MATERIALS and METHODS

• HA was employed to detect changes in Pg clonal types in a ligature-induced periodontitis vaccine study by comparing pre-vaccination numbers of Pg clonal types at baseline with samples collected post-vaccination at week 16 and post-ligation at week 32.

• HA PROTOCOL - Method established by Leys et al. (3) was employed. The procedure is briefly described below and exceptions to the referenced protocol are noted.

• Principle of HA: the ISR is predominately a non-coding region between the highly conserved 16S and 23S ribosomal genes. Lack of selective pressure results in accumulation of mutations and greater sequence diversity thus allowing for discrimination between strains of a species. When PCR amplified ISR DNA is melted and slowly cooled, double-stranded DNA is formed. Identical strands reanneal to form homoduplexes and mismatched strands form heteroduplexes that migrate more slowly than homoduplexes during polyacrylamide gel electrophoresis.

• IMMUNOGEN: A Pg-derived peptide-based immunogen, Rgp-Kgp, was used in this study.

• RELEVANT TIME POINTS to SAMPLING:

Temporal Changes in Porphyromonas gingivalis Clonal Types During a Macaca fascicularis Ligature-Induced Periodontitis Vaccine StudyP. H. Braham*, R. P. Darveau, T. J. Sims, F. A. Roberts, G. R. Persson, L. S. Houston, and R. C. Page

University of Washington, Seattle, WA 98195

2211

ABSTRACT

In a previous Porphyromonas gingivalis (Pg) vaccine study, protection of alveolar bone loss in a Macaca fascicularis (Mf) ligature induced periodontitis model was reported. Although Pg was suppressed, it was not eliminated in vaccinated animals. Objective: To determine if the observed persistence of Pg might be related to the acquisition of different Pg clonal types (CTs) following vaccination. Method: Heteroduplex analysis (HA) of the Pg 16S/23S ribosomal intergenic spacer region (ISR) has successfully been used to detect multiple Pg CTs in the oral cavities of humans and Mf. This method was chosen to initially examine 2 experimental and 3 control animals for changes in Pg CTs in another Pg-based vaccine study of 20 animals. Pooled subgingival plaque and tongue scrapings were taken at baseline, week 16 (prior to ligature placement), and week 32. DNA was extracted and nested PCR was performed to amplify the ISR. HA was used to detect differences in the ISR fragments and therefore differences in Pg CTs. Results: Preliminary results showed no change in Pg CTs in 1 experimental and 2 control animals. However, a second clonal type was acquired after ligation in 1 experimental animal, and 1 of 2 clonal types was apparently lost in 1 control animal by week 16. Conclusion: Heteroduplex analysis revealed that Porphyromonas gingivalis clonal types may be both lost and acquired during a longitudinal vaccine study using the Macaca fascicularis ligature-induced periodontitis model. Patterns of acquisition and loss of Pg CTs, and any correlations to vaccination will be determined upon analysis of all 20 animals. Supported by NIH/NIDCR R01 DE12939.

INTRODUCTION

Periodontitis is a chronic inflammatory disease that is a major cause of tooth loss. Porphyromonas gingivalis (Pg) is strongly implicated in the pathogenesis of the disease.

Previously, we have employed a ligature-induced periodontitis model in the nonhuman primate, Macaca fascicularis (Mf), to examine a whole-cell based Pg vaccine, which was subsequently shown to be protective against alveolar bone loss.

It has been reported that in the oral cavity of Mf harboring Pg, the number of Pg clonal types that may be detected ranges from 1 to 3 using the method of Heteroduplex Analysis (HA) of the Pg 16S/23S ribosomal intergenic spacer region (2 ) as originally described by Leys et al. in their studies on differentiation and detection of multiple Pg clonal types in humans (3 ).

In our previous study, immunized animals that obtained high serum antibody titers to the vaccine strain of Pg (Pg 5083 originally isolated from Mf ) demonstrated suppression, but not elimination of Pg (1).

In contrast to suppression effected by vaccination, the placement of ligatures in the animal model enhances the numbers of total bacteria including Pg.

Therefore we asked if Pg clonal types were affected by conditions which altered Pg growth in vivo (vaccine suppression or ligature enhancement).

Fig.2

S = sample collection for HAV = vaccinationL = ligature placement

SV V V

S

L

SV

Base WK 6 WK16 WK 32WK 3

• SAMPLE COLLECTION: paperpoint subgingival plaque samples from all teeth + tongue scrapings

• DNA ISOLATION and PURIFICATION• AMPLIFICATION of ISR using NESTED POLYMERASE CHAIN REACTION (PCR)

2 kb

500 bp

1 kb

Fig. 2. 1% agarose gel analysis of the “Pg-specific” and ISR PCR products. Lane 1: “Pg-specific PCRproduct (~1.7 Kb). Lane 2: ISR PCR product (~800 bp). Lane 3: 1 Kb DNA ladder.

