RESUMO
BACKGROUND: Staphylococcus aureus is unrestrictedly found in humans and in animal species that maintain thermal homeostasis. Inadequate cleaning of processing equipment or inappropriate handling can contaminate processed food and cause severe food poisoning. Staphylococcal enterotoxin B (SEB), a potent superantigenic exotoxin, is produced by 50% of clinical isolates of S. aureus and is associated with massive food poisoning and with the induction of toxic shock syndrome. RESULTS: A gene sequence encoding a recombinant SEB (rSEB), devoid of superantigenic activity, was successfully cloned and expressed in a cytoplasmic or a secreted form in the food-grade lactic acid bacterium Lactococcus lactis. The recombinant protein detected in the cytoplasm or in the culture medium exhibited the expected molecular mass and was recognized by a SEB-polyclonal antibody. Oral immunization with the recombinant L. lactis strains induced a protective immune response in a murine model of S. aureus infection. Immunized mice survived intraperitoneal challenge with an S. aureus SEB-producer strain. Counts of S. aureus in the spleen of rSEB-immunized mice were significantly reduced. The rSEB-immunized mice showed significant titers of anti-SEB IgA and IgG in stools and serum, respectively. Both recombinant L. lactis strains were able to elicit cellular or systemic immune responses in mice, with no significant difference if rSEB was produced in its cytoplasmic or secreted form. However, recombinant L. lactis expressing the cytoplasmic rSEB increased the survival rate of the challenged mice by 43%. CONCLUSIONS: These findings show the vaccine efficacy of L. lactis carrying an attenuated SEB, in a murine model, following lethal S. aureus challenge.
Assuntos
Enterotoxinas/metabolismo , Lactococcus lactis/imunologia , Administração Oral , Animais , Anticorpos/metabolismo , Enterotoxinas/genética , Lactococcus lactis/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/metabolismoRESUMO
Using the natural killer (NK) cell-surface marker CD56 to study NK T cells in peripheral blood, we found that their frequency in mononuclear cells among healthy individuals was 1%-20% (average, 7.3%) and sporadically increased 4-5-fold within individuals over the course of 8 months. Infection of mononuclear cells in vitro with Venezuelan equine encephalitis virus replicon particles (VRPs) resulted in a significant increase in CD56(+) T cells and in the expression of interferon-alpha, tumor necrosis factor (TNF)-alpha, and interferon-gamma by CD56(+) but not CD56(-) T cells. NK and CD56(+) T cells expressed higher levels of Toll-like receptor (TLR)-3 and TLR4 after infection with VRPs, whereas only NK cells expressed inducible TNF-alpha and TLR2. Most of these effects were duplicated by activating mononuclear cells with double-stranded RNA. These expression patterns indicate that T cells coexpressing NK markers respond like NK cells to viral infection or double-stranded RNA, potentially fulfilling innate and adaptive immune functions.
Assuntos
Antígeno CD56/imunologia , Citocinas/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Células Matadoras Naturais/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Linfócitos T/imunologia , Animais , Cricetinae , Citocinas/biossíntese , Citocinas/genética , Vírus da Encefalite Equina Venezuelana/genética , Citometria de Fluxo , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/virologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , RNA/química , RNA/genética , RNA de Cadeia Dupla/imunologia , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Replicon/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Linfócitos T/virologia , Receptor 2 Toll-Like , Receptor 3 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
A candidate vaccine against staphylococcal enterotoxin B (SEB) was developed using a Venezuelan equine encephalitis (VEE) virus vector. This vaccine is composed of a self-replicating RNA, termed "replicon," containing the VEE nonstructural genes and cis-acting elements and a gene encoding mutagenized SEB (mSEB). Cotransfection of baby hamster kidney cells with the mSEB replicon and 2 helper RNA molecules resulted in the release of propagation-deficient mSEB-VEE replicon particles (mSEB-VRPs). Mice inoculated subcutaneously with mSEB-VRPs were protected (15 of 20 mice) from a challenge with 5 median lethal dose units of wild-type (wt) SEB. T cells from mice vaccinated with mSEB-VRP responded normally both in vitro to wt SEB and in recall response to the inactivated mSEB polypeptide. The profile of cytokines measured after challenge with wt SEB suggested that the mode of protection was predominantly Th1 dependent. Our results suggest that the VEE replicon is a practical and convenient model system for evaluating efficacy of vaccines for the control of bacterial diseases.