THE JOURNAL OF INFECTIOUS DISEASES • VOL. 132, NO.4· OCTOBER 1975 © 1975 by the University of Chicago. All rights reserved.
Hepatitis B Virus Infection in Chimpanzees: Titration of Subtypes
Thirty-four chimpanzees were inoculated with sera containing the adw, ayw, adr, or ayr subtype of hepatitis B surface antigen (HB s Ag). Twenty-nine of the animals became infected with hepatitis B virus, and in every instance the subtype of HB s Ag in the infected animal was the same as the subtype in the inoculum. Infectivity titers were established for the adw and ayw inocula. The patterns of serologic events varied in the infected animals but included most of the typical patterns of serologic change seen in human cases of type B hepatitis. Mild disease, manifested by elevated concentrations of serum enzymes and changes detected by liver biopsy, occurred in 23 of the 29 infected animals.
The finding of hepatitis B surface antigen (HB s Ag) and its antibody (anti-Hfs.) in chimpanzees was followed by reports from several laboratories of successful induction of hepatitis B virus (HBV) infections in this species [1-81. The obvious potential value of these animals to further our understanding of type B hepatitis prompted a collaborative study involving laboratories at the National Institutes of Health, the Center for Disease Control (C DC), and the Food and Drug Administration to evaluate the sensitivity of these animals to HBV infection. Accordingly, we undertook to infect chimpanzees with each of the four major antigenic subtypes of HBV [9, 101 and have commenced titrations of sera containing each subtype known to be infectious for chimpanzees. This report describes the current status of these studies, which
have resulted in successful infection with each of the four subtypes and have demonstrated a broad spectrum of serologic events and disease manifestations in 34 animals inoculated with these infectious materials. The ultimate objective of this phase of the studies is to make available pedigreed infectious materials of known titer for use as challenge inocula in future experimental studies of active and passive immunization of chimpanzees against HBV infection. Materials and Methods
Animals. Chimpanzees born in the United States were obtained from the chimpanzee colonies at Delta Regional Primate Research Center (Covington, La.) and at Holloman Air Force Base (Alamagordo, N. M.). Animals imported from Africa were obtained from the Delta Regional Primate Research Center and Primate Imports, Port Washington, N. Y. Inoculation studies were done in isolation quarters at the C DC- Phoenix Laboratory, the Delta Regional Primate Research Center, Hazelton Laboratories, (Vienna, Va.), and Meloy Laboratories (Springfield, Va.). All animals were caged individually and were held for periods ranging from two to 12 months for observation and
Received for publication April 4, 1975. We are grateful to Drs. Dan W. Dalgard, Dale Boyd, Robert Wolf, and William Allen for providing expert care and handling of the chimpanzees, and to Drs. James Vickers and Hans Smetana for interpretation of liver biopsies. Dr. S. Rastogi performed the statistical analyses. Please address requests for reprints to Dr. Lewellys F. Barker, Bureau of Biologics, 8800 Rockville Pike, Bethesda, Maryland 20014. 451
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From the Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Rockville, Maryland; the Bureau of Epidemiology, Center for Disease Control, Phoenix, Arizona; the Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, and the Infectious Diseases Branch, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland
LeweUys F. Barker, James E. Maynard, Robert H. Purcell, Jay H. Hoofnagle, Kenneth R. Berquist, William T. London, Robert J. Gerety, and Donald H. Krushak
Barker et al.
452
Table 1. Titer of hepatitis B surface antigen in sera used for inoculation of chimpanzees. Titer by indicated method*
Inoculum subtype
CEP
CF
RIA
adw ayw adr ayr
320 80 20 2
800 400 80 20
256,000 20,000 16,000 1,000
Chimpanzee infectivity titer/ml
107 •5 107.5 ~106
~
100, < 104
* CEP = counterimmunoelectrophoresis; CF = complement fixation; RIA = radioimmunoassay.
and in HB s Ag-positive chimpanzee sera was done by agar gel diffusion and counterimmunoelectrophoresis methods [9, 10, 15, 16]. Levels of transaminases, expressed in international units, were determined for all serum samples [6, 8], and complete blood cell counts were obtained at approximately one-month intervals as a monitor of the animals' hematologic status. Histology. Needle biopsies of the liver were performed at frequent intervals when serum enzyme levels were elevated and less frequently when there was no biochemical evidence of disease; biopsy specimens were stained with hematoxylin and eosin for microscopic examination for evidence of histopathologic changes. Liver tissue was also prepared for immunofluorescence microscopy and electron microscopy by previously described methods [8]. Results
Subtype ayw of HBV (MS-2 strain). The results of inoculations of 14 animals with dilutions of serum containing the MS-2 strain of HBV (subtype ayw) and offour additional animals with acute-phase HB s Ag-reactive sera from animals infected with the MS-2 strain of HBV are shown in table 2. Evidence of HBV infection appeared in all animals inoculated with dilutions of up to 10-7 ; however, no evidence of infection appeared in one animal (no. 783) that received the 10-8 dilution. In a later experiment this animal was given the 10-4 dilution of the MS-2 strain and subsequently developed typical type B hepatitis. The usual course of serologic and biochemical changes in infected animals is shown in figure 1, which depicts the HBV infec-
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serologic monitoring prior to inoculation. The animals were fed commercial monkey chow (Purina) and a mixture of fruits daily and given water ad lib. During the quarantine period all animals were tested and found to be nonreactive for tuberculosis. Complete blood cell counts and determinations of serum enzymes (glutamicoxalacetic and glutamic-pyruvic transaminases; SGOT and SGPT) were obtained on several occasions, and at least one aspiration needle biopsy of the liver was obtained before inoculation. The animals were anesthetized with phencyclidine hydrochloride (Sernylan; Parke, Davis, Detroit, Mich.) for weekly bleedings and, where indicated, for liver biopsies. Inocula. HB s Ag-positive sera or plasma containing each of the four major antigenic subtypes were obtained. Subtype ayw consisted of serum from serial bleedings from a patient with type B hepatitis; this material had been shown to be infectious for humans and was kindly provided by Dr. Saul Krugman (New York University , New York, N. Y.), who called it the MS-2 strain of HBV. Subtypes adw and adr were obtained from asymptomatic chronic carriers and were provided by Dr. Martin Goldfield (New Jersey Department of Health, Trenton, N.J .); Dr. Paul Holland (National Institutes of Health, Bethesda, Md.) provided the ayr subtype, which was also obtained from a chronic carrier. Serial to-fold dilutions of infectious sera were made in fetal calf serum (Flow Laboratories, Rockville, Md.) that was nonreactive for HB s Ag and anti-Hll.; multiple aliquots of each dilution were stored at -70 C. Inoculations consisted of I-ml volumes injected iv. The titers of HB s Ag in the inocula, as tested by several different methods, are listed in table 1. Serology. Sera obtained from the chimpanzees at weekly intervals for at least six months after inoculation were tested for HB s Ag by counterimmunoelectrophoresis, C F, and the solid-phase radioimmunoassay technique (RIA; Ausria.s Abbott Laboratories, North Chicago, Ill.) [11J and for anti- HB s by passive hemagglutination and/or radioimmunoprecipitation [12,13]. Sera collected prior to inoculation and six months after inoculation from each animal were tested for antibodies to hepatitis B core antigen (anti-Hll.) by CF [14]. Subtyping of HB s Ag in the inocula
Titrations of HBV in Chimpanzees
Table 2.
