Silvia Pietrobon

Serological Evidence of an Early Seroconversion to Simian Virus 40 in Healthy Children and Adolescents

At present Simian virus 40 (SV40) infection in humans appears to be transmitted independently from early contaminated vaccines. In order to test the spread of SV40 infection in children, an immunologic assay employing specific SV40 synthetic peptides corresponding to its viral protein (VP) antigens was employed to estimate the seroprevalence of this polyomavirus in Italian infants and adolescents. Serum samples from 328 children and adolescents, up to 17 years, were investigated. Serum antibodies against SV40 VPs were detected by indirect enzyme-linked immunosorbent assays. The seroprevalence of this polyomavirus was calculated after stratifying the subjects by age. Anti-viral capsid protein 1-2-3 SV40 IgG antibodies were detected in 16% of the study participants. The prevalence of antibodies against SV40 VPs tended to increase with age in children, up to 10 year old (21%). Then, in the cohort of individuals aged 11–17 years, the prevalence decreased (16%). A higher prevalence rate (23%) of SV40 VP antibodies was detected in the cohorts of 1–3 year and 7–10 year old children, than in children and adolescents of the other age groups. This age corresponds to children starting nursery and primary school, respectively, in Italy. IgM antibodies against SV40 VP mimotopes were detected in 6–8 month old children suggesting that SV40 seroconversion can occur early in life. SV40 VP antibodies are present at low prevalence in Italian children (16%), suggesting that SV40 infection, although acquired early in life, probably through different routes, is not widespread. The low SV40 seroprevalence suggests that SV40 is less transmissible than other common polyomaviruses, such as BKV and JCV. Alternatively, our immunologic data could be due to another, as yet undiscovered, human polyomavirus closely related to SV40.

Specific Antibodies Reacting with SV40 Large T Antigen Mimotopes in Serum Samples of Healthy Subjects

Simian Virus 40, experimentally assayed in vitro in different animal and human cells and in vivo in rodents, was classified as a small DNA tumor virus. In previous studies, many groups identified Simian Virus 40 sequences in healthy individuals and cancer patients using PCR techniques, whereas others failed to detect the viral sequences in human specimens. These conflicting results prompted us to develop a novel indirect ELISA with synthetic peptides, mimicking Simian Virus 40 capsid viral protein antigens, named mimotopes. This immunologic assay allowed us to investigate the presence of serum antibodies against Simian Virus 40 and to verify whether Simian Virus 40 is circulating in humans. In this investigation two mimotopes from Simian Virus 40 large T antigen, the viral replication protein and oncoprotein, were employed to analyze for specific reactions to human sera antibodies. This indirect ELISA with synthetic peptides from Simian Virus 40 large T antigen was used to assay a new collection of serum samples from healthy subjects. This novel assay revealed that serum antibodies against Simian Virus 40 large T antigen mimotopes are detectable, at low titer, in healthy subjects aged from 18–65 years old. The overall prevalence of reactivity with the two Simian Virus 40 large T antigen peptides was 20%. This new ELISA with two mimotopes of the early viral regions is able to detect in a specific manner Simian Virus 40 large T antigen-antibody responses.

Circulating microRNAs found dysregulated in ex-exposed asbestos workers and pleural mesothelioma patients as potential new biomarkers

Malignant pleural mesothelioma (MPM), a fatal cancer, is an occupational disease mostly affecting workers ex-exposed to asbestos fibers. The asbestos, a cancerogenic mineral of different chemical composition, was widely employed in western Countries in industrial manufactures of different types. MPM may arise after a long latency period, up to five decades. MPM is resistant to conventional chemo- and radio-therapies. Altogether, these data indicate that the identification of new and specific markers are of a paramount importance for an early diagnosis and treatment of MPM. In recent years, microRNAs expression was found dysregulated in patients, both in cancer cells and sera, affected by tumors of different histotypes, including MPM. Cell and circulanting microRNAs, found to be dysregulated in this neoplasia, were proposed as new biomarkers. It has been reported that circulating microRNAs are stable in biological fluids and could be employed as potential MPM biomarkers. In this investigation, circulating microRNAs (miR) from serum samples of MPM patients and workers ex-exposed to asbestos fibers (WEA) and healthy subjects (HS) were comparatively analyzed by microarray and RT-qPCR technologies. Our results allowed (i) to select MiR-3665, an endogenous stable microRNA, as the internal control to quantify in our analyses circulating miRNAs; to detect (ii) miR-197-3p, miR-1281 and miR 32-3p up-regulated in MPM compared to HS; (iii) miR-197-3p and miR-32-3p up-regulated in MPM compared to WEA; (iv) miR-1281 up-regulated in both MPM and WEA compared to HS. In conclusion, three circulating up-regulated microRNAs, i.e. miR-197-3p, miR-1281 and miR-32-3p are proposed as potential new MPM biomarkers.

