Characterization Of The Immuno-protection Of Plague Vaccine And Q Fever Vaccine Via Aerosolized Intratracheal Inoculation

Plague,which is caused by Yersinia pestis,is one of the most dangerous infectious diseases.Plague has brought worldwide epidemic in the past,which led to over 200 million human deaths.Pneumonic plague is considered the most dangerous infectious disease because the pathogen can disseminate through aerosol droplets.Human Q fever is recognized as a worldwide public health problem.It often occurs by inhalation of airborne aerosols contaminated with Coxiella burnetii,a gram-negative intracellular bacterium,mainly from domestic livestock.Human Q fever usually manifests as an acute flu-like and often self-limiting illness or less frequently as a chronic disease in the form of endocarditis or hepatitis.Both Yersinia pestis and Coxiella burnetii can be used for bioterrorism,and developing effective vaccine is the main way for the prevention of Yersinia pestis and Coxiella burnetii infections.The exploration of safer and more convenient immune route is necessary for large scale vaccination and emergency immunization condition.Here we developed Yersinia pestis live attenuated vaccine and Yersinia pestis nanocarrier subunit vaccine,and evaluated the immune safety,immunogenicity and protective effect of these two vaccines in mice model via aerosolized intratracheal inoculation.We also evaluated the immune safety,immunogenicity and protective effect of chloroform-methanol residue(CMR)vaccine of Q fever in mice model via the aerosolized intratracheal inoculation route.No FDA-approved vaccine against plague is available for human use at present.Liveattenuated vaccines are able to induce long-lasting humoral and cellular immune responses,making them the preferable vaccine type.The pigmentation locus(pgm)-lacking live-attenuated Yersinia pestis EV76 vaccine strain can induce protection against bubonic and pneumonic plague,and it was used during plague endemic periods throughout the world.However,the ?pgm mutant of Yersinia pestis caused side effects of varying severity and caused fatal infection of an individual with hemochromatosis,indicating that pgm deletion-based live-attenuated vaccines still need to be improved.In this study,a plasminogen activator protease(pla)gene deletion mutant of the attenuated Yersinia pestis strain EV76-B-SHU was constructed using the CRISPRCas12a-assisted lambda RED recombineering system,and its residual virulence,immunogenicity and protective efficacy were evaluated in a BALB/c mouse model via aerosolized intratracheal inoculation(i.t.)or via subcutaneous injection(s.c.).As a result,the residual virulence of EV76-B-SHU?pla was significantly reduced compared to that of the parental strain EV76-B-SHU following i.t.and s.c.infection.The EV76-B-SHU?pla induced higher levels of mucosal antibody s Ig A in the bronchoalveolar lavage fluid of mice immunized by i.t.but not by s.c..Moreover,after lethal challenge with Yersinia pestis strain 201(isolated from vole and is avirulent in human),the survival rate,the degree of pathological lesions,and the levels of bacterial load in the EV76-B-SHU?pla-i.t.group were comparable to those of the EV76-B-SHU?pla-s.c.and EV76-B-SHU immunized groups.Thus,the EV76-B-SHU?pla represents an excellent live-attenuated vaccine candidate against pneumonic plague.It has been reported that nanomaterials can be used as vaccine adjuvants to effectively stimulate cellular immune response.In this study,we analyzed the possibility of using nanomaterial as an adjuvant for i.t.delivery of Yersinia pestis subunit vaccine.The antigen protein F1 of Yersinia pestis was expressed in Escherichia coli,and the protein r F1 was coupled to the nano material GO-PEG to obtain the Yersinia pestis nanocarrier subunit vaccine GO-PEG-r F1,then the physical characteristics of nanocarrier subunit vaccine were characterized,the cytotoxicity of the vaccine was evaluated in mouse macrophage cells,and the immunogenicity and protective effect of the vaccine were evaluated in mouse model via i.t.route.The Yersinia pestis nanocarrier subunit vaccine showed no detectable cytotoxic on mouse macrophage cells,and could induce significantly higher level of humoral immune antibody than r F1 immunized group.Also,the Yersinia pestis nanocarrier subunit vaccine provided higher protective efficacy than r F1 immunized group after challenge with Yersinia pestis live-attenuated strain 201.The whole-cell formalin-inactivated Q fever vaccine(Q-vax)was reported with the possibility of adverse reactions when used in humans.A Q fever CMR vaccine developed by the US Army Research Institution was thought to be an efficacious vaccine alternative to Q-vax.In this study,we analyzed the possibility to establish mucosal and systemic immunity against Coxiella burnetii infection using a pulmonary delivery of CMR vaccine.Mice were immunized by the intratracheal inoculation of CMR(IT-CMR)or the subcutaneous injection of CMR(SC-CMR),and the immunized mice were challenged with Coxiella burnetii by the intratracheal route.Then the level of humoral immunity,mucosal immunity and cellular immunity and the protective efficacy after challenge were evaluated.The levels of IFN-?,IL-12p70,IL-5,and IL-4 in the IT-CMR group in splenic T cells stimulated ex vivo were significantly higher than in the SC-CMR group.Significantly elevated s Ig A to Coxiella burnetii was detected in the bronchoalveolar lavage fluid of mice immunized by IT-CMR but not by SC-CMR,which might have contributed to the significant reduction in Coxiella burnetii load and pathological lesions in the lungs of the mice after the challenge of Coxiella burnetii.These results suggest that compared with SC-CMR in mice,IT-CMR was more efficient to elicit cellular and lung mucosal immune responses against aerosol infection of Coxiella burnetii.In summary,the Yersinia pestis live attenuated EV76-B-SHU?pla and nanocarrier subunit vaccine GO-PEG-r F1 developed in this study show excellent protection effect against pneumonic plague infection.According to the results of immune safety and protective efficacy evaluation of Yersinia pestis vaccine and Q fever vaccine via aerosolized i.t.,i.t.immune route can induce comprehensive immune responses including humoral immunity,cell-mediated immunity,and mucosal immunity,and plays crucial roles in protection against pathogen infection,suggesting that i.t.represents a promising immune route.