Journal of General Virology (1990), 71, 2185-2190.
Printed in Great Britain
Increased antibody responses to human papiHomavirus type 16 L1 protein
expressed by recombinant vaccinia virus lacking serine protease inhibitor
genes
Jian Zhou,1 Lionel Crawford, 1. Lia McLean, 2 Xiao-yi Sun, 1 Margaret Stanley, 2 Nell Almond 1f
and Geoffrey L. Smith2:~
I l C R F Tumour Virus Group, Department of Pathology and 2Department of Pathology, University of Cambridge,
Tennis Court Road, Cambridge CB2 IQP, U.K.
The L1 gene of human papillomavirus type 16 (HPV-
16) driven by the vaccinia virus major late 4b gene
promoter has been inserted into three different sites of
the vaccinia virus genome. Insertion into the thymidine
kinase (TK) gene was achieved by selection of TK-
mutants in BUdR on TK- cells. Insertion into two
vaccinia virus serine protease inhibitor (serpin) genes
was achieved by co-insertion of the Escherichia coli
xanthine guanine phosphoribosyltransferase gene
linked to the vaccinia virus 7-5K promoter and
selection of mycophenolic acid-resistant recombinant
A major problem with research with human papilloma-
viruses (HPV), particularly type 16 (Diirst et al., 1985), is
the lack of any system for producing virus particles in
quantity. Virion proteins, such as the major coat protein
L1, must therefore be produced in expression vectors.
For immunological purposes eukaryotic vectors are
preferable as they are more likely to generate proteins
with correct post-translational modifications including
phosphorylation and glycosylation and these may be
important for the recognition and processing of the
antigen. Vaccinia virus recombinants have been widely
used to express eukaryotic proteins including HPV
antigens (Mackett et al., 1984; Browne et al., 1988) and
have the advantage over some other eukaryotic vectors
in that infection of an animal with recombinant virus
presents the native protein to the immune system over an
extended period.
Expression of the HPV-16 L1 gene in vaccinia virus
poses conflicting requirements. Firstly, this gene con-
i" Present address: National Institute of Biological Standards and
Control, Blanche Lane, Potters Bar, Hertfordshire EN6 2QG, U.K.
:~ Present address: Sir William Dunn School of Pathology, Univer-
sity of Oxford, South Parks Road, Oxford OX1 3RE, U.K.
0000-9550 © 1990 SGM
2185
viruses. Each recombinant virus expressed a 57K L1
protein at similar levels and with similar kinetics.
However, immunization of mice with these recombin-
ant viruses induced different levels of antibody to the
L1 protein. Viruses lacking serpin genes B13R and
B24R induced significantly higher antibody levels than
did viruses lacking the TK gene. The presence of
functional B13R and B24R gene products is therefore
somehow immunosuppressive at least for antibody
responses to the L1 protein of HPV-16.
tains the sequence TTTTTNT twice within its coding
region (Seedorf et al., 1985). This sequence functions as a
termination signal for the vaccinia virus DNA-depen-
dent RNA polymerase early during infection (Rohr-
mann et al., 1986; Yuen & Moss, 1987) so that full-length
L1 transcripts would not be produced from exclusively
early vaccinia virus promoters. Consistent with this,
much greater levels of L1 expression were obtained from
the 4b (major late) rather than 7-5K (early and late)
vaccinia virus promoter (Browne et al., 1988). Secondly,
stimulation of T cell responses may be better if the
antigen is expressed from early rather than from late
vaccinia virus promoters, whereas B cell responses may
result from the use of either class of promoter (Coupar et
al., 1986). Early promoters may therefore be preferable if
stimulation of both humoral and cell-mediated immunity
is required.
The vaccinia virus thymidine kinase (TK) gene has
been a widely used site for insertion of foreign D N A
since this is both non-essential for virus replication and
enables genetic selection of T K - recombinant viruses.
