Serological Markers in Chimpanzees

Transcription

1 INFECTION AND IMMUNITY, May 1979, p Vol. 24, No /79/ /05$02.00/0 Hepatitis B e-antigen and Its Correlation with Other Serological Markers in Chimpanzees CHUNG-MEI LING,* ISA K. MUSHAHWAR,1 LACY R. OVERBY,' KENNETH R. BERQUIST,2 AND JAMES E. MAYNARD2 Diagnostics Division, Abbott Laboratories, North Chicago, Illinois and Hepatitis Laboratories Division, Bureau of Epidemiology, Center for Disease Control (World Health Organization Collaborating Center for Reference and Research on Viral Hepatitis), Phoenix, Arizona Received for publication 29 January 1979 Three chimpanzees experimentally infected with hepatitis B virus, and another three chimpanzees that were hepatitis surface antigen carriers, were studied for the presence of viral antigens and humoral immune responses. Quantitative analyses of hepatitis B surface and e-antigens in sequential serum samples at early acute stages revealed cyclic oscillations of these two antigens following a synchronous pattern. Similar analyses of anti-e-antigen and anti-hepatitis B core antigen antibodies from the three experimentally infected primates indicated that peak titers of these two antibodies occurred as surface antigen decreased to undetectable levels. Of the three surface-antigen carriers, two were positive for e- antigen and one was positive for e-antigen antibody for the entire course of surveillance (8, 9, and 22 months, respectively). Three distinct antigen-antibody systems associated with hepatitis B virus (HBV) infection have now been established: hepatitis B surface antigen (HBsAg) and its antibody (anti-hbs); hepatitis B core antigen and its antibody (anti- HBc); and the more recently discovered hepatitis B e-antigen (HBeAg) and its antibody (anti- HBe) (8). The presence of HBsAg in serum has been used as a practical marker for HBV infection. Anti-HBs is observed most frequently in convalescent serum, whereas the presence of anti-hbc indicates past or current infection with HBV. HBeAg is antigenically distinct from HBsAg and the core antigen (7, 9). Recent studies have suggested a positive correlation between HBeAg, virus-specific deoxyribonucleic acid polymerase activity, and circulating Dane particles (2, 6, 14, 18). Thus, the presence of HBeAg in serum has been proposed as a marker of ongoing liver damage in patients and a high risk of transmission of hepatitis B. The presence of anti-hbe is indicative of lower infectivity and a better clinical prognosis (4, 5, 13). Complete and detailed analyses of the appearance, disappearance, or persistence of all the above HBV markers in an infected animal are not readily available in the literature. The recent availability of specific and very sensitive solidphase radioimmunoassays (RIAs) for the detection of anti-hbc (15), HBeAg, and anti-hbe (11) made possible an assessment of temporal patterns of these serological markers in chronic and acute self-limited HBV infection in six chimpanzees. MATERIALS AND METHODS The chimpanzees were inoculated and bled as described previously by Berquist and co-workers (3). Serum samples from the various consecutive bleedings were tested with commercial RIA kits, Ausria II for HBsAg and Ausab for anti-hbs (Abbott Laboratories, North Chicago, Il.). The procedures for HBeAg, anti- HBe, and anti-hbc were described previously (11, 15). All serial dilutions were made in normal human serum devoid of all known hepatitis B markers. RESULTS Chronology of HBV antigenic markers after induced HBV infections. In the three chimpanzees experimentally infected with HBV, five antigenic markers were shown to appear sequentially during the course of disease. In general, these markers appeared in the following sequential order: HBsAg, HBeAg, anti-hbc, anti-hbe, and anti-hbs. The titers and time course of the antigens and antibodies of one animal (CC) are shown graphically in Fig. 1. HBsAg and HBeAg became detectable almost simultaneously or only a few days apart. HBeAg was eventually replaced by anti-hbe, but a short period of coexistence of the antigen and antibody was observed. The appearance of anti-hbe also signaled the declining and eventual resolution of HBsAg. In contrast to the rapid seroconversion 352

