Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992), pp. 641--644

Detection of Hepatitis C Virus-Specific Antigens in Semen from Non-A, Non-B Hepatitis Patients G I R I S H J. K O T W A L , PhD, VINOD K. R U S T G I , MD, and B A H I G E M. B A R O U D Y , PhD

Semen samples from nine patients clinically diagnosed as having non-A, non-B hepatitis (NANBH) were tested by an ELISA using antibodies raised in rabbits against HCVspecific antigens. The semen from all nine patients had elevated levels o f HCV-specific antigen in comparison to semen from five healthy donors. Semen from five o f the nine patients had significant levels of the HCV-specific antigen. Seven of the eight serum samples from these patients were reactive with the standard C-IO0 HCV ELISA. Eight of these nine patients had serum reactive for HCV-specific antibodies in our ELISA using HCV-specific antigens. This more direct evidence for viral presence supports the earlier epidemiological data suggesting that HCV could be transmitted sexually. KEY WORDS: hepatitis C; antigens; semen; non-A, non-B hepatitis.

The majority of parenterally transmitted non-A, non-B hepatitis ( P T - N A N B H ) cases are believed to be caused by the hepatitis C virus (HCV) (1-3). Possible sources of infection for a substantial number of remaining cases, known as sporadic hepatitis C, have been controversial. It has been suggested that heterosexual (4-6), and possibly homosexual activity (7), may be important, perhaps accounting for up to an additional 11% of all cases seen. In order to obtain more direct evidence for the potential of sexual transmission of HCV, we utilized an enzyme-linked immunosorbent assay (ELISA) consisting o f structural epitopes of H C V to detect its presence in semen samples.

Manuscript received September 16, 1991; accepted October 30, 1991. From the Division of Molecular Virology, James N. Gamble Institute of Medical Research, Cincinnati, Ohio 45219; and INOVA Institute of Research and Education, 3300 Gallows Road, Falls Church, Virginia 22046. Address for reprint requests: Dr. Vinod K. Rustgi, Office of Medical Research, Department of Medicine, Fairfax Hospital, Falls Church, Virginia 22046.

MATERIALS AND METHODS The available nucleotide sequence information (8-11) reveals that the genome organization of hepatitis C virus is similar to the flavi- and pestivirus families. It encodes a single continuous open reading frame (ORF) for a polypeptide of at least 3000 amino acids consisting of the structural region located at the NH 2 terminus and a nonstructural region at the carboxy terminus. The antigenicity of the sequence of the first 20% from the NH 2 terminus of the putative polypeptide was analyzed. Two antigens, HCV-GK and HCV-BB, whose sequence was derived from within this region, were synthesized. Each antigen was conjugated to KLH and injected in New Zealand rabbits along with Freund's adjuvant using standard protocols (12). In order to determine the titers of the antibodies made, ELISA plates coated separately with 100 ng of the two antigens were treated with the serial dilutions of the prebleed and the three-month bleeds from the rabbits for 4-6 hr at room temperature. The plates then were washed five times with phosphate-buffered saline containing 0.1% Tween and treated with goat anti-rabbit IgG conjugated with horse radish peroxidase (diluted 1:200) for 2-4 hr. The washed plates were then treated with a substrate solution prepared by dissolving four OPD (1,2-phenylenediamine dihydrochloride) tablets (2 mg/tablet) in 12 ml of 0.1 M citric acid-phosphate, pH 5.0, and further adding 5

Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992)

0163-2116/92/0500-0641506.50/09 1992PlenumPublishingCorporation

641

K O T W A L ET AL TABLE 1. ANTIBODYTITRATION Serum dilution

HCV-GK 1/10,000 1/25,000 1/50,000 1/75,000 1/100,000 1/250,000 1/375,000 1/500,000 1/750,000 1/1,000,000 HCV-BB 1/10,000 1/25,000 1/50,000 1/75,000 1/100,000 1/250,000 1/375,000 1/500,000 1/750,000 1/1,000,000

