An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Article (PDF Available)inArchives of Virology 153(3):585-9 · February 2008with18 Reads
DOI: 10.1007/s00705-007-0025-2 · Source: PubMed
Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size ( approximately 55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb.
An investigation into the use of human papillomavirus type 16
virus-like particles as a delivery vector system for foreign
proteins: N- and C-terminal fusion of GFP to the L1 and L2
capsid proteins
Oliver P. Windram Æ Brandon Weber Æ
Mohamed A. Jaffer Æ Edward P. Rybicki Æ
Dionne N. Shepherd Æ Arvind Varsani
Received: 2 October 2007 / Accepted: 17 December 2007 / Published online: 4 January 2008
Ó Springer-Verlag 2007
Abstract Development of vaccine strategies against
human papillomavirus (HPV), which causes cervical can-
cer, is a priority. We investigated the use of virus-like
particles (VLPs) of the most prevalent type, HPV-16, as
carriers of foreign proteins. Green fluorescent protein (GFP)
was fused to the N or C terminus of both L1 and L2, with L2
chimeras being co-expressed with native L1. Purified chi-
maeric VLPs were comparable in size (*55 nm) to native
HPV VLPs. Conformation-specific monoclonal antibodies
(Mabs) bound to the VLPs, thereby indicating that they
possibly retain their antigenicity. In addition, all of the
VLPs encapsidated DNA in the range of 6–8 kb.
Human papillomaviruses (HPVs; family Papillomaviridae)
are non-enveloped viruses, of which over 100 types have
been classified. Of these, genital HPVs fall into two
groups: low-risk, causing benign anogenital warts, and
high-risk, causing cervical cancers. Of the high-risk group,
HPV type 16 (HPV-16) alone is responsible for up to 50%
of diagnosed cervical cancers worldwide [3], making it an
important model from which to develop appropriate pro-
phylactic or therapeutic vaccination strategies.
The HPV virion consists of an *8 kb double-stranded
circular DNA genome encoding six early (E) proteins
involved in viral replication and host cell malignant
transformation, and two late (L) structural proteins, L1
(major capsid protein, making up the capsid) and L2
(minor capsid protein, incorporated within the capsid).
Analysis of various L2 mutant constructs has localized the
carboxyl region of L2 (residues 396–439) as a critical L1–
L2 interaction domain which allows incorporation of L2
into the capsid [2]. The presence of L2 within the capsid is
also suggested as a prerequisite for DNA encapsulation [9].
Recombinant expression of L1, either on its own or with
L2, results in the formation of virus-like particles (VLPs)
that resemble native papillomavirus (PV) particles, both
morphologically and immunologically [1, 5, 16].
Despite the development of native HPV VLPs as can-
didate prophylactic vaccines, chimaeric VLPs are
increasingly being researched and synthesised to accom-
modate more viral antigen targets. An extensive study
investigating chimaeras of HPV-16 L1 and E7 (oncopro-
tein) was conducted [10] where either the 34-residue C-
terminus of L1 was replaced with various segments of E7
or the E7 segments were inserted into the L1 residue region
295–302. Most of the chimaeric VLPs were able to induce
a neutralising immune response [10]. HPV-16 E7 chima-
eras of both L1 and L2 were shown to elicit a CD8+ CTL
response against E7 in mice and to protect against E7-
expressing tumors [4]. Similar results were obtained with
chimaeric HPV-16 L1 fused at the C-terminus with a
truncated E7 protein [15], E7 peptides [12] and a string of
HIV CTL epitopes [7]. In addition, there are a number of
reports describing the use of PV-like particles to deliver/
display foreign epitopes [4, 7, 8, 10, 11, 19].
Electronic supplementary material The online version of this
article (doi:10.1007/s00705-007-0025-2) contains supplementary
material, which is available to authorized users.
O. P. Windram E. P. Rybicki D. N. Shepherd A. Varsani
Department of Molecular and Cell Biology,
University of Cape Town, Rondebosch,
Cape Town 7701, South Africa
B. Weber M. A. Jaffer A. Varsani (&)
Electron Microscope Unit, University of Cape Town,
Rondebosch, Cape Town 7701, South Africa
Arch Virol (2008) 153:585–589
DOI 10.1007/s00705-007-0025-2
Despite such research on using chimaeric PV VLPs as a
vaccine/epitope delivery system, there have been no sys-
tematic studies comparing the fusion of a foreign protein to
the N- and C-termini of L1 and L2. Our study uses GFP
fused to the N- and C-termini of both the HPV-16 L1 and
L2 (GenBank accession # AY177679 and EU118173,
respectively), with L2 chimeras being co-expressed with
native L1, to compare the effects of N- and C-terminal
fusions on VLP formation.