453
Summary of data on chimpanzees inoculated with sera containing hepatitis B virus, subtype ayw. Week indicated finding in sera* Dilution
HB s Ag
Anti-HB s
Xat Yon Zeba Gogi 23 777* 778* 783* 22 1764 920 1858 Butch 783 1 1 Coco# Tody# Red** Bat**
Undiluted Undiluted Undiluted Undiluted 10- 1 10-4 10-4 10-4 10-6 10-6 10-1 10- 1 10- 1 10- 8 Undiluted Undiluted Undiluted Undiluted
9-14
15 5 40 7 30
3-34 3-6 4-27 9->24 7->24 11->24 8-25 10-25 12-25 13-27 11-14 5-31 4-48 .'\-25 5-23
25
+§ 32 33 19 46
60 25 26
Elevated SGPT
Anti-HB e
Histopathologic changes
7-34(389)t 5-11(116) 13-29(120)
+ + + + +
+ + +
15->24(225) 12-28(174) 11-25(1,000) 21-27(60) 20-27(310) 11-17(484)
+ + + +
+
8-31(135) 8-45(98) 14-29(86) 18-24(164)
+ + + +
+ + + +
+ +
+ + + +
NOTE. Definitions of abbreviations used in tables 2-5: HB s Ag = hepatitis B surface antigen; anti-Hfl, = antibody to HB s Ag; SGPT = serum glutamic-pyruvic transaminase; anti-H Be = antibody to hepatitis B core antigen; - = negative findings; + = positive findings. * Number of weeks after inoculation of virus. Anti-Hls , was present during the indicated week and thereafter. t Numbers in parentheses are the peak concentrations of S G PT expressed in international units. HB s Ag was still in the bloodstream six months after inoculation. § This animal developed anti-Hll, more than six months after inoculation, but the exact time of appearance of antibody was not documented. II This animal had no evidence of infection after inoculation with the 10 -8 dilution but was infected later with the 10- 4 dilution. # Received acute-phase serum from Zeba and Gogi (first passage). ** Received acute-phase serum from Coco and Tody (second passage).
*
tion in animal no. 920. This animal received the 10-7 dilution of the MS-2 strain. As seen in table 2, HB s Ag became detectable in 16 of the 17 infected animals, and enzyme elevations (SOPT, > 30 international units) developed in 13 animals. Histopathologic changes typical of acute viral hepatitis in humans, as described previously [8], were consistently seen in needle biopsy specimens during periods of enzyme elevation. There appeared to be a correlation between the dilution of the inoculum and the incubation period as measured by time between inoculation and appearance of HB s Ag detectable in serum by RI A and also by time between inoculation and elevation of serum enzymes. Thus, the average incubation time for appearance of HB s Ag in the animals that received the undiluted and 10- 1
dilution of the MS-2 serum was 4.8 weeks, which contrasted with 10.8 weeks for the animals that received the 10-6 and 10-7 dilutions of this serum. The incubation period from inoculation to elevation of SO PT level averaged 8.3 weeks in animals receiving the undiluted and 10- 1 dilutions, as compared with 15 weeks in those animals receiving the 10 -6 and 10-7 dilutions. Similar correlations were seen in animals receiving dilutions of the adw and adr subtypes (see below). There was a wide range (four to> 24 weeks) in the duration of circulating HB sAg, with persistence throughout the observation period in one animal (Tody) that became a carrier of HB sAg. The HB s Ag in the sera of infected animals was subtyped in every instance as ayw. Severity of infection as judged by duration of HB s Ag persis-
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Animal
454
Barker et al.
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Figure 1. Course of hepatitis B virus infection in chimpanzee no. 920, inoculated with I ml of the 10- 7 dilution of the ayw subtype (MS-2 strain). SGPT = serum glutamic-pyruvic transaminase; anti-Hls , (CF) = CF test for antibody to hepatitis B core antigen; HB s Ag (RIA) = radioimmunoassay for hepatitis B surface antigen; anti-Hls, (PHA) = passive HA assay for antibody to hepatitis B surface antigen.
tence or by duration or magnitude of elevated S GPT level did not correlate with the dose of virus inoculated. Subtype adw of HBV. Seven animals received dilutions of a single human serum containing the adw subtype of HBV (table 3). Evidence of HBV infection appeared in four of these animals and consisted of the development of transient circulating HB s Ag (subtype adw) in three, prolonged circulating HB s Ag in one, and elevated levels of serum enzymes in three. No evidence of infection developed in one of two animals given the 10-6 dilution or in the two animals that received the 10-8 and 10-9 dilutions. The Table 3. adw.
Summary of data on chimpanzees inoculated with human serum containing hepatitis B virus, subtype
913 15 1396
1M 581 950
17 NOTE.
See table 2 for definition of abbreviations. after inoculation of virus. Anti-Hls , was present during the indicated week and thereafter. t Numbers in parentheses are peak concentrations expressed in international units. :/: This animal appears to have become a chronic carrier of HB sAg.
