Vol. 12, No. 3 Printed in U.S.A.
INFECTION AND IMMUNITY, Sept. 1975, p. 564-570 Copyright ( 1975 American Society for Microbiology
Cell-Mediated Immunity in Guinea Pigs to Subunits Derived from Hepatitis B Surface Antigen GUY A. CABRAL, RUBEN CHAIREZ, 1 FRANCINE MARCIANO-CABRAL, MONICA SUAREZ, GORDON R. DREESMAN, JOSEPH L. MELNICK, AND F. BLAINE HOLLINGER* Department of Virology qnd Epidemiology, Baylor College of Medicine, Houston, Texas 77025 Received for publication 22 April 1975
Guinea pigs immunized with hepatitis B $urface antigen (HB5Ag). types adu and ayw, and with two polypeptides (40,000 and 24,000 molecular weight) developed cell-mediated immunity (CMI), as determined by the macrophage inhibition assay, and humoral immunity, as determined by radioimmunoassay. Peritoneal exudate (PE) cells from guinea pigs immunized with the 40,000molecular-weight polypeptide migrated poorly (30 to 88%> inhibition) after challenge with the immunizing subunit or with purif'ied HB.Ag, type adu or ayw. Response of the same PE cells to the 24,000-molecular-weight subunit was significantly reduced. Similar but less striking evidence for CMI was observed with PE cells derived from guinea pigs inoculated with purified tvpe-specif'ic HB.Ag or with the 24,000-molecular-weight polypeptide. Humoral responses were predictable and showed a reasonable degree of correlation with the CMI response. PE cells from control animals inoculated with normal human serum or polyacrylamide gel were not inhibited af'ter challenge with purified preparations of HB.Ag or with the 40,000-molecular-weight polypeptide, but did show CMI with their respective immunogens. In addition, PE cells f'rom guinea pigs inoculated with normal human serum were inhibited f'rom migrating after challenge with the 24,000-molecular-weight subunit, suggesting that the latter may contain an antigenic determinant related to a human serum protein.
Hepatitis B is a serious public health problem and deoxyribonucleic acid polymerase. This of international concern which is associated suggests that these particles are not infectious with significant morbidity and mortality. It is and could be used as immunizing agents. Some particularly prevalent among certain high-risk theoretical objections have been raised to the populations. These groups include individuals proposed use of' the whole 20-nm particle as the who have recently received blood transfusions, immunogen since antigenic determinants reusers of illicit drugs, homosexuals, health care lated to human plasma proteins have been personnel (surgeons, physicians, dentists, reported to be constituent components of the nurses, laboratory technicians, plasmaphoresis, 20-nm HB.Ag-associated particles (115, 16). and blood bank staff), hemodialysis patients These determinants were distinct from groupAnd their contacts, offspring of mothers who specif'ic (a) and type-specific (d or y) sites and become infected in the last trimester of preg- appeared to be related to antigenic specif'icities nancy, and residents of crowded institutions. on prealbumin, albumin, apolipoproteins C and Thus, the need for development of a suitable D, and the y-chain of immunoglobulin G. Such vaccine is evident. preparations may lead to the production of Efforts in vaccine development, hampered bv antibodies directed against human serum proan inability to grow hepatitis B virus in tissue teins. and organ cultures, have been directed towards Tihe use of purified polypeptides derived from use of modified hepatitis B surface antigen HB8Ag as immunizing agents rather than other (HB8Ag) preparations. Plasma from persist- preparations may have some advantages in that ently infected carriers contains an abundance of' the preparation would exclude genes and comthe small 20-nm spherical particles which, after pcnents of viral or host cell origin. In addition, purification, are free of' detectable nucleic acid major polypeptides derived from the 20-nm 'Present address: Department of Biology. George Mason particles have elicited vigorous antibody reUniversity, Fairfax, Va. 22030. sponses in guinea pigs (6a). 564
VOL. 12, 1975
Recent evidence suggests that not only humoral but also cell-mediated immunity plays a role in preventing or aborting hepatitis B infection (6, 9, 19). In this paper we report the measurement of cell-mediated immunity to HB8Ag and to HB8Ag-derived polypeptides using a migration inhibition assay. MATERIALS AND METHODS Purification and isolation of HB,Ag polypeptides. Spherical, 20 ± 2-nm particles containing HB.Ag were purified from human serum as previously described (7) with slight modifications (12). Briefly, the particles were pelleted for 15 h at 100,000 x g and 4 C, resuspended in saline, and repelleted for 5 h at 200,000 x g and 4 C. The resulting pellet was acidified to pH 2.4 with 0.05 M potassium phthalate-hydrochloride buffer, incubated for 1 h at room temperature, and then twice banded in CsCl at 286,000 x g and 4 C for 18 h. The second isopycnic density centrifugation was followed by a rate-zonal centrifugation for 4 h at 286,000 x g and 4 C in a preformed 5 to 35% (wt/wt) CsCl gradient. After extensive dialysis against physiological saline to remove the CsCl, the purified antigen was sterilized by filtration through a 0.22-tim cellulose acetate filter. Polypeptides of purified HB8Ag were prepared by disc polyacrylamide gel electrophoresis as described in detail by this laboratory (3, 6a) and lyophilized. Briefly, the antigen-containing particles were solubilized by heating at 60 C for 10 min in the presence of 6 M urea, 1% sodium dodecyl sulfate, and 1% 2-mercaptoethanol. The subunits were initially fractionated in a preparative polyacrylamide gel electrophoresis apparatus (Buchler Instruments, Fort Lee, N.J.) using a 13.2% separating gel. After elution, the fractions were divided into six pools and refractionated by analytical polyacrylamide gel electrophoresis. The polypeptide products were detected by staining with Coomassie brilliant blue or by scanning the unstained gel in a Gilford gel scanner. Molecular weights of each polypeptide were estimated by comparing distances migrated to that observed using a series of known molecular weight markers. We selected the 24,000- and 40,000-molecular-weight subunits for this study. The smaller-molecular-weight polypeptide has been characterized as a glycoprotein
(4).
Immunization of animals. The specific polypeptides were excised from the gel, emulsified with an equal volume of complete Freund adjuvant, and injected into the footpads of adult (400 to 500 g) Hartley strain guinea pigs (12). All animals received a booster dose between days 14 to 16. Additional guinea pigs were inoculated with 5 to 10 gg of purified HB8Ag/adw or HB8Ag/ayu. Control animals received an inoculum containing either 1.4 or 140 ,g of normal human serum (NHS) or polyacrylamide gel (PAG). Animals were evaluated between day 28 and day 35 postinoculation for the development of humoral and
cell-mediated immunity. Assay for antibody. Antibody titers were determined by radioimmunoassay for each antigenic type using staphylococcal protein A to separate the bound
CMI TO HBSAG SUBUNITS
['251]HB.Ag/anti-HB.
565
complex from the free
[125I]HB.Ag fraction (6a, 10). Serial dilutions of antiserum were prepared in phosphate-buffered saline, pH 7.4, containing 12.5% NHS and incubated with labeled HB.Ag in a 37 C water bath for 18 h. Staphylococcal protein A (Sigma, St. Louis, Mo.) was added to each tube and incubation was continued for 30 min at room temperature. The pellets were washed twice by centrifugation with phosphate-buffered saline and residual radioactivity was determined in an auto-gamma counter. A specimen was considered to be positive if the counts per minute exceeded 2.0 times the mean of a panel of normal guinea pig sera which were negative for anti-HB,. Cell-mediated immunity assays. Cell-mediated immunity was measured in vitro by the macrophage inhibition assay (1, 5, 14). In these experiments, immunized guinea pigs were inoculated intraperitoneally with 30 ml of sterile paraffin oil. After 3 days the animals were anesthetized with ether and exsanguinated by cardiac puncture. A midline abdominal incision was made and several 50-ml aliquots of ice-cold (6 to 10 C) Hanks solution containing 0.075% NaHCO3 and 0.5 units of heparin per ml were introduced into the peritoneal cavity, and the abdominal contents were gently mixed. Peritoneal fluid rich in mononuclear cells was aspirated and pooled in 250-ml centrifuge bottles kept in an ice bath. The fluid was centrifuged at 900 rpm for 15 min and the oil and liquid phases were discarded. The peritoneal exudate (PE) cells were resuspended in 2 ml of Hanks medium (4 C) containing 0.075% NaHCO3 and were transferred to a 15-ml centrifuge tube. After washing the cells twice in this same medium, they were resuspended to approximately 10% by volume in Eagle minimal essential medium containing penicillin/streptomycin and 15% heat-inactivated normal guinea pig serum. Cells were counted and the concentration was adjusted to 107 cells/ml. Equal volumes (0.4 ml) of cells and medium (with and without antigen) were placed in culture tubes (12 by 75 mm) and allowed to interact for 30 min at room temperature. After this incubation period, the suspensions were centrifuged at 200 x g and 4 C for 5 min, and 0.5 ml of the supernatant was removed before resuspending the cells. Sterile 20-,ul capillary pipettes (Drummond Scientific Co., Broomall, Pa.) were filled with the cell suspensions to approximately 5 mm from the top and were sealed at one end with Critoseal (Sherwood Medical Industries, St. Louis, Mo.). The micropipettes were placed in test tubes and centrifuged at 200 x g for 5 min at 4 C. The capillary tubes were broken at the cell-medium interface and were placed in sterile Mackaness-type chambers. These chambers were filled with culture medium, sealed with silicone grease, and incubated in a humidified CO2 incubator at 37 C for 48 h. Tests were performed in triplicate and the chambers were coded to eliminate observer bias. The area of migration was projected and measured after 24 and 48 h of incubation as follows: average migration of PE cells with antigen x 100 % migration = average migration of PE cells without antigen
566
INFECT. IMMUN.
CABRAL ET AL. % inhibition
=
100%
-
% migration.
Using this procedure, an inhibition of 20% or greater arbitrarily selected as indi.-ative of delayed hypersensitivity. was
RESULTS
Preliminary evaluation of the macrophage inhibition assay. Initial studies indicated that relatively large concentrations of purified HB.Ag or polypeptides would be required to significantly inhibit the migration of sensitized PE cells if the antigen was added directly into the Mackaness-type chambers with the medium. Because the supply of polypeptides was limited, it was necessary to modify the procedure. Antigen and PE cells were preincubated in a small volume for 10 and 60 min at 4, 25 (room temperature), and 37 C before preparing the capillary tubes for the chambers. Table 1 shows the results of this experiment. Although no significant differences were observed, migration patterns appeared to be less stable after preincubation at 4 and 37 C, especially when the incubation period was continued for 48 h. A 30-min preincubation period at room temperature was selected for subsequent tests. An optimal immunization schedule was ascertained. Guinea pigs were inoculated with 4 or 16 ,ug of purified HB.Ag. One-half of' the animals received a booster injection containing the same quantity of antigen on day 14. On day 28 the animals were sacrificed and the PE cells were challenged with varying concentrations of purified HB5Ag ranging from 0.4 to 50 gg/ml. PE cells from guinea pigs immunized and boostered with 16 ,g of HB.Ag and challenged with the same protein failed to migrate a significant distance from the capillary tubes over a 24-h incubation period when compared to unchallenged control PE cells from the same animal (Table 2). Lesser but equally significant inhibition of' macrophage migration was observed in all other HB.Ag-immunized animals except those obtained from guinea pigs immunized once with 4 Mg of HB8Ag and challenged with higher concentrations of HB ,Ag (10 and 50 mg/ml). However, significant inhibition (29 and 28%, respectively) was observed within this same experimental group after 48 h of incubation. From these preliminary data, we concluded that the optimal immunization schedule was the administration of the immunogen in two equally divided doses at 14-day intervals with subsequent measurement of cell-mediated immunity on day 28. A similar observation was made in other studies in which delayed cutane-
ous hypersensitivity was evaluated (unpublished data). Cellular immunity. Cell-mediated immunity in guinea pigs was measured by the macrophage inhibition assay. Guinea pigs were immunized (i) with purified 20 + 2-nm particles containing type-specif'ic aduw or avw HB8Ag, (ii) with the 40,000- or 24,000-molecular-weight polypeptides derived from HB8Ag/adw, (iii) with NHS, or (iv) with PAG. The results are summarized in Table 3. PE cells obtained from four guinea pigs inoculated with the 40,000-molecular-weight polypeptide and challenged with the same antigen exhibited a 35 to 88% decrease in macrophage migration. Inhibition ranging from 49 to 58% was observed when the same cells were challenged with purified HB.Ag/adw. Correspondingly, macrophages from three of the four animals were inhibited from migrating when challenged with purified HB8Ag/ayw, and PE cells from the fourth guinea pig showed 14% inhibition. In contrast, significant inhibition of macrophage migration above 20% was observed in only one guinea pig (no. 9) when the same PE cells were challenged with the 24,000-molecular-weight polypeptide preparation. Macrophages from two other guinea pigs were inhibTABLE 1. Preincubation of immune guinea pig PE cells with 10 ug of HB8Ag Preincubation time (min)
10 60 a
Migration inhibition (%) after preincubation at :a 4C
25 C
37 C
78 76
78 84
76 72
Results read after a 24-h incubation period.
TABLE 2. Migration inhibition of immune and nonimmune PE cells after incubation with varying amounts of HBAg at room temperature for 30 min Migration inhibition (%) after Concn of HB,Ag used to inoculate guinea pigs None (control) 4 gg, no booster 16 Mg, no booster 4 ug, boostered on day 14 16 gIg, boostered on day 14
preincubation with varying concn of HB.Ag (mg)a 0.4 2 10 50 Ob
44 59 68 94
5 34 48 69 94
0 oc
45 71 82
0 O0 66 71 71
Macrophage migration determined after 24 h of incubation. 'No inhibition when compared with unchallenged PE cells. In some instances, migration was enhanced. c Significant migration inhibition observed after 48 h of incubation. a
VOL. 12, 1975
CMI TO HB AG SUBUNITS
567
TABLE 3. Migration inhibition of guinea pig PE cells by HB.Ag or by HB,Ag-derived polypeptides Inoculum
40,000-mol-wt polypeptide (HB.Ag/adw)
24,000-mol-wt polypeptide (HB.Ag/adw)
HB.Ag/adw HB,Ag/ayw NHS, 1.4ug
140,gg
PAG a
Migration inhibition (%) after incubation with: Animal no. 40000-mol wt 24,000-mol wt HB3Ag/ HB.Ag/ polypeptide polypeptide adw ayw
1 9 11 12 18 19 13 10 2 6 7 15 16 14
88 54 36
35 97 13 67 49 71 53 0 0 0 19
17 55 Oa 19 95 57 22 0 29 11 0 13 26 10
52 55 58 49 81 6 43 74 43 33 0 0 0 2
41 14 30 51 0 4 52 64 10 35 22 0 0 0
No inhibition when compared with unchallenged PE cells. In some instances, migration was enhanced.
ited at a level slightly below our arbitrary cut-off value of 20%. Mononuclear cells obtained from three additional animals immunized with the 24,000molecular-weight polypeptide were inhibited 22, 57, and 95% when challenged with the same polypeptide preparation. Furthermore, cells from two of these animals (no. 13 and 18) exhibited evidence for cell-mediated immunity to the 40,000-molecular-weight subunit and to the purified whole particle containing HB.Ag/ adw. PE cells procured from one animal, guinea pig no. 13, were inhibited 52% after challenge with HB,Ag/ayw, implying a certain degree of type specificity to this polypeptide preparation
quently challenged with PAG (data not shown). Minimal to absent inhibition was observed among guinea pigs challenged with the two polypeptides or with the whole particles. When PAG was used to challenge PE cells from the other groups of guinea pigs, no inhibition was observed. Significant macrophage inhibition was observed in guinea pigs inoculated and challenged with 1.4 or 140 qg of NHS. In addition, macrophage migration was inhibited 13 and 26%, respectively, when challenged with the 24,000-molecular-weight polypeptide. It is noteworthy that mononuclear cells from one of two guinea pigs inoculated with the 24,000molecular-weight polypeptide were inhibited 97% after challenge with 100 ,g of NHS (data not shown), whereas none of the other groups of guinea pigs exhibited such evidence of delayed hypersensitivity to this protein. Figure 1 illustrates typical PE migration inhibition patterns obtained after challenge of sensitized PE cells with homologous and heterologous antigens. Humoral immunity. Humoral immunity was evaluated in all guinea pigs by radioimmunoassay, and the results are summarized in Table 4. Antibody titers to HB8Ag, types adw and ayw, ranged from 1:300 to 1:290,000. The antisera were type specific and homologous titers exceeded heterologous reactions for each immunogen. No anti-HB. was detected in sera obtained from guinea pigs inoculated with NHS or with PAG.
(6a). Guinea pigs inoculated with purified particles containing HB8Ag/adw exhibited evidence for the development of delayed hypersensitivity when their PE cells were challenged with HB.Ag/adw or with the 40,000-molecularweight subunit. PE cells from one of the guinea pigs were inhibited by the 24,000-molecularweight polypeptide, whereas those from the other animal were responsive to HB.Ag/ayw. A positive inhibitory response to homologous protein was observed with macrophages from guinea pigs sensitized to HB8Ag/ayw. However, cells from only one animal (no. 6) responded to the 40,000-molecular-weight subunit (derived from HB.Ag/adw) and to the whole particle containing HB.Ag/adw. Neither animal exhibited cell-mediated immunity to the 24,000DISCUSSION molecular-weight polypeptide. One approach to the development of a vacMigration of PE cells was significantly inhibited (69%) in animals inoculated and subse- cine for the prevention of type B hepatitis
568
INFECT. IMMUN.
CABRAL ET AL.
;-
Ls-e{jtI2l24 X p iTape;
;
K
L
FIG. 1. Capillary tube migration patterns. (A-D): Guinea pig peritoneal exudate (PE) cells sensitized to the 40,000-molecular-weight polypeptide and challenged with (A) medium, (B) the 40,000-molecular-weight polypeptide, (C) HBqAg/adu, and (D) HBAg/ayw. (E, F): PE cells sensitized to the 24,000-molecular-weight polypeptide and challenged with (E) medium and (F) the 24,000-molecular-weight polypeptide. (G, H): PE cells sensitized to HB9Ag/ayw and challenged with (G) medium and (H) HBAg/ayw. (I-L): PE cells sensitized to NHS and challenged with (I) medium, (J) NHS, (K) the 40,000-molecular-weight polypeptide, and (L) HB,Ag/ayw. Note that in all cases significant inhibition of migration occurred when sensitized PE cells were challenged with the homologous antigen (B, F, H, and J). TABLE 4. Determination of anti-HB, titers in guinea pig serum by radioimmunoassay Reciprocal antibody titera Animal no.
Inoculum
62,500
Anti-HB/ayu' 5,000
900 3,500 1,500 850 300 700 290,000 16,000 3,000 5,000
INFECTION AND IMMUNITY, Sept. 1975, p. 564-570 Copyright ( 1975 American Society for Microbiology
Cell-Mediated Immunity in Guinea Pigs to Subunits Derived from Hepatitis B Surface Antigen GUY A. CABRAL, RUBEN CHAIREZ, 1 FRANCINE MARCIANO-CABRAL, MONICA SUAREZ, GORDON R. DREESMAN, JOSEPH L. MELNICK, AND F. BLAINE HOLLINGER* Department of Virology qnd Epidemiology, Baylor College of Medicine, Houston, Texas 77025 Received for publication 22 April 1975
Guinea pigs immunized with hepatitis B $urface antigen (HB5Ag). types adu and ayw, and with two polypeptides (40,000 and 24,000 molecular weight) developed cell-mediated immunity (CMI), as determined by the macrophage inhibition assay, and humoral immunity, as determined by radioimmunoassay. Peritoneal exudate (PE) cells from guinea pigs immunized with the 40,000molecular-weight polypeptide migrated poorly (30 to 88%> inhibition) after challenge with the immunizing subunit or with purif'ied HB.Ag, type adu or ayw. Response of the same PE cells to the 24,000-molecular-weight subunit was significantly reduced. Similar but less striking evidence for CMI was observed with PE cells derived from guinea pigs inoculated with purified tvpe-specif'ic HB.Ag or with the 24,000-molecular-weight polypeptide. Humoral responses were predictable and showed a reasonable degree of correlation with the CMI response. PE cells from control animals inoculated with normal human serum or polyacrylamide gel were not inhibited af'ter challenge with purified preparations of HB.Ag or with the 40,000-molecular-weight polypeptide, but did show CMI with their respective immunogens. In addition, PE cells f'rom guinea pigs inoculated with normal human serum were inhibited f'rom migrating after challenge with the 24,000-molecular-weight subunit, suggesting that the latter may contain an antigenic determinant related to a human serum protein.
Hepatitis B is a serious public health problem and deoxyribonucleic acid polymerase. This of international concern which is associated suggests that these particles are not infectious with significant morbidity and mortality. It is and could be used as immunizing agents. Some particularly prevalent among certain high-risk theoretical objections have been raised to the populations. These groups include individuals proposed use of' the whole 20-nm particle as the who have recently received blood transfusions, immunogen since antigenic determinants reusers of illicit drugs, homosexuals, health care lated to human plasma proteins have been personnel (surgeons, physicians, dentists, reported to be constituent components of the nurses, laboratory technicians, plasmaphoresis, 20-nm HB.Ag-associated particles (115, 16). and blood bank staff), hemodialysis patients These determinants were distinct from groupAnd their contacts, offspring of mothers who specif'ic (a) and type-specific (d or y) sites and become infected in the last trimester of preg- appeared to be related to antigenic specif'icities nancy, and residents of crowded institutions. on prealbumin, albumin, apolipoproteins C and Thus, the need for development of a suitable D, and the y-chain of immunoglobulin G. Such vaccine is evident. preparations may lead to the production of Efforts in vaccine development, hampered bv antibodies directed against human serum proan inability to grow hepatitis B virus in tissue teins. and organ cultures, have been directed towards Tihe use of purified polypeptides derived from use of modified hepatitis B surface antigen HB8Ag as immunizing agents rather than other (HB8Ag) preparations. Plasma from persist- preparations may have some advantages in that ently infected carriers contains an abundance of' the preparation would exclude genes and comthe small 20-nm spherical particles which, after pcnents of viral or host cell origin. In addition, purification, are free of' detectable nucleic acid major polypeptides derived from the 20-nm 'Present address: Department of Biology. George Mason particles have elicited vigorous antibody reUniversity, Fairfax, Va. 22030. sponses in guinea pigs (6a). 564
VOL. 12, 1975
Recent evidence suggests that not only humoral but also cell-mediated immunity plays a role in preventing or aborting hepatitis B infection (6, 9, 19). In this paper we report the measurement of cell-mediated immunity to HB8Ag and to HB8Ag-derived polypeptides using a migration inhibition assay. MATERIALS AND METHODS Purification and isolation of HB,Ag polypeptides. Spherical, 20 ± 2-nm particles containing HB.Ag were purified from human serum as previously described (7) with slight modifications (12). Briefly, the particles were pelleted for 15 h at 100,000 x g and 4 C, resuspended in saline, and repelleted for 5 h at 200,000 x g and 4 C. The resulting pellet was acidified to pH 2.4 with 0.05 M potassium phthalate-hydrochloride buffer, incubated for 1 h at room temperature, and then twice banded in CsCl at 286,000 x g and 4 C for 18 h. The second isopycnic density centrifugation was followed by a rate-zonal centrifugation for 4 h at 286,000 x g and 4 C in a preformed 5 to 35% (wt/wt) CsCl gradient. After extensive dialysis against physiological saline to remove the CsCl, the purified antigen was sterilized by filtration through a 0.22-tim cellulose acetate filter. Polypeptides of purified HB8Ag were prepared by disc polyacrylamide gel electrophoresis as described in detail by this laboratory (3, 6a) and lyophilized. Briefly, the antigen-containing particles were solubilized by heating at 60 C for 10 min in the presence of 6 M urea, 1% sodium dodecyl sulfate, and 1% 2-mercaptoethanol. The subunits were initially fractionated in a preparative polyacrylamide gel electrophoresis apparatus (Buchler Instruments, Fort Lee, N.J.) using a 13.2% separating gel. After elution, the fractions were divided into six pools and refractionated by analytical polyacrylamide gel electrophoresis. The polypeptide products were detected by staining with Coomassie brilliant blue or by scanning the unstained gel in a Gilford gel scanner. Molecular weights of each polypeptide were estimated by comparing distances migrated to that observed using a series of known molecular weight markers. We selected the 24,000- and 40,000-molecular-weight subunits for this study. The smaller-molecular-weight polypeptide has been characterized as a glycoprotein
(4).
Immunization of animals. The specific polypeptides were excised from the gel, emulsified with an equal volume of complete Freund adjuvant, and injected into the footpads of adult (400 to 500 g) Hartley strain guinea pigs (12). All animals received a booster dose between days 14 to 16. Additional guinea pigs were inoculated with 5 to 10 gg of purified HB8Ag/adw or HB8Ag/ayu. Control animals received an inoculum containing either 1.4 or 140 ,g of normal human serum (NHS) or polyacrylamide gel (PAG). Animals were evaluated between day 28 and day 35 postinoculation for the development of humoral and
cell-mediated immunity. Assay for antibody. Antibody titers were determined by radioimmunoassay for each antigenic type using staphylococcal protein A to separate the bound
CMI TO HBSAG SUBUNITS
['251]HB.Ag/anti-HB.
565
complex from the free
[125I]HB.Ag fraction (6a, 10). Serial dilutions of antiserum were prepared in phosphate-buffered saline, pH 7.4, containing 12.5% NHS and incubated with labeled HB.Ag in a 37 C water bath for 18 h. Staphylococcal protein A (Sigma, St. Louis, Mo.) was added to each tube and incubation was continued for 30 min at room temperature. The pellets were washed twice by centrifugation with phosphate-buffered saline and residual radioactivity was determined in an auto-gamma counter. A specimen was considered to be positive if the counts per minute exceeded 2.0 times the mean of a panel of normal guinea pig sera which were negative for anti-HB,. Cell-mediated immunity assays. Cell-mediated immunity was measured in vitro by the macrophage inhibition assay (1, 5, 14). In these experiments, immunized guinea pigs were inoculated intraperitoneally with 30 ml of sterile paraffin oil. After 3 days the animals were anesthetized with ether and exsanguinated by cardiac puncture. A midline abdominal incision was made and several 50-ml aliquots of ice-cold (6 to 10 C) Hanks solution containing 0.075% NaHCO3 and 0.5 units of heparin per ml were introduced into the peritoneal cavity, and the abdominal contents were gently mixed. Peritoneal fluid rich in mononuclear cells was aspirated and pooled in 250-ml centrifuge bottles kept in an ice bath. The fluid was centrifuged at 900 rpm for 15 min and the oil and liquid phases were discarded. The peritoneal exudate (PE) cells were resuspended in 2 ml of Hanks medium (4 C) containing 0.075% NaHCO3 and were transferred to a 15-ml centrifuge tube. After washing the cells twice in this same medium, they were resuspended to approximately 10% by volume in Eagle minimal essential medium containing penicillin/streptomycin and 15% heat-inactivated normal guinea pig serum. Cells were counted and the concentration was adjusted to 107 cells/ml. Equal volumes (0.4 ml) of cells and medium (with and without antigen) were placed in culture tubes (12 by 75 mm) and allowed to interact for 30 min at room temperature. After this incubation period, the suspensions were centrifuged at 200 x g and 4 C for 5 min, and 0.5 ml of the supernatant was removed before resuspending the cells. Sterile 20-,ul capillary pipettes (Drummond Scientific Co., Broomall, Pa.) were filled with the cell suspensions to approximately 5 mm from the top and were sealed at one end with Critoseal (Sherwood Medical Industries, St. Louis, Mo.). The micropipettes were placed in test tubes and centrifuged at 200 x g for 5 min at 4 C. The capillary tubes were broken at the cell-medium interface and were placed in sterile Mackaness-type chambers. These chambers were filled with culture medium, sealed with silicone grease, and incubated in a humidified CO2 incubator at 37 C for 48 h. Tests were performed in triplicate and the chambers were coded to eliminate observer bias. The area of migration was projected and measured after 24 and 48 h of incubation as follows: average migration of PE cells with antigen x 100 % migration = average migration of PE cells without antigen
566
INFECT. IMMUN.
CABRAL ET AL. % inhibition
=
100%
-
% migration.
Using this procedure, an inhibition of 20% or greater arbitrarily selected as indi.-ative of delayed hypersensitivity. was
RESULTS
Preliminary evaluation of the macrophage inhibition assay. Initial studies indicated that relatively large concentrations of purified HB.Ag or polypeptides would be required to significantly inhibit the migration of sensitized PE cells if the antigen was added directly into the Mackaness-type chambers with the medium. Because the supply of polypeptides was limited, it was necessary to modify the procedure. Antigen and PE cells were preincubated in a small volume for 10 and 60 min at 4, 25 (room temperature), and 37 C before preparing the capillary tubes for the chambers. Table 1 shows the results of this experiment. Although no significant differences were observed, migration patterns appeared to be less stable after preincubation at 4 and 37 C, especially when the incubation period was continued for 48 h. A 30-min preincubation period at room temperature was selected for subsequent tests. An optimal immunization schedule was ascertained. Guinea pigs were inoculated with 4 or 16 ,ug of purified HB.Ag. One-half of' the animals received a booster injection containing the same quantity of antigen on day 14. On day 28 the animals were sacrificed and the PE cells were challenged with varying concentrations of purified HB5Ag ranging from 0.4 to 50 gg/ml. PE cells from guinea pigs immunized and boostered with 16 ,g of HB.Ag and challenged with the same protein failed to migrate a significant distance from the capillary tubes over a 24-h incubation period when compared to unchallenged control PE cells from the same animal (Table 2). Lesser but equally significant inhibition of' macrophage migration was observed in all other HB.Ag-immunized animals except those obtained from guinea pigs immunized once with 4 Mg of HB8Ag and challenged with higher concentrations of HB ,Ag (10 and 50 mg/ml). However, significant inhibition (29 and 28%, respectively) was observed within this same experimental group after 48 h of incubation. From these preliminary data, we concluded that the optimal immunization schedule was the administration of the immunogen in two equally divided doses at 14-day intervals with subsequent measurement of cell-mediated immunity on day 28. A similar observation was made in other studies in which delayed cutane-
ous hypersensitivity was evaluated (unpublished data). Cellular immunity. Cell-mediated immunity in guinea pigs was measured by the macrophage inhibition assay. Guinea pigs were immunized (i) with purified 20 + 2-nm particles containing type-specif'ic aduw or avw HB8Ag, (ii) with the 40,000- or 24,000-molecular-weight polypeptides derived from HB8Ag/adw, (iii) with NHS, or (iv) with PAG. The results are summarized in Table 3. PE cells obtained from four guinea pigs inoculated with the 40,000-molecular-weight polypeptide and challenged with the same antigen exhibited a 35 to 88% decrease in macrophage migration. Inhibition ranging from 49 to 58% was observed when the same cells were challenged with purified HB.Ag/adw. Correspondingly, macrophages from three of the four animals were inhibited from migrating when challenged with purified HB8Ag/ayw, and PE cells from the fourth guinea pig showed 14% inhibition. In contrast, significant inhibition of macrophage migration above 20% was observed in only one guinea pig (no. 9) when the same PE cells were challenged with the 24,000-molecular-weight polypeptide preparation. Macrophages from two other guinea pigs were inhibTABLE 1. Preincubation of immune guinea pig PE cells with 10 ug of HB8Ag Preincubation time (min)
10 60 a
Migration inhibition (%) after preincubation at :a 4C
25 C
37 C
78 76
78 84
76 72
Results read after a 24-h incubation period.
TABLE 2. Migration inhibition of immune and nonimmune PE cells after incubation with varying amounts of HBAg at room temperature for 30 min Migration inhibition (%) after Concn of HB,Ag used to inoculate guinea pigs None (control) 4 gg, no booster 16 Mg, no booster 4 ug, boostered on day 14 16 gIg, boostered on day 14
preincubation with varying concn of HB.Ag (mg)a 0.4 2 10 50 Ob
44 59 68 94
5 34 48 69 94
0 oc
45 71 82
0 O0 66 71 71
Macrophage migration determined after 24 h of incubation. 'No inhibition when compared with unchallenged PE cells. In some instances, migration was enhanced. c Significant migration inhibition observed after 48 h of incubation. a
VOL. 12, 1975
CMI TO HB AG SUBUNITS
567
TABLE 3. Migration inhibition of guinea pig PE cells by HB.Ag or by HB,Ag-derived polypeptides Inoculum
40,000-mol-wt polypeptide (HB.Ag/adw)
24,000-mol-wt polypeptide (HB.Ag/adw)
HB.Ag/adw HB,Ag/ayw NHS, 1.4ug
140,gg
PAG a
Migration inhibition (%) after incubation with: Animal no. 40000-mol wt 24,000-mol wt HB3Ag/ HB.Ag/ polypeptide polypeptide adw ayw
1 9 11 12 18 19 13 10 2 6 7 15 16 14
88 54 36
35 97 13 67 49 71 53 0 0 0 19
17 55 Oa 19 95 57 22 0 29 11 0 13 26 10
52 55 58 49 81 6 43 74 43 33 0 0 0 2
41 14 30 51 0 4 52 64 10 35 22 0 0 0
No inhibition when compared with unchallenged PE cells. In some instances, migration was enhanced.
ited at a level slightly below our arbitrary cut-off value of 20%. Mononuclear cells obtained from three additional animals immunized with the 24,000molecular-weight polypeptide were inhibited 22, 57, and 95% when challenged with the same polypeptide preparation. Furthermore, cells from two of these animals (no. 13 and 18) exhibited evidence for cell-mediated immunity to the 40,000-molecular-weight subunit and to the purified whole particle containing HB.Ag/ adw. PE cells procured from one animal, guinea pig no. 13, were inhibited 52% after challenge with HB,Ag/ayw, implying a certain degree of type specificity to this polypeptide preparation
quently challenged with PAG (data not shown). Minimal to absent inhibition was observed among guinea pigs challenged with the two polypeptides or with the whole particles. When PAG was used to challenge PE cells from the other groups of guinea pigs, no inhibition was observed. Significant macrophage inhibition was observed in guinea pigs inoculated and challenged with 1.4 or 140 qg of NHS. In addition, macrophage migration was inhibited 13 and 26%, respectively, when challenged with the 24,000-molecular-weight polypeptide. It is noteworthy that mononuclear cells from one of two guinea pigs inoculated with the 24,000molecular-weight polypeptide were inhibited 97% after challenge with 100 ,g of NHS (data not shown), whereas none of the other groups of guinea pigs exhibited such evidence of delayed hypersensitivity to this protein. Figure 1 illustrates typical PE migration inhibition patterns obtained after challenge of sensitized PE cells with homologous and heterologous antigens. Humoral immunity. Humoral immunity was evaluated in all guinea pigs by radioimmunoassay, and the results are summarized in Table 4. Antibody titers to HB8Ag, types adw and ayw, ranged from 1:300 to 1:290,000. The antisera were type specific and homologous titers exceeded heterologous reactions for each immunogen. No anti-HB. was detected in sera obtained from guinea pigs inoculated with NHS or with PAG.
(6a). Guinea pigs inoculated with purified particles containing HB8Ag/adw exhibited evidence for the development of delayed hypersensitivity when their PE cells were challenged with HB.Ag/adw or with the 40,000-molecularweight subunit. PE cells from one of the guinea pigs were inhibited by the 24,000-molecularweight polypeptide, whereas those from the other animal were responsive to HB.Ag/ayw. A positive inhibitory response to homologous protein was observed with macrophages from guinea pigs sensitized to HB8Ag/ayw. However, cells from only one animal (no. 6) responded to the 40,000-molecular-weight subunit (derived from HB.Ag/adw) and to the whole particle containing HB.Ag/adw. Neither animal exhibited cell-mediated immunity to the 24,000DISCUSSION molecular-weight polypeptide. One approach to the development of a vacMigration of PE cells was significantly inhibited (69%) in animals inoculated and subse- cine for the prevention of type B hepatitis
568
INFECT. IMMUN.
CABRAL ET AL.
;-
Ls-e{jtI2l24 X p iTape;
;
K
L
FIG. 1. Capillary tube migration patterns. (A-D): Guinea pig peritoneal exudate (PE) cells sensitized to the 40,000-molecular-weight polypeptide and challenged with (A) medium, (B) the 40,000-molecular-weight polypeptide, (C) HBqAg/adu, and (D) HBAg/ayw. (E, F): PE cells sensitized to the 24,000-molecular-weight polypeptide and challenged with (E) medium and (F) the 24,000-molecular-weight polypeptide. (G, H): PE cells sensitized to HB9Ag/ayw and challenged with (G) medium and (H) HBAg/ayw. (I-L): PE cells sensitized to NHS and challenged with (I) medium, (J) NHS, (K) the 40,000-molecular-weight polypeptide, and (L) HB,Ag/ayw. Note that in all cases significant inhibition of migration occurred when sensitized PE cells were challenged with the homologous antigen (B, F, H, and J). TABLE 4. Determination of anti-HB, titers in guinea pig serum by radioimmunoassay Reciprocal antibody titera Animal no.
Inoculum
62,500
Anti-HB/ayu' 5,000
900 3,500 1,500 850 300 700 290,000 16,000 3,000 5,000
