Simian virus recombinant that directs the synthesis of hepatitis B surface antigen

A process for producing a recombinant between simian virus 40 (SV40) and hepatitis B virus (HBV) is given. When tissue culture cells are infected with the recombinant, hepatitis B surface antigen is produced. Because a single cloned gene is used, the surface antigen produced is homogeneous and can be produced without conventional dependence on human sera. The antigen is excreted into the culture medium as 22 nm particles with the same physical properties, antigenic composition and constituent polypeptides as those found in the sera of patients with Type B hepatitis. The antigen is useful for the preparation of vaccines.

STATEMENT OF PRIOR ART 
DuBois et al, "Excretion of hepatitis B surface antigen particles from 
mouse cells transformed with cloned viral DNA," Proc. Natl. Acad. Sci., 
Vol. 77, No. 8, pp. 4549-4553, August, 1980. The surface antigen is 
excreted into the cell culture medium when a plasmid with two cloned 
hepatitis B viral genomes is introduced into mouse cells using 
cotransformation. No SV40 vector is used in this procedure. Moreover, in 
these experiments HBsAg expression depended upon the presence of two 
complete, tandem copies of the HBV genome rather than upon a defined 
subfragment. 
Charnay et al, "Biosynthesis of hepatitis B virus surface antigen in E. 
coli," Nature, Vol. 286, August, 1980. This hybrid antigen is neither 
glycosylated, assembled into particles, nor excreted from the bacterial 
host. 
Conventional preparations of hepatitis B surface antigen are given, for 
example, in U.S. Pat. Nos. 4,113,712; 4,118,479; 4,138,287 and 4,186,193. 
STATEMENT OF DEPOSIT 
SV40 vector cleaved with BamHI at 0.14 map units and EcoRI at 0.00 map 
units and designated Y182 has been filed with the American Type Culture 
Collection and received ATCC Number 31964. 
BACKGROUND 
At least half of the world population shows evidence for past or present 
infection by hepatitis B virus (HBV), and the approximately 200 million 
carriers in the world today are at serious risk to chronic liver disease 
and, possibly, primary liver cancer. The classic marker for chronic 
infection by this virus is the surface antigen, HBsAg, which circulates in 
the serum of HBV carriers in three morphological forms: 22 nm spherical 
particles, 22 nm filaments of various lengths and the 42 nm spherical form 
known as the Dane particle. The 22 nm particles and filaments are subviral 
forms containing two predominant polypeptides, with apparent molecular 
weights of about 23,000 and 29,000, together with several minor 
polypeptides of larger size. The two predominant species, which are 
probably identical except that the larger is glycosylated, carry both the 
group (a) and the subtype (d/y) antigenic determinants of HBsAg. The Dane 
particle, which represents the infectious virion, consists of a 
lipoprotein coat (HBsAg) surrounding an internal core particle which 
contains a DNA polymerase and the 3200 base pair (bp) circular DNA genome. 
The 22 nm particle is the predominant form in the sera of chronic 
carriers and circulates at concentrations as high as 100-200 .mu.g/ml. 
Characterization of the life cycle and biology of HBV has been hampered by 
its narrow host range, which is restricted to humans and a few other 
primates, and by its inability to grow in cultured cells. Problems 
associated with conventional purification and isolation from human serum 
are discussed, for example, in U.S. Pat. Nos. 4,113,712; 4,138,287 and 
4,186,193. 
Recently, however, several groups have succeeded in cloning the viral 
genome in Escherichia coli phage lambda (Charnay et al, Proc. Natl. Acad. 
Sci. USA 76, 2222-2226, 1979) and plasmid vector (Burrell et al, Nature 
279, 43-47, 1979, and Sninsky et al, Nature 279, 346-348, 1979) and in 
determining its primary structure (Valenzuela et al, Nature 280, 815-819, 
1979; Pasek et al, Nature 282, 575-579, 1979; and Galibert et al, Nature 
281, 646-650, 1979). This has allowed the identification of a continuous 
892 bp sequence that could encode surface antigen, a 549 bp sequence that 
may specify the core antigen and several additional open sequences of 
unknown function. 
