# Patent

## Title
Methods to diagnose and immunize against the virus causing human Merkel cell carcinoma

## Abstract

The present invention provides isolated or substantially purified polypeptides, nucleic acids, and virus-like particles (VLPs) derived from a Merkel cell carcinoma virus (MCV), which is a newly-discovered virus. The invention further provides monoclonal antibody molecules that bind to MCV polypeptides. The invention further provides diagnostic, prophylactic, and therapeutic methods relating to the identification, prevention, and treatment of MCV-related diseases.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This patent application is a U.S. National Phase of International Patent Application No. PCT/US08/86895, filed Dec. 15, 2008. This patent application claims the benefit of U.S. Provisional Patent Application No. 61/013,772 filed Dec. 14, 2007, which is incorporated by reference.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with Government support under Grant Number NIH R33CA120726 awarded by the National Institutes of Health. The Government has certain rights in this invention.


BRIEF SUMMARY OF THE INVENTION
The present invention provides isolated or substantially purified polypeptides, nucleic acids, and virus-like particles (VLPs) derived from a Merkel cell carcinoma virus (MCV), which is a newly-discovered virus. The invention further provides monoclonal antibody molecules that bind to MCV polypeptides. The invention further provides diagnostic, prophylactic, and therapeutic methods relating to the identification, prevention, and treatment of MCV-related diseases. These aspects, and other inventive features, will be apparent upon reading the following detailed description in conjunction with the accompanying figures and sequence listing.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
FIG. 1. A) Merkel cell carcinoma (MCC). MCC is an aggressive small, round-cell skin cancer of Merkel mechanoreceptor cells with higher mortality rates than most other skin cancers (Left panel: H&E, Right panel: cytokeratin 20 staining, 40×). B) 3′-RACE mapping of MCV T antigen-human PTPRG fusion transcript. The viral transcript discovered by DTS in MCC347 was extended by 3′-RACE. Three mRNA sequences were isolated, two of which terminate in human PTPRG intron 1 on Chromosome 3p14. The two viral-human transcripts were generated by read-through of a weak polyadenylation signal in the viral T antigen gene.
FIG. 2. Neighbor-joining trees for MCV putative large T (T-1, see FIG. 3), small T (T-2), VP1 and VP2 proteins. The four known human polyomaviruses (BK, JC, KI and WU) cluster together in the SV-40 subgroup while MCV is most closely related to MuPyV subgroup viruses. Both subgroups are distinct from the avian polyomavirus subgroup.
FIG. 3. Northern and 3′-, 5′-RACE mapping of MCV T antigen transcripts in MCC tumors and during expression in 293 cells. Genomic T antigen fragments from MCV350 and 339 were expressed in pcDNA vector in 293 cells. The left panel shows northern blotting of the 293 cell RNAs allowing individual transcripts to be assigned to four distinct bands. Higher molecular weight bands in this northern blot exceed the genome fragment size and are artifactually produced from vector expression (note difference sizes between pcDNA339 and pcDNA350 result from a 201 nt. deletion in MCV339 T antigen sequence). T-1 is similar to polyomavirus large T antigens and possesses highly conserved cr1, DnaJ (HPDKGG), LXCXE, origin binding domain (OBD) and helicase/ATPase features. Stop codons and deletions prevent full-length large T protein expression in tumor derived viruses. T-2 possesses a large intronic sequence resulting in a protein similar to small T antigens from other polyomaviruses. An additional 0.8 kbase band is expressed in 293 cells but no corresponding tumor RACE product was isolated.
FIG. 4. A) Clonal MCV integration in MCC tumors detected by direct Southern hybridization. DNA digests with BamHI (left) or EcoRI (right) and Southern blotted with seven MCV DNA probes reveals different banding patterns in each tumor, including >5.4 kbase bands. Open arrow shows the expected position for MCV episomal or concatenated-integrated genome (5.4 kbase) with corresponding bands present in tumors MCC344 and 350. Tumors MCC339, 347/348, and 349 have different band sizes and doublet bands (closed arrows) consistent with genomic monoclonal integration. MCC352 has both prominent episomal/concatenated bands (EcoRI), and higher and lower molecular weight integration bands (BamHI). Tumors MCC337, 343 and 346 have no MCV DNA detected by Southern blotting (bands at 1.5 kbase (kidney) and 1.2 kbase (MCC346) are artifacts). B) Viral and cellular monoclonality in MCC347. Tumor MCC347 and its metastasis MCC348 were digested with SacI and NheI, and Southern blotted with unique human flanking sequence probe (Chr3 (red), left panel) or viral probes (LT1 and LT2 (yellow), right panel). The wild-type human allele is present in all samples at 3.1 kbase (left panel). The MCC tumors, however, have an additional 3.9 kbase allelic band from MCV insertion into 3p14. Probing with for MCV T antigen sequence (right panel) generates an identical band.
FIG. 5. Representative results of MCC and control tissue PCR-Southern blotting. (Top panel) Agarose gel of amplification products from 16 randomized control and MCC tissue samples using LT1 primers (see Supplementary Table 4) (Bottom panel). Specific hybridization of PCR products to a (α32P) dCTP-labeled M1-M2 internal probe (see Supplementary Table 4) after transfer of DNA to nitrocellulose membrane. Sample identities are as follows: MCC tissue samples in lanes 1 (MCC346), 5 (MCC348), 6 (MCC344), 9 (MCC339), and 15 (MCC343); negative control (H2O) samples in lanes 2, 10 and 11; and surgical control tissue samples in lanes 3, 4, 7, 8, 12, 13, 14 and 16. Weak signal in lane 13 (control gall bladder tissue) is positive only after Southern blotting of the PCR product compared to robust PCR amplification for MCC348, 344, 339 tissues. MCC346 and 343 are negative.
FIG. 6. BamHI-EcoRI double-digestion and Southern hybridization of the MCV T antigen locus. BamHI-EcoRI should generate a single  ̃1.5-1.7 kbase fragment from T antigen (see FIG. 7) unless genomic integration or deletion occurs in this region. Marked variation in band sizes (MCC339, 344, 345, 347, 348) are consistent with either human genomic integrations or deletions within T antigen locus. Open arrow indicates expected BamHI-EcoRI viral fragment (1.7 kbase) for MCV350 and closed arrow, expected T antigen fragment (1.5 kbase) for MCV 339.
FIG. 7. MCV genome diagram showing large T, small T, VP1, and overlapping VP2 and VP3 genes and DTS1 and DTS2. The former was used to identify MCV and latter is a spliced transcript having low polyomavirus homology.
FIG. 8 graphically illustrates the location of the ORFS within the genome of MCV 350.
FIG. 9 presents data showing CM2B4 mAb is specific for MCV LT. (A) A MCV T antigen-EGFP fusion protein colocalizes with CM2B4 staining in 293 cells transfected with pMCV TAg-EGFP or pEGFP, an empty vector. (B) CM2B4 does not react with T antigens of human polyomaviruses belonging to SV40 subgroup. Constructs encoding LT genes for JCV, BKV and MCV were expressed in 293 cells and stained with CM2B4 or PAb416 antibodies. PAb416 cross reacts with JCV and BKV LT proteins but not with MCV LT. (C) Immunoblotting for expressed polyomavirus LT with PAb416 and CM2B4 antibodies in cell lysates described in (B). CM2B4 recognizes an MCV 120 kDa (LT) protein and a shorter 60 kDa T antigen isoform. No cross-reactivity is apparent for PAb416 with MCV LT or CM2B4 with SV40-group polyomavirus proteins. (D) Brain tissue with progressive multifocal leukoenchephalopathy show JCV infection of oligodendroglial cells by JCV specific in situ hybridization (left panel), and CM2B4 shows no reactivity to JCV antigens (right panel). (E) Detection of truncated LT protein by CM2B4 in MCV positive MKL-1 cell line. Proteins from MKL-1 cells and MCV negative UISO, MCC13 and MCC26 cells were immunoblotted with CM2B4. LT antigen bands are only present in MKL-1 cells.
FIG. 10 presents data showing specific and uniform expression of MCV LT protein in MCC. (A) Uniform expression of MCV LT in MKL-1 cell line. Representative sections showing MCV LT and CK20 protein expression in MKL-1 and UISO cells. (B) MCC tissue specific expression of LT protein. Representative sequential sections from MCC showing histological phenytype (H&E) and immunostaining for MCV LT (CM2B4) and CK20 proteins. Expression of MCV LT protein expression is precisely localized in nucleus of MCC cells but not in surrounding tissues including the epidermis, adnexal epithelium (arrow), endothelial cells, or dermal fibroblasts.
FIG. 11 presents data demonstrating the construction of MCV VLPs.
FIG. 12 presents data demonstrating the construction of MCV VLPs. The top panel shows an anti-MCV Western blot of 293TT cells after transfection with the VP1 expression construct shown, together with an appropriate VP2 expression construct. In the far right lane of the Western, 5-fold more cell lysate was applied to the gel. The bottom panel shows a SYPRO Ruby-stained SDS-PAGE gel analysis of Optiprep gradients used to purify VLPs out of cell lysates. For MPyV and MCV399, 2.5 μl each of fractions 6-9 was loaded onto the gel. For MCV350, 12.5 μl each of fractions 6-9 was loaded. Fractions were screened for the presence of encapsidated DNA using Picogreen reagent.
FIG. 13 presents data showing the determination of serum sample working dilution for competitive ELISA for MCV VLP. This figure represents a single experiment with one serum sample. Working dilution was estimated as lowest dilution at which OD was greater than 1.
FIG. 14 presents data showing examples of VP1 peptides screen (A-B) of MCV-positive sera (A) and negative sample (B) diluted 1:500.
FIG. 15 presents data showing the extent of positive reaction to MCV VLP ELISA among Langerhan's cell histiocytosis patients of various age groups.
FIG. 16 presents data showing the results of the reactivity (ELISA) of 12 sera samples to BKV VLPs.
FIG. 17 presents data showing competitive ELISA with BK- and MCV-VLPs. This figure demonstrates results of the typical experiment with MCV-positive serum.
FIG. 18 presents data showing the seroreactivity of 12 serum samples to HPV and CRPV VLPs. A—HPV VLP, B—CRPV VLP ELISA.
FIG. 19 presents the results of ELISA assays for MCV VLPs in various cohorts (confirmed MCC patients positive with MCV, lupus patients, Langerhan's cell histiocytosis patients, blod from commercial sources, and serum form blood donors).
FIG. 20 presents data showing the OD values of 20 confirmed MCC patients positive with MCV (left) and from blood donors.
FIG. 21 presents data showing validation of a MCV neutralization assay. The ▾ line shows an MCV neutralization curve for IgG purified out of the pooled human serum using protein G resin (starting concentration 1 mg/ml). The ♦ line shows results using serum after passage over protein G resin. The X and O points display, respectively a general lack of neutralization of an MPyV reporter vector by the pooled human serum and complete neutralization of the MPyV vector by MPyV-specific rabbit serum.
FIG. 22 compares the sequence of VP1 proteins for strains of MCV against a consensus polyomavirus sequence.
FIG. 23 presents sequences discussed herein.



DETAILED DESCRIPTION OF THE INVENTION
Within the context of the present invention, a nucleic acid sequence is considered to be “selectively hybridizable” to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, “maximum stringency” typically occurs at about Tm−5° C. (5° C. below the Tm of the probe); “high stringency” at about 5-10° C. below the Tm; “intermediate stringency” at about 10-20° C. below the Tm of the probe; and “low stringency” at about 20-25° C. below the Tm. Functionally, maximum stringency conditions may be used to identify sequences having strict identity or near-strict identity with the hybridization probe; while high stringency conditions are used to identify sequences having about 80% or more sequence identity with the probe. This is especially true for polynucleotides having a minimum of from about 18-22 nucleic acids, but those of ordinary skill in the art are also able to apply these principals to larger or smaller polynucleotides.
Moderate and high stringency hybridization conditions are well known in the art (see, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Press, Plainview, N.Y., 1989, especially chapters 9 and 11; and Ausubel F M et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1993). An example of high stringency conditions includes hybridization at about 42° C. in 50% formamide, 5×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured carrier DNA followed by washing two times in 2×SSC and 0.5% SDS at room temperature and two additional times in 0.1×SSC and 0.5% SDS at 42° C.
Furthermore, within the context of the present invention, it will be understood that a protein (such as an antibody molecule, but also applicable to other proteins (e.g., in the context of binding other proteins or DNA)) “binds selectively” to a target (e.g., an antigen, target DNA, etc.) if it binds to the target to a significantly higher degree than it binds to another substance (e.g., another protein or a non-specific DNA sequence). Those of ordinary skill will understand this concept as being a hallmark of immunoglobulin-antigen interaction or the interaction between a DNA-binding protein and its target consensus sequence.
One aspect of the present invention relates to proteins and nucleic acids (DNAs or RNAs) derived from the newly-identified MCV. In this respect, one embodiment of the present invention provides an isolated or substantially purified nucleic acid molecule comprising at least about 6 contiguous nucleotides of a MCV genomic DNA sequence. The complete genomic DNA sequence of MCVs from four tissue sources (MCV 350 (SEQ ID NO:1), MCV 339 (SEQ ID NO:2), MCV 352 (SEQ ID NO:3) and MCV MKLI (SEQ ID NO:4) are set forth in the Sequence Listing. Amino acid sequences for the VP1, VP2, VP3, T-1, T-2, T-3, T-4, and T-5 proteins, as well as their coding DNA sequences, are set forth in the Sequence Listing as SEQ ID NOs:5-20. DNA sequences for MCVs isolated from small cell lung carcinomas are set forth as SEQ ID NOs:21-45 in the Sequence Listing. Furthermore, possible ORFs of the MCV 350 genome (SEQ ID NO:1) are displayed in FIGS. 7 and 8. With reference to the genome itself (SEQ ID NO:1), these ORFs are further identified in the following table A:










TABLE A







Sense Strand
Antisense Strand









124 to 756 length = 633
C 5386 to 5294 length = 93



330 to 410 length = 81
C 5324 to 5064 length = 261



362 to 469 length = 108
C 5266 to 5141 length = 126



470 to 595 length = 126
C 5148 to 4393 length = 756



591 to 695 length = 105
C 5068 to 4973 length = 96



623 to 715 length = 93
C 4846 to 4760 length = 87



716 to 865 length = 150
C 4718 to 4620 length = 99



780 to 860 length = 81
C 4433 to 3156 length = 1278



835 to 1536 length = 702
C 4429 to 4250 length = 180



861 to 1403 length = 543
C 4249 to 4094 length = 156



893 to 1132 length = 240
C 4182 to 4090 length = 93



1319 to 1501 length = 183
C 3975 to 3874 length = 102



1404 to 3080 length = 1677
C 3742 to 3638 length = 105



1537 to 1626 length = 90
C 3222 to 3016 length = 207



1834 to 1959 length = 126
C 3205 to 3122 length = 84



2023 to 2175 length = 153
C 3125 to 2949 length = 177



2132 to 2227 length = 96
C 3073 to 2993 length = 81



2237 to 2323 length = 87
C 3006 to 2914 length = 93



2275 to 2364 length = 90
C 2965 to 2834 length = 132



2365 to 2445 length = 81
C 2862 to 2749 length = 114



2414 to 2491 length = 78
C 2735 to 2652 length = 84



2753 to 2881 length = 129
C 2703 to 2530 length = 174



2818 to 2973 length = 156
C 2623 to 2354 length = 270



2986 to 3129 length = 144
C 2529 to 2410 length = 120



3047 to 3133 length = 87
C 2441 to 2361 length = 81



3134 to 3319 length = 186
C 2349 to 2233 length = 117



3162 to 3311 length = 150
C 2284 to 2135 length = 150



3283 to 3381 length = 99
C 2232 to 2128 length = 105



3382 to 3465 length = 84
C 2114 to 2019 length = 96



3450 to 3584 length = 135
C 2014 to 1919 length = 96



3523 to 3657 length = 135
C 1998 to 1906 length = 93



3642 to 3770 length = 129
C 1905 to 1804 length = 102



3802 to 3885 length = 84
C 1873 to 1640 length = 234



3863 to 4012 length = 150
C 1667 to 1533 length = 135



4006 to 4128 length = 123
C 1588 to 1400 length = 189



4017 to 4097 length = 81
C 1392 to 1222 length = 171



4195 to 4443 length = 249
C 1387 to 857 length = 531



4209 to 4301 length = 93
C 1274 to 1182 length = 93



4370 to 4567 length = 198
C 1221 to 922 length = 300



4467 to 4544 length = 78
C 911 to 825 length = 87



4545 to 4646 length = 102
C 856 to 761 length = 96



4589 to 4873 length = 285
C 760 to 677 length = 84



4737 to 4850 length = 114
C 725 to 645 length = 81



4935 to 5030 length = 96
C 711 to 601 length = 111



4946 to 5134 length = 189
C 652 to 560 length = 93



5131 to 5292 length = 162
C 641 to 474 length = 168



5205 to 5336 length = 132
C 591 to 466 length = 126



5216 to 5320 length = 105
C 473 to 318 length = 156




C 465 to 310 length = 156




C 309 to 208 length = 102




C 263 to 147 length = 117




C 256 to 134 length = 123




C 201 to 67 length = 135




C 146 to 63 length = 84




C 85 to 8 length = 78










Exemplary MCV genomic DNA sequences from which the inventive nucleic acid can be derived from include, but are not limited to, SEQ ID NOs: 1-5, 7, 9, 11, 13, 15, 17, 19, and 21-45. Thus, for example, the inventive DNA can include from about 10 to about 20 contiguous nucleotides of such MCV genomic DNA sequences, the majority of contiguous nucleotides of such MCV genomic DNA sequences, substantially all of such MCV genomic DNA sequences, or even including the complete sequence set forth in SEQ ID NOs: 1-5, 7, 9, 11, 13, 15, 17, 19, or 21-45 or other MCV genomic DNA sequence. It will be understood that the invention also includes the complement of such sequences. Furthermore, the invention also includes a nucleic acid that hybridizes under high stringency conditions to such sequences.
As minor differences in sequence are tolerated, so long as they do not impede the function of the nucleic acids, the invention further provides an isolated or substantially purified nucleic acid molecule consisting essentially of at least about 6 contiguous nucleotides of a MCV genomic DNA sequence, such as those discussed herein. Thus, for example, the inventive DNA can consist essentially of from about 10 to about 20 contiguous nucleotides of such MCV genomic DNA sequences, the majority of contiguous nucleotides of such MCV genomic DNA sequences, substantially all of such MCV genomic DNA sequences, or even consisting essentially of the complete sequence set forth in SEQ ID NOs: 1-5, 7, 9, 11, 13, 15, 17, 19, or 21-45 or other MCV genomic DNA sequence. It will be understood that the invention also includes the complement of such sequences as well a nucleic acid that hybridizes under high stringency conditions to such sequences.
In one respect, the inventive isolated or substantially purified nucleic acids can be employed as probes, for example in diagnostic assays for identifying MCV. In this respect, while it has been recited that the inventive nucleic acid can comprise at least about 6 contiguous nucleic acids from an MCV genomic sequence, somewhat shorter contiguous residues can be permitted, if the molecule is nonetheless capable of hybridizing under high stringency to MCV genomic DNA or its complement. Moreover, the length of the probe can vary to be as long as useful for the assay-in-question. Thus, the probe can comprise about 12-15 nucleotides, or can comprise longer sequences, such as about 20 or about 25 nucleotides, if desired. Of course, a probe also can have sequences other than MCV sequences, such as restriction endonuclease consensus recognition sequences to facilitate cloning.
In other respects, the inventive the inventive isolated or substantially purified nucleic acids can be employed as agents to interfere with viral replication. Thus, the inventive nucleic acid can be or comprise an oligodeoxynucleotide, siRNA molecule, or other suitable type of polynucleic acid. Such molecules can include standard modifications to structure or employed modified sequences/nucleotides (e.g., generation of hairpins, use of triphosphate modified dNTPs, etc.) to enhance activity or stability of such molecules.
In yet further aspects, certain of the inventive nucleic acids encode MCV proteins and polypeptides, and the invention provides such encoding nucleic acids, as well as nucleic acids which complement such or which hybridize to such under high stringency. Thus, for example, the inventive isolated or substantially purified nucleic acid molecule can encode all or a portion of an MCV polypeptide. Examples of some such polypeptides include SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and any open reading frame (ORF) of SEQ ID NOs: 1-4. ORFs of SEQ ID NO:1 are represented in table A, but putative ORFs of SEQ ID NOs: 2-4 can be deduced by those of ordinary skill in the art. Typically, the nucleic acid will encode a polypeptide that includes at least about 4 or about 5 contiguous nucleic acid residues of an MCV protein, and often substantially more contiguous nucleic acids from such proteins (e.g., at least about 10 contiguous nucleic acids or at least about 25 contiguous nucleic acids or even at least about 50 contiguous nucleic acids from such proteins). In some preferred embodiments, the amino acid encodes an MCV protein comprising at least about 4 or about 5 or at least about 10 contiguous amino acids of the amino-terminal 258 sequence of an MCV T-1 polypeptide (one example of which is set forth at SEQ ID NO:12). Of course, the nucleic acid can encode a polypeptide comprising the majority of contiguous amino acids from such proteins, such as all or substantially all of such MCV proteins.
For embodiments in which expression of the inventive nucleic acid is desire, the nucleic acid molecule can be placed into a suitable genetic context to promote expression. Thus, in one embodiment, the invention provides a composition of matter comprising an expression cassette comprising a nucleic acid as herein described in operable linkage to a second nucleic acid having an expression control sequence. An “expression control sequence” is any nucleic acid sequence that promotes, enhances, or controls expression (typically and preferably transcription) of another nucleic acid sequence. Suitable expression control sequences include constitutive promoters, inducible promoters, repressible promoters, and enhancers. Examples of suitable promoters include the human cytomegalovirus (hCMV) promoters, such as the hCMV immediate-early promoter (hCMV IEp), promoters derived from human immunodeficiency virus (HIV), such as the HIV long terminal repeat promoter, the phosphoglycerate kinase (PGK) promoter, Rous sarcoma virus (RSV) promoters, such as the RSV long terminal repeat, mouse mammary tumor virus (MMTV) promoters, or the herpes thymidine kinase promoter, promoters derived from SV40 or Epstein Barr virus, and the like.
The expression cassette can be placed within a larger nucleic acid and can include other elements (e.g., sequences for controlling replication, polyadenylation sequences, restriction endonuclease cleavage cites, IRES sites, other expression cassettes (such as encoding proteins conferring resistance to a toxin or other selectable marker) and the like. The expression cassette can be constructed by any suitable methodology, which is known to ordinary skill in the art. For example, a polynucleotide encoding an MCV protein as herein described can be ligated within a suitable distance of a promoter, and the entire cassette can be further ligated into a desired plasma backbone. Thereafter the construct can be propagated, further engineered, and/or expressed within a suitable expression system as desired.
It will be further understood that the inventive polynucleic acid (including expression cassettes) can be incorporated within gene transfer vector. Such a vector can facilitate transfer of an expression cassette into a cell, for example. Alternatively, the vector can facilitate transfer of an interfering oligonucleotide or siRNA into a cell, in conjunction, for example, with a protocol for inhibition of viral replication or expression. The inventive polynucleotide can be incorporated into any suitable vector system, such as plasmids, cosmids, YACs, viral vector systems (e.g., adenovectors, HSV vectors, retroviral vectors, etc.), which are known to those of ordinary skill in the art. Also, methods of constructing such vectors (e.g., via recombinant DNA technology) and of growing and propagating such vectors (e.g., using suitable host cells) are known to those of ordinary skill in the art.
Another embodiment of the present invention provides isolated or substantially purified MCV proteins and polypeptides. The isolated and substantially purified proteins and polypeptides of the present invention can be employed to develop antibodies, or as reagents in diagnostic assays. Examples of some such polypeptides include SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and that encoded any open reading frame (ORF) of SEQ ID NOs: 1-4. ORFs of SEQ ID NO:1 are represented in table A, and encoded proteins are set forth in FIG. 8, but putative ORFs of SEQ ID NOs: 2-4 and their encoded proteins can be deduced by those of ordinary skill in the art. Typically, the inventive polypeptide includes at least about 5 contiguous nucleic acid residues of an MCV protein, and often substantially more contiguous nucleic acids from such proteins (e.g., at least about 10 contiguous nucleic acids or at least about 25 contiguous nucleic acids or even at least about 50 contiguous nucleic acids from such proteins). In some preferred embodiments, the inventive protein comprises at least about 5 or at least about 10 contiguous amino acids of the amino-terminal 258 sequence of an MCV T-1 polypeptide (one example of which is set forth at SEQ ID NO:12). Of course, the polypeptide can comprise the majority of contiguous amino acids from such proteins, such as all or substantially all of such MCV proteins.
The proteins and nucleic acids of the present invention are isolated or substantially purified in the sense that they are separated from cellular components or mature MCV virions. Thus, in one example, isolated proteins and nucleic acids can exist in substantially (e.g., 90% or more) purified form away from other proteins, polypeptides, and/or nucleic acids. However, it is possible for the inventive proteins and polypeptides to be present in a combination other than found in natural cellular infection or as a mature virion. Thus, for example, the inventive proteins and polypeptides can be present in an artificial virus-like particle (VLP).
The proteins/polypeptides and nucleic acids of the present invention can be produced by standard technologies. For example, particularly with shorter sequences, the inventive proteins/polypeptides and nucleic acids can be produced by solid-state synthesis. However, it will be understood than an efficient method of synthesis involves recombinant DNA technology coupled with (in the case of polypeptides) in vitro or in vivo translation technology. In many aspects, it is preferable for the inventive polypeptides and proteins to be synthesized using a eukaryotic synthesis system (e.g., CHO cells), to achieve desirable folding of the amino acid chain and desirable glycoslation patterns.
As noted herein, the MCV proteins and polypeptides can be employed to produce antibody molecules, which are useful regents for diagnostic assays and potential therapeutic agents. Thus, in another aspect, the present invention provides an antibody preparation containing antibody molecules directed against MCV proteins, polypeptides, and virions. The antibody molecule that binds specifically to a polypeptide consisting essentially or comprising an amino acid sequence selected from the group of sequences consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and any open reading frame (ORF) of SEQ ID NOs: 1-4 or otherwise to an MCV virion.
The antibody molecules of the present invention typically are immunoglobulins, and can be of any isotype (e.g., IgA, IgE, IgG, IgI, IgM, and the like) or an active fragment that retains specific binding activity (such as Fab fragments and the like). Both polyclonal preparations and monoclonal antibodies can be prepared by standard techniques. Following production, the antibody molecules can be isolated and/or substantially purified, and the nventino provides such antibodies in isolated or substantially purified form.
While not wishing to be bound by theory, it is believed that the T1 antigen ORF acquires a mutation during integration into the host cell chromosome that may be responsible for carcinogenesis. The same mutation results in a truncation of the encoded T1 protein. Thus, it is sometimes desirable for the antibody to bind specifically to the N-terminal portion of the MCV T-1 protein (e.g., to one or more epitopes contained within the N-terminal-most 258 amino acids or so of the MCV T-1 protein. In other embodiments, it is desirable for the antibody molecule to bind specifically to MCV Large T polypeptide (T-1) without specifically binding to the MCV Small T polypeptide (T-2). For diagnostic assays, it also is desirable for the inventive antibodies not to bind specifically to proteins or polypeptides of other polyomaviruses, such as SV-40 Large T antigen.
In another aspect, the invention provides compositions, which include the inventive nucleic acid, polypeptide, and/or antibody. In one embodiment, such a composition can include the inventive nucleic acid, polypeptide, and/or antibody in lyophilized form. Of course, if desired, such a lyophilized composition can include a lyoprotectant, such as sucrose or other agent known to those of ordinary skill in the art. Also, the invention provides a composition comprising the inventive nucleic acid, polypeptide, and/or antibody and a carrier, diluent, or buffer. Such carriers; diluents, and buffers are known to persons of ordinary skill in the art.
In another aspect, the invention provides a virus-like particle (LVP) comprising one or more polypeptides MCV polypeptides selected from the group of polypeptides consisting of VP1, VP2, and VP3. Such particles can be produced by expressing one or more of the VP1, VP2, and/or VP3 proteins in a suitable cell. The protein(s) thereafter will assemble to form VLPs, which can be purified from the producing cells by methods such as employed with purifying VLPs of other polyomaviruses.
While not wishing to be bound by any particular theory, it is believed that MCV VLPs form more efficiently if they comprise VP1 molecules from a MCC strain that has consensus-like polyomavirus VP1 sequences at 288(Asp), 316(Arg) and/or 366(Asp) (See FIG. 22). In this respect, it has been observed that the MCV 350 strain does not form VLPs as efficiently as the MCV 339 strain (MCV 350 has differences at 288(His), 316(Ile) and 366(Asn)). Also, the presence of MCV339-like residues 185(Gln) and 422(Glu) might also be important for efficient formation of VLPs, although these positions are not broadly conserved among polyomaviruses.
The VLPs can be used as carriers for foreign DNA, for example, to facilitate transfection of cells. Thus, the invention provides a composition of matter comprising a MCV VLP and a non-MCV nucleic acid (e.g., DNA). Such compositions can be made by exposing a VPL to the non-MCV nucleic acid under conditions suitable for the VLP to bind the nucleic acid. The inventive VLPs, and their use as vectors, can be accomplished by methods such as are known in the art in connection to other polyomaviruses (see, e.g., Goldmann et al., J Viral Methods. 2000 October; 90(1):85-90, Goldmann et al., J. Virol. 1999 May; 73(5):4465-9, Kosukegawa et al., Biochim Biophys Acta. 1996 May 21; 1290(1):37-45, Lundstig et al., Adv Exp Med. Biol. 2006; 577:96-101, Tegerstedt et al., Anticancer Res. 2005 July-August; 25(4):2601-8, Tegerstedt et al., Cancer Immunol Immunother. 2007 September; 56(9):1335-44, Viscidi et al., Adv Exp Med. Biol. 2006; 577:73-84, Viscidi et al., Clin Diagn Lab Immunol. 2003 March; 10(2):278-85, Yokoyama et al., J Biochem (Tokyo). 2007 February; 141(2):279-86, and Zielonka et al., Virus Res. 2006 September; 120(1-2):128-37).
In another aspect, the invention provides a pharmaceutical preparation comprising a composition including the inventive nucleic acid, protein, antibody, and/or VLP and one or more pharmaceutically-acceptable excipient. Suitable preparations can be formulated for delivery by oral, nasal, transdermal, parenteral, or other routes by standard methodology. In this respect, the excipient can include any suitable excipient (e.g., lubricant, diluent, buffer, surfactant, co-solvent, glidant, etc.) known to those of ordinary skill in the art of pharmaceutical compounding (see, e.g., “Handbook of Pharmaceutical Excipients” (Pharmaceutical Press), Rowe et al., 5th Ed. (2006)).
In another embodiment, the invention provides a method of assaying for MCV exposure in a patient, which can be used to assess past exposure, primary infection, or possibly an MCV-associated cancer in the patient. In accordance with this method, a tissue or fluid sample is obtained from the patient. The sample can be, for example, tissue biopsy, blood, plasma, urine, or other fluid. The sample is then assayed for the presence of one or more MCV molecule(s). Such molecules can be MCV DNA, an MCV polypeptide, or an antibody that binds specifically to an MCV polypeptide or VLP. The assay for DNA can be facilitated by Northern or Southern hybridization or PCR. Assaying for an MCV polypeptide, or an antibody that binds specifically to an MCV polypeptide can be facilitated using common immunohistochemical methods. In any event, a positive test for the presence of the MCV molecule within the sample is indicative of exposure of the patient to MCV. Where the test is conducted on tissue obtained from a tumor, the test can facilitate diagnosis of a carcinoma in the patient. Such assays can be used to diagnose patients with MCV-induced cancers or predict which individuals are at greater risk of developing MCV-induced cancers. It is possible that high-level MCV infection causes non-cancer disease symptoms. If so, VLPs might be used as a diagnostic tool for primary MCV disease.
A preferred assay for MCV exposure in a patient is an ELISA, in which the sample from the patient is exposed to one or more purified MCV polypeptides, such as VLPs containing VP1, VP2, and/or VP3. Such assays can be facilitated by high-throughput screening methods employing multi-well places. In this sense, a multi-well place can be coated with MCV protein or VLPs by standard methods, and the invention provides a multi-well (e.g., 96 well) plate coated with MCV protein and/or VLPs.
Another type of assay (a neutralization assay) is facilitated by infectious VLPs. In accordance with such an assay, MCV VLPs are produced such that they encapsulate a reporter construct (e.g., alkaline phosphatase or Gaussia luciferase). It will be observed, that when such VLPs infect cells, the reporter is expressed in the cells and can be readily detected. However, upon exposure of the VLPs to neutralizing antibodies that target MCV prior to exposure to the cells, the titer of VLPs is substantially reduced, leading to the infection of fewer infected cells (and fewer cells expressing the reporter). The neutralization assay can be about 40-fold more sensitive than ELISA for detection of MCV sero-responses. Accordingly, the assay involves producing VLPs that contain a reporter construct, exposing the VLPs to a sample, and then exposing the preparation to cells and assaying for expression of the reporter within the cells. Reduction of reporter expression in comparison to a control indicates the presence of neutralizing antibodies in the sample. In this context, the sample can be obtained from a patient (such as described herein) or a sample of a candidate antibody for clinical use. In this sense, the neutralizing assay can be employed clinically to ascertain patients having immunoreactivity to MCV, or it can be alternatively employed to screen for potential therapeutically-relevant agents targeting MCV (such as immunoglobulins).
In another embodiment, the invention provides a method of identifying an agent that attenuates MCV infection. In this context, attenuation can involve the reduction of likelihood of infection, or reduction in magnitude. In some applications, the reduction can amount to complete prophylaxis. In accordance with this method, target DNA is exposed to an MCV protein (e.g., VP1, VP2, VP3, T-1, T-2, T-3, T-4, and T-5). The target DNA should include a sequence to which the MCV protein can specifically bind relative a negative control DNA. The assay is conducted in the presence of a test agent, which is a putative agent under investigation to assess whether it can attenuate the MCV infection. Thus, the MCV protein and the target DNA are exposed to each other under conditions which, except for the test substance, are suitable for the MCV protein and target DNA to bind. It will be understood that, as a result of this assay, the ability of the test substance to attenuate binding of the MCV protein to the target DNA identifies the test substance as a candidate agent for use as an anti-MCV therapeutic agent. An example of this type of assay is a gel-shift assay, which is known to those of ordinary skill in the art. Also, while the test agent can be identified as a candidate MCV therapeutic agent by this method, other tests likely will be needed to assess whether the agent is safe and effective for clinical use.
In another embodiment, the invention provides a method of identifying an agent that attenuates MCV infection by employing triplex DNA technology. In accordance with this method, a test agent is exposed to MCV DNA, wherein the ability of the test substance to promote the formation of triplex structure within the MCV DNA identifies the test substance as a candidate agent for use as an anti-MCV therapeutic agent. The promotion of triplex DNA can be assessed by standard methods (see, e.g., Havre et al., J Viral. 1993 December; 67(12):7324-3). While the test agent can be identified as a candidate MCV therapeutic agent by this method, other tests likely will be needed to assess whether the agent is safe and effective for clinical use.
In other aspects, the invention involves prophylactic and therapeutic methods against MCV diseases. In this context, the MCV disease can be primary MCV infection or a carcinoma (such as Merkel cell carcinoma, small cell lung carcinoma, or other carcinoma associated with MCV infection). For example, the invention provides a method of vaccinating a patient against an MCV disease. In accordance with this method, a patient is vaccinated with MCC DNA and/or a MCV polypeptide under conditions suitable for the patient to generate an immune response to the MCV DNA and/or MCC polypeptide. A preferred agent for serving as the vaccine is a polypeptide comprising at least 10, and preferably at least the majority of, contiguous amino acids from the N terminus of the MCV T1 protein, particularly contiguous amino acids from among the N-terminal approximately 258 amino acids (see SEQ ID NO:12). Another preferred agent is a VLP as herein described. Indeed, rabbits and mice immunized with MCV can exhibit very high anti-MCV antibody responses, with 50% neutralizing titers in the million-fold dilution range. It will be understood that MCV VLPs could be combined with other viral subunit vaccines such as the current vaccines against hepatitis B virus and human papillomavirus, for combined vaccination protocols.
In another aspect, the invention provides a method for treating a patient suffering from an MCV disease involving adoptive immunotherapy. In accordance with this method, a population of T lymphocytes is first obtained from the patient. Thereafter, the population of T lymphocytes is exposed ex vivo to an MCV polypeptide, including a VLP (such as described herein) under conditions suitable to activate and expand the population of T lymphocytes. For example, the T lymphocytes can be exposed to cells in vitro, which express an MCV polypeptide (e.g., having been transfected with an expression cassette encoding the MCV polypeptide). A preferred MCV polypeptide includes at least 10, and preferably at least the majority of, contiguous amino acids from the N terminus of the MCV T1 protein, particularly contiguous amino acids from among the N-terminal approximately 258 amino acids (see SEQ ID NO:12). In other aspects, the method can be practices using standard techniques. (see, e.g., June, J. Clin. Invest., 117(6) 1466-76 (2007)). After they have been activated, at least some of the T lymphocytes are re-introduced into the patient. Such a method can attenuate the severity of the MCV disease within the patient. It should be understood that the method need not eradicate the MCV disease within the patient to be effective as a therapy. The method can be deemed effective if it lessens symptoms, improves prognosis, or augments other modes of therapy if used adjunctively.
It is believed that the newly-discovered MCV should respond to agents that interferes with the replication of other polyomaviruses. Thus, the invention provides a method of treating an MCV disease by administering such an agent to a patient suffering from an MCV disease. As noted, the MCV disease can be primary MCV infection, Merkel cell carcinoma, small cell lung carcinoma, or another carcinoma that is caused by MCV. It is believed that the administration of some such agents can attenuate the severity of the MCV disease within the patient. Examples of such agents are cidofovir and vidarabine, and other agents that interfere with polyomavirus replication known to those of ordinary skill may be useful in treating such conditions as well. Additional agents include interferons and mTOR inhibitors (e.g., sirolimus and tacrolimus).
It will be understood that the diagnostic therapeutic methods described herein to be performed on a patient can include human patients as well as animals. In this respect, the diagnostic and therapeutic methods can be performed in the veterinary context, i.e., on domestic animals, particularly mammals (e.g., dogs, cats, etc.) or agriculturally-important animals (e.g., horses, cows, sheep, goats, etc.) or animals of zoological importance (apes, such as gorillas, chimpanzees, and orangutans, large cats, such as lions, tigers, panthers, etc., antelopes, gazelles, and others).
