SV40 Detection and Transmission: Does SV40 Circulate in Human Communities?

Transcription

1 SV40 Detection and Transmission: Does SV40 Circulate in Human Communities? Keerti Shah, Dana Rollison and Raphael Viscidi Johns Hopkins Medical Institutions Baltimore, MD

2 Background A large number of cancer patients whose tumors were reported to contain SV40 sequences were born after the polio vaccines were required to be free of SV40. How did these individuals acquire SV40 infection? There are three possibilities.

3 Three Possibilities The vaccines continued to be contaminated with SV40. SV40 infection became established in human communities and is circulating by person-to-person transmission. SV40 was a human infection preceding the exposure in the vaccine.

4 Poliovirus vaccines not found to contain SV40 Sierra-Honigmann and Krause (Biologicals, 2000, 28:1-4) did not detect SV40 sequences in live oral polio vaccines manufactured in the U.S. between 1972 and Sanger et al. (Biologicals, 1999, 27:1-10) did not detect SV40 sequences in live oral polio vaccines used in the U.K. between 1966 and now.

5 Polyomaviruses Viruses co-evolved with their hosts Highly species-specific, adapted to a single species or to a group of closely related species Highly tissue-specific Infections asymptomatic, pathogenic if the host is immunocompromised

6 Human Polyomaviruses BKV and JCV Isolated in 1971, from immunocompromised patients Infections common in childhood and asymptomatic; 50% of children infected with BKV by 3 years, with JCV by years Viruses latent in kidney, shed in urine JCV causes PML in immunosuppressed patients, BKV causes hemorrhagic cystitis in bone marrow transplant recipients and BKV nephropathy in renal transplant recipients

7 Simian Virus 40 Natural infection of some macaque species, for example, the rhesus macaque in North India is infected in nature but the bonnet macaque in South India is not. Infections asymptomatic, virus latent in kidney, shed in urine. Rhesus can be experimentally infected by respiratory, intragastric or subcutaneous route SIV-infected animals develop a PML-like illness. Shares 80% homology with BKV and JCV.

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11 Questions Is SV40 shed in human urines? Is there serologic evidence of SV40 infection in the community? Do patients with cancers linked in the literature to SV40 have serologic evidence of SV40 infection?

12 Search for BKV, JCV and SV40 in sewage The best-documented shedding of human polyomaviruses is in urine. Bofill-Mas et al. (Appl Environ Microbiol 2000, 66: ) examined 28 concentrated sewage samples for the viruses; samples from Spain (n=16), Sweden (n=4), France (n=4) and South Africa (n=4) were tested by PCR. Positive for: n (%) Adenoviruses 26 (93%) JCV 26 (93%) BKV 17 (62%) SV40 0

13 Search for BKV, JCV and SV40 in human urines (Shah et al., J Infect Dis 176: , 1997) Urine samples from 166 homosexual men, 88 of them HIVseropositive, 78 HIV-seronegative Collected , median age of donor, years 3-10 ml of urine centrifuged at 49000g, for 4 hours, pellet resuspended and tested by PCR Masked SV40-positive controls, each containing 200 SV40- transformed cells

14 Results of urine tests Number (%) positive HIVnegative (n=78) HIVpositive (n=88) Total (n=166) BKV 4 (5%) 19 (22%) 23 (14%) JCV 29 (37%) 27 (31%) 56 (34%) SV40* *All 17 masked SV40-positive controls were positive

15 Human response to documented exposure to SV40 (from review, Shah &Nathanson 1976, Am J Epidemiol 103: 1-12) By oral route small amounts of virus excreted intermittently for five weeks no antibody response By respiratory route virus excreted intermittently in throat secretions antibody response low level By subcutaneous route high level antibody response, antibodies persist 8-13 years no studies on virus shedding

16 SV40-reactive antibodies in human sera Two categories Antibodies induced by SV40 in inactivated poliovaccines Antibodies unrelated to documented SV40 exposure

