Download Assembled baculovirus-expressed human papillomavirus type 11

Journal of General Virology (1994), 75, 2271-2276. Pr&ted in Great Britain
2271
Assembled baculovirus-expressed human papillomavirus type 11 L1 capsid
protein virus-like particles are recognized by neutralizing monoclonal
antibodies and induce high titres of neutralizing antibodies
Neil D. Christensen, ~* Reinhard H6pfl, 3 Susan L. DiAngelo, ~ N a n c y M. Cladel, ~
Susan D. Patrick, 1 Patricia A. Welsh, 1 Lynn R. Budgeon, ~ Cynthia A. Reed ~
and John W. Kreider 1,2
Departments o f ~Pathology, 2 Microbiology and Immunology, The Milton S. Hershey Medical Center, Hershey,
Pennsylvania 17033, U.S.A. and 3 Department of Dermatology, University o f Innsbruck, Innsbruck, Austria
Baculovirus-expressed human papillomavirus type 11
(HPV-11) major capsid protein (L1) virus-like particles
(VLPs) were produced in insect cells and purified on
CsC1 density gradients. The VLPs retained conformational neutralizing epitopes that were detected by a
series of HPV-11-neutralizing monoclonal antibodies.
Electron microscopy determined that the HPV-11 L1
VLPs were variable in size with a surface topography
similar to that of infectious HPV-I 1. The VLPs were
very antigenic, and induced high titres of neutralizing
antibodies in rabbits and mice when used as an
immunogen without commercial preparations of adjuvant. These VLP reagents may be effective vaccines for
protection against HPV infections.
Introduction
al., 1990; Rumenapf et al., 1991; Jones et al., 1990;
Martinez et al., 1992; Kirnbauer et al., 1993). The
production of VLPs for some viruses such as polyomavirus and parvovirus has even been accomplished
successfully by expression of the major coat protein
alone (Montross et al., 1991; Brown et al., 1991;
Martinez et al., 1992). Recently, VLPs of various PVs
have been developed using recombinant vaccinia viruses
(Zhou et al., 1991, 1993; Hagensee et al., 1993), or
recombinant baculoviruses (Kirnbauer et al., 1992, 1993 ;
Rose et al., 1993). As found for polyomavirus and
parvovirus, successful PV VLP formation was possible
by overexpression of the major coat protein L1 in the
absence of the minor coat protein L2 (Kirnbauer et al.,
1992, 1993; Zhou et al., 1993; Rose et al., 1993;
Hagensee et al., 1993). For the formation of some PV
VLPs however, dual expression of both L1 and L2 was
necessary (Zhou et aL, 1991).
In this report we developed a recombinant baculovirus
producing human PV-11 L1 that self-assembled into
VLPs in insect cells. These VLPs were screened by a set
of neutralizing MAbs to determine whether they retained
conformational neutralizing epitopes, and were also used
as an antigen in the immunization of mice and rabbits.
The results demonstrated that HPV-11 L1 VLPs were
recognized by the neutralizing MAbs and induced high
titres of neutralizing antibodies when used as immunogen
without adjuvant.
Conformational, strongly neutralizing epitopes on papillomaviruses (PVs) have been identified by a series of
monoclonal antibodies (MAbs) (Christensen et al.,
1990b; Christensen & Kreider, 1991, 1993). Although
several weakly neutralizing linear epitopes have also
been identified on both the major (L1) and minor (L2)
capsid proteins of PVs, immunization against PV
infection has been successful in some cases (Jarrett et al.,
1991; Campo et al., 1993), but weak or ineffectual in
others (Jin et al., 1990; Christensen et al., 1991; Ghim et
al., 1991 ; Lin et al., 1992). In contrast the conformational
epitopes located on intact virions induce high titres of
neutralizing antibodies with subsequent strong protection against viral challenge (Olson, 1963; Christensen
& Kreider, 1990; Jarrett et al., 1990a, b). Strategies to
protect against human PV (HPV) infection therefore
require the development of antigenic reagents that mimic
the conformational neutralizing epitopes located on
intact virions.