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. In summary, they support the possibility that MCV plays a role in MCC tumorigenesis:
1. MCV is integrated into the tumor cell genome. Precisely identical integration sites (confirmed by sequencing) occur in a metastasis (MCC348) and its primary tumor (MCC347). The most likely explanation for this is that the metastatic tumor arose from a single tumor cell already having the virus integration allele.
2. There is an association between MCV and Merkel cell carcinoma. Examination of two different groups of MCC tumors finds them statistically more likely to be positive for virus than control tissues. In our initial analysis, MCV was present in MCC 43-fold more commonly than a convenience sample of tissues from different body sites from different persons. Virus is found only in classic MCC cell lines suggesting that it may be associated with the classic but not variant form of disease. These analyses are based on three independent PCR primers in a blinded and randomized analysis using stringent PCR segregation precautions, and confirmed through two additional qPCR assays.
3. Southern blots can detect MCV in tumors. Direct Southern blotting confirms presence of abundant genome in 8 of 12 tumors and in MCV-positive cell lines. This data indicates that MCV genome is present at sufficient levels to play a biological role in these tumors. Similar data is not available for other polyomaviruses in human cancer. This data is experimentally independent from PCR-based studies and confirms them, demonstrating that the association is not caused by PCR contamination.
4. MCV is monoclonally integrated into MCC genome in 6 of the 8 MCV-positive tumors. Cellular monoclonality is also shown for two tumors (MCC347 and its metastasis MCC348) in which the integration site has been mapped. This suggests MCV infected cells prior to developing into MCC tumors that subsequently underwent monoclonal expansion. This is not consistent with MCV being a passenger virus that secondarily infects MCC tumors.
5. MCV T antigens are expressed in tumor cells. DTS and RACE studies demonstrate viral T antigen gene expression in tumors infected with MCV. Mutations in the T antigen oncoprotein are consistent with animal models of polyomavirus-induced tumors and are not found in non MCC tissues positive for MCV.
EXAMPLE 1
This example demonstrates the identification of a previously-unknown polyomavirus and its integration in Merkel cell carcinoma.
We performed DTS on MCC tissue mRNA and identified T antigen transcripts from a previously undescribed polyomavirus. This virus is closely related to the murine polyoma virus group that includes primate viruses such as the lymphotrophic African green monkey (AGM) polyomavirus (20), and is more distantly related to all known human polyomaviruses including WUV and KIV. The virus is somatically and monoclonally integrated into the human genome, suggesting that it was present prior to tumor cell clonal expansion.
Methods and Materials
Human Tissue Sample Testing:
Human Merkel cell carcinoma tissues were obtained from the Cooperative Human Tissue Network as frozen excess biopsy samples (Supplementary Table 1). All MCC tumors except MCC352 were reconfirmed in our laboratory by H&E and with cytokeratin 20 immunostaining. All MCC tissues except MCC350 were positive for cytokeratin 20. MC350 is MCC metastatic to a lymph node and due to sampling issues we were unable to identify MCC tumor cells the portion of tissue taken for our examination. We relied on the original pathology report as evidence for MCC. Excess surgical tissues used as controls were collected as a consecutive series of anonymized pathology collections from a single operating day (Supplementary Table 1). Confidentiality issues limited the availability of diagnosis for these tissues. Four cases (MCC347, MCC337, MCC343, and MCC346) from 4 men ranging in age from 38 to 84 years were used for DTS.
Generation of cDNA Library for Pyrosequencing:
Total RNA was extracted from MCC tissues using RNEASY MIDI kit (Qiagen, Alameda, Calif.) and treated with DNase I (Ambion, Austin, Tex.) to remove genomic DNA. Integrity of tissue RNAs was analyzed by the AGILENT 2100 bioanalyzer (Quantum Analytics, Foster City, Calif.) using the RNA 6000 Nano reagent kit. mRNA was purified with DYNABEADS mRNA purification Kit (Invitrogen). Double strand cDNA was synthesized with oligo (dT) primer using the SUPERSCIRPTTM Double-strand cDNA Synthesis kit (Invitrogen). Five microgram of MCC cDNA was used for pyrosequencing after confirming cDNA quality on an AGILENT bioanalyzer (Quantum Analytics) at 454 Life Sciences (Roche). The cDNA sample was fractionated into small fragments (300-500 bp) and blunted for ligation of two different adaptors at both ends. These two adaptors provide unique priming sequences for both amplification and sequencing, forming the basis of the single-strand template library for pyrosequencing accordingly. Finally, by GENOME SEQUENCER GS20 system (Roche Diagnostic), large scale sequencing was performed on two cDNA libraries from a single (MCC347) and pooled (MCC337, 343 and 346) cases, respectively (M. Margulies et al., Nature 437, 376 (Sep. 15, 2005)).
Digital Transcriptome Subtraction:
The sequences data from large scale library sequencing were first trimmed using Lucy (H. H. Chou, M. H. Holmes, Bioinformatics 17, 1093 (December, 2001)) with similar Phred scores of 20 or higher (−error 0.01 0.01), and long read over 50 bp (−m 50). Only high quality sequences obtained after Lucy trimming were used for further subtraction with SeqClean. First, Poly(A/T), dust (low-complexity), human repeat and primer adaptors sequences were removed to obtain high fidelity (HiFi) datasets. These HiFi sequences were then aligned against human databases, including human Refseq RNA, mitochondrial and assembled chromosomes, and human immunoglobulin variable sequences with a minimum hit length of 30 bp. The remaining sequences were then aligned to online GenBank nonredundant (NR) using BLASTX program in netblast package.
RACE Analysis on MCV Transcripts:
Both rapid amplification of 5′ and 3′ cDNA ends (RACE) were performed with GENERACER Kit (Invitrogen) according to the manufacturer's instructions. Primers used for RACE are listed in supplementary table 2. To capture the large T antigen, M1-L primer and M3 were used in 5′RACE. M2-L primer and M4 were used as primers in 3′RACE. To capture the small T antigen, small.t.R in intron 1 was used in 5′RACE. Small.t.F and small.t.F.nest were used in 3′RACE. The PCR fragments were isolated from agarose gel and extracted with QIAEX II gel extraction Kit (Qiagen), and ligated in pCR 2.1 vector (Invitrogen) for DNA sequencing.
Consensus PCR for VP1:
Consensus PCR for the VP1 region of Polyomavirus was previously described (R. Johne, D. Enderlein, H. Nieper, H. Muller, J Virol 79, 3883 (March, 2005)). The genomic DNAs from MCC339, MCC344, MCC347, and MCC350 were subjected to PCR amplification by PLATINUM Taq DNA polymerase (Invitrogen) using two sets of VP1 consensus primers, VP1-1 and VP1-2, as in Supplementary Table 2. The cycling conditions for the first PCR was 5 min at 95° C., followed by 45 cycles each of 94° C. for 30 sec, 46° C. for 1 min and 72° C. for 1 min, and final elongation at 72° C. for 10 min. For nested PCR, 4 μl of the first PCR product was used as the template in a similar reaction at 95° C. for 5 min, 45 cycles of 94° C. for 30 sec, 56° C. for 30 min and 72° C. for 30 sec, and 72° C. for 10 min. PCR fragments were recovered from gel, cloned in pCR2.1 cloning vector (Invitrogen) and subjected to nucleotide sequencing. Specific primers (VP1-iF and VP1-iR) for MCV350 VP1 region were designed based on the sequencing results.
MCV Genome Sequencing:
The genome was bidirectionally sequenced with at least 3 fold coverage. Successive outward PCR was performed from the 3′ end of the T antigen sequence to a conserved VP1 site with primer M6 and VP1_iR, and 5′ end of the T antigen sequences to conserved VP1 site with Primer M5 and VP1_iF. Walking primer set (W1 ̃W10) was used to sequence the long PCR product. Second and third rounds of sequencing used 13 pairs of primers (contig 1 ̃contig 13) designed to encircle the entire genome. All PCR reactions were performed with HIGH FIDELITY PLATINUM Taq DNA polymerase (Invitrogen). Primers for genome sequencing are listed in Supplementary Table 3.
Northern Blotting:
Total RNA from 293 cells transfected with pcDNA R339 or R350 containing large T genomic region, were extracted by the TRIZOL (Invitrogen). Northern blotting was performed using 5 μg of total RNA for each sample. RNAs were electrophoresed through 1.2% formaldehyde-agarose gels and transferred onto nitrocellulose membranes (Amersham) with 10×SSC. DNA probes were generated by random prime labeling of (α32P) dCTP (Amersham) on the P1 MCV DNA fragment which contains the exon-1 sequence of MCV T antigen (Supplementary Table 4). Hybridization was performed at 42° C. in 5×SSC, 50% formamide, 5×Denhardt's solution, 2% SDS, 10% dextran sulfate, and 100 mg/ml of denatured salmon sperm DNA (Stratagene). Final rinse of the blots were conducted in 2×SSC/0.1% SDS at 60° C. for 30 min. RNA ladder marker (Sigma) was used as length control.
MCV Detection by PCR-Southern Blotting:
Genomic DNA was extracted by standard phenol-chloroform technique and the quality of the DNA was ascertained by PCR with β-actin primers. One hundred nanograms of genomic DNA was amplified using Taq DNA polymerase (Invitrogen) in a final volume of 50 μl. The cycling condition was 3 min at 94° C., followed by 31 cycles each of 94° C. for 45 sec, 58° C. for 30 sec and 72° C. for 45 sec, and final elongation of 15 min at 72° C. Three different primer sets for the T antigen locus (LT1 and LT3) and VP1 gene (VP1) and corresponding primers for the internal probes of T antigen (M1-M2 and LT5) and VP1 gene (VP1.3) used in Southern blotting are listed in Supplementary Table 4. To avoid potential contamination of template DNA, PCR mixtures were prepared in an isolated room and template DNA was prepared or added in an UV-irradiated clean hood. Recombinant DNA harboring MCV DNA sequence was not amplified at the same time as tissue samples to avoid cross contamination between PCR samples. Negative controls contained all components except DNA template. Both case and control tissue samples were randomized and blinded to the scientist throughout the PCR-Southern testing for MCV positivity.
Genomic DNA Southern Blotting:
Fifteen microgram of each sample, digested overnight with 60 units of restriction endonucleases, were separated on 0.7% agarose gels at 80 volts. Completion of digestion was checked with ethidium bromide staining. Gels were transferred onto nitrocellulose membrane (Amersham) with 10×SSC and hybridized overnight with (α32P) dCTP-labelled probe (2.7×107 d.p.m./ml) at 42° C. Membranes were rinsed in 0.2×SSC/0.5% SDS at 60° C. or 72° C. PCR fragments used for the probe synthesis are listed in Supplementary Table 4. The MCV DNA fragments (LT 1, LT2, P1, P3, P6, P9, and P12), which cover 2.5 kb of non-overlapping MCC 350 genome, were used for Southern blotting in FIG. 4A. For Southern blotting in FIG. 4B, a probe specific for the intron 1 region of the human PTPRG gene (Chr3) was used.
Results and Discussion
Digital Transcriptome Subtraction from Merkel Cell Carcinoma:
To perform DTS, we isolated mRNA from four anonymized MCC tumors from the Cooperative Human Tissue Network (21). One case mRNA (MCC347) was examined separately, while three case mRNAs were pooled (MCC337, 343 and 346) to increase the likelihood for virus discovery (Table S1). We pyrosequenced 216,599 and 179,135 cDNA sequences (−150-200 bp) from these two libraries, respectively. This allowed us to use all high-confidence sequence reads in contrast to our previous DTS analyses with serial analysis of gene expression (SAGE) tags (15). These 395,734 cDNA sequences were trimmed with LUCY stringency equivalent to PHRED scores of 20 or higher (22). Poly(A/T), dust (low-complexity), human repeat and primer adaptor sequences were then removed, leaving 382,747 sequences to form the HiFi dataset. Of these, 380,352 (99.4%) aligned to human Refseq RNA, mitochondrial, assembled chromosomes or immunoglobin NCBI databases. The remaining 2395 sequences were then aligned to GenBank NR using BLASTX.
One transcript (DTS1) from MCC347 aligned over 111 nt. to the DNA binding domain of human BK polyomavirus T antigen [gi:113204635] with 54% identity. The full 201 nt. sequence from this DTS transcript prior to LUCY trimming has highest homology to AGM PyV T antigen [gi:135284] (59% identity over 170 nt., 1×e−12). A second DTS transcript (DTS2) from the T antigen locus was subsequently identified after alignment of candidate HiFi sequences to the full-length viral genome. This fragment corresponds to a unique viral T antigen region with low polyomavirus homology. These two sequences define a new human polyomavirus that we call Merkel cell virus (MCV) because of its close association with Merkel cell carcinoma.
MCV Genome Cloning:
3′-Rapid amplification of cDNA ends (3′-RACE) extended the DTS transcript from case MCC347 to three different cDNAs (FIG. 1B); one transcript terminated at a poly(A) site in the T antigen sequence and two cDNAs read through this poly(A) site to form different length fusions with intron 1 of the human receptor tyrosine phosphatase, type G (PTPRG) gene [gi:18860897] at chromosome 3p14.2. Genomic integration at this site was confirmed by sequencing DNA PCR products from a viral and a PTPRG primer. Identical 3′-RACE cDNA transcripts were independently obtained from MCC348, a metastatic lymph node from MCC347, suggesting that the metastasis was seeded from a clonal tumor cell having the T antigen-PTPRG fusion.
Viral genome walking was successful on DNA from tumor MCC350 (Table S2) providing the complete closed circular genome (5387 bp, prototype) and a second genome, MCV339 (5201 bp), was then cloned and sequenced using specific primers (Table S3). Both viruses have high homology to polyomavirus T antigen, VP1, VP2/3 and the replication origin sequences (FIG. 7). The principal differences between MCV350 and MCV339 being a 201 bp (1994-2184 nt) deletion in T antigen, and a 2 bp (5215-5216 nt) deletion in MCV339 late promoter and a 7 bp deletion (5222-5228 nt.) in the MCV350 late promoter. Excluding these sites, 41 (0.8%) nucleotides differ between MCV350 and 339. In comparison, 1558 HiFi sequences comprising 179,301 nucleotides from the MCC347 dataset were aligned without gaps to known cellular genes in RefSeq RNA database. Only 130 polymorphic nucleotides were found (99.93% concordance), suggesting that high mutation rates are not present in this tumor.
Features of the MCV Genome:
MCV has an early gene expression region (196-3080 nt.) containing the T antigen locus, with large T and small T open reading frames, and a late gene region containing VP1 and VP2/3 open reading frames between 3156 and 5118 nt (FIG. 7). The MCV350 replication origin (5360-69 nt) is highly conserved with seven pentameric T antigen binding sites, including pentanucleotide palindrome and tandem pentanucleotide boxes, a homopolymeric T tract and semiconserved inverted repeats. Comparison of four MCV genes to those of other polyomaviruses show MCV to be highly divergent from known human polyomaviruses and SV40 (FIG. 2). MCV has highest homology to viruses belonging to the MuPyV subgroup and is closely related to AGM PyV (23).
MCV T Antigen Expression:
To examine MCV T antigen transcription, 3′- and 5′-RACE products were sequenced from MCC 339, 347, 348, 349, 350 and 352 RNAs (FIG. 3). These products were compared to results of northern blots and RACE products from 293 cells expressing pcDNA-cloned genomic T antigen (48-3695 nt.) fragments from MCV350 and MCV339. Four T antigen spliced products were identified in tumors that can be assigned to transcripts expressed from the T antigen expression cassettes in 293 cells.
A predicted 2.3 kbase large T transcript (T-1) has near-precise sequence homology to large T domains from SV40 and other polyomaviruses, including pRB1-binding, DnaJ, Bub1-binding and origin-binding domains as well as C-terminal helicase/ATPase A, B1 and B2 motifs (24). Surprisingly, stop codons are present in all large T antigens so far sequenced from tumors (MCV339, 347, 348, 349, 350, 352) that will prematurely terminate this protein at different lengths after the pRB1-binding LXCXE motif (FIG. 3), generally deleting origin-binding and helicase functions. The deletion in MCV339 produces a frameshifted large T antigen, eliminating expression of the highly-conserved helicase/ATPase domain. Given the importance of the origin-binding and helicase domains for replicating episomal virus, these mutations most likely arose after viral integration. This transcript forms the fusion to PTPRG in MCC347/348 but is unlikely to generate a fusion protein due to a stop codon in large T exon 2.
Shorter T antigen proteins are likely to be expressed from other T antigen transcripts that splice out origin-binding and helicase motifs, but all retain the 5′ cr1, DnaJ and LXCXE domains. A small T antigen transcript (T-2) reads through the first splice site. Another T transcript (T-3) generates two downstream splice junctions, reminiscent of SV40 17KT (25). Transcripts with obvious, unique homology to rodent virus middle T sequences were not identified. Genomic integration at different sites within the T antigen locus could also be expected to disrupt full-length gene expression but may still allow protein expression from smaller viral transcripts (e.g., T-2, T-3) predicted to target cell cycle regulatory pathways. Defining the actual T antigen proteins expressed in MCC requires specific antibody panels that do not currently exist. But this initial analysis reveals that MCC tumors have an unexpected level of mutational variation affecting T antigen protein expression.
Merkel Cell Virus in Merkel Cell Carcinomas:
To determine whether MCV is commonly found in human tissue, 59 control DNA samples from various body sites were compared to 12 tissues from 10 MCC patients (Tables 1, S1). All case and control samples were randomized and blinded to the scientist testing and scoring two PCR primer sets in the T antigen locus (LT1 and LT3) and one in the VP1 gene (VP1), followed by Southern blotting with internal probes (Table S4). None of these primer sets amplify plasmid cloned human BK or JC genomic DNA (26, 27).
Of 10 MCC tumors, 7 were positive by PCR using one or more primer sets without Southern hybridization to amplify detection (Table 1). One additional tumor was positive only after PCR-Southern hybridization. In comparison, none of the control tissues were positive by PCR alone but 5 of 59 (8.5%) samples tested weakly positive after PCR-Southern hybridization, giving an odds ratio of 43 (95% confidence interval 7 to 261) for detecting MCV in MCC tumors compared to non MCC tissue samples. Both MCC348 (the metastatic lymph node from tumor MCC347) and MCC338 (tumor infiltrating adjacent skin from MCC339) were positive for MCV genome using multiple PCR primer sets.
In addition to patient samples, common cell lines were tested for the presence of MCV genome (Table 2). Of four available MCC cell lines, only one cell line (MLK-1) grows in suspension culture and is positive for MCV genome by PCR. The three negative cell lines (MCC1, MCC13, MCC26) are adherent “variant” cell lines that have been shown to have distinct gene expression profiles from classical, suspension MCC cells (18). None of the nonMCC cells show evidence for MCC infection including COS-7 cells containing SV40 genome.
MCV Genomic Integration:
MCV integration can be expected to destroy transmissible virus replication capacity and thus should be a relatively rare event that does not contribute to viral replication fitness. Integration nonetheless is frequent in polyomavirus-induced tumors, for example (28), suggesting that this biological accident contributes to polyomavirus tumorigenesis—similar to well-characterized papillomavirus integration in cervical cancer (29).
Genomic integration can be exploited to examine the origins of MCV-infected tumor cells. If tumor DNA is digested with single-cutter restriction endonucleases, such as EcoRI or BamHI, and Southern blotted with viral sequence probes, four different patterns can be predicted: 1) If virus exists as closed circular episomes or integrated viral concatemers, then a  ̃5.4 kbase band will be present, 2) if MCV integrates polyclonally—as might to occur during secondary infection of the tumor—then diffuse hybridization representing different band sizes is expected, 3) if MCV preferentially integrates at one or a few sites, then tumors will have identical or near identical non-5.4 kbase banding patterns, or 4) if MCV integrates at different places in the human genome prior to tumor clonal expansion, distinct bands of different sizes will be present (monoclonal viral integration).
Eight of 11 MCC DNA (including MCC348 metastasis from MCC347) show robust MCV hybridization after BamHI and EcoRI digestion (FIG. 4). These same tumors are also positive by MCV PCR (Table 1). Monoclonal viral integration is evident with one or both enzymes in six tumors: MCC339, 345, 347, 348, 349 and 352 (closed arrows). EcoRI digestion of MCC339, for example, results in two distinct 7.5 and 12.2 kbase bands that can only arise if MCV is integrated at a single site in the bulk of the tumor mass. MCC344 and 350 bands, in contrast, are consistent with episomal virus (open arrow), whereas MCC352 has a predominantly episomal or concatenated-integrated pattern but also clear monoclonal integration bands on BamHI digestion (T antigen sequencing from MCC350, 352 failed to identify a replication-competent T antigen). The banding patterns for MCC347 and its metastasis, MCC348, are identical, consistent with 3′-RACE results (FIG. 1B). Of the three Southern blot negative cases, two were negative by PCR-Southern (MCC343 and 346) and the third was weakly positive with only one PCR primer set (MCC337).
Mapping the human genomic integration site (PTPRG locus on chromosome 3p14) for MCC347 and 348 allows us to directly confirm these results. NheI-SacI digestion of MCC347 is predicted to generate a 3.1 kbase fragment from the wild-type allele and a 3.9 kbase fragment from the MCV-integrated allele. As seen in FIG. 4B, the virus-integrated allele is present in MCC347 and MCC348 DNA, but not control tissues, when probed with a flanking human PTPRG sequence probe. Hybridization with a MCV T antigen sequence probe generates the same 3.9 kbase band in MCC347 and MCC348, consistent with both cellular and viral monclonality in this tumor. These results together with those in FIG. 4A indicate that MCV infection and genome integration often occurs prior to clonal expansion of the MCC tumor.
A Potential Role for MCV in MCC: Our results demonstrate an intimate association between this new human polyomavirus and Merkel cell carcinoma. Determination of causality must await confirmation but our results suggest that MCV is present in most MCC tumors prior to their monoclonal expansion. There are a number of unresolved and interesting questions. If MCV plays a role in MCC tumorigenesis, we do not know whether MCV T antigen expression, insertional mutagenesis or both are contributing to the tumor phenotype. The PTPRG gene is suspected to be a tumor suppressor locus (30) and MCV integration may disrupt its expression. Clonality studies (FIG. 4A) indicate that MCV integration occurs at other sites and it is possible that dysregulation of the PTPRG pathway is one of several complementing mutations that contribute to MCC.
The potential role for MCV T antigen in tumorigenesis is complex but may lead to critical insights into viral carcinogenesis and immunity. Virus-induced tumors are generally rare biological accidents that do not benefit viral transmission (31). We have not yet characterized freely infectious MCV but mutations in tumor-derived MCV paradoxically prevent full-length large T protein from being expressed. This is likely to destroy origin-binding and helicase activities required for free virus replication but unlikely to affect integrated virus. Further, Southern blotting for the viral BamHI-EcoRI fragment spanning the T antigen locus reveals deletions and insertions for several viruses that is also likely to disrupt full large T expression. But these mutations may not affect expression of smaller T antigens (e.g., T-2 and T-3) that retain domains suspected to play a role in cancer cell transformation such as pRB1-interaction and DnaJ domains. These findings demonstrate that tumors strongly select against retention of intact MCV large T antigen. Since polyomavirus T antigens provoke robust cytotoxic immune responses (32), these may represent immune escape mutants selected during tumor evolution.
Identifying a new human tumor virus opens diagnostic, therapeutic and prevention possibilities for tumors like MCC that respond poorly to current therapies. In our study, only eight of 10 MCC tumors had evidence for MCV infection suggesting that MCC may arise from two or more etiologies. This is supported by gene expression and cell culture studies that define MCC into classical and variant types (18). At present, we do not know if this accounts for heterogenous MCV infection in MCC. We also do not know if MCV is a common or uncommon infection of humans. Our PCR analysis only shows that MCV is far more common in MCC tissues than an assortment of nonMCC tissues. Addressing the MCC prevalence in human populations requires development of a sensitive and specific serologic test. Intriguingly, serologic studies led Brade et al. in 1981 to speculate that a virus related to AGM PyV circulates in the human population (23). Caution is needed in interpreting this, however, since polyomavirus serologic cross-reactivity has been a source of confusion (5). PCR contamination has similarly plagued human tumor virology studies. Direct Southern blotting in our study, however, shows that MCV genome is present at high copy numbers in most MCC tumors without amplification (FIGS. 4A-B).
Sequencing technology and databases have matured so that direct high-throughput sequencing now can be used to characterize human viral infections. DTS does not depend on sequence homology and we had no a priori expectation that polyomavirus RNA would be present in MCC. This is only practical for hosts, such as humans, in which validated whole genome sequencing has been accomplished. DTS also has the advantage of placing a quantitative upper limit on the abundance of distinguishable viral transcripts when none are found. This does not rule out all forms of infection but it does help to define the possibilities for infection in a tissue sample.
We chose to study an immune-related tumor based on the concept that direct infectious carcinogens express antigenic viral transcripts in each tumor cell (33). MCC is one of the few tumors significantly elevated, a 13-fold increase, among AIDS patients in population-based database cross-matching for AIDS and cancers (33). DTS is less likely to be useful for tumors caused by chronic inflammation or by viruses that do not generate mRNA.
Of the four tumors we chose to initially study, only one was found retrospectively to have significant MCV virus (Table 1). MCV transcripts in this tumor (MCC347) are present at 9 transcripts per million or approximately 2 transcripts per cell. We and others have found that some latent tumor virus infections retard viral protein synthesis and turnover as a means to evade antigenic peptide processing, with correspondingly reduced mRNA transcription (34, 35). Our experience with MCC illustrates that sequencing to <10 transcript per million level on multiple tissue samples should be used, whenever possible, in searching for new human viruses.