To create the fusion constructs GFP-L1 and GFP-L2
(GFP fused to the N-terminus of L1 and L2, respectively);
L1-GFP and L2-GFP (GFP fused to the C-terminus of L1
and L2, respectively), the capsid protein genes were PCR-
amplified from pSKSAL1 and pSKSAL2 plasmids [native
L1 and L2 in pSK (Stratagene), respectively] using the
proofreading DNA polymerase Accuzyme
(Bioline) and
relevant primers (Supplementary Table 1). The PCR
products were A-tailed and inserted into either of the
vectors of the pdDNA3.1/NT-GFP-TOPO
TA (N-termi-
nal fusions) and pcDNA3.1/CT-GFP-TOPO
TA (C-
terminal fusions) expression kit (Invitrogen) to create the
fusion constructs. The fusion constructs were amplified
using a second set of primers (Supplementary Table 1) and
subcloned into the suicide vector of the Gene JET
(Fermentas). GFP-L1 and L1-GFP were then inserted into
the XbaI and XbaI/PstI site, respectively, of the pFast-
1 vector (Invitrogen). For the L2-GFP chimaeras to
be co-expressed with L1, the native full-length L1
sequence was excised from pSKSAL1 using SalI/XbaI and
inserted into the same sites of the primary multiple cloning
site of the pFastBac
Dual vector (Invitrogen), creating
L1-pFastBacDual. The L2-GFP fusion constructs were then
inserted into the NheI/NsiI and NcoI/NsiI site, respectively,
of the secondary multiple cloning site of L1-pFastBacDual,
creating L1:GFP-L2 and L1:L2-GFP fusion constructs.
DH10Bac E. coli cells (Invitrogen) were transfected with
the four pFastBac1
clones, and Spodopetra fungiperda
(Sf21) (Invitrogen) cells were transfected with the resulting
bacmid DNA using Cellfectin (Life Technologies). The
Bac-to Bac
manufacturer-recommended protocol was
followed to amplify the recombinant virus.
For each construct, 10 llml
of the amplified virus
was used to infect five 75 cm
flasks of 6 9 10
Sf21 cells
seeded in 10 ml of complete TC100 and incubated at 27°C
for 48–72 h. Infected cells were harvested as described [19,
20]. To isolate the expressed constructs using a CsCl gra-
dient, Sf21 cell pellets were resuspended in high-salt
phosphate-buffered saline (PBS) with 0.5 M NaCl, 0.4 g
CsCl and protease EDTA-free inhibitor cocktail
(Roche diagnostics). The suspension was sonicated for
4 min with 15 s treatments interspersed with 15 s intervals.
The sonicated material was matured overnight at 4°C and
then centrifuged at 35,000 rpm for 24 h at 16°C in a SW-
55 Ti Beckman rotor. Two distinct bands were observed for
constructs GFP-L1 and L1-GFP, and three bands for
L1:GFP-L2 and L1:L2-GFP. Only the GFP-containing
band that fluoresced upon excitation with a long-wave UV
lamp was extracted for each of the constructs (Supple-
mentary Figure 1). The extracted bands were verified by
western blot analysis as described previously [20], using
primary antibody H16:J4 (courtesy of Dr. Neil Christen-
sen) and anti-GFP (Roche), secondary antibody anti-mouse
conjugated to glutathione peroxidase (Sigma), and detected
using SIGMA Fast NBT/BCIP. The L1 and L2 GFP fusion
constructs were confirmed by the ca. 95 kDa products that
bound H16:J4 and anti-GFP antibodies, respectively
(Fig. 1). In addition, the native L1 in the dual expression
vector was confirmed by the ca. 60 kDa product. Further
confirmation was achieved using an ELISA protocol
described by Varsani et al. [19, 20]. First, the L1 concen-
tration in each sample was normalized using the
monoclonal antibody H16:J4, which recognizes a linear
epitope of L1, independent of protein conformation. The
four normalised samples were then analysed by ELISA
using a host of well-characterized monoclonal antibodies
(Mabs) recognizing either linear or conformation-specific
epitopes (courtesy of Dr Neil Christensen). This was done
in order to investigate the structural integrity of L1 with
GFP fused to the different termini of the two capsid pro-
teins. An ELISA was performed in triplicate for each Mab
on all four fusion constructs as well as positive (native
HPV L1 VLPs) and negative (cell lysate) controls, and
average values were calculated for each triplet (Fig. 2).
The analysis indicates that L1/GFP fusions not only bind
Mabs recognizing linear epitopes (H16:J4 and H16:I23) but
also bind conformation-specific Mabs (H16:U4, H16:V5,
H16:E70 and H16:9 A), indicating that L1 maintained its
structural conformation in the presence of all types of GFP
fusion chimeras. This indicates that the fused GFP did not
alter or interfere with the VLP conformational epitopes.
That the fusion constructs formed VLPs was confirmed
using electron microscopy. Samples were prepared using
immunoelectron microscopy and negative stain techniques.