However, T K - recombinants are severely attenuated
(Buller et al., 1985) so that the immune response evoked
against a foreign antigen may be reduced. We have
2186 Short communication

investigated the consequence of insertion of HPV-16 L1 C KF E 1 GJH D A B

J
into other sites in the virus genome and compared the
immunogenicity of recombinant viruses expressing this
,-~ /'x ~ ' N,,
Sail" / B 13R\ FB~4R
gene. The insertion sites chosen are the serine protease
inhibitor (serpin) genes B13R and B24R (Kotwal &
Moss, 1989; Smith et al., 1989) (Fig. 1). These genes are
expressed early during infection (Smith et al., 1989) and • Virus DNA
• Virus promoter
in cowpox virus the homologue of the vaccinia virus [ ] gpt gene
E. coligpt HPV-16 L1 • HPV ORF
B13R gene is responsible for a haemorrhagic pock B24R P7-5 P4b B24R
phenotype (Pickup et al., 1986) and influences the
inflammatory response (Palumbo et al., 1989; Chua et
al., 1990).
Recombinant vaccinia viruses which express the
HPV-16 L1 gene and which are defective for TK or
serpin genes B13R or B24R were constructed as follows.
The serpin coding regions were disrupted by the HPV L 1
7 B13R P7.5
pSX5

E. coli gpt HPV-16 L1

P4b B 13R

gene linked to the vaccinia virus strong late 4b promoter

(Rosel & Moss, 1985; Kent, 1988) and the Escherichia coli
xanthine guanine phosphoribosyltransferase (Ecogpt)
gene driven by the constitutively active 7.5K promoter
(Boyle & Coupar, 1988) (Fig. 1). Insertion of HPV-16 L1
into the TK gene was similar to that described (Browne
et al., 1988). The 2.5kb SfaNI-AvalI D N A fragment
from HPV-16-pAT153 (Dfirst et al., 1983), which starts
10 nucleotides upstream of the first ATG of the L1 open
reading frame and terminates 865 nucleotides down-
stream of the L1 TAA stop codon, was modified by
addition of BglII linkers and cloned into the BamHI site
of pRK19 (Kent, 1988) downstream of the 4b promoter
to form plasmid pRK19/16 L1. Plasmid pSX5 was
constructed by the insertion of a 1479 bp PvuII-SalI
fragment containing serpin B24R (derived from the
vaccinia virus Sail fragment) into SmaI- and SalI-
cut pUC9. The Ecogpt gene joined to the vaccinia
virus 7-5K promoter was isolated as "an EcoRI fragment
from plasmid pGpt07/14 (Boyle & Coupar, 1988), treated
with Klenow enzyme to create blunt ends and cloned into
the coding region of B24R at the unique KpnI site, which
had been treated with T4 DNA polymerase, to form
plasmid pSX3. An XbaI-SmaI fragment containing the
4b promoter linked to HPV L1 was then isolated from a
modified version of pRK19/16 L1, treated with Klenow
polymerase to create blunt ends and cloned into pSX3,
which had been cleaved with BamHI and treated with
Klenow enzyme, to form pSX5. Plasmid pLC19 was
constructed by cloning a 5652 bp EcoRI-SalI fragment
(derived from the vaccinia virus SalI G fragment) into
EcoRI- and Sa/I-cut pUC13. This plasmid, pSTH1,
contains most of serpin B 13R. The complete serpin gene
was re-formed by cleavage of the vaccinia virus SalI I
fragment with BglII, treatment with Klenow enzyme to
form blunt ends, cleavage with SalI, isolation of a 1221
bp fragment and insertion of this into pSTH1 that had
been cleaved with HindIII, treated with Klenow enzyme
pLCl9I ~' F
TK P4b HPV-16 L1 TK
pRK19/16 L1
Fig. 1. Upper panel, location of the serpin genes B13R and B24R and
the TK gene on the vaccinia virus genome. The serpin genes and TK
gene are in the HindIIl B and J fragments, respectively. The I-IindlII
map is shown on the top line and the SalI sites are indicated below. The
arrows indicate the direction of transcription. Lower panel, plasmid
vectors used to construct recombinant vaccinia viruses. Abbreviation:
ORF, open reading frame.