Antibodies reacting with Simian Virus 40 capsid protein mimotopes in serum samples from patients affected by uveal melanoma

The uveal melanoma (UM) is the most common human intraocular tumour. Simian Virus 40 (SV-40) is a small DNA tumor virus detected in some malignancies, including the cutaneous melanoma. In this study an indirect ELISA using synthetic peptides that mimic SV-40 antigens, was employed to detect antibodies against SV-40 in serum samples from UM patients. Our report indicates a significant higher prevalence of antibodies against SV-40 capsid protein antigens in serum samples from UM patients compared to controls. Our data suggest an association between UM and SV-40, indicating that patients affected by uveal melanoma tested SV-40-positive could be treated by innovative therapies.

Specific Detection of Serum Antibodies against BKPyV, A Small DNA Tumour Virus, in Patients Affected by Choroidal Nevi

Ocular or choroidal nevus (CN) is a rare benign neoplastic lesion of the eye. The cause of CN onset/progression, which arises from the transformation of ocular melanocytes, is not known. A fraction of CN patients may develop uveal melanoma. The objective of this study was to investigate the association between CN and BK polyomavirus (BKPyV), a small DNA tumor virus. Serum IgG antibodies which react with BKPyV antigens were analyzed. An indirect E.L.I.S.A. using synthetic peptides that mimic BKPyV antigens was employed. Serum antibodies against BKPyV were also investigated by haemagglutination inhibition (HAI) assay. Sera were from CN patients and healthy subject (HS) were the control. A statistically significant higher prevalence of antibodies against BKPyV capsid protein antigens in serum samples from CN patients was detected, compared to HS, using two independent techniques, indirect E.L.I.S.A. and HAI (87.3% CN vs. 62.1% HS and 91.5% CN vs. 64.4% HS, respectively; p < 0.005). Our data suggest an association exists between CN and BKPyV indicating that this small DNA tumor virus could be responsible in the onset of this benign neoplastic lesion affecting eye melanocytes. This investigation reports the association between choroidal nevi and BKPyV infection for the first time. These data are innovative in this field and may represent a starting point for further investigation into the putative role of BKPyV in CN onset/progression.

Antibodies Against Mimotopes of Simian Virus 40 Large T Antigen, the Oncoprotein, in Serum Samples From Elderly Healthy Subjects

Simian Virus 40 (SV40), a monkey polyomavirus, was administered to human populations by early anti‐poliomylitis vaccines contaminated by this small DNA tumor virus. Data on SV40 infection in humans remain controversial. Elderly subjects represent an interesting cohort to investigate, because they were not immunized with SV40‐contaminated vaccines. Taking advantage of the Italian population, the second oldest worldwide, elderly subjects (n = 237) up to 100 years old were enrolled in this study. Their sera were analyzed, by ELISA tests with synthetic peptides mimicking the viral epitopes, for IgG antibodies reacting with SV40 large Tumor antigen (Tag), the viral oncoprotein. An overall seroprevalence of 22% was revealed in subjects aged 66–100 years, ranging from 19% in individuals 66–74 years old, to 24% in subjects 82–100 years old, with a lower SV40 titer detected in the oldest group. Our data show that: (i) SV40 infection is not frequent in old individuals; (ii) the infection rate increases in elderly with the age; (iii) the antibody titer of SV40 Tag decreases with the age. In conclusion, SV40 infection seems to spread in old subjects independently from SV40‐contaminated vaccines. This study seems to confirm that SV40 is also a human virus. J. Cell. Physiol. 232: 176–181, 2017.

Specific IgG Antibodies React to Mimotopes of BK Polyomavirus, a Small DNA Tumor Virus, in Healthy Adult Sera

BK polyomavirus (BKPyV) was isolated in 1971 from the urine of a kidney transplant patient. Soon after its identification, BKPyV was characterized as a kidney-tropic virus, which is responsible of a significant fraction of the rejection of transplant kidney in the host. Moreover, in experimental conditions, BKPyV is able to transform different types of animal and human cells and to induce tumors of different histotypes in experimental animals. BKPyV DNA sequences have been detected in healthy individuals and cancer patients using polymerase chain reaction/Shouthern blot hybridization methods. Serum antibodies against this polyomavirus were revealed using immunological techniques, which, however, cross-react with other polyomaviruses such as JC (JCPyV) and Simian Virus 40. These non-specific data indicate the need of novel immunological methods and new investigations to check in a specific manner, BKPyV spread in humans. To this aim, mimotopes from BKPyV structural capsid protein 1 (VP1) were employed for specific immunological reactions to IgG antibodies of human serum samples. An indirect enzyme-linked immunosorbent assay with synthetic peptides mimicking immunogenic epitopes of BKPyV VP1 was set up and employed to test sera of healthy adult subjects. Data from this innovative immunological assay indicate that serum antibodies against BKPyV VP1 mimotopes are detectable in healthy subjects ranging from 18 to 90 years old. The overall prevalence of serum samples that reacted to BKPyV VP1 mimotopes was 72%. The strong points from this investigation are the novelty of the immunological method, its simplicity of the approach, and the specificity of BKPyV antibody reaction to VP1 mimotopes.