Fig.2

PRIMERS:

Schematic representation of the P. gingivalis ribosomal operon with DNA sequence areas targeted for PCR amplification with specifically designed primers as described in Leys et al. (3). Fig. 1(modified).

1st PCR: “UNIVERSAL” (Template = Sample DNA)

785422

Universal primers (Fig. 1). This amplification yields a PCR productcontaining the ISR and its flanking sequences from numerous bacterialspecies that may be present in the sample including Pg.

2nd PCR: “Pg-specific” (Template = 1st PCR product)

PG3RL189*

Pg-specific (PG3R) and universal (L189) primers (Fig. 1). Only thespecies determined by the specific primer is amplified which yieldsa Pg-specific fragment of ~ 1.7 Kb (Fig. 2, Lane 1) containing the ISR(Fig. 1). This product confirms the presence of Pg in the sample.

3rd PCR: ISR (Template = 2nd PCR product or “agar stab” from 1.7 Kb Pg-specifics fragment in agarose gel)

PG7RERICS**

Pg-specific (PG7R) and ERICS primers (Fig. 1). Amplify the ISRwhich yields a fragment of ~800 bp (Fig. 2, Lane 2). This PCRproduct is used for HA.

* sequence modified from original L189 by one nucleotide substitution** sequence modified from original ERICS by 2 nucleotide substitutions

NESTED PCR

• HETERODUPLEX ANALYSIS:

The ISR PCR product was heated (950C for 5 min.) to denature double-stranded DNA, and slowly cooled (to 25oC at 1oC/min.) so as to allow for duplex formation as described.

Schematic Examples of Duplex Formations

1 strain (a) Homoduplex (runs to bottom of gel)

2 strains (a & b) Homoduplexes (frun to bottom of gel)

Heteroduplexes 1-2 bands

(doublet may not be resolved)

+

3 strains Homoduplexes+

Heteroduplexes 3-6 bands

+

a1a2

a1a2

b2b1

Double strandmatch

Reciprocalstrandsa2

b1

Double strandmismatch

a1b2

(NOT SHOWN)

16S 23S 5S785 PG3R PG7R

EricS L189 422

ILE-tRNA ALA-tRNA

Fig. 1ISR

RESULTS

Figures 3, 4, and 5 show examples of Heteroduplex analysis of ISR samples duplexed alone from each of 3 animals (A, B, and C respectively ) at baseline, week 16, and week 32. Positive and negative controls and a DNA standard ladder ( DNA/EcoR 1 + Hind III) are included in each TBE 10% polyacrylamide gel.

W50

/Hg1

6 91

Fig. 4Fig. 4 shows samples from animal B with multiple strains of Pgat baseline, wk16, and wk 32. Each sample wasduplexed by itself. Lane 1: baseline. Lane 2: wk 16.Lane 3: wk 32. Three heteroduplex bands and homoduplexesare formed indicating detection of 3 Pg strains in eachsample. The heteroduplex bands have identical mobilitiesindicating all 3 samples most likely have the same 3 Pgstrains present. Lane 4: Negative control. Lane 5:Positive control (W50 duplexed with Hg 1691). Lane 6:DNA Ladder.1 2 3 4 5 6

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Background:non-hybridized DNA

3 Heteroduplexbands (= 3 strains)

Homoduplexes

Fig. 5 shows animal C with temporal changes in Pg strains detected. Each sample wasduplexed by itself. Lane 1: baseline - 1 heteroduplexband (unresolved) and homoduplexes - 2 Pg strainsdetected. Lane 2: wk 16 - homoduplex only - 1 Pg straindetected. Lane 3: wk 32 - 1 heteroduplex band(unresolved) and homoduplexes - 2 Pg strains detected.Lane 4: Negative control. Lane 5: (W50 duplexedwith Hg1691). Lane 6: DNA Ladder.

W50

/Hg1

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Homoduplexes

Heteroduplex

Background:non-hybridized DNA

Fig. 5

Differentiation of Pg Strains by Heteroduplex Analysis with Pg Reference Strains(e.g.: The ISRs of 2 given strains of Pg duplexed together form heteroduplexes with a specific mobility pattern)

Fig. 6 shows samples, baseline, wk 16, and wk 32from animal A (previously shown to have 1 strain of Pg at each timepoint - see Fig. 3), duplexed with each of 2 Pg reference strains, ATCC 49417 and Hg1691. Lanes 1, 2 , and 3: Baseline, wk 16, wk 32, respectively, duplexed with 49417. All 3 lanes display heteroduplexes with identical mobilities indicating all 3 unknown Pg strains are most likely identical. Lanes 4, 5, and 6: Samples from each respective timepoint duplexed with Hg1691. Again all heteroduplexes display identical mobilities. Lane 7: negative control. Lane 8: positive control. Lane 9: DNA ladder.

