THE JOURNAL OF !:\FECTIOUS DISEASES. VOL. 136, :'\0. 4 • OCTOBER 1977 © 1977 by the University of Chicago. All rights reserved,
Detection of e Antigen during Acute and Chronic Hepatitis B Virus Infections in Chimpanzees Edward Tabor, Robert J. Gerety, and Lewellys F. Barker
From the Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland
The e antigen of hepatitis B (HB e Ag) is intimately associated with infection due to hepatitis B virus (HBV), but this antigen is distinct from the other known particle-associated antigens, i.e., hepatitis B surface antigen (HB R Ag) and hepatitis B core antigen (HB c Ag). The HB e Ag was first described by Magnius and Espmark in 1972 [1] and has been found in some patients with chronic persistent or chronic aggressive type 13 hepatitis [2, 3]. In contrast, antibody to HB e Ag (anti-Hfs.) is found more frequently in the sera of asymptomatic carriers of HB s Ag [2, 3]. The high prevalence of HB e Ag among carriers of HB s Ag in hemodialysis units and the increased infectivity attributed to their sera [1] suggested that HB e Ag might be an indicator of increased infectivity of HBV. Recent studies have shown an association between HB e
Ag, the presence of intact HBV particles, the presence of DNA polymerase [4, 5], and increased risk of transmission of HBV from infected mothers to their newborn infants [6]. Data on incubation period and the time of appearance of HB e Ag are not readily available from humans. The well-documented suitability of the chimpanzee as a model for infection of humans with HBV [7,8] led us to undertake the present study. Serial samples of serum from HBVinfected chimpanzees were studied to ascertain the pattern of appearance and disappearance of HB e Ag and anti-H'B, and to evaluate the meaning of HB e Ag in determination of the prognosis in hepatitis type B.
Received for publication February 3, 1977, and in revised form April25, 1977. The authors thank Mr. A. J. Shawver, Mr. D. Gilbert, and Mr. F. Mitchell for technical assistance. Please address requests for reprints to Dr. Edward Tabor, Hepatitis Branch, Division of Blood and Blood Products, Bureau of Biologics, Building 29, Room 311, 8800 Rockville Pike, Bethesda, Maryland 20014.
Chimpanzees and inocula. Chimpanzees (35) that acquired HBV infection experimentally or during quarantine were studied. Further detailed investigations were carried out on four of these chimpanzees (no. 883, 788, 16, and 197). Chimpanzee no. 883 received 1 ml of a 10-1 dilution of a known infectious HBV inoculum iv; this inoc-
Materials and Methods
541
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Sera from 35 chimpanzees that developed infections with hepatitis B virus (HBV) were tested for the pre~ence of hepatitis B e antigen (HB e Ag) and its antibody (antiHB e ) . HB e Ag was detected early in the course of the disease in the sera of six of eight chimpanzees, which became chronic carriers of hepatitis B surface antigen (HB s Ag), and in the sera of three of 27 chimpanzees, which recovered from acute infection with HBV. Of the nine chimpanzees with HB e Ag, all had the previously described HB e Agj 1 and HB e Agj2 specificities, and one appeared to have a third specificity. None of the 35 chimpanzees developed detectable titers of anti-Hb., Weekly samples of serum from four chimpanzees (three of which developed chronic HBV infections and one with an acute infection) were further studied to determine the pattern of acquisition of HB e Ag. In each animal, HB e Ag appeared eight to 12 weeks after the appearance of HB s Ag detected by radioimmunoassay and up to four weeks before elevation of levels of liver enzymes. In the three chronically infected chimpanzees, HB e Ag was undetectable after four, nine, and 95 weeks, although HB R Ag persisted. In the animal with acute hepatitis, HB e Ag was undetectable before clearance of HB s Ag. The observed pattern of HB e Ag detectability suggests that the appearance of HB e Ag in the course of HBV infection in chimpanzees may signal the progression of the disease to chronic infection.