2 VOL. 24, 1979 of HBeAg and anti-hbe, "windows" of 1 to 3 months were observed between the loss of HBsAg and the emergence of anti-hbs. Once the three antibodies anti-hbc, anti-hbe, and anti-hbs appeared, they persisted throughout the entire experimental periods. Correlation between HBsAg and HBeAg. In general, HBeAg was detected either simultaneously with or slightly after the appearance of HBsAg. The titer of HBeAg by RIA was always lower than that of HBsAg, but the appearance and disappearance of these two antigens during early acute stage followed a very similar pattern. As shown in Fig. 2, both chimpanzees RD and BT showed several cycles of periodic oscillation between HBsAg positive and negative before this antigen finally became persistent. The same pattern held true for HBeAg. Correlation between anti-hbe and antiz 0 - cz MONTH AFTR INOCULATION FIG. 1. Time course of various markers of HBV infection in chimpanzee CC. The animal was inoculated with serum positive for HBsAg at day 0. Consecutive blood samples were taken, and sera were assayed for various serological markers as indicated. z 0 a 0 HEPATITIS B e-antigen IN CHIMPANZEES 353 HBc. Although the HBc system and the HBe system are antigenically distinct, the quantitative fluctuations of anti-hbc and anti-hbe appeared to follow identical patterns. The maximum titers of both antibodies appeared just before the resolution of surface antigenemia. As shown in Fig. 3, chimpanzees CC and RD showed steady rises of anti-hbc and anti-hbe until HBsAg became undetectable. The titers of both antibodies then declined over a period of several months. In the third chimpanzee, BT, both anti-hbc and anti-hbe reached their respective maximum titers about a month before HBsAg became undetectable, and remained at constant levels thereafter. HBsAg carrier chimpanzees. Three chimpanzees (Fig. 4) were HBsAg positive at the beginning of this study. They remained HBsAg positive with almost constant titers throughout the entire course of the surveillance, ranging from 8 to 22 months. Thus, they were classified as HBsAg carriers. Two of these three primates, CM and NTS, had very high levels of HBsAg (RIA titer 1:500,000) and were positive for HBeAg with almost constant titers of 1:1,000 (CM) and 1:10,000 (NTS). Chimpanzees CM and NTS were also anti-hbc positive, but they showed different trends in the titers of this antibody. Chimpanzee CM at first showed a stable level of anti-hbc, which was followed by a steady descending phase until the end of the surveillance. This chimpanzee received immune suppressive drugs, and the declining titer of anti- HBc coincided with the period of immune suppression. A 40-fold drop in anti-hbc titer was observed during this period. The anti-hbc titer in chimpanzee NTS increased throughout the DAYS AFTER INOCULATION FIG. 2. Correlation of serum HBsAg and HBeAg in chimpanzees RD (A) and BT (B) early in the course of HBV infection. The animals were inoculated with serum positive for HBsAg at day 0. Consecutive blood samples were taken, and sera were assayed for HBsAg and HBeAg as indicated Before day 30, the RIA results for both antigens were negative. 130

3 354 LING ET AL. INFECT. IMMUN. VW C9 'U z I- 0 P MONTHS AFTER INOCULATION FIG. 3. Correlation of serum anti-hbc and anti-hbe levels in chimpanzees CC (A), BT (B), and RD (C) The animals were inoculated with serum during acute and early convalescent stages of HBV infection. positive for HBsAg at day 0. Consecutive blood samples were taken, and sera were assayed for various serological markers as indicated. %A I- 0/73 D f/74 A J A 0 0/76 D F/77 A J JUL/75 M/76 M J S N J/77 M MONTH/YEAR FIG. 4. Time course of various markers of HBV in chronic HBsAg carrier chimpanzees, CM (A), NTS (B), and JK (C). The animals were bled periodically on the dates indicated. Sera were assayed by RIA for HBsAg, HBeAg, anti-hbc, anti-hbe and anti-hbs. All were HBsAgpositive when the first samples were taken. Only positive serological markers are shown in the figure. course of the surveillance, reaching a titer of 6,000 in 8 months. The third HBsAg carrier chimpanzee, JK, had about a 10-fold lower HBsAg titer than the other two. It was both anti-hbc and anti-hbe positive. The titers of all three hepatitis B markers in JK remained constant throughout the 22 months of surveillance. DISCUSSION The patterns of appearance, disappearance, and persistence of hepatitis B markers in chimpanzees with acute self-limited hepatitis were basically similar in three chimpanzees experimentally infected with HBV. HBsAg appeared 1 or 2 weeks earlier than HBeAg in CC and BT, but in chimpanzee RD the two markers appeared at the same time. These findings differed from those of Tabor et al. (17), who found that HBeAg appeared in HBV-infected chimpanzees 8 to 12 weeks after the appearance of HBsAg.