and digested with SalI. The plasmid formed, pGS124,
was digested with EcoRI and BglII, treated with Klenow
enzyme and the large fragment was isolated and self-
ligated to form pYC 15. pYC 15 was digested with HincII
and ligated with a 2-1 kb fragment containing the Ecogpt
gene joined to the vaccinia virus 7.5K promoter (isolated
as described above) to form pYC16, pYC16 was digested
with PstI, treated with T4 DNA polymerase and ligated
with the XbaI-SmaI fragment containing the HPV L1
gene joined to the 4b promoter (above) rendered blunt-
ended with Klenow enzyme.
Plasmids pLC19 and pSX5 were transfected into cells
infected with wild-type (wt) vaccinia virus strain WR
(Mackett et al., 1984). Recombinant viruses were
selected by plaque assay in the presence of mycophenolic
acid, xanthine and hypoxanthine (Falkner & Moss,
1988; Boyle & Coupar, 1988). For cloning of HPV L1
into the TK gene, plasmid pRK19/16 L1 was transfected
into wt-infected cells and T K - recombinant virus was
selected by plaque assay in the presence of BUdR on

human T K - 143 cells and screened for the presence of
HPV L1 D N A (Mackett et al., 1984). The recombinant
vaccinia viruses containing the HPV L1 gene inserted
into the TK, B 13R or B24R genes were termed p R K 19i 16
L1 vv, pLC19 vv and pSX5 vv, respectively. Stocks of
recombinant viruses were grown and titrated in CV-1
cells as described (Mackett et al., 1985a).
The genomes of these recombinant viruses were
analysed by restriction enzyme digestion, agarose gel
electrophoresis and Southern blotting (data not shown).
In the case of pRKI9/16 L1 vv, the 5 kb HindlII J
fragment containing the T K gene had increased in size to
7.7 kb (the predicted size with addition of the HPV L1
gene) and this band also hybridized with HPV L1 DNA.
For pSX5 vv, the Sail I fragment, containing serpin
B24R, had increased in size consistent with addition of
both the HPV L1 and Ecogpt genes. Serpin B13R crosses
the Sail G and Sail I junction so that after SalI digestion
of pLC19 vv the Sail G fragment had increased in size
due to insertion of L1, and the Sail I fragment had
increased in size due to insertion of the Ecogpt gene. In
summary, the viruses all had genome structures consis-
tent with the insertion of foreign D N A as predicted.
Expression of the L1 gene was analysed by Western
blot analysis using an anti-HPV L1 monoclonal antibody
Camvir 1 (McLean et al., 1990; Fig. 2). Lysates from cells
infected with wt or recombinant vaccinia viruses were
separated by electrophoresis on 10~ polyacrylamide gels
and the proteins blotted onto nitrocellulose filters. The
filters were incubated in 3~o bovine serum albumin
(BSA) in phosphate-buffered saline (PBS) at 37 °C for 1 h
and then with Camvir 1. After a 30 rain wash in PBS
containing 1 ~ NP40, the filter was incubated with anti-
mouse ~25I-labelled IgG, washed again, dried and
autoradiographed. In lysates from cells infected with
each of the three recombinant viruses there was a
polypeptide of 57000 Mr that was absent from lysates of
cells infected with wt virus. This polypeptide was of the
predicted size for HPV L1 and was synthesized at the
same level and with the same kinetics in each case. The
expression and nuclear location of the L1 protein in
recombinant virus-infected cells was also demonstrated
by immunofluorescence (data not shown).
