construction and immunogenic characterization of a fusion anti-caries

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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.



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.



BACKGROUNDBACKGROUNDOF THE STUDYOF THE STUDY Dental caries

S treptococcus mutans

Cell-surface Protein A (PAc)

Glucosyltransferase (GTF)

DNA Vaccine



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.



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.



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)



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)



Due to their importancein the cariogenicity of S .mutans, these proteins

have been rationaltargets for the

development of an anti-caries vaccine.



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.



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



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



THETHEEXPERIMENTEXPERIMENT Objectives

Materials and Methods

Results

Discussion



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



MATERIALS AND METHODS

PlasmidConstruction

Fusion Protein

Expression inCultured Cells

Immunizationof Rats

Antibody

Analysis

Statistical Analysis



Plasmid Construction



W e verified itsviability by

restrictiondigestion and by sequencingthe completely

inserted DNA .



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



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



Immunization of Rats



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.



Immunization of Rats



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).



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



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



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



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



RESULTS

Expression of Recombinant Fusion Protein

Antibody Responses to PAc

Antibody Responses to GTF

Caries Protection



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.



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




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




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.)





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.)




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).





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.)





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




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.)





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



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.)


Caries Protection



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.)


Caries Protection



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.)


Caries Protection



Caries Protection



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



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)



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



Anti PAc Antibodies

suppressed the adhesion of S. Mutans in the absence of sucrose

Inhibit sucrose-dependent adhesion of the organism



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



Antibodies against Recombinant Protein

P A c A P A

GTFCould not inhibit

glucan synthesis

A. PAc protein

B. GTF



GTF

A. PAc protein

B. GTF

CAT

P A c A P A

markedly inhibit

glucan synthesis

Antibodies against Recombinant Protein



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



Fusion anti-caries DNA vaccine was also foundto enhance or promote secretion of IgA antibodies

Recall:

MUCOS AL IMMUNITY

-lysozymes-lactoferrin

-IgA

pGLUA-P



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



CRITIQUE ANDCRITIQUE ANDRECOMMENDATIONRECOMMENDATION



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



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.

construction and immunogenic characterization of a fusion anti-caries

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