The Construction Of Invasive Lactic Acid Bacteria Based On FnBPA And ScFv-FnBPA And Its Molecular Immunological Mechanisms

Swine influenza?SI?is an acute,highly contagious respiratory infectious disease caused by Swine influenza virus?SIV?,which is characterized by a sudden cough,dyspnea,fever,and rapid resolution.SIV is a single,negative strand and segmental RNA virus with spherical structure.Hemagglutinin?HA?is an important membrane protein of SIV,which could stimulate the production of neutralizing antibody.Currently,the influenza vaccines mainly include inactivated vaccines,attenuated vaccines,live vector vaccine,et al,and the use of lactic acid bacteria as the carrier of mucosal vaccine delivery has also been studied,but the efficiency of antigen delivery still needs to be improved.In this study,Lactobacillus plantarum?L.plantarum?NC8 was used as the host strain and two kinds of invasive L.plantarum were constructed.One was the expression of invasion from Staphylococcus aureus,named fibronectin binding protein A?FnBPA?,the other was a single-chain variable fragment against CD11c,scFv-CD11c or aCD11c.To explore the molecular immunological mechanisms,these two novel invasive lactic acid bacteria vectors were used to express HA antigen from SIV.Firstly,we constructed a novel L.plantarum strain with surface displayed FnBPA,named NC8-pSIP409-FnBPA.After that the strain was used to infect a porcine intestinal epithelial cell line?IPEC-J2?,the adhesion and invasion ratios were 1.41%and 0.15%,respectively,which were about two-fold and five-fold increase compared with empty vector group.The above strain was co-cultured with bone marrow-derived dendritic cells?BMDCs?and the activation of BMDCs were determined by flow cytometry?FCM?and then the expression levels of selected mRNA were detected by quantitative reverse transcription PCR?qRT-PCR?.The results suggested that the strain NC8-pSIP409-FnBPA significantly improved the expression levels of CD40and CD80 on BMDCs surface?P<0.001?,and the mRNA levels of IL-6 and MyD88genes were also significantly increased?P<0.001?.Similarly,the above strains were used to immunize BALB/c mice to evaluate their immunomodulatory characteristics.The results showed that NC8-pSIP409-FnBPA significantly stimulated the differentiation of DCs in the Peyer's patch?PPs??P<0.001?,increased the percentages of CD4⁺IL-4⁺?1.72%,P=0.0037?and CD4⁺IL-17A⁺?2.07%,P=0.0079?T cells of splenocytes.The levels of IL-4?P=0.0012?and IL-17A?P<0.001?in serum as measured via ELISA were also increased in mice treated with FnBPA⁺L.plantarum.Compared to the empty vector group,NC8-pSIP409-FnBPA significantly increased the production of B220⁺B cells in mesenteric lymph nodes?MLN??P<0.001?and PPs?P<0.001?,the titers of FnBPA-specific IgG?1.72 fold?and sIgA?2.41 fold?antibodies.Secondly,we also constructed a L.plantarum strain with surface displayed aCD11c,named NC8-pSIP409-aCD11c.The adhesion and invasion ratios of NC8-pSIP409-aCD11c on BMDCs were 36.17%and 2.23%,respectively,which were1.56 fold and 2.42 fold increased compared with empty vector group.Furthermore,NC8-pSIP409-aCD11c strain harboring a eukaryotic plasmid pValac-GFP was co-cultured with BMDCs for 36h and GFP expression in BMDCs was detected by FCM to determine the delivery efficiency of pValac-GFP.The results showed that the strain could significantly enhance GFP expression in BMDCs?P=0.0110?.The mice were immunized using the above strains harboring pValac-GFP and the oral administration resulted in efficient expression of GFP in both PPs and MLN at different times after the last administration.In addition,the NC8-pSIP409-aCD11c strain significantly promoted the differentiation and maturation of DCs,the percentages of IL-4⁺and IL-17A⁺of CD4⁺T cells in MLN,as well as production of B220⁺IgA⁺B cells in the PPs.Next,we used the above two invasive vectors to express the HA antigen from SIV and constructed three strains of L.plantarum,named NC8-pSIP409-pgsA'-HA?HA?,NC8-pSIP409-FnBPA-pgsA'-HA?FnBPA-HA?and NC8-pSIP409-aCD11c-pgsA'-HA?aCD11c-HA?,respectively.The above strains were used to immunize BALB/c mice and the results showed that all the three strains expressing HA could promote the activation of DCs in the spleen and the expression of IFN-?