Research Paper
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Acta Biochim Biophys Sin
2005,37: 743–750
doi:10.1111/j.1745-7270.2005.00109.x
Construction of Prophylactic Human Papillomavirus Type 16 L1 Capsid
Protein Vaccine Delivered by Live Attenuated Shigella flexneri
Strain sh42
Xiao-Feng YANG1,3#, Xin-Zhong QU3#,
Kai WANG1, Jin ZHENG1, L?-Sheng SI1, Xiao-Ping
DONG2*, and Yi-Li WANG1*
1 Key Laboratory of
Biomedical Information Engineering of Ministry of Education, Institute for
Cancer Research, Xian Jiaotong University, Xian 710061, China;
2 National Institute
for Viral Disease Control and Prevention, Chinese Center for Disease Control
and Prevention, Beijing 100052, China;
3 Department of
Obstetrics and Gynecology, First Hospital of Xi’an Jiaotong University, Xian
710061, China
Received: April 4,
2005
Accepted: September
7, 2005
This work was
supported by the grants from the National High Technology Research and
Development Program of China (No. 2001AA215221) and the National Natural Science
Foundation of China (No. 30271184)
# These authors
contributed equally to this work
*Corresponding
authors:
Yi-Li WANG: Tel,
86-29-82655499; Fax, 86-29-82655499; E-mail, [email protected]
Xiao-Ping DONG:
Tel, 86-10-83534616; Fax, 86-10-63529809; E-mail, [email protected]
Abstract To express human
papillomavirus (HPV) L1 capsid protein in the recombinant strain of Shigella
and study the potential of a live attenuated Shigella-based HPV
prophylactic vaccine in preventing HPV infection, the icsA/virG fragment
of Shigella-based prokaryotic expression plasmid pHS3199 was
constructed. HPV type 16 L1 (HPV16L1) gene was inserted into plasmid pHS3199 to
form the pHS3199-HPV16L1 construct, and pHS3199-HPV16L1 was electroporated into
a live attenuated Shigella strain sh42. Western blotting analysis showed
that HPV16L1 could be expressed stably in the recombinant strain sh42-HPV16L1.
Sereny test results were negative, which showed that the sh42-HPV16L1 lost
virulence. However, the attenuated recombinant strain partially maintained the
invasive property as indicated by the HeLa cell infection assay. Specific IgG,
IgA antibody against HPV16L1 virus-like particles (VLPs) were detected in the
sera, intestinal lavage and vaginal lavage from animals immunized by
sh42-HPV16L1. The number of antibody-secreting cells in the spleen and draining
lymph nodes were increased significantly compared with the control group. Sera
from immunized animals inhibited murine hemagglutination induced by HPV16L1
VLPs, which indicated that the candidate vaccine could stimulate an efficient
immune response in guinea pigs mucosal sites. This may be an effective
strategy for the development of an HPV prophylactic oral vaccine.
Key words human papillomavirus type 16; attenuated Shigella flexneri;
vaccine; cervical cancer; prophylactic
Cervical cancer is the second most common cause of cancer-related
deaths in women worldwide. More than 450,000 cases are diagnosed each year,
resulting in nearly 250,000 deaths. It has been extensively confirmed that
high-risk human papillomaviruses (HPVs), particularly types 16, 18, 33, 45 and
58, are the initiators of the vast majority of cervical cancers. HPV16 is the
most prevalent, accounting for more than half of cervical cancer cases.
Moreover, HPV can induce malignant disease at other sites, such as the oral
cavity, esophagus and lung [1,2]. It is therefore reasonable to assume that
vaccines that protect against HPV infection would theoretically prevent women,
especially those in developing countries, from developing cervical cancer and
other HPV-related malignancies in later life.
HPVs can not be grown in the laboratory as a source of antigen for
serological tests and conventional killed vaccine development, and they do not
cause diseases in animals, so vaccine development is difficult. Therefore, HPV
vaccines currently under development employ genetic engineering technology. The
main requirement for prophylactic HPV vaccines is to induce neutralizing
antibodies against natural structural viral capsid proteins to prevent virus
entry into the host cell. The prerequisite to obtain this effect is that the
immunogen should possess the natural structure to induce the
conformation-dependent antibodies, and the route of immunization should favor
the activation of mucosal immunity. A major breakthrough in HPV vaccine
research came with the discovery that the capsid proteins L1 and L2 (or L1
alone) self-assemble into virus-like particles (VLPs) when expressed in
appropriate host cells. VLPs closely resemble native HPV particles. They
include the conformational epitopes that induce virus-neutralizing antibodies
[3], and the phase I/II clinical trials have shown promising results. VLPs are
not only immunogenic and safe, but also able to induce strong cell-mediated and
humoral immune responses [4,5]
in a controlled trial of HPV16 vaccine, and nearly 100%
effectiveness was achieved [6]. However, the costly production and distribution
of current VLP vaccines, for example, by the use of recombinant baculoviruses,
will prevent their widespread application in developing countries. Moreover, we
are not sure whether intramuscular injection is the optimal route, although the
HPV VLP intramuscular administration can induce a stronger antibody response in
the serum and can prevent the infection of HPV. A cheaper vaccine with a better
delivery system in stimulating mucosal immunity is needed.