4 VOL. 24, 1979 The presence or absence of detectable antigenic markers is related to the sensitivity of the analytical procedures. The RIA reagents we used for HBsAg determinations were more refined than those for HBeAg determinations. The higher sensitivity of the HBsAg RIA over HBeAg RIA resulted in the much higher calculated titers of HBsAg than of HBeAg in these chimpanzees. This might account for the delayed detections of HBeAg compared to HBsAg in chimpanzees CC and BT. Since Tabor et al. (17) used an agar gel diffusion method for HBeAg detection, the difference between their results and ours may be explained on the basis of test sensitivity. It is possible that HBeAg and HBsAg were actually synthesized and released in comparable quantities at about the same time. This point of view is strengthened by the fact that, in chimpanzees BT and RD (Fig. 2), the oscillations of HBsAg and HBeAg parallelled each other in the early acute stages of HBV infection. Although it is possible that this is a coincidence of unrelated events, the more reasonable explanation for our observations would be that the syntheses of HBsAg and HBeAg were under the same translational controls. Recently, Neurath and Strick (12) presented experimental data suggesting that HBeAg had the identity of immunoglobulin G, specifically a G4 subclass. They further postulated that HBeAg could be the product of an idiotypic immunological response by the host upon prolonged stimulation by HBsAg. These possibilities are not commensurate with the appearance of HBeAg in our experimentally infected chimpanzees. Our observation of HBsAg and HBeAg oscillations early in the course of HBV infection is not unique. Berquist et al. (3) previously reported that serum HBsAg in these experimentally infected chimpanzees oscillated between positive and negative levels in the early stage of infection. Mitamura and co-workers reported (10) that HBeAg reappeared in a hepatitis B patient after it had changed from positive to undetectable. They suggested that this might possibly reflect significant production of viral material after a reinfection. The cyclic appearance of HBsAg and HBeAg suggests an initial clearing of the antigen by body defense mechanisms and an eventual failure of these mechanisms, resulting in persistent antigenemia. In this study, circulating anti-hbe appeared shortly before or after HBeAg became undetectable. Previous reports (1, 18) showed lags of several weeks between the two events. Differences in the sensitivities of HBeAg and anti- HBe tests between these other studies and ours must again account for this discrepancy. Synchronous trends of anti-hbc and anti- HEPATITIS B e-antigen IN CHIMPANZEES 355 HBe were observed in all three experimentally infected chimpanzees (Fig. 3). It was further noted that both anti-hbc and anti-hbe reached their peak titers at the time of or shortly before the resolution of HBsAg from the blood. After that, the titers of both antibodies either decreased sharply before reaching stabilized levels or remained at the peak level without declining. These results suggested that detectable levels of HBsAg or, more likely, a relatively active viral replication were required for the persistence of anti-hbc and anti-hbe biosyntheses. The titers of anti-hbc were always higher than anti-hbe. Our anti-hbe RIA procedure had a greater inherent sensitivity than our anti-hbc RIA. The nearly 100-fold difference in the titers observed for these two antibodies probably reflects a greater immunogenicity of hepatitis B core antigen. A long-term coexistence of HBsAg and HBeAg was observed in two of these chronic HBsAg carrier chimpanzees. The third animal was anti-hbe positive at the first observation. For a period of 22 months this chimpanzee (JK) maintained constant titers of HBsAg, anti-hbc, and anti-hbe, suggesting