542
after inoculation and 49 weeks (no. 788) after onset of observation. Dilutions of serum were made in Veronal-buffered saline, pH 8.2, to determine titers of HB e Ag. Agarose plates were made with a concentration of 0.6% agarose (Indubiose A 37; L'industrie Biologique Francaise, Gennevilliers, France), with one drop of 10% sodium azide/ 500 ml of agarose. Melted agarose preparation (3 ml) was poured into small (35- X 10-mm) tissue culture plates (no. 0001; Falcon Plastics, Oxnard, Calif.). Wells were punched in a hexagonal configuration [19]; each well was 5 mm in diameter, and the centers of the wells were 8 mm apart. Reagent anti-H'B, (--0.07 ml) was placed in the center well, and --0.07 ml of reagent HB e Ag and samples to be tested were placed in peripheral wells. (HB e Ag and anti-Hls, reagents were standardized with use of reagents provided by Dr. George L. Le Bouvier and Mr. Alan Williams, Yale University School of Medicine, New Haven, Conn.) The plate was incubated in a moist chamber at room temperature (about 24 C) and read at 24-hr intervals for seven days. The presence of HB e Ag was determined by the appearance of a precipitin line with reagent anti-Hb, and confirmed by a line of identity with reagent HB e Ag. Specificities of HBe Ag (HBeAgj 1 and HB eAg/2) were detected as described by Williams and Le Bouvier [20]. An apparent third specificity was detected as an additional precipitin line resulting from a reaction between the sample and two reagent sera containing anti-H'B, from asymptomatic human chronic carriers of HB s Ag (provided by Dr. Shirley L. Rivers, American National Red Cross, Atlanta, Ga.): this precipitin line was parallel to the lines formed by HB e Agfl or HB e Ag/2 but was distinct from them. The presence of anti-H'B, was determined by the appearance of a precipitin line with reagent HB e Ag and a line of identity with reagent anti-Hb., Selected samples of serum from the four chimpanzees investigated further were tested for HBVassociated DNA polymerase before, during, and after HB e Ag was detectable (done by Drs. John Gerin and Paul Kaplan, Molecular Anatomy Program, Rockville, Md.) with use of radiolabeled precursors [21]. A positive test yielded a value
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ulum contained HB s Ag subtype adr and was derived from the serum of an asymptomatic, HB e Ag-negative, anti-Hb.-negative, human chronic carrier [7]. Chimpanzee no. 788 developed laboratory evidence of HBV infection detected in weekly samples of serum after exposure to serum from chimpanzee no. 883, which was infected with HBV. Chimpanzee no. 16 received I ml of a 10-7dilution of a known infectious HBV inoculum iv; this inoculum contained HB s Ag subtype adw derived from the serum of an asym P' tomatic, HB e Ag-negative, anti-Hb.-negative, human chronic carrier [7]. Chimpanzee no. 197 received 2 ml of an icterogenic, HB e Ag-negative, anti-Hb.-negative, plasma pool (NIH-6; National Institutes of Health, Bethesda, Md.) sc; this inoculum consisted of plasma from human blood donors implicated in cases of post-transfusion hepatitis and was known to contain the two more common subtypes of HB s Ag found among carriers in the United States, adw and ayw [810]. (The absence of HB e Ag in this inoculum may have resulted from the pooling of plasma units with resultant dilution of any plasma units containing HB e Ag or from the possible inactivation of HB e Ag by plasma units containing antiHB e .) Serologic testing. Each of the 35 chimpanzees was monitored through tests of serum samples taken weekly before and after exposure to HBV. Serum glutamic-oxalacetic transaminase (SGOT; also known as aspartate aminotransferase) and glutamic-pyruvic transaminase (SGPT; also known as alanine aminotransferase) were determined by the Sigma-Frankel method [8]; the upper limit of normal was 40 Karmen units [8]. Sera were tested for HB s Ag by radioimmunoassay (RIA) [11] and counterelectrophoresis [12]. HB s Ag subtyping was performed by rheophoresis [10, 13] and by a modified RIA [14]. AntiHB s was detected by RIA [15] and by passive HA [16]. Antibody to HBc Ag (anti-Hb.) was detected by CF [17]. At least six samples of serum obtained during active infection in each chimpanzee were tested for HB e Ag and anti-Hls, by agar gel diffusion [1, 18]; weekly tests were performed on the four chimpanzees studied further for 117 weeks (no. 16), 44 weeks (no. 197), and 40 weeks (no. 883)
Tabor, Gerety, and Barker
e Antigen in Hepatitis B
Results