TABLE 1





MCV PCR on human tumor (MCC) and control tissues with LT1,


LT3 and VP1 primers, followed by Southern blot hybridization


with internal probes







MCC Cases (n = 10)












Patient
Tissue ID
LT1
LT3
VP1
Summary





1
MCC337
+/−*
−
−
+/−


2
MCC338**
+
+
+
+



MCC339
+
+
+
+


3
MCC343
−
−
−
−


4
MCC344
+
+
+
+


5
MCC345
−
+
−
+


6
MCC346
−
−
−
−


7
MCC347
+
+
−
+



MCC348***
+
+
−
+


8
MCC349
+
+
+/−
+


9
MCC350
+
+
+
+


10 
MCC352
ND****
+
+
+











No. of Positives (%)
6/9(66.7)
7/10(70)
7/10(70)
8/10(80)










Control Cases (n = 59)











Positive Tissues
LT1
LT3
VP1
Summary





Appendix
+/−
+/−
+/−
+/−


Appendix
−
+/−
+/−
+/−


Gall Bladder
+/−
−
−
+/−


Bowel
−
+/−
+/−
+/−


Hemorrhoid
−
−
+/−
+/−


No. of Positives (%)
2/59(3.4)
3/59(5.1)
4/59(6.8)
5/59(8.5)





* +/−: Signal positive only after Southern hybridization of PCR products.