* Number of weeks
+ +
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MONTHS AFTER EXPOSURE
incubation periods in the animals infected with the 10-6 and 10-7 dilutions (13, 14, and 17 weeks) were considerably longer than the two-week incubation period prior to appearance of HB s Ag in the animal given the undiluted serum. HB s Ag persisted from four to > 26 weeks in the four infected animals; three of these animals had elevations of S GPT level, and histopathologic changes were seen in the two that carried HB sAg and had elevated enzyme levels for longer than four weeks. Subtype adr of H BV. Six animals received dilutions of serum containing the adr subtype (table 4).- Inoculation of undiluted serum containing the adr subtype resulted in circulating HB s Ag of the same subtype in each of three animals after a three-week incubation period. HB s Ag appeared after incubation periods of six and 10 weeks in two animals inoculated with a 10-3 dilution of this serum and after 10 weeks in a single animal inoculated with a 10-6 dilution. In five animals that were followed in this group, HB s Ag persisted for one to 47 weeks; each of these animals had changes detectable by liver biopsy and enzyme elevations typical of acute viral hepatitis. The sixth animal (no. 784) died of an unrelated cause near the time ofonset of hepatitis . Subtype ayr of HBV. Inoculation of two animals with undiluted serum containing the ayr subtype resulted in appearance of HB s Ag of the same subtype in one animal 10 weeks later. The second animal had an illness manifested by elevated enzyme levels during weeks 15 and 16 without detectable HB s Ag in the serum. This animal became positive for anti-Hls, during
Titrations of HBV in Chimpanzees
455
Table 4. Summary of data on chimpanzees inoculated with human serum containing hepatitis B virus, subtypes adr and ayr.
Animal
adr, undiluted adr, undiluted adr, undiluted adr, 10-3 adr, 10-3 adr, 10-6 ayr, undiluted ayr, undiluted ayr, 10--4
Week indicated finding in sera* HB s Ag
Anti-HB,
Elevated SGPT
Anti-HB c
3-21
43 57 29 33
9-26(165)t 5-52(125) 11-25(205) 15-23(90)
+ + + +
13 14 23
11-13(30) 15-16(54) 11-18(654)
+ +
3~9
3-23 6-23 10-12 10-11 10-14
Histopathologic changes
+ + +
NOTE. See table 2 for definition of abbreviations. * Number of weeks after inoculation of virus. Anti-HB, was present during the indicated week and thereafter. t Numbers in parentheses are peak concentrations expressed in international units. + This animal died of an unrelated cause two weeks after acquisition of HB s Ag in serum.
week 14, just prior to the elevation of enzymes. In every other animal that developed biochemical evidence of disease, the illness was accompanied by the presence of HB s Ag in the serum. A third animal inoculated with a 10-4 dilution of the serum containing the ayr subtype of HB V did not develop evidence of infection during the six months after inoculation. Discussion
The disease induced in chimpanzees by HB V has been consistently mild, but it has encompassed all of the typical biochemical and serologic patterns of mild type B hepatitis in humans. None of the animals became icteric or demonstrated clinical evidence of overt disease such as lethargy, decreased food intake, weight loss, or vomiting. Table 5 summarizes the results obtained in the animals exposed to the four major subtypes of HB V. The serologic responses were varied but not distinguishable from one another for the different subtypes. The infectivity titers oftheadw, ayw, and adr inocula appear to be similar (table 1), although the end point has not been determined for the adr inoculum. It is obvious that there is not a direct correlation between titer of HB s Ag and infectivity titer; this fact is consistent with the hypothesis that the infectious units are the intact 40- to 42-nm Dane particles and with the observation that the ratio of these particles to HB s Ag particles varies widely among sera. Almost all (93%) of the infected animals de-
veloped HB sAg; in all but two animals the antigen did not persist. As expected, the HB s Ag subtype bred true in all of the infected animals with detectable antigen [17]. The two animals that did not have detectable HB s Ag in any of their weekly bleedings (Yon and no. 392) experienced primary antibody responses commencing four and 14 weeks after inoculation, respectively. Anti-Hfl , was detected in 79% of infected animals. Anti-HB s was detected in all of the infected animals studied after the disappearance of HB sAg, although frequently there was a considerable lag period between the disappearance of HB s Ag and the appearance of detectable antibody; the longest such lag period was 22 weeks (no. 921). All of the above serologic response patterns to HBV infection in humans have been seen in patients who were followed with serial Table 5. Summary of serologic and biochemical changes in chimpanzees infected with indicated subtypes of hepatitis B virus. No. with each finding
Subtype
No. infected
HB s Ag
adw ayw adr ayr Total
4 17 6 2 29
4 16 6 1 27
AntiHB, 3 14 5 2 24*
AntiHB" 4 13 4 2 23
Elevated SGPT
3 13 5 2 23
NOTE. See table 2 for definition of abbreviations. * Includes all animals studied after disappearance of H B s
Ag.
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921 Susy 1640 13 784+ 1854 392 Rosy 1825
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Figure 3. Relationship between initial elevation of serum glutamic-pyruvic transaminase (SGPT) and dilutions of adw(_), ayw(e), and adr(A) sera inoculated. Correlation coefficient = 0.73; P < 0.01. Plus signs indicate average of all values at that dilution.
