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Introduction
Pertussis or whooping cough is a highly contagious disease of the human respiratory tract. There are approximately 45
million cases of whooping cough worldwide per year, of these 400 000 result in fatalities. Most cases of severe symptoms and
the majority of fatalities are observed in early
infancy[1]. Pertussis causes significant economic costs for families, hospitals
and communities[2].
Whole-cell and acellular pertussis vaccines have been effective in providing protection against
Bordetella pertussis infection in
humans[3,4]. However, whole-cell vaccines have been associated with serious side effects such as convulsions
and encephalopathy[5]. As a result, acellular vaccines have replaced whole-cell vaccines in many developed countries.
Unfortunately, acellular vaccines are too expensive for routine use, especially in developing countries which have the
greatest need. In the last 10 years, there has been a resurgence of pertussis in many developing countries.
B pertussis secretes several virulence factors, including toxins such as pertussis toxin (PT), tracheal cytotoxin and
adhesins such as filamentous hemagglutinin (FHA) and pertactin
(PRN)[6]. Protection for pertussis has been
associated with higher levels of antibodies against PT, PRN and
FHA[4]. PT is a protein exotoxin which is central to the pathogenesis of
whooping cough. Detoxified PT is considered to be essential for acellular pertussis
vaccines[3]. It is the S1 subunit of PT
which possesses enzymatic capabilities of the ADP ribosyltransferase. A mutant containing a single amino acid substitution
Arg9-Lys of pertussis toxin subunit 1 (PTS1) reduced the enzymatic activity of wild PTS1 by approximately 0.02%, while
retaining the protective epitope[7]. The Arg9-Lys and Glu129-Gly analog was found to be significantly more immunogenic
than other mutation analogs[8]. PRN is another important pertussis vaccine antigen in multicomponent vaccines. Studies
have shown that PRN is a critical component of pertussis
vaccines[2]. The interaction between the mammalian cells and PRN
can be specifically inhibited by an Arg-Gly-Asp (RGD)-containing synthetic peptide that is homologous with a region found
between positions 939 and 1014 in the PRN gene
sequence[9]. FHA is the major adhesin of
B pertussis for bronchial epithelial
cells[10]. Monoclonal antibodies of amino acid residues following the RGD peptide were shown to inhibit binding of
B pertussis to ciliated rabbit cells in the same fashion as antibodies elicited by whole
FHA[11]. In this study, mutated PTS1 and
the fragments of PRN (amino acid residues 248_364) and FHA (amino acid residues 1099_1210) were used as combined and
recombinant vaccines.
DNA vaccines make use of the fact that plasmid DNA can directly transfect animal cells
in vivo[12]. This discovery made
it possible to induce immune responses by direct injection of DNA plasmids encoding for antigenic proteins into animal cells.
This method, referred to as DNA immunization, has now been used to elicit protective antibody and cell-mediated immune
responses for viral, bacterial and parasitic
diseases[5].
Being an extracellular pathogen,
B pertussis was thought to be a humoral pathogen only, whereas evidence suggests that
cellular and humoral immunity may act in concert to eliminate
B pertussis
infection[13]. Thus B pertussis
is now considered to be a facultative intracellular pathogen. Defence against intracellular pathogens is usually dependent on the activation of
a strong cellular immune response[11]. Furthermore, cellular immunity memory lasts longer than humoral immunity, and may
play a role in protective immunity after infection with
B pertussis[2]. DNA vaccines are simple to make and deliver, and can
elicit both humoral and cellular
immunity[14]. Many studies suggest that DNA vaccines induce a broad range of protective
immunities, including antibodies, CD8+CTL,
CD4+Th cells that challenge the
pathogens[15]. Immune responses generated by
DNA vaccination are initiated by professional antigen-presenting cells (APC). APC presentation of antigens in the context
of major histocompatibility complexes (MHC) class I and MHC class II molecules lead to cell-mediated and humoral
immunity[16]. Commonly, interleukin (IL)-4 and IL-10 are mainly elicited by Th2 cells and are typically used as markers for humoral
immunity, IFN-g and TNF-α are mainly produced by Th1 cells and are considered cellular
immune[14].