Detection of HB e Ag. HB e Ag was detected in the sera of nine of 35 chimpanzees during infection with HBV. Six of these nine became chronic carriers of HB s Ag (the chronic carrier state was defined by the presence of HB s Ag for longer than six months) [22]. The other three animals with HB e Ag developed acute HBV infection and recovered. Whereas six of nine HB e Ag-positive chimpanzees became chronic carriers of HB s Ag, only two of the 26 animals without detectable HB e Ag developed chronic HBV infection (P < 0.001). Anti-Hb, was not detected in any of the chimpanzees. All nine chimpanzees wi th HB e Ag had both the HB e Agj 1 and HB e Agj2 specificities in their sera coincident with peak titers of HB e Ag. In some cases, HB e Agj 1 appeared before HB e Agj2, and in some cases these antigens appeared simultaneously. HB e Agj2 was never detected in the absence of HB e Agj 1. The apparently distinct third specificity could only be detected in the serum of one of the nine chimpanzees with HB e Ag (no. 16), which also contained the HB e Agj 1 and HB e Agj2 specificities. Appearance and disappearance of HB e Ag in four chimpanzees. In the four chimpanzees studied further, HB e Ag became detectable eight to 12 weeks after the appearance of HB s Ag (figure 1); three of these chimpanzees subsequently became chronic carriers of HB s Ag. HB e Ag rose to peak titers on the relatively insensitive agar gel diffusion system (range, 1:4-1 :64) and became undetectable between four to 95 weeks aft-
er the antigen was first detected. In no case did HB e Ag reappear once it became undetectable. In the three chronic carriers, HB s Ag remained detectable after the disappearance of HB e Ag. AntiHB c appeared subsequent to or at the same time as the appearance of HB e Ag in all four of these chimpanzees (peak titers, 1:128-1 :2,048). In one animal (no. 883), anti-His, was not detected until six weeks after HB e Ag could no longer be detected. DNA polymerase values remained within the normal range (chimpanzees no. 883, 788, and 16) or were slightly elevated (chimpanzee no. 197), but these values did not vary in selected samples from these four chimpanzees. Detailed analyses of four chimpanzees. Chimpanzee no. 883 (chronic carrier). HB s Ag subtype adr first appeared six weeks after inoculation of HBV and persisted in this animal for longer than 34 weeks (figure 1, top left). HB e Ag was detectable from week 18 to week 21, with a peak titer of 1:4 at week 19. Levels of liver enzymes were elevated from week 19 to week 21. AntiHB c appeared at week 28. Liver biopsies remained normal at all times. Anti-Hls, was never detected. Chimpanzee no. 788 (chronic carrier). HB s Ag subtype adr appeared 12 weeks after exposure to the serum of chimpanzee no. 883 that was infected with HBV, and the antigen persisted for longer than 37 weeks (figure 1, top right). HB e Ag was detected from week 24 to week 32, with a peak titer of 1:32 at week 24. Levels of liver enzymes were elevated intermittently from week 28 until week 49. Inflammatory changes were present, as detected by liver biopsy, from week 19 to week 42; subsequent biopsies were normal. Anti-Hb, was first detected at week 24. AntiHB e was never detected. Chimpanzee no. 16 (chronic carrier). HB s Ag subtype adw appeared 13 weeks after inoculation of HBV and persisted for longer than 104 weeks (figure 1, bottom left). Both the HB e Agj 1 and HB e Agj2 specificities were detected, as well as an apparent third specificity. HB e Ag became detectable at week 22 (peak titer, 1:64 at week 30) and became undetectable at week 117. Levels of liver enzymes were elevated intermittently from week 24 to and including week 117, the time of the disappearance of HBe
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equal to a ratio of number of positive cpm to number of negative cpm (P:N) of ==5.00; a borderline positive test had a P:N ratio of 3.00-4.99. Liver biopsies. Serial liver biopsies were performed on chimpanzees no. 883, 788, and 197 at intervals of one to four weeks and less frequently on chimpanzee no. 16. Specimens were stained with hematoxylin and eosin and were examined by light microscopy. Criteria for defining histopathological changes have been described [8]. Statistical analyses. The relationship between HB e Ag and progression to chronicity was analyzed by the X2 method using contingency tables.
543
544
Tabor, Gerety, and Barker
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Figure 1. Appearance of e antigen (e e) of hepatitis B, surface antigen (HB s Ag) of hepatitis B (~), antibody to HB s Ag (0), antibody to core antigen of hepatitis B (~), abnormal levels of serum glutamic-oxalacetic transaminase (SCOT; also known as aspartate aminotransferase) and glutamic-pyruvic transaminase (SGPT; also known as alanine aminotransferase) (S), and abnormal liver biopsy (~) in chimpanzees no. 883 (top left), no. 788 (top right), no. 16 (bottom left), and no. 197 (bottom right). Chimpanzee no. 883 never had an abnormal liver biopsy. Chimpanzees no. 883,788,16, and 197 were infected with hepatitis B virus subtypes adr, adr, adw, and adyw, respectively.