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weeks. The correlation coefficients between log dilutions and week of onset of HB s Ag and of SOPT elevations are 0.86 and 0.73, respectively, yielding a highly significant linear relationship in both cases (P < 0.01). A similar significant correlation between size of the HB s Ag inoculum (the NIH infectious plasma pool) and incubation time before onset of disease has been described [22]. In contrast, there appeared to be no correlation between dose of virus and severity of disease, as measured by duration of HB s Ag in the serum or duration of SOPT elevations. These data for animals infected with the adw, ayw, and adr subtypes are plotted in figures 4 and 5. The correlation coefficients are -0.02 and -0.05, respectively, indicating no significant association. Furthermore, peak levels of serum enzymes did not appear to be particularly associated with either low or high dilutions of the infectious inocula. Finally, it is noteworthy that failure to observe infection after inoculation of a serum dilution that could be expected to contain infectious
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bleedings [1,2, 12-14, 18-20]. To date it has not been possible to delineate clear-cut associations between clinical or serologic features of human HBV infections and the infecting subtype [17, 21]. Of particular interest is the direct relationship between the dose of inoculated virus for the three relatively high-titered subtypes (adw, ayw, and adr) and the incubation periods before appearance of HB s Ag and SOPT elevations. These relationships are summarized in figures 2 and 3. The undiluted serum and the 10 -1 dilution induced appearance of HB s Ag in eight recipients after an average incubation period of 3.8 weeks and elevated enzyme levels in six animals after an average incubation period of 8.3 weeks. In contrast, the 10-6 and 10-7 dilutions resulted in an average incubation period (HB s Ag appearance) of 12 weeks in nine animals and an average incubation period (SOPT elevation) of )6.4
Titrations of HBV in Chimpanzees
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Figure 4. Relationship of duration of circulating hepatitis B surface antigen (HB sAg) to dilutions of adw(_), ayw(e), and adr(.A.) sera inoculated. Correlation coefficient = -0.02 (not significant). Plus signs indicate average of all values at that dilution.
HBV has been documented in only one animal (no. 1396). This animal received the 10-6 dilution of the adw serum, which infected both animals inoculated with the 10-7 dilution of the same serum. Thus it would appear that chimpanzees are quite sensitive to HBV infection, a vitally
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Figure 5. Relationship of duration of elevated level of serum glutamic-pyruvic transaminase (S OPT) to dilution of adw(_), ayw(e), and adr(.A.) sera inoculated. Correlation coefficient = -0.05 (not significant). Plus signs indicate average of all values at that dilution.
I. Blumberg, B. S., Sutnick, A. 1., London, W. T. Hepatitis and leukemia: their relation to Australia antigen. Bull. N.Y. Acad. Med. 44:1566-1586, 1968. 2. Hirschman, R. J., Shulman, N. R., Barker, L. F., Smith, K. O. Virus-like particles in sera of patients with infectious and serum hepatitis. J.A.M.A. 208:1667-1670, 1969. 3. Lichter, E. A. Chimpanzee antibodies to Australia antigen. Nature (Lond.) 224:810-811, 1969. 4. Desmyter, J., Uu, W., DeSomer, P., Mortelmans, J. Epidemic clustering of anti-HAA (Australia) antibodies in chimpanzees. Bacteriol. Proc., 1971, p. 168. 5. Maynard, J. E., Hartwell, W. V., Berquist, K. R. Hepatitis-associated antigen in chimpanzees. J. Infect. Dis. 123:660-664, 1971. 6. Maynard, J. E., Berquist, K. R., Krushak, D. H., Purcell, R. H. Experimental infection of chimpanzees with the virus of hepatitis B. Nature (Lond.) 237:514-515, 1972. 7. Prince, A. M. Infection of chimpanzees with hepatitis B virus. In G. N. Vyas, H. A. Perkins, and R. S. Schmid [ed.]. Hepatitis and blood transfusion. Grune and Stratton, 1972, p. 403-406. 8. Barker, L. F., Chisari, F. V., McGrath, P. P., Dalgard, D. W., Kirschstein, R. L., Almeida, J. D., Edgington, T. S., Sharp, D. G., Peterson, M. R. Transmission of type B viral hepatitis to chimpanzees. J. Infect. Dis. 127:648-662, 1973. 9. Le Bouvier, G. L. The heterogeneity of Australia antigen. J. Infect. Dis. 123:671-675, 1971. 10. Bancroft, W. H., Mundon, F. K., Russell, P. K. Detection of additional antigenic determinants of the hepatitis B antigen. J. Immunol. 109:842-848, 1972. II. Ling, C. M., Overby, L. R. Prevalence of hepatitis B virus antigen as revealed by direct radioimmune assay with 125I-antibody. J. Immunol. 109:834-841, 1972. 12. Vyas, G. N., Shulman, N. R. Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science 170:332-333, 1970. 13. Lander, J. J., Alter, H. J., Purcell, R. H. Frequency of antibody to hepatitis-associated antigen as measured by a new radioimmunoassay technique. J. Immunol. 106:1166-1171, 1971. 14. Hoofnag1e, J. H., Gerety, R. J., Barker, L. F. Antibody to hepatitis B virus core in man. Lancet 2:869-873, 1973. 15. Holland, P. V., Purcell, R. H., Smith, H., Alter, H. J. Subtyping of hepatitis-associated antigen (HB Ag);
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is
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important asset in their use for evaluation of the safety and effectiveness of experimental vaccines and passive immunization. In fact, the titers obtained with the adw, ayw, and adr inocula approximate the titer of the infectious National Institutes of Health plasma pool that was studied in humans many years ago [22].
458
19. Krugman, S., Giles, J. P. Viral hepatitis, type B (MS2-strain): further observations on natural history and prevention. N. EngI. J. Med. 288:755-760, 1973. 20. Barker, L. F., Peterson, M. R., Shulman, N. R., Murray, R. Antibody responses in viral hepatitis, type B. J.A.M.A. 223:1005-1008, 1973. 21. Gerety, R. J., Hoofnagle, J. H., Nortman, D. F., Barker, L. F. Hepatitis B surface antigen (HB s Ag) subtypes and indices of clinical disease. Gastroenterology 68: 1253-1261, 1975. 22. Barker, L. F., Murray, R. Relationship of virus dose to incubation time of clinical disease and time of appearance of hepatitis-associated antigen. Am. J. Med. Sci. 263:27-33, 1972.
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simplified technique with counterelectrophoresis. J. Immunol. 109:420-425, 1972. 16. Gerety, R. J., Hoofnagle, J. H., Markenson, J. A., Barker, L. F. Exposure to hepatitis B virus and development of the chronic HB Ag carrier state in children. J. Pediatr. 84:661-665, 1974. 17. Le Bouvier, G. L., McCollum, R. W., Hierholzer, W. J., Jr., Irwin, G. R., Krugman, S., Giles, J. P. Subtypes of Australia antigen and hepatitis B virus. J.A.M.A. 222:928-930, 1972. 18. Prince, A. M., Brotman, B., Cherubin, C. E. Immune response in patients who received blood containing serum hepatitis antigen: studies with the passive hemagglutination technique. Am. J. Dis. Child. 123:415-418, 1972.
