Method of preparing hepatitis B core antigen

HB.sub.c Ag is prepared by isolating Dane particles by isopycnic banding of fluid from HB.sub.s Ag positive donors, optionally but preferably pelleting the Dane particles, and then removing the surface antigen by contacting the Dane particles with a nonionic surfactant having from about 15 to about 35 oxyethylene units in the presence of a reducing agent such as mercaptoethanol. The isolated core antigen is used as a diagnostic and immunologic agent.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to hepatitis B core antigen (HB.sub.c Ag) and, more 
particularly, to a process for preparing hepatitis B core antigen in high 
yield and purity. 
Hepatitis B is one of the types of viral hepatitis which results in a 
systemic infection with the principal pathologic changes occurring in the 
liver. This disease affects mainly adults and is maintained chiefly by 
transfer of infection from long term carriers of the virus. Usual methods 
of spread are by blood transfusion, contaminated needles and syringes, 
through skin breached by cuts or scratches, by unsterilized dental 
instruments as well as by saliva, veneral contact or exposure to 
aerosolized infected blood. 
The incubation period of type B hepatitis is relatively long: from 6 weeks 
to 6 months may elapse between infection and the onset of clinical 
symptoms. The illness usually begins with fatigue and anorexia, sometimes 
accompanied by myalgia and abdominal discomfort. Later jaundice, dark 
urine, light stools and tender hepatomegaly may appear. In some cases, the 
onset may be rapid, with appearance of jaundice early in association with 
fever, chills, and leukocytosis. In other cases jaundice may never by 
recognized and the patient may be aware of a "flu-like" illness. It is 
estimated that the majority of hepatitis infections result in a mild, 
anicteric illness. 
Serum obtained from patients with hepatitis B infections usually contains 
three distinct morphologic forms. The largest of these morphologic forms, 
a 42-nm to 45-nm double shelled spherical particle, often referred to as 
the Dane particle (HBV), is believed to be the virus of hepatitis B. The 
outer surface or envelope of the Dane particle (HB.sub.s Ag) surrounds a 
27-nm inner core which does not react with antibody against HB.sub.s Ag 
and which contains a distinct antigen, the core antigen (HB.sub.c Ag). 
Antibody to HB.sub.c Ag appears after acute hepatitis B infection, and 
also can be demonstrated consistently in chronic carriers of HB.sub.s Ag. 
Highly sensitive techniques are now available for detection of the 
HB.sub.c Ag system. A deterrent to the more widespread use of such 
techniques, however, is the absence of a simple yet practical and 
effective method for obtaining HB.sub.c Ag. The methods proposed 
heretofore generally involve the use of selected plasma which contains 
exceptionally high amounts of Dane particles. 
2. Objects of the Invention 
It is, accordingly, an object of the present invention to provide a 
practical and effective method for obtaining HB.sub.c Ag. Another object 
is to provide an improved method for concentrating and purifying HB.sub.c 
Ag. Still another object is to provide a method for obtaining HB.sub.c Ag 
from biological fluid found positive for HB.sub.s Ag rather than from 
selected high titer HB.sub.s Ag plasma. A further object is to provide 
immunogenic and therapeutic compositions containing HB.sub.c Ag. These and 
other objects of the present invention will be apparent from the following 
description. 
SUMMARY OF THE INVENTION 
HB.sub.c Ag is prepared by isolating Dane particles by isopycnic banding of 
biological fluid from human HB.sub.s Ag positive donors, optionally but 
preferably pelleting the Dane particles, and then removing the surface 
antigen by contacting the Dane particles with a nonionic surfactant having 
from about 15 to about 35 oxyethylene units in the presence of a reducing 
agent such as mercaptoethanol. 
