Identification Of Secretory Fractions Of Enterococcus Faecalis For Inducing Trained Immunity And Its Adjuvant Effect

Trained immunity is a stronger response of innate immune cells to a second stimulus,homologous or heterologous,after an initial stimulus or infection,i.e.natural immune memory.In response to an infection or danger signal,innate immune cells recognize and respond to clear the infection or danger signal and the immune response returns to basal levels,while at the same time,innate immune cells undergo medium to long-term reprogramming changes in metabolism and epigenetics and become more responsive upon re-stimulation.Adjuvants are often required in vaccines such as inactivated or subunit to enhance the immune response.The discovery and mechanism of activating ligands based on trained immunity will provide the basis for the development of novel adjuvants and dual memory vaccines.This thesis therefore screens and identifies bacterial products that induce trained immunity and analyses their adjuvant effects by constructing a model for trained immunity studies.In vivo studies that monitor the biological response of an organism,as opposed to cellular level studies,allow for a global evaluation of the functional effects of exogenous stimuli.As the Galleria mellonella lack an adaptive immune system,and as the innate immune system also has a high degree of structural and functional similarity to that of vertebrates,this project team explored the development of a model for the study of trained immunity in the Galleria mellonella.In this study,we first determined the growth curve of the Candida albicans seed organism and conducted a screening of the optimal infective dose of Candida albicans to determine the optimal trained induction dose and the minimum lethal dose.Further we used the survival rate,degree of skin darkening,motility and insect haemolymph loadings of the Galleria mellonella evaluation indicators for the trained immunity model.Candida albicans is known to have a trained immunity induction effect due to its cell wall component β-glucan,which enhances the responsiveness of innate immune cells to reinfection with Candida albicans,and therefore Candida albicans was chosen as the initial stimulating and restimulating factor for the trained immunity model.Galleria mellonella were injected with different doses of Candida albicans and received lethal doses of Candida albicans infection after a 3 or 7 d interval.The results showed that pre-stimulation with Candida albicans significantly increased the survival and motility of Galleria mellonella and reduced the degree of body surface darkening and haemolymph load,suggesting that Candida albicans induces domestication immunity in Galleria mellonella.Using an immune screening model for the trained immunity of Galleria mellonella,a self-constructed symbiotic bacterium of livestock origin from this laboratory was screened for trained immunity-inducing activity and Enterococcus faecalis WY-15 was found to have a trained immunity-inducing effect.Further studies showed that both live Enterococcus faecalis WY-15 and culture supernatant were trained immunity-inducing.To isolate the trained immunity-inducing material produced by Enterococcus faecalis,we used saturated ammonium sulphate precipitation for crude extraction,ion exchange chromatography for preliminary purification,and preparative high performance liquid chromatography(HPLC)for fine purification.The F4-2 fraction obtained was identified by MALDI-TOF/TOF mass spectrometry as ribosomal protein S11 and its coding gene.The activity of ribosomal protein S11 was analysed using mouse peritoneal macrophages.The results showed that macrophages pre-stimulated with ribosomal protein S11 showed increased responsiveness to secondary stimuli,including increased release of nitric oxide,increased secretion levels of TNF-α and IL-6,and enhanced phagocytosis and killing of Staphylococcus aureus.In order to improve the yield of ribosomal protein S11 and to fully validate the trained immunity induction of ribosomal protein S11,a recombinant expression system was constructed by applying p ET-28 a and E.coli BL21(DE3)to obtain recombinant ribosomal protein S11,and the recombinant and purified ones had comparable trained immunity induction activity.Ribosomal protein S11 has the potential to be used as an adjuvant due to its trained immunity-inducing properties.A preliminary analysis of its safety was performed by cytotoxicity assays.The results showed no significant cytotoxicity of ribosomal protein S11 against mouse peritoneal macrophages and RAW 264.7 cells at effective working concentrations.In vitro haemolysis assays showed that ribosomal protein S11 did not significantly haemolysis erythrocytes in mice and chickens.Stability assays showed that ribosomal protein was thermally and p H stable and could be hydrolysed by trypsin and proteinase K.The adjuvant effect of ribosomal protein S11 was analysed in mice immunised with ovalbumin(OVA)as the model antigen.Compared with aluminium adjuvant,the OVA and ribosomal protein S11 group produced higher levels of OVA-specific Ig G and had a significantly enhanced humoral immune response,with significant weight gain in splenic tissues.The proliferation of lymphocytes was significantly enhanced by CCK8 assay,indicating that ribosomal protein S11 could induce cellular immune responses.These results suggest that ribosomal protein S11 has great potential as a novel adjuvant.In summary,in this study,the Galleria mellonella were applied to a trained immunity model study to screen for symbiotic bacteria that induce trained immunity.In vitro safety experiments showed that ribosomal protein S11 has good biosafety and stability.The adjuvant effect showed that ribosomal protein S11 enhances OVA-specific humoral immune response compared to aluminium adjuvant,and therefore has great potential as an excellent adjuvant.