Comparison Of The Differences Between Monomeric Apa2H1 And Trimetric Apa2H1 In Activation Of BMDCs In Vitro And Immunoprotectionin Vivo

Actinobacillus pleuropneumoniae(APP) is a causative agent of porcine pleuropneumonia, which is a highly contagious disease and has become one of the most common diseases that lead to death of fattening and breeding pigs. There are 15 serotypes of APP, but the serotypes 1, 3, 5 and 7 plaied the most predominated role in causing this disease in China. However, no cross-protective effects were found during vaccination, resulting in great difficulties in prevention and treatment of pleuropneumonia. Trimeric Autotransporter Adhesins(TAAs) is a kind of new found bacterial virulence factor on many pathogenic gram negative bacteria surface, whose Yad A-like head domain is their main functional areas that largely mediated bacterial adhesion and colonization and immune evasion. Although previous studies have shown that TAAs have immunogenicity, which functional domain performs this protective effects remains poorly understood. Meanwhile, the natural state of the TAAs is a trimeric structure, whether this trimeric antigen has better performance in immunogenicity and protective immunity than monomer antigen still made no consensus. Therefore, this study selected the previous reported APP serotype 5b strain L20 surface TAA protein, Apa2, as a represent, selected the forecasted functional domain Apa2H1 as the research object, to study the difference in immunogenicity between trimetric antigen and monomeric antigen, providing a new theoretical basis for the preparation of novel subunit vaccines.In this study, we fused the Apa2H1 to C-terminal domain of Hia or cloned to p ET-28 a plasmid to produce natural trimetric Apa2H1 and monomeric Apa2H1 respectively in the prokaryotic expression systems, then purified these two types of Apa2H1 by taking advantage of their HAT and 6x His tags, respectively. Determined the direct adhesion ability of trimetric and monomeric Apa2H1 by adding them to porcine lung epithelial cell line(SJPL) and porcine alveolar macrophage cell line(CRL), both are the host cells of APP; Measured their functions in mediating bacterial adhesion and invasion by co-culturing the trimetric or monomeric Apa2H1 expressing E.coli with trimetric or monomeric Apa2H1 pretreated or common SJPL and CRL cell lines; finally, conformed the function of Apa2H1 in mediating adhesion and invasion of APP using wild type APP to interact with trimetric or monomeric Apa2H1 pretreated or common SJPL and CRL cell lines. The results showed two types of Apa2H1 can adhere to host cells direct and significant increased the capacity of bacterial adhesion and invasion, Apa2H1 pretreatment, however, restored this promotional ability. Therefore, these data demonstrated that Apa2H1 is a key functional domain of Apa2.In order to determine whether Apa2H1 has immunogenicity and whether can be developed into subunit vaccine or not, we firstly measured the ability to activate Dendritic cells(DCs) by co-culturing Apa2H1 with bone marrow dendritic cells(BMDCs) in vitro; then we detect the specific antibodies and splenic T cell activation in trimetric Apa2H1 and Freund’s adjuvant immunized mice to determine the immunogenicity in vivo; Finally, we measured the bacterial burden in lung and circulation, lung inflammatory cells infiltration, survival rates and other indicators of Apa2H1 immunized mice after challenged with lethal dose of APP to value immune protection ability of Apa2H1. Our results indicated Apa2H1 activates BMDCs and induces expression of several proteins that contributed to proliferation, differentiation and activation of T and B lymphocytes in vitro;Immunization of Apa2H1 induce boost humoral immunity and moderate Th1/Th2 mixed T cell response, significantly(p<0.05) and extremely significant(p<0.01) decreasing bacterial burden in lungs and blood at the early and later phase of infection, respectively, reducing the degree of lung inflammation and improving the survival rate of mice(p<0.01). Thus proved that Apa2H1 has profound immunogenicity and great immune protective ability, can be, therefore, treated as a vaccine candidate protein.To investigate the differences of trimetric antigen and monomeric antigen in antigenicity, we chose Apa2H1 as a representative and co-cultured trimetric or monomeric Apa2H1 with BMDCs in vitro to measured their stimulating ability difference; further immunized mice with these two kinds of Apa2H1 with addition or without addition of adjuvants, to value their difference of immune protective ability in vivo. Our results found trimetric antigen can stimulated the activation of BMDCs more effective than monomeric antigen(p<0.05); When immunized with adjuvants, there was no difference in specific antibodies production and immune protection between two forms of antigen(P > 0.05), but trimetric Apa2H1 immunization could induce significant higher specific antibodies compared to monomeric Apa2H1 in the absence of adjuvants, which significantly reduced lung inflammatory cell infiltration and significantly delayed death of lethal dose of APP infected mice((P < 0.05). Therefore, this study suggested the immunogenicity of trimetric antigen is stronger than that of monomeric antigen from in vitro and in vivo experiments.This study elucidated that Apa2H1 can not only promote APP adhere and invade to host cells adhesion, but also has good antigenicity and can be used as a vaccine candidate protein against APP, provided a novel method to prepare of trimetric proteins and proved trimetric antigens have stronger immunogenicity compared to the monomeric forms of antigen. The results not only can be directly used for porcine contagious pleuropneumonia vaccine research, helping to promote the control and treatment of APP infection, thus to reduce economic losses in pig industry worldwide, but more important, provide theoretical and experimental basis for the development of novel trimetric protein vaccines.