Ag. Inflammatory changes detected on liver biopsy were present from week 29 to week 35; subsequent biopsies were normal. Anti-Hll, became detectable at week 27. Anti-Hb, was never detected. Chimpanzee no. 197 (acute hepatitis). HB s Ag diplotypic subtype adyw appeared at week 10 after infection with HBV [8] and was detectable for only 19 weeks (figure 1, bottom right). HB e Ag was detected from week 21 to week 28, with a peak liter of 1:4 at week 22. Levels of liver enzymes were elevated intermittently from week 22 to week 42, and inflammatory changes detected on liver biopsy were present from week 25 to week 44. Anti-Hls, became detectable at week 26. Anti-HB, appeared at week
30 and ultimately reached a titer of 1:400 (not shown). Anti-HB, was never detected. Discussion
Of 35 chimpanzees with HBV infection, six of eight chimpanzees that became chronic carriers of HB s Ag had HB e Ag detectable in their serum early in the course of their disease. Three additional chimpanzees also developed HB e Ag but recovered from acute infection with HBV. In no animal could anti-H'B, be detected. Animals with HB e Ag could not be distinguished from those without HB e Ag by their age, sex, weight, or origin (animal colony or jungle), nor could their disease be distinguished by inoculum, incubation
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WEEKS UNDER OBSERVATION
e Antigen in Hepatitis B
545
Table 1. Appearance of e antigen (HB e Ag) in 35 chimpanzees infected with hepatitis B virus (HBV), according to characteristic. Characteristic (no. of animals)
No. without HBeAg
4 5
8 18
7 2
15 11
6 3
12 14
4 5
8 18
3 3 3
10 12 4
3 6
24 2
*Hepatitis B surface antigen. tIndeterminate incubation periods occurred in seven chimpanzees infected by exposure to other animals or in chimpanzees that developed serologic evidence of infection with HBV after two inoculations.
period, or clinical severity. There was, however, increased frequency of development of chronic infection with HBV in the HBe Ag-positive chimpanzees (table I). Other authors also have shown that the presence of detectable HB e Ag is associated with an increased likelihood of progression to chronic active or chronic persistent hepatitis
[23-27]. The pattern of appearance of HBe Ag was similar in each of the four chimpanzees studied further, despite the fact that these animals acquired hepatitis from three separate sources, were exposed to three different subtypes of HB s Ag, and had clinically dissimilar courses of infection. HB e Ag appeared eight to 12 weeks after the appearance of HB s Ag detected by RIA and one to four weeks before the initial elevations in levels of SGOT and SGPT. Magnius et al. [2] studied serial samples of serum from five patients from the Willowbrook State School, Staten Island, N.Y., and found that HB e Ag appeared when HB s Ag
29]. Other authors have suggested an association between HBe Ag, HB c Ag, intact HBV, DNA polymerase, and increased infectivity of HBV [4, 5, 23, 26, 30-32]. In the present study, no association could be demonstrated between HB e Ag and the presence of DNA polymerase, a finding which suggested that the association between DNA polymerase and HB e Ag in the chimpanzee may not be consistently present. Unlike the djy and w jr SUbtype determinants of HB s Ag, to which the infection always "breeds true" (i.e., the same subtype appears in the donor and recipient of transmitted HBV infection [7]), HB e Ag may be present in a recipient's serum even when the antigen was not detectable in the donor's serum. This situation has occurred in humans where HBe Ag-negative mothers appeared to transmit HB e Ag-positive HBV infection to their newborn offspring [33]. The inocula to which three of the intensively studied chimpanzees were exposed (no. 883, 16, and 197) were of previously documented infectivity [7-9], despite the absence of HB e Ag, and these inocula transmitted HB e Ag-positive HBV infection to these chimpanzees. Thus, HB e Ag may become detectable when conditions of the host, virus, or disease are appropriate, regardless of the detectability of HB e Ag in the infectious inoculum, with use of the relatively insensitive
