Immunological Cross-Reactivities of Woodchuck and Hepatitis

Transcription

1 INFECTION AND IMMUNITY, Feb. 1982, p /82/ $02.00/0 Vol. 35, No. 2 Immunological Cross-Reactivities of Woodchuck and Hepatitis B Viral Antigens IRVING MILLMAN,* THERESA HALBHERR, AND HEIDI SIMMONS Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania Received 5 August 1981/Accepted 29 September 1981 Woodchuck sera were tested for antigens and antibodies with tests which detect human hepatitis virus antigens and antibodies. Data on 264 woodchuck sera are presented. Summers et al. (5), Werner et al. (6), Cummings et al. (1), and Snyder and Summers (4) have described viruses similar to hepatitis B virus (HBV) in woodchucks (woodchuck hepatitis virus lwhv]), Marmota monax. Werner et al. (6) reported that antigens found on the cores of human and woodchuck viruses are crossreactive but that there is a lack of cross-reactivity between the surface antigens of the two viruses in immunodiffusion experiments, suggesting that the major antigenic determinants of the viral surfaces are different. More sensitive passive hemagglutination tests indicate that there are common minor surface determinants. These findings are in agreement with their liquid hybridization experiments, in which they showed 3 to 5% homology. Galibert et al. (G. Galibert, T. N. Chen, and E. Mandart, Proc. Natl. Acad. Sci. U.S.A., in press) compared the section of HBV DNA which codes for the hepatitis B surface antigen (HBsAg) with the equivalent section of WHV DNA. They found that 487 of 678 residues were identical; that is, the homology for the surface antigen genes was 78%. Feitelson et al. (2) compared the surface antigens of human and ground squirrel hepatitis virus and WHV both serologically and by tryptic peptide mapping. Although only two woodchuck sera were studied, these authors concluded that there is a close relationship among all three. These findings predict that immunological crossreactivity should be greater than that reported by Werner et al. (6). This paper includes data on the cross-reactivity with HBV of 264 sera from woodchucks trapped in Pennsylvania and Delaware. These sera were assayed with commercial reagents (Abbott Laboratories, North Chicago, Ill.) designed to detect HBV antigens and antibodies in human sera. These included the Ausria II radioimmunoassay (RIA) and Auszyme enzyme immunoassay (EIA) kits for the detection of HBsAg, the Ausab RIA kit for the detection of antibody to hepatitis B surface antigen (anti- HBs), the Corab RIA kit for the detection of antibody to hepatitis B core antigen (anti-hbc) or hepatitis B core antigen (HBcAg), and RIA kits for the detection of the e antigen that is closely associated with hepatitis B infection (HBeAg) and antibody to the e antigen (anti- HBe; Abbott Laboratories). Woodchuck sera were collected from three sources. Our primary source was Cocalico Woodchuck Farms, Reinhold, Pa., where 216 sera were collected. These sera were collected mainly in Delaware. Our second source was a colony of M. monax maintained by our laboratory at the New Bolton Center of the University of Pennsylvania School of Veterinary Medicine in Chester County, Pennsylvania, where 37 sera were collected. Lastly, 11 sera were from the woodchuck colony maintained by Robert Snyder at the Penrose Laboratories of the Philadelphia Zoological Gardens. Guinea pig anti-woodchuck surface antigen (anti-whs) was produced by centrifuging woodchuck serum containing WHV at 160,500 x g for 4 h in a Ti 60 rotor (Beckman Instruments, Inc., Fullerton, Calif.) at 2 C. The pellet containing virus was suspended in physiological saline and mixed with complete Freund adjuvant (three parts of Freund adjuvant with one part of the antigen suspension). Twelve guinea pigs were injected subcutaneously with this suspension at multiple sites. At the end of 6 weeks, a booster of the WHV suspension was injected subcutaneously. Animals were bled by cardiac puncture 7 days after the last injection and then at 4-week intervals. Several precipitating antibodies were found by immunodiffusion (ID), including one which reacted solely with woodchuck sera positive for WHV. All antibodies which did not react with WHV were removed by absorption with one or two parts of woodchuck serum which did not contain WHV. The absorbed guinea pig antiserum produced a heavy precipitin band forming a line of identity with that of antiserum against WHV which occurs naturally in woodchucks. This guinea pig antiserum did not react by ID with purified woodchuck hepatitis core antigen 752