DETAILED DESCRIPTION 
The starting material for the purified hepatitis B core antigen (HB.sub.c 
Ag) of the present invention is plasma obtained from donors positive to 
HB.sub.s Ag. The plasma is obtained in conventional manner, e.g., by 
plasmaphoresis. The level of HB.sub.s Ag may be measured in known manner 
by any suitable means, e.g., reversed passive hemagglutination or 
complement fixation. Optionally, the plasma may be cooled and the 
cryoprecipitate which forms is removed by light centrifugation. The Dane 
particles in the resulting plasma are isolated by isopycnic banding. The 
Dane particle-rich fraction is treated to remove Dane core antibody, 
preferably by pelleting, and then treated to remove the surface antigen 
and liberate the core antigen. Removal of the surface antigen is effected 
by contacting the Dane particle with a nonionic surfactant having from 
about 15 to about 35, preferably about 18 to about 33, oxyethylene units 
in the molecule in the presence of a mercaptan reducing agent, for 
example, mercaptoethanol, dithiothreitol, dithioerythritol, and 
dithiooctanoic acid. Suitable nonionic surfactants are oxyethylated alkyl 
phenols, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene 
acids, polyoxyethylene alcohols, polyoxyethylene oils and polyoxyethylene 
oxypropylene fatty acids. Some specific examples are the following: 
alkylphenoxypolyethoxy (30) ethanol 
polyoxyethylene (20) sorbitan monolaurate 
polyoxyethylene (20) sorbitan monopalmitate 
polyoxyethylene (20) sorbitan monostearate 
polyoxyethylene (20) sorbitan tristearate 
polyoxyethylene (20) sorbitan monooleate 
polyoxyethylene (20) sorbitan trioleate 
polyoxyethylene (20) palmitate 
polyoxyethylene (20) lauryl ether 
polyoxyethylene (20) cetyl ether 
polyoxyethylene (20) stearyl ether 
polyoxyethylene (20) oleyl ether 
polyoxyethylene (25) hydrogenated castor oil 
polyoxyethylene (25) oxypropylene monostearate. 
In isopycnic banding the partially purified concentrate is contacted with a 
liquid medium having a density gradient therein which includes the density 
of the specific antigen being isolated. The liquid medium is then 
subjected to ultracentrifugation to attain an equilibrium distribution of 
the serum components through the density gradient according to their 
individual densities. Successive fractions of the medium are displaced and 
those containing the desired antigen, i.e. the fractions having a density 
of from about 1.26 to about 1.30 g/cc, are separated. The concentrations 
of the solutions forming the gradient are selected so as to encompass the 
density range of from about 1.0 to about 1.41 g/cc. The liquid medium may 
be employed in the form of a linear gradient or a step gradient. 
Preferably it is employed in the form of a step gradient due to its 
inherently higher capacity for fractionation. 
The liquid media used in the isopycnic banding step may be any density 
gradient in the appropriate ranges, e.g. sucrose, potassium bromide, 
cesium chloride, potassium tartrate, or sodium bromide. Sodium bromide is 
preferred.

EXAMPLE 1 
A. Preparation of Dane Particles (HBV) 
The rotor of a centrifuge, Electronucleonics K is filled with 8,400 ml of 
phosphate buffer. After running the rotor up to 10,000 rpm to degas the 
system, the following step gradient is pumped into the bottom of the 
stationary rotor: 
1. 2,400 ml of 10% NaBr, .rho. = 1.08 
2. 1,000 ml of 20% NaBr, .rho. = 1.17 
3. 1,500 ml of 30% NaBr, .rho. = 1.28 
4. 3,500 ml of 40% NaBr, .rho. = 1.41 
Plasma containing HB.sub.s Ag, 1,750 ml, is pumped into the top of the 
stationary rotor displacing 1,750 ml of 40% NaBr from the bottom of the 
rotor. The rotor is accelerated to 30,000 rpm and run at this speed for 4 
hours. The rotor is then stopped and 1,750 ml of 40% NaBr are pumped into 
the bottom of the rotor forcing the plasma out the top. An additional 
1,750 ml of fresh plasma containing HB.sub.s Ag are pumped into the top of 
the rotor displacing an equal volume of 40% NaBr out the bottom of the 
rotor. The rotor is then run at 30,000 rpm for 18 hours. After stopping 
the rotor 1,000 ml of Dane particle rich material in the 1.26-1.30 density 
region is collected. 