One such strategy to generate the correct surface
topography of viruses synthetically is to produce artificial
virus-like particles (VLPs) by overexpression of the viral
coat proteins in various expression systems (Urakawa et
al., 1989; Saliki et al., 1992; Brown et al., 1991 ; Kajigaya
et aI., 1991; Montross et al., 1991; French et al., 1990;
Spehner et al., 1991 ; Zhou et al., 1991, 1992; Haffar et
0001-2370 © 1994SGM
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N . D. C h r i s t e n s e n a n d o t h e r s
Methods
Production of H P V-I1 L1 recombinant baculovirus and purification o f
VLPs. A recombinant baculovirus expressing the HPV-11 L1 protein
was produced by recombination of wild-type baculovirus DNA with
the transfer vector pVL1392 (Invitrogen) containing an HPV-11 L1
insert. HPV-11 DNA cloned from the Hershey isolate (Kreider et al.,
1985) was digested with restriction enzymes EcoRI and Bsu36I to
obtain the fragment spanning 5678 bp to 7718 bp containing the entire
L1 open reading frame. The fragment was blunt-ended using Klenow
enzyme, then ligated into the transfer vector pVL 1392 and recombinant
baculoviruses were then produced by standard methods (Summers &
Smith, 1988). Isolated recombinants were screened for LI protein
production by Western blotting using anti-PV group-specific antigen
(GSA) antibody (Dako). The method for VLP purification was
identical to that used to purify infectious PV from cysts produced in the
athymic mouse xenograft system (Kreider et al., 1985). HPV-II L1
VLPs were produced by infection of Spodoptera frugiperda-derived
insect cells (sfg) at an m.o.i, of 10, and the infected cells were lysed 72 h
later by one freeze-thaw cycle. The lysed cells were homogenized in a
Virtis homogenizer for 5 rain at 30000 r.p.m., cellular debris was
removed by low speed centrifugation and then the supernatant was
placed onto a CsC1 gradient for banding of VLPs. Aliquots of the
gradient were removed and their density was determined from the
measured refractive indices. Fractions of the gradients were analysed
for VLPs and LI protein by Western blotting and by ELISA using
MAbs (Christensen et al., 1990b).
of complete virus neutralization, and cysts morphologically transformed (HPV-11 DNA-positive) as non-neutralized. Positive controls
for neutralization included previously tested rabbit anti-HPV-I 1 sera,
and neutralizing MAb (Christensen et al., 1990b). Negative control
sera included preimmune rabbit sera and non-immune mouse sera.
Results
Production
Immunizations. Rabbits and mice were immunized with HPV-11 L1
VLPs diluted in PBS. Rabbits received 50 gg protein per injection
intramuscularly and mice received 1 gg protein per injection subcutaneously at 2 week intervals. The L1 protein content of VLP
preparations was estimated by densitometric scanning of Coomassie
blue-stained gels using BSA as a standard. Approximately 23 % of total
VLP protein was calculated to be L1 protein. Serum samples were
collected 3 to 4 days after each booster immunization, heat-inactivated
at 56 °C for 30 min, then tested in ELISA for reactivity to intact
infectious HPV-I1 (Christensen et al., 1990b).
Neutralization o f HPV-11 by anti- VLP sera. Neutralization of HPV11 was assayed using the athymic xenograft system (Kreider et al.,
1985) as previously described (Christensen et al., 1990b). Dilutions of
rabbit sera were added to aliquots of infectious HPV-11 and incubated
for 1 h at 37 °C, then foreskin chips were added and incubated for a
further 1 h at 37 °C. The foreskin chips were transplanted under the
renal capsule of athymic mice and were examined 70 days later for
morphological transformation and HPV-11 DNA by in situ hybridization (Christensen et al., 1990b). Morphologically normal epithelial
cysts that were negative in situ were considered experimental evidence
L1 VLPs
in i n s e c t cells
A r e c o m b i n a n t b a c u l o v i r u s t h a t e x p r e s s e d H P V - 1 1 L1 i n
i n s e c t cells w a s d e v e l o p e d . CsC1 g r a d i e n t f r a c t i o n s o f
l y s a t e s o f i n f e c t e d Sf9 cells w e r e t e s t e d i n E L I S A u s i n g a
neutralizing MAb to test the hypothesis that the VLPs
r e t a i n e d c o n f o r m a t i o n a l n e u t r a l i z i n g e p i t o p e s ( F i g . 1).