The antigen delivery system of the vaccine is the key factor which
determines the effectiveness of a given vaccine. The recombinant attenuated enteropathogenic
bacteria, such as Salmonella or Shigella, may represent ideal
antigen delivery systems, as they efficiently cross all mucosal surfaces to
gain access to both mucosa-associated lymphoid tissue and draining lymph nodes.
It has been demonstrated that the attenuated Salmonella can express
HPV16L1 protein and stimulate strong neutralizing antibodies in mucosa sites
[7,16]. Compared with Salmonella, several intrinsic advantages of Shigella
strains make them ideal vehicles to deliver HPVL1 protein to mucosal sites. (1)
Shigella bacilli are also enteropathogenic bacteria, only the ileum and
colonic epithelium of humans and primates are their natural hosts. In
principal, the invasion of HPV16L1 carried by the recombinant Shigella
strain can cross the lumen of the gut by way of the M cells of Peyers patches
and then be taken up by macrophages and dendritic cells at local sites. Because
of the establishment of a short-lived infection after their delivery, an innate
immune response can be generated to promote the development of adaptive immune
responses against HPV16L1 protein. These responses triggered by mucosal
delivery can be effective at both mucosal and systemic sites [17,18]. (2) Shigella
infection, unlike other attenuated live vectors such as Bacille Calmette Gu?rin
(BCG) and Salmonella typhimurium [19], is localized at the infection
site and can not disseminate into circulation. Therefore, the attenuated Shigella
can be used safely as a mucosa-tropic vaccine vehicle in non-immune
compromised and immunocompromised hosts, such as those with HIV infection.
Therefore, in the present study, we expressed HPV16L1 in a strain of
live recombinant attenuated Shigella strain sh42. The production of
conformationally dependent and neutralizing antibodies in serum and body lavage
fluid was assessed after immunization of guinea pigs with the live recombinant
bacteria.
Materials and Methods
Bacterial strains and plasmids
Attenuated Shigella flexneri strain sh42 and its wild-type
progenitor M90Ts (S. flexneri 5a serotype) were generously provided by
Dr. Jun YU (Imperial College, London, UK). S. flexneri strains were
routinely grown at 37 ?C on Luria-Bertani (LB) agar plates containing 0.01%
Congo red. Red colonies were implanted into LB broth and grown to an appropriate
turbidity at 37 ?C with vigorous shaking. Escherichia coli strain Top10
was purchased from Invitrogen (Carlsbad, USA) and routinely grown at 37 ?C in
LB medium (broth or plate containing 1.5% agar). Antibiotics were supplemented
with the following final concentrations when needed: 100 mg/ml of
streptomycin; 200 mg/ml of ampicillin and 50 mg/ml of gentamycin. Plasmid pBR322 was
purchased from Invitrogen.
Construction of pHS3199-HPV16L1 plasmid
A 3.3 kb DNA fragment of gene icsA/virG of the S. flexneri
strain was amplified by polymerase chain reaction (PCR) from M90Ts, the wild
type of Shigella flexneri 5a strain, with forward primer 5‘-GGGAATTCGCATGAATCAAATTCA-3‘
and reverse primer 5‘-GCGGATCCTCAGAAGGTATAT-3‘, which
contained an EcoRI restriction site at the 5‘ end and a BamHI
site at the 3‘ end. The icsA/virG gene was then directionally
inserted into pBR322 to form a novel plasmid pHS3199 with ampicillin
resistance. A fragment of HPV16L1 gene (5637–7154 nt, 1518 nt) was
amplified by PCR from pFast-bacHPV16L1 with forward primer 5‘-GCTCTAGACAGGAGCTATTTAATGTCTCTTTGGCTGCCT-3‘
(XbaI restriction site in italic and SD sequence underlined) and reverse
primer 5‘-CCCTTAAAGCTTAATTACAGCTTACGTTTTTT-GCGTTTA-3‘
(HindIII restriction site in italic). An ATG start codon and a TTA stop
codon were included in the forward and reverse primers, respectively. The
HPV16L1 fragment and plasmid pHS3199 digested by XbaI/HindIII
were ligated using T4 ligase (TaKaRa, Dalian, China) and the ligation product
was designated pHS3199-HPV16L1 (Fig. 1).