** MCC338, non-tumorous skin tissue adjacent to MCC339.


***MCC348 Metastatic lymph node from MCC347.


****ND Not Determined













TABLE 2







MCV PCR detection in various cell lines


Cell Lines












Name
Origin
LT1
LT3
VP1
Summary





293
Human embryonic kidney
−
−
−
−


COS7
SV40-transfected African
−
−
−
−



green monkey kidney


HT1080
Human fibrosarcoma
−
−
−
−


MCF7
Human breast cancer
−
−
−
−


MCC1
MCC
−
−
−
−


MCC13
MCC
−
−
−
−


MCC26
MCC
−
−
−
−


MKL1
MCC
+
+
+
+
















SUPPLEMENTARY TABLE 1







Clinicopathological data for MCC patients.


MCC Cases



















Cyto-


Patient
Tissue ID
Age
Sex
Race
Phenotype
keratin 20
















1
MCC337
84
Male
White
Malignant
—


2
MCC338*
79
Male
White
Normal
—



MCC339



Malignant
—


3
MCC343
79
Male
White
Malignant
—


4
MCC344
57
Male
White
Malignant
—


5
MCC345
77
Male
Black
Malignant
—


6
MCC346
38
Male
Unknown
Malignant
—


7
MCC347
56
Male
White
Malignant
—



MCC348**



Malignant
—


8
MCC349
58
Female
White
Malignant
—


9
MCC350
58
Male
White
Malignant
—


10
MCC352
58
Male
White
Malignant
ND***





*MCC338 is non-tumorous skin tissue adjacent to MCC339 tumor.


**MCC348: Metastatic lymph node from MCC347.


***ND: Not Determined
















Control tissue types used in the study (MCV PCR-):


















Colon
5



Small Bowel
  3 (1)



Hemorrhoid
  1 (1)



Gall Bladder
  7 (1)



Appendix
  9 (2)



Mouth
1



Vein
2



Heart
1



Kidney
1



Skin
9



Hernia
2







Hematolymphoid tissues










Lymph node
1



Tensil
5



B cell CLL
1



Myeloid hyperplasia
1



Posttransplant
1



lymphoma



HIV+ large cell
1



lymphoma







Miscellaneous tissues










Lipoma
1



Fibrous tissue
2



Fistula track
1



Meningioma
1



Breast cancer
1



Lung cancer
1



Prostate
1

















SUPPLEMENTARY TABLE 2







Primers used for the MCV cloning.















SEQ






ID


Name
Position*
Purpose
Sequence
NO














M1L
1894-1864
5′-RACE
TTCTCTTGCAGTAATT
46





TGTAAGGGGACTTAC



 


M3
1848-1827
5′-RACE
TTTCAGGCATC
47





TTATTCACTCC



 


M2L
1707-1734
3′-RACE
AGCAGGCATGCCTG
48





TGAATTAGGATGTA



 


M4
1784-1805
3′-RACE
TTTTTGCTCTA
49





CCTTCTGCACT



 


small.t.R
562-530
5′-RACE
TAATACAAGCGCACTTA
50





GAATCTCTAAGTTGCT



 


small.t.F
442-473
3′-RACE
TTTCCTTGGGAAGAAT
51





ATGGAACTTTAAAGGA



 


small.t.
496-517
3′-RACE
GCTAGATTTTG
52


F.nest


CAGAGGTCCTG



 


VP1-1F

VP1 
CCAGACCCAACT
53




Consensus 
ARRAATGARAA





PCR




 


VP1-1R

VP1 
AACAAGAGACACA
54




Consensus 
AATNTTTCCNCC





PCR




 


VP1-2F

VP1 
ATGAAAATGGGGTTG
55




Consensus 
GCCCNCTNTGYAARG





PCR




 


VP1-2R

VP1 
CCCTCATAAACCCGA
56




Consensus 
ACYTCYTCHACYTG





PCR




 


M6
1827-1848
Genome 
GGAGTGAATAA
57




Cloning
GATGCCTGAAA



 


VP1iR
3480-3461
Genome 
ATGGGTGAAA
58




Cloning
AACCCCTACC



 


M5
1796-1770
Genome 
GGTAGAGCAAAAAT
59




Cloning
TCTTAATAGCAGA



 


VP1iF
3508-3527
Genome 
CTAGGCAACC
60




Cloning
CATGAAGAGC





*Nucleotide position is based on MCV350 genome













SUPPLEMENTARY TABLE 3







Primers used for genome sequencing















SEQ






ID


Name
Position*
Purpose
Sequence
NO














W1
 411-4130
Primer
ACTCTTGCCA
61




walking
CACTGTAAGC



 


W2
1290-1272
Primer
CAGGGGAGGA
62




walking
AAGTGATTC



 


W3
4268-4288
Primer
GGGTAATGCTA
63




walking
TCTTCTCCAG



 


W4
946-929
Primer
TATTCGTAT
64




walking
GCCTTCCCG



 


W5
4293-4316
Primer
CACAGATAATAC
65




walking
TTCCACTCCTCC



 


W7
5260-5278
Primer
TTATCAGTCA
66




walking
AACTCCGCC



 


W8
5294-5312
Primer
TCAATGCCAG
67




walking
AAACCCTGC



 


W9
166-148
Primer
AACAGCAGAG
68




walking
GAGCAAATG



 


W10
96-78
Primer
TCTGCCCTTA
69




walking
GATACTGCC



 


contig1f
5344-5363
overlapping
TTGGCTGCCT
70




contigs
AGGTGACTTT



 


contiglr
518-499
overlapping
CCAGGACCTC
71




contigs
TGCAAAATCT



 


contig2f
354-373
overlapping
GGAATTGAAC
72




contigs
ACCCTTTGGA



 


contig2r
879-860
overlapping
ATATAGGGGC
73




contigs
CTCGTCAACC



 


contig3f
730-749
overlapping
TGCTTACTGC
74




contigs
ATCTGCACCT



 


contig3r
1287-1268
overlapping
GGGAGGAAAG
75




contigs
TGATTCATCG



 


contig4f
1132-1151
overlapping
AGGAACCCAC
76




contigs
CTCATCCTCT



 


contig4r
1641-1619
overlapping
AAATGGCAAAAC
77




contigs
AACTTACTGTT



 


contig5f
1538-1561
overlapping
AAACAACAGAGA
78




contigs
AACTCCTGTTCC



 


contig5r
2088-2069
overlapping
GAGCCTTGTG
79




contigs
AGGTTTGAGG



 


contig6f
1934-1953
overlapping
AGAGGCCAGC
80




contigs
TGTAATTGGA



 


contig6r
2437-2418
overlapping
GCAGCAAAGC
81




contigs
TTGTTTTTCC



 


contig7f
2328-2349
overlapping
TTTGAAAAGAA
82




contigs
GCTGCAGAAAA



 


contig7r
2885-2866
overlapping
TGTATCAGGC
83




contigs
AAGCACCAAA



 


contig8f
2763-2783
overlapping
CACTTTTTCCC
84




contigs
AAAGGCAAAT



 


contig8r
3282-3263
overlapping
TTACCCAAAG
85




contigs
CCCTCTGTTG



 


contig9f
3187-3206
overlapping
GAGGCCTTTT
86




contigs
GAGGTCCTTT



 


contig9r
3687-3667
overlapping
TCAGACAGGCT
87




contigs
CTCAGACTCC



 


contig10f
3599-3618
overlapping
ATAGAGGGCC
88




contigs
CACTCCATTC



 


contig10r
4107-4088
overlapping
TCTGCCAATG
89




contigs
CTAAATGAGG



 


contig11f
3949-3969
overlapping
CCTGACACAGG
90




contigs
AATACCAGCA



 


contig11r
4504-4485
overlapping
GCAAACTCCA
91




contigs
GATTGGCTTC



 


contig12f
4329-4349
overlapping
TTTTGGAACTG
92




contigs
AGGCAACATT



 


contig12r
4829-4810
overlapping
TAACTGTGGG
93




contigs
GGTGAGGTTG



 


contig13f
4765-4784
overlapping
TACCCACGAA
94




contigs
ACATCCCTGT



 


contig13r
5386-5367
overlapping
AGCCTCTGCC
95




contigs
AACTTGAAAA





*Nucleotide position is based on MCV350 genome













SUPPLEMENTARY TABLE 4







PCR Primers and Probes used for MCV detection










Name
Position*
Sense (SEQ ID NO)
Antisense (SEQ ID NO)










Primers for diagnostic PCR










LT1
 1514-1953
TACAAGCACTCCACCAAAGC (96)
TCCAATTACAGCTGGCCTCT (97)


 


LT3
 571-879
TTGTCTCGCCAGCATTGTAG (98)
ATATAGGGGCCTCGTCAACC (99)


 


VP1
 4137-3786
TTTGCCAGCTTACAGTGTGG (100)
TGGATCTAGGCCCTGATTTTT (101)


 







PCR Primers for probes in Northern or Southern hybridizations










M1-M2
 1711-1889
GGCATGCCTGTGAATTAGGA (102)
TTGCAGTAATTTGTAAGGGGACT (103)


 


LT5
 253-855
GCTCCTAATTGTTATGGCAACA (104)
TGGGAAAGTACACAAAATCTGTCA (105)


 


VP1.3
 4107-3599
TCTGCCAATGCTAAATGAGG (106)
ATAGAGGGCCCACTCCATTC (107)


 


P1
5344-518
TTGGCTGCCTAGGTGACTTT (108)
CCAGGACCTCTGCAAAATCT (109)


 


P3
  730-1287
TGCTTACTGCATCTGCACCT (110)
GGGAGGAAAGTGATTCATCG (111)


 


P6
 1934-2437
AGAGGCCAGCTGTAATTGGA (112)
GCAGCAAAGCTTGTTTTTCC (113)


 


P9
 3187-3687
GAGGCCTTTTGAGGTCCTTT (114)
TCAGACAGGCTCTCAGACTCC (115)


 


P12
 4329-4829
TTTTGGAACTGAGGCAACATT (116)
TAACTGTGGGGGTGAGGTTG (117)


 


LT2
 1054-1428
CTGGGTATGGGTCCTTCTCA (118)
TGGTGAAGGAGGAGGATCTG (119)


 


Chr.3
 61563308-61563830
TTTCAGACGGAAGCGAAGTT (120) 
ACCACGATTTGGAAAACAGC (121)





*Nucleotide position is based on MCV350 genome


** Nucleotide position is based on NT_022517.17






The publications referenced in this Example are as follows:

    1. L. Gross, Proc Soc Exp Biol Med 83, 414 (1953).
    2. K. A. Crandall, M. Perez-Losada, R. G. Christensen, D. A. McClellan, R. P. Viscidi, Adv Exp Med Biol 577, 46 (2006).
    3. T. Allander et al., J Virol 81, 4130 (2007).
    4. A. M. Gaynor et al., PLoS Pathog 3, e64 (2007).
    5. D. L. Poulin, J. A. DeCaprio, J Clin Oncol 24, 4356 (2006).
    6. J. A. DeCaprio et al., Cell 54, 275 (1988).
    7. D. P. Lane, L. V. Crawford, Nature 278, 261 (1979).
    8. D. I. Linzer, A. J. Levine, Cell 17, 43 (1979).
    9. D. C. Pallas et al., Cell 60, 167 (1990).
    10. M. Cotsiki et al., Proc Natl Acad Sci USA 101, 947 (2004).
    11. D. R. Kaplan, D. C. Pallas, W. Morgan, B. Schaffhausen, T. M. Roberts, Biochim Biophys Acta 948, 345 (1989).
    12. S. M. Dilworth, Nat Rev Cancer 2, 951 (2002).
    13. D. Ahuja, M. T. Saenz-Robles, J. M. Pipas, Oncogene 24, 7729 (2005).
    14. Y. Chang et al., Science 265, 1865 (1994).
    15. H. Feng et al., J Virol 81, 11332 (2007).
    16. Y. Xu et al., Genomics 81, 329 (2003).
    17. B. Lemos, P. Nghiem, J Invest Dermatol 127, 2100 (2007).
    18. M. Van Gele et al., Oncogene 23, 2732 (2004).
    19. E. A. Engels, M. Frisch, J. J. Goedert, R. J. Biggar, R. W. Miller, Lancet 359, 497 (2002).
    20. M. Pawlita, A. Clad, H. zur Hausen, Virology 143, 196 (1985).
    21. V. A. LiVolsi et al., Cancer 71, 1391 (1993).
    22. H. H. Chou, M. H. Holmes, Bioinformatics 17, 1093 (2001).
    23. L. Brade, N. Muller-Lantzsch, H. zur Hausen, J Med Virol 6, 301 (1981).
    24. J. M. Pipas, J Virol 66, 3979 (1992).
    25. J. Zerrahn, U. Knippschild, T. Winkler, W. Deppert, Embo J 12, 4739 (1993).
    26. P. M. Howley et al., J Virol 36, 878 (1980).
    27.1. Seif, G. Khoury, R. Dhar, Cell 18, 963 (1979).
    28. D. Hollanderova, H. Raslova, D. Blangy, J. Forstova, M. Berebbi, Int J Oncol 23, 333 (2003).
    29. M. Durst, A. Kleinheinz, M. Hotz, L. Gissman, Journal of General Virology (1985).
    30. D. M. Pitterle, E. M. Jolicoeur, G. Bepler, In Vivo 12, 643 (1998).
    31. P. S. Moore, Y. Chang, Annu Rev Microbial 57, 609 (2003).
    32. T. D. Schell et al., J Virol 73, 5981 (1999).
    33. J. Parsonnet, in Microbes and Malignancy J. Parsonnet, Ed. (Oxford University Press, New York, 1999) pp. 3-18.
    34. Y. Yin, B. Manoury, R. Fahraeus, Science 301, 1371 (2003).
    35. H. J. Kwun et al., J Viral 81, 8225 (2007).


EXAMPLE 2
This example demonstrates that MCV is significantly more likely to be present in MCC tumors than in control tissues
A second independent set of 8 pathologically-confirmed MCC were randomized and blindly tested; all 8 (100%) were positive for MCV genome, assuring reproducibility of our findings. To examine skin MCV infection, we examined 25 control skin or skin tumor samples from 20 HIV-negative and 5 HIV-positive persons without MCC including another 9 normal skin samples, Kaposi's sarcoma (n=15) and malignant melanoma (n=1). Four tissues (16%, p<0.001) were positive including one normal skin and three KS lesions, all from HIV negative patients. Thus, MCV is significantly more likely to be present in MCC tumors (80-100%) than in control tissues from patients without MCC (8-16%, p<0.001) (see Table 2).







TABLE 3







MCV in MCC and Control Patients by PCR and PCR-Southern









Test Group
PCR only
PCR-Southen





1. MCC (n = 10)*
7/10
8/10 (80%)


2. MCC (n = 8)
8/8
 8/8 (100%)


3. Control, various body sites (n = 59)
0/59
4/59 (8%)**


4. Control, skin and skin tumor (n = 25)
0/25
4/25 (16%)***





*Two additional metatastatic tumors from these patients were also MCV positive


**p < 0.0001 vs. MCC#1; positive tissues included appendix (2/9), gall bladder (1/7), bowel (1/3), hemorrhoid (1/1). Other tissues (all negative) included 9 skin, 6 other GI, 10 lymphoid, 15 other miscellaneous tissues including organ sites (brain, heart, kidney, lung).