2 VOL. 35, 1982 NOTES 753 (WHcAg) isolated from woodchuck liver. Since we were able to absorb all precipitating antibody with purified woodchuck hepatitis surface antigen (WHsAg), we concluded that our guinea pig antiserum contained only anti-whs. Of the 264 woodchucks in this survey, 18 had naturally acquired anti-whs in their sera. Orcein staining (3) produced a magenta coloration similar to that produced by HBsAg-anti-HBs precipitin bands. Woodchuck sera which reacted with reagents designed to detect HBsAg were considered presumptive positives for WHsAg. As a rule, either RIA or EIA (peroxidase-linked assay) was used. To confirm these presumptive-positive tests, the woodchuck sera in question were incubated with an equal volume of either woodchuck anti-whs or guinea pig anti-whs for 2 h at 37 C and 18 h at 5 C. A drop in count (by RIA) or optical density (by EIA) of 50% or more compared with controls (same woodchuck serum combined with either woodchuck or guinea pig serum known to contain no anti-whs) was considered a positive confirmed test. Woodchuck sera which reacted with reagents designed to detect anti-hbs were considered presumptive positives for anti-whs. These were confirmed in the same manner as were the presumptive positives for WHsAg, except these presumptive-positive woodchuck sera were incubated with woodchuck sera known to contain WHsAg. A drop in count of 50% or more (by RIA) indicated a positive confirmed test for anti-whs. Presumptive-positive tests for HBcAg, anti-hbc, HBeAg, and anti-hbe were not confirmed but were considered positive solely on the basis of reacting with the reagents designed to detect the equivalent antigens and antibodies in human sera. Confirmation assays were also conducted by immunodiffusion, where the presumptive-positive woodchuck sera incubated with their counterparts, antibodies or antigens, were shown to no longer produce a precipitin reaction against either constituent of the mixture. Since results with Ausria II and Auszyme did not differ when any of our sera were tested for HBsAg or WHsAg, we used either test and, more often, both. To determine the sensitivity of Ausria II or Auszyme for the detection of WHsAg, 18 positive sera were diluted to 1: 20,000, using normal human sera as diluent. A ratio of 2.1 or more was considered positive for HBsAg. We assumed the same conditions for WHsAg. The results of this evaluation (Table 1) show that 5 of 17 positive sera in the 1:10 dilution could be detected in a dilution of 1: 20,000. The number of positives detected increased with an increase in concentration. The results of testing 24 woodchuck sera for other HBV markers are shown in Table 2. Other antigens and antibodies detected by cross-reactivity (Table 2) were anti-woodchuck core antigen (anti-whc) (by Abbott's Corab), anti-whs (by Abbott's Ausab), and woodchuck hepatitis e antigen (WHeAg) and anti-woodchuck e antigen (anti-whe) (using Abbott's HBeAg and anti-hbe assays). We found (Tables 1, 2, and 3) that there appeared to be no association of anti-whc assayed by RIA with WHsAg assayed by RIA. Unlike human hepati- TABLE 1. Cross-reactivity of woodchuck sera with HBsAg, using RIA (Ausria II) Ratio (cpm test/cpm controls) with following dilutiona: Woodchuck serum 1:1 1:10 1:100 1:1000 1:2000 1:4000 1:8000 1:10,000 1:20, ND NDb ND ND ND ND ND ND ,1.9C ND a A ratio of 2.1 or more was considered positive. Numbers b represent the mean of seven normal human sera. ND, Not done. c Assayed twice, 1 month apart.

