CLINICAL
IMMUNOLOGY
AND
IMMuNOPATHOLOGY
4, 52x-537
(1975)
Delayed Hypersensitivity and Arthus Reaction to Purified Hepatitis B Surface Antigen (HBsAg) in Immunized Chimpanzees C. TREPO, ‘Z J. VNEK,~
AND
A. M.
PRINCE’
‘Laborutory of Virology, The Lindsley F. Kimboil Kcrearch InstituIe c!f The Ne~7 York Blood Center, New York. NOI. York. and “Division of Gustrornteroloyy. Depurtment of Medicine, The New York Hospital-Cornell Medic-al College. Nr~c, I’ork. NW, >‘urk Keceived
February
IX. 1975
To further investigate the specific immunopathogenic role of hepatitis B surface antigen (HBsAg), we have histologically documented the sequential appearance of both delayed hypersensitivity and Arthus reactions following skin testing with purified HBsAg of chimpanzees. These findings were correlated with anti-HBs and anti-HBc antibody titers and with results of lymphocyte migration inhibition (LMII tests to purified HBsAg. Positive cutaneous delayed hypersensitivity (DH) reaction developed in all six recently immunized chimpanzees, with evidence of in Llitro DH to HBsAg by LMI in five of six. Arthus reactions following skin testing with HBsAg were observed in all seven animals immunized I yr earlier. which had high circulating anti-HBs titers. The LMI test was positive in only two of these. No cutaneous reactions. LMI. or anti-HBs were detected in two healthy chronic HBsAg carrier chimpanzees. By contrast, anti-HBc was detected only in this latter group. These data suggest that specific cellular as well as humoral response to HBsAg can develop in immune chimpanzees. In contrast. chronic carriers do not react in these tests. suggesting a form of immunologic paralysis.
Hepatitis B virus (HBV) is most likely devoid of direct cytopathogenic effect. Both humoral and cell mediated immune mechanisms have been suspected to operate in the immunopathogenesis of HBV infection. Two antigen-antibody systems have now been recognized to be associated with HBV: The hepatitis B surface antigen (HBsAg) and antibody (anti-HBs) and the recently characterized hepatitis B core antigen (HBcAg) and antibody (anti-HBc). Careful evaluation should thus differentiate the respective role of these two independent antigenic systems in the triggering of immunological processes. In the present study we have histologically documented the sequential appearance of both delayed hypersensitivity and Arthus reactions following immunization of chimpanzees with purified HBsAg. These findings were correlated with the level of circulating anti-HBs and anti-HBc antibodies and the results 01 lymphocyte migration inhibition (LMI) tests to purified HBsAg as an in rlitro correlate of specific cell mediated immunity. MATERIALS
AND METHODS
Antigens. HBsAg subtype ad was isolated from the plasma of chimpanzee No. 30, a healthy carrier of the antigen for several years. Purification procedure was 528 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved
SKIN
FIG. testing. nm.
1. Electron Only small
TESTING
micrograph of the purified spherical particles average
WITH
HBsAg diameter
HBS
ANTIGEN
529
preparation used for immunization and skin 20 nm can be seen (X63,000) bar length IO0
carried out by a combination of polyethylene glycol precipitation (l), isopycnic banding, and rate zonal centrifugation (2). The resulting preparation contained small spherical particles approximately 20 nm when studied by electron microscopy. There was no trace of plasma protein detectable by immunoelectrophoresis with polyvalent antiserum against normal human serum proteins. Repeated immunization of guinea pigs with the same preparation in complete Freund’s adjuvant provided high titer anti-HBs gamma globulins which did not react with normal human serum. Electron microscopy revealed that the preparation contained only small spherical particles (Fig. I). Purified protein derivative of tuberculin (PPD) without preservative was obtained from The Central Veterinary Laboratory, Ministry of Agriculture, Waybridge, Surrey, England and used as a control antigen for DH reactions. The buffer in which purified HBsAg was diluted (0.02, phosphate buffer pH 7.2) was also used as a negative control.
530
TREPO,
VNEK
AND
PRINCE
Chimpanzees. Both male and female chimpanzees between the ages of 3 and 10 yr and weighing 35 to 160 lb were used in this experiment. The chimpanzees were housed in separate cages in a special facility at the Laboratory for Experimental Medicine and Surgery in Primates of the New York University Medical Center, Sterling Forest, New York. Three groups of chimpanzees differing by previous history of immunization were studied. Six chimpanzees (Group I) immunized 3 to 4 weeks earlier with 50 lg of purified polyvalent HBsAg (i.e., demonstrating a, d, y, and w specificities) in complete Freund’s adjuvant composed Group 1. Group II consisted of seven animals which had been immunized with the same protocol and then boosted repeatedly with HBsAg in incomplete Freund’s adjuvant. These animals had been skin tested for HBsAg reactivity in a previous study (3). Group 111 was represented by two chronic HBsAg carrier chimpanzees. Sernylan (0.25 mg/kg body wt) was used intramuscularly to anesthetize the animals before skin testing and withdrawal of 50 ml heparinized blood. Skin tests. The test for DH was carried out by duplicate intradermal injection (on both sides of the chest) of 100 ~1 of various dilutions of purified HBsAg, PPD, or buffer. Animals were skin tested with two doses of 100 and 200 pg of HBsAg in Groups I and III and with seven different doses increasing from 15 to 100 pg in Group II. The skin tests were read at 6 hr for immediate reactivity and at 24 and 48 hr for DH response. The cutaneous reactivity was quantitated by measuring with calipers the diameter of erythema in millimiters, and degree of induration by measuring skin fold thickness in millimiters at the test site minus the normal adjacent skin fold thickness. Skin biopsy specimens were taken on one side of the chest for any positive reaction. The specimen was immediately fixed in formalin and processed for light microscopy examination. In vitro leukocyte migration iahibition tests. The capillary migration inhibition test (4) was performed on peripheral blood leukocytes with 100 pg of HBsAg in medium 199 with Hanks’ salts (Grand Island Biological Company). Leukocyte migration tests were performed in duplicate each time, with one capillary in each chamber containing HBsAg. Similarly, duplicate tests were performed in the absence of antigen. After incubation at 37°C for 20 hr, migration chambers were projected by a photograph projector (Durst M300, Durst-Nedoneg, Italy), traced on paper, and measured by planimetry. The migration index was derived from the ratio of the average area of migration in the presence and absence of antigen. Agreement between the duplicate capillaries was usually within 29% of average area of migration. A migration index < 0.80 was considered significant, Antibody assays for anti-HBs and anti-HBc. Tests for humoral immune response to HBsAg were performed by passive hemagglutination assay for antiHBs (5). Anti-HBc was assayed by the indirect immunofluorescence test as described by Brzosko et al. (6) using 3-pm cryostat sections of liver obtained at autopsy from a chronic HBsAg carrier hemodialysis patient.
o/o 6/O 913 813 713
0 0
0 0
613 614
1014 9110
815 1514 813 1115 914
15/S 1014 1415 1314 1414 1716
24 hr
o/o o/o o/o o/o 010 010
6 hr
in Vitro
PMN” PMN PMN PMN PMN -
+ PMN
+ PMNr + PMN + PMN
PMN
0
-
PNM -
PMN + LIM PMN + LIM
LIM LIM LIM LIMd LIM LIM
24 hr
1
CELLULAR
TABLE AND
infiltration.
Histology
OF HUMORAL
PMN + LIM PMN + LIM
-0 0 -
6 hr
TESTING
a Expressed as mm erythema/mm induration. * Not studied. c LIM + PMN = mixed mononuclear and polymorphonuclear d LIM = predominantly mononuclear infiltration. e PMN = predominantly polymorphonuclear infiltration.
33 47 61 43 46 Group III 30 31
Group I 15 17 19 21 24 26 Group II 53 6.5
CH no.
AND
Skin reaction”
In Viva
1.08 0.92
0.80 1.05 0.98 1.07 1.03
0.88 0.64
1.20 0.99 1.22 0.72 1.21 0.67
Migration inhibition index
RESPONSES
IN
32000 >32000 >32000
16000 8192
8192 16384 1024 512 1024 8
>I/1000 >l/lOOO
IMMUNOLOGY
AND
IMMuNOPATHOLOGY
4, 52x-537
(1975)
Delayed Hypersensitivity and Arthus Reaction to Purified Hepatitis B Surface Antigen (HBsAg) in Immunized Chimpanzees C. TREPO, ‘Z J. VNEK,~
AND
A. M.
PRINCE’
‘Laborutory of Virology, The Lindsley F. Kimboil Kcrearch InstituIe c!f The Ne~7 York Blood Center, New York. NOI. York. and “Division of Gustrornteroloyy. Depurtment of Medicine, The New York Hospital-Cornell Medic-al College. Nr~c, I’ork. NW, >‘urk Keceived
February
IX. 1975
To further investigate the specific immunopathogenic role of hepatitis B surface antigen (HBsAg), we have histologically documented the sequential appearance of both delayed hypersensitivity and Arthus reactions following skin testing with purified HBsAg of chimpanzees. These findings were correlated with anti-HBs and anti-HBc antibody titers and with results of lymphocyte migration inhibition (LMII tests to purified HBsAg. Positive cutaneous delayed hypersensitivity (DH) reaction developed in all six recently immunized chimpanzees, with evidence of in Llitro DH to HBsAg by LMI in five of six. Arthus reactions following skin testing with HBsAg were observed in all seven animals immunized I yr earlier. which had high circulating anti-HBs titers. The LMI test was positive in only two of these. No cutaneous reactions. LMI. or anti-HBs were detected in two healthy chronic HBsAg carrier chimpanzees. By contrast, anti-HBc was detected only in this latter group. These data suggest that specific cellular as well as humoral response to HBsAg can develop in immune chimpanzees. In contrast. chronic carriers do not react in these tests. suggesting a form of immunologic paralysis.
Hepatitis B virus (HBV) is most likely devoid of direct cytopathogenic effect. Both humoral and cell mediated immune mechanisms have been suspected to operate in the immunopathogenesis of HBV infection. Two antigen-antibody systems have now been recognized to be associated with HBV: The hepatitis B surface antigen (HBsAg) and antibody (anti-HBs) and the recently characterized hepatitis B core antigen (HBcAg) and antibody (anti-HBc). Careful evaluation should thus differentiate the respective role of these two independent antigenic systems in the triggering of immunological processes. In the present study we have histologically documented the sequential appearance of both delayed hypersensitivity and Arthus reactions following immunization of chimpanzees with purified HBsAg. These findings were correlated with the level of circulating anti-HBs and anti-HBc antibodies and the results 01 lymphocyte migration inhibition (LMI) tests to purified HBsAg as an in rlitro correlate of specific cell mediated immunity. MATERIALS
AND METHODS
Antigens. HBsAg subtype ad was isolated from the plasma of chimpanzee No. 30, a healthy carrier of the antigen for several years. Purification procedure was 528 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved
SKIN
FIG. testing. nm.
1. Electron Only small
TESTING
micrograph of the purified spherical particles average
WITH
HBsAg diameter
HBS
ANTIGEN
529
preparation used for immunization and skin 20 nm can be seen (X63,000) bar length IO0
carried out by a combination of polyethylene glycol precipitation (l), isopycnic banding, and rate zonal centrifugation (2). The resulting preparation contained small spherical particles approximately 20 nm when studied by electron microscopy. There was no trace of plasma protein detectable by immunoelectrophoresis with polyvalent antiserum against normal human serum proteins. Repeated immunization of guinea pigs with the same preparation in complete Freund’s adjuvant provided high titer anti-HBs gamma globulins which did not react with normal human serum. Electron microscopy revealed that the preparation contained only small spherical particles (Fig. I). Purified protein derivative of tuberculin (PPD) without preservative was obtained from The Central Veterinary Laboratory, Ministry of Agriculture, Waybridge, Surrey, England and used as a control antigen for DH reactions. The buffer in which purified HBsAg was diluted (0.02, phosphate buffer pH 7.2) was also used as a negative control.
530
TREPO,
VNEK
AND
PRINCE
Chimpanzees. Both male and female chimpanzees between the ages of 3 and 10 yr and weighing 35 to 160 lb were used in this experiment. The chimpanzees were housed in separate cages in a special facility at the Laboratory for Experimental Medicine and Surgery in Primates of the New York University Medical Center, Sterling Forest, New York. Three groups of chimpanzees differing by previous history of immunization were studied. Six chimpanzees (Group I) immunized 3 to 4 weeks earlier with 50 lg of purified polyvalent HBsAg (i.e., demonstrating a, d, y, and w specificities) in complete Freund’s adjuvant composed Group 1. Group II consisted of seven animals which had been immunized with the same protocol and then boosted repeatedly with HBsAg in incomplete Freund’s adjuvant. These animals had been skin tested for HBsAg reactivity in a previous study (3). Group 111 was represented by two chronic HBsAg carrier chimpanzees. Sernylan (0.25 mg/kg body wt) was used intramuscularly to anesthetize the animals before skin testing and withdrawal of 50 ml heparinized blood. Skin tests. The test for DH was carried out by duplicate intradermal injection (on both sides of the chest) of 100 ~1 of various dilutions of purified HBsAg, PPD, or buffer. Animals were skin tested with two doses of 100 and 200 pg of HBsAg in Groups I and III and with seven different doses increasing from 15 to 100 pg in Group II. The skin tests were read at 6 hr for immediate reactivity and at 24 and 48 hr for DH response. The cutaneous reactivity was quantitated by measuring with calipers the diameter of erythema in millimiters, and degree of induration by measuring skin fold thickness in millimiters at the test site minus the normal adjacent skin fold thickness. Skin biopsy specimens were taken on one side of the chest for any positive reaction. The specimen was immediately fixed in formalin and processed for light microscopy examination. In vitro leukocyte migration iahibition tests. The capillary migration inhibition test (4) was performed on peripheral blood leukocytes with 100 pg of HBsAg in medium 199 with Hanks’ salts (Grand Island Biological Company). Leukocyte migration tests were performed in duplicate each time, with one capillary in each chamber containing HBsAg. Similarly, duplicate tests were performed in the absence of antigen. After incubation at 37°C for 20 hr, migration chambers were projected by a photograph projector (Durst M300, Durst-Nedoneg, Italy), traced on paper, and measured by planimetry. The migration index was derived from the ratio of the average area of migration in the presence and absence of antigen. Agreement between the duplicate capillaries was usually within 29% of average area of migration. A migration index < 0.80 was considered significant, Antibody assays for anti-HBs and anti-HBc. Tests for humoral immune response to HBsAg were performed by passive hemagglutination assay for antiHBs (5). Anti-HBc was assayed by the indirect immunofluorescence test as described by Brzosko et al. (6) using 3-pm cryostat sections of liver obtained at autopsy from a chronic HBsAg carrier hemodialysis patient.
o/o 6/O 913 813 713
0 0
0 0
613 614
1014 9110
815 1514 813 1115 914
15/S 1014 1415 1314 1414 1716
24 hr
o/o o/o o/o o/o 010 010
6 hr
in Vitro
PMN” PMN PMN PMN PMN -
+ PMN
+ PMNr + PMN + PMN
PMN
0
-
PNM -
PMN + LIM PMN + LIM
LIM LIM LIM LIMd LIM LIM
24 hr
1
CELLULAR
TABLE AND
infiltration.
Histology
OF HUMORAL
PMN + LIM PMN + LIM
-0 0 -
6 hr
TESTING
a Expressed as mm erythema/mm induration. * Not studied. c LIM + PMN = mixed mononuclear and polymorphonuclear d LIM = predominantly mononuclear infiltration. e PMN = predominantly polymorphonuclear infiltration.
33 47 61 43 46 Group III 30 31
Group I 15 17 19 21 24 26 Group II 53 6.5
CH no.
AND
Skin reaction”
In Viva
1.08 0.92
0.80 1.05 0.98 1.07 1.03
0.88 0.64
1.20 0.99 1.22 0.72 1.21 0.67
Migration inhibition index
RESPONSES
IN
32000 >32000 >32000
16000 8192
8192 16384 1024 512 1024 8
>I/1000 >l/lOOO