TABLE2. SEMENANALYSIS

Prebleed A450

Average o f 3 too. dup-prebleed

0.i12 0.088 0.102 0.083 0.074 0.067 0.068 0.085 0.071 0.084

2.175 2.248 2.145 2.079 2.130 1.710 1.427 1.350 1.083 0.721

0.226 0.142 0.169 0.154 0.134 0.149 0.167 0.193 0.185 0.252

2.459 2.521 2.480 2.475 2.470 2.036 1.746 1.341 0.997 0.527

ixl of 30% H20 2. The plate then was incubated in the dark for 5 min at room temperature. To stop the reaction, 50 t~l of 1 M H2SO 4 was added to each well and absorbance of the orange-colored end-product was read by a microweU plate reader at 450/492 nm. Table 1 shows the absorbance values obtained for the serial dilution of the prebleed and the three-month bleed for each of the two antigens. The p r e s e n c e o f antibodies in a v e r y high dilution (1: 1,000,000) suggested that both antigens were highly immunogenic and that the antibody was likely to be a very sensitive reagent for the detection of structural components of the HCV. Blood-free semen samples from nine patients with clinical manifestation of N A N B H and from five healthy donors were homogenized and treated with 0.5% NP-40 in phosphate-buffered saline to obtain a final concentration of 0.1% NP-40 at room temperature. The contents were centrifuged at 12,000 rpm for 15 min at 4 ~ C in a microfuge. E L I S A plates then were coated with 100-125 ~l of the supernatant for 8-12 hr at 4 ~ C. The plates were then washed five times with PBS containing 0.1% Tween 20 and treated with the rabbit antibody at a dilution of 1:100 for 6-8 hr at room temperature. The plate was washed once again and treated as described above with goat anti-rabbit antibody and further with the substrate solution; the absorbance values obtained at 450/492 nm are shown in Table 2. In order to verify whether patients whose semen contained HCV-specific antigen had HCV-specific antibodies in their serum, we tested the serum from eight of the nine patients. We coated ELISA plates with 50 ng of either HCV-GK or HCV-BB and added 100 txl of the eight serum samples and five healthy donors diluted 1:5 in PBS-Tween to individual wells. The procedure was identical to the one described above except that a mixture of goat anti-human and rabbit anti-human IgG conjugated 642

Patient

1 2 3 4 5 6 7 8 9 Mean of 5 Control Semen Range of Control Semen

HCV-GK

HCV-BB

1.519 2.7% 2.363 1.930 2.399 2.045 1.622 1.420 1.513

0.813 2.052 1.420 1.110 1.910 1.339 1.224 0.854 0.709

0.900

0.500

0.700-1.200

0.380-0.640

with horse radish peroxidase was used as a second antibody in place of goat anti-rabbit. The absorbance values are shown in Table 3 under the columns of HCV-GK and HCV-BB. The cutoff values were obtained by multiplying by a factor of 1.5 the mean absorbance value obtained by averaging the absorbance values for the five healthy donors. The serum samples were also tested with commercially available Ortho test kit, and the values are also shown in Table 3. RESULTS

All nine s e m e n samples o b t a i n e d f r o m N A N B H patients had a b s o r b a n c e values higher than the values o b t a i n e d f r o m the h e a l t h y d o n o r s e m p l o y i n g antibodies to e a c h o f the t w o antigens. Significantly higher a b s o r b a n c e values (greater than twice the m e a n a b s o r b a n c e value for h e a l t h y d o n o r s ) w e r e o b t a i n e d in five o f the nine s e m e n samples (Table 2). Available s e r u m f r o m eight patients w a s reactive. Patient 4 w a s n o n r e a c t i v e with b o t h the O r t h o test and an E L I S A using H C V - B B but w a s r e p e a t e d l y positive (A492 ,mI.669 at a c u t o f f o f 0.447) with the E L I S A using H C V - G K as antigen. A n a l y s i s o f a large n u m b e r o f s e r u m samples has s h o w n that an E L I S A using H C V - G K has a higher sensitivity and TABLE3. SERUMANALYSIS Patient

