Note: Descriptions are shown in the official language in which they were submitted.
~7388~
The invention relate~ to a recombinant deoxyribonucleic
acid (DNA) which contains the genetic code for an antigenic
protein from herpes viruse~, a cell which contains that
recombinant deoxyribonucleic acid and produces this antigenic
protein, the use of that recombinant deoxyribonucleic acid or
this cell for the preparation of this protein and processes for
their preparation, and to the use of this antigenic protein.
A protein which causes formation of antibodies against
herpes viruses is of great interest for the preparation of
vaccines for the prophylaxis and therapy of diseases caused by
these viruses.
Investigations carried out recently give rise to the
as~umption that certain types of tumor may be caused by herpes
viruses (HV). A requirement of a vaccine, for example against
herpes simplex virus type I (HSV-l) or type II (HSV-2) is
therefore that this vaccine must be absolutely free from HSV-DNA
(deoxyribonucleic acid)~ or that the viral DNA must be
inactivated, in order to exclude possible transformations.
The conventional preparation of a vaccine corresponding
to these requirements would necessitate expensive purification
steps.
Some glycoproteins which are integrated in the envelope
of the virus (so-called envelope proteins) and to which it has
been possible to allocate genetic loci on the HSV-DNA have so far
been described as antigenic components of HSV.
There was therefore the ob~ect of preparing a herpes
antigen which is free from the nucleic acid of herpes viruses and
$8 suitable for th0 preparation of a vaccine against diseases
caused by herpes viruses.
This ob~ect has now been achieved by isolating a genome
section (DNA) from a herpes simplex virus and transferring this
to a microorganism in which this DNA iS replicated and expressed.
Cloning of the gene section, which provides the code for the
glycoprotein C (gC) is described in the following text. The
process can also be carried out analogously for the other
envelope proteins gA, gB, gD and gE, which have already been
described, and for any other viral protein.
'
. - .
i ~ 73 88
- 2 -
European Patent Application 0,013,828 published August
6, 1980 described a process for the preparation of a polypeptide
with the antigenic properties of hepatitis B virus, in which a
DNA sequence from the DNA of the virus which provides the code
for an antigenic determinant of the virus is bonded to ~he DNA
of a so-called "cloning vehiclel~ or 'vector , and a host cell is
invaded with this combined deoxyribonucleic acid 80 that it
produces the polypeptide with the antigenic properties of
hepatitis B viru~.
A process suitable for achieving the stated object is
described in the following text. However, variants of this
process can also be used for the purpose according to the
invention, for example those with other vector/host cell
combinations.
1. Obtaining the virus-specific nucleic acid
1.1 Choice of the HSV strain
Infections with high infection doses lead in many virus
systems to the formation of defective virus genomes. To avoid
such artifacts, a cloned virus strain with which host cells, for
example vero cells, were infected in low multiplicity was
therefore used. We used HSV-1 strain F. The procedure would be
the same if other strains were used (for example cos or
McIntyre).
1.2 Isolstion of the HSV-DNA
To avoid the losses arising on purification of a virus,
the viral DNA was obtained directly from the cell lysate.
Centrifugation in a high-resolution density gradient (KI, NaI)
wa~ used for this; this centrifugation permits substantial
removal of the cell DNA from the viral DNA, the cell DNA having
a significantly different flotation density. The addition of
intercalating dyestuffs, for example ethidium bromide, permitted
direct visual monitoring of the separation result and the
isolation of the viral DNA (Walbromers and Scheggat, Virology
(1976) 74, 256-258).
35 2. Isolation of a gsnome section which provides the code
for an antigenic protein from the virus envelope.
~'
.: .
': '
.
~73~38~
-- 3 --
HSVl-DNA was hydrolyzed with restriction endonucleases
and the DNA fragments were separated electrophoretically in an
agarose gel. The nuclease ~indIII is suitable for this, since,
after digestion of the DNA with this enzyme, the complete
information for gC is localized on a specific fragment (HindIII-
L fragment). The HindIII-L fragment (DNA sections: 0.592 -
0.647 about 8,200 bp, compare also Figure 1) was extracted from
the agarose gel (J. Langridge et al., Analyt. Biochem. 103, 264-
271 (1980). The DNA can also be isolated by methods such as
tho3e described by H.O. Smith (Meth. Enzymol. 65, 371-380
(1980)).
3. Preparation of a plasmid which has the HSV-DNA HindIII-
L section, and transformation of bacteria ~ith this
recomb~nant plasmid.
The HindIII-L fraqment was ligated (F. Bolivar and K.
Backman, Meth. Enzymol. 65, 245 (1980) (Fig. 1)) in the
tetracycline-gene (Tet-gene) of the plasmid pBR 322 (F. Bolivar
et al., Gene 2, 95 (1977)) and transformed into Escherichia coli
strain HB 101.
In addition to plasmid pBR322, it is also possible to
use other suitable vectors, such as, for example, pBR327, pBR328
or pBR329 (L. Covarrubias and F. Bolivar, Gene 17, 79 - 89
(1982)) or pUC7, pUC8 or pUC 5 (J. Messing, Recombinant DNA, A.G.