The Dane particles (HBV) are separated from the NaBr zonal fraction in the 
following procedure. The zonal fraction (1000 ml) is diluted to 3000 ml 
using phosphate buffered saline. This material is then placed into twelve 
type 19 rotor plastic bottles (ea. 250 ml/bottle). The material is then 
centrifuged using a type 19 rotor (Beckman). The rotor is spun at 17,000 
rpm (45,000 .times. g) for 24 hours in order to pellet the Dane particles. 
The rotor is then stopped and the supernate from each bottle is decanted. 
The pellet material from all 12 bottles is recovered in a total volume of 
5-7 ml of Tris-saline buffer and stored at -70.degree. C. This material is 
the Dane particle concentrate. 
B. Purification of Dane Particles 
1 ml of concentrated Dane particles from part A is layered over 4 ml of 20% 
sucrose - 1% bovine serum albumin (BSA) in Tris buffer (pH 7.6) in a SW 65 
rotor with 1/2 .times. 2 inch cellulose nitrate tubes. The particles are 
centifuged at 35,000 rpms for 4 hours. Post centrifugation, the supernate 
fluid is decanted and the pellet is gently resuspended in 0.5 ml of Tris 
buffer with 1% BSA using a cotton tipped swab (pre-moistened with buffer). 
The cotton swab is then rinsed with 0.5 ml of buffer. The final volume of 
Dane particle material is 1 ml. The Dane particles are stored at 
-70.degree. C. 
C. Preparation of HB.sub.c Ag (Core Antigen) 
The material from part B, 1 ml, is added to 1 ml of a 1% (v/v) solution of 
2-mercaptoethanol is deionized water, and 1 ml of a 1% (v/v) solution of 
polyoxyethylene (20) sorbitan monooleate in deionized water. The resulting 
mixture is agitated gently and placed in a 37.degree. C. water bath. After 
1 hour the mixture is diluted with TMN-1% BSA (a solution containing 0.08 
M Tris, 0.008 M MgCl.sub.2 and 0.14 M NaCl, and 1% BSA) using a previously 
calculated quantity of diluent until if contains 32 IAHA units per ml. The 
solution is then dispensed into plastic 2 ml screw-cap serum tubes (0.5 
ml/tube) and stored in a liquid nitrogen freezer. 
EXAMPLE 2 
To each of twelve 6 ml glass vials there are added 1 ml of purified Dane 
particles prepared as described in Example 1, 1 ml of a 1% (v/v) solution 
of 2-mercaptoethanol in deionized water, and 1 ml of a 1% (v/v) solution 
in deionized water of the materials listed below. The mixtures are 
agitated gently and placed in a 37.degree. water bath. After 1 hour the 
resulting mixtures are assayed for HB.sub.c Ag by the immune adherence 
hemagglutination assay. 
______________________________________ 
Nonionic Surfactant IAHA Units 
______________________________________ 
polyoxyethylene (20) sorbitan monooleate 
1000 
polyoxyethylene (20) sorbitan monolaurate 
1000 
polyoxyethylene (23) lauryl ether 
1000 
polyoxyethylene (20) cetyl ether 
1000 
alkyl phenoxypolyethoxy (30) ethanol 
1000 
alkyl phenoxypolyethoxy (9) ethanol 
240 
alkyl phenoxypolyethoxy (40) ethanol 
240 
polyoxyethylene (9) octaphenol 
60 
sorbitan monolaurate 30 
sorbitan monostearate 30 
sodium dodecyl sulfate 30 
polyalkylaryl sulfonic acid 
30 
______________________________________ 
The foregoing results show that nonionic surfactants containing 9 or 40 
oxyethylene units are significantly inferior to those containing from 20 
to 30 oxyethylene units while those without any oxyethylene units are 
almost totally ineffective. 
EXAMPLE 3 
To a first 1 ml sample of purified Dane particles from the same lot used in 
Example 2 there are added 1 ml of polyoxyethylene (20) sorbitan 
monooleate, and 1 ml of 2-mercaptoethanol. A second 1 ml sample is treated 
similarly except substituting 1 ml of deionized water for the 
2-mercaptoethanol. Each sample is mixed, incubated for 1 hour at 
37.degree. and assayed for HB.sub.c Ag by the immune adherence 
hemagglutination assay. The second sample without 2-mercaptoethanol is 
found to contain less than half the amount of HB.sub.c Ag in the first 
sample. 