***p < 0.001 vs. MCC#1; positive tissues include KS (3/15), normal skin (1/9). Other negative tissue included malignant melanoma (1).






EXAMPLE 3
This example demonstrates that MCV has a lymphotropic infection in asymptomatic individuals
Because of MCV's similarity to African green monkey lymphotropic polyomavirus, we sought to determine if MCV can be detected in peripheral blood from asymptomatic individuals. We have developed qPCR primers that amplify a region of the T antigen and VP1 genes and can be quantified by comparison to cellular RnasP primers.
Using plasmid dilutions, we find precise linearity over a 4-log DNA dilution for these primers (not shown). We performed a pilot study of 29 Multicenter AIDS Cohort Study (MACS) PBMC from HIV-positive participants. Of these 29 PBMC samples, 4 (14%) have >3.5 genome copies/300 ng DNA indicating asymptomatic infection. Sera from these 4 individuals are available in our serum bank and it is evident that additional DNA positive individuals can be readily identified by testing additional individuals with paired PBMC-serum from the MACS repository. A second group of 65 anonymous PBMC collected during routine clinical studies (hence HIV status is unknown) shows that 10 (15%) are MCV positive. Thus, it is likely that MCV is a lymphotropic virus like LPyV. These are initial pilot studies and more detailed and rigorous analysis is needed to determine population rates of infection and possible role of HIV infection in MCV positivity.
EXAMPLE 4
Common cell lines (293, COST, HT1080 and MCF7) were tested and found negative for MCV genome. Five MCC cell lines were tested and two are positive for MCV, providing a renewable source of virus for in vitro studies. Both MCV-positive cell lines have a classic phenotype whereas the three negative MCC cells all belong to the variant phenotype, suggesting the possibility that only classic MCC is infected with MCV (Van Gele et al., Oncogene 2004; 23(15):2732-42). This would be analogous to KSHV in Castleman's disease in which plasmacytic multicentric Castleman's disease are KSHV-positive but not hyaline-vascular Castleman's disease (Soulier et al., Blood 1995; 86:1276-80), qPCR and Southern blotting reveal monoclonal integration of a single MCV copy into the genome of the MCV positive cell lines. Additional patient studies are needed to confirm a relationship between MCV and classic MCC but this may explain why two MCC tumors were found to be negative in our PCR analysis (Table 3).
EXAMPLE 5
This example demonstrates the construction of MCV VLPs.
To produce MCC VLPs, 293TT cells are co-transfected with expression vectors encoding VP1 and VP2 from either MCV 339 or MCV 350. VLPs are then extracted from the cells and purified by ultracentrifugation through an OPTIPREP density gradient; gradient fractions are collected at the bottom of the tube (See FIG. 11). Fractions 6, 7, and 8, depicted in FIG. 11 were selected for the presence of nuclease-resistant encapsidated DNA detected using QUANT-IT PICOGREEN dsDNA reagent (Invitrogen).
FIG. 12 demonstrates the production of VLPs for MCV 339 relative to MCV 350. The top panel shows an anti-MCV Western blot of 293TT cells after transfection with the VP1 expression construct shown, together with an appropriate VP2 expression construct. In the far right lane of the Western, 5-fold more cell lysate was applied to the gel. The bottom panel shows a SYPRO Ruby-stained SDS-PAGE gel analysis of OPTIPREP gradients used to purify VLPs out of cell lysates. For MPyV and MCV399, 2.5 μl each of fractions 6-9 was loaded onto the gel. For MCV350, 12.5 μl each of fractions 6-9 was loaded. Fractions were screened for the presence of encapsidated DNA using PICOGREEN reagent.
These results reveal the production of MCV VLPs from at least one strain of MCV (MCC 339).
EXAMPLE 6
This example demonstrates the construction of a mouse monoclonal antibody (mAb) specific to the MCV large T (LT) antigen.
Methods
Human Tissue Samples.
DNA samples were obtained from excess clinical specimens. All the DNA samples were obtained from fresh frozen tissues. For reasons of confidentiality, minimal patient identification and demographic data are available for most of these specimens. For Merkel cell carcinoma, fresh frozen tumor samples were obtained from the Cooperative Human Tissue Network (CHTN). An MCC tissue core microarray consisting of 36 MCC specimens was generated from archival paraffin-embedded tissues from the pathology departments at Hospital Universitari del Mar and the Hospital Universitari Germans Trias i Pujol, Barcelona, Spain as previously described (17). Tissue microarrays for lymphoid malignancies and normal controls were purchased commercially (US Biomax, Inc.). Genomic DNA samples from consecutive hematolymphoid tumor tissues were collected and archived by the late Dr. Anne Matsushima, Columbia University, from excess tissue submitted for diagnostic pathology. This was supplemented with additional hematolymphoid tissues obtained from tissue banks at the University of Pittsburgh Department of Pathology. PBMC specimens were obtained from two sources: 1) excess samples submitted to the Division of Molecular Diagnostics, University of Pittsburgh Medical Center for genetic screening, and 2) PBMC collected from HIV-positive persons participating in Kaposi's sarcoma epidemiologic studies (13). None of these study subjects were diagnosed with Merkel cell carcinoma. All specimens were tested under University of Pittsburgh Institutional Review Board-approved guidelines.
Isolation of Genomic DNA.
Genomic DNA was extracted using proteinase K/lysis buffer (0.1 M NaCl, 10 mM Tris-HCl pH 8.0, 25 mM EDTA pH 8.0, SDS 0.5%, 200 μg/ul proteinase K) for up to 3 days at 56° C., followed by phenol-chloroform extraction and ethanol precipitation. DNA amount and quality were determined by spectroscopy followed by PCR amplification for cellular β-actin or RNaseP. Real time quantitative PCR (qPCR). qPCR was performed using primers amplifying the MCV T antigen, TAg (1051 to 1131 nt; forward: 5′-cctctgggtatgggtccttctca-3′ (SEQ ID NO:122), reverse: 5′-atggtgttcgggaggtatatc-3′ (SEQ ID NO:123)) and VP2 (4563 to 4472 nt, forward: 5′-agtaccagaggaagaagccaatc-3′ (SEQ ID NO:124), reverse: 5′-ggccttttatcaggagaggctatattaatt-3′ (SEQ ID NO:125)) loci with internal TaqMan probes (TAg: 5′-cccaggcttcagactc-3′ (SEQ ID NO:126), VP2: 5′-gcagagttcctc-3′ (SEQ ID NO:127)) labeled with FAM and MGB quencher (Applied Biosystems). For the additional 10 peripheral blood samples with CLL, primers designed against MCV T antigen promoter region (98 to 184 nt forward: 5′-cccaagggcgggaaactg-3′ (SEQ ID NO:128), reverse: 5′-gcagaaggagtttgcagaaacag-3′ (SEQ ID NO:129)) and internal probe (5′-ccactccttagtgaggtagctcatttgc-3′ (SEQ ID NO:130)) labeled with FAM and BHQ quencher (Biosearch Technologies) was used. Primers were chosen to maximize specificity to MCV and minimize any cross-reactivity with other polyomaviruses. Copy numbers were established from standard curves of Ct values from serial dilutions of known concentrations of MCV DNA originally amplified by PCR using contig 3 andcontig 12 primer sets for TAg and VP2 detections, respectively (1). Water was used as control to detect template contamination. No evidence of PCR template contamination was observed in the PCR reactions with water control. RNaseP (Applied Biosystems) or 13-actin primer-probe mixtures (forward: 5′-cactggctcgtgtgacaagg-3′ (SEQ ID NO:131), reverse: 5′-cagacctactgtgcgcctacttaa-3′ (SEQ ID NO:132), probe: 5′-tggtgtaaagcggccttggagtgtgt-3′ (SEQ ID NO:133)) (Biosearch Technologies) were used to determine cell genome copy number. qPCR reactions were performed using PRISM 7700 Detection System, PRISM 7900HT Fast Real-Time PCR System (Applied Biosystems) and/or SMART CYCLER 5RX4Z01 (Cepheid) with TaqMan reagents (UNG(+) TaqMan Universal PCR Master Mix). All the primers and probes were aliquoted and stored until in an isolated, clean PCR facility to avoid template DNA contamination. Amplification reactions of all target genes were performed in reaction volumes of 20 μl with following condition: 50° C. for 2 min, denaturing at 95° C. for 10 min, then denaturing at 95° C. for 15 s followed by annealing and extension at 60° C. for 1 min, 40 cycles. Results were expressed as numbers of viral copies per cell calculated from Ct values of viral and cellular gene standards (Table 6-1). Cellular viral DNA copy number below 1.0×10 ̃3 per cell was considered to be negative.
Cell Lines and Transfection Conditions.
Human embryonic kidney 293 cells (American Type Culture Collection (ATCC)) used for transfection experiment were grown in DMEM medium supplemented with 10% fetal calf serum. For protein expression analysis, cells were transfected with expression constructs using LIPOFECTAMINE 2000 (Invitrogen) following manufacturer's instructions on 90% confluent cells. Cells were harvested 48 h after transfection for analysis.
Plasmids.
To generate the pMCV TAg-EGFP expression constructs, pcDNA6 gLT206 encoding wild type full length genomic T antigen (4) was digested with Nhe I and Sac II and cloned into pEGFP-N1 (Clonetech) in frame to C terminus GFP using same restriction sites. LT expression constructs for JCV and BKV were kindly provided by Dr. James DeCaprio (18). SV40 T antigen cDNA cloned in pCMV vector is described elsewhere (19).
Generation of CM2B4 mAb.
Monoclonal antibody CM2B4 (IgG2b isotype) was generated by standard methods of immunizing mice with KLH-derivatized SRSRKPSSNASRGA (SEQ ID NO: 134) peptide from the MCV T antigen exon 2 with a C-terminal cysteine (Epitope Recognition Immunoreagent Core facility, University of Alabama). Immunofluorescence and immunohistochemistry. For immunofluorescence staining, cells were spotted on glass slides by CYTOSPIN3 (Shandon), fixed with 10% buffered formalin for 20 min, and permeabilized with phosphate-buffered saline (PBS) with 0.1% Triton X-100. After blocking with 10% normal donkey serum (Jackson ImmunoResearch Laboratories), cells were reacted with CM2B4 (1:100 dilution) at 4° C. overnight followed by secondary antibody (Alexa-595-conjugated anti-mouse, 1:1000 Invitrogen) for one hour at room temperature. Stained cells were mounted in aqueous medium containing DAPI (Vector Laboratories, CA). For immunohistochemical staining of paraffin embedded tissues, epitope retrieval was performed using EDTA antigen retrieval buffer (Dako, Glostrup, Denmark) at 126° C. for 3 min after deparaffinization and hydrogen peroxide treatment. After blocking with PROTEIN BLOCK (Dako), samples were reacted to primary antibody for 30 min at room temperature with dilutions described below. After washing, samples were incubated with Mouse ENVISION Polymer (Dako) for 30 min at room temperature for subsequent deaminobenzidine (DAB) reaction. mAbs used for immunohistochemistry were: CM2B4 (1:10-1:50 hybridoma supernatant), CK20 (Dako; 1:50), Chromogranin A (Dako, 1:600), Synaptophysin (Biogenex, San Ram6n, Ca, USA; 1:100), and CD56 (Novocastra, Newcastle upon Tine, UK; 1:50).
In-Situ Hybridization.
Tissue sections were deparaffinized, dehydrated, incubated at 95° C. for 20 min, cooled for 5 min at room temperature and reacted over night at 37° C. with JC virus BIOPROBE labeled probe (Enzo Life Sciences) diluted in hybridization buffer. Excess probe was washed with 2×SSC/0.75% BSA followed by PBS. To visualize signal, samples were treated with ABC elite solution (Vector Laboratories, CA) for 30 min, washed twice with PBS and reacted with DAB solution. Samples were counterstained with Shandon hematoxylin.
Immunoblotting.
Transfected cells from 6 well plate were lysed in 120 μl of lysis buffer (10 mM Tris-HC1 pH8.0, 0.6% SDS) containing proteinase inhibitor cocktail (Roche). 12.5% of lysate was electrophoresed in 10% SDS-PAGE, transferred to nitrocellulose membrane (Amersham). Membranes were blocked in 5% skim milk for 1 h, reacted with Pab416 (1:10) or CM2B4 mAb (1:10) for overnight at 4° C., followed by anti-mouse IgG-HRP conjugates (Amersham, 1:5000) for 1 h at room temperature. Detection of peroxidase activity was performed by WESTERN LIGHTNING PLUS-ECL reagent (Perkin Elmer).
Cell Sorting.
5.0×106-107 PBMC were washed with PBS twice, stained with CD3-FITC, CD20-PE and CD14-PC5 (IOTest) (2 μl/10×106 cells) and incubated on ice for 20 minutes. The cells were then washed and resuspended in 400 μl of PBS containing 8.0 μg/ml DAPI and sorted by the MOFLOW High Speed Sorter (Cytomation). Single antibody stained and unstained cells were used as controls for compensation purposes. DNA from the sorted cell fractions was extracted using QIAAMP BLOOD MINI KIT (Qiagen).
Results
MCV and T Antigen Expression in Merkel Cell Carcinoma Tumors.
We developed a mouse monoclonal antibody (mAb) (CM2B4) to the peptide epitope (SRSRKPSSNASRGA (SEQ ID NO:134)) in exon 2 of the MCV T antigen. This epitope is N-terminal to an LFCDE motif previously found to bind retinoblastoma protein and is likely to be conserved in viruses from both tumor and nontumor tissues (4). There was precise nuclear colocalization of CM2B4 staining with MCV LT-GFP fusion protein fluorescence when a MCV LT-GFP plasmid was expressed in 293 cells (FIG. 9A).
CM2B4 was highly specific for MCV and did not react to T antigens from JCV or BKV by immunofluorescence (FIG. 9B) or to T antigens from JCV, BKV or SV40 by immunoblotting (FIG. 9C). In contrast, an anti-SV40 T antigen mAb, PAb416, cross-reacts with T antigens from other SV40-group viruses including JCV and BKV, but not with MCV T antigen. We examined 22 other anti-SV40 T antigen mAbs (Table S1), and none showed reactivity to MCV T antigen on immunoblotting (data not shown). CM2B4 also did not react to JCV T antigen in a brain biopsy of JCV-infected progressive multifocal leukoencephalopathy (PML) (FIG. 9D).
The MCV LT protein was detected at 120 kDa on immunoblotting of lysates from cells transfected with the genomic T antigen expression construct (FIG. 9C). An additional 60 kDa band may represent an alternatively spliced T antigen isoform (4, data not shown). Lysates of the MCV positive MKL-1 cell line were positive for T antigen expression while T antigen bands were absent from MCV negative UISO, MCC13, and MCC26 cell lines (4) (FIG. 9E).
Immunohistochemical staining of MKL-1 cells showed expression of LT protein predominantly in a diffuse nuclear pattern (FIG. 10A). Examination of a MCV positive MCC biopsy (MCC349) showed similar strong reactivity with CM2B4 among tumor cells, but not surrounding tissues including the epidermis, adnexal epithelia, endothelial cells, or dermal fibroblasts (FIG. 10B). CK20, a low molecular weight cytokeratin marker for MCC (9, 10) was present in a characteristic perinuclear dot-like pattern in CM2B4 positive cells (FIGS. 10A and B).
These results were extended by examining a tissue microarray containing 30 CK20-positive MCC, 6 CK20-negative but clinically-suspect MCC and 4 CK20-negative neuroendocrine control tumors (2 small bowel, one bladder and one lung derived). Of the 30 CK20 positive MCC, 21 (70%) were positive for LT protein expression. Of the 6 CK20-negative tumors diagnosed as MCC, none were positive with CM2B4. These six tumors had clinical appearances consistent with MCC and expressed neuroendocrine markers CD56, synaptophysin, or chromogranin (Table 6-1). These results suggest that most CK20-positive MCC express MCV LT in tumor cells.
To screen tissues for MCV infection, we next developed a quantitative real-time PCR (qPCR) assay to determine the burden of virus infection in MCC tumors (Table 6-2). Ten tumors previously examined by PCR and Southern blotting (1) were examined with primers designed to amplify regions of the T antigen and VP2. Seven of these Southern blot positive tumors had an average of 5.2 (range 0.8 to 14) T antigen DNA copies per cell. Consistent results were found using VP2 qPCR except for MCC345 and MCC347. This tumor had robust T antigen qPCR positivity (5 copies per cell) but minimal amplification of the VP2 gene, which may reflect integration and loss of this late viral capsid gene region. These findings were confirmed by CM2B4 staining in CK20+ MCCs, which was concordant with qPCR results for all cases except MCC344 (Table 6-2). This case showed abundant viral DNA but was negative with CM2B4 staining.
PBMC Infection with MCV.
83 whole PBMC DNA samples collected from persons undergoing genetic testing for Factor V Leiden deficiency were tested by qPCR. These samples were collected mainly from adults (average of 60 yrs, range 1-78 yrs) with 73 (88%) samples from persons over 18 years of age. None were positive for viral gene PCR products. Among 21 PBMC collected from adult HIV/AIDS patients without MCC, 2 (9.5%) were positive by either T antigen (2.8×10−3 copies per cell) or VP2 (8.8×10−3 copies per cell) primers and one (5%) was positive with both primers (T antigen, 7.9×10−3 copies per cell; VP2, 6.0×10−3 copies per cell). These levels approach the technical limit of reproducibility of our assay (>10−3 copies per cell).
This should not be interpreted as evidence against current infection among these participants. PBMC from two MCV-positive MCC patients and from three healthy blood donors were sorted into CD20+ (B cell), CD3+ (T cell), CD14+ (monocyte) and CD20−/CD3−/CD14− (remainder) fractions and tested for T antigen and VP2 DNA. CD20+ B cell fractions from both MCC patients were positive for both T antigen and VP2 (2.7−9.8×10−3 DNA copies per CD20+ cell). In contrast, CD20+ cell fractions from two of three blood donors were only positive for the VP2 locus (1.2−2.1×10−3 DNA copies per CD20+ cell) but not MCV T antigen. CD3+ T cells from one MCC patient were positive at lower levels for both viral loci (1.0−2.0×10−3 copies per CD3+ cell). Either T antigen or VP2 DNA but not both were also detected in CD14+ cells from one MCC patient (T antigen: 1.2×10−3 copies per CD14+ cell), CD20+ cells from two blood donors (VP2: 1.2−2.1×10−3 copies per CD20+ cell). All other fractions from MCC and blood donor patients were either negative or below the threshold value for a positive result. We interpret these results to indicate that mainly B cell fractions from infected persons can harbor low copy MCV but dilution of this fraction in whole PBMC reduces MCV below our reliable threshold for detection.
Survey of Hematolymphoid Malignancies for MCV Infection.
qPCR was performed on DNA from 104 T cell-associated and 161 B cell-associated malignancies, 19 myeloid disorders and 41 other tumors including Hodgkin lymphoma and post-transplant lymphoproliferative disorders (Table 6-3). Of these 325 tumors, 7 (2.2%) were positive for either T antigen or VP2 DNA and two were positive for both. No consistent pattern of virus infection was found among these malignancies: 1 (3%) of 33 chronic lymphocytic leukemia, 1 (7.1%) of 14 non-Hodgkin lymphoma, not otherwise specified (NOS), 2 (3.1%) of 65 diffuse large B cell lymphoma, 1 (11%) of 9 marginal zone lymphoma and 1 (3.3%) of 30 Hodgkin lymphoma (Table 6-3). Copy numbers for these positive hematolymphoid malignancies (Table 6-4), however, were all 2-4 logs lower than MCV-positive MCC tumors (Table 6-2).
These results were confirmed by CM2B4 staining of commercial tissue microarrays of hematolymphoid tumors. Of 122 B cell lymphomas, 17 T cell lymphomas, one myeloid disorder and 2 Hodgkin lymphomas examined, none showed evidence for LT protein expression (Table 6-5). Thirty-one healthy lymphoid control tissues were also negative for MCV T antigen.
MCV and Chronic Lymphocytic Leukemia.
Given the epidemiological relationship between MCC and CLL, we examined additional CLL cases for evidence of MCV infection. Ten peripheral blood samples with CLL (WBC counts ranging from 13.2×109-84.3×109 cells per L) were harvested and tested for the presence of MCV DNA. One displayed low MCV positivity VP2 (2.0×10−3 copies per cell). Twelve additional paraffin-embedded biopsies with CLL were examined by CM2B4 staining. All CLL cases were uniformly negative for MCV T antigen protein expression.







