Field of invention

The present invention refers to the identification and use of new specific synthetic peptides of the SV40 early region, which encodes the large T antigen (Tag), to reveal the presence of antibodies against the oncogenic Polyomavirus SV40, in human serum and other fluids, by employing the immunologic technique known as indirect E.L.I.S.A (Enzyme-Linked Immunosorbent Assay).

State of the art

At present, the International Committee of the Taxonomy of Viruses (ICTV) has listed twelve viruses which belong to the Polyomaviruses. Among these viruses the most- fittingly characterized are the five human Polyomaviruses, which are known as the BK virus (BKV), the JC virus (JCV), the KI polyomavirus (KIPyV), the WU polyomavirus (WUPyV), the Merkel cell polyomavirus (MCPyV) and their simian homolog, SV40. SV40 was isolated for the first time in 1960 from the kidney cells of the Asian macaque (Rhesus macaque), which had been employed to produce the first vaccines against poliomyelitis. Human Polyomaviruses BKV and JCV, on the other hand, were isolated in 1971. In 2007, two new human Polyomaviruses, KIPyV and WUPyV, were discovered. More recently, in the 2008, the fifth human Polyomavirus, MCPyV, was discovered in cases of Merkel cell carcinoma, a rare but aggressive skin tumor. The morpho- structural characteristics and the genomic organization of the three viruses, SV40, BKV and JCV, are very similar, indicating their tight evolutive relation. These viruses are ubiquitous in the human population. SV40, BKV and JCV are not oncogenic in their natural hosts, but are oncogenic in experimental animals such as rodents and monkeys, and are able to transform different animal and human cells to the neoplastic phenotype.

Many articles published in recent years by prestigious international scientific Journals have reported on the presence of SV40 in human tumors, including mesotheliomas, gliomas, sarcomas and lymphoproliferative disorders. In addition, our research group has found SV40 sequences in blood samples and sperm fluids from healthy individuals, though with less prevalence compared to neoplastic tissues. SV40 Polyomavirus, for its transforming and oncogenic properties, is considered a possible cofactor in the onset of the aforementioned human tumors, whose incidence has increased over the last twenty years. At present, there are no commercially available standardized methods employing immunologic, virologic, or molecular biology techniques to verify the presence of SV40 in humans. However, for scientific purposes, some groups including our team, have been able to detect SV40 sequences in different human samples by employing molecular biology techniques such as Southern blot hybridization and polymerase chain reactions (PCR and Real Time PCR). Other groups, using immunologic techniques, which are not specific for SV40 have tried to detect antibodies against SV40 in human sera. However, doubts on these results have been raised due to the cross-reactivity of the antigens employed, which in part overlap between SV40 and the other two human Polyomaviruses, BKV and JCV. The lack of specific assays for SV40 indicates the need to produce new and specific methods of analysis. These methods should be sensitive, rapid, standardized and low cost, but at the same time must allow the detection of antibodies against SV40 in serum samples from oncologic patients, patients affected by other diseases, and healthy individuals including blood and organ donors.

Detailed description of the invention

The present invention allows the problem of the lack of appropriate analytical methods for revealing the presence of specific antibodies against SV40 in human serum and other fluids to be solved. We set up an immunologic method with new synthetic peptides in an indirect ELISA. This approach allowed us to detect specific SV40 antibodies. Polyomavirus large T antigen (Tag), which is a viral oncoprotein, is a good immunogenic protein which induces specific antibodies in the infected host. The gene encoding this viral oncoprotein maps in the early region of viral DNA. There are few differences in the Tag amino acid sequence of SV40 and the other two Polyomaviruses BK and JC. We took advantage of this variability to select unique amino acid segments belonging to SV40 which are specific immunologic targets, known as epitopes, for the antibodies present in human serum and other fluids. The early genomic region of the three Polyomaviruses SV40, BKV and JCV is highly conserved, with a sequence homology among the three genomes of approximately 70-80%. However, inside this early region there are short variations of the encoding sequences which allowed us to design specific peptides corresponding to the unique epitope for each of the three Polyomaviruses. Materials and methods

Human samples

Human serum samples from normal individuals and oncologic patients, both males and females of different ages, were taken from different Institutions in Italy represented by Hospitals, Research Centers, University Clinics and Transfusion Centers.

