Failure of Preexisting Antibody Against Hepatitis B Surface Antigen to Prevent Subsequent Hepatitis B Infection

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1983, p Vol. 18, No /83/ $02.00/0 Copyright C 1983, American Society for Microbiology Failure of Preexisting Antibody Against Hepatitis B Surface Antigen to Prevent Subsequent Hepatitis B Infection PAUL D. SWENSON,tt MARIO R. ESCOBAR,'* ROBERT L. CARITHERS, JR.,2 AND THOMAS J. SOBIESKI III2 Departments of Pathology' and Medicine,2 Medical College of Virginia, Health Sciences Division of Virginia Commonwealth University, Richmond, Virginia Received 15 November 1982/Accepted 6 April 1983 We studied a patient who developed acute hepatitis B virus (HBV) infection despite the presence of preexisting antibody to the surface antigen of HBV (anti- HBs). Anti-HBs has been reported to consist primarily of antibody against the common a determinant of HBV. Antibody directed against this major determinant appears to confer protection against HBV, regardless of the subtype. Our patient was shown to have had preexisting anti-hbs of anti-d but not anti-a specificity. She subsequently developed non-a, non-b viral hepatitis followed by an episode of acute hepatitis B after exposure to HBV of the ayw subtype. Acute hepatitis B virus (HBV) infection is usually characterized by the appearance of circulating hepatitis B surface antigen (HBsAg) followed by the development of its corresponding antibody (anti-hbs) (7). Four major HBsAg subtypes (ayw, ayr, adw, and adr) have been reported, based on the presence of the common group-reactive antigenic determinant a and the mutually exclusive antigenic specificities d or y (10) and w or r (1). The number of HBsAg subtypes has been expanded to eight (ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, and adr), depending on which of the w subdeterminants can be demonstrated (2). The production of anti- HBs after HBV infection is generally believed to confer protection against reinfection with either homologous or heterologous HBV subtypes, presumably by virtue of the common a determinant (11, 12). The development of acute hepatitis B despite the previous existence of anti-hbs has been reported in only one patient (9). In this patient, the preexisting anti-hbs was shown to consist of anti-w antibody of restricted subspecificity which permitted reinfection by HBV with a heterologous w subdeterminant. In this report, we describe a patient in whom preexisting anti- HBs with predominantly anti-d specificity failed to prevent acute HBsAg-positive hepatitis upon reexposure to HBV with ayw specificity. CASE REPORT The patient, a 22-year-old female with no previous history of clinical hepatitis, developed symptoms of acute hepatitis in July The peak alanine aminot Present address: Division of Infectious Diseases, Department of Medicine, North Shore University Hospital, Manhasset, NY transferase level was 2,424 U/liter, and the peak bilirubin level was 19.8 mg/dl. Serological tests for HBsAg, antibody to hepatitis B core antigen (anti- HBc), hepatitis B e antigen (HBeAg) and antibody (anti-hbe), and antibody to hepatitis A virus were negative. Anti-HBs was detectable, however, with a counts-per-minute ratio of 34 times the negative-control mean of the Ausab test (Abbott Laboratories, North Chicago, Ill.). The presence of anti-hbs without HBsAg or anti-hbc suggested that this individual had HBV infection long in the past or was immunized with HBsAg rather than infected with the virus. The patient was believed to have acute non-a, non-b hepatitis on the basis of these clinical and laboratory findings and the lack of known exposure to hepatotoxic drugs. She had an uneventful recovery, becoming asymptomatic in 4 weeks with the return of alanine aminotransferase values to within normal limits in 8 weeks (Fig. 1). HBsAg was first detected in the patient in November Symptoms of acute hepatitis recurred in January Anti-HBc appeared shortly before the onset of clinical symptoms and persisted. HBeAg appeared briefly thereafter and was followed by anti- HBe seroconversion. Based on these serological studies, a diagnosis of acute hepatitis B was made. The peak alanine aminotransferase level was 1,950 U/liter, and the peak bilirubin level was 8.5 mg/dl. Anti-HBs disappeared 1 week after the onset of symptoms, reappeared 3 weeks later, and was still detectable 7 months later. The patient again had an uneventful recovery, becoming asymptomatic in 8 weeks with disappearance of HBsAg in 5 months (Fig. 1). The patient denied use of intravenous drugs, exposure to blood or its derivatives, and vaccination against HBV and revealed no possible exposure to HBV except for sexual contact with her boyfriend, who was an intravenous-drug abuser. His serum, collected 6 weeks after the onset of the patient's acute hepatitis B episode, was found to be positive for anti- HBc and anti-hbs.