The major potential disadvantage of DNA vaccination is the low immune responses. Consequently, many methods have
been attempted to enhance the responses, including incorporation into liposomes, co-administration with adjuvants such as
monophosphoryl lipid A, alum or co-administration of plasmids encoding
cytokines[17].
In this study, we investigated the immunogenicity of a prime-boost strategy, co-injection with murine
granulocyte-macrophage-colony stimulating factor (mGM-CSF) plasmids and the boost with protein for a recombinant pertussis DNA
vaccine.
Materials and methods
Animals, bacterial strains and plasmids
Balb/c mice (4 weeks of age) were purchased from Shanghai Laboratory Animal
Co Ltd (Shanghai, China). Bordetella
pertussis CS was from the Chinese National Institute for the Control of Pharmaceutical
and Biological Products (Beijing, China); plasmid pQE31, pGEX-5X-1 were from the Department of Medical Microbiology and
Parasitology, Shanghai Jiao Tong University School of Medicine (Shanghai, China). These plasmids could be used for
His-tag (pQE31) and GST-tag (pGEX-5X-1) purification, respectively. Plasmid pVAX1 and pCEP4/mGM-CSF (pGM-CSF) were
generous gifts from Dr David D HO from the Aaron Diamond AIDS Research Center, Rockefeller University, USA and Dr
Mi-hua TAO from the Institute of Biomedical Sciences, Academia Sinica (Taipei, China), respectively. Plasmid pVAX1 contains
a CMV promoter allowing high-copy number replication
in Escherichia coli and high-level transient expression of the protein
in most mammalian cells.
PCR-based site-directed mutagenesis of PTS1
Primers P1 and P2 (see Table 1) were designed to amplify gene PTS1, and
other primers P3, P4 and P5 were designed to mutate PTS1 at amino acid 9 (aa9) and aa129 following
Burnette's[7] and
Loosmore's[8] studies. PCR were conducted with Pfu DNA polymerase (Shanghai Invitrogen, Shanghai, China), synthetic
oligonucleotide primers (Shanghai Invitrogen, Shanghai, China) on GeneAmp PCR System 9600 (Perkin-Elmer, Wellesley,
MA, USA). PCR products were purified by Qiaquick gel extraction kit (Qiagen, Hilden, Germany).
Construction and preparation of the plasmids
Primers P6 and P7 were designed to amplify the fragment of gene PRN;
primers P8 and P9 were designed to amplify the fragment of gene
FHA. These 2 fragments were cloned to plasmid
pGEX-5X-1, as the mutated PTS1 whole gene described above was cloned to plasmid pQE31.
Primers P1, P10, P11, P12, P13 and P14 were designed to recombine gene
PTS1, PRN and FHA fragments to a recombinant
fragment (PPF). First, PTS1, the PRN and FHA fragments were amplified with primers P1 and P10, P11 and P12, P13 and P14,
respectively. Then 3 PCR products were puri-fied, mixed and used as templates in the next PCR reaction which used P1 and
P14 as primers. There were overlaps between P10 and P11, P12 and P13, thus a full-length recombinant DNA,
PTS1_PRN_FHA (PPF) could be produced.
Purified PCR products and plasmid pQE31, pGEX-5X-1, pVAX1 were digested by restriction enzymes (Takara Bio Inc,
Otsu, Shiga, Japan), ligated (T4 ligase, Takara Bio Inc, Otsu, Shiga, Japan) and sequenced with ABI DNA sequencer
(Perkin-Elmer, Wellesley, MA, USA). pQE31/PTS1, pQE31/PPF, pGEX-5X-1/PRN, and pGEX-5X-1/FHA were prepared for protein
expression, pVAX1/PTS1, pVAX1/PRN, pVAX1/FHA and pVAX1/PPF were prepared for DNA injection with Qiagen Plasmid
Mega Kit (Qiagen, Hilden, Germany).
Expression and purification of proteins
Expression of proteins PTS1 and PPF: The plasmid pQE31/PTS1 and pQE31/PPF
were isolated and transformed into the lac inducible expression system of
E coli M15. An overnight culture was diluted
50-fold into fresh medium at 37oC in Luria-Bertani medium containing 100
mg ampicillin (per mL) and 30 mg kanamycin (per mL).