Selected peptides for the indirect ELISA

The designated peptides reported herein were synthesized with standard instruments and techniques. The two different synthetic peptides employed in ELISA were used to reveal the presence of antibodies against the SV40 Tag in human sera. The two amino acid sequences were as follows:

peptide A, with 21 amino acids:

NH2-G S F Q A P Q S S Q S V H D H N Q P Y H I -COOH

and

peptide D, with 24 amino acids:

NH2-H E T G I D S Q S Q G S F Q A P Q S S Q S V H D- COOH.

These two selected peptides are part of the early region encoding Tag oncoprotein; peptide A contains amino acids 669-689, and peptide D contains amino acids 659-682. Peptide A from SV40 has 14 different amino acids compared to the corresponding amino acid sequences from BKV, Dunlop strain and 16 different amino acids from JCV, Mad 1 strain. Moreover, 2 amino acids from SV40 peptide A are absent in BKV. Peptide D from SV40 has 18 and 17 different amino acids compared to the corresponding sequences of BKV, Dunlop strain and JCV, Mad 1 strain, respectively, while 5 amino acids are absent in BKV. The specificity of the immunologic reaction was evaluated using appropriate controls which were represented by hyperimmune sera anti-BKV and anti-JCV produced in animal models.

Identification of antibodies against SV40 in human sera with the immunologic assay, indirect ELISA.

This assay is composed of different phases:

1. Coating. The plate contains 96 wells with a flat bottom. Each well was coated with 5 μg of peptide, solubilized in 100 μΐ of Coating Buffer (Candor Bioscience, Weissensberg, Germany). The plate was incubated at 4°C for 16 hours in order for the peptide to completely cover the bottom of the well.

2. Blocking reactions. After 16 hours, the plate was rinsed three times with the Washing Buffer (Candor Bioscience, Germany). This step eliminated the peptide which had not adhered to the well. Then, a blocking reaction was performed with 200 μΐ of Blocking Solution (Candor Bioscience, Germany) at 37°C for 90 min. After incubation, the plate was rinsed three more times with Washing Buffer (Candor Bioscience, Germany) to eliminate the Blocking Solution.

3. Primary antibody adding. 100 μΐ of the following sera were added to the wells: one positive control, represented by the anti-SV40 antibodies (2 wells), 2 negative control sera with anti-BKV (2 wells) and anti-JCV antibodies (2 wells) and human sera under analysis for the presence of anti-SV40 antibodies, diluted at 1:20 in Low Cross-Buffer (Candor Bioscience, Germany). In each plate, in addition to the aforementioned samples, two additional controls were added. Two wells contained the secondary antibody, while both primary and secondary antibodies were omitted from the other two wells. Each serum sample was analyzed twice. The plate was incubated at 37°C for 90 min. At the end of the incubation, a new cycle of rinsing with three washes was performed with the same solution as described in step 2.

4. Secondary antibody adding. The specific secondary antibody conjugated with the enzyme peroxidase, diluted at 1: 10,000 in Low Cross-Buffer (Candor Bioscience,

Germany), was added to the sample followed by incubation at room temperature for 90 min. At the end of this incubation phase, a new cycle of three washes was performed.

5. ABTS [2,2 'Azin-bis (3-ethylbenzthiazoline-6-sulfonic acid)] treatment and spectrophotometric reading. ABTS (100 μΐ) was added to each well. This substrate is necessary for enzyme peroxidase to generate the reaction which generates the color. After 45 min, the reaction was stopped with 100 μΐ of citric acid 0.1 M. Then, the spectophotometric reading was performed at a 405 nm wavelength to determine the intensity of the reaction, as optical density (O.D.) inside the wells, where the immune- complex was formed because of the presence of the specific antibodies was able to bind the specific peptide.

6. Determination of the "cut-off. The cut-off was determined in each assay, by an OD reading of two negative controls, added to the standard deviation and multiplied three times (+3SD). Sera with antibodies against SV40 were considered Tag positive upon reacting to both peptides of the early region and when sera which had been analyzed three times by indirect ELISA testing gave the same positive result.

RESULTS

Specificity assay of the two synthetic peptides, used as antigens, to detect the presence of antibodies against SV40 in human serum samples.

Indirect ELISA was employed to analyze 1,837 serum samples.

Sera were obtained from 91 children, between 0 and 11 months old, and from 225 children and adolescents, between 1 and 17 years old. These sera were from the Pediatric Clinic, University of Ferrara and Clinical Laboratories, Ferrara City Hospital and Delta County Hospital, Province of Ferrara.