Construction of recombinant Shigella strain sh42-HPV16L1
The pHS3199-HPV16L1 construct was transferred into the attenuated
sh42 competent cell by electroporation (Multiporator, Eppendorf, Germany) at
2500 V, one pulse, for 5 ms. The ampicillin-resistant colonies were picked up from the agar
plate and grown in LB broth to the mid-logarithmic phase. The cells were
collected by centrifugation and resuspended in phosphate-buffered saline (PBS).
The cell lysates were separated by 12% sodium dodecyl sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride
membrane (Invitrogen) for Western blotting. The HPV16L1 protein was identified
with a mouse monoclonal antibody against HPV16L1 (DAKO A/S, Glostrup, Denmark),
and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse polyclonal
antibody as the second antibody. Color development was carried out by the
addition of the diaminobenzidine (DAB) substrate-chromogen solution.
Genetic stability of recombinant strain sh42-HPV16L1
The genetic stability of the recombinant strain sh42-HPV16L1 was
determined by consecutive passage culture. The single sh42-HPV16L1 colony
picked up from the LB agar plate containing 50 mg/ml ampicillin was
incubated in LB broth containing ampicillin at 37 ?C overnight, then 10–4–10–6
folds dilution was made in antibiotic-free LB broth. The appropriate volume of
the diluted bacterial suspension was incubated overnight and further diluted
in the same folds as above with antibiotic-free LB broth. This procedure was
repeated until the 140th generation was obtained.
For each generation, part of the remaining diluted bacterial
suspension was used for detecting the expression of HPV16L1 protein by Western
blot; the rest of the diluted bacterial suspension was used for counting the
frequency of ampicillin-resistant colonies. For the frequency analysis, the
diluted bacterial suspension of each generation was transferred onto
non-ampicillin LB agar plates at 37 ?C overnight. One hundred colonies were
picked up randomly and seeded on ampicillin-containing LB agar plates at 37 ?C
overnight, then the number of colonies was counted and the frequency was
analyzed.
Safety test of recombinant strain sh42-HPV16L1
The virulence of recombinant strains was tested with the classical
Sereny test [8]. Briefly, 6–8-week-old outbred female Hartley guinea pigs were challenged by
the recombinant strain sh42-HPV16L1: 25 ml PBS containing 5?108 CFU
sh42-HPV16L1 was inoculated into each eye through the conjunctival sac, and the
eyes were observed for 7 d for the development of keratoconjunctivitis.
Development of disease was rated as follows: grade 0, no disease or mild
irritation; grade 1, mild conjunctivitis or late development and/or rapid
clearing of symptoms; grade 2, keratoconjunctivitis without purulence; grade 3,
fully developed keratoconjunctivitis with purulence.
Invasion ability of recombinant strain sh42-HPV16L1
HeLa cell infection assay was performed to test the invasion ability
of the recombinant strain sh42-HPV16L1. In brief, HeLa cell monolayers were
incubated on 35 mm plates in 5% CO2 at 37 ?C to
half-confluence in antibiotic-free Dulbeccos minimal essential medium (DMEM)
containing 10% fetal calf serum, then 25 ml of mid-logarithmic phase
bacteria was overlaid, spun down to adhere the HeLa cells at 1500 rpm for 10
min, and incubated together in humidified 5% CO2 at 37 ?C for
30 min. The HeLa cells were then washed six times with DMEM, and treated with
DMEM containing 50 mg/ml gentamicin for 90 min to kill the extracellular bacteria. After
washing with PBS, the HeLa cells were fixed with 4% formalin and Giemsa
staining was carried out. The number of intracellular bacteria was counted
under microscopy [9].
Immunogenicity of recombinant strain sh42-HPV16L1
The efficacy of sh42-HPV16L1 to evoke mucosal immunity against
HPV16L1 was tested by immunization through mucosal routes as previously
described [10]. The red colony of sh42-HPV16L1 was picked from the LB plate
containing 0.01% Congo red and cultured to the mid-logarithmic phase.
