Construction Of A Ferritin Nanoparticle Pseudomonas Aeruginosa Vaccine Demonstrating PcrV-OprI And Its Protection Mechanism

Background:Pseudomonas aeruginosa（PA）is one of the common agents of nosocomial infections,particularly in intensive care units.The main clinical presentations of PA infections are ventilator-associated pneumonia,cystic fibrosis with infectious pneumonia and systemic infections.Multidrug-resistant strains of PA are resistant to aminoglycosides,quinolones and beta-lactam antibiotics,which not only increases morbidity and mortality rates,but also adds to the financial burden of patients.There is an urgent need to develop a safe and effective vaccine.In recent years many protective antigens have been identified and evaluated in human clinical trials,such as Pseudomonas aeruginosa V-antigen（Pcr V）and outer membrane protein I（Opr I）.So far there is no licensed PA vaccine available.One possible reason for the limited immune response is the lack of an effective delivery system.The injection-like Type 3 secretion system（T3SS）is the key protein machine for the secretion of toxins from bacteria into the host cell.Pcr V is one of the key components of T3SS and is highly conserved in sequence in different strains.Opr I is a lipoprotein located in the outer membrane and has an important role in stabilising the outer cell membrane and can have an adjuvant effect when immunised in combination with other protein antigens.In previous studies,the group has constructed re PO（re Pcr V-Opr I）,a fusion protein vaccine of Pcr V and Opr I,and found that it has good immunogenicity and immune protection.Self-assembled Ferritin nanoparticles are suitable carriers of antigens,which significantly enhance the response to immunity.Therefore,we assume that ferritin enhances the immunogenicity and protective effect of Pcr V and Opr I antigens.Based on the above analysis,ferritin nanoparticles displaying the fusion protein antigen Pcr V-Opr I on the surface（re Pcr V-Opr I-Ferritin,re PO-FN）were constructed by the Spy Tag/Spy Catcher protein linker system in this study.It also evaluated immunogenicity and its protective mechanisms in an acute pneumonia model mouse,providing the basis for the successful development of a PA vaccine.Methods:1.Design,preparation and physicochemical property analysis of nanoparticle vaccine re PO-FN.2.Study of the protective effect and mechanism of nanoparticle vaccine re PO-FN.3.Ex vivo safety evaluation of nanoparticle vaccine re PO-FN.Results:1.Successful construction of p ET28a-re PO-ST and p ET28a-SC-FN recombinant plasmids.The recombinant proteins were recombinantly expressed in E.coli BL21 and purified by Ni²⁺affinity chromatography and other purifications to obtain the proteins of re PO-ST and SC-FN with purity of about 93.2%and 91.2%;SC-FN was combined with re PO-ST in a molar ratio（1:20）for 18 h.SDS-PAGE,molecular sieving,dynamic light scattering,transmission electron microscopy showed that re PO-ST and SC-FN proteins were successfully linked into homogeneous and stable nanoparticles re PO-FN.2.Intramuscular injection of immune re PO-FN protein significantly improves survival in mice in a lethal dose PA XN-1 infection model;In a sub-lethal dose PA XN-1infection model,intramuscular injection of immune re PO-FN protein reduced the degree of weight loss and attenuated the somatic infection status of mice.It inhibits the colonization of PA in mouse lung,effectively protects the tissue structure of mouse lung and reduces the inflammatory response in mouse lung.Compared to re PO alone,re PO-FN not only enhances the intensity of the humoral immune response but also accelerates the rate of the humoral immune response.3.In a lethal dose PA XN-1 infection model,intranasal immunization with re PO-FN protein showed a significant increase in survival in mice,with a significantly higher protective effect than intramuscular immunization;In a sub-lethal dose PA XN-1 infection model,intranasal immunization with re PO-FN protein reduced the degree and rate of weight loss in mice.It also reduced the infection status,reduced the amount of bacterial colonisation in the lungs,protected the tissue structure of the lungs,reduced the degree of neutrophil infiltration and reduced the inflammatory response in the lungs of mice.Similarly,compared to monomeric re PO,adjuvant-free nasal drops of immune re PO-FN not only significantly increased the intensity of the immune response,but also accelerated it and induced the production of secretory Ig A antibodies.4.The nanoparticle vaccine re PO-FN does not cause haemolysis of red blood cells.It has no significant damage or inflammatory infiltration to the heart,liver,spleen,lungs and kidneys,and has a good safety profile in vitro and in vivo.Conclusions:1.We successfully developed the fusion protein re PO-FN with surface display of Pcr V-Opr I,which is homogeneous and stable nanoparticles in solution and has good safety.2.The self-assembled re PO-FN significantly enhanced the immunogenicity of re PO and showed promising immunoprotective effects in an acute pneumonia model of PA infection in mice.