Strong reactivity with the intact (but not disrupted)
VLPs by the neutralizing MAb Hl l.B2 was obtained
i
1-0
(a)
•
•
•
•
0.8
0-6
Electron microscopy (EM) ofVLPs. HPV-11 L1 VLPs were examined
by EM to determine whether their surface topography was similar to
that of infectious PVs (Kirnbauer et al., 1992). CsC1 gradient-purified
infectious HPV-11 and VLPs were dialysed against PBS then 5 lal of
this suspension was applied to formvar-coated EM grids and the excess
liquid was drawn off with a paper tissue. The sample was then stained
with 1% phosphotungstic acid and viewed under a Philips EM400
electron microscope.
EL1SA and Western blot. Intact and disrupted HPV-11 L1 VLPs
were tested in ELISA for reactivity to HPV-11-neutralizing MAbs by
previously published methods utilizing infectious PV (Cowsert et al.,
1987; Christensen et al., 1990b). Intact VLPs were attached to ELISA
plate wells at neutral pH, and disrupted VLPs were attached in alkaline
and reducing conditions (0.2 M-Na2COa, 0'01 M-DTT pH 10"5) (Favre
et al., 1975; Christensen et al., 1990b). Western blot analysis of
gradient fractions of CsCl-banded VLPs were also examined using antiPV GSA antibody as previously described (Christensen et al., 1991).
of HPV-11
0-4
0-2
1
4
0.5
5
9
13
17
21
(
0-4
0.3
0.2
0-1
n
1
rn
5
n
~
9
~
m
13
m
rn
17
~
~
,~,
21
Gradient fraction number
Fig. 1. ELISA reactivity of CsCI gradient fractions of lysed Sf9 cells
infected with recombinant baculovirus expressing HPV-11 L1. One
microlitre of individual fractions of the CsC1 gradient were added to
duplicate wells of an ELISA plate and screened with MAb H 11. B2 (IS])
and polyclonal anti-6L1 peptide antisera (A) for reactivity to intact (a)
or disrupted (b) VLPs. A41o values are expressed as the mean _+S.E.M. of
duplicate wells. Gradient densities (V) from left to right: 1.30, 1.32,
1-34 and 1.36 g/ml. A visible band was detected in fractions l l to 13.
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H P V - 1 1 L1 VLPs induce neutralizing antibody
Table 1. E L I S A reactivity o f M A b s to CsCl-banded H P V - 1 1 L1 VLPs
obtained f r o m SJ'9 cell lysates infected with recombinant baculovirus
ELISA reactivityt
MAb/
antiserum*
H l l .A3
H I I .B2
H 11. F 1
H l l .G5
H 11. H3
CRPV-IA
2795C-4
2975A-4
NRS~
Antigen
HPV-11
HPV-11
HPV- 11
HPV-11
HPV- 11
CRPV
HPV-6 L1
HPV-16 L1
-
Neutralizing
No
Yes
Yes
Yes
Yes
Yes
No
No
-
Intact
VLPs
0.263
0.528
0.404
0.707
0.784
0-001
0.007
0.006
0"000
(0-017)
(0-011)
(0.049)
(0.065)
(0.024)
(0-001)
(0.000)
(0.001)
(0.001)
Disrupted
VLPs
0-001 (0.001)
0.002 (0.001)
0.002 (0.000)
0.001 (0.000)
0.002 (0-000)
0-001 (0-000)
0.971 (0.002)
0.470 (0.042)
0.006 (0.001)
* MAbs and polyclonal rabbit antisera. Antisera 2795C-4 and 2975A-4 were generated against
HPV-6b and HPV-16 L1 peptides and fusion proteins (Firzlaff et al., 1988; Christensen et al.,
1990a).
t Results are shown as A410 values, the S.E.M. is shown in parenthesis. Values shown underlined
are considered reactive.