One mmol/L Isopropyl-b-D-thiogal-actopy-ranoside (IPTG) was added when the culture reached the optical density of 0.6 to
0.8 at 600 nm. The cells were harvested and resuspended in phosphate-buffered saline (PBS) 4 h later. The cell lysate
containing the proteins was analyzed in SDS-PAGE and Western blotting with monoclonal antibody to
6*His-tag[18].
Protein PRN and FHA was prepared in a similar method as PTS1 and PPF, except that the expression system
was E coli BL21; the concentration of IPTG was 0.15 mmol/L, and the monoclonal antibody used in the Western blotting was GST-tag.
Purification of protein PTS1 and PPF was fulfilled according to the protocol of Qiaexpressionist (Qiagen, Hilden, Germany)
and protein PRN and FHA to the protocol of Glutathione Sepharose 4B (Amersham Biosciences, Little Chalfont, UK).
Animal immunization Five groups of 10 Balb/c mice were used in this study. Plasmid pVAX1/PTS1, pVAX1/PRN and
pVAX1/FHA were mixed and named pVAX1/m. These 3 fragments were constructed together to a recombinant fragment PPF
and then cloned in the same plasmid pVAX1 and named pVAX1/PPF. The animal immunization procedure is shown in Table
2. The volume of each injection of 50 mg was intramuscularly administered into the thigh quadriceps muscle per mouse for
each plasmid or 10 mg intraperitoneally administered per mouse for protein. On d 35, serum was collected from each mouse.
ELISA assay for antigen-specific antibody production
The wells of the polystyrene microdilution plates were coated
overnight with 50 µL (4 µg/mL) of PT , PRN or FHA per well, respectively. The plates were washed 3 times with PBS. PBS
containing 2% bovine serum albumin was used to block nonspecific binding sites for 2 h. After the plates were washed with
PBS, 50 µL of serum was added to each well for 30 min at room temperature to bind to PT, PRN and FHA. After the addition
of anti-murine IgG conjugated with alkaline phosphatase (Sigma-Aldrich, St Louis, MO, USA), the plates were incubated for
1 h at room temperature and then washed. The color reaction was developed by the addition of enzyme substrate
p-Nitrophenyl Phosphate (Sigma-Aldrich, USA) and was quenched with the addition of 3
mol/L NaOH. The optical density was measured at 405 nm with an ELISA reader.
ELISA assay for cytokine induction
The collected sera were used to detect cytokine IL-10, IL-4,
IFN-g and TNF-α with homologous ELISA kits (Boster, Wuhan, China). Serum 100 µL was added to each well and the plates were incubated for 90
min (for IL-10, IL-4 and TNF-α) or 120 min (for
IFN-g) at 37oC. The serum was discarded from each well; 100
µL biotin-antibody reagent was added to each well, then the plates were incubated for 60 min at 37oC and washed 3 times with PBS. With 100 µL
avidin-biotin-complex reagent added to each well, the plates were incubated for another 30 min at 37oC and washed 5 times with PBS. The plates were incubated for 20 min and protected from light after TMB reagent was added to each well. The
reaction was quenched with the addition of 100 µL TMB stop reagent to each well. The optical density was measured at 450
nm with an ELISA reader.
Splenocyte-proliferation assay
Lymphocytes were prepared from mouse spleens. The isolated cell suspensions were
resuspended to a concentration of
2×104 cells/mL. A 100 mL aliquot containing
2×103 cells was added immediately to each well
of a 96-well flat bottom microtitre plate in triplicate. rIL-2 (R&D, Minneapolis, MN, USA) and homologous proteins were
added to the wells to the final concentration of 4 ng/mL (rIL-2) and 10
mg/mL (homologous proteins). Another triple well was
added (rIL-2) as the control. After a period of 72 h incubation at 37oC in 5% CO2, splenocyte proliferation was assayed with
a MTT kit (Beyotime, Haimen,
China). The ratio (test/control) of the optical density at 570 nm was described as the splenocyte-proliferation value.
Statistical analysis Statistical analysis was performed using Student-Newman-Keuls test. Values were compared
between different immunization groups. P values <0.05 or
0.01 were considered statistically significant.