Significant Low Prevalence of Antibodies Reacting with Simian Virus 40 Mimotopes in Serum Samples from Patients Affected by Inflammatory Neurologic Diseases, Including Multiple Sclerosis

Many investigations were carried out on the association between viruses and multiple sclerosis (MS). Indeed, early studies reported the detections of neurotropic virus footprints in the CNS of patients with MS. In this study, sera from patients affected by MS, other inflammatory (OIND) and non-inflammatory neurologic diseases (NIND) were analyzed for antibodies against the polyomavirus, Simian Virus 40 (SV40). An indirect enzyme-linked immunosorbent assay (ELISA), with two synthetic peptides, which mimic SV40 antigens, was employed to detect specific antibodies in sera from patients affected by MS, OIND, NIND and healthy subjects (HS). Immunologic data indicate that in sera from MS patients antibodies against SV40 mimotopes are detectable with a low prevalence, 6%, whereas in HS of the same mean age, 40 yrs, the prevalence was 22%. The difference is statistically significant (P = 0.001). Significant is also the difference between MS vs. NIND patients (6% vs. 17%; P = 0.0254), whereas no significant difference was detected between MS vs OIND (6% vs 10%; P>0.05). The prevalence of SV40 antibodies in MS patients is 70% lower than that revealed in HS.

High prevalence of serum IgG antibodies reacting to specific mimotopes of BK polyomavirus, a human oncogenic polyomavirus, in patients affected by uveal melanoma: BONONI et al.

The uveal melanoma (UM) is the most common human intraocular tumor. The BK polyomavirus (BKPyV) is a small DNA tumor virus whose footprints have been detected in different human cancers. BKPyV has oncogenic potential. Indeed, BKPyV, when inoculated into experimental animals, induces tumors of different histotypes, whereas in vitro, it transforms mammalian cells, including human cells from distinct tissues. In this investigation, the association between UM and BKPyV was studied employing indirect enzyme‐linked immunosorbent assays (ELISAs) using synthetic peptides that mimic BKPyV viral capsid 1 (VP1) antigens. Indirect ELISAs were used to detect serum IgG antibodies against this polyomavirus with oncogenic potential in samples from patients with UM and controls, represented by healthy subjects (HS). It was found that serum samples from patients with UM had a higher prevalence of BKPyV antibodies, 85% (51/60), compared with that detected in HS1, 62% (54/87), and HS2, 57% (68/120). The different prevalence of BKPyV antibodies detected in UM versus the two control groups, HS1 and HS2, is statistically significant (p < 0.005). Our immunologic data suggest a significantly higher prevalence of antibodies against BKPyV VP1 epitopes in serum samples from patients with UM compared with HS. These results indicate an association between UM and BKPyV, suggesting that this small DNA tumor virus may be a cofactor in the UM onset or progression.

Serum IgG antibodies from healthy subjects up to 100 years old react to JC polyomavirus

JC polyomavirus (JCPyV) was identified in 1971 in the brain tissue of a patient (J.C.) affected by the progressive multifocal leukoencephalopathy (PML). JCPyV encodes for the oncoproteins large T antigen (Tag) and small t‐antigen (tag). These oncoproteins are responsible of the cell transformation and tumorigenesis in experimental animals. JCPyV is ubiquitous in human populations. After the primary infection, which is usually asymptomatic, JCPyV remains lifelong in the host in a latent phase. Its reactivation may occur in heathy subjects and immunocompromised patients. Upon reactivation, JCPyV could reach (i) the CNS inducing the PML, (ii) the kidney of transplant patients causing the organ rejection. Association between JCPyV, which is a small DNA tumor virus, and gliomas and colorectal carcinomas has been published. In the present investigation, we report on a new indirect ELISA with two specific synthetic peptides mimicking JCPyV VP1 immunogenic epitopes to detect specific serum IgG antibodies against JCPyV. Serum samples of healthy subjects (n = 355) ranging 2–100 years old, were analyzed by this new indirect ELISA. The linear peptides VP1 K and VP1 N resemble the natural JCPyV VP1 capsidic epitopes constituting a docking site for serum antibodies. Data from this innovative immunologic assay indicate that the overall prevalence of JCPyV‐VP1 antibodies in healthy subjects is at 39%. The innovative indirect ELISA with JCPyV VP1 mimotopes seems to be a useful method to detect specific IgG antibodies against this virus, without cross‐reactivity with the closely related SV40 and BKPyV polyomaviruses.