Recombinant fimbriae protein of Porphyromonas gingivalis induces an inflammatory response via the TLR4/NF­κB signaling pathway in human peripheral blood mononuclear cells.

Porphyromonas gingivalis (P. gingivalis) is a periodontal pathogen that may accumulate with other organisms in subgingival plaque biofilms and is associated with periodontal disease. P. gingivalis fimbriae (FimA) is a filamentous structure on the surface of bacteria that is closely associated with bacterial adhesion to and colonization of host tissues, and serves an essential role in biofilm formation. The present study aimed to construct P. gingivalis FimA prokaryotic expression plasmids, purify a FimA fusion protein and explore the effect of a recombinant FimA protein on the inflammatory response in human peripheral blood mononuclear cells (PBMCs). P. gingivalis FimA prokaryotic expression plasmids were constructed by gene cloning and recombination technology. SDS­PAGE was used to evaluate the purified recombinant FimA protein. The cell proliferation rate and inflammatory cytokine expression of PBMCs treated with the FimA fusion protein with or without transfection with toll­like receptor 4 (TLR4) small interfering (si)RNA were detected by CCK­8 assays and ELISAs, respectively. The expression levels of TLR4, nuclear factor kappa­light­chain­enhancer of activated B cells (NF­κB) and myeloid differentiation primary response 88 (MyD88) in PBMCs were detected by western blot analysis and reverse transcription quantitative polymerase chain reaction. A FimA fusion protein with high purity was obtained. FimA fusion protein treatment significantly increased PBMC proliferation and promoted the release of tumor necrosis factor­α (TNF­α), interleukin (IL)­6, matrix metalloproteinase (MMP)­8 and MMP­9 in PBMCs. TLR4 interference reversed the effects of the FimA fusion protein on PBMC proliferation and inflammatory cytokine release. The expression levels of TLR4, NF­κB and MyD88 in PBMCs were significantly increased following treatment with the FimA fusion protein, while the expression levels of these genes at the mRNA and protein levels decreased significantly in PBMCs following FimA fusion protein treatment and TLR4 interference. The FimA fusion protein increased PBMC proliferation and promoted the release of the inflammatory cytokines TNF­α, IL­6, MMP­8 and MMP­9 via the TLR4/NF­κB signaling pathway. FimA may serve as a promising therapeutic strategy for periodontal disease.

Construction and phase I clinical evaluation of the safety and immunogenicity of a candidate enterotoxigenic Escherichia coli vaccine strain expressing colonization factor antigen CFA/I.

Oral delivery of toxin-negative derivatives of enterotoxigenic Escherichia coli (ETEC) that express colonization factor antigens (CFA) with deletions of the aroC, ompC, ompF, and toxin genes may be an effective approach to vaccination against ETEC-associated diarrhea. We describe the creation and characterization of an attenuated CFA/I-expressing ETEC vaccine candidate, ACAM2010, from a virulent isolate in which the heat-stable enterotoxin (ST) and CFA/I genes were closely linked and on the same virulence plasmid as the enteroaggregative E. coli heat-stable toxin (EAST1) gene. A new suicide vector (pJCB12) was constructed and used to delete the ST and EAST1 genes and to introduce defined deletion mutations into the aroC, ompC, and ompF chromosomal genes. A phase I trial, consisting of an open-label dose escalation phase in 18 adult outpatient volunteers followed by a placebo-controlled double-blind phase in an additional 31 volunteers, was conducted. The vaccine was administered in two formulations, fresh culture and frozen suspension. These were both well tolerated, with no evidence of significant adverse events related to vaccination. Immunoglobulin A (IgA) and IgG antibody-secreting cells specific for CFA/I were assayed by ELISPOT. Positive responses (greater than twofold increase) were seen in 27 of 37 (73%) subjects who received the highest dose level of vaccine (nominally 5 x 10(9) CFU). Twenty-nine of these volunteers were secreting culturable vaccine organisms at day 3 following vaccination; five were still positive on day 7, with a single isolation on day 13. This live attenuated bacterial vaccine is safe and immunogenic in healthy adult volunteers.