TABLE 6-1







Summary of Merkel cell carcinoma tissue microarray staining












Case number
CM2B4
CK20
CD56
Chromogranin
Synaptophysin















1
+
+
+
+
+


2
+
+
+
−
+


3
+
+
+
+
+


4
+
+
+
+
+


5
+
+
NDA
ND
ND


6
+
+
+
−
ND


7
+
+
+
+
+


8
+
+
+
+
+


9
+
+
+
+
+


10
+
+
+
+
+


11
+
+
+
+
+


12
+
+
+
+
+


13
+
+
+
+
+


14
+
+
+
+
+


15
+
+
+
+
+


16
+
+
−
+
+


17
+
+
+
+
+


18
+
+
−
+
+


19
+
+
+
+
+


20
+
+
+
−
+


21
+
+
+
+
ND


22
−
+
+
+
+


23
−
+
+
+
ND


24
−
+
+
+
+


25
−
+
+
+
+


26
−
+
+
+
+


27
−
+
+
−
+


28
−
+
+
+
+


29
−
+
+
−
+


30
−
+
+
+
+


31
−
−
−
−
−


32
−
−
+
+
+


33
−
−
−
−
−


34
−
−
−
+
+


35
−
−
+
−
−


36
−
−
+
−
+







Control: Neuroendocrine small cell carcinoma












37
−
−
−
−
+


38
−
−
+
−
+


39
−
−
+
+
+


40
−
−
+
−
+





AND, Not determined.













TABLE 6-2







qPCR detection of MCV genome in MCC











MCV genome




MCC
copies/cella
Genomic
Immunostaining












Tissue
T Ag
VP2
Southernb
CM2B4
CK20d





MCC337
<10−3 
0  
−
−
+


MCC339
5.2
11.1 
+
+
+


MCC343
0  
0  
−
−
+


MCC344
6.3
13.7 
+
−
+


MCC345
4.9
<10−3 
+
+
+


MCC346
<10−3 
0  
−
−
+


MCC347
1.6
0  
+
+
+


MCC349
3.3
8.0
+
+
+


MCC350
 0.83
3.0
+
NTC
NT


MCC352
14.3 
47.5 
+
+
+





aRNaseP copy number was divided by two to determine cellular equivalent of DNA.


bMCV positivity was previously examined by Southern blotting (1).


CNT, No paraffin embedded MCC tissues to evaluate.


CCK20 expression was previously examined by immunostaining (1).













TABLE 6-3







qPCR detection of MCV genome in hematolymphoid malignancies.











No. MCV positive


Hematopathological samples studied
No. Tested
(% MCV Positive)












B cell-associated lymphomas




Chronic lymphocytic leukemia
33
1 (3.0)


Non-Hodgkin lymphoma, NOS
14
1 (7.1)


Diffuse large B cell lymphoma
65
2 (3.1)


Follicular lymphoma
14
0


Acute lymphoblastic leukemia
11
0


Primary effusion lymphoma
2
0


Mucosa-associated lymphoid tissue
5
0


lymphoma




Mantle cell lymphoma
8
0


Marginal zone lymphoma
9
1 (11)


T cell-associated lymphomas




Acute lymphoblastic leukemia
10
0


Large granular lymphocyte leukemia
1
0


Mycosis fungoides
11
0


T cell lymphoma, unspecified
82
1 (1.2)


Myeloid disorders




Chronic myelogenous leukemia
5
0


Acute myeloid leukemia
11
0


Myelodysplastic syndrome
3
0


Others




Hodgkin lymphoma
30
1 (3.3)


Post transplant lymphoproliferative
11
0


disorder




Total
325
7 (2.2%)
















TABLE 6-4







qPCR detection of MCV genome in hematolymphoid malignancies.









Copies per cell









Hematolymphoid malignancies positive for MCV
T Ag
VP2





Chronic lymphocytic leukemia (#354A)
1.2 × 10−2
8.4 × 10−3


Non-Hodgkin lymphoma (#351)
1.5 × 10−3
<10−3


Diffuse large B cell lymphoma (#229)
1.1 × 10−3
0


Diffuse large B cell lymphoma (#500)
3.8 × 10−3
1.1 × 10−3


Marginal zone lymphoma (#781)
5.8 × 10−3
0


T cell lymphoma (#18)
3.2 × 10−3
0


Hodgkin lymphoma (#86)
1.8 × 10−3
2.9 × 10−3





ABlinded testing number.













TABLE 6-5





MCV LT protein detection in hematolymphoid malignancies


















B cell malignancies
0/122



T cell malignancies
0/17



Myeloid disorders
0/1



Hodgkin Lymphoma
02



Normal lymphoid tissues



Normal splenic tissue
0/18



Normal lymph node
0/13







AThese cases are derived from tissue microarray slides #SP482t, #LM801t, #NHL801t from BioMax.













TABLE S1







A list of SV40 T antigen specific monoclonal antibodies screened for cross reactivity with MCV T


antigen.








SV40 T antigen



Monoclonal Antibodies
Reference(s)





PAb101, 108
Gurney, E. G., Tamowski, S., and Deppert, W. 1986. Antigenic binding sites of monoclonal



antibodies specific for simian virus 40 large T antigen.J Virol 57: 1168-1172.



Tack, L. C., Wright, J. H., and Gurney, E. G. 1989. Alterations in the structure of new and old



forms of simian virus 40 large T antigen (T) defined by age-dependent epitope changes: new



T is the same as ATPase-active T.J Virol 63: 2352-2356.


PAb 204, 210, 211, 216
Mole, S. E., Gannon. J. V., Ford, M. J., and Lane, D. P. 1987. Structure and function of SV40



large-T antigen.Philes Trans R Soc Lond B Biol Sci 317: 455-469.



Gannon, J. V., and Lane, D. P. 1990. Interactions between SV40 T antigen and DNA



polymerase alpha. New Biol 2: 84-92.


PAb 405, 409, 407, 416, 419,
Harlow, E., Crawford, L. V., Pim, D. C., and Williamson, N. M. 1981. Monoclonal antibodies


423,430, 431,433, 441, 442
specific for simian virus 40 tumor antigens.J Virol 39: 861-869.


PAb 602, 603, 605, 606
Mole, S. E., Gannon. J. V., Ford, M. J., and Lane. D. P. 1987. Structure and function of SV40



large-T antigen.Philes Trans R Soc Lond B Biol Sci 317: 455-469.


PAb 901, 902
Karjalainen, H. E., Tevethia, M. J., and Tevethia, S. S. 1985. Abrogation of simian virus 40



DNA-mediated transformation of primary C57BL/6 mouse embryo fibroblasts by exposure



to a simian virus 40-specific cytotoxic T-lymphocyte clone.J Virol 56: 373-377.



Thompson, D. L., Kalderon, D., Smith, A. E., and Tevethia, M. J. 1990. Dissociation of Rb-



binding and anchorage-independent growth from immortalization and tumorinenicity using



SV40 mutants producing N-terminally truncated large T antigens.Virology 178: 15-34.



Fu, T. M., Bonneau, R. H., Epler, M., Tevethia, M. J., Alam, S., Verner, K., and Tevethia, S. S.



1996. Induction and persistence of a cytotoxic T lymphocyte (CTL) response against a



herpes simplex virus-specific CTL epitope expressed in a cellular protein.Virology 222: 269-274.









The publications referenced in this Example are as follows:

    1. Feng, H., Shuda, M., Chang, Y., and Moore, P. S. 2008. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science 319:1096-1100.
    2. Kassem, A., Schöpflin, A., Diaz, C., Weyers, W., Stickeler, E., Werner, M., and Zur Hausen, A. 2008. Frequent Detection of Merkel Cell Polyomavirus in Human Merkel Cell Carcinomas and Identification of a Unique Deletion in the VP1 Gene. Cancer Res 68:5009-5013.
    3. Becker, J. C., Houben, R., Ugurel, S., Trefzer, U., Pfohler, C., and Schrama, D. 2008. MC Polyomavirus Is Frequently Present in Merkel Cell Carcinoma of European Patients. J Invest Dermatol. advance online publication PMID: 18633441
    4. Shuda, M., Feng, H., Kwun, H. J., Rosen, S. T., Gjoerup, O., Moore, P. S., and Chang, Y. 2008. T antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus. Proc Natl Acad Sci USA 105:16272-16277.
    5. Leonard, J. H., Bell, J. R., and Kearsley, J. H. 1993. Characterization of cell lines established from Merkel-cell (“small-cell”) carcinoma of the skin. Int J Cancer 55:803-810.
    6. Quaglino, D., Di Leonardo, G., Lalli, G., Pasqualoni, E., Di Simone, S., Vecchio, L., and Ventura, T. 1997. Association between chronic lymphocytic leukaemia and secondary tumours: unusual occurrence of a neuroendocrine (Merkell cell) carcinoma. Eur Rev Med Pharmacol Sci 1:11-16.
    7. Howard, R. A., Dares, G. M., Curtis, R. E., Anderson, W. F., and Travis, L. B. 2006. Merkel cell carcinoma and multiple primary cancers. Cancer Epidemiol Biomarkers Prey 15:1545-1549.
    8. zur Hausen, H., and Gissmann, L. 1979. Lymphotropic papovaviruses isolated from African green monkey and human cells. Med Microbiol Immunol 167:137-153.
    9. Moll, R., Schiller, D. L., and Franke, W. W. 1990. Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns. J Cell Biol 111:567-580.
    10. Moll, R., Lowe, A., Laufer, J., and Franke, W. W. 1992. Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol 140:427-447.
    11. Pope, J. H., and Rowe, W. P. 1964. Detection of Specific Antigen in Sv40-Transformed Cells by Immunofluorescence. J Exp Med 120:121-128.
    12. Diamandopoulos, G. T. 1972. Leukemia, lymphoma, and osteosarcoma induced in the Syrian golden hamster by simian virus 40. Science 176:173-175.
    13. Moore, P. S., Kingsley, L. A., Holmberg, S. D., Spira, T., Gupta, P., Hoover, D. R., Parry, J. P., Conley, L. J., Jaffe, H. W., and Chang, Y. 1996. Kaposi's sarcoma-associated herpesvirus infection prior to onset of Kaposi's sarcoma. AIDS 10:175-180.
    14. Whitby, D., Howard, M. R., Tenant-Flowers, M., Brink, N. S., Copas, A., Boshoff, C., Hatziouannou, T., Suggett, F. E. A., Aldam, D. M., Denton, A. S., et al. 1995. Detection of Kaposi's sarcoma-associated herpesvirus (KSHV) in peripheral blood of HIV-infected individuals predicts progression to Kaposi's sarcoma. Lancet 364:799-802.
    15. Dorries, K., Vogel, E., Gunther, S., and Czub, S. 1994. Infection of human polyomaviruses JC and BK in peripheral blood leukocytes from immunocompetent individuals. Virology 198:59-70.
    16. Stolt, A., Sasnauskas, K., Koskela, P., Lehtinen, M., and Dillner, J. 2003. Seroepidemiology of the human polyomaviruses. J Gen Virol 84:1499-1504.
    17. Fernandez-Figueras, M. T., Puig, L., Musulen, E., Gilaberte, M., Lerma, E., Serrano, S., Fernandez, C., and Ariza, A. 2007. Expression profiles associated with aggressive behavior in Merkel cell carcinoma. Mod Pathol 20:90-101.
    18. Poulin, D. L., Kung, A. L., and DeCaprio, J. A. 2004, p53 targets simian virus 40 large T antigen for acetylation by CBP. J Virol 78:8245-8253.
    19. Campbell, K. S., Mullane, K. P., Aksoy, I. A., Stubdal, H., Zalvide, J., Pipas, J. M., Silver, P. A., Roberts, T. M., Schaffhausen, B. S., and DeCaprio, J. A. 1997. DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes efficient viral DNA replication. Genes Dev 11:1098-1110.