Results
Clone, expression and purification of plasmids and antigens
Thegene PTS1 was mutated by a series of PCR reactions
and cloned to plasmid pQE31. Then the recombinant gene PPF was constructed by PCR and cloned to plasmid pQE31;
protein PTS1 and PPF were expressed and purified. Similarly, the other gene fragments, PRN and FHA were cloned to
pGEX-5X-1 and the proteins were produced (Figures 1A, C, D). Proteins PTS1, PRN and FHA were purified for coating the ELISA
plates used for measuring the antibody production and MTT assay, whereas the fusion protein PPF was purified for the
boosts during the animal experiment.
The plasmids we constructed and used in the animal immunizations are shown in Figure 1B. pVAX1/PTS1, pVAX1/PRN,
pVAX1/FHA and pVAX1/PPF were digested with restriction enzyme
HindIII and BamHI.
ELISA assay for antigen-specific antibody production
ELISA assays were used to display antigen-specific antibody
production (Figure 2). The results are similar in the 3 ELISA assays. Group pVAX1/m and group pVAX1/PPF showed more
specific antibodies than control group pVAX1. The group protein boost and group CSF prime-protein boost showed a
significant increase in antibodies compared to group pVAX1/m and group pVAX1/PPF. Especially in anti-PTS1 and
anti-FHA, group CSF prime-protein boost showed a higher absorbance than group protein boost. These results indicate that mice
boosted with protein elicited an increased humoral immune response than those boosted with DNA plasmids.
ELISA assay for cytokine induction
To reveal cellular and humoral response in the mice, ELISA assays were used to
describe cytokines production (Figure 3). IL-10 and IL-4 were used to indicate the level of humoral immune response,
IFN-g and TNF-α were used to reveal the level of cellular immune response. For IL-10 and
IFN-g, all the vaccine groups showed significant increases compared to the control group pVAX1. For IL-4 and
TNF-α, only group CSF prime-protein boost showed more cytokine production than group pVAX1. These cytokine results show that better humoral and cellular immune
responses were elicited when the mice immunized with DNA and cytokine plasmid pGM-CSF boosted with the corresponding
proteins.
Splenocyte-proliferation assay
MTT test was used to detect the splenocyte-proliferation reaction. The proliferation
ratio (test/control) analyzed at 570 nm with ELISA reader are shown in Table 3. All the test groups, except the CSF
prime-protein boost group, showed significant differences compared to the control group pVAX1
(P<0.05). The CSF prime-protein boost group showed a more significant difference compared to the pVAX1 group
(P<0.01). There was no difference between
any 2 test groups.
Discussion
B pertussis is a pathogen for humans only. Immunization is the most effective method for the prevention and control of
this disease and has been used successfully for
decades[19]. That primary vaccination of infants against pertussis does not
always afford long-term protection suggests a need for a better immune strategy to maintain adequate levels of specific
immunity to B
pertussis[20].
In the present study, the mutated PTS1 gene and the 2 gene fragments described earlier were cloned into plasmid pQE31,
pGEX-5X-1 for protein expression and pVAX1 for DNA immunization. The 3 plasmids (pVAX1/PTS1, pVAX1/PRN and
pVAX1/FHA) were mixed as a mixed vaccine. These 3 genes were also constructed to a recombinant fragment PPF, and then
constructed to another DNA plasmid pVAX1/PPF.
There are several benefits of multiple antigens being
encoded as DNA vaccines. For example, different components may have different functions. Another advantage of using
multiple genes is the ability to target more than one stage of the life cycle of a
pathogen[14]. For pertussis, the first stage of
the cycle is the bacterium entering the host via the airways, adhering to ciliated epithelial cells in the trachea and nasopharynx
with FHA and PRN actions. In the next stage, PT and other toxins are secreted by the micro-organism damaging the epithelial
lining and resulted in the loss of ciliated cells which induce
symptoms[11]. In the work described here, either the mixed
plasmids or the recombinant one can elicit specific antibodies: anti-PTS1, anti-PRN and anti-FHA. These antigen-specific
antibodies may play a role in protection
against B pertussis in these different stages.
In the cytokine assays, all of the pertussis DNA recombinant plasmids elicited IL-10,
IFN-g and specific antibodies. The assay showed that these plasmids can elicit both humoral immunity and cellular immunity. The relatively poor efficacy of
DNA vaccines in immune responses, especially in large animals, has limited their practical use. Here, we provide a strategy
to improve the immune response induced by DNA vaccines by supplementing it with the GM-CSF expression vector and
boosting it with protein antigens.