A -

bas

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bas

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49417 Hg1691

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Heteroduplexes (resolved)Heteroduplexes (unresolved)

Homoduplexes

1 2 3 4 5 6 7 8 9

(Background)Fig.6

(Fig. 6 reveals the 3 Pg strainsto be of one clonal type by HA)

Fig. 3 shows samples from animal A with only 1 strain of Pg in each sample for all time points. Each sample wasduplexed by itself. Lane 1: baseline. Lane 2: wk 16.Lane 3: wk 32. Only homoduplexes are formedindicating 1 Pg strain detected in each sample. Lane 4: negative control (no DNA). Lane 5: Positive control (Pg reference strains W50 and Hg1691 duplexed together - heteroduplex and homoduplex bands formed). Lane 6: DNA ladder.

Fig.3A -

Bas

eA

- w

k 16

A -

wk

32N

eg c

ont r

olW

50/H

g169

1D

NA

Lad

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Homoduplex only ( = 1 strain)

Note: heteroduplexin pos control Lane 5

Background:non-hybridized DNA

1 2 3 4 5 6

Fig. 3

REFERENCES1. Persson, R.G., D. Engel, C. Whitney, R. Darveau, A. Weinberg, M. Brunsvold, and R. C. Page. 1994.

Immunization against Porphyromonas gingivalis Inhibits Progression of Experimental Periodontitis in Nonhuman Primates. Infect. Immun. 62:1026-1031.

2. Braham, P., T. Sims, F. Roberts, R. Darveau, E. Leys, S. Lyons, and R. Page. 2001. Porphyromonas gingivalis Colonization of Macacca fascicularis: Enumeration of Clonal Types by Heteroduplex Analysis. J. Dent. Research. 80:167.

3. Leys, E. J., J.H. Smith, S.R. Lyons, and A.L. Griffen. 1999. Identification of Porphyromonas gingivalis Strains by Heteroduplex Analysis and Detection of Multiple Strains. J. Clin. Microbiol. 37:3906-3911.

The goal of this study was to investigate the effects of vaccination and ligation on predominant Pgclonal types in the ligature-induced periodontitis monkey model. The previous examples of 3 animals demonstrated that Pg clonal types as detectedby HA may remain stable or may exhibit apparent changes from baseline. The table below is a summary from all animals in the vaccinated and control groups analyzedto date.

Table 1. Results from 14 animals analyzed for post-vaccination(at week 16 and/or week 32) changes in Pg clonal typesdetected as compared to Pg clonal types detected at baseline.

Test Group # of animals with change from baseline

# of animals withno change from baseline

Controls 3 3

Vaccinated 4 4

Vaccination or ligation did not affect the acqusition or loss of Pg clonal types in this study.

Heteroduplex analysis of the P. gingivalis ribosomal intergenic spacer region, based on the protocol established by Leys et al. (3), proved to be a useful tool for studying temporal changes in Pg clonal types in M. fascicularis during a longitudinal vaccine study.

The oral cavity of M. fascicularis may harbor single or multiple clonal types of Pg that over time in some animals appear to remain stable and in other animals may exhibit apparent changes by either loss or acquisition/emergence of different Pg clonal types.

Vaccination nor ligation effected changes in predominate Pg clonals types in the 14 animals analyzed for this study.

The evidence in this study suggests that vaccination with the immunogen, Kgp-Rgp, does not select for novel clonal types of Pg.

We believe that temporal changes as revealed by heteroduplex analysis are effected by natural fluctuations in Pg populations combined with sampling procedures.

Final analysis of all 20 animals in this study has yet to be completed.

CONCLUSIONS

49417Fig. 7 shows each sample from baseline, wk 16, and wk 32 from animal C (previously shown tohave temporal changes in Pg strains detectedafter baseline - see Fig. 5) duplexed with Pg reference strain ATCC 49417. Lanes 1 and 3: Fiveheteroduplex bands are evident (3 bands appear faint in Lane 1 due to the lower concentrationof 1 of the animal Pg strains) indicating 3 Pg strainsdetected (2 animal strains + reference strain). All heteroduplexbands have identical mobilities indicating all Pgstrains are likely the same at baseline and wk 32.Lane 2: Two heteroduplex bands are present indicating2 strains detected (1 animal strain + reference strain).These 2 bands correspond to 2 of the bands in Lanes 1and 3 indicating that the Pg strain at wk 16 is alsopresent at baseline and wk 32. Lane 4: negative control. Lane 5: positive control. Lane 6: DNA ladder.

HeteroduplexesLanes 1 and 3 : 5 bands. Lane 2: 2 bands.

Homoduplexes

1 2 3 4 5 6

C -

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C -

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ontr

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(Background)

Fig. 7