Barker et ai.
Hepatitis B Virus Infection in Chimpanzees: Titration of Subtypes
Thirty-four chimpanzees were inoculated with sera containing the adw, ayw, adr, or ayr subtype of hepatitis B surface antigen (HB s Ag). Twenty-nine of the animals became infected with hepatitis B virus, and in every instance the subtype of HB s Ag in the infected animal was the same as the subtype in the inoculum. Infectivity titers were established for the adw and ayw inocula. The patterns of serologic events varied in the infected animals but included most of the typical patterns of serologic change seen in human cases of type B hepatitis. Mild disease, manifested by elevated concentrations of serum enzymes and changes detected by liver biopsy, occurred in 23 of the 29 infected animals.
The finding of hepatitis B surface antigen (HB s Ag) and its antibody (anti-Hfs.) in chimpanzees was followed by reports from several laboratories of successful induction of hepatitis B virus (HBV) infections in this species [1-81. The obvious potential value of these animals to further our understanding of type B hepatitis prompted a collaborative study involving laboratories at the National Institutes of Health, the Center for Disease Control (C DC), and the Food and Drug Administration to evaluate the sensitivity of these animals to HBV infection. Accordingly, we undertook to infect chimpanzees with each of the four major antigenic subtypes of HBV [9, 101 and have commenced titrations of sera containing each subtype known to be infectious for chimpanzees. This report describes the current status of these studies, which
have resulted in successful infection with each of the four subtypes and have demonstrated a broad spectrum of serologic events and disease manifestations in 34 animals inoculated with these infectious materials. The ultimate objective of this phase of the studies is to make available pedigreed infectious materials of known titer for use as challenge inocula in future experimental studies of active and passive immunization of chimpanzees against HBV infection. Materials and Methods
Animals. Chimpanzees born in the United States were obtained from the chimpanzee colonies at Delta Regional Primate Research Center (Covington, La.) and at Holloman Air Force Base (Alamagordo, N. M.). Animals imported from Africa were obtained from the Delta Regional Primate Research Center and Primate Imports, Port Washington, N. Y. Inoculation studies were done in isolation quarters at the C DC- Phoenix Laboratory, the Delta Regional Primate Research Center, Hazelton Laboratories, (Vienna, Va.), and Meloy Laboratories (Springfield, Va.). All animals were caged individually and were held for periods ranging from two to 12 months for observation and
Received for publication April 4, 1975. We are grateful to Drs. Dan W. Dalgard, Dale Boyd, Robert Wolf, and William Allen for providing expert care and handling of the chimpanzees, and to Drs. James Vickers and Hans Smetana for interpretation of liver biopsies. Dr. S. Rastogi performed the statistical analyses. Please address requests for reprints to Dr. Lewellys F. Barker, Bureau of Biologics, 8800 Rockville Pike, Bethesda, Maryland 20014. 451
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From the Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Rockville, Maryland; the Bureau of Epidemiology, Center for Disease Control, Phoenix, Arizona; the Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, and the Infectious Diseases Branch, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland
LeweUys F. Barker, James E. Maynard, Robert H. Purcell, Jay H. Hoofnagle, Kenneth R. Berquist, William T. London, Robert J. Gerety, and Donald H. Krushak
Barker et al.
452
Table 1. Titer of hepatitis B surface antigen in sera used for inoculation of chimpanzees. Titer by indicated method*
Inoculum subtype
CEP
CF
RIA
adw ayw adr ayr
320 80 20 2
800 400 80 20
256,000 20,000 16,000 1,000
Chimpanzee infectivity titer/ml
107 •5 107.5 ~106
~
100, < 104
* CEP = counterimmunoelectrophoresis; CF = complement fixation; RIA = radioimmunoassay.
and in HB s Ag-positive chimpanzee sera was done by agar gel diffusion and counterimmunoelectrophoresis methods [9, 10, 15, 16]. Levels of transaminases, expressed in international units, were determined for all serum samples [6, 8], and complete blood cell counts were obtained at approximately one-month intervals as a monitor of the animals' hematologic status. Histology. Needle biopsies of the liver were performed at frequent intervals when serum enzyme levels were elevated and less frequently when there was no biochemical evidence of disease; biopsy specimens were stained with hematoxylin and eosin for microscopic examination for evidence of histopathologic changes. Liver tissue was also prepared for immunofluorescence microscopy and electron microscopy by previously described methods [8]. Results
Subtype ayw of HBV (MS-2 strain). The results of inoculations of 14 animals with dilutions of serum containing the MS-2 strain of HBV (subtype ayw) and offour additional animals with acute-phase HB s Ag-reactive sera from animals infected with the MS-2 strain of HBV are shown in table 2. Evidence of HBV infection appeared in all animals inoculated with dilutions of up to 10-7 ; however, no evidence of infection appeared in one animal (no. 783) that received the 10-8 dilution. In a later experiment this animal was given the 10-4 dilution of the MS-2 strain and subsequently developed typical type B hepatitis. The usual course of serologic and biochemical changes in infected animals is shown in figure 1, which depicts the HBV infec-
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serologic monitoring prior to inoculation. The animals were fed commercial monkey chow (Purina) and a mixture of fruits daily and given water ad lib. During the quarantine period all animals were tested and found to be nonreactive for tuberculosis. Complete blood cell counts and determinations of serum enzymes (glutamicoxalacetic and glutamic-pyruvic transaminases; SGOT and SGPT) were obtained on several occasions, and at least one aspiration needle biopsy of the liver was obtained before inoculation. The animals were anesthetized with phencyclidine hydrochloride (Sernylan; Parke, Davis, Detroit, Mich.) for weekly bleedings and, where indicated, for liver biopsies. Inocula. HB s Ag-positive sera or plasma containing each of the four major antigenic subtypes were obtained. Subtype ayw consisted of serum from serial bleedings from a patient with type B hepatitis; this material had been shown to be infectious for humans and was kindly provided by Dr. Saul Krugman (New York University , New York, N. Y.), who called it the MS-2 strain of HBV. Subtypes adw and adr were obtained from asymptomatic chronic carriers and were provided by Dr. Martin Goldfield (New Jersey Department of Health, Trenton, N.J .); Dr. Paul Holland (National Institutes of Health, Bethesda, Md.) provided the ayr subtype, which was also obtained from a chronic carrier. Serial to-fold dilutions of infectious sera were made in fetal calf serum (Flow Laboratories, Rockville, Md.) that was nonreactive for HB s Ag and anti-Hll.; multiple aliquots of each dilution were stored at -70 C. Inoculations consisted of I-ml volumes injected iv. The titers of HB s Ag in the inocula, as tested by several different methods, are listed in table 1. Serology. Sera obtained from the chimpanzees at weekly intervals for at least six months after inoculation were tested for HB s Ag by counterimmunoelectrophoresis, C F, and the solid-phase radioimmunoassay technique (RIA; Ausria.s Abbott Laboratories, North Chicago, Ill.) [11J and for anti- HB s by passive hemagglutination and/or radioimmunoprecipitation [12,13]. Sera collected prior to inoculation and six months after inoculation from each animal were tested for antibodies to hepatitis B core antigen (anti-Hll.) by CF [14]. Subtyping of HB s Ag in the inocula
Titrations of HBV in Chimpanzees
Table 2.