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Estimated age ..,;;4 years (12) >4 years (23) Sex Male (22) Female (13) Weight 10-20 kg (18) 21-40 kg (17) Origin Colony-born (12) Jungle-caught (23) Incubation time to appearance of HB s Ag*
Detection of e Antigen during Acute and Chronic Hepatitis B Virus Infections in Chimpanzees Edward Tabor, Robert J. Gerety, and Lewellys F. Barker
From the Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland
The e antigen of hepatitis B (HB e Ag) is intimately associated with infection due to hepatitis B virus (HBV), but this antigen is distinct from the other known particle-associated antigens, i.e., hepatitis B surface antigen (HB R Ag) and hepatitis B core antigen (HB c Ag). The HB e Ag was first described by Magnius and Espmark in 1972 [1] and has been found in some patients with chronic persistent or chronic aggressive type 13 hepatitis [2, 3]. In contrast, antibody to HB e Ag (anti-Hfs.) is found more frequently in the sera of asymptomatic carriers of HB s Ag [2, 3]. The high prevalence of HB e Ag among carriers of HB s Ag in hemodialysis units and the increased infectivity attributed to their sera [1] suggested that HB e Ag might be an indicator of increased infectivity of HBV. Recent studies have shown an association between HB e
Ag, the presence of intact HBV particles, the presence of DNA polymerase [4, 5], and increased risk of transmission of HBV from infected mothers to their newborn infants [6]. Data on incubation period and the time of appearance of HB e Ag are not readily available from humans. The well-documented suitability of the chimpanzee as a model for infection of humans with HBV [7,8] led us to undertake the present study. Serial samples of serum from HBVinfected chimpanzees were studied to ascertain the pattern of appearance and disappearance of HB e Ag and anti-H'B, and to evaluate the meaning of HB e Ag in determination of the prognosis in hepatitis type B.
Received for publication February 3, 1977, and in revised form April25, 1977. The authors thank Mr. A. J. Shawver, Mr. D. Gilbert, and Mr. F. Mitchell for technical assistance. Please address requests for reprints to Dr. Edward Tabor, Hepatitis Branch, Division of Blood and Blood Products, Bureau of Biologics, Building 29, Room 311, 8800 Rockville Pike, Bethesda, Maryland 20014.
Chimpanzees and inocula. Chimpanzees (35) that acquired HBV infection experimentally or during quarantine were studied. Further detailed investigations were carried out on four of these chimpanzees (no. 883, 788, 16, and 197). Chimpanzee no. 883 received 1 ml of a 10-1 dilution of a known infectious HBV inoculum iv; this inoc-
Materials and Methods
541
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Sera from 35 chimpanzees that developed infections with hepatitis B virus (HBV) were tested for the pre~ence of hepatitis B e antigen (HB e Ag) and its antibody (antiHB e ) . HB e Ag was detected early in the course of the disease in the sera of six of eight chimpanzees, which became chronic carriers of hepatitis B surface antigen (HB s Ag), and in the sera of three of 27 chimpanzees, which recovered from acute infection with HBV. Of the nine chimpanzees with HB e Ag, all had the previously described HB e Agj 1 and HB e Agj2 specificities, and one appeared to have a third specificity. None of the 35 chimpanzees developed detectable titers of anti-Hb., Weekly samples of serum from four chimpanzees (three of which developed chronic HBV infections and one with an acute infection) were further studied to determine the pattern of acquisition of HB e Ag. In each animal, HB e Ag appeared eight to 12 weeks after the appearance of HB s Ag detected by radioimmunoassay and up to four weeks before elevation of levels of liver enzymes. In the three chronically infected chimpanzees, HB e Ag was undetectable after four, nine, and 95 weeks, although HB R Ag persisted. In the animal with acute hepatitis, HB e Ag was undetectable before clearance of HB s Ag. The observed pattern of HB e Ag detectability suggests that the appearance of HB e Ag in the course of HBV infection in chimpanzees may signal the progression of the disease to chronic infection.