2 306 SWENSON ET AL. co >D M ID w x od sn a o 2 tl: a: nl i )Oil Onse of Acute NANO I1 Onset of Acute HB HH sag I anti - HBe anti - HBs ] HBeAg 0E I I anti - HBea :10 u Aug Sep p4 ()g 5_ I July Ag. Sept. Oct. Nov. Dec. Jon. Feb. Mar. Apr. May Juw July Aug TIME (months) FIG. 1. HBV serological markers and alanine aminotransferase (ALT) before and after onset of acute hepatitis B (HB) in the serum of a patient who had previous non-a, non-b (NANB) hepatitis followed by an episode of acute HBV infection. MATERIALS AND METHODS Detection of serological markers of viral hepatitis. HBsAg, anti-hbs, anti-hbc, HBeAg, anti-hbe, and antibody to hepatitis A virus were detected by radioimmunoassay (RIA) (Ausria II, Ausab, Corab, Abbott- HBe, and Havab, respectively; all from Abbott Laboratories). HBsAg subtype reagents. The eight HBsAg reagents (ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, and adr) were obtained from the Research Resources Branch, National Institute of Allergy and Infectious Disease, Bethesda, Md. Each reagent was diluted in negativecontrol serum (NCS) that was nonreactive for both HBsAg and anti-hbs to an HBsAg titer of 1:256 when tested by Ausria II. The subtyping reagents were found to be most sensitive at this concentration for demonstrating the subtype specificity of anti-hbspositive samples, provided that the latter were diluted in NCS to yield counts per minute approximately 10 times that of the negative-control mean of the Ausab test. The diluted HBsAg subtype reagents were also used to confirm the monospecificity of the anti-d, antiy, and anti-w reagents. Preparation of monospecific antisera. Monospecific anti-d, anti-y, and anti-w reagents were prepared from individual anti-hbs-positive human sera (kindly provided by Ali Hossaini, Medical College of Virginia, Richmond) by absorption with excess HBsAg-positive human sera containing the antigenic determinants not desired, as previously described (8). These monospecific-antibody reagents were then diluted in NCS to yield counts per minute approximately 10 times that of the negative-control mean in the Ausab test. This resulted in monospecific-antibody reagents after dilutions of 1:128 for anti-d, 1:4,096 for anti-y, and 1:512 for anti-w. HBsAg and anti-hbs subtyping procedures. HBsAg and anti-hbs subtype determinants were demonstrated by means of a previously described modification (8) of the Ausab RIA procedure. Reagents from the same --- i J. CLIN. MICROBIOL. Ausab lot number were used for all HBsAg and anti- HBs subtype determinations. RESULTS Characterization of monospecific antisera. The monospecificity of the anti-d, anti-y, and anti-w reagents was confirmed by demonstrating that the anti-hbs reaction was significantly inhibited by homologous but not by heterologous HBsAg subtypes (data not shown). Thus, HBsAg subtype reagents possessing the d determinant inhibited the anti-hbs reactivity of the anti-d reagent (mean > 80%) but not that of the anti-y reagent (mean < 40%o); those with the y determinant inhibited the reactivity of the anti-y reagent (mean > 80%) but not that of the anti-d reagent (mean < 40%); and those with the w determinant inhibited the reactivity of the anti-w reagent (mean > 60%o), whereas those with the r determinant failed to inhibit the latter (mean < 30%). HBsAg subtyping. The RIA subtyping results for HBsAg-positive serum collected from the patient 3 weeks after the onset of the acute hepatitis B are shown in Table 1. This HBsAg was of subtype ayw since it significantly inhibited the reactivity of the anti-y (96%) and anti-w (100%) reagents but not that of the anti-d reagent (14%). It is likely that the w subdeterminant specificity of this HBsAg was either ayw3 or ayw2, which are the most common y strains of HBV in Western countries (17). Anti-HBs subtyping. The RIA subtyping results for anti-hbs-positive sera collected from the patient 6 months before as well as 5 and 7 months after the onset of acute hepatitis B are shown in Table 2. The subtype specificity of anti-hbs 6 months before the acute hepatitis B episode was predominantly anti-d. HBsAg subtype reagents possessing the d determinant inhibited the patient's anti-hbs reactivity (mean > 98%), but those with the y determinant did not (mean < 14%). In addition, the HBsAg-positive serum collected from the patient 3 weeks after the onset of acute hepatitis B did not inhibit her preexisting anti-hbs reactivity (17%). TABLE 1. Subtyping of patient's HBsAg 3 weeks after onset of acute hepatitis B Sample cpm % Inhibition NCS 92a Positive-control serum 7,353b NCS + anti-d 1,471 0 NCS + anti-y 1,334 0 NCS + anti-w 1,791 0 Patient HBsAg + anti-d 1, Patient HBsAg + anti-y Patient HBsAg + anti-w a Mean of seven determinations. b Mean of three determinations.