The immunogenicity of the three recombinant viruses
was compared by immunization of groups of 10 female
BALB/c mice with 1 x 107 p.f.u, of virus by intraperito-
neal (i.p.) or subcutaneous (s.c.) injection (2 x 106 p.f.u.
s.c., 8 x 106 p.f.u.i.p.). Inoculations were repeated after
2 weeks and sera collected 7 days later and tested for
antibodies to H P V L1 by ELISA (Fig. 3). HPV16 LI
protein was produced in E. coli by expression vector
pKK223-3 (Pharmacia) and was kindly provided by Dr
D. H. Davis. One-hundred gl samples of diluted L1
protein (containing approximately 1 gg of protein per ml)
Short communication 2187
1 2 3 4
92K- -
30K--
Fig. 2. Western blot analysis of HPV-16 L1 expressedby recombinant
vaccinia viruses. CV-1 cellswere infectedat 10 p.f.u./cellwith wt (lane
1) or recombinantvaccinia viruses pRK19/16 L1 vv (lane 2), pSX5 vv
(lane 3) or pLCI9 vv (lane 4) and harvestedat 48 h p.i. The positionsof
Mr markers are shown on the left.
were placed in fiat-bottomed microtitre plate wells and
left overnight at 4 °C. After washing three times with
borate-buffered saline and blocking with 1 ~ BSA, the
antigen-fixed wells were reacted with test antiserum,
followed by peroxidase-linked sheep anti-mouse IgG.
The wells were then reacted with o-phenylenediamine
(800 gg/ml) plus H 2 0 : (0.025~o) and the absorbance of
the supernatants was measured by an ELISA Kinetic
Microplate Reader (Molecular Devices) at 490 nm. The
data are expressed as kinetic rates (mOD/min) corrected
for the control serum values. All mice immunized with
recombinant virus expressing HPV L1 showed antibody
responses against this antigen; sera from mice immu-
nized with wt virus remained at background level.
However, the antibody titres in the pLC19 vv and pSX5
vv groups were higher than in those receiving pRK19/16
LI vv (Fig. 3) and could be detected at 1:500 dilution.
These differences could have been due to serpin gene
inactivation. Alternatively, these different titres may be
due to virus attenuation resulting from loss of the T K
gene in virus pRK19/16 L1 vv. To address the latter
possibility, T K - virus (derived from wt virus by passage
in BUdR) was used for construction of additional serpin-
negative viruses. These T K - and serpin-negative viruses
induced similar levels of anti-HPV L1 antibody to their
T K + counterparts (Fig. 3) demonstrating that the
increased levels of antibodies are due to serpin gene
inactivation. Interestingly, antiserum induced by pLC 19
2188 Short communication
30--
T response of mice immunized with these different viruses.
= 20-
T T
©
T B24R genes (Pickup et al., 1986; Kotwal & Moss, 1989),
10-
t-~
Fig. 3. ELISA m e a s u r e m e n t of anti-HPV LI antibody levels from
vaccinated mice. From left to right the data are the m e a n of sera from
10 mice immunized with pRK19/16 L1 vv, pSX5 vv (B24R),
pSX5)TK- (B24R), pLC19 vv (B13R) or p L C 1 9 / T K - (B13R). The bar
on each column indicates the standard deviation of the m e a n value.
vv (B13R insertion) and pRK19/16 L1 vv (TK insertion)
recognized different epitopes of LI (D. H. Davies & M.
Wishart, personal communication).
Many recombinant vaccinia viruses have been used to
immunize animals and generate good immune responses
against a foreign gene product, for example the influenza
virus haemagglutinin (Smith et al., 1983b), hepatitis B
virus surface antigen (Smith et al., 1983a), herpes
simplex virus glycoprotein D (Paoletti et al., 1984;
Cremer et al., 1985) and the Epstein-Barr virus gp340
(Mackett & Arrand, 1985). In these experiments neutral-
izing antibodies were produced and experimental ani-
mals have subsequently been protected against challenge
with the appropriate viruses (Moss et al., 1984). Besides
the humoral immune response, cell-mediated reactions
were also found against the foreign antigens (Bennink et
al., 1984, 1986; Wiktor et al., 1984; McMichael et al.,
1986). The ability of recombinant vaccinia viruses to
stimulate both humoral and cell-mediated responses
against the expressed foreign antigens greatly enhances
the potential uses of the vaccinia viruses to prevent these
virus infections.
