Commensal oral streptococci based designer vaccine against tooth and gum diseases: a proof of principle study

Anokhi Patel; Kiyonobu Honma, Ph.D.; Ashu Sharma, Ph.D.School of Dental Medicine, University at Buffalo, Buffalo, NY, USA

ABSTRACT

PRELIMINARY DATA

CONCLUSIONS

INTRODUCTION

REFERENCES

The Cause: Tannerella forsythia – Major Culprit of Periodontitis• Periodontitis is a progressive inflammation of the periodontium, which

often leads to tooth loss.• Periodontitis is a poly-microbial disease in which the red-complex

bacteria Tannerella forsythia, Porphyromonas gingivalis, andTreponema denticola are strongly implicated.

• In this study, we delved into T. forsythia since it is one of the majorbacteria known to enhance the deterioration of tooth attachment inperiodontitis.

•Immunization with the recombinant BspA protein is able to induce specific immune response in mice.•Vaccination with the recombinant BspA polypeptide confers protection against T. forsythia infection in a mouse model.•In this proof-of-concept experiment, BspA polypeptide encoding gene was successfully cloned.•Immunization with Sg-BspA recombinants may reduce T. forsythia associated alveolar bone loss in mice.•S. gordonii vectors could be utilized for the development of a periodontal vaccine.

1. Sharma, A., and K. Honma. "Expression of Saliva-BindingEpitopes of The Porphyromonas Gingivalis FimA Proteinon the Surface Of Streptococcus Gordonii." Biochemicaland Biophysical Research Communications 258.1 (1999):222-26

2. Sharma, A., K. Honma, and R. T. Evans. "OralImmunization with Recombinant Streptococcus GordoniiExpressing Porphyromonas Gingivalis FimA Domains."Infection and Immunity 69.5 (2001): 2928-934

3. Sharma, A. "Virulence Mechanisms of TannerellaForsythia." Periodontology 54 (2010): 106-16

BspA gene was amplified by PCR (Polymerase Chain Reaction) from the T. forsythia genomic DNA asa template using synthetic primers. The amplified fragments were cloned into a plasmid vector forchromosomal integration into S. gordonii by genetic transformation.

OBJECTIVES

3000 bp2500 bp2000 bp

• To genetically modify non-pathogenic oral streptococci, Streptococci gordonii, to heterologouslyexpress the BspA protein of the periodontal pathogen Tannerella forsythia.

• BspA is a potential target antigen of T. forsythia, the immunization by which protect mice against T.forsythia infection.

• We will test in a mouse model of periodontitis if oral colonization with such modified streptococciprovides protection against T. forsythia.

Immunization(Sg-BspA)

Antibodies (IgG)

Cytokines

Inflammation

Tissue Destruction

T. forsythia

Epithelial cells

Fibrinogen

T. forsythia

DCsDCs MΦ

RESULTS

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

Bone Loss (mean mm/site)

Sham IF(Gr.A )

Tf43037 (Gr.B)

BFM571 (Gr.C)

rBspAImm+Tf43037(Gr. D )Sham Imm +Tf43037 (GrE)

0

200

400

600

800

1000

1200

1400

Whole-Cell-Specific IgG(µg/ml)

0.001

0.01

0.1

1

10

100

1000

10000

BspA-Specific Serum IgG(µg/ml)

We attempt to genetically modify oral non-pathogenic streptococci (Streptococcus gordonii) to express a virulence factor of the periodontal pathogen Tannerella forsythia. We will test in a mouse model to see if streptococci expressing surface antigen BspA provides protection against T. forsythia infection. My objectives during the past research period were to generate the recombinant DNA constructs for BspA-expressing S. gordonii. The bspA gene was amplified by PCR from the T. forsythia genomic

DNA and cloned into a plasmid vector for chromosomal integration into S. gordonii by genetic transformation. Our preliminary data showed that immunization with the recombinant BspA protein in mice elicits BspA-specific serum IgG response, and protects mice against T. forsythia infection. As a

proof of principle concept of recombinant vaccine for periodontitis and other infections, genetically modified S. gordonii expressing BspA (Sg-BspA) will be tested against T. forsythia infection in a mouse

model of periodontitis. We predict that oral immunization with Sg-BspA vaccine would reduce T. forsythia infection and the associated alveolar (jaw) bone loss in mice.

The Problem: Periodontal Disease

• Expensive and painful dental procedures and surgeries• Sore teeth and swollen, bleeding, sensitive gums• Embarrassing bad breath• Increased risk of heart attack and stroke• Digestive disorders and bone loss

The Solution: Streptococcus gordonii for Vaccine Delivery• Streptococcus gordonii is a non-pathogenic commensal organism of the

human oral cavity.• It can be genetically engineered to surface-express or secrete heterologous

vaccine antigens or therapeutic proteins.• Genetically modified live bacteria can colonize mucosal surfaces and

deliver vaccines or therapeutic molecules; these vectors can also provide‘herd immunity.’

• It does not require special storage/handling which makes it easy to use.

Figure 2: T. forsythia Figure 3: S. gordonii

Figure 7: A genetic fragment of BspA

gene can be successfully cloned

(2200 bp).

Figure 8: White colonies show successful transformation of the BspA gene.

Figure 1: Progression of PeriodontitisSource: Oramd.com

BspA

BspA

BspA

BspA

Plasmid (vector)

BspA

Forward primer

Reverse primer

BspA genesite

BspA

(1) Gene amplification by PCR (copy) 

(2) Sub‐cloned into vector (fuse)(2) Sub‐cloned into vector (fuse)

(3) Linearization (cut)(3) Linearization (cut)

(4) Ligation (glue)(4) Ligation (glue)

(5) TA cloning into Sg (insert)(5) TA cloning into Sg (insert)

Figure 4 (a,b,c): T. forsythia mechanisms Figure 5: Producing recombinant S. gordonii for vaccineFigures courtesy of Summar Amin and Stephanie Chummar

BspA

BspA

BspA

(1)

(2)

(3)

(4)(5)T. forsythia

Y

YMacrophage

MATERIALS & METHODS

4 (b)

(c)

5(a)

S. gordonii

Figure 6: BspA is an important virulence factor that can act as a protective antigen in potential periodontal vaccination.