Serum samples (no. =960) of blood donors from different geographical areas of Italy were collected at the Istituto Superiore di Sanita (ISS) in Rome, the blood bank in Novara City Hospital and Casale Monferrato City Hospital, the Cancer Bioimmunotherapy Unit, Oncology Centre, Aviano (C. R. O), Clinical Laboratory, City Hospital of the Republic of San Marino; Delta County Hospital, Province of Ferrara. The serum samples (no =94) from pregnant women were from the ISS in Rome. The sera from adults (no. =90) exposed to the asbestos fibers, were from the Section of Occupational Medicine, School of Medicine and Surgery, University of Pisa. The sera from oncologic patients (no.=95) affected by malignant pleural mesothelioma were from the City or University Hospitals of Casale Monferrato, Brescia and Pisa. The sera from oncologic patients (no. =47) affected by osteosarcoma were from the "Rizzoli" Orthopedic Institute, Bologna. The sera from oncologic patients (no. =89) affected by Non-Hodgkin Lymphoma (NHL) were from University Hospital, Modena. The sera from oncologic patients (no. =44) affected by glioblastoma multiforme were from University Hospital, Verona. The sera from oncologic patients (no. =38) affected by breast cancer and (no. =64) undifferentiated naso-pharyngeal carcinoma (U.N.P.C.) were from C.R.O., Aviano.

Prevalence of antibodies against SV40 determined by indirect ELISA.

In our assay, samples were considered SV40-positive with a reaction to both specific peptides used as antigens at the same time.

Children between 1 and 11 months old had a 14% (13/91) prevalence of SV40 antibodies. None of the serum samples from children less than 6 months old was SV40-positive. In children and adolescents between 1 and 17 years of age, the prevalence of SV40 antibodies was 20% (44/225). Specifically, the prevalence of SV40 antibodies was 25% (30/121) in children between 1 and 10 years of age, and of 13% (14/104) in adolescents between 11 and 17 years of age.

The prevalence of antibodies against SV40 in blood donors between 18-65 years of age was 19%, overall (180/960). In subjects between 18 and 50 years of age the prevalence of SV40 antibodies did not vary consistently and was around 20% (120/612). In blood donors between 51-65 years of age, the prevalence was 15% (36/243). The prevalence of SV40 antibodies in serum samples from elderly people between 66-90 years of age, was 23% (24/105).

In serum sample from 94 pregnant women, the prevalence of SV40-positive samples was 13% (12/94). This result is probably due to the natural immune depression/immune tolerance which occurs in all pregnant women. Then, we analyzed serum samples from former workers who had been exposed to asbestos fibers. These sera were SV40-positive with a prevalence of 10% (9/90).

Serum samples from 47 patients affected by osteosarcoma were SV40-positive with a prevalence of 57% (27/47). It is interesting to note that a statistically significant (p<0.0001) difference was observed between the prevalence of SV40-antibodies in serum samples from normal individuals and patients affected by osteosarcoma.

Serum samples from 95 patients affected by malignant pleural mesothelioma were SV40- positive with a prevalence of 32% (30/95). A statistically significant (p<0.0033) difference was observed between the prevalence of SV40-antibodies in serum samples from normal individuals and patients affected by malignant pleural mesothelioma.

Serum samples from 89 patients affected by Non-Hodgkin Lymphoma (NHL) were SV40- positive with a prevalence of 51% (45/89). A statistically significant (p<0.0001) difference was observed between the prevalence of SV40-antibodies in serum samples from normal individuals and patients affected by NHL.

Serum samples from 44 patients affected by glioblastoma multiforme were SV40-positive with a prevalence of 32% (14/44). A statistically significant (p<0.0117) difference was observed between the prevalence of SV40-antibodies in serum samples from normal individuals and patients affected by glioblastoma multiforme. Serum samples from 38 patients affected by breast cancer and 64 affected by UNPC were SV40-positive with a prevalence of 21% (8/38) and 23% (15/64), respectively. SV40- positive serum samples from these oncologic patients (breast cancer and UNPC) did not statistically differ from the prevalence revealed in the control group represented by blood donors.

Our immunologic data suggest that SV40 is circulating in the human populations and that SV40 infection occurs during the first months of life. Our results are in agreement with data from molecular biology studies. SV40 sequences have been found by PCR techniques with a high prevalence in many human tumors, including NHL, mesothelioma, osteosarcoma, and glioblastoma multiforme. These results, which have been published by many scientific journals, have been reported by more than 50 different laboratories from different countries such as: the U.S.A., France, the UK, Japan, Australia, New Zealand and Italy.