To attempt to increase the immunogenicity of vaccinia
virus recombinants we have inserted a foreign gene, in
this case the HPV LI gene, into different sites in the
vaccinia virus genome and compared the immune
Serpin genes B13R and B24R which are located between
10 and 17 kb from the right end of the genome (Kotwal &
Moss, 1989; Smith et al., 1989) were chosen for insertion
sites. A third serpin gene is located in the H i n d l I I K
fragment near the opposite end of the virus genome
(Boursnell et al., 1988). The function and essentiality of
these genes in vaccinia virus was unknown, but in
cowpox virus which has homologues of the B13R and
the BI3R gene i'~ non-essential for virus growth and is
responsible for a haemorrhagic pock phenotype (Pickup
e t a / . , 1986). Recently, it has been suggested that this
haemorrhagic phenotype is attributable to the serpin
gene product directly or indirectly inhibiting the infiltra-
tion of the infected area by white cells, and it is the
presence of these cells in pocks formed by serpin-
deficient virus that prevents haemorrhage (Palumbo et
al., 1989). Extracts from cells infected with serpin-
positive cowpox virus also prevent the migration of
lymphocytes in vitro (Chua et al., 1990). Whether the
serpin causes haemorrhage by direct inhibition of blood
clotting is unknown. However, our observation that
antibody responses to a foreign antigen are reduced by
serpin genes B13R or B24R is consistent with a model in
which the serpin gene product prevents the infiltration of
the infected area by inflammatory cells, resulting in a
diminished immune response. Another proposed func-
tion for the serpin genes is the inhibition of cellular
proteases which are involved in the proteolytic degrada-
tion of intracellular antigens (Smith et al., 1989). The
observation that vaccinia virus may block the presenta-
tion of some antigens to class I major histocompatibility
complex-restricted cytotoxic T cells (Coupar et al., 1986)
and that this blockage may be overcome by expressing
unstable proteins (Townsend et al., 1988) or peptide
fragments (Gould et al., 1989) is consistent with this
proposal.
The potential for successful immunoprophylaxis of
HPV infections is encouraged by the fact that bovine
papillomavirus type 1 L1 expressed in E. coli protects
cattle from development of warts due to virus infection
(Pilacinski et al., 1985). Vaccinia virus recombinants
might offer some advantages for immunization against
HPV due to the vaccine's stability, low cost, ease of
administration and ability to stimulate T cell and
antibody responses. Although the HPV L1 protein is
nuclear, other nuclear virus antigens expressed by
vaccinia virus have induced good immune responses in
vaccinated animals (Mackett et al., 1985b; Yewdell et
al., 1985). Moreover, immunization of animals with

recombinant viruses expressing nuclear antigens of
polyoma virus have caused the prevention or regression
of polyoma virus-induced tumours (Lathe et al., 1987). It
remains to be determined Whether deletion of the serpin
genes can influence the immune responses to other
foreign proteins expressed by vaccinia virus but it is
possible that immune responses (antibody and T cell)
may be generally increased by the deletion of these genes.
We are indebted to Professor H. zur Hausen for the HPV-16 plasmid,
to Dr D. H. Davies for the L1 protein produced by the pKK223-3
expression vector, to Dr D. B. Boyle for the Ecogpt gene and to Mrs
Helen Wilson for preparing the manuscript. G. L. S is a Lister Institute-
Jenner Research Fellow.
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(Received 28 February 1990; Accepted 24 May 1990)