3 VOL. 18, 1983 TABLE 2. Subtyping of patient's anti-hbs 6 months before and 5 and 7 months after onset of acute hepatitis B % Inhibition with serum collected: Preincubation with: 6 months 5 months 7 months before onset after onset after onset NCS HBsAg/aywl HBsAg/ayw HBsAg/ayw HBsAg/ayw HBsAg/ayr HBsAg/adw HBsAg/adw HBsAg/adr Patient HBsAg/ayw Five months after the onset of acute hepatitis B, the subtype specificity of the patient's anti- HBs appeared to consist of both anti-d and antiy, since the sample was either not inhibited or only minimally inhibited by HBsAg subtype reagents possessing the d and y determinants. Seven months after the onset of acute hepatitis B, the subtype specificity of the patient's anti-hbs was predominantly anti-y. HBsAg subtype reagents possessing the y determinant inhibited the patient's anti-hbs reactivity (mean > 74%), but those with the d determinant exhibited only minimal or no inhibition (mean < 47%). In addition, the patient's HBsAg-positive serum also inhibited her anti-hbs reactivity (79%). The RIA subtyping results for the anti-hbspositive serum collected from the patient's heterosexual partner 6 weeks after the clinical onset of the patient's acute hepatitis B episode are illustrated in Table 3. The subtype specificity of his anti-hbs was predominantly anti-a since the anti-hbs reaction was significantly inhibited by both d and y subtype reagents (mean = 91%). The percent inhibition by y subtype reagents (mean = 94%) was slightly greater than that by the d subtype reagents (mean = 85%), suggesting the presence of some anti-y but no anti-d reactivity. His anti-hbs reactivity was also inhibited by the patient's HBsAg-positive serum (96%). DISCUSSION Anti-HBs produced after exposure to HBV has been reported to consist primarily of anti-a specificity (4). The development of antibody to the common a determinant appears to confer protection against reinfection with HBV, regardless of the subtype (11, 12). The anti-hbs present in our patient's serum 6 months before the clinical onset of acute hepatitis B was predominantly of anti-d specificity, with little or no anti- FAILURE OF ANTI-HBs TO PREVENT HEPATITIS B 307 a reactivity (Table 2). This patient developed serologically confirmed acute hepatitis B despite the presence of anti-hbs. The source of our patient's HBV infection appears to have been her heterosexual partner, who had a history of intravenous drug abuse and was seropositive for anti-hbc and anti-hbs 6 weeks after the onset of the patient's acute hepatitis B episode. Since the partner had no past clinical history of hepatitis, we presume that he had an asymptomatic acute HBV infection. The subtyping results for this man's anti- HBs, which revealed anti-a and anti-y reactivity, correspond to the ayw HBsAg subtype detected in the patient during her acute hepatitis B infection. The increase in anti-y reactivity observed in the patient's serum 5 and 7 months after her clinical onset of HBV infection is also consistent with an HBV infection of the y specificity. The relative frequencies of the d subtype (85%) and the y subtype (15%) in the United States (3) and the reported association of the y subtype with intravenous drug abuse (5, 6, 16, 18) further suggest that the patient's heterosexual partner was the source of her HBV infection. After acute infection with the ayw subtype of HBV, our patient's anti-hbs response exhibited an increase in anti-y reactivity and a corresponding reduction in anti-d reactivity, but again little or no anti-a reactivity. Therefore, her unresponsiveness to the a antigenic determinant of HBsAg persisted during reinfection with another strain of