542
after inoculation and 49 weeks (no. 788) after onset of observation. Dilutions of serum were made in Veronal-buffered saline, pH 8.2, to determine titers of HB e Ag. Agarose plates were made with a concentration of 0.6% agarose (Indubiose A 37; L'industrie Biologique Francaise, Gennevilliers, France), with one drop of 10% sodium azide/ 500 ml of agarose. Melted agarose preparation (3 ml) was poured into small (35- X 10-mm) tissue culture plates (no. 0001; Falcon Plastics, Oxnard, Calif.). Wells were punched in a hexagonal configuration [19]; each well was 5 mm in diameter, and the centers of the wells were 8 mm apart. Reagent anti-H'B, (--0.07 ml) was placed in the center well, and --0.07 ml of reagent HB e Ag and samples to be tested were placed in peripheral wells. (HB e Ag and anti-Hls, reagents were standardized with use of reagents provided by Dr. George L. Le Bouvier and Mr. Alan Williams, Yale University School of Medicine, New Haven, Conn.) The plate was incubated in a moist chamber at room temperature (about 24 C) and read at 24-hr intervals for seven days. The presence of HB e Ag was determined by the appearance of a precipitin line with reagent anti-Hb, and confirmed by a line of identity with reagent HB e Ag. Specificities of HBe Ag (HBeAgj 1 and HB eAg/2) were detected as described by Williams and Le Bouvier [20]. An apparent third specificity was detected as an additional precipitin line resulting from a reaction between the sample and two reagent sera containing anti-H'B, from asymptomatic human chronic carriers of HB s Ag (provided by Dr. Shirley L. Rivers, American National Red Cross, Atlanta, Ga.): this precipitin line was parallel to the lines formed by HB e Agfl or HB e Ag/2 but was distinct from them. The presence of anti-H'B, was determined by the appearance of a precipitin line with reagent HB e Ag and a line of identity with reagent anti-Hb., Selected samples of serum from the four chimpanzees investigated further were tested for HBVassociated DNA polymerase before, during, and after HB e Ag was detectable (done by Drs. John Gerin and Paul Kaplan, Molecular Anatomy Program, Rockville, Md.) with use of radiolabeled precursors [21]. A positive test yielded a value
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ulum contained HB s Ag subtype adr and was derived from the serum of an asymptomatic, HB e Ag-negative, anti-Hb.-negative, human chronic carrier [7]. Chimpanzee no. 788 developed laboratory evidence of HBV infection detected in weekly samples of serum after exposure to serum from chimpanzee no. 883, which was infected with HBV. Chimpanzee no. 16 received I ml of a 10-7dilution of a known infectious HBV inoculum iv; this inoculum contained HB s Ag subtype adw derived from the serum of an asym P' tomatic, HB e Ag-negative, anti-Hb.-negative, human chronic carrier [7]. Chimpanzee no. 197 received 2 ml of an icterogenic, HB e Ag-negative, anti-Hb.-negative, plasma pool (NIH-6; National Institutes of Health, Bethesda, Md.) sc; this inoculum consisted of plasma from human blood donors implicated in cases of post-transfusion hepatitis and was known to contain the two more common subtypes of HB s Ag found among carriers in the United States, adw and ayw [810]. (The absence of HB e Ag in this inoculum may have resulted from the pooling of plasma units with resultant dilution of any plasma units containing HB e Ag or from the possible inactivation of HB e Ag by plasma units containing antiHB e .) Serologic testing. Each of the 35 chimpanzees was monitored through tests of serum samples taken weekly before and after exposure to HBV. Serum glutamic-oxalacetic transaminase (SGOT; also known as aspartate aminotransferase) and glutamic-pyruvic transaminase (SGPT; also known as alanine aminotransferase) were determined by the Sigma-Frankel method [8]; the upper limit of normal was 40 Karmen units [8]. Sera were tested for HB s Ag by radioimmunoassay (RIA) [11] and counterelectrophoresis [12]. HB s Ag subtyping was performed by rheophoresis [10, 13] and by a modified RIA [14]. AntiHB s was detected by RIA [15] and by passive HA [16]. Antibody to HBc Ag (anti-Hb.) was detected by CF [17]. At least six samples of serum obtained during active infection in each chimpanzee were tested for HB e Ag and anti-Hls, by agar gel diffusion [1, 18]; weekly tests were performed on the four chimpanzees studied further for 117 weeks (no. 16), 44 weeks (no. 197), and 40 weeks (no. 883)
Tabor, Gerety, and Barker
e Antigen in Hepatitis B
Results