3 754 NOTES INFECT. IMMUN. TABLE 2. Cross-reactivity of woodchuck sera with different HBV assay reagents Presence of Presence of Presence of Woodchuck serum HBsAg by anti-hbc by Ausria II Coraba Anti-HBs by Ausubb HBeAgC anti-hbed ND NDe ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 2.7 ND ND 3.2 ND ND 3.3 ND ND 1.2 ND ND 1.8 ND ND 2.2 ND ND 2.1 ND ND 1.8 ND ND 2.7 ND ND 2.Of ND (weak) - ND ND a The presence of anti-hbc is determined by comparing the net counts per minute of the specimen to a cutoff value. The cutoff value is the mean of the negative controls plus the mean of the positive controls divided by 2. Specimens whose counts per minute are less than the cutoff value are Corab positive. b A ratio of 2.1 or greater is considered positive. c A ratio of 2.1 or greater is considered positive. d The presence of anti-hbe is determined by comparing the net counts per minute of the specimen to a cutoff value. The cutoff value is the mean of the negative controls plus the mean of the positive controls divided by 2. Specimens whose counts per minute are less than the cutoff value are anti-hbe positive. ' ND, Not done. f At 1:5 dilution. tis B, where all sera containing HBsAg also contain anti-hbc, sera from woodchucks with WHsAg did not always contain anti-whc. We are confident of the reliability of the crossreactivity assay for WHsAg (Table 3). The reliability of the cross-reactivity assay for anti-whc has not been tested because there are no commercial assays for woodchuck antigens. If we assume that the cross-reactivity assay for anti- WHc is reliable, then there is no association between anti-whc and WHsAg. Another way to determine this is by use of a direct test with ID and purified WHcAg. A total of 45 WHsAgpositive sera were selected from our 264 woodchuck sera collection and tested for anti-whc. All 45 were negative. While we would have expected some positives on the basis of the data shown in Table 3, we could attribute these results to a lack of sensitivity of the ID assay. Of 253 sera tested for anti-whs, 14 (Table 3) proved positive by RIA for anti-hbs and 9 by ID for anti-whs. Of the original 264 sera, 11 could not be included because of insufficient quantity. Of 253 sera, 235 proved negative by both assays, whereas only 5 assayed positive by both assays. These five and the others (nine positive by RIA and four positive by ID only) were confirmed as positive by absorption with sera containing WHsAg. Anti-WHs was mixed with an equal volume of woodchuck serum containing

4 VOL. 35, 1982 WHsAg. After an incubation of 2 h at room temperature, no precipitin band formed by immunodiffusion. Those positive by ID also produced lines of identity with woodchuck anti- WHs. When ID and Ausria II or Auszyme were compared for the detection of WHsAg, using 253 woodchuck sera (Table 3), a strong and significant association of the two assays was found when analyzed by the chi-square test. One woodchuck serum was positive by ID (reacting with both guinea pig and woodchuck anti-whs) and negative by both Ausria II and Auszyme. Woodchuck sera positive for WHsAg by Ausria II and Auszyme could be confirmed by neutralization with both guinea pig or woodchuck anti- WHs. Confirmation was shown by incubating woodchuck positive sera with either guinea pig or woodchuck anti-whs for 2 h at 37 C before testing or by the procedure recommended by Abbott Laboratories in which guinea pig or woodchuck anti-whs is incubated with polystyrene beads containing a complex of antibody and antigen before the addition of "SI-labeled anti- HBs. In either case, a drop in count of 50% or more was considered a positive confirmation. (6 I, It.4.. I, I/ I t i 0 5..dI_. I NOTES 755 Confirmations were never achieved when human anti-hbs was used in the above procedures, nor could WHsAg be neutralized by human anti-hbs when tested by ID (Fig. 1). However, complete neutralization was achieved with guinea pig and woodchuck anti-whs. However, when human serum containing HBsAg was incubated with woodchuck or guinea pig anti- WHs, anti-whs activity was still present, and HBsAg disappeared by RIA or ID. This indicates that anti-whs may contain antibodies to all determinants of WHsAg, whereas, anti-hbs may lack antibodies for some determinants of WHsAg. These data are in accord with those of Galibert et al. (in press) and indicate high immunological cross-reactivities of WHsAg and HBsAg which could be expected with 78% homology. Furthermore, high cross-reactivities can also be found with WHeAg and anti-whe. These close antigenic relationships between antigens and antibodies of WHV and human HBV should make it relatively easy for investigators to look for markers of WHV infection with established HBV assays. The differences observed when testing for anti-whs, using Ausab and ID, may i o J L 3 X > ell, f 4 FIG. 1. ID pattern showing that human anti-hbs does not block the reaction between guinea pig anti-whs and WHsAg. The center well (0) contains guinea pig anti-whs. Wells 1 and 4 contain serum from a positive woodchuck. Wells 2 and 3 contain serum of the positive woodchuck absorbed with two different human anti-hbs sera. Well 5 is a control containing serum of a positive woodchuck absorbed with serum from a normal woodchuck. Well 6 contains serum of a normal woodchuck which was used for the absorption in well 5.