17 Antibody response to contaminated vaccine (Gerber P, Proc Soc Exp Biol Med 125: , 1967) Number of sera with SV40 antibody titer Postvaccine 1:5 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1 month years

18 Antibody profile in individuals not exposed to contaminated vaccine (Shah KV, J Natl Cancer Inst 42: , 1969) Titer of positive sera No. tested No. positive 1:1 1:2 1:4 1:8 1:16 1:32 Cancer patients, North India (5.3%)

19 SV40 reactivity in human sera in individuals not exposed to SV40 Prevalence of 5-10% Positive sera at very low titers and scattered in different age groups In India, positivity not related to geographic proximity of infected monkeys Cross-reactivity with human polyoma-viruses considered

20 Serological cross-reactivity between BKV and SV40 Brown et al. (Am J Epidemiol, 1975, 192:331) examined this question in their serum collections from isolated populations. BKV antibody No. of sera No. (%) reactive to SV40 Present (35%) Absent (5%)

21 Serum antibodies to JCV, BKV, SV40 and the risk of incident primary malignant brain tumors in a Maryland cohort Dana E. M. Rollison Kathy J. Helzlsouer Keerti V. Shah Eugene O. Major

22 Study Rationale The polyomaviruses JCV, BKV, and SV40 can produce brain tumors in laboratory animals DNA sequences from all three viruses have been found in human brain tumors Most previous epidemiologic findings for SV40 infection and brain tumors are null and based on surrogate measures of potential exposure to SV40 There are no epidemiologic studies of JCV or BKV and brain tumors The nested case-control design has been successfully used in previous studies of serum antibodies to infections and cancer

23 JCV in Glioblastomas Date First # tested # positive % positive Author Greenlee Dorries Arthur Huang Calderelli Del Valle Includes data from Del Valle, 2001; Calderelli-Stefano, 2000; Huang, 1999; Arthur, 1994; Greenlee, 1978; Dorries, 1987;

24 BKV in Glioblastomas Date First # tested # positive % positive Author Greenlee Corallini Dorries Arthur Martini Huang Calderelli Includes data from Calderelli-Stefano, 2000; Huang, 1999 ; Martini, 1996; Arthur, 1994; Greenlee, 1978; Corallini, 1987; Dorries, 1987;

25 SV40 in Glioblastomas Date First Author 1 # tested # positive % positive 1978 Greenlee Tabuchi Krieg Dorries Martini Huang Zhen Calderelli Ohgaki Kouhata Includes data from Calderelli-Stefano, 2000; Ohgaki, 2000; Huang, 1999; Zhen, 1999; Martini, 1996; Arthur, 1994; Krieg, 1981; Tabuchi, 1978; Greenlee, 1978; Dorries, 1987; Kouhata, 2001

26 Washington County, MD

27 Study Design and Methods A nested case-control study was conducted in the context of two research serum banks established in Washington Co., MD in 1974 and incident cases of brain tumors were identified from this cohort and 88 cancer-free controls were matched to the cases on age and gender Serum antibodies to JCV and BKV were measured using a virion ELISA in Eugene Major s lab at the NINDS Serum neutralizing antibodies to SV40 were measured using a plaque neutralization assay in Keerti Shah s lab at the Johns Hopkins University

28 Characteristics of Study Participants Characteristic Gender Cases (n=44) % Controls (n=88) % Male Female Education 12 grades > 12 grades Smoking Never Ever Age at dx* (years) Mean, SD 52.6, , 18.6 D.E.M. Rollison, unpublished data

29 Proportion of Cases (n=44) and Controls (n=88) Who Expressed Antibodies to JCV, BKV, and SV % Cases Controls 0 JCV BKV SV40 positive defined as >= 640 Weak or strong positive D.E.M. Rollison, unpublished data

30 SV40 Neutralizing Antibodies in the Context of JCV and BKV Capsid Ab s Measured by Virion ELISA No. of SV40 plaque + specimens # % p-value BKV BKV JCV JCV D.E.M. Rollison, unpublished data

