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MATERIALS and METHODS

S. S. S. V. V. V. V. L. Base. WK 6. WK16. WK 32. WK 3. 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). RESULTS.

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MATERIALS and METHODS

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  1. S S S V V V V L Base WK 6 WK16 WK 32 WK 3 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). RESULTS 49417 Fig. 7 shows each sample from baseline, wk 16, and wk 32 from animal C (previously shown to have temporal changes in Pg strains detected after baseline - see Fig. 5) duplexed with Pg reference strain ATCC 49417. Lanes 1 and 3: Five heteroduplex bands are evident (3 bands appear faint in Lane 1 due to the lower concentration of 1 of the animal Pg strains) indicating 3 Pg strains detected (2 animal strains + reference strain). All heteroduplex bands have identical mobilities indicating all Pg strains are likely the same at baseline and wk 32. Lane 2: Two heteroduplex bands are present indicating 2 strains detected (1 animal strain + reference strain). These 2 bands correspond to 2 of the bands in Lanes 1 and 3 indicating that the Pg strain at wk 16 is also present at baseline and wk 32. Lane 4: negative control. Lane 5: positive control. Lane 6: DNA ladder. 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. DNA ladder Neg control Pos control ILE-tRNA C - wk 16 C - wk 32 ALA-tRNA C - base PG7R PG3R 785 Fig. 7 16S 23S 5S (Background) Fig. 1 L189 EricS 422 Fig. 3shows samples from animal A with only 1 strain of Pg in each sample for all time points. Each sample was duplexed by itself. Lane 1: baseline. Lane 2: wk 16. Lane 3: wk 32. Only homoduplexes are formed indicating 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. Heteroduplexes Lanes 1 and 3 :5 bands. Lane 2: 2 bands. W50/Hg1691 ISR DNA Ladder Neg control A - wk 16 A - wk 32 A - Base Fig.3 Homoduplexes Background: non-hybridized DNA Fig. 3 NESTED PCR 1 2 3 4 5 6 Note: heteroduplex in pos control Lane 5 Homoduplex only ( = 1 strain) (Fig. 6 reveals the 3 Pg strains to be of one clonal type by HA) The goal of this study was to investigate the effects of vaccination and ligation on predominant Pg clonal types in the ligature-induced periodontitis monkey model. The previous examples of 3 animals demonstrated that Pg clonal types as detected by 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 analyzed to date. 1 2 3 4 5 6 DNA Ladder W50/Hg1691 Neg control B - wk 16 B - wk 32 B - Base Fig. 4shows samples from animal B with multiple strains of Pg at baseline, wk16, and wk 32. Each sample was duplexed by itself. Lane 1: baseline. Lane 2: wk 16. Lane 3: wk 32. Three heteroduplex bands and homoduplexes are formed indicating detection of 3 Pg strains in each sample. The heteroduplex bands have identical mobilities indicating all 3 samples most likely have the same 3 Pg strains present. Lane 4: Negative control. Lane 5: Positive control (W50 duplexed with Hg 1691). Lane 6: DNA Ladder. Fig. 4 Background: non-hybridized DNA 1st PCR: “UNIVERSAL”(Template = Sample DNA) Table 1. Results from 14 animals analyzed for post-vaccination (at week 16 and/or week 32) changes in Pg clonal types detected as compared to Pg clonal types detected at baseline. 3 Heteroduplex bands (= 3 strains) Universal primers (Fig. 1). This amplification yields a PCR product containing the ISR and its flanking sequences from numerous bacterial species that may be present in the sample including Pg. 785 422 # of animals with no change from baseline Homoduplexes # of animals with change from baseline Test Group 1 2 3 4 5 6 3 Controls 3 2nd PCR: “Pg-specific”(Template = 1st PCR product) * sequence modified from original L189 by one nucleotide substitution ** sequence modified from original ERICS by 2 nucleotide substitutions 4 Vaccinated 4 Pg-specific (PG3R) and universal (L189) primers (Fig. 1). Only the species determined by the specific primer is amplified which yields a 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. Fig.2 W50/Hg1691 DNA Ladder Neg control PG3R L189* C - wk 32 C - wk 16 C - base Fig. 5shows animal C with temporal changes in Pg strains detected. Each sample was duplexed by itself. Lane 1: baseline - 1 heteroduplex band (unresolved) and homoduplexes - 2 Pg strains detected. Lane 2: wk 16 - homoduplex only - 1 Pg strain detected. Lane 3: wk 32 - 1 heteroduplex band (unresolved) and homoduplexes - 2 Pg strains detected. Lane 4: Negative control. Lane 5: (W50 duplexed with Hg1691). Lane 6: DNA Ladder. Vaccination or ligation did not affect the acqusition or loss of Pg clonal types in this study. Fig. 5 Background: non-hybridized DNA CONCLUSIONS Fig. 2. 1% agarose gel analysis of the “Pg-specific” and ISR PCR products. Lane 1: “Pg-specific PCR product (~1.7 Kb). Lane 2: ISR PCR product (~800 bp). Lane 3: 1 Kb DNA ladder. 2 kb • 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. 1 kb 3rd PCR: ISR(Template = 2nd PCR product or “agar stab” from 1.7 Kb Pg-specifics fragment in agarose gel) Heteroduplex 500 bp Homoduplexes Pg-specific (PG7R) and ERICS primers (Fig. 1). Amplify the ISR which yields a fragment of ~800 bp (Fig. 2, Lane 2). This PCR product is used for HA. 1 2 3 4 5 6 PG7R ERICS** • 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. 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) Schematic Examples of Duplex Formations 49417 Hg1691 Fig. 6 shows samples, baseline, wk 16, and wk 32 from 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. a1 Double strand match 1 strain (a) Homoduplex (runs to bottom of gel) a2 DNA Ladder Neg control Pos control A - wk 16 A - wk 16 A - wk 32 A - wk 32 A - base A - base REFERENCES 1. 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 Colonizationof 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. a1 a2 2 strains (a & b)  Homoduplexes (frun to bottom of gel) b1 Fig.6 b2 (Background) + Heteroduplexes (resolved) a1 Double strand mismatch b2 Heteroduplexes (unresolved) Heteroduplexes 1-2 bands (doublet may not be resolved) b1 Reciprocal strands Homoduplexes a2 3 strains  Homoduplexes + Heteroduplexes 3-6 bands (NOT SHOWN) + 1 2 3 4 5 6 7 8 9 2211 Temporal Changes in Porphyromonas gingivalis Clonal Types During a Macaca fascicularis Ligature-Induced Periodontitis Vaccine Study P. 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 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. Fig.2 • 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 ifPg clonal types were affected by conditions which altered Pg growth in vivo (vaccine suppression or ligature enhancement). • 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: S = sample collection for HA V = vaccination L = ligature placement • SAMPLE COLLECTION: paperpoint subgingival plaque samples from all teeth + tongue scrapings • DNA ISOLATION and PURIFICATION • AMPLIFICATION of ISR using NESTED POLYMERASE CHAIN REACTION (PCR)

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