Normal rabbit serum.
0-3
i
i
i
i
0.7
T
0-6
o5
l
0-1
.~ 0.4
0-3
Serum dilution (log10)
0.2
Fig. 2. ELISA titration of rabbit (B0567) anti-VLP sera against intact
infectious HPV-11 particles. Dilutions of rabbit sera from pre-immune
sera (@) and after 1 (11), 2 (O), 3 ([]) and 4(A) immunizations at 2
week intervals with 50 !ag/VLP protein in PBS. Antibody titres (50%
binding) reach 1:20000 to 1:50000 by the second immunization. A410
values represent mean _+S.E.M. of duplicate readings and error bars are
given unless smaller than the symbols. One of two similar rabbit
responses to VLP immunizations is shown.
(Fig. 1 a). VLP disruption was confirmed by positive
reaction with polyclonal anti-6L 1 peptide sera (Firzlaff et
al., 1988; Christensen et al., 1990a), and by loss of
binding by MAb H l l . B 2 (Fig. l b). All five MAbs
(Christensen et al., 1990b) that recognized surface
conformational epitopes (four neutralizing and one nonneutralizing) showed strong reactivity to intact but not
disrupted HPV-11 L1 VLPs by ELISA (Table 1).
EM of the VLPs was conducted to determine whether
their gross surface morphology was similar to that of
infectious virus as has been observed by others
(Kirnbauer et al., 1992, 1993; Rose et al., 1993 ; Hagensee
et al., 1993). The results showed that VLPs of variable
0.1
0
=
(-)
1
' [l
2
3
4
Immunization number
Fig. 3. ELISA reactivity of mouse sera (1:200 dilution) to intact HPV11 following immunizations every 2 weeks with 1 gg/VLP protein in
PBS. Serum samples were obtained 3 to 4 days after injection of VLP
antigen from two mice (O, []), and tested in duplicate. Preimmnne
serum (1[) and M A b Hl l.B2 (@) were included as negative and
positive controls, respectively. A410 values are shown as the
mean±s.E.M, of duplicate wells.
size were produced that had features similar to those of
infectious virions and the HPV VLPs produced by other
researchers (Kirnbauer et al., 1993) (data not shown).
Immunization with H P V - 1 1 L1 V L P s
Rabbits and mice were immunized intramuscularly and
subcutaneously respectively with HPV-11 L1 VLPs
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N. D. Christensen and others
Table 2. Neutralization o f HPV-11 by antisera generated against HPV-11 L1
VLPs
Antiserum
B0567 preimmune
1:100
B0567 immune
1 : 100
1 : 1000
1:10000
Preimmune mouse
serum 1 : 100
Mouse immune
sera 1:100
1:1000
639 1 : 100
H l l .B2
1 : 100
1 : 1000
Antigen
Cyst size~ Morphology:~
HPV- 11
DNA§
-
3.3 (0.4)
6/7/8
6/7/8
HPV-11 L1 VLP
HPV-11 L1 VLP
HPV-11 L1 VLP
1.7 (0.4)
18 (0-3)
2.2(0.3)
0/6/7
0/5/8
0/6/8
2/6/6
0/5/6
0/6/7
3.5 (0.5)
5/5/8
5/5/7
HPV-11 L1 VLP
HPV-11 El VLP
Infectious HPV-11
2.6(0.5)
1.7 (0.2)
1-9 (0-3)
0/4/8
1/6/8
0/5/7
0/4/7
1/5/6
0/5/6
Infectious HPV-11
Infectious HPV-11
1-4 (0-2)
2.0 (0"2)
0/7/7
0/5/7
0/7/7
1/5/7
* Antisera were as follows: B0567, Dutch belted rabbit B0567; 639, New Zealand white rabbit
polyclonal HPV- 11-neutralizing antisera (Christensen et al., 1990 b); H 11. B2, HPV- 11-neutralizing
MAb supernatant (Christensen et al., 1990b).
t Surviving cysts only, results are given as geometric mean diameter (mm), the S.E.M. is given in
parenthesis. Underlined values have P < 0.005 compared to cyst sizes developing in response to
preimmune rabbit or mouse serumtreatment.