HCV-GK

HCV-BB

Ortho

Cutoff 1 2 3 4 5 6 7 8 9

0.220 2.669 0.320 2.545 0.681 0.829 2.713 NA 2.761 2.423

0.768 1.042 1.340 3.298 0.633 1.100 2.662 NA 1.306 1.112

0.140 2.215 0.300 2.208 0.079 2.262 2.287 NA 0.961 0.672

lnterp. *

R R R R R R R NA R R

*Interp. = Interpretation; R = Reactive; NA = Not available. Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992)

DETECTION OF HCV ANTIGENS specificity in c o m p a r i s o n to E L I S A using the other two antigens (13). Based upon our results, all patients tested were infected with H C V , and their s e m e n contained varying a m o u n t s of H C V viral antigen. DISCUSSION Well-recognized parenteral routes for transmission of H C V do not account for a substantial n u m b e r of clinical cases. While m a n y patients are reluctant to admit a history of k n o w n risk factors, particularly intravenous drug use, epidemiologic data m a y suggest a role for sexual transmission. Alter et al (4) d e m o n s t r a t e d an association between multiple heterosexual partners and incidence of hepatitis C. A higher than e x p e c t e d incidence of 8% was reported in h o m o s e x u a l men in Spain by Esteban et al (14) and in D e n m a r k b y Melbye et al (6). Other studies h a v e shown no direct evidence of H C V transmission sexually or in household contacts (15). Hepatitis C virus transmission seems to be associated with transmission of hepatitis B virus as well as H I V (16). This study detected the p r e s e n c e of HCV-specific viral antigens in the s e m e n of nine patients with well-documented H C V infection. In contrast, sem e n f r o m five healthy donors did not show antigen presence. The p r o s p e c t of using P C R to detect viral presence has b e e n considered. S o m e investigations have not found H C V in s p e r m by this technique (17). A potential p r o b l e m in using P C R m a y result f r o m the e x t r e m e l y low n u m b e r of virus particles in the s e m e n of hepatitis C patients. The hepatitis C virus contains only one R N A molecule. This, coupled with an inefficient reverse-transcription step, m a y result in the lack of a clear signal. In contrast, there m a y be a several-fold higher n u m b e r of structural protein molecules present leading to a higher sensitivity of the techniques described here. Our findings support the potential for sexual transmission of hepatitis C virus. While sexual transmission of H C V m a y be an inefficient mechanism, the role of other factors in determining sexual transmission m u s t be considered. T h e s e factors include local antibody, (ie, IgA antibody in the female genital tract), mucosal abrasions, and the i m p o r t a n c e of a critical a m o u n t of viral inoculum. L y m p h o c y t e s are present in s e m e n in reduced numbers (18), and further e x p e r i m e n t s to localize w h e t h e r the virus is present cell-free or attached to Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992)

s p e r m cells are needed. It also remains to be determined whether h u m a n spermicides such as benzalkonium chloride are needed to stop transmission and w h e t h e r they are protective. R e c o m m e n dations m a y also be needed for testing of sperm banks and potential h u m a n donors for artificial insemination programs.

ACKNOWLEDGMENTS The authors are grateful to Dr. Paul Russell of the Division of Reproduction and Fertility at Christ Hospital for his assistance in providing semen from healthy donors, to Ms. Frances McDonald for assistance in manuscript preparation, and to Dr. Gilbert Schiff for support and valuable suggestions for revising the manuscript.