It is well known that parenterally-delivered DNA vaccines do not efficiently activate specific secreted antibody
responses at mucosal sites, further reducing the applicability of effective vaccines against mucosal bacterial
pathogens[21]. However, with a boost, the systemic immune response can be largely
improved[22]. In this study, a recombinant protein in
adjuvant was used as a boosting agent and significantly enhanced immune response. All the antibodies elicited in the group
protein boosted showed significant increases than those in group non protein boosted.
Another solution to improve the immune response of DNA vaccines is the co-injection of cytokine plasmids. Cytokines
have been used as genetic adjuvants to enhance the efficacy of DNA immunization. The co-injection of cytokine genes for
DNA vaccines can modulate antigen-specific immune responses and augment immune
response[23]. GM-CSF is produced by T and B cells, endothelial cells, fibroblasts and macrophages. Although the mechanism through which GM-CSF enhances
immunity is not completely understood, evidence suggests that GM-CSF may work on
APC[24]. GM-CSF increases expression of major histocompatibility complex class II molecules, enhances antigen presentation, and augments antigen-specific
T-cell proliferative response[25]. Thus GM-CSF expression plasmid plays an important role in the amplification of humoral and
cell-mediated immune responses[26]. In this study, GM-CSF plasmid was co-injected with recombinant fragment plasmids to
elicit a better immune response.
Specific antibodies and IL-4 and IL-10 were detected to investigate humoral immune responses in this study. For specific
antibodies, the specific antibodies in group protein boost and group CSF prime-protein boost were significantly increased
compared to the other groups. For IL-10, all 4 test groups showed significant increases compared to the control group
pVAX1, and there was no significant difference between any 2 test groups. For IL-4, only the CSF prime-protein boost group
showed significant increase compared to the control group. Hence, only in the CSF prime-protein boost group were all the
antibodies and cytokines significantly different compared to the pVAX1 group, which indicates that when primed with
pGM-CSF and boosted with homologous proteins, a much more enhanced humoral immune response was produced.
In the present study, splenocyte-proliferation assay along with
IFN-g and TNF-α detection was used to describe cellular
immune responses. The proliferation ratio from the splenocyte-proliferation assay was used to analyze the specific spleen T
cell proliferation. All the test groups showed increases compared to the control. Only the CSF prime-protein boost group
elicited a significant increase with similar conditions in the
TNF-α and IL-4 assay. However, for IFN-g, the CSF prime-protein
boost group showed a significant increase compared to the control groups and group pVAX1/PPF, but
IFN-g produced in group CSF prime-protein boost was less than those in the mixed DNA plasmids group pVAX1/m. Indeed, group CSF
prime-protein boost is the only group where all parameters showed a significant increase compared to the pVAX1 group, indicating
that an enhanced cellular immune response was elicited with the GM-CSF plasmid prime-protein boost.
Usually DNA vaccination is particularly effective in priming an immune response, but the low amount of protein antigens
synthesized in the host limited this type of
immunization[27]. Otherwise, DNA prime-protein boost immunization can enhance
both antigen-specific antibody and Th1-type cellular immune responses in other
studies[28]. Many cyto-kines, such as
IL-2[15,29,30], IFN-g[17],
GM-CSF[29], IL-12[15,31] have been co-injected to provoke better immune and protection responses. In our
experiment, we combined these 2 strategies and demonstrated the increased immunogenicity of a recombinant pertussis DNA
vaccine.
In conclusion, a multiple antigen-recombinant pertussis DNA vaccine can elicit the homologous specific protection
antibodies and cellular immune response. When the recombinant DNA plasmid is co-injected with another plasmid pGM-CSF
as the prime, and boosted with homologous purified protein, it showed significant increased specific humoral and cellular
immune responses. The results reported here provide a useful strategy of DNA immunization and may be applied in the
future.
Acknowledgements
We are extremely grateful to Dr David D HO(Rockefeller University, USA) and Dr Mi-Hua TAO (Institute of Biomedical
Sciences, Academia Sinica, China) for kindly providing the plasmids. We also thank Dr Jia-yan CHEN (University of Southern
California, USA) for the critical reading of the manuscript.
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