453
Summary of data on chimpanzees inoculated with sera containing hepatitis B virus, subtype ayw. Week indicated finding in sera* Dilution
HB s Ag
Anti-HB s
Xat Yon Zeba Gogi 23 777* 778* 783* 22 1764 920 1858 Butch 783 1 1 Coco# Tody# Red** Bat**
Undiluted Undiluted Undiluted Undiluted 10- 1 10-4 10-4 10-4 10-6 10-6 10-1 10- 1 10- 1 10- 8 Undiluted Undiluted Undiluted Undiluted
9-14
15 5 40 7 30
3-34 3-6 4-27 9->24 7->24 11->24 8-25 10-25 12-25 13-27 11-14 5-31 4-48 .'\-25 5-23
25
+§ 32 33 19 46
60 25 26
Elevated SGPT
Anti-HB e
Histopathologic changes
7-34(389)t 5-11(116) 13-29(120)
+ + + + +
+ + +
15->24(225) 12-28(174) 11-25(1,000) 21-27(60) 20-27(310) 11-17(484)
+ + + +
+
8-31(135) 8-45(98) 14-29(86) 18-24(164)
+ + + +
+ + + +
+ +
+ + + +
NOTE. Definitions of abbreviations used in tables 2-5: HB s Ag = hepatitis B surface antigen; anti-Hfl, = antibody to HB s Ag; SGPT = serum glutamic-pyruvic transaminase; anti-H Be = antibody to hepatitis B core antigen; - = negative findings; + = positive findings. * Number of weeks after inoculation of virus. Anti-Hls , was present during the indicated week and thereafter. t Numbers in parentheses are the peak concentrations of S G PT expressed in international units. HB s Ag was still in the bloodstream six months after inoculation. § This animal developed anti-Hll, more than six months after inoculation, but the exact time of appearance of antibody was not documented. II This animal had no evidence of infection after inoculation with the 10 -8 dilution but was infected later with the 10- 4 dilution. # Received acute-phase serum from Zeba and Gogi (first passage). ** Received acute-phase serum from Coco and Tody (second passage).
*
tion in animal no. 920. This animal received the 10-7 dilution of the MS-2 strain. As seen in table 2, HB s Ag became detectable in 16 of the 17 infected animals, and enzyme elevations (SOPT, > 30 international units) developed in 13 animals. Histopathologic changes typical of acute viral hepatitis in humans, as described previously [8], were consistently seen in needle biopsy specimens during periods of enzyme elevation. There appeared to be a correlation between the dilution of the inoculum and the incubation period as measured by time between inoculation and appearance of HB s Ag detectable in serum by RI A and also by time between inoculation and elevation of serum enzymes. Thus, the average incubation time for appearance of HB s Ag in the animals that received the undiluted and 10- 1
dilution of the MS-2 serum was 4.8 weeks, which contrasted with 10.8 weeks for the animals that received the 10-6 and 10-7 dilutions of this serum. The incubation period from inoculation to elevation of SO PT level averaged 8.3 weeks in animals receiving the undiluted and 10- 1 dilutions, as compared with 15 weeks in those animals receiving the 10 -6 and 10-7 dilutions. Similar correlations were seen in animals receiving dilutions of the adw and adr subtypes (see below). There was a wide range (four to> 24 weeks) in the duration of circulating HB sAg, with persistence throughout the observation period in one animal (Tody) that became a carrier of HB sAg. The HB s Ag in the sera of infected animals was subtyped in every instance as ayw. Severity of infection as judged by duration of HB s Ag persis-
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Animal
454
Barker et al.
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Figure 1. Course of hepatitis B virus infection in chimpanzee no. 920, inoculated with I ml of the 10- 7 dilution of the ayw subtype (MS-2 strain). SGPT = serum glutamic-pyruvic transaminase; anti-Hls , (CF) = CF test for antibody to hepatitis B core antigen; HB s Ag (RIA) = radioimmunoassay for hepatitis B surface antigen; anti-Hls, (PHA) = passive HA assay for antibody to hepatitis B surface antigen.
tence or by duration or magnitude of elevated S GPT level did not correlate with the dose of virus inoculated. Subtype adw of HBV. Seven animals received dilutions of a single human serum containing the adw subtype of HBV (table 3). Evidence of HBV infection appeared in four of these animals and consisted of the development of transient circulating HB s Ag (subtype adw) in three, prolonged circulating HB s Ag in one, and elevated levels of serum enzymes in three. No evidence of infection developed in one of two animals given the 10-6 dilution or in the two animals that received the 10-8 and 10-9 dilutions. The Table 3. adw.
Summary of data on chimpanzees inoculated with human serum containing hepatitis B virus, subtype
913 15 1396
1M 581 950
17 NOTE.
See table 2 for definition of abbreviations. after inoculation of virus. Anti-Hls , was present during the indicated week and thereafter. t Numbers in parentheses are peak concentrations expressed in international units. :/: This animal appears to have become a chronic carrier of HB sAg.