Walton, ed. 143-153 (1982), Elsevier Scientific Publishing Comp.,
Amsterdam) or the plasmid pUR 222 (U. R~ther et al., Nucl. Acids
Res. 9, 4087 - 4098 (1981)).
Besides the strain HB 101, other strains of E. coli,
such as C600 (B. Bachmann, Bacteriol. Rev. 36, 525 - 557 (1972)),
RRl, SF8 or SX1592 are transformable by similar methods to that
used by ourselves ~S.L. Peacock et al., Biochim. Biophys. Acta
655, 243-250, (1981); and M.G.M. Brown et al., FENS Microbiol.
Lett. 5, 219 - 222 (1979)).
For example, the following vectors could be used in
transformation of eucaryotic cellss bovine papilloma virus DNA
35 (N., Sarver et al., Nol. Cell. Biol. 1, 486 to 496 (1981); SV40
(J.T. Elder et al., Ann. Rev. Genetics 15, 295 - 340 (1981)) or
:
. ~i
- : , ' . : -
1;~73~4
chimeric plasmids, such as pSG ~R.N. Sendai-Goldin et al., Mol.
Cell. Biol. I, 743-752 (1981)).
4. Selection of suitable clones
The cells transformed with recombinant plasmids were
streaked out on Petri dishes with L broth as a source of
nutrients, with addition of Na ampicillin (50 mg/liters) and were
incubated at 37C for 1 day. Clones which proved to be resistant
to ampicillin and sensitive to tetracycline were hybridized
against radioactive HSV-1 HindIII-L fragment (E. Southern, ~eth.
Enzymol. 65, 152 - 176 (1980)).
Positive clones were bred in 5.0 ml portions each of
liquid culture and the DNA was isolated (H.M. Goodman and R.J.
MacDonald, Meth. Enzymol. 65, 75 - 90 (1980); and D.S. Holmes and
M. Quigley, Anal. Biochem. 114, 193 - 197 (1981)).
Clones which had integrated the HindIII-L fragment into
the Hind III position of pBR322 were unambiguously identified by
digestion of the recombined plasmids with the endonucleases Hind
III, ~am HI, Sal I, Pvu I, Pvu II and Mst II and comparison with
the restriction card of HSV-I strain COS. Orientation of the L-
fragment and the gC-mRNA in pNB HSV 9 is shown in Figure 1.
5. Shortening of the HindIII-L fragment and subcloning of
the fragment.
gC is a late (r) protein, the mRNA of which is localized
on the right section of the HindIII-L fragment (Fink et al., J.
Virol. in pres~, 1983). Since gC-mRNA, which provides the code
of the peptide part of gC, is probably not spliced, the genomic
information can be expressed in procaryotes. It is worth
endeavoring to remove the unnecessary DNA portions of the
HindIII-L fragment for the synthesis of gC. This can be
achieved, inter alia, by digestion of the DNA with suitable
endonucleases. ~y digestion of the Hind III-L fragment upstream
from the translation start with the exonuclease Bal 31 up to the
translation start and bonding of the shortened L-fragment behind
an efficient promoter (such as, for example, a Trp-, Lac- or Tac-
promoter), a high rate of ~ynthesis of the gC-polypeptide can be
achieved in procaryotic systems (Figure 2).
- .
,- ~ , .
73~
5 _
In order to be able to induce antibody production, not
all the protein qC is necessarily required, but only a part of
the protein carrying the antigenic determinants. Cloned DNA
fragments in which degradation has been effected in the coded
region of the gC with exonucleases, such as Bal 31, or sub-
fragments of the HindIII-L fragment, for example the Eco-Hind I-
L fragment, can be used for the production of such polypeptides.
Bonding of the Eco-Hind I -L fragment in the Eco RI
position behind the lac W 5 promoter in pUC 8 permits synthesis
of a fusion protein with the first NH2-terminating amino acids of
~-galactosidase and most of the gC-polypeptide (Figure 3).
Cloning and expression of antigenic glycoproteins of HSV
other than gC can be carried out analogously to the procedures
listed here.
If the corresponding mRNAs are spliced, the
complementary DNA (cDNA) can be used for expression of the
glycoproteins, in which case small cloned DNA fragments, such as
the Eco-Hind I-L fragment, to which only the gCmRNA of HSV-l
bonds, can be used for selecting the corresponding mRNAs from a
lysate of cells infected with HSV. However, cloning of the
genomic DNA is also conceivable in such cases, since the
corresponding gene product formed in procaryotes can, in spite
of the absence of splicing mechanisms here, be a protein which
carries some or all of the antigenic determinants. Linking of
DNA fragments from different genes of only one or of two
different types of herpes viruses and expression of such a
hydrbid protein is even possible.
Expression in eucaryotic cells (yeast, animal cell
cultures) is also possible, especially if glycosilation of the
protein is desirable.
Because of its antigenic properties, such a protein is
suitable for isolating antisera and/or vaccines, if necessary
using ad~uvants and conventional auxiliaries.
It can also be used for diagnostic purposes, for example
for the detection of antibodies against herpes viruses.
Antisera against such a protein can themselves be used
for detection of herpes viruses.
:
:' ,
'
