| Listeria monocytogenes(Lm)is a Gram-positive intracellular bacterium.It can break through the intestinal tract,blood and blood-brain barrier of human or animal,and then cause clinical symptoms such as gastroenteritis,meningitis,sepsis and abortion.Its mortality rate can reach 20%-30%,it is a pathogen of zoonosis.The bacterium poses a great threat to human health and is currently treated with antibiotics for listeriosis.Vaccine immunization is one of the most important measures to prevent and control infectious diseases.Although the vaccine against listeriosis has been studied internationally for decades,there is no effective vaccine for clinical use.In view of the intracellular infection characteristics of Lm,a live attenuated vaccine strain with better stimulation of cellular and humoral immunity possesses research and development potential.In this study,we chosen a serotype 4b strain of Listeria monocytogenes NTSN that caused the outbreak of sheep listeriosis as the parental strain,constructed the mutant strain NTSNA actA/plcB/orfX with deletion three adjacent virulence genes act A,plcB and orfX,using the homologous recombination technique.The safety,protective immune response and differentiation of infected from vaccinated animals(DIVA)of vaccine candidate strain were explored in both mouse and sheep models.The three gene-deleted live attenuated Listeria monocytogenes vaccine strain can effectively reduce the bacteria loads and infection rate,thus contributes to improve the safety of animal-derived products from farm to table,potentially reduce the risk of human listeriosis,which is a powerful means to ensure the development of animal husbandry and human health,and has important public health significance.1.Construction,identification and stability of Listeria monocytogenes NTSNA actA/plcB/orfXUsing the homologous recombination technique,the three virulence genes actA,plcB and orfX genes in the Lm NTSN genome were knocked out.The results of RT-PCR,Western blotting and phospholipase activity showed that the deletion strain lost expression capability of actin aggregation factor ActA,phospholipase PC-PLC and dissolved bilayer membrane activity OrfX.Favorable genetic stability is a prerequisite for practical vaccine strains.In this study,the stability of vaccine candidate strain was studied by in vitro passage experiments and the results showed that the 5th,15th and 30th generation strains were compared with the primary vaccine candidate strain.Compared with the primary vaccine strain,the phenotype,biochemical characteristics,target gene and virulence gene sequence of the strain after passage were not changed.This indicates that the deletion strain has good stability and lays a foundation for studying the safety and immunological characteristics of the deletion strain.2.Safety and protective immune response characteristics of Listeria monocytogenes NTSNA actA/plcB/orfX in a murine modelSafety is one of the main considerations in the use of vaccines.In this study,we used 6-week-old female BALB/c mice as an animal model to evaluate the virulence of vaccine candidates by intraperitoneal inoculation.The LD50 showed that the NTSN ?actA/plcB/orfX virulence was 794-fold lower than that of the wild-type NTSN.The vaccine candidate strain was intraperitoneally inoculated with a dose 100 times higher than that of the wild strain,and the results showed that the vaccine candidate strain could be completely cleared by the body in a short time.However,more than 100 CFU/g of Listeria was still colonized in the spleen and liver of the wild-strained mice.Thus the infection kinetics experiment indicated that the three-gene deletion strain could be completely eliminated by the body in a short time.Histopathological results showed that there was no obvious pathological change in the liver,spleen,heart,lung and brain of NTSN?actA/plcB/orfX immunized group comparing with the control group,suggesting that the safety of the mutant was significantly improved.The level of antibody in mouse serum was determined by indirect ELISA,and the antibody level induced by the immunized group reached 5×104 U,which was significantly higher than that of the control group,indicating that the vaccine candidate strain can induce a good humoral immune response.In addition,to explore the immune response types induced by vaccine candidate,the serum titer of IgG2a and IgG1 was determined,it showed that the titer of IgG2a in the immunized group was significantly higher than that of IgG1,and the ratio value IgG2a/IgG1 was greater than 1,indicating NTSN?actA/plcB/orfX induced a Thl-biased cellular immune response in mice.The results of cytokine secretion assay showed that the levels of IFN-y,TNF and IL-6 produced by spleen lymphocytes in the immunized group were significantly increased,and the content of IL-4 was decreased.The mice were induced to produce a Thl-type cellular response.After booster immunization at intervals of two weeks,challenge experiments were carried out with lethal doses of 4b and 1/2b virulent strains.The results showed that this vaccine candidate could not only afford protection against the challenge of virulent 4b strain,and also confer protection against the infection of serotype 1/2b virulent strain.In summary,the highly attenuated strain HTSN?actA/plcB/orfX has high safety,can stimulate good humoral immune characteristics,and Thl type cellular immune response,cross-immunoprotective against the infection of both serotypes 4b and 1/2b virulent strains.Therefore,this deletion strain with high safety and protective immune response characteristics is a vaccine candidate strain for preventing listeriosis of animals,and has potential application prospects.The safety and immunogenic characteristics need to further transgenic evaluation by field experiment.3.Safety,immunoprotection and DIVA characteristics of Listeria monocytogenes,NTSNA actA/plcB/orfX in sheep modelSheep is the most susceptible host of Listeria monocytogenes,and sheep listeriosis is one of the important factors affecting the development of animal husbandry.Due to these reasons,Hu sheep was used as an animal model to further evaluate the potential application of the vaccine candidate strain NTSNAactA/plcB/orfX for the prevention of listeriosis.We studied the safety,efficacy and DIVA characteristics of this vaccine candidate by subcutaneous immunization.Experiment of colonization showed that the bacteria loads in organs of sheep vaccinated with NTSNA actA/plcB/orfX gradually decreased,and was completely elimination by the host on the 23rd day after immunization.Histopathological observation showed that the vaccine strain NTSNA actA/plcB/orfX did not cause significant lesions in sheep tissues,which indicated that the vaccine candidate strain had higher safety.Primer-boost immunizations via subcutaneous route were performed on the 15 kg Hu sheep at intervals of 21 days.The serum assay using LLO as the coating antigen showed that the antibody level of sheep induced by the vaccine candidate strain was no significantly different comparing with the NTSN infection group.The indirect ELISA assay using ActA as coating antigen showed that the antibody level stimulated by the vaccine candidate strain was significantly lower than that of the NTSN infection group.The above results indicated that lost expression capability of PlcB encoded by plcB gene in the vaccine candidate strain can be used as a marker(phospholipase activity),and as a serological marker for distinguishing wild-type infection from vaccinated sheep,and the expression product of LLO encoded by hly gene is available for the detection of humoral immunity,antigens and cellular immune stimulatorsAfter 21 days of the second immunization,the challenge was carried out with the wild-type strain NTSN,and the survival rate of the immunized group sheep was 78%,while the control group all died.The pathological observation showed that NTSN ?actA/plcB/orfX could be immunized.Significantly reduced the tissue lesions of the sheep against the NTSN.This indicates that the vaccine can better immunoprotect the infection of the homologous virulent strain.Taken together,our studies indicate that the three-genes-deletion attenuated strain NTSN AactA/plcB/orfX has high safety,affords effective protection against challenges of serotype 4b and 1/2b strains,and meets the requirements of the DIVA program. |
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