Twenty-five microliters of the bacterial culture harvested in 1?PBS were
inoculated into the guinea pigs conjunctival sac (2.5–5?108 CFU
per eye) on day 0, 2, 4, 14 and 15. Animals inoculated with PBS or sh42-pHS3199
were used as the control group (6–8 guinea pigs in each group). Two weeks after
the last immunization, the animals were killed and their blood samples were
drawn. Vaginal and intestinal fluids were collected in lavage buffer composed
of phenylmethylsulfonylfluride (PMSF) inoptine and NaN3,
and the spleens and draining lymph nodes were harvested.
Enzyme-linked immunosorbent assay (ELISA) was used to measure
antibodies against HPV16 VLP in the serum, and vaginal and intestinal lavage
fluid. Each well of the polyvinyl microtiter plates was coated with 100 ml 50 mM
carbonate buffer (pH 9.6) with or without 1 mg HPV16L1 VLP [11]. Guinea
pigs serum (1:10 dilution), vaginal and intestinal lavage fluid without
dilution, HRP-conjugated anti-guinea pig IgG (DaKo) and IgA (1:1200; Bethyl,
Montgomery, USA) were added consecutively. Absorbance was read at 450 nm.
The frequency of HPV16L1-specific antibody-secreting cells (ASCs)
in the immunized animals was determined using a modified enzyme-linked
immunospot (ELISPOT) assay based on the method of Czerkinsky [13]. The
splenocytes and lymphocytes from draining lymph nodes were prepared for ELISPOT
as described previously [13]. Briefly, each well of U-bottomed 96-well
microtiter plates was coated with 1 mg HPV16L1 VLP, and antigen-specific ASCs were
visualized as blue spots. The number of ASCs was counted under stereomicroscopy
and the data were recorded as ASCs per 106 cells.
Murine erythrocyte inhibition hemagglutination assay
HPV16 VLP causes hemagglutination of murine erythrocytes, which can
be inhibited by conformation-dependent neutralizing antibodies against VLP
[14]. Hence, we tested whether the serum from candidate vaccine-immunized mice
could inhibit VLP-induced hemagglutination of murine erythrocytes (HAI). The
whole assay was performed as described previously [15].
Results
Identification of recombinant plasmid pHS3199-HPV16L1
The PCR of HPV16L1 gene with specific primers generated a 1.5 kb
product (sequencing confirmed, data not shown). After having been cleaved by XbaI/HindIII,
the fragment was inserted into the plasmid pHS3199 cleaved with the same
enzymes. The insertion of the HPV16L1 fragment into pHS3199 plasmid was
confirmed by XbaI/HindIII digestion and 1% agarose
electrophoresis. The results showed the HPV16L1 fragment was successfully
cloned into pHS3199 plasmid (Fig. 2). The construct was designated
pHS3199-HPV16L1.
Identification of the expression of HPV16L1 protein in recombinant
strain sh42-HPV16L1
After the plasmid pHS3199-HPV16L1 was transferred into attenuated
sh42 by electroporation, the expression of HPV16L1 protein in the recombinant
strain sh42-HPV16L1 was analyzed by SDS-PAGE and Western blotting. The SDS-PAGE
result showed a single protein band with a molecular weight of 58 kDa
corresponding to that of HPV16L1 protein, which reacted specifically with the
anti-HPV16L1 monoclonal antibody as proved by Western blotting (Fig. 3).
The recombinant strain was designated sh42-HPV16L1.
Genetic and expression stability of recombinant strain sh42-HPV16L1
The genetic stability of the recombinant strain sh42-HPV16L1 was
measured by its ampicillin resistance. The level of resistance was maintained
in the 140th generation, and the growth rate of colonies was up to 100%. The
target protein HPV16L1 was expressed stably in the recombinant strain in the
140th generation, as demonstrated by Western blotting (Fig. 4), and
there was no apparent difference in the level of protein expression.
Safety of recombinant strain sh42-HPV16L1
The virulence of the recombinant strain sh42-HPV16L1 was determined
by the Sereny test. None of the eyes inoculated with sh42-HPV16L1 developed
keratoconjunctivitis. The Sereny test results also indicated that sh42-pHS3199
and sh42-HPV16L1 had lost virulence compared with the wild-type strain M90Ts (Table
1).