Detection of HB e Ag. HB e Ag was detected in the sera of nine of 35 chimpanzees during infection with HBV. Six of these nine became chronic carriers of HB s Ag (the chronic carrier state was defined by the presence of HB s Ag for longer than six months) [22]. The other three animals with HB e Ag developed acute HBV infection and recovered. Whereas six of nine HB e Ag-positive chimpanzees became chronic carriers of HB s Ag, only two of the 26 animals without detectable HB e Ag developed chronic HBV infection (P < 0.001). Anti-Hb, was not detected in any of the chimpanzees. All nine chimpanzees wi th HB e Ag had both the HB e Agj 1 and HB e Agj2 specificities in their sera coincident with peak titers of HB e Ag. In some cases, HB e Agj 1 appeared before HB e Agj2, and in some cases these antigens appeared simultaneously. HB e Agj2 was never detected in the absence of HB e Agj 1. The apparently distinct third specificity could only be detected in the serum of one of the nine chimpanzees with HB e Ag (no. 16), which also contained the HB e Agj 1 and HB e Agj2 specificities. Appearance and disappearance of HB e Ag in four chimpanzees. In the four chimpanzees studied further, HB e Ag became detectable eight to 12 weeks after the appearance of HB s Ag (figure 1); three of these chimpanzees subsequently became chronic carriers of HB s Ag. HB e Ag rose to peak titers on the relatively insensitive agar gel diffusion system (range, 1:4-1 :64) and became undetectable between four to 95 weeks aft-
er the antigen was first detected. In no case did HB e Ag reappear once it became undetectable. In the three chronic carriers, HB s Ag remained detectable after the disappearance of HB e Ag. AntiHB c appeared subsequent to or at the same time as the appearance of HB e Ag in all four of these chimpanzees (peak titers, 1:128-1 :2,048). In one animal (no. 883), anti-His, was not detected until six weeks after HB e Ag could no longer be detected. DNA polymerase values remained within the normal range (chimpanzees no. 883, 788, and 16) or were slightly elevated (chimpanzee no. 197), but these values did not vary in selected samples from these four chimpanzees. Detailed analyses of four chimpanzees. Chimpanzee no. 883 (chronic carrier). HB s Ag subtype adr first appeared six weeks after inoculation of HBV and persisted in this animal for longer than 34 weeks (figure 1, top left). HB e Ag was detectable from week 18 to week 21, with a peak titer of 1:4 at week 19. Levels of liver enzymes were elevated from week 19 to week 21. AntiHB c appeared at week 28. Liver biopsies remained normal at all times. Anti-Hls, was never detected. Chimpanzee no. 788 (chronic carrier). HB s Ag subtype adr appeared 12 weeks after exposure to the serum of chimpanzee no. 883 that was infected with HBV, and the antigen persisted for longer than 37 weeks (figure 1, top right). HB e Ag was detected from week 24 to week 32, with a peak titer of 1:32 at week 24. Levels of liver enzymes were elevated intermittently from week 28 until week 49. Inflammatory changes were present, as detected by liver biopsy, from week 19 to week 42; subsequent biopsies were normal. Anti-Hb, was first detected at week 24. AntiHB e was never detected. Chimpanzee no. 16 (chronic carrier). HB s Ag subtype adw appeared 13 weeks after inoculation of HBV and persisted for longer than 104 weeks (figure 1, bottom left). Both the HB e Agj 1 and HB e Agj2 specificities were detected, as well as an apparent third specificity. HB e Ag became detectable at week 22 (peak titer, 1:64 at week 30) and became undetectable at week 117. Levels of liver enzymes were elevated intermittently from week 24 to and including week 117, the time of the disappearance of HBe
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equal to a ratio of number of positive cpm to number of negative cpm (P:N) of ==5.00; a borderline positive test had a P:N ratio of 3.00-4.99. Liver biopsies. Serial liver biopsies were performed on chimpanzees no. 883, 788, and 197 at intervals of one to four weeks and less frequently on chimpanzee no. 16. Specimens were stained with hematoxylin and eosin and were examined by light microscopy. Criteria for defining histopathological changes have been described [8]. Statistical analyses. The relationship between HB e Ag and progression to chronicity was analyzed by the X2 method using contingency tables.
543
544
Tabor, Gerety, and Barker
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Figure 1. Appearance of e antigen (e e) of hepatitis B, surface antigen (HB s Ag) of hepatitis B (~), antibody to HB s Ag (0), antibody to core antigen of hepatitis B (~), abnormal levels of serum glutamic-oxalacetic transaminase (SCOT; also known as aspartate aminotransferase) and glutamic-pyruvic transaminase (SGPT; also known as alanine aminotransferase) (S), and abnormal liver biopsy (~) in chimpanzees no. 883 (top left), no. 788 (top right), no. 16 (bottom left), and no. 197 (bottom right). Chimpanzee no. 883 never had an abnormal liver biopsy. Chimpanzees no. 883,788,16, and 197 were infected with hepatitis B virus subtypes adr, adr, adw, and adyw, respectively.