Carbon-coated copper grids coated with L1 H16:J4 anti-
body (diluted 1/50), were used to trap the constructs, after
which they were stained with 2% uranyl acetate. Grids
were visualized and photographed using a 2 9 2 k CCD
camera using a LEO Omega 912 (Zeiss) transmission
electron microscope. Figure 3 shows the VLPs present in
the prominent fluorescing bands from the CsCl gradients.
The VLPs in these samples looked well-formed and mor-
phologically uniform. The chimaeric L1 VLPs (containing
GFP-L1 and L1-GFP monomers) appeared to have added
density in the capsid shell wall. L1-GFP VLPs particularly
indicate the added density as being present on the inner
surface of the capsid shell. Also, the overall L1-GFP VLP
586 Arch Virol (2008) 153:585–589
size remains comparable to other chimaeric VLPs (Fig. 3).
In terms of VLP size, VLPs of all four chimaeric constructs
were on average 55 nm in diameter, which is similar to the
size determined previously for native VLPs.
The development of papillomavirus (PV) VLPs as DNA
delivery vectors for genetic immunisations is at a relatively
early stage. Bovine papillomavirus 1 (BPV-1) and HPV-33
VLPs, expressed by a recombinant vaccinia virus, have
been shown to encapsidate a plasmid containing the
b-galactosidase (b-Gal) reporter gene [18, 21]. BPV gen-
omes autonomously replicating in a mammalian cell line
have been encapsidated by BPV-1 and HPV-16 L1 protein
expressed by recombinant Semliki forest virus [13]. DNA
encapsidated in pseudovirions can also be generated from
assembly of previously disassembled particles in the pres-
ence of different unrelated plasmids containing a reporter
gene [6, 17]. Assembly of pseudovirions and encapsidation
of plasmid DNA in S. cerevisiae-expressed HPV-16 L1/L2
has recently been shown, and in vitro infection of mam-
malian cells with these pseudovirions resulted in the
delivery of the reporter gene [14]. These studies support the
possibility of generating PV pseudovirions that package
DNA in vitro and that such pseudovirions are able to deliver
the packaged DNA to various cell lines.
To determine whether the VLPs had the potential to
incorporate DNA into their structure during their assembly,
VLP samples were electrophoresed through 0.6% agarose
gels. Based on differential analyses using ethidium bro-
mide staining, we distinguished GFP fluorescence at
254 nm from DNA with inter-chelated ethidium bromide.
All four types of the chimaeric VLPs prepared by CsCl
density gradient purification appeared to package random
fragments of genomic DNA ranging from 6 to 8 kb in size
(Supplementary Figure 2). This contradicts previous
reports that suggest that L2 is a prerequisite for DNA
incorporation [9], as we found DNA was also incorporated
into VLPs composed purely of L1-GFP fusion proteins.
However, Touze and Coursaget [17] reported the packag-
ing in vivo of random plasmid DNA and its delivery to
eukaryotic cells by HPV-16 L1 VLPs. The intrinsic capa-
bility of these VLPs to package DNA during their initial
formation may predispose them towards development as
DNA vaccine delivery vehicles.
In summary, L1, which is a major constituent of HPV
VLPs, maintains its structural conformation in the presence
of N- and C-terminal GFP fusions. In addition, L2 N- and
Fig. 1 Western blot analysis of fluorescing CsCl bands using
HPV16:J4 for GFP-L1/L1-GFP (ca. 95 kDa) and L1 in the L1:GFP-
L2/L1: L2-GFP (ca. 60 kDa) protein products
H16:V5 H16:E70 H16:U4 H16:J4 H16:I23 H16:9A
HPV-16 Specific Antibody
GFP-L1 L1-GFP L1:GFP-L2 L1:L2-GFP Negative control Positive control
Absorbance at 405nm (ODU)
Fig. 2 Antibody-binding
characterisation by ELISA of
the purified GFP fusion products
(confirmed by western blot
analysis) using a panel of
monoclonal antibodies. H16:V5,
H16:E70, H16:U4 and H16:9 A
are conformation-specific Mabs
that bind neutralising epitopes.
H16:J4 and H16:I23 bind linear
eptiopes. Error bars represent
standard deviation for each
Arch Virol (2008) 153:585–589 587
C-terminal GFP fusions were incorporated into VLPs made
up of native L1. ELISA results suggest that the GFP does
not interfere with or alter the conformation-specific epi-
topes of L1 in VLPs. HPV 16 VLPs exhibit the potential
for the development of multivalent vaccines that are
capable of presenting one or even more than one type of
antigen concurrently to the immune system by potentially
humoral or cellular means. Furthermore, the potential
exists to use the system as a DNA vaccine delivery vector,
possibly even coupling this strategy with the above-men-
tioned systems to generate vaccines capable of stimulating
both arms of the immune system simultaneously.
Acknowledgments We would like thank to Dr. Neil Christensen for
providing the monoclonal antibodies used in this study. This research
was funded by the Poliomyelitis Research Foundation of South Africa
(grant #04/25) and the University of Cape Town start up grant. AV is
supported by the Carnegie Corporation of New York.
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