Cytotoxic T cell adjuvant effects of three Salmonella enterica flagellins

Abstract

Bacterial flagellins are important virulence-associated factors and strong inducers of inflammatory responses in mammalian hosts. Flagellins have also been investigated as potential vaccine adjuvants, either for induction of humoral or cellular immune responses, to different target antigens. In this study we investigated the adjuvant properties of three Salmonella enterica flagellins types (FliCd, FliCi and FljB) to an ovalbuminderived CD8 + T cell-restricted epitope (OVA257-264). Although mice immunized with the three tested flagellins elicited antigen-specific activated CD8 + T cells, only animals immunized with FliCi and FliCd flagellins admixed with ovalbumin mounted specific in vivo cytotoxic responses to peptide-pulsed target cells. The present results indicate that Salmonella flagellins are endowed with type-specific adjuvant effects toward murine CD8 + T cells, a feature that may impact their use as adjuvants for prophylatic or therapeutic vaccines.

Functional Diversity of Heat-labile Toxins (LT) Produced by Enterotoxigenic Escherichia coli

“…Cytokine Measurements-For evaluation of cytokine production, the animals were euthanized 2 weeks after the immunization protocol, and spleen cells were harvested according to a previously reported protocol (42). Spleen cells (1 ϫ 10 7 viable cells/ml) were cultured in a CO 2 -containing atmosphere at 37°C in the presence or absence of OVA (5 g/ml), , named OVA 265-280 ) (1.2 g/ml).…”

“…Enzyme-linked Immunosorbent Spot (ELISPOT) AssayThe INF-␥ ELISPOT assay was performed to detect activated CD8 ϩ T lymphocytes specific for the OVA 257-264 peptide, as described previously (42). Two weeks after the immunization regimen, cells (2 ϫ 10 5 ) derived from spleens of vaccinated mice were placed into wells of a 96-well microtiter plate (MultiScreen; Millipore) previously coated with 10 g/ml of rat anti-mouse mAb specific to INF-␥ (BD PharMingen).…”

“…In Vivo Determination of Cytotoxic CD8 ϩ T LymphocytesThe in vivo cytotoxic activity of CD8 ϩ T cells in OVA 257-264 -pulsed spleen cells was determined according to the method described previously (42). Splenocytes from naive mice were stained with 0.5 or 5 M carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen) during 15 min of incubation at 37°C.…”

“…The following day, the inoculated animals were euthanized, and individual spleens were isolated to identify the two CFSE-labeled cell populations by FACS. The percentages of specific target cell killing were calculated by the relative reduction of peptide-pulsed cells in regard to the nonpulsed cells, as described previously (42).…”

Avaliação terapêutica da formulação vacinal baseada no peptídeo P10 derivado da glicoproteína (gp43) de Paracoccidioides brasiliensis e na flagelina de Salmonella (FliCd).

“…Após a remoção das células por centrifugação, o sobrenadante foi coletado e adicionado 3 a 4 volumes de acetona gelada para precipitação da flagelina presente. Após 30 min de repouso a -20 ºC, o material foi centrifugado e o precipitado ressuspenso em aproximadamente 0,5 mL de PBS (BRAGA et al, 2008). As flagelinas purificadas passaram por um tratamento (65 ºC por 30 min) para dissociação dos monômeros.…”