31 Development of VLP-based Serology for JCV, BKV and SV40 Raphael Viscidi Dana Rollison Keerti Shah

32 Advantages of VLP-Based ELISA Virus Like-Particles (VLPs) are empty virions formed by self-assembly of capsid proteins VLPs resemble native virions and display conformationally dependent surface epitopes VLPs can be produced in large quantities using commercially available expression systems VLPs can be prepared as highly purified reagents, free of mammalian pathogens ELISAs are economical for large scale, high throughput testing ELISAs are versatile: quantitative antibody determinations, measurement of class and subclass immunoglobulins, and competitive binding experiments

33 Electron Micrograph of Purified SV40 VLPs PREPARATION AND PURIFICATION of VLPs: SV40, BK, and JC VP1 expressed in insect cells from recombinant baculoviruses VLPs purified by density gradient ultracentrifugation and column chromatography R. Viscidi, unpublished data

34 Reactivity of Rhesus Sera

35 Reactivity of SV40 Plaque Inhibition Assay Positive and Negative Monkey Sera In SV40, BK and JC VLP-Based ELISA A B OD 405nm Value SV40 BK JC SV40 BK JC VLP ELISA Plaque Negative Sera N = 11 Plaque Positive Sera N = 17 R. Viscidi, unpublished data

36 Serial Dilution of Two SV40 Plaque Inhibition Assay Positive Monkey Sera in SV40, BK and JC VLP-based ELISA SV40 VLP BK VLP JC VLP OD 405nm Value SV40 VLP BK VLP JC VLP /- cutpoint Reciprocal Dilution R. Viscidi, unpublished data

37 Correlation Between SV40 and BK Seroreactivity For SV40 Plaque Positive Monkey Sera 0.6 BK VLP ELISA (OD 405nm Value) Spearman Rank Test r = 0.68; P = SV40 VLP ELISA (OD 405nm Value) R. Viscidi, unpublished data

38 Correlation Between SV40 and JC SeroreactivityFor SV40 Plaque Positive Monkey Sera Spearman Rank Test r = 0.71, P = <0.001 JC VLP ELISA (OD 405nm Value) SV40 VLP ELISA (OD 405nm Value) R. Viscidi, unpublished data

39 Percent Blocking of SV40 Reactivity of 10 Monkey Sera by SV40 and BK VLPs SV40 VLPs BK VLPs Percent Blocking Serum Sample R. Viscidi, unpublished data

40 Percent Blocking of BK Reactivity of 10 Monkey Sera by SV40 and BK VLPs Percent Blocking Serum Samples SV40 VLPs BK VLPs R. Viscidi, unpublished data

41 Reactivity of Human Sera

42 Reactivity of Humans Serum Samples in SV40, BK and JC VLP-Based ELISA OD 405nm Value SV 40 (N = 130) BK (N = 130) VLP ELISA JC (N = 123) R. Viscidi, unpublished data

43 Reactivity of SV40 Plaque Inhibition Assay Negative and Positive Human Sera in SV40 VLP ELISA OD 405nm Value Negative (N = 115) Plaque Inhibition Assay Positive (N = 13) R. Viscidi, unpublished data

44 Agreement Between SV40 Plaque Inhibition Assay and SV40 VLP- Based ELISA SV40 VLP ELISA SV40 Plaque Inhibition Assay Positive Negative Total Positive Negative Total Sensitivity = 79% Specificity = 74% R. Viscidi, unpublished data

45 Reactivity of 130 Human Sera in JC VLP-Based ELISA By Quartile of SV40 Antibody Reactivity ANOVA on Ranks P = OD 405nm Value lo w e s t quartile (N = 30) second quartile (N = 31 ) th ird quartile (N = 30) highest quartile (N = 30) SV40 Reactivity Groups Correlation analysis of JC and SV40 Reactivity Spearman rank coefficient: r = 0.175, P = 0.06 R. Viscidi, unpublished data