Although the DNA sequence provides crucial structural information, it 
clearly is not sufficient to establish all of the HBV gene products nor to 
understand how these products interact during infection of the target 
cell. For this purpose it would be useful to develop a system for 
introducing defined portions of the viral genome into cultured cells. 
Simian virus 40 (SV40), a small DNA tumor virus that can lytically infect 
cultured monkey cells, provides a useful vector for this purpose. The 
present invention provides for the construction and propagation of an SV40 
recombinant carrying a 1350 bp fragment of HBV DNA that includes the 
structural sequences for surface antigen. Monkey kidney cells infected 
with this recombinant synthesize surface antigen that is excreted into the 
culture media as 22 nm particles. These results set an upper limit on the 
amount of HBV genetic information required for 22 nm particle formation 
and demonstrate the feasibility of using SV40 recombinants to study HBV 
gene expression in cultured primate cells. Ultimately, the antigen is 
useful in the preparation of vaccines, as a standard antigen reagent for 
testing hepatitis B surface antigen and antibody and as an antigen for 
immunizing animals to obtain a highly specific and strong antibody. 
Because the antigen is derived from a single cloned gene, it is 
homogeneous. Moreover, this unique process is free of contaminating serum 
components that conventional human serum processing cannot avoid. 
SUMMARY OF THE INVENTION 
Hepatitis B surface antigen is produced by amplifying the hepatitis B virus 
genome containing the hepatitis B surface antigen coding sequence by 
cloning the HBV genome in a plasmid, preparing an SV40 vector and ligating 
the HBV genome to the SV40 vector plasmid to produce a double recombinant 
HBV-SV40 plasmid, and transfecting the double recombinant in monkey kidney 
cells with helper virus and thereby producing hepatitis B surface antigen. 
An SV40-HBV recombinant is constructed that retains about 70% of the SV40 
genome and 40% of the HBV genome. This hybrid virus is replicated and 
packaged into SV40 virions in cultured monkey kidney cells, the permissive 
host for SV40, that have been coinfected with a complementing SV40 helper 
virus. The expression of the inserted HBV sequences was examined by both 
immunological and biochemical techniques. This showed that the recombinant 
directs the synthesis of surface antigen but no other known HBV-specific 
antigens. Furthermore, blocking assays and immunoprecipitation with 
monospecific antibodies demonstrated that the SVHBV (complex mixture of 
virus)-derived HBsAg had the same subtype (ad) as the antigen from the 
original donor of the HBV DNA. These observations demonstrate that both 
group and subtype-specific determinants of HBsAg are encoded within a 
limited portion of the viral genome. 
The HBsAg encoded by SVHBV is excreted into the culture medium as 22 nm 
particles with the same buoyant density, sedimentation properties and 
morphology as the particles from human serum. These particles are produced 
at a rate of 2.5 .mu.g/10.sup.7 cells/2 days or approximately 
3.times.10.sup.4 particles/infected cell/day. This compares favorably with 
HBsAg production by the Alexander cell line derived from a human 
hepatocellular carcinoma. It is concluded that monkey kidney cells possess 
all of the functions required for particle formation and that the failure 
of HBV to grow in tissue culture is not due to a block at this stage of 
the viral life cycle. 
The ability to propagate defined portions of the HBV genome in cultured 
primate cells raises several interesting experimental opportunities. In 
addition to the obvious possibility of vaccine production, it should be 
feasible to construct viruses that encode useful diagnostic reagents such 
as surface antigen peptides bearing single, highly specific immunological 
determinants. SV40-HBV hybrids might also be useful for identifying new 
HBV gene products; e.g., non-structural proteins that are not excreted 
into the serum. Finally, the availability of SV40-HBV recombinants 
provides an experimental system to investigate various mechanisms for the 
persistence and pathogenicity of HBV in liver cells.