REFERENCES 1. Choo Q-L, Kuo G., Weiner AJ, Overby LR, Bradley DW, Houghton M: Isolation of a cDNA clone derived from a

blood-borne non-A, non-B viral hepatitis genome. Science 244:359-362, 1989 2. Kuo G, Choo Q-L, Alter HF, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, Dienstag JL, Alter MJ, Stevens CE, Tegtmeier GE, Bonino F, Colombo M, Lee W-S, Kuo C, Berger K, Shuster JR, Overby LR, Bradley DW, Houghton M: An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 244:362-364, 1989 3. Choo Q-L, Weiner AJ, Overby LR, Bradley DW, Houghton M: Hepatitis C virus: The major causative agent of viral non-A, non-B hepatitis. Br Med Bull 46:423-441, 1990 4. Alter MJ, Coleman PJ, Alexander WJ, Kramer E, Miller JK, Mandel E, Handler SC, Margolis HS: Importance of heterosexual activity in the transmission of hepatitis B and non-A, non-B hepatitis. JAMA 262:1201-1205, 1989 5. Alter MJ, Hadler SC, Judson FN, Mares A, Alexander WJ, Hu PY, Miller JK, Moyer LA, Fields HA, Bradley DW, Margolis HS: Risk factors for acute non-A, non-B hepatitis in the United States and association with hepatitis C virus infection. JAMA 264:2231-2235, 1990 6. Melbye M, Bigggar RJ, Wantzin P, Krogsgaard K, Ebbesen P, Becket NG: Sexual transmission of hepatitis C virus: Cohort study (1981-9) among European homosexual or household contact with chronic carriers. Br Med J 301:210212, 1990 7. Tedder RS, Gilson RJC, Briggs M, et al: Hepatitis C virus: Evidence for sexual transmission. Br Med J 302:1299-1302, 1991 8. Okamoto H, Okada S, Sugiyama Y, Yotsumoto S, Tanaka T, Yoshizawa H, Tsuda F, Miyakawa Y, Mayumi M: The 5'-terminal sequence of hepatitis C virus genome. Jpn J Exp Med 607:167-177, 1990 9. Kato N, Hijikata M, Ootsuyama Y, Nakagawa M, Ohkoshi S, Sugimura T, Shimotohno K: Molecular cloning of the human hepatitis C virus genome isolated from human carriers. Proc Natl Acad Sci USA 87:9524-9528, 1990 10. Choo Q-L, Richman KH, Han JH, Berger K, Lee C, Dong C, GaUegosC, Coit D, Medina-Selby A, BaITPJ, Weiner AJ,

643

KOTWAL ET AL Bradley DW, Kuo G, Houghton M: Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci USA 88:2451-2455, 1991 11. Takamizawa A, Mori C, Fuke I, Manabe S, Murakami S, Fujita J, Onishi E, Andoh T, Yoshida I, Okayama H: Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol 65:1105-1113, 1991 12. Green N, Alexander H, Olson S, Alexander S, Shinnick TM, Sutcliffe JG, Lerner R: Immunogenic structure of the influenza virus hemagglutinin. Cell 28:477-487, 1982 13. Kotwal GJ, Baroudy BM, Holland PV, Kuramoto KI, Zeldis J: A new structural epitope for the detection of hepatitis C virus infection. Gastroenterology 100(5):762A, 1991

644

14. Esteban JI, Esteban R, Viladomiu L, et al: Hepatitis C virus antibodies among risk groups in Spain. Lancet 2:294-297, 1989 15. Everhart JE, DiBisceglie AM, Murray LM, Alter HJ, Melpolder JJ, Kuo G, Hoofnagle JH: Risk for non-A, non-B (type C) hepatitis through sexual or household contact with chronic carders. Ann Intern Med 1i2(7):544-545, 1990 16. Tor J, Llibre JM, Carbonell M, Muga R, Ribera A, Soriano V, Clofet B, Sabria M, Foz M: Sexual transmission of hepatitis C virus and its relation with hepatitis B virus and HIV. Br Med J 301:1130-1133, 1990 17. Hsu HH, Wright TL, Luba D, et al: Is hepatitis C virus in human secretions? Gastroenterology 100(5):754A, 1991 18. Bagasra O, Freund M, WeidmannJ, Harley G: Interaction of human immunodeficiency virus with human sperm in vitro. J Acq Immune Deficiency Syndromes 1:431-435, 1988

Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992)