* Number of weeks
+ +
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MONTHS AFTER EXPOSURE
incubation periods in the animals infected with the 10-6 and 10-7 dilutions (13, 14, and 17 weeks) were considerably longer than the two-week incubation period prior to appearance of HB s Ag in the animal given the undiluted serum. HB s Ag persisted from four to > 26 weeks in the four infected animals; three of these animals had elevations of S GPT level, and histopathologic changes were seen in the two that carried HB sAg and had elevated enzyme levels for longer than four weeks. Subtype adr of H BV. Six animals received dilutions of serum containing the adr subtype (table 4).- Inoculation of undiluted serum containing the adr subtype resulted in circulating HB s Ag of the same subtype in each of three animals after a three-week incubation period. HB s Ag appeared after incubation periods of six and 10 weeks in two animals inoculated with a 10-3 dilution of this serum and after 10 weeks in a single animal inoculated with a 10-6 dilution. In five animals that were followed in this group, HB s Ag persisted for one to 47 weeks; each of these animals had changes detectable by liver biopsy and enzyme elevations typical of acute viral hepatitis. The sixth animal (no. 784) died of an unrelated cause near the time ofonset of hepatitis . Subtype ayr of HBV. Inoculation of two animals with undiluted serum containing the ayr subtype resulted in appearance of HB s Ag of the same subtype in one animal 10 weeks later. The second animal had an illness manifested by elevated enzyme levels during weeks 15 and 16 without detectable HB s Ag in the serum. This animal became positive for anti-Hls, during
Titrations of HBV in Chimpanzees
455
Table 4. Summary of data on chimpanzees inoculated with human serum containing hepatitis B virus, subtypes adr and ayr.
Animal
adr, undiluted adr, undiluted adr, undiluted adr, 10-3 adr, 10-3 adr, 10-6 ayr, undiluted ayr, undiluted ayr, 10--4
Week indicated finding in sera* HB s Ag
Anti-HB,
Elevated SGPT
Anti-HB c
3-21
43 57 29 33
9-26(165)t 5-52(125) 11-25(205) 15-23(90)
+ + + +
13 14 23
11-13(30) 15-16(54) 11-18(654)
+ +
3~9
3-23 6-23 10-12 10-11 10-14
Histopathologic changes
+ + +
NOTE. See table 2 for definition of abbreviations. * Number of weeks after inoculation of virus. Anti-HB, was present during the indicated week and thereafter. t Numbers in parentheses are peak concentrations expressed in international units. + This animal died of an unrelated cause two weeks after acquisition of HB s Ag in serum.
week 14, just prior to the elevation of enzymes. In every other animal that developed biochemical evidence of disease, the illness was accompanied by the presence of HB s Ag in the serum. A third animal inoculated with a 10-4 dilution of the serum containing the ayr subtype of HB V did not develop evidence of infection during the six months after inoculation. Discussion
The disease induced in chimpanzees by HB V has been consistently mild, but it has encompassed all of the typical biochemical and serologic patterns of mild type B hepatitis in humans. None of the animals became icteric or demonstrated clinical evidence of overt disease such as lethargy, decreased food intake, weight loss, or vomiting. Table 5 summarizes the results obtained in the animals exposed to the four major subtypes of HB V. The serologic responses were varied but not distinguishable from one another for the different subtypes. The infectivity titers oftheadw, ayw, and adr inocula appear to be similar (table 1), although the end point has not been determined for the adr inoculum. It is obvious that there is not a direct correlation between titer of HB s Ag and infectivity titer; this fact is consistent with the hypothesis that the infectious units are the intact 40- to 42-nm Dane particles and with the observation that the ratio of these particles to HB s Ag particles varies widely among sera. Almost all (93%) of the infected animals de-
veloped HB sAg; in all but two animals the antigen did not persist. As expected, the HB s Ag subtype bred true in all of the infected animals with detectable antigen [17]. The two animals that did not have detectable HB s Ag in any of their weekly bleedings (Yon and no. 392) experienced primary antibody responses commencing four and 14 weeks after inoculation, respectively. Anti-Hfl , was detected in 79% of infected animals. Anti-HB s was detected in all of the infected animals studied after the disappearance of HB sAg, although frequently there was a considerable lag period between the disappearance of HB s Ag and the appearance of detectable antibody; the longest such lag period was 22 weeks (no. 921). All of the above serologic response patterns to HBV infection in humans have been seen in patients who were followed with serial Table 5. Summary of serologic and biochemical changes in chimpanzees infected with indicated subtypes of hepatitis B virus. No. with each finding
Subtype
No. infected
HB s Ag
adw ayw adr ayr Total
4 17 6 2 29
4 16 6 1 27
AntiHB, 3 14 5 2 24*
AntiHB" 4 13 4 2 23
Elevated SGPT
3 13 5 2 23
NOTE. See table 2 for definition of abbreviations. * Includes all animals studied after disappearance of H B s
Ag.
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921 Susy 1640 13 784+ 1854 392 Rosy 1825
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weeks. The correlation coefficients between log dilutions and week of onset of HB s Ag and of SOPT elevations are 0.86 and 0.73, respectively, yielding a highly significant linear relationship in both cases (P < 0.01). A similar significant correlation between size of the HB s Ag inoculum (the NIH infectious plasma pool) and incubation time before onset of disease has been described [22]. In contrast, there appeared to be no correlation between dose of virus and severity of disease, as measured by duration of HB s Ag in the serum or duration of SOPT elevations. These data for animals infected with the adw, ayw, and adr subtypes are plotted in figures 4 and 5. The correlation coefficients are -0.02 and -0.05, respectively, indicating no significant association. Furthermore, peak levels of serum enzymes did not appear to be particularly associated with either low or high dilutions of the infectious inocula. Finally, it is noteworthy that failure to observe infection after inoculation of a serum dilution that could be expected to contain infectious
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bleedings [1,2, 12-14, 18-20]. To date it has not been possible to delineate clear-cut associations between clinical or serologic features of human HBV infections and the infecting subtype [17, 21]. Of particular interest is the direct relationship between the dose of inoculated virus for the three relatively high-titered subtypes (adw, ayw, and adr) and the incubation periods before appearance of HB s Ag and SOPT elevations. These relationships are summarized in figures 2 and 3. The undiluted serum and the 10 -1 dilution induced appearance of HB s Ag in eight recipients after an average incubation period of 3.8 weeks and elevated enzyme levels in six animals after an average incubation period of 8.3 weeks. In contrast, the 10-6 and 10-7 dilutions resulted in an average incubation period (HB s Ag appearance) of 12 weeks in nine animals and an average incubation period (SOPT elevation) of )6.4
Titrations of HBV in Chimpanzees
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Figure 4. Relationship of duration of circulating hepatitis B surface antigen (HB sAg) to dilutions of adw(_), ayw(e), and adr(.A.) sera inoculated. Correlation coefficient = -0.02 (not significant). Plus signs indicate average of all values at that dilution.