Number of grafts morphologically transformed/number of surviving grafts/number
attempted.
§ Number of grafts HPV-I1 DNA-positiveby in situ hybridization/numbersurviving/number
attempted.
diluted in PBS. Two Dutch belted rabbits were immunized with 50 lag VLP protein in PBS at 2 week intervals.
Serum samples were prepared from blood collected 3 to
4 days after each booster immunization and the sera were
tested in ELISA for reactivity to intact HPV-11 (Fig. 2).
High titres of serum antibodies reactive to HPV- 11 were
obtained after the second immunization, demonstrating
that the VLPs are highly antigenic. To demonstrate
further the antigenicity of the VLPs, BALB/c mice were
immunized subcutaneously four times with 1 pg VLP
protein in PBS. Strong antibody reactivity to HPV-11
developed after three immunizations (Fig. 3).
Neutralization o f H P V - 1 1 infectivity by a n t i - V L P sera
Antibody reactions to intact infectious PVs have been
shown to be predominantly neutralizing (Christensen et
al., 1990b; Jarrett et al., 1990b; Ghim et al., 1991).
However, the induction of neutralizing antibodies to an
HPV type by HPV VLPs has not been documented. We
therefore tested the polyclonal anti-VLP sera for HPV11 neutralizing activity in the xenograft system. Dilutions
of rabbit and mouse anti-VLP sera were mixed with
aliquots of infectious HPV-11 then foreskin chips were
added and transplanted subrenally. The presence of
normal epithelial cysts (Kreider et al., 1985) that were
HPV-11 DNA-negative by in situ hybridization was
evidence for the presence of neutralizing antibody. The
anti-VLP sera were strongly neutralizing, with complete
protection against HPV-11 infection at dilutions greater
than 1 : 10000 for the rabbit sera (Table 2). Sera obtained
from mice immunized four times with 1 lag VLP protein
were also strongly neutralizing with partial protection
against HPV-11 infection at dilutions greater than
l:1000. Positive controls for neutralization included a
rabbit anti-HPV-11 serum previously shown to be
neutralizing and a neutralizing MAb (Christensen et aL,
1990b).
Discussion
In this study, we have shown that HPV-11 L1 when
overexpressed in insect cells self-assembles into VLPs
that retain conformational neutralizing epitopes as
identified by neutralizing MAbs (Christensen et al.,
1990b). These results are in agreement with other
published studies showing that the major capsid protein,
L1, of PVs can self-assemble in isolation from other PV
proteins (Kirnbauer et al., 1992, 1993; Rose et al., 1993;
Hagensee et al., 1993) and induce neutralizing antibodies
(Kirnbauer et al., 1992). All four neutralizing MAbs that
were generated against infectious HPV-11 in a previous
study (Christensen et al., 1990b) reacted with the intact
VLPs demonstrating that neutralizing antibodies against
conformation-dependent epitopes are directed predominantly against L1.
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H P V - 1 1 L1 V L P s induce neutralizing antibody
In several animal/PV model systems, intact virus
(containing conformational neutralizing epitopes) has
induced high titres of neutralizing antibodies and strong
protection against experimental virus challenge (Olson,
1963; Jarrett et aI., 1990a, b; Christensen et al., 1991).