a balance between HBsAg biosynthesis and production of these antibodies. Such a balance might be important in the maintenance of the carrier status. In chimpanzee NTS, the very low level of anti-hbc at first and the subsequent steady increases in titer might indicate that this chimpanzee represented a recently acquired HBV infection. In chimpanzee CM, the declining of anti-hbc titer was probably the result of immunosuppression. It was also evident that immunosuppression for nearly 4 months did not disturb the steady state of viral antigen synthesis as indicated by the constant levels of HBsAg and HBeAg. LITERATURE CITED 1. Aikawa, T., H. Sairenji, S. Furuta, K. Kiyosawa, T. Shikata, T. Mitsunobo, Y. Miyakawa, Y. Yanase, and M. Mayumi Seroconversion from hepatitis B e-antigen to anti-hbe in acute hepatitis B virus infection. N. Engl. J. Med. 298: Alter, H. J., L B. Seef, P. M. Kaplan, V. J. McAuliffe, E. C. Wright, J. L. Gerin, R. H. Purcell, P. V. Holland, and H. J. Zimmerman Type B hepatitis: the infectivity of blood positive for e-antigen and DNA polymerase after accidental needlestick exposure. N. Engl. J. Med. 295: Berquist, K. R., J. M. Peterson, B. L Murphy, J. W. Ebert, J. E. Maynard, and R. H. Purcell Hepatitis B antigens in serum and liver of chimpanzees acutely infected with hepatitis B virus. Infect. Immun. 12: Eleftheriou, N., J. Heathcoat, H. C. Thomas, and S. Sherlock Incidence and clinical significance of e-antigen and antibody in acute and chronic liver disease. Lancet ii: El Sheikh, N., I. L. Woolf, R. M. Galbraith, A. L. Eddleston, L. W. Dymock, and R. Williams

5 356 LING ET AL. INFECT. IMMUN. "e" Antigen-antibody systems as indicator of liver damage in patients with hepatitis B antigen. Br. Med. J. 4: Hindman, S. H., C. R. Gravelle, B. L. Murphy, D. W. Bradley, W. R. Budge, and J. E. Maynard "e" Antigen, Dane particles and serum DNA polymerase activity in HBsAg carriers. Ann. Intern. Med. 85: Magnius, L Characterization of a new antigenantibody system associated with hepatitis B. Clin. Exp. Immunol. 20: Magnius, L. O., and J. A. Espmark New specificities in Australia antigen positive sera distinct from LeBouvier determinants. J. Immunol. 109: McAuliffe, V. J., R. H. Purcell, and G. L. LeBouvier e: a third hepatitis B antigen. N. Engl. J. Med. 294: Mitamura, K., H. Suzuki, Y. Endo, B. Oda, M. Inai, and M. Mayumi e-ag, anti-e and DNA polymerase in HBsAg carriers in Japan. Acta Hepatol. Jpn. 17: Mushahwar, I. K., L. R. Overby, G. Frosner, F. Deinhardt, and C. M. Ling Prevalence of hepatitis B e-antigen and its antibody as detected by radioimmunoassays. J. Med. Virol. 2: Neurath, A. R., and N. Strick Host specificity of a serum marker for hepatitis B: evidence that "e antigen" has the properties of an immunoglobulin. Proc. Natl. Acad. Sci. U.S.A. 74: Nielsen, J. O., 0. Dietrichson, and E. Juhl Incidence and meaning of the "e" determinant among hepatitis B antigen positive patients with acute and chronic liver diseases. Lancet ii: Nordenfelt, E., and L. Kjellen Dane particles, DNA polymerase, and e-antigen in two different categories of hepatitis B antigen carriers. Intervirol. 5: Overby, L. R., and C. M. Ling Radioimmune assay for anticore as evidence for exposure to hepatitis B virus. St. Lukes Med. Bull. 15: Shikata, T., T. Karasawa, K. Abe, T. Uzawa, H. Suzuki, T. Oda, M. Mayumi, and M. Moritsugo Hepatitis B e-antigen and infectivity of hepatitis B virus. J. Infect. Dis. 136: Tabor, E., R. J. Gerety, and L. F. Barker Detection of e-antigen during acute and chronic hepatitis B virus infections in chimpanzees. J. Infect. Dis. 136: Takahashi, K., M. Imai, F. Tsuda, T. Takahashi, Y. Miyakawa, and M. Mayumi Association of Dane particles with e-antigen in serum of asymptomatic carriers of hepatitis B surface antigen. J. Immunol. 117: Downloaded from on May 8, 2018 by guest