⁺and perforin in CD8⁺T cells in the spleen and MLN as well as the production of B220⁺IgA⁺B cells in the PPs,compared with the empty vector group.Meanwhile,all the three recombinant strains expressing HA could promote the production of specific IgG in serum?about 2.28 fold?and sIgA in fecal?2.37 fold?and BALF?2.39 fold?compared with the empty vector group by ELISA,with the production of neutralizing antibodies as well.BALB/c mice were infected with10×LD₅₀ influenza A/PuertoRico/8/1934?H1N1?,and on the 7th day after challenge,compared with the HA group and FnBPA-HA group,the aCD11c-HA group significantly increased the expression of IFN-?⁺?P<0.001?and perforin?P<0.001?in CD8⁺T cells in MLN,which had a certainly antiviral effect.The protection rates of IV in the FnBPA-HA group and the aCD11c-HA group were 60%,while that of the HA group was 40%.Finally,the molecular immunological mechanisms of aCD11c-HA strain against H1N1 subtype IV was explored.At 12 h after the co-incubation of L.plantarum expressing aCD11c-HA with BMDCs,the effects of the strains on the differentiation of BMDCs were detected by FCM.The results showed that the aCD11c-HA strain could significantly promote the activation of BMDCs.The expression of cytokines in the above cells supernatants were detected by ELISA and the results suggested that the aCD11c-HA strain could significantly increase the production of IL-12P70 and inhibit the secretion of IL-6.CD4⁺and CD8⁺T cells from the spleen of non-immunized mice were sorted and cultured with the above activated BMDCs for48 h,and the expressions of IFN-?⁺and perforin in the cells and the contents of IFN-?in the supernatants were determined by FCM and ELISA,respectively.The results showed that the aCD11c-HA strain increased the expression of IFN-?⁺?4.33%?of CD4⁺T cells and IFN-?⁺?7.68%?and perforin?17.50%?of CD8⁺T cells,as well as the secretion of cytokines IFN-?.The expression of IFN-?⁺?P<0.001?and perforin?P<0.001?from HA-specific CD8⁺T cells of spleen and MLN were detected in vivo and the aCD11c-HA group was significantly increased.Moreover,the aCD11c-HA group significantly promoted the proliferation of T cells in the spleen?P<0.001?.The magnetic beads were used to sort CD8⁺T cells from the spleen of immunized mice in each group,and then adoptive transfer of the cells to NOD/Lt-SCID mice by caudal vein was performed.5×LD50 IV was infected after 24 h.The results showed that CD8⁺T cells of the aCD11c-HA group could extend the life of NOD/Lt-SCID mice to2 days compared with the HA group and had a certainly protective effect.In this study,two invasive L.plantarum strains based on FnBPA protein and scFv-CD11c were constructed,respectively,co-expressing HA protein of SIV.The recombinant strains were then used to immunize BALB/c mice,and the results showed that both co-expressed strains significantly increased the protective rates against IV challenge compared with strain expressing HA alone,in particular,the presence of scFv-CD11c performed better in inducing humoral immune response.Furthermore,the aCD11c-HA strain could promote the production of IFN-?⁺and perforin of CD8⁺T cells shown by FCM,flow sorting and adoptive transfer to NOD/Lt-SCID mice,which revealed the molecular mechanism of immune response induced by the DCs targeting invasive LAB strain,laying the foundation for developing the new LAB vaccine.