HBV. Her failure to develop detectable anti-a antibody after recovery from HBV infection apparently represents an abnormal host response to HBsAg. This pattern has been reported to occur in 2.8% of anti-hbs-positive sera from the Blood Bank of the National Institutes of Health Clinical Center (Bethesda, Md.) (4) Ḋifferent strains of inbred mice recently were found to vary in their ability to mount antibody responses to the a and d determinants of HBsAg TABLE 3. Subtyping of heterosexual partner's anti- HBs-positive serum collected 6 weeks after patient's onset of acute hepatitis B Preincubation with: % Inhibition with partner's serum NCS... 0 HBsAg/aywl HBsAg/ayw HBsAg/ayw HBsAg/ayw HBsAg/ayr HBsAg/adw HBsAg/adw HBsAg/adr Patient HBsAg/ayw

4 308 SWENSON ET AL. (14). The observation that CBA (H-2k) mice, in contrast to SWR/J(H-2q) mice, produce an anti-d but not an anti-a response after primary immunization with HBsAg/ad is of particular interest. More recently, the same investigators have reported that the relative affinity of the anti-a response in the H-2q haplotype (C3H.Q) was 8.4-fold that observed in the H-2k haplotype (C3H) after secondary immunization with HBsAg/ad (15). Similar allogeneic differences in humoral immune responsiveness to HBsAg in humans may account for the development of anti-d in the absence of a detectable anti-a response. In an accompanying paper, it was shown that SJL (H-25) nonresponder mice produce an anti-d response after a single intraperitoneal dose of HBsAg/ad-coupled sheep erythrocytes emulsified in complete Freund adjuvant but produce no anti-a response after primary or secondary immunization (13). However, immunization of SJL (H-2s) mice with HBsAg via the hind footpads was found to result in both anti-a and anti-d specific antibody production. These findings may have implications with respect to the mechanisms of determinant-specific nonresponsiveness to HBsAg, in terms of antibody production, in humans. The disappearance of the patient's anti-hbs 1 week after the onset of her acute hepatitis B episode, followed by its reappearance 3 weeks later, is difficult to explain. Since the preexisting anti-hbs was predominantly anti-d and the patient became infected with HBV of the y subtype, the anti-hbs reactivity would be expected to remain detectable. High levels of circulating immune complexes composed of HBsAg/ayw and anti-y might have blocked the reactivity of uncomplexed anti-d antibody in the Ausab test. The hepatitis B vaccine (Heptavax-B) containing only the HBsAgIad subtype has been found to protect humans against infection with homologous (20, 21) and, more recently, heterologous subtypes of HBV (19), presumably by the development of anti-a antibody. Since anti- HBs-positive sera have occasionally been found to have anti-d with no detectable anti-a reactivity after natural HBV infection, it is conceivable that the antibody response to the HBsAg/ad vaccine might occasionally consist of anti-d in the absence of anti-a reactivity. The development of monospecific anti-d antibody in the absence of an anti-a response after vaccination with monovalent HBsAg/ad vaccine has been reported in one recipient (12a). A determinantspecific anti-d response after immunization with the HBsAg/ad vaccine is presumably a rare event, if it occurs at all, in view of the recent demonstration of vaccine efficacy and subtype cross-protection in staff members of hemodialysis units where infection with the HBsAg/ay J. CLIN. MICROBIOL. subtype of HBV predominates (19). We are currently determining the subtype specificity of anti-hbs in vaccinated individuals to clarify this possibility. ACKNOWLEDGMENTS This study was supported in part by General Clinical Research grant 5MOlRR00065 from the National Institutes of Health. LITERATURE CITED 1. Bancroft, W. H., F. K. Mundon, and P. K. Russell Detection of additional antigenic determinants of hepatitis B antigen. J. Immunol. 