construction and immunogenic characterization of a fusion anti-caries
TRANSCRIPT
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Construction and Immunogenic
Characterization of a FusionAnti-caries DNA vaccine against
PAc and Glucosyltransferase I of
Streptococcus mutans
J.H. Guo, R. Jia, M.W. Fan, Z. Bian, Z. Chen and B. Peng
2004, China This is the first report where a DNA vaccine encoding gene
fragments of two virulence factors has been found toresult in better protection against dental caries than a
vaccine encoding one factor alone.
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Abstract
Glucosyltransferases (GTFs) and A cell-surface protein(PAc) are two important virulence factors of the cariogenicorganism S treptococcus mutans. They may mediate sucrose-
independent or sucrose-dependent attachment of S
treptococcusmutans to tooth surfaces, respectively. Thus, inhibiting both virulencefactors is predicted to provide better protection against caries thaninhibiting a single factor. To develop a highly efficient vaccine againstcaries, we constructed a fusion DN A v accine, pGLU A -P, by cloning the GLU region of GTF into a DNA vaccine, pCIA-P, which
encodes two highly conservative regions of PAc. In this report, weprovide evidence that fewer caries lesions were obser v ed in ratsfollowing subcutaneous injection of pGLUA-P, compared with pCIA-P,near the submandibular gland. Our findings suggest that a multigenicDNA vaccine may be more caries-preventive than a single-gene DNA vaccine.
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BACKGROUNDBACKGROUNDOF THE STUDYOF THE STUDY Dental caries
S treptococcus mutans
Cell-surface Protein A (PAc)
Glucosyltransferase (GTF)
DNA Vaccine
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DENTAL CARIES The mouth is a jungle. It is a home to bacteria,
viruses, fungi, and protozoa. Among these microbes,the bacteria are the most numerous with over 100million in every milliliter of saliva from more than
600 different species. These little capitalizinginhabitants in our mouth are responsible for dentalcaries, a disease that remains highly commonamong humans. Probably the most infective of these
is the S treptococcus mutans.
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STREPTOCOCCUS MUTANS
Streptococcus mutans ( S . mutans) has beenstrongly implicated as a causative organism of dental caries because their colonization on tooth
surfaces is thought to be an important step forthe initiation of dental caries. Two mechanisms,one sucrose-independent and the other sucrose-dependent, mediate this process.
Glucosyltransferases (GTFs) and A cell-surfaceprotein (PAc) are two important virulencefactors of this bacteria.
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CELL-SURFACE PROTEIN A (PAc)
Involved in sucrose-independent colonization
Mediates the initial adherence of S . mutans to
acquired pellicles on tooth surfaces 2 adherence/immunogenic region:
N-terminal alanine rich region ( A region)
Proline rich region (P region)
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GLUCOSYLTRANSFERASE (GTF)
Involved in sucrose-dependent colonization
Catalyze water-soluble and water-insolubleglucan from sucrose
Play important roles in dental plaque formation
2 adherence/immunogenic region:
N-terminal catalytic (CAT region)
C-terminal glucan binding (GLU region)
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Due to their importancein the cariogenicity of S .mutans, these proteins
have been rationaltargets for the
development of an anti-caries vaccine.
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DNA VACCINE
An anti-caries DNA vaccine, pCIA-P, encodestwo regions of Pac and induces protective anti-caries immune responses. This lead to a new
experiment which was evaluated in comparison with pCIA-P of the resulting systemic andmucosal immune responses and anti-cariesprotection in a rat model.
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Compared with traditional vaccines, DNA vaccines have obvious advantages:
long-term and stable expression of endogenously
produced antigenic protein (similar inconformation to the natural)
stronger antigenicity, with the capacity to induce both cellular and humoral immune responses
the possibility for creation of a polyvalent vaccineagainst several kinds of pathogens
DNA VACCINE
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Antibody against a protein fusing the A region of PAc and the GLU region of GTF-I, whichsynthesize water-insoluble glucan, inhibited
adhesion of S . mutans to saliva-coatedhydroxyapatite (S-HA) and glucan synthesis by GTFs. A chimeric protein consisting of the two
virulence determinants enhanced mucosal
immune responses. Thus, inhibiting both v irulence factors is predicted to pro v ide better protection against caries.
DNA VACCINE
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THETHEEXPERIMENTEXPERIMENT Objectives
Materials and Methods
Results
Discussion
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OBJECTIVES
To develop a highly efficient vaccine againstcaries via construction of a fusion DNA vaccine,
pGLUA-P To clone the GLU region of GTF into a DNA vaccine, pCIA-P
To assess the effectiveness of the fusion DNA
vaccine against caries by immunizing rats andanalyzing its resulting effects
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MATERIALS AND METHODS
PlasmidConstruction
Fusion Protein
Expression inCultured Cells
Immunizationof Rats
Antibody
Analysis
Statistical Analysis
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Plasmid Construction
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W e verified itsviability by
restrictiondigestion and by sequencingthe completely
inserted DNA .