Invasion ability of recombinant strain sh42-HPV16L1
HeLa cells were incubated with sh42-HPV16L1 and the wild-type strain
M90Ts, and the number of bacilli intruding into HeLa cells was enumerated. The
results of HeLa cell infection assay showed that the intracellular bacterial
number in the recombinant strain was less compared with the wild-type strain
M90Ts, which reflected that the invasion ability of the recombinant strain sh42-HPV16L1
was diminished but not completely abolished. This is a prerequisite for a
candidate vaccine (Fig. 5).
Immunogenicity of sh42-HPV16L1
To evaluate the immunogenicity of sh42-HPV16L1, specific IgG and IgA
levels in the serum and body fluid (vaginal and intestinal lavage) of immunized
animals were tested by ELISA. Antibody secreted cells in the spleen and
draining lymph nodes specifically against HPV16 VLP were detected by ELISPOT.
Compared with the control group (immunized by PBS and sh42-pHS3199), the IgG
level was much higher than that of IgA in the serum of immunized animals (P<0.05). The IgG and IgA levels in the lavages of both the intestine and vagina showed no apparent differences (P>0.05); the IgA level in the lavages of the
intestine and vagina were slightly higher than that in serum, but the
difference was not significant. More significantly, after immunization through
the mucosal route, the immune response in other mucosal sites, such as
intestine and vagina, reached a similar level (Fig. 6).
The frequency of HPV16L1-specific IgG/IgA ASCs in the spleen,
mandibular lymph nodes (MDLN), mesenteric lymph nodes (MSLN), peyers patches (PP)
and superficial ventral cervical lymph nodes (SCVLN) of immunized animals were
inspected by ELISPOT (Fig. 7). Both the specific IgA and IgG ASCs in
spleen cells were much higher than those in other tested tissues. MDLN, the
nearest local draining lymph nodes to the conjunctival sac, also contained
higher frequencies of the specific ASCs, which were slightly lower than that in
the spleen. The other lymph nodes tested also showed increased levels of ASCs
compared with the control sh42-pHS3199 or PBS.
HAI induced by sera of immunized animals
HAI assay demonstrated that the sera from immunized animals could
significantly inhibit the hemagglutination activity induced by HPV16L1 VLPs,
which indicated that the anti-sera were conformation-dependent (Fig. 8).
Discussion
Although the attenuated Shigella is theoretically an ideal
vehicle for a mucosal predominant vaccine, the key point was to confirm that
HPV16L1 protein can be expressed in Shigella strains. We used a new
prokaryotic expression plasmid based on the invasive plasmid icsA/virG
of Shigella, in which the HPV16L1 gene fragment was inserted. The new
construct carrying the HPV16L1 coding fragment was introduced into the
attenuated S. flexneri strain sh42. It was demonstrated that HPV16L1
protein could be stably expressed in sh42. Recombinant sh42-HPV16L1 lost
virulence completely, but retained its partially invasive ability, which
warranted that HPV16L1 protein could be presented to a mucosal site, mimicking
the process of natural infection. The results of animal tests showed that
sh42-HPV16L1 exhibited good immunogenicity, and it could elicit specific
antibodies against HPV16 VLP in systemic (serum) as well as at mucosal sites
(intestinal and vaginal lavage fluids). The IgA level, which is more important
to evaluate the efficacy of a vaccine against a mucosal pathogen, was
significantly higher than in control groups. The frequency of VLP-specific
ASCs in the regional draining lymph nodes and spleen were measured by ELISPOT
assay, and the results indicated that the number of ASCs at these sites was
significantly higher than that in control groups.
The HPV neutralizing antibody is conformation-dependent, and only
the immunogen with the natural conformational epitopes can stimulate such an
antibody. HPV VLP cause hemagglutination of murine erythrocytes, which can be
inhibited by specific neutralizing antibodies, referred to as HAI, which can
well reflect the properties of functional neutralizing antibodies [20]. The
result of HAI in the experiment showed that IgG in the sera of immunized
animals could block VLP-induced hemagglutination of murine erythrocytes. This
demonstrated that sera from immunized animals with the candidate vaccine were
conformation-dependent.In conclusion, the current study provides evidence that an
attenuated Shigella strain expressing the major capsid protein of HPV16
represents a promising vaccine candidate against HPV16 infection.
Conformational-dependent VLP-specific antibodies, which correlate with
protection from experimental challenge in animal virus models, were generated
both systemically and locally at genital and intestinal mucosal sites. The
vaccine could be cheaply produced, be given non-invasively and potentially
induce long-lasting protection after a single inoculation. These
characteristics are particularly desirable in improving recipients compliancy
in vaccination and in a vaccine targeted for use in developing countries,
where cervical cancer is the leading cause of cancer-related deaths in women.
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