Ag. Inflammatory changes detected on liver biopsy were present from week 29 to week 35; subsequent biopsies were normal. Anti-Hll, became detectable at week 27. Anti-Hb, was never detected. Chimpanzee no. 197 (acute hepatitis). HB s Ag diplotypic subtype adyw appeared at week 10 after infection with HBV [8] and was detectable for only 19 weeks (figure 1, bottom right). HB e Ag was detected from week 21 to week 28, with a peak liter of 1:4 at week 22. Levels of liver enzymes were elevated intermittently from week 22 to week 42, and inflammatory changes detected on liver biopsy were present from week 25 to week 44. Anti-Hls, became detectable at week 26. Anti-HB, appeared at week
30 and ultimately reached a titer of 1:400 (not shown). Anti-HB, was never detected. Discussion
Of 35 chimpanzees with HBV infection, six of eight chimpanzees that became chronic carriers of HB s Ag had HB e Ag detectable in their serum early in the course of their disease. Three additional chimpanzees also developed HB e Ag but recovered from acute infection with HBV. In no animal could anti-H'B, be detected. Animals with HB e Ag could not be distinguished from those without HB e Ag by their age, sex, weight, or origin (animal colony or jungle), nor could their disease be distinguished by inoculum, incubation
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WEEKS UNDER OBSERVATION
e Antigen in Hepatitis B
545
Table 1. Appearance of e antigen (HB e Ag) in 35 chimpanzees infected with hepatitis B virus (HBV), according to characteristic. Characteristic (no. of animals)
No. without HBeAg
4 5
8 18
7 2
15 11
6 3
12 14
4 5
8 18
3 3 3
10 12 4
3 6
24 2
*Hepatitis B surface antigen. tIndeterminate incubation periods occurred in seven chimpanzees infected by exposure to other animals or in chimpanzees that developed serologic evidence of infection with HBV after two inoculations.
period, or clinical severity. There was, however, increased frequency of development of chronic infection with HBV in the HBe Ag-positive chimpanzees (table I). Other authors also have shown that the presence of detectable HB e Ag is associated with an increased likelihood of progression to chronic active or chronic persistent hepatitis
[23-27]. The pattern of appearance of HBe Ag was similar in each of the four chimpanzees studied further, despite the fact that these animals acquired hepatitis from three separate sources, were exposed to three different subtypes of HB s Ag, and had clinically dissimilar courses of infection. HB e Ag appeared eight to 12 weeks after the appearance of HB s Ag detected by RIA and one to four weeks before the initial elevations in levels of SGOT and SGPT. Magnius et al. [2] studied serial samples of serum from five patients from the Willowbrook State School, Staten Island, N.Y., and found that HB e Ag appeared when HB s Ag
29]. Other authors have suggested an association between HBe Ag, HB c Ag, intact HBV, DNA polymerase, and increased infectivity of HBV [4, 5, 23, 26, 30-32]. In the present study, no association could be demonstrated between HB e Ag and the presence of DNA polymerase, a finding which suggested that the association between DNA polymerase and HB e Ag in the chimpanzee may not be consistently present. Unlike the djy and w jr SUbtype determinants of HB s Ag, to which the infection always "breeds true" (i.e., the same subtype appears in the donor and recipient of transmitted HBV infection [7]), HB e Ag may be present in a recipient's serum even when the antigen was not detectable in the donor's serum. This situation has occurred in humans where HBe Ag-negative mothers appeared to transmit HB e Ag-positive HBV infection to their newborn offspring [33]. The inocula to which three of the intensively studied chimpanzees were exposed (no. 883, 16, and 197) were of previously documented infectivity [7-9], despite the absence of HB e Ag, and these inocula transmitted HB e Ag-positive HBV infection to these chimpanzees. Thus, HB e Ag may become detectable when conditions of the host, virus, or disease are appropriate, regardless of the detectability of HB e Ag in the infectious inoculum, with use of the relatively insensitive
Downloaded from http://jid.oxfordjournals.org/ at Indiana University Library on July 16, 2015
Estimated age ..,;;4 years (12) >4 years (23) Sex Male (22) Female (13) Weight 10-20 kg (18) 21-40 kg (17) Origin Colony-born (12) Jungle-caught (23) Incubation time to appearance of HB s Ag*