EXAMPLE 4 
HB.sub.c Ag as prepared in Example 1 is adsorbed on alum as follows. Ten ml 
of HB.sub.c Ag (Type Ad) containing 16-32 IA units/ml are mixed with 0.85 
ml of 10% alum solution KAl (SO.sub.4).sub.2.12H.sub.2 O. While stirring 
0.1 N NaOH is added slowly to adjust the pH to 6.8. Mixing is continued 
for 1 hour at room temperature. The solution is then centrifuged at 1500 
.times. g for 10 minutes. The supernate is decanted and the pellet is 
resuspended with saline solution to the original volume (10 mls). The 
solution is then mixed for 5-10 minutes prior to use as an antigen. 
EXAMPLE 5 
The procedure of Example 4 is employed except using 10 ml of HB.sub.c Ag 
(Type Ay). 
EXAMPLE 6 
Alum antigens prepared in Examples 4 and 5 are used to make high titer 
HB.sub.c Ab serums. The guinea pigs are divided into two groups which are 
used to produce the HB.sub.c Ab antiserum. The first group is administered 
intramuscular injections of 3 doses of 0.5 ml at monthly intervals of the 
product of Example 4. The second group is treated similarly with the 
product of Example 5. High titered hepatitis B antibody serums are 
produced in each group. 
EXAMPLE 7 
Enzyme-linked immunosorbant assay (ELISA) 
The HB.sub.c Ag obtained in Example 1 is purified by centrifugation on a 
20-60% sucrose gradient at 200,000 .times. g for 2.5 hours. The HB.sub.c 
Ag is then assayed to determine protein content. 
The HB.sub.c Ag is diluted to 1 .mu.g/ml with 0.1M carbonate buffer pH 9.7 
for use in the ELISA assay. 
The solid phase used in the assay is a 96 well, Cooke microtiter, U-bottom, 
polystyrene plate. 
The enzyme-conjugate is alkaline phosphatase conjugated goat anti-human 
immunoglobulin (Engvall et al.)..sup.1 . Use level is determined by 
titration. 
FNT .sup.1 The Journal of Immunology 109, 129 (1972) 
The enzyme substrate is 0.01% p-nitrophenyl phosphate in 0.1M carbonate 
buffer pH 9.8, containing 0.001M MgCl.sub.2. 
The assay method is described by E. Nassau et al..sup.2 This method is used 
to detect HB.sub.c Ab in plasma and serum samples. 
FNT .sup.2 Tubercle 57, 67-70 (1976) 
EXAMPLE 8 
The final product of Example 1 is treated under aseptic conditions with 
1:4000 formalin at 37.degree. C. for 72 hours. Excess formalin is then 
neutralized with sodium bisulfite. The core antigen is then adsorbed on 
alum by following the procedure of Example 4. 
Individuals positive for HB.sub.c Ab and having an HB.sub.c Ab antibody 
titer (as measured by the immune adherence hemagglutination assay, IAHA) 
of 32 IAHA units/ml or greater are administered 1 ml (40 .mu.g) doses of 
vaccine intramuscularly. Additional injections are given 1 month and 3 
months following the first injection. One week after the third injection 
the individuals are plasmapheresed and HB.sub.c Ab titers are run on the 
individual plasma using the immune adherence hemagglutination assay. The 
majority of the individuals experience an increase in their HB.sub.c Ab 
titer compared to their initial titer. Those plasmas having an antibody 
titer of 2000 or higher are processed to yield gamma globulin having high 
HB.sub.c Ab titer. 
EXAMPLE 9 
The material from part B of Example 1, 1 ml, is added to 1 ml of a (v/v) 
solution of 2-mercaptoethanol in deionized water, and 1 ml of a 1% (v/v) 
solution of polyoxyethylene (20) sorbitan monooleate in deionized water. 
The resulting mixture is agitated gently and placed in a 37.degree. C. 
water bath. After 1 hour the mixture is diluted with SPGA using a 
previously calculated quantity of diluent until it contains 32 IAHA units 
per ml. The solution is then dispensed into plastic 2 ml screw-cap serum 
tubes (0.5 ml/tube) and stored in a liquid nitrogen freezer.