DETAILS OF INVENTION 
Materials and Methods: 
The following methods have been described: general procedures for the 
construction of recombinant plasmids and viruses (Hamer, D. H. (1980) in 
Genetic Engineering, eds. Setlow & Hollander, Plenum Publishing Corp., New 
York, pp. 83-107); growth of African green monkey kidney cells and 
propagation of virus stocks (Hamer et al (1977), J. Mol. Biol., 112, 
155-182); preparation of plasmid (Clewell et al (1969), Proc. Natl. Acad. 
Sci. USA, 62, 1159-1166); and intracellular SV40 DNA (Hirt, J. Mol. Biol., 
26, 365-369); analysis of DNA by restriction endonuclease cleavage (Hamer 
et al (1977), J. Mol. Biol., 112, 155-182) and agarose gel electrophoresis 
(Hayward et al (1972), J. Mol. Biol, 63, 383-395, and Cummings et al 
(1980), Proc. Natl. Acad. Sci. USA, 77, 1842-1846); and transformation of 
EK2 E. coli strain HB 101 (Cohen et al (1972), Proc. Natl. Acad. Sci. USA, 
69, 2110-2114). 
Restriction endonucleases and T4 DNA ligase were purchased from Bethesda 
Research Laboratories (Bethesda, MD) and reaction conditions were 
according to the supplier. 
The source of HBV DNA was plasma, subtype adw, from an HBsAg-positive 
donor. Dane particles were purified by the method of Robinson (Robinson 
(1975), Am. J. Med. Sci., 270, 151-159), incubated in the endogenous DNA 
polymerase reaction (Kaplan et al (1973), J. Virol., 12, 995-1005) with 
all four deoxynucleotide triphosphates prior to DNA extraction. 
The 22 nm form of HBsAg was purified from the plasma of chronic carriers as 
described previously (Gerin et al (1975), J. Immunol. 115, 100-105). 
Hyperimmune guinea pig antiserum to HBsAg/ad was that supplied by the 
Research Resources Branch, NIAID (#V801-502-058) and monospecific 
antibodies to the HBs/a and HBs/d determinants were prepared from this 
serum by affinity chromatography (Shih et al (1978) J. Immunol., 120, 
520-525). Fluorescein isothiocyanate conjugated rabbit anti-guinea pig IgG 
was obtained from Cappel Laboratories (Cockranville, PA). 
Radioimmunoassays for hepatitis B core antigen (Purcell et al (1973/1974) 
Intervirology, 2, 231-243) delta antigen (Rizzetto et al (1980), J. 
Immunol., 125, 318-324) and e antigen (HBeAg test kit, Abbott Laboratories 
(N. Chicago, IL) have been described. HBsAg was detected by the Ausria II 
radioimmunoassay (Abbott Laboratories) and quantitated by a parallel-line 
assay using a known standard (BoB HBsAg/adw vaccine, Reference Lot 1, 40 
.mu.g/ml). The d/y subtype of HBsAg was determined by the competition 
radioimmunoassay method of Hoofnagle (Hoofnagle et al, Gastroenterology, 
72, 290-296). 
Construction and Progapation of the SV40-HBV Recombinant 
The SV40-HBV recombinant described here carried a 1350 bp fragment of HBV 
DNA, representing about 40% of the HBV genome, inserted into the late gene 
region of SV40. The first step in the construction of this recombinant was 
to amplify the HBV genome by cloning it in an E. coli plasmid vector. Dane 
particles were purified from the serum of a chronic HBsAg carrier, subtype 
adw, and the partially single-stranded viral genome was repaired by an 
endogenous DNA polymerase reaction. Two fragments of sizes 1350 and 1850 
bp were obtained after cleavage of this DNA with Bam HI. Partial digestion 
with Bam HI generated a full HBV genome which was ligated to Bam 
HI-cleaved plasmid pBR322 DNA then cloned in E. coli. 