Included in these studies are several epithelial tissuespecific PVs [bovine PVs (BPVs)-4 and -6 and cottontail
rabbit PV (CRPV)] PVs inducing fibropapillomas (BPV1 and -2), and a PV that infects mucosal epithelia (BPV4). Neutralizing epitopes have also been identified on the
capsid proteins L1 and L2 (Pilacinski et al., 1984; Jin et
al., 1990; Christensen et al., 1991 ; Lin et al., 1992). These
epitopes, however, induced low titres of neutralizing
antibodies and were only weakly protective. Recent
studies have demonstrated that fragments of L1 proteins
are incapable of triggering neutralizing antibodies,
indicating that linear epitopes on CRPV L1 are not
neutralizing (Lin et al., 1993). The weakly neutralizing
epitopes on L2 have not been characterized in the same
manner, but are likely to be linear because denatured L2
has been used for immunizations (Christensen et al.,
1991 ; Lin et al., 1992). It is not yet known whether these
weakly neutralizing epitopes are capable of protecting
hosts against natural PV infections.
Assembled VLPs have recently attracted attention in
PV research because they provide an antigen source
which retains surface conformational epitopes that are
lost when individual and subunit capsid antigens are
used. A major group of antigens that are provided by the
VLPs are conformational neutralizing epitopes and
evidence has accumulated that these epitopes provide the
dominant antibody response to capsid proteins in
experimental and natural infections (Jarrett et al.,
1990a, b; Ghim et al., 1991; Christensen & Kreider,
1990). An important additional result that is demonstrated in this study is the strong antigenicity of these
VLPs for the induction of neutralizing antibodies. The
VLPs are potent antigens and trigger high titres of
neutralizing antibodies in the absence of commercial
preparations of adjuvant. This observation is of significance for the development of effective vaccines for
humans because commercial adjuvants and their associated problems can be bypassed.
Neutralization of HPV-11 using rabbit anti-HPV-11
L1 VLP antisera indicated that at low dilutions (1 : 100)
there was some breakthrough of infectivity as determined
by in situ hybridization (Table 2). This result is intriguing
because higher dilutions of rabbit anti-VLP antisera
(1:10000) afforded complete protection. One possible
explanation is that very high concentrations of anti-VLP
antibodies agglutinated the virus into large aggregates
which formed an infectious' unit' with the central virions
having minimal bound antibody. Another possibility is
that a second, higher affinity protective (non-
2275
neutralizing) antibody response was also generated that
was of lower titre than the neutralizing antibodies but
afforded partial protection for infectious virus at low
serum dilutions by steric hindrance of the binding of the
neutralizing antibodies. A MAb analysis of the antibody
profile to VLP immunizations may help to resolve these
possibilities, and the outcome may have important
consequences for the development of the VLP strategy
for vaccines against HPV infections.
Although serological cross-reactivity of HPV types at
the level of conformational neutralizing epitopes is
unknown, further studies with the HPV-11-neutralizing
MAbs (Christensen et al., 1990b) have indicated that
HPV-11 and HPV-6 L1 can be immunoprecipitated by
the HPV-11-neutralizing MAbs, but HPV-16 L1 cannot
(B. Jenson, personal communication). Future studies
with L1 VLPs of the different HPV types may provide
data to indicate that HPVs (at the level of neutralizing
epitopes) are either antigenically type-specific or are
grouped into several major serological subsets. If such
subsets are identified, then a limited number of HPV L1
VLPs representing each serological subgroup could
provide cross-protection to most HPV types if used
together as a composite vaccine.
In summary, we have shown that HPV-11 L1 selfassembles into VLPs which are highly antigenic in the
absence of adjuvant and which induce high titres of
serum neutralizing antibodies. The conformationally
correct neutralizing epitopes on these VLPs were further
confirmed by a set of HPV-11-neutralizing MAbs
prepared against infectious HPV-11. These VLPs have
the potential to be safe (non-infectious) vaccines for
humans and to serve as an antigen source for serological
studies to identify serum neutralizing antibodies following vaccination and natural HPV infection.
We thank Norman Olson (Purdue University, West Lafayette, Ind.,
U.S.A.) for the photomicrographs of the VLPs. This work was
supported by Public Health Service grants CA47622 (J.W.K.) and
CA56460 (N. D. C.) from the National Cancer Institute and by the Jake
Gittlen Memorial Golf Tournament.
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(Received 18 January 1994; Accepted 25 March 1994)
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