5 756 NOTES INFECT. IMMUN. 4)~~~~~~~~~~~~~~~~~~~~~~~~~1 N4)~~~~~~~~~~~~~~~~1 ~~~U~ ~ W)WI)~~~~~~~~,1 40 ~ ~ ~ ~ ~~~06C~ ~.o 40~~~~~~~~~~~~0 ~~~~~~~~0~ ~ ~ ~ ~ O 4) 44) U~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4,U 4) 4.) U' 0. 4)~~~~~~~~~~~~~~~~~~~~~~~. 40~~~~~~~~~ -~~~~ 4)~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~0 co.- 0,,u~~ ~ ~ ~ ~ ~ ~ ~ ~ ~

6 VOL. 35, 1982 NOTES 757 reflect differences in subtypes of WHsAg, since nine woodchuck sera were found positive by Ausab but negative by ID, and four were positive by ID and negative by Ausab (Table 3). Furthermore, the inability to completely absorb all WHsAg antibodies with HBsAg indicates determinants on WHsAg which are missing on HBsAg. The low incidence of anti-whc in woodchuck sera containing surface antigen should make the isolation of WHc particles from woodchuck livers easier than it has been to isolate HBcAg from human livers. This work was supported by Public Health Service grants CA-06551, RR-05539, and CA from the National Institutes of Health and by an appropriation from the Commonwealth of Pennsylvania. LITERATURE CITED 1. Cummigs, I. W., J. K. Browne, W. A. Salsis, G. V. Tyler, R. L. Snyder, J. M. Smolec, and J. Summers Isolation, characterization and comparison of recombinant DNAs derived from genomes of human hepatitis B virus and woodchuck hepatitis virus. Proc. Natl. Acad. Sci. U.S.A. 77: Feitelson, M. A., P. L. Marion, and W. S. Robinson Antigenic and structural relationships of the surface antigens of hepatitis B virus, ground squirrel hepatitis virus, and woodchuck hepatitis virus. J. Virol. 39: Shikata, T., T. Uzawa, N. Yoshiwara, T. Akatsuka, and S. Yamayold Staining methods of Australia antigen in paraffin sections. Detection of cytoplasmic inclusion bodies. Jpn. J. Exp. Med. 44: Snyder, R. L., and J. Summers Woodchuck hepatitis virus and hepatocellular carcinoma. p In M. Essex, G. Todaro, and H. zur Hausen (ed.), Viruses in naturally occurring cancers. Cold Spring Harbor Conferences on Cell Proliferation, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 5. Summers, J., J. M. Smolec, and R. L. Snyder A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proc. Natl. Acad. Sci. U.S.A. 75: Werner, B. G., J. M. Smolec, R. Snyder, and J. Summers Serological relationship of woodchuck hepatitis virus to human hepatitis B virus. J. Virol. 32: Downloaded from on June 27, 2018 by guest