EXAMPLE 7
This example demonstrates the use of MCV VLPs as reagents in assays for the detection of MCV infection in human subjects.
Methods
Cases:
22 MCV positive cases were obtained from persons with biopsy-confirmed MCC and qPCR-confirmed MCV infection. Seroprevalence among different age groups was tested using sera from patients with Langerhans Cell Histiocytosis (LCH) (n=151, age of patients: 1 month to 72 years old) were obtained from Dr. Frank Jenkins.
Control:
seroprevalence among adult population was tested using control sera (n=167) were obtained from the New York City Blood Bank (NYCBB) and the Columbia University Blood Bank. All sera were tested for HIV, HCV, HBV, syphilis and were found negative for these infections.
Informed consent from all study participants and IRB approval were received in accordance with the guidelines for human experimentations of the University of Pittsburgh.
ELISA Assay:
an EIA based on purified VLPs was used to detect presence of specific antibodies in sera samples. Sera were tested using 96-well 2HB Immulon plates (Thermo Scientific), coated with codon-optimized MCV VLP, which are based on two major capsid proteins, VP1 and VP2. In addition, 12 sera samples were tested by CRPV-, BK-, and HPV-VLP ELISA.
For all viruses, 100 μl of purified VLPs at concentration of 1 μg/ml were added to the wells. After overnight incubation, plates were washed with PBS and blocked with PBS/0.5% milk for 2 hours at room temperature (see [1] for plate set up).
Serum samples diluted 1:500 were added to 4 wells (2 wells coated with VLP and 2 wells without VLPs) and incubated for 2 hours at room temperature. After one-hour reaction with rabbit anti-human immunoglobulin G horseradish peroxidase (diluted 1:6000, Dako, Carpenteria, Calif.). Following another washing step 3,3′,5,5′-tetramethyl-benzidine (Sigma) substrate was added and incubated for 45 minutes in the dark at room temperature. Reaction was stopped adding 2N sulfuric acid. Optical density was measured on a MRX plate reader (Dynex Technologies, Chantilly, Va.) at a 405 nm wavelength with reference at 620 nm.
Quality Control Testing.
For testing each plate included two test sera standards (MCV high- and medium-reactive sera), which were tracked over time. These sera were aliquoted and used through out the testing. If results of these standards were different by greater than 2 standard deviations from the mean optical density, then the results were discarded and the plate was repeated. All sera were tested in duplicates, and average ODs for a given sample were calculated as an average of OD of the wells containing VLPs minus the average of OD of the wells without antigen. Duplicate tests were performed independently, and testing was done in blinded sets.
Competitive ELISA.
12 sera (four high-positive, four-medium, and four negative according to results of MCV-VLP testing) samples were tested by BK-, and HPV-VLP competitive ELISA. Each sera sample was tested by MCV VLP ELISA in serial dilutions: 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, and 1:32000. For each sample, working dilutions were determined to perform competitive ELBA (FIG. 13).
BK-Competitive ELISA.
200 μl of sera at working dilutions as determined above were incubated with 2 ug of BK-VLPs for one hour at room temperature. After incubation, sera with VLPs were added to two wells on the plates, previously coated with 100 ng BK-VLPs. Also, two wells were filled with sera without BK-VLPs as a control. After two hours of incubation at room temperature, plates were washed three times with PBS and reacted with 100 ul of rabbit anti-human immunoglobulin G horseradish peroxidase (diluted 1:6000, Dako, Carpenteria, Calif.). Following another washing step 100 ul of 3,3′,5,5′-tetramethyl-benzidine (Sigma) substrate was added for a 45 minute incubation in the dark at room temperature. Reaction was stopped by adding 100 ul of 2N sulfuric acid.
HPV-Competitive ELISA.
HPV VLP ELISA was performed the same way as BK-competitive ELISA, except sera samples were incubated with HPV VLPs and added to the plates, precoated with HPV VLPs.
Peptide Mapping of LT-, MT-, and VP1-Antigens.
In total, 183 peptides (LT), 66 peptides (MT), and 103 peptides (VP1) biotinylated (N-terminal SGSK) 17 mer offset by 5 were synthesized by Mimotop (Clayton Victoria, Australia). An ELISA was performed according to the manufacturer's protocol by using a panel of human serum diluted 1:500. 52 samples from LCH patients and 4 positive serum samples were tested for all three antigens peptides in order to identify specific seroreactive linear epitope for MCV infection.
Results
Peptide ELISA.
Analysis of all 352 peptides studied identified 12 potential immunoreactive peptides. An example of the peptide screen by ELISA is shown in FIG. 14. However, when screened against serum from 9 MCC patients, no specific linear epitope was identified.
MCV VLP ELISA.
Total of 340 serum samples were tested by VLP ELISA. Based on results of the test OD value of 0.5 was determined as cutoff. All samples with OD>0.5 were classified as positive.
Among samples derived from MCV-positive individuals 95.5% were positive with OD>2. Only one sera was negative on the test (4.5%) with OD equal to 0.1. To evaluate prevalence among blood donors two groups of sera samples were tested. In the group of NYCBB (n=105) prevalence was 54.3%. Among Columbia University blood donors (n=62) positive results of the test were 27.4% of the samples. Absence of demographic data for these two groups does not allow us to interpret this differences in prevalence.
In order to evaluate prevalence of MCV infection in various age groups we performed testing of 151 serum samples from LCH patients. According to the test results, 29.8% of all samples were positive. Samples were divided in the 5 groups based on age data: group 1—from 0 to 4 years old (y.o.) (n=29), group 2—from 5 to 9 y.o. (n=32), group 3—from 10 to 14 y.o. (n=22), group 4—from 15 to 20 y.o. (n=17), and group 5—from 21 to 72 y.o. (n=51).
These serum studies suggest that approximately one-half of normal blood donors have serum antibodies against MCV, implying that a high proportion of the population has been exposed to MCV. We observed an age-dependent increase in MCV seroprevalence, with about 70% of individuals over age 50 showing detectable MCV antibody responses. The results of testing are presented in FIG. 15.
VLP Competitive ELISA. BK-Competitive ELISA.
Since cross-reactivity between antibodies to SV40, BKV and JCV VLP has been previously reported, we performed testing for BKV VLP ELISA on 12 samples (high-, medium-, and low-positive to MCV VLPs). Results of the testing demonstrate positive reactivity to BKV (FIG. 16).
To determine if MCV VLP reactivity is due to cross-reaction to BKV, BK competitive ELISA was performed. In order to compete out AB to BKV we incubated 100 μl of diluted serum samples with 1 ug of BK VLPs before adding to the plates precoated with MCV VLPs. Results of testing demonstrate that after incubation with BKV VLPs samples were still reactive to MCV (FIG. 17). Serum samples incubated with BK VLPs showed no reactivity on BK precoated plates. This suggests that MCV reactivity is not due to BKV AB.
CRPV and HPV VLP Competitive ELISA.
12 samples were also tested for CRPV and HPV VLP reactivity. Results of the tests are shown in FIG. 18 (A and B).
Prevalence of HPC VLP Positive Reactivity.
As can be seen in FIG. 19, the MCV VLP assay resulted in a nearly 100% positive result for patients who had been diagnosed with MCC. In contrast, patients with unrelated conditions (lupus, Langerhan's cell histiocytosis) and from commercial blood sources as well as blood donors tested positive for MCV VLPs at a rate of about 50%. A comparison between the OD from the Elisa assays for MCV+ MCC patients and blood donors (1:500 dilution) is presented in FIG. 20. The arrow in the right panel represents the mean value for the MCV+ MCC patients (2.3 OD) less 5× the standard deviation for a value of 0.285. While a different threshold can be ascertained, these results suggest that an OD value above 0.285 might be useful diagnostically.
The publication referenced in this Example are as follows:

    1. A. S. Laney, J. S. Peters, S. M. Manzi, L. A. Kingsley, Y. Chang, P. S. Moore. 2006 Use of multiantigen detection algorithm for diagnosis of Kaposi's sarcoma-associated herpesvirus infection. J. Clin. Microbiol. 44(10):3734-3741.


EXAMPLE 8
This example demonstrates the development of a neutralizing assay based on MCV VLPs.
Neutralization assays were based on the infection of 293TT cells with MCV and MPyV reporter vectors carrying an expression plasmid encoding Gaussia luciferase, a secreted reporter protein. Using these MCV reporter vectors, a high-sensitivity MCV neutralization assay was developed. The neutralization assay is about 40-fold more sensitive than ELISA for detection of MCV sero-responses. 12 MCC patients' MCV sero-responses are, on average, greater in magnitude than responses found in 35 normal individuals (p<2.4×10−5). A small number of normal individuals were found to exhibit very high MCV sero-responsiveness comparable to the MCC patients. Validation of the assay is shown in FIG. 21 based on titration of MCV-reactive pooled human serum and MPyV-specific rabbit serum. The values for ELISA or neutralizaiton assay (neut) were standardized to calculated maximum optical density (OD) or maximum relative light units (RLUs), respectively.
Hypothetically, these very high sero-responses may be a record of a prior period of sustained high-level MCV replication. Since MCC tumors typically do not express the viral capsid genes, it is likely that this putative episode of high-level virus replication occurred prior to development of the cancer, and may have contributed to the cancer's development. Also is expected that the neutralization assay will be less likely than the ELISA to detect antibody responses to other human polyomavirus types.
EXAMPLE 9
This example demonstrates the development of a neutralizing assay based on MCV VLPs.
It is believed that polyomaviruses require two minor capsid proteins, VP2 and VP3 for full infectivity. In previously-characterized polyomaviruses, VP3 is an internally-initiated, N-truncated isoform of VP2. MCVs do not encode a VP2 methionine codon homologous to the codon that initiates translation of the VP3 ORF of other known polyomaviruses. We therefore produced constructs encoding possible alternative MCV VP3 ORFs initiated from VP2 methionine codons 46 or 129. In pilot experiments, inclusion of expression plasmids encoding MCV VP346 enhanced vector infectious titer yield only very modestly, while inclusion of VP3129 modestly reduced titer yield (data not shown). The putative MCV VP3 genes were therefore omitted from the MCV vector production scheme. For MPyV, an expression plasmid encoding the standard VP3 ORF was incorporated into the reporter vector production process.
A substantial majority of the VLPs used for the ELISA studies above were found to contain  ̃5 kb fragments of cellular DNA (data not shown). We therefore employed a previously-described procedure for enriching reporter vector stocks for capsids containing reporter plasmids, as opposed to cellular DNA fragments. This strategy resulted in a major improvement in particle to infectivity ratios for the reporter vector stocks (data not shown).
Infection of 293TT cells with an MCV-Glue reporter vector dose of 400 pg/ml (roughly 8 picomolar with respect to VP1 or roughly 100 VLPs per cell) resulted in a robust luminescent signal 72 hours after infection. Typical assay conditions resulted in the appearance of roughly 500,000 relative light units (RLUs) with a background of roughly 500 RLUs in control wells.
To validate the neutralization assay, we tested the ability of pooled human sera (PHS) to neutralize the MCV and MPyV Glue vector stocks. The PHS neutralized the MCV-Glue reporter vector, with 50% neutralization (EC50) occurring at a calculated serum dilution of 1:44,000 (95% CI 1:32,000-1:60,000). In terms of EC50 values, the neutralization assay was >100-fold more sensitive than the ELISA. This improved sensitivity is presumably due to the 2,500-fold lower dose of virions used in the neutralization assay relative to the ELISA.
In an additional set of assay validation experiments, we found that IgG purified out of the PHS neutralized the vector with an EC50 of about 90 ng/ml). Conversely, PHS stripped of IgG using protein G resin neutralized the MCV reporter vector with an EC50 of only 1:600 (data not shown).
PHS diluted 1:100 failed to neutralize the MPyV reporter vector, whereas an MPyV-specific rabbit serum neutralized the MPyV reporter vector titer by >99% at the same dose. The MPyV-specific rabbit serum only partially neutralized the MCV vector at a 1:100 dilution. The results confirm that MCV and MPyV are not serologically cross-reactive and that neutralization is due primarily to virus-specific antibodies in various sera.
We used the MCV neutralization assay to compare serial dilutions of sera from MCV+ MCC patients (age 14+ years) to sera from a subset of the 48 oldest plasma donors (age 47-74 years). We also tested sera from LCH patients age 47-72 years in the neutralization assay. A small number of MCC patients whose tumors were found not to contain MCV were also tested. MCV+ MCC patients displayed very high titer MCV-neutralizing responses that were not typical among control donors. This difference was statistically significant. Although the apparent difference between the neutralizing titers of MCV+ and MCV− MCC patients was not statistically significant, several of the MCV-patients displayed neutralizing titers much lower than titers observed for MCV+ individuals, suggesting that a minority of MCC cases are MCV-independent.
88% ( 42/48) of the plasma donor sera detectably neutralized the infectivity of the MCV reporter vector at the lowest serum dilution tested (1:100). In contrast, only half ( 24/48) of this subset of sera scored seropositive in the VLP ELISA, To address this discrepancy, we retested this subset of sera at a more concentrated dilution (1:40) in the VLP ELISAs. For the re-testing, any serum whose raw OD against MCV VLPs was at least three-fold greater than its raw OD against MPyV VLPs was defined as seropositive. By this standard, 75% ( 36/48) of the plasma donor sera displayed MCV-specific ELISA reactivity, in a pattern generally correlating with neutralizing titers. Taken together, the results suggest that, in addition the roughly 50% of donors who display robust anti-MCV antibody responses, an additional 25% of donor sera have weak, but detectable, MCV-specific reactivity.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

## Claims

1. A method of assaying for Merkel cell carcinoma virus (MCV) exposure in a patient, the method comprising:
obtaining a tissue or fluid sample from the patient, and assaying the sample for the presence of at least one MCV molecule,
wherein the assaying comprises a process of exposing the sample to an agent that binds to at least one MCV molecule, and
wherein at least one MCV molecule is selected from the group of MCV molecules consisting of (a) an MCV nucleic acid (b) an MCV polypeptide, and (c) an antibody that binds specifically to an MCV polypeptide,
wherein a positive test for the presence of the MCV molecule within the sample is indicative of exposure of the patient to MCV.

2. The method of claim 1, wherein the assaying comprises an ELISA assay comprising exposing the sample to an MCV polypeptide.

3. The method of claim 2, wherein the MCV polypeptide is VP1, VP2, and/or VP3 and is within a virus-like particle (VLP).

4. A method of assaying for MCV neutralization, the method comprising:
(a) obtaining MCV VLPs that contain a reporter construct,
(b) exposing the VLPs to a sample to produce a preparation,
(c) exposing the preparation to cells amenable to infection by MCV, and
(d) assaying for the expression of the reporter within the cells,

wherein a lower level of expression of the reporter in the cells relative to a control indicates the presence of a neutralizing factor within the sample.

5. The method of claim 4, wherein the sample is a fluid or tissue sample obtained from a patient.

6. The method of claim 4, wherein the sample contains a candidate anti-MCV immunoglobulin.

7. The method of claim 1, wherein the patient is human.

8. The method of claim 1, wherein the patient is a non-human animal.

9. The method of claim 1, wherein the sample is a tissue sample and the assay comprises in situ hybridization.

10. The method of claim 1, wherein at least one MCV molecule comprises at least one MCV RNA.

11. The method of claim 1, wherein at least one MCV molecule comprises at least one MCV DNA.

12. The method of claim 1, wherein at least one MCV molecule comprises at least one MCV polypeptide.

13. The method of claim 12, wherein at least one MCV polypeptide is VP1.

14. The method of claim 12, wherein at least one MCV polypeptide is VP2.

15. The method of claim 12, wherein at least one MCV polypeptide is VP3.

16. The method of claim 12, wherein at least one MCV polypeptide is a large T polypeptide.

17. The method of claim 12, wherein at least one MCV polypeptide is a small T polypeptide.

18. The method of claim 12, wherein at least one MCV polypeptide is one or more of T-1, T-2, T-3, T-4, and/or T-5.

19. The method of claim 1, wherein at least one MCV molecule is an antibody that binds specifically to an MCV polypeptide.

20. The method of claim 19, wherein the MCV polypeptide to which the antibody binds is VP1.

21. The method of claim 19, wherein the MCV polypeptide to which the antibody binds is VP2.

22. The method of claim 19, wherein the MCV polypeptide to which the antibody binds is VP3.

23. The method of claim 19, wherein the MCV polypeptide to which the antibody binds is a large T polypeptide.

24. The method of claim 19, wherein the MCV polypeptide to which the antibody binds is a small T polypeptide.

25. The method of claim 19, wherein the MCV polypeptide to which the antibody binds is one or more of T-1, T-2, T-3, T-4, and/or T-5.