109: Courouce-Pauty, A. M., and J. P. Soulier Further data on HBs antigen subtypes-geographical distribution. Vox Sang. 27: Dodd, R. Y., P. V. Holland, L. Ni, H. M. Smith, and T. J. Greenwalt Hepatitis B antigen: regional variation in incidence and subtype ratio in the American Red Cross donor population. Am. J. Epidemiol. 95: Gold, J. W. M., H. J. Alter, P. V. Holland, J. L. Gerin, and R. H. Purcell Passive hemagglutination assay for antibody to subtypes of hepatitis B surface antigen. J. Immunol. 112: Gust, I. D., M. Dimitrakakis, and C. R. Lucas Changing patterns in the distribution of hepatitis B subtypes. Vox Sang. 38: HeUtink, R. A., J. van Hattum, S. W. Schalm, and N. Massurel Co-occurrence of HBsAg and anti-hbs: two consecutive infections or a sign of advanced chronic liver disease. J. Med. Virol. 10: Hoofnagle, J. H Serologic markers of hepatitis B infection. Annu. Rev. Med. 32: Hoofnagle, J. H., R. J. Gerety, L. A. Smallwood, and L. F. Barker Subtyping of hepatitis B surface antigen and antibody by radioimmunoassay. Gastroenterology 72: Koziol, D. E., H. J. Alter, J. P. Kirchner, and P. V. Holland Development of HBsAg-positive hepatitis despite the previous existence of antibody to HBsAg. J. Immunol. 117: LeBouvier, G. L The heterogeneity of Australia antigen. J. Infect. Dis. 123: Markenson, J. A., R. J. Gerety, J. H. Hoofnagle, and L. F. Barker Effect of cyclophosphamide on hepatitis B virus infection and challenge in champanzees. J. Infect. Dis. 131: McAuliffe, V. J., R. H. Purcell, and J. L. Gerin Type B hepatitis: a review of current prospects for a safe and effective vaccine. Rev. Infect. Dis. 2: a.McAuliffe, V. J., R. H. Purcell, J. L. Gerin, and F. J. Tyeryar Current status of NIAID hepatitis B vaccines, p In W. Szmuness, H. J. Alter, and J. E. Maynard (ed.), Viral hepatitis. Franklin Institute Press, Philadelphia, Pa. 13. Milich, D. R., H. Alexander, and F. V. Chisari Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). III. Circumvention of nonresponsiveness in mice bearing HBsAg nonresponder haplotypes. J. Immunol. 130: Milich, D. 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5 VOL. 18, 1983 FAILURE OF ANTI-HBs TO PREVENT HEPATITIS B Neilson, J. O., and G. L. LeBouvier Subtypes of Australia antigen among patients and healthy carriers in Copenhagen. A relationship between subtypes and the degree of liver damage in acute viral hepatitis. N. Engl. J. Med. 288: Shorey, J., I. K. Mushahwar, J. Shorey, and L. R. Overby Reexamination of hepatitis B virus subtypes and e-antigen expression by radioimmunoassays. J. Med. Virol. 10: SkinboJ, P Hepatitis-associated antigen, subtypes d and y. Scand. J. Infect. Dis. 5: Szmuness, W., C. E. Stevens, E. J. Harley, E. A. Zang, J. J. Alter, P. E. Taylor, A. DeVera, G. T. S. Chen, A. Kellner, and the Dialysis Vaccine Trial Study Group Hepatitis B vaccine in medical staff of hemodialysis units. Efficacy and subtype cross-protection. N. Engi. J. Med. 307: Szmuness, W., C. E. Stevens, E. J. Harley, E. A. Zang, W. R. Oleszko, R. Sadovsky, J. M. Morrison, and A. Kellner Hepatitis B vaccine: demonstration of efficacy in a controlled clinical trial in a high-risk population in the United States. N. Eng. J. Med. 303: Szmuness, W., C. E. Stevens, E. A. Zang, E. J. Harley, and A. Keilner A controlled clinical trial of the efficacy of the hepatitis B vaccine (Heptavax B): a final report. Hepatology 1: