Monoclonal antibodies against hepatitis A virus.

Vol. 18, No. 5

JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1983, p. 1237-1243 0095-1137/83/111237-07$02.00/0 Copyright © 1983, American Society for Microbiology

Monoclonal Antibodies Against Hepatitis A Virus ANDREW MAcGREGOR,l* MYKOLA KORNITSCHUK,' JOHN G. R. HURRELL,1 NOREEN 1. LEHMANN,2 ANTHONY G. COULEPIS,2 STEPHEN A. LOCARNINI, AND IAN D. GUST2 Immunochemistry Department, Research and Dev,elopment Division, Commonwealth Serum Laboratories, Parkville, Victoria, 3052, Australia1; and Virology Department, Fairfield Hospital, Fairfield, Victoria, 3078, Australia2

Received 3 May 1983/Accepted 25 July 1983

Three monoclonal antibodies (K2-4F2, K3-2F2, and K3-4C8) of the immunoglobulin G2a class were raised against hepatitis A virus. The specificity of these antibodies was confirmed by immune electron microscopy, solid-phase radioimmunoassay, and in vitro neutralization in cell culture. Binding studies suggested that they all recognize closely related antigenic determinants. These monoclonal antibodies should prove to be of great value as diagnostic and research reagents.

Hepatitis A virus (HAV) is a 27 to 32-nm virus (8) whose biophysical and biochemical characteristics (5) have resulted in its classification as a member of the genus enterovirus (lOa) within the family Picornaviridae (2). HAV has proved difficult to isolate in vitro (22), and relatively small quantities of virus have been recovered from the feces of hospitalized patients (4) and experimentally infected animals (21). The difficulty of obtaining large quantities of virus has hampered the production of antisera for diagnostic and research purposes. In this paper we report the development and characterization of three monoclonal antibodies which are specific for HAV, and which may be of value as diagnostic reagents and research tools. MATERIALS AND METHODS Virus preparation. (i) Mouse inoculations. HAV for mouse inoculation was obtained from a fecal specimen (HM-790) collected from a patient with naturally acquired hepatitis in whom the clinical diagnosis of hepatitis A was supported by liver function tests and confirmed by the presence of specific immunoglobulin M (IgM) in the acute-phase sera (17). Virus was

identified by solid-phase radioimmunoassay (SPRIA) and immune electron microscopy (IEM) (19) and then purified by a process of differential centrifugation, chloroform extraction, column chromatography with agarose gel filtration, and isopycnic ultracentrifugation in cesium chloride as described previously (3). During purification, the presence of HAV was monitored by SPRIA. The final identity of purified virus was assessed by direct and IEM with human pre- and postinfection hepatitis A sera (16), and no adventitious agents were isolated after routine viral culture (3). HAV particle counts in this preparation exceeded 1,000 per electron microscope 400-mesh grid square. (ii) Reagent antigen. HAV as reagent antigen comprised four fecal preparations and three cell culture isolates. The four fecal preparations were derived from different patients with hepatitis A and were three

individual specimens (HM-947, HM-838, and HM-952) and a pool (pool A) of six fecal specimens known to contain HAV. The three cell culture isolates (HM790/7P, HM-165/1OP, and HM-172/5P) had been passaged in monkey embryonic kidney (MEK) (12), Buffalo green monkey kidney (BGM) (6), and fetal rhesus kidney (FRhK-4) (22) and were at the seventh, tenth, and fifth passage, respectively. HAV extracted from samples HM-952, pool A, HM-165/10P, and HM790/7P were used for IEM. The reagent antigen from both feces and cell culture was purified by differential centrifugation and chloroform extraction (16). Immunization. Four female BALB/c mice, 6 weeks of age, were obtained from and maintained at the breeding colony at the Commonwealth Serum Laboratories, Melbourne. The limited quantities of antigen available permitted the immunization of two mice with each preparation of HAV (Table 1). Retroorbital bleedings were obtained from the mice by using tribromoethanol administered intraperitoneally as an anaesthetic (25). Bleedings were done the day before the primary inoculation (day 0) and at days 15 and 90 after the immunization schedule was begun. The development of specific anti-HAV in the mice was monitored by SPRIA as described below. Further intraperitoneal doses of HAV were given to animals that responded poorly to the initial immunization. When required for fusion, the animal with the highest serum antibody level at the previous bleeding was selected and boosted. Cell hybridization. Spleen cells were fused with NS1 cells (13) by methods similar to those of Galfre et al. (10). Briefly, spleen cells were dispersed through a stainless steel screen (0.3-mm mesh), mixed with 107 NS-1 cells per spleen, and washed three times by centrifugation with Dulbecco modified Eagle mediumhigh glucose (MA Bioproducts) containing 2.4 g of NaHCO3 per liter (DME). The cell pellet was suspended in 30% (vol/vol) polyethylene glycol 1000 (Sigma Chemical Co.) in DME (pH 7.0 to 7.2) at 37°C, pelleted after 3 min by low-speed centrifugation, and diluted with a large volume of DME at 7.5 min. The fused cells were centrifuged again and diluted in growth medium

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MACGREGOR ET AL.

J. CLIN. MICROBIOL.

TABLE 1. Immunization schedule Mouse

Primary dose (day 1), intraperitoneal

Kl

500-p inoculum consisting of 250 pul of HAV in PBS, pH 7.2 (5 p.g of protein), mixed with 250 pR1 of Freund complete adjuvanta b

K2

Inoculum as for mouse Kl

K3

500 pL. inoculum consisting of 250 RI1 of HAV in PBS, pH 7.2 (5 p.g of protein), mixed with 250 p.l of Freund complete adjuvantb'

b

Secondary dose, intraperitoneal

Boost given 3 days before fusion, intravenous (via tail vein)

None

Day 34, 500-pul inoculum consisting of 250 pAl of HAV in PBS, pH 7.2 (5 pug of protein), mixed with 250 pI of PBSa b

None

Day 70, inoculum as for mouse K1'

None

Day 119, 400-p.l inoculum consisting of 200 p.l of HAV in PBS, pH 7.2 (4 p.g of protein), mixed with 200 p.1 of PBS"

400-p.l inoculum consisting of Day 179, 600-p.l inoculum conDay 201, 500-p.l inoculum con200 p.l of HAV in PBS, pH sisting of 300 pL. of HAV in sisting of 300 p.l of HAV in 7.2 (4 p.g of protein), mixed PBS, pH 7.2 (6 p.g of protein), PBS, pH 7.2 (6 p.g of prowith 200 p.l of Freund commixed with 300 p.l of Freund tein), mixed with 200 pL. of plete adjuvantb' complete adjuvantb.' PBSa a Mice were immunized (primary dose or booster) with a preparation of HM-790 containing approximately 1012 HAV particles per ml. The HAV had been purified by differential centrifugation, chloroform extraction, agarose gel filtration, and isopycnic ultracentrifugation. b Inocula also contained 0.05% mouse serum. ' Mice received primary or secondary doses (or both) of a preparation of HM-790 containing approximately 1010 HAV particles per ml. The HAV had been purified by the first three purification steps only. K4

(DME containing 1 mM sodium pyruvate, 100 U of penicillin G per ml, 100 p.g of streptomycin sulfate per ml, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, [pH 7.5], 1% 100x Eagle nonessential amino acids solution [CSL, Australia] and 15% [vol/vol] fetal calf serum [CSL]). For initial plating, the growth medium was supplemented with 0.1 mM hypoxanthine (Calbiochem) and 0.4 mM thymidine (Calbiochem) (15). The cell suspension was plated out in 96-well tissue culture plates (Costar). Every 24 h, 50% of the medium was replaced with growth medium containing hypoxanthine and thymidine. From 48 h after fusion, 0.04 mM aminopterin (Sigma) was also included. After 14 days, aminopterin was omitted from the fresh growth medium; 7 days later, growth medium alone was used for subsequent cell cultivation. Hybridomas were visible from day 8 after fusion, and supernatants were assayed for antibody from day 11. Cells from wells positive for specific antibody production were cloned twice by limiting dilution on 3T3 BALB/c feeder layers in 96-well plates (1). Detection of anti-HAV by SPRIA. Antibody activity in cell culture supernatants was detected by using a modified SPRIA (14). Wells of polyvinyl microtiter plates (Cooke Engineering Inc.) were coated with a 1:1,000 dilution of human convalescent-phase hepatitis A serum containing anti-HAV (18) in 0.85% NaCl (saline) and 0.1% mouse serum for 4 h at 20°C. The wells were then washed with phosphate-buffered saline (PBS), pH 7.4, and incubated with HAV (pool A) overnight at 4°C. Wells were then washed three times with PBS (pH 7.4) and drained. Test samples (50 p.1) were inoculated into the wells and incubated for 4 h at

20°C. Fifty microliters of 1251I-labeled human anti-HAV serum was then added to each well, and the incubation was continued overnight at 4°C. The wells were then washed three times with PBS, cut out with a hot wire, and counted. Two positive and two negative controls were included in each assay. The result was expressed as percent counts bound ([counts per minute in sample well/mean counts per minute in negative controls] x 100). A figure of 60% reduction in the binding of labeled antibodies per well or less was taken as positive for antibody activity. Isotyping. The three monoclonal antibodies were isotyped by immunodiffusion with isotype-specific antisera (Litton Bionetics) (20). Monoclonal antibody production. Monoclonal antibodies were produced both in ascites fluid and in cell culture. For ascites fluid production, BALB/c mice older than 6 weeks were inoculated intraperitoneally with 0.5 ml of Pristane (Aldrich Chemical Co.). After 10 to 14 days, 5 x 105 hybridoma cells in 0.5 ml of Dulbecco PBS were inoculated intraperitoneally into each mouse. When abdominal swelling occurred, ascites fluid was harvested daily using a 20-gauge needle. EDTA (2.5 mg/ml) and NaN3 (0.2 mg/ml) were added to the pooled harvest. Cells and precipitates were removed by centrifugation. For cell culture supernatant production, hybridoma cells were grown in 1-liter volumes of growth medium from an initial density of 2 x 104 cells to a final density of 2 x 10" cells per ml, and the supernatants were harvested by centrifugation. Purification of monoclonal antibody from ascites fluid. Ascites fluid pools were clarified by centrifugation at 12,000 x g for 20 min and filtered through a 0.2-p.m

MONOCLONAL ANTIBODIES AGAINST HAV

VOL. 18, 1983

membrane. The filtrate was adjusted to pH 8.1 and passed through an 8-ml protein A-Sepharose CL-4B (Pharmacia, Uppsala, Sweden) column which had been equilibrated with 0.1 M phosphate buffer, pH 8.1 (washing buffer). Washing buffer was passed through the column until optical density at 280 nm of the eluate was less than 0.05. The monoclonal antibody was eluted with 0.1 M phosphate buffer in 0.9% [wt/vol] NaCl (pH 3.0), and 1-ml fractions (optical density at 280 nm of greater than 0.1) were pooled and immediately adjusted to pH 7 (7). Protein concentration was estimated by the biuret method and adjusted to 1 mg/ml. lodination of antibody. Antibodies were iodinated by adding 100 ,ug of antibody in 0.1 ml of PBS (pH 7.4) to 1.2 ,ug of lodogen (Pierce Chemical Co.). 1251 (1 mCi; Amersham Corp., Amersham, United Kingdom) in 10 ,ul of distilled water was added and reacted for 10 min at room temperature; 0.2 ml of 0.1% NaN3 in PBS was added to stop the reaction, and free 125I was separated from the iodinated antibody by passage over a Sephadex G-25 (Pharmacia) column that had been preequilibrated with PBS (9). The proportion of specific antibody in iodinated preparations was estimated by incubating serial dilutions on HAV attached to wells and measuring the percentage of counts bound at the asymptote in a plot against dilution. IEM. HAV was centrifuged at 55,000 x g for 4 h at 4°C in a SW60 rotor (Beckman Instruments, Inc.). Pellets were incubated with 100 ,u1 of cell culture supernatant containing each monoclonal antibody for 1 h at 34°C. After overnight incubation at 4°C the samples were centrifuged for 2 h at 34,000 x g, suspended in approximately 30 ,ul of PBS, and stained with 3% phosphotungstic acid (pH 7.4). The preparations were examined for immune complexes in a Philips EM301 electron microscope at a plate magnification of 44,000. Virus neutralization. Cell lysates of FRhK-4 cells infected with HAV isolate HM-165/1OP were diluted in 10-fold steps from 10-1 to 10-5, and 0.1-ml volumes were incubated with 0.1-ml volumes of monoclonal antibody (1 mg/ml) at 37°C for 2 h and then inoculated onto monolayers of FRhK-4 cells in 25-cm2 plastic disposable flasks (Costar). Control flasks were inoculated with 0.1-ml volumes of each virus dilution which had previously been incubated with 0.1 ml of PBS. Four sets of flasks were set up and incubated at 37°C. At weekly intervals cells from one set of flasks were stripped off by a brief exposure at 37°C to a trypsinversene solution consisting of 0.12% (wt/vol) trypsin (Difco Laboratories) and 0.2% (wt/vol) versene-EDTA in calcium- and magnesium-free Hanks balanced salt

1239

solution. The cells were disrupted by three cycles of freeze-thawing, and the presence of HAV was monitored by SPRIA (14). Specificity testing. Each monoclonal antibody was tested for its ability to capture both cell culture and fecally derived HAV by SPRIA. Ascites fluid containing each of the monoclonal antibodies, purified by protein A and diluted 1:1,000 to give a final protein concentration of 25 ,ug/ml, was used to coat the wells of polyvinyl microtiter plates (Cooke Engineering Inc.) for 4 h at 20°C. The wells were then washed with PBS (pH 7.4), and 50-,ul volumes of the different HAV preparation were added and incubated for 4 h at 20°C. The wells were washed once again with PBS before the addition of 50 ,ul of 1251I-labeled human anti-HAV convalescent serum. Wells were incubated overnight at 4°C and washed with PBS, and counts bound were calculated. The human coating antibody was purified as described previously (17). Competitive binding assay. The SPRIA for the detection of anti-HAV was carried out on serial 10-fold dilutions of each monoclonal antibody. However, instead of 125I-labeled human anti-HAV, labeled monoclonal antibody K3-4C8 was added to the wells as the competing probe. Wells were incubated overnight at 4°C and washed with PBS, and the counts bound were calculated. The competitive binding assay was repeated with 125I-labeled K2-4F2 and 125I-labeled K3-2F2. RESULTS

Hybridomas. Spleens from four mice (Kl through K4) were used in this study (Table 2). After fusion, of the 1,250 wells seeded, 842 (67.4%) contained hybridomas, and of these only 4 (0.5%) prodticed anti-HAV. One of the four hybridomas lost its ability to produce antiHAV on subsequent passaging. The three remaining parent hybridoma lines (K2-4F2, K32F2, and K3-4C8) were each cloned twice (K24F2-3G2-1D9, K3-2F2-2C7-1C8, and K3-4C81E8-3F6, respectively) to ensure their monoclonality. Isotyping of the three antibodies showed that they were all of the IgG2a class. IEM. Anti-HAV-containing supernatants from the three positive hybridoma cell lines were tested by IEM. Supernatants from hybridomas that were negative by SPRIA were used as negative controls. All three monoclonal antibodies produced immune complexes with HAV

TABLE 2. Hybridoma production Development of anti-HAV on day:

Mouse

0

15

++ Kl + K2 ++ K3 + K4 a One hybridoma failed to maintain antibody

No. Day of

of wells

No. of wells with

No. of hybridomas producing anti-

fusion

seeded

hybridomas

HAV

37 73 122 204

350 300 300 300

212 300 300 30

0 2a 2 0

production.

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A

lOOnrrt-. :.

j100nm FIG. 1. (A) Immune electron micrograph of HAV strain HM-925 with a cell culture supernatant from a hybridoma line not producing anti-HAV. The specimen was stained with 3% (wt/vol) phosphotungstic acid, pH 7.4. Immune complexes and individual virus particles coated with antibody could not be visualized. (B) Immune electron micrograph of HAV strain HM-925 with a cell culture supernatant from hybridoma K2-4F2. The specimen was stained with 3% (wt/vol) phosphotungstic acid, pH 7.4. Immune complexes were commonly seen in this preparation.


VOL. 18, 1983

TABLE 3. Neutralization of HAV in cell culture with monoclonal antibodies P/N ratio at weeka:

HAV-

Antibody

Control

165/1OP dilutions

1

2

0.9 1.0 1.3 0.8 1.3

2.4 (+) 4.1 (+) 1.8 1.6 1.2

3.5 (+) 4.4 (+) 4.3 (+) 4.9(+) 4.0(+ 5.0(+ 4.2 ()5.5(+ 3.3 (+) 4.3 (+)

1.1 1.0 0.9 1.0

1.2 1.1

2.0 1.1

1.0 1.3 1.0

1.0 1.3 1.2

1.1 1.2

1.3 0.9

2.3 0.9

10-3 10-4 lo-5

1.0 0.8 1.3 1.0 0.9

1.2 1.0 0.9

1.0 0.9 0.9

10-

1.0 1.2 1.2 0.9 0.9 1.0 1.3 0.8 1.2

1.1 1.0

2.7(+)

1010-2

10-3 10-4 10-5 K2-4F2

1010-2

10-3

10-4 10-5 K3-2F2

K3-4C8

1010-2

10-2 10-3

1.2 0.9 1.0 1.2

1.0

3

1.2 1.1 1.0

4

2.9 (+) 2.7 (+) 1.5

1.2 1.3

2.6(+) 2.3 (+) 1.8

10-4 1.4 10-5 a Values are expressed as positive/negative (P/N) ratios where P represents the counts per minute of bound 125I-anti-HAV human convalescent serum in test wells and N represents the counts per minute bound in the control wells. Ratios of 2.1 or greater are regarded as positive for HAV.

strain HM-952 which were not visualized on reaction of HAV with control supernatants. Figure 1 shows representative results of these IEM experiments with monoclonal antibody K2-4F2. Similar findings were obtained with a number of other HAV preparations, including HM165/1OP, HM-790/7P, and pool A. Virus neutralization. The results of HAV neutralization by the monoclonal antibodies are shown in Table 3. HAV was not detected in any of the dilutions of control and test flasks after the first week. At week 2, HAV was only detected at 10-1 and 10-2 in the control flasks. At week 3, all the control flasks were positive, but virus was still undetectable in the test flasks. However, at week 4 all three test flasks showed signs of HAV activity. The monoclonal antibodies appeared to reduce infectivity rather than completely neutralize the virus. Specificity testing. The ability of each of the three monoclonal antibodies to recognize HAV purified from both feces and cell culture is shown in Table 4. In most cases the use of monoclonal antibodies resulted in higher positive/negative ratios than those obtained with polyclonal human convalescent sera. The reac-

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tivity of the monoclonal antibodies K2-4F2 and K3-4C8 appeared to be similar and generally higher than that of K3-2F2 for both the cell culture and the fecally derived virus, except that K3-4C8 failed to recognize cell culture isolate HM-175/5P. Competitive binding assay. All three monoclonal antibodies inhibited the binding of 125I-labeled K3-4C8. Similar results were obtained with 125I-labeled K3-2F2 and K2-4F2. The kinetics of binding inhibition of K3-4C8 for all three monoclonal antibodies (Fig. 2) suggest that they bind to antigenically similar or sterically related determinants. DISCUSSION The percentage of hybridomas recognized as producing HAV-specific antibody was very low, being less than 1%. This was probably due to the screening method used in this study, namely, a "blocking"-type SPRIA with human polyclonal convalescent-phase anti-HAV. This system would miss any specifically reacting monoclonal antibodies that were not effective in blocking labeled polyclonal anti-HAV and would select for antibodies that react with the major surface antigens of HAV. There can be little doubt regarding the specificity of the three monoclonal antibodies for

TABLE 4. Ability of each of the three monoclonal antibodies to bind HAV in an SPRIA P/N ratio with the following coating HAV antibodya: Human K24F2 K3-2F2 K3-4C8

Fecal strains Pool A HM-947 HM-972 Cell culture isolates HM-790 (7th passage)b HM-165

(10th passage)c

17.1 7.5 2.2

47.0 25.4 3.1

27.6 20.0 2.2

40.0

23.1

32.2

12.0

23.1

11.0

13.9

7.8

13.3

23.8 3.5

HM-172 3.7 5.3 2.4 1.2 (Sth passage)d a Values are expressed as positive/negative (P/N) ratios where P represents the counts per minute of bound 125I-anti-HAV human convalescent serum in the test wells and N represents the counts per minute bound in the control wells. Ratios of 2.1 or greater are regarded as positive for HAV. Negative controls (PBS, complete medium, hybridoma supernatant, and ascites) all had P/N values of less than 2.1. b Passaged in monkey kidney cells (MEK) (6). Passaged in buffalo green monkey kidney cells (BGM) (12). d Passaged in fetal Rhesus kidney cells (FRhK) (22).

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MACGREGOR ET AL.

$100' -

0

C.)

2 "ft.

o? K2-4F2

W' 50C) I..

: K2.3F2

0

X

K3-4C8

g,~~~~~ D~ 10000

1000

100

10

1

CONCENTRATION OF ANTIBODY (ng)/WELL FIG. 2. Inhibition of binding of 1251I-labeled K3-4C8 monoclonal antibody by each of the three monoclonal antibodies.

HAV. First, the virus used to produce the antibodies was prepared by a method known to produce material of high purity (3, 16), and mice immunized with this material produced specific anti-HAV. Second, all three monoclonal antibodies produced immune complexes with HAV by IEM. Third, the antibodies recognized both cell culture-derived and fecally derived HAV when used in a SPRIA test. Fourth, they partially neutralized the infectivity of HAV in cell culture. The inability of the monoclonal antibodies to completely neutralize cell culture-derived HAV may reflect either the presence of virus aggregates before neutralization (11) or inadequacies of the neutralization conditions used in this study (23). The suppression of neutralization by the presence of virus aggregates has been observed with other enteroviruses that were grown in cells of monkey origin (12). Monodispersion of viral aggregates with an agent such as deoxycholate before neutralization may be required before total neutralization can be demonstrated (11). In addition, many enteroviruses require prolonged incubations with antibody (several hours at room temperature or 370C followed by overnight incubation at 4°C) before total neutralization can be achieved (23). The competitive binding data of Fig. 2, in which the binding of labeled monoclonal antibody K3-4C8 to HAV in the SPRIA test was blocked by the other monoclonal antibodies, suggest that these antibodies recognize closely

related (antigenically similar or sterically associated) antigenic determinants. In general, the monoclonal antibodies yielded higher positive/negative ratios when reacted with HAV than those observed with human antiHAV used at optimal conditions. The choice of monoclonal antibodies and the optimization of conditions for their use could therefore prove valuable in generating diagnostic reagents that would be free of the inherent difficulties of quality and supply involved in the use of human convalescent serum. Other possible applications for the monoclonal antibodies include searching for antigenic variants of HAV, differentiation of wild-type from attenuated strains, comparison with future monoclonal antibodies against cell culture isolates of HAV, purification of cell culture-derived HAV by affinity chromatography, and detection of HAV-specific antigen producing colonies of DNA recombinant organisms into which elements of the HAV genome have been cloned

(24). ACKNOWLEDGMENTS This work was supported by the Commonwealth Serum Laboratories, Melbourne, Australia, and by grants from the National Health and Medical Research Council of Australia. We acknowledge the assistance of Linda E. Smith, David A. Harrison, and Zhuang Hui in cell handling, John C. Cox for isotyping the monoclonal antibodies, Alan R. Coulter for assistance in iodination, Kathleen Gavin in preparation of materials, John Marshall for the IEM, and Robert Pringle for helpful discussions.

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