46 Reactivity of 130 Human Sera in BK VLP-Based ELISA By Quartile of SV40 Antibody Reactivity ANOVA on Ranks P = < OD 405nm Value lowest quartile (N = 32) second quartile (N = 33) thrid quartile (N = 33) highest quartile (N = 32) SV40 Reactivity Groups Correlation analysis of BK and SV40 Reactivity Spearman rank coefficient: r = 0.60, P = <0.001 R. Viscidi, unpublished data

47 Community-based Survey Raphael Viscidi Aimee Kreimer Maura Gillison

48 Screening Study Serum specimens were collected in from three groups for an HPV-related project. The serum donors were drug users (n=151, median age 35 yrs), HIVseropositive individuals (n=184, median age 44 yrs) and individuals recruited at a Community Health Clinic (n=159, median age 43 yrs). There were 285 males and 209 females. VLP ELISA results are available for 491 of the 494 specimens.

49 491 Human Sera from 2001 Blood Draw Tested in SV40 and BK VLP-Based ELISA OD 405nm Value SV40 VLP ELISA BK R. Viscidi, unpublished data

50 Histogram of Reactivity of 491 Human Sera in BK and SV40 VLP-based ELISA Histogram of Reactivity of 491 Human Sera in BK and SV40 VLP-Based ELISA BK SV40 Number of Sera OD Values From to Units BIN Size OD Units R. Viscidi, unpublished data

51 Reactivity of 491 Human Sera in BK VLP-based ELISA By Quartile of SV40 VLP Antibody Reactivity Kruskal-Wallis ANOVA p < OD 405nm Value Lowest Quartile Second Quartile Third Quartile Highest Quartile SV40 Reactivity Group R. Viscidi, unpublished data

52 Studies in Progress To determine whether the SV40 reactivity can be explained by cross-reactivity with JCV and BKV with competitive binding assays

53 SV40 Neutralising Antibody in Humans in England (W. Knowles, unpublished data) 2435 sera; 1991; 1 to 69 years Overall seroprevalence 3.2% Seroprevalence not related to age (p = 0.52) Only 10% of titres >1 in 16 Titre not related to age (p = 0.99) Seroprevalence and titre not related to sex (p = 0.44; p = 0.89) Significant association with BK (p < 0.001) 3.8% positive for SV40 in BK positives; 0.9% in BK negatives Significant association with JC (p = 0.009) 4.5% positive for SV40 in JC positives; 2.5% in JC negatives

54 Age Seroprevalence of BKV, JCV and SV40 in England (1991) (W. Knowles, unpublished data) SV40 BKV JCV Seroprevalence 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 1 to 4 5 to 9 10 to to to to to to to 69 Age group (years)

55 Distribution of anti-sv40 positive titres D Number with titre Titre W. Knowles, unpublished data

56 Comparison of cases and controls for SV40 prevalence

57 SV40 antibodies in sera of patients and controls Donors (Strickler et al., CEPB 5: , 1996) Number of sera Number with antibodies* Mesothelioma patients 34 3 Osteosarcoma patients 33 1 Non-cancer controls 35 1 *antibodies in lower titers, in SV40 plaque neutralization assays

58 Polyomavirus antibodies in sera from osteosarcoma patients (J Carter, G Wipf, D Galloway, FHCRC, Seattle, unpublished data) VLPs expressed for BKV, JCV and SV40, and confirmed to be of correct size by electronmicroscopy In direct ELISA, sera from a population-based control group (n=300) were compared with sera from osteosarcoma patients (n=112) % with antibodies BKV 64.7 JCV 36.0 BKV and JCV 19.7 SV40 1.2

59 Polyomavirus antibodies in sera from osteosarcoma patients (J Carter, G Wipf, D Galloway, FHCRC, Seattle, unpublished data) Continued: The five sera which reacted with SV40 also reacted with BKV, JCV or both Reactivity to SV40 eliminated by preadsorption with heterologous VLPs Studies in progress to characterize further specificity of SV40 reactivity

60 Conclusions The virologic and serologic data do not indicate that SV40 infection is widely disseminated in the community It will not be possible to evaluate the etiologic link between SV40 and human tumors until the virus transmission is characterized