HBV has been documented in only one animal (no. 1396). This animal received the 10-6 dilution of the adw serum, which infected both animals inoculated with the 10-7 dilution of the same serum. Thus it would appear that chimpanzees are quite sensitive to HBV infection, a vitally
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Figure 5. Relationship of duration of elevated level of serum glutamic-pyruvic transaminase (S OPT) to dilution of adw(_), ayw(e), and adr(.A.) sera inoculated. Correlation coefficient = -0.05 (not significant). Plus signs indicate average of all values at that dilution.
I. Blumberg, B. S., Sutnick, A. 1., London, W. T. Hepatitis and leukemia: their relation to Australia antigen. Bull. N.Y. Acad. Med. 44:1566-1586, 1968. 2. Hirschman, R. J., Shulman, N. R., Barker, L. F., Smith, K. O. Virus-like particles in sera of patients with infectious and serum hepatitis. J.A.M.A. 208:1667-1670, 1969. 3. Lichter, E. A. Chimpanzee antibodies to Australia antigen. Nature (Lond.) 224:810-811, 1969. 4. Desmyter, J., Uu, W., DeSomer, P., Mortelmans, J. Epidemic clustering of anti-HAA (Australia) antibodies in chimpanzees. Bacteriol. Proc., 1971, p. 168. 5. Maynard, J. E., Hartwell, W. V., Berquist, K. R. Hepatitis-associated antigen in chimpanzees. J. Infect. Dis. 123:660-664, 1971. 6. Maynard, J. E., Berquist, K. R., Krushak, D. H., Purcell, R. H. Experimental infection of chimpanzees with the virus of hepatitis B. Nature (Lond.) 237:514-515, 1972. 7. Prince, A. M. Infection of chimpanzees with hepatitis B virus. In G. N. Vyas, H. A. Perkins, and R. S. Schmid [ed.]. Hepatitis and blood transfusion. Grune and Stratton, 1972, p. 403-406. 8. Barker, L. F., Chisari, F. V., McGrath, P. P., Dalgard, D. W., Kirschstein, R. L., Almeida, J. D., Edgington, T. S., Sharp, D. G., Peterson, M. R. Transmission of type B viral hepatitis to chimpanzees. J. Infect. Dis. 127:648-662, 1973. 9. Le Bouvier, G. L. The heterogeneity of Australia antigen. J. Infect. Dis. 123:671-675, 1971. 10. Bancroft, W. H., Mundon, F. K., Russell, P. K. Detection of additional antigenic determinants of the hepatitis B antigen. J. Immunol. 109:842-848, 1972. II. Ling, C. M., Overby, L. R. Prevalence of hepatitis B virus antigen as revealed by direct radioimmune assay with 125I-antibody. J. Immunol. 109:834-841, 1972. 12. Vyas, G. N., Shulman, N. R. Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science 170:332-333, 1970. 13. Lander, J. J., Alter, H. J., Purcell, R. H. Frequency of antibody to hepatitis-associated antigen as measured by a new radioimmunoassay technique. J. Immunol. 106:1166-1171, 1971. 14. Hoofnag1e, J. H., Gerety, R. J., Barker, L. F. Antibody to hepatitis B virus core in man. Lancet 2:869-873, 1973. 15. Holland, P. V., Purcell, R. H., Smith, H., Alter, H. J. Subtyping of hepatitis-associated antigen (HB Ag);
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is
10"
important asset in their use for evaluation of the safety and effectiveness of experimental vaccines and passive immunization. In fact, the titers obtained with the adw, ayw, and adr inocula approximate the titer of the infectious National Institutes of Health plasma pool that was studied in humans many years ago [22].
458
19. Krugman, S., Giles, J. P. Viral hepatitis, type B (MS2-strain): further observations on natural history and prevention. N. EngI. J. Med. 288:755-760, 1973. 20. Barker, L. F., Peterson, M. R., Shulman, N. R., Murray, R. Antibody responses in viral hepatitis, type B. J.A.M.A. 223:1005-1008, 1973. 21. Gerety, R. J., Hoofnagle, J. H., Nortman, D. F., Barker, L. F. Hepatitis B surface antigen (HB s Ag) subtypes and indices of clinical disease. Gastroenterology 68: 1253-1261, 1975. 22. Barker, L. F., Murray, R. Relationship of virus dose to incubation time of clinical disease and time of appearance of hepatitis-associated antigen. Am. J. Med. Sci. 263:27-33, 1972.
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simplified technique with counterelectrophoresis. J. Immunol. 109:420-425, 1972. 16. Gerety, R. J., Hoofnagle, J. H., Markenson, J. A., Barker, L. F. Exposure to hepatitis B virus and development of the chronic HB Ag carrier state in children. J. Pediatr. 84:661-665, 1974. 17. Le Bouvier, G. L., McCollum, R. W., Hierholzer, W. J., Jr., Irwin, G. R., Krugman, S., Giles, J. P. Subtypes of Australia antigen and hepatitis B virus. J.A.M.A. 222:928-930, 1972. 18. Prince, A. M., Brotman, B., Cherubin, C. E. Immune response in patients who received blood containing serum hepatitis antigen: studies with the passive hemagglutination technique. Am. J. Dis. Child. 123:415-418, 1972.
Barker et ai.