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Fusion Protein Expression
Recombinant fusion protein was performed in a
transient transfection assay with the use of (1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propyl-amid
Briefly, Chinese hamster ovary cells were
incubated with DNA-liposome complexes for 5hrs and cultured overnight with CHO medium
Evaluated with a fluorescent immunoassay withavidin-biotin-Cy3 Complex
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The slides were incubated with anti-PAc IgG
antibody or anti-GTF antibody at 37°C for 30min and kept at 4°C overnight.
They were then incubated with biotinylated goatanti-rabbit IgG and avidin-biotin-Cy3 complex
in turn and viewed under a fluorescencemicroscope.
Fusion Protein Expression
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Immunization of Rats
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S. mutans Ingbritt
injection of pGLU A -Pinto thequadricepsfemorismuscle
(GLUA-P/i.m.)
subcutaneousinjection of pGLU A -Pnear thesubmandibular gland
(GLUA-P/s.c.)
injection of pCI A -P intothequadricepsfemorismuscle ( A-
P/i.m.)
subcutaneousinjection of pCI A -P nearthesubmandibular gland
( A-P/s.c.)
injection of 100-L pCI v ector (1g/L) intothequadriceps
femorismuscle.
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Immunization of Rats
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Antibody Analysis
For measurement of anti-PAc or anti-GTF IgA and IgG antibody responses in saliva and serum
Each well of an ELIS A plate was coated with P A c (10g/mL in carbonate buffer, pH9.6) or rGTF (10 g/mL incarbonate buffer, pH 9.6)
o v ernight at 4°C and then blocked with phosphate- buffered saline (PBS, pH7.2) containing 3% bo v ineserum albumin (BS A).
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After samples were washed with PBS containing 0.1%T ween (PBST, pH 7.2), a 100-L quantity of diluted sali v aor sera was added to each well and incubated for 1.5 hrsat 37°C.
Antibody Analysis
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Each well was washed again with PBST, and then treated with 100-L quantities of goatanti-rat IgG or goat anti-rat Ig A (1:1000; Sigma),incubated for 2 hrs at 37°C,and washed again.
Antibody Analysis
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Next, a 100-L quantity of alkaline-phosphatase-conjugated rabbit anti-goat IgG (1:10000; Sigma) was added to each well and
incubated for 5 hrs at 37°C,followed by phosphatesubstrate (-nitrophenylphosphate) for30 min at 37°C.
Antibody Analysis
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Optical density (OD) readings were taken at
405 nm. The end-point titer was defined as thehighest dilution with an absorbance 0.1 abovethat of the sham control.
Antibody Analysis
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RESULTS
Expression of Recombinant Fusion Protein
Antibody Responses to PAc
Antibody Responses to GTF
Caries Protection
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Recombinant Fusion Protein
Recombinant fusion protein detected by anti-GTF antibody in the cytoplasm of CHOcells transfected with pGLU A -P.
Recombinant fusion protein detected by anti-PAc antibody in the cytoplasm of CHOcells transfected with pGLU A -P.
CHO cells transfected with pCI A -P,
detected by anti-PAc antibody.
CHO cells transfected with pCI v ector. Bar,5 m.
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Many cells positively stained with a fluorescentred were found in the cytoplasm of:
pGLUA-P-transfected CHO cells incubated with both anti-PAc IgG and anti-GTF antibody
pCIA-P-transfected CHO cells incubated withanti-PAc IgG antibody
No such specific products could be found in cells
transfected with pCI vector.
Analysis of the data indicates that the plasmidpGLU A -P has the ability to express both P A c and GTF protein in eukaryotic cells.
Recombinant Fusion Protein
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Antibody Responses to PAc
The anti-PAc IgG ELIS A end-point titers in theserum of rats immunized DNA vaccines weresignificantly higher than those of control rats (p< 0.01).
(GLU A -P/i.m.)
(GLU A -P/s.c.)
( A -P/i.m.) ( A -P/s.c.) Control
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Antibody Responses to PAc
Mean serum PAc-specific IgG antibody titersin the GLUA-P/i.m. and A-P/i.m groups weresignificantly higher than those in the GLUA-P/s.c. and A-P/s.c. groups (p < 0.05).
(GLU A -P/i.m.)
(GLU A -P/s.c.)
( A -P/i.m.) ( A -P/s.c.) Control
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Antibody Responses to PAc
Sali v ary anti-PAc Ig A antibody responses inthe GLUA-P/s.c. and A-P/s.c. groups weresignificantly higher than those in the control,GLUA-P/i.m., or A-P/i.m. group (p < 0.01).
(GLU A -P/i.m.)
(GLU A -P/s.c.)
( A -P/i.m.) ( A -P/s.c.) Control
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There was no notable difference in serum anti-PAc IgG antibody responses between the GLUA-P/i.m. group and the A-P/i.m. group (p > 0.05).
There was no notable difference in serum andsaliva anti-PAc antibody responses between the
GLUA-P/s.c. group and the A-P/s.c. group (p >0.05).
Antibody Responses to PAc
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Antibody Responses to PAc
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Antibody Responses to GTF
No GTF-specific antibody response was detectedin serum or saliva from rats immunized withpCIA-P.
(GLU A -P/i.m.)
(GLU A -P/s.c.)