From the published sequence data, it is anticipated that the HBsAg coding 
sequence would be located within the 1350 bp Bam HI fragment. This 
fragment was purified, ligated to a Bam HI-cleaved pBR322-SV40 vector 
plasmid and cloned in E. coli. 
The SV40 vector is SV40 DNA cleaved with Bam HI at 0.14 map units and Eco 
RI at 0.00 map units. The fragment extending clockwise from the Bam site 
to the EcoRI site is cloned into plasmid pBR322 between a single Bam site 
and a single EcoRI site. Map units and orientation are based on the 
standard SV40 map where the EcoRI site is designated as map position 0. 
The direction clockwise is the same direction as late transcription. 
Details of other recombinant DNA and SV40 genomes which are cloned and 
propagated in bacteria are given in copending application Ser. No. 309,110 
filed Oct. 6, 1981. The advantages of this method are given and the 
expression of other proteins by the recombinant in eukaryotic cells by 
transformation or transfection is illustrated. 
Digestion of the resultant pBR322-SV40-HBV "double recombinant" plasmid 
with Hae II removed all but 143 bp of the pBR322 DNA and yielded a 
homogenous preparation of 4950 bp SV40-HBV linear recombinant molecules. 
These molecules retain the SV40 origin of DNA replication and the complete 
SV40 early gene region but lack most of the SV40 late gene region and 
hence are defective. Nevertheless, they could be packaged into SV40 coats 
and propagated as virions by making a mixed DNA infection of monkey kidney 
cells with an SV40 temperature-sensitive early gene mutant 
(SV40tsA.sub.239) as helper. This mixed infection was performed at the 
non-permissive temperature (39.degree. C.) to insure that progeny virions 
would be produced only by cells doubly infected with the SV40-HBV 
recombinant, which supplies functional SV40 early gene products, and with 
the helper, which supplies all of the required SV40 late gene products. 
To determine if the SV40-HBV recombinant was encapsidated into SV40 
virions, it was acknowledged that only those genomes incorporated into 
viral particles during the original DNA infection would be transferred and 
replicated in a subsequent viral infection. Accordingly, a fresh culture 
of monkey cells was infected with the virus stock from the DNA infection, 
intracellular viral DNA was prepared three days later and examined by 
restriction endonuclease cleavage and agarose gel electrophoresis. This 
showed that the stock contained approximately 75% helper genomes and 5% 
SV40-HBV recombinant genomes retaining the complete 1350 bp HBV fragment. 
The remaining 20% of the DNA was found in a heterogenous collection of 
genomes with lengths ranging from about 3000 to 4900 base pairs. Although 
the structure of these molecules were not examined in detail, they may 
have arisen due to illegitimate intramolecular recombination of the linear 
DNA used for infection. This complex mixture of virus is referred to as 
SVHBV. 
Monkey Kidney Cells Infected with SVHBV Synthesize HBsAg 
Specific immunological assays were used to show that monkey kidney cells 
infected with SVHBV synthesized HBsAg but no other established HBV 
antigens. Immunofluorescence analysis revealed that approximately 45% of 
the cells infected with SVHBV expressed cytoplasmic HBsAg by 72 hours post 
infection, whereas uninfected and wild-type SV40 infected controls were 
negative. Quantitative radioimmunoassays showed that a culture of 
2.times.10.sup.7 cells produced a total of 2.5 .mu.g of HBsAg. Of this, 
40% was found in the medium and 60% was released from the cells by 
freeze-thawing and sonication. Subtype analysis showed that SVHBV-HBsAg 
had the same antigenic composition (d+, y-) as the antigen from the 
original donor of the HBV DNA. SVHBV infected cells were negative for 
HBcAg and .delta. antigen by immunofluorescence. The culture medium was 
negative (less than 2.1 P/N) for HBcAg, HBeAg and .delta. antigen by solid 
phase radioimmunoassay. 
