group of peptides of SEQ ID NO: 1 , 2, 5, 7, 9, 15, 21 , 29, 30-32, 40, 46-51 , 62, 65, 66, 74, 80, 81 and 97.

6. Recombinant peptide according to claim 1 or 2 comprising the peptide of SEQ ID NO: 4 and at least one further epitope selected from the group of peptides of SEQ ID NO:1 to 3,

5 to 97, and 121 -164.

7. Recombinant peptide according to claim 1 comprising a peptide of SEQ ID NO: 98-1 17.

8. Recombinant peptide according to claim 7 comprising the peptide of SEQ ID NO: 98, 106 or 1 12.

9. Recombinant peptide according to claim 7 comprising the peptide of SEQ ID NO: 1 12- 1 17.

10. Recombinant peptide according to claim 1 or 2 comprising two, three, four, five or six optionally overlapping epitopes selected from the group of peptides of SEQ ID NO: 1 , 2, 12, 39, 40, 48, 49, 72 and 97.

1 1 . Recombinant peptide according to claim 1 or 2 comprising the peptide of SEQ ID NO: 39 and one, two, three, four or five optionally overlapping epitopes selected from the group of peptides of SEQ ID NO:1 to 38, 40 to 97 and 121 to 164.

12. Recombinant peptide according to claim 1 or 2 comprising the peptide of SEQ ID NO: 72 and one, two, three, four or five optionally overlapping epitopes selected from the group of peptides of SEQ ID NO:1 to 71 , 73 to 97 and 121 to 164.

13. Recombinant peptide according to claim 1 or 2 comprising the peptide of SEQ ID NO: 34 and one, two, three, four or five optionally overlapping epitopes selected from the group of peptides of SEQ ID NO:1 to 33, 35 to 97 and 121 to 164.

14. Recombinant peptide according to claim 1 or 2 comprising the peptide of SEQ ID NO: 48 and one, two, three, four or five optionally overlapping epitopes selected from the group of peptides of SEQ ID NO:1 to 47, 49 to 97 and 121 to 164.

15. Recombinant peptide according to claim 1 or 2 comprising the peptide of SEQ ID NO: 2 and one, two, three, four or five optionally overlapping epitopes selected from the group of peptides of SEQ ID NO: 1 , 3 to 97 and 121 to 164.

16. A carrier comprising a recombinant peptide according to any one of claims 1 to 15.

17. A nucleic acid encoding a recombinant peptide according to any one of claims 1 to 1 15.

18. A nucleic acid according to claim 17 comprising a DNA of SEQ ID NO: 165 to 327.

19. A vaccine comprising a recombinant peptide according to any one of claims 1 to 15 and an adjuvant.

20. Use of a recombinant peptide according to any one of claims 1 to 15 or of a nucleic acid of claim 17 or 18 in a diagnostic method.

21 . A method of prophylaxis or treatment of polyomavirus infection comprising administering an effective amount of a vaccine according to claim 19 to a patient thereof.