( A -P/i.m.) ( A -P/s.c.) Control
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Antibody Responses to GTF
Anti-GTF IgG ELIS A end-point titers in serumof rats immunized with pGLUA-P weresignificantly higher than those of control rats (p< 0.01).
(GLU A -P/i.m.)
(GLU A -P/s.c.)
Control
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Antibody Responses to GTF
Mean serum GTF-specific IgG antibody titer inthe GLUA-P/i.m. group was significantly higherthan that in the GLUA-P/s.c. group (p < 0.05).
(GLU A -P/i.m.)
(GLU A -P/s.c.)
( A -P/i.m.) ( A -P/s.c.) Control
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Antibody Responses to GTF
Sali v ary anti-GTF IgA levels in the GLUA-P/s.c. group were significantly higher than thosein either the GLUA-P/i.m. group or the controlgroup (p < 0.01).
(GLU A -P/i.m.)
(GLU A -P/s.c.)
Control
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Antibody Responses to GTF
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Groups of rats immunized with anti-cariesDN A v accines had significantly less cariesexperience than was observed in the controlgroup (p < 0.01).
(GLU A -P/i.m.)
(GLU A -P/s.c.)
( A -P/i.m.) ( A -P/s.c.) Control
Caries Protection
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Subcutaneous administration of DNA vaccinesnear the mandibular gland afforded betterprotection against caries than did intramuscularinjection (p < 0.01).
(GLU A -P/i.m.)
(GLU A -P/s.c.)
( A -P/i.m.) ( A -P/s.c.) Control
Caries Protection
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Group GLU A -P/s.c. rats displayed thefewest enamel lesions (p < 0.01), while slightdentinal lesions (p < 0.05) were seen in allgroups.
(GLU A -P/i.m.)
(GLU A -P/s.c.)
( A -P/i.m.) ( A -P/s.c.) Control
Caries Protection
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Caries Protection
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Conventional vs. DNA Vaccine
Traditional Vaccines DN A Vaccines
stimulate only the antibody
response
stimulates both the antibody and
cell-mediated immune responseLess effective and shoretr durationof immunity
More effective and longer period of immunity
Shorter shelf life Easy to store and longer shelf life
Limited Large-scale manufacturingprocedures available
More complicated; vaccinesmanufactured under certainconditions
Allows a more simplified andeffective quality control process
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2 Virulence Factors of Streptococcus Mutans
1. PAc
Involved in the Sucrose-independent mechanism
Mediates initial adherence of S . Mutans to pellicles
2 adherence/immunogenic region:
a. N-terminal alanine rich region ( A region)
b. Proline rich region (P region)
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2. GTF (Glucosyltransferase)
involved in the sucrose-dependent mechanism
Catalyzing water-soluble and water-insoluble glucanfrom sucrose
Play important roles in dental plaque formation
2 adherence/immunogenic region:a. N-terminal catalytic (CAT region)
b. C-terminal glucan binding (GLU region)
2 Virulence Factors of Streptococcus Mutans
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Anti PAc Antibodies
suppressed the adhesion of S. Mutans in the absence of sucrose
Inhibit sucrose-dependent adhesion of the organism
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Antibodies against GTFs
antibodies against synthesized peptides derived fromCAT and GLU region could inhibit glucan synthesis by GTFs
A NTIBODY A CTION
antibodies against
synthesized peptides derived
from CAT region only
could not inhibit glucansynthesis
antibodies against
synthesized peptides derived
from GLU region only
Recognize S. Sobrinusglucan-binding protein whichimprove colonization of organism to adhere glucans
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Antibodies against Recombinant Protein
P A c A P A
GTFCould not inhibit
glucan synthesis
A. PAc protein
B. GTF
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GTF
A. PAc protein
B. GTF
CAT
P A c A P A
markedly inhibit
glucan synthesis
Antibodies against Recombinant Protein
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pGLUA-P
Rats immunized with pGLUA-P displayedsignificantly fewer enamel lesions
DNA vaccine encoding gene fragments of both virulence factors has been found to protect better than vaccines against one factor
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Fusion anti-caries DNA vaccine was also foundto enhance or promote secretion of IgA antibodies
Recall:
MUCOS AL IMMUNITY
-lysozymes-lactoferrin
-IgA
pGLUA-P
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CONCLUSION
Anti-caries DNA vaccine, the pGLUA-P couldexpress GLUA-P protein in eukaryotic cells andcan induce specific immune response
Immunization of polyvalent DNA vaccine
provided better protection than single gene DNA
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CRITIQUE ANDCRITIQUE ANDRECOMMENDATIONRECOMMENDATION
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CRITIQUE
Overall, the study is good. They have coveredeverything under their focus.
The main focus of the study is to construct afusion anti-caries DNA vaccine against PAc andGlucosyltransferase I of Streptococcus mutans
Coverage: Anti-caries DNA vaccine
Streptococcus mutans
Immunization
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RECOMMENDATION
Immunization before presence of S treptococcusmutans
Multigenic DNA vaccine may be more caries-preventive than a single-gene DNA vaccine.
Recombinant protein DNA vaccine against root
surface caries.