The HBsAg is Secreted from Monkey Kidney Cells as a Particle 
SVHBV-HBsAg found in the tissue culture fluid is a 22 nm particle whose 
physical characteristics are those of the particles found in human serum. 
Isopycnic banding in CsCl of SVHBV-HBsAg revealed a buoyant density value 
(1.2 g/cm.sup.3) identical to purified HBsAg run in parallel. The 
SVHBV-HBsAg material was further characterized by rate zonal 
centrifugation. Most of the SVHBV-specific antigen sedimented as a 
particle indistinguishable from the 22 nm particles found in human era. 
The morphology of the antigen was examined by electron microscopy of the 
pooled, concentrated peak fractions from the sucrose gradient. This 
revealed 22 nm spherical particles with the same appearance as the 
predominant form of HBsAg from human sera. Examination of the less highly 
purified material from the CsCl gradient also showed 22 nm filaments of 
variable lengths. No Dane particles were observed. 
HBsAg Polypeptides 
The polypeptide composition of the excreted SVHBV-HBsAg was analyzed by 
immunoprecipitation and gel electrophoresis of both chemically and 
biosynthetically labelled antigen. SVHBV-HBsAg from the rate sedimentation 
step was radioiodinated, resedimented, and immunoprecipitated by anti-HBs 
serum and antibodies against the group (a) and d-subtype specific 
determinants (Table 1 ). 
TABLE 1 
______________________________________ 
Immunoprecipitation of .sup.125 I-HBsAg from SVHBV- 
Infected Cells 
% Precipitation by Antiserum 
Sample Pre 
HBs/ad 
HBs/a 
HBs/d 
______________________________________ 
Serum HBsAg/ad 
1.2 83.5 76.6 82.8 
SVHBV-HBsAg 5.9 64.1 62.2 59.1 
______________________________________ 
The peak fractions, 300 .mu.l each, were pooled and concentrated 15-fold by 
pelleting. The concentrate was iodinated by the chloramine-T procedure and 
further purified by rate zonal centrifugatio in sucrose. A standard 
HBsAg/ad preparation isolated from human serum was iodinated and purified 
in parallel. Radioiodinated SVHBV-HBsAg and HBsAg/ad, 100 .mu.l each, were 
incubated with 10 .mu.l of guinea pig antisera at room temperature for 2 
hr and precipitated with rabbit anti-guinea pig IgG, 140 .mu.l, in 0.05% 
Tween 20/PBS. After further incubation at 4.degree. C. for 18 hr, the 
mixture was centrifuged at 2300 xg for 30 min. and the precipitates were 
washed twice with 30 .mu.l Tween-20/PBS. The supernatants were pooled and 
the percent precipitation was calculated. 
Greater than 60% of the iodinated antigen was precipitated by anti-HBs and 
by both of the monospecific antibodies. SDS-gel electrophoresis of the 
denatured and reduced precipitates revealed two predominant polypeptides 
(P-1 and P-2) with the same mobilities as those isolated from human serum. 
These proteins are thought to be identical except that P2 is glycosylated. 
In addition, the iodinated SVHBV-HBsAg contained at least one of the minor 
polypeptides of higher molecular weight (P-5). 
In a companion experiment, SVHBV-specific proteins were biosynthetically 
labelled by incubating the infected cells with .sup.35 S-methionine for 4 
hours late in the lytic cycle. Direct examination of the medium from SVHBV 
infected cells revealed two proteins with the same mobilities as P-1 and 
P-2, both of which were precipitated by anti-HBs serum. In contrast, no 
such proteins were found in the media from uninfected or wildtype SV40 
infected cells. This experiment also demonstrated that HBsAg is actively 
excreted rather than merely released by cell lysis because only a 
restricted subpopulation of the total cell proteins was found in the 
media.