Fundamental Study Of The Recombinant Bacillus Subtilis Spore-Based Influenza Candidate Vaccine

Influenza is an acute respiratory infectious disease caused by influenza virus,which seriously endangers human health.Vaccination is one of the effective measures to prevent and control influenza.At present,influenza vaccines on the market are categorized as those based on inactivated virus,live attenuated virus,proteins,and DNAs.In view of the continuous variation of influenza virus,especially the highly variant influenza A virus,researchers are developing universal influenza vaccines that can solve the problem of influenza virus variation.However,most of the currently approved influenza vaccines or those under development need low-temperature storage,formulation with adjuvants,or needle injection,leading to a number of problems and inconvenience during production,storage,transportation,or use of vaccines.In this regard,vaccines in the form of Bacillus subtilis?B.subtilis?spores are the key to solve above problems.B.subtilis is non-pathogenic,and maintains the properties of good safety profile,clear genetic background,facile construction of recombinant spores,and convenient and safe oral delivery.In addition,it has strong stability contributing to its spores,as well as adjuvanticity,capable of inducing mucosal immune responses.Using recombinant B.subtilis spores as an effective surface display vehicle,and development of B.subtilis spore-based vaccines will well meet the needs for production and application of influenza vaccines.Another key issue in vaccine research is the selection of antigens.The high variability of influenza virus significantly affects the broad-spectrum protective immunity of influenza vaccines.Therefore,selection of the conserved antigen sequences as the vaccine targets is a hot topic in recent influenza vaccine studies.Influenza virus hemagglutinin fusion protein?HA-FP?and M2 protein extracellular domain?M2e?are highly conserved in influenza virus sequences,and thus can be used as antigen targets to develop universal candidate vaccines.In this study,B.subtilis spores were used as vaccine antigen presenting vectors.Based on highly variable influenza A virus,we designed an influenza virus conservative antigen suitable for carrying and delivering on the surface of B.subtilis spores.We then constructed recombinant B.subtilis spores that can display recombinant antigens on the surface,and analyzed their biological characteristics.This study further investigated the function and mechanisms of action of B.subtilis spore vaccines in intestinal mucosa.We analyzed and evaluated the immunogenicity and protective efficacy of these vaccines by performing animal experiments.These recombinant spores can use as a new type of influenza candidate vaccine based on B.subtilis spore display system.1.Design and functional study of influenza candidate vaccine antigen based on B.subtilis spores?1?In order to design a universal influenza vaccine target antigen for presentation of B.subtilis,this study used literature research and bioinformatics analysis of the sequence of H5N1 and H7N9 subtypes of influenza virus HA-FP and M2e,and designed four short?<30 amino acids?and conserved epitope peptides without containing glycosylation but targeting the conserved antigens of influenza virus.These antigen peptides are named H5N1-M2e,H5N1-FP,H7N9-M2e,H7N9-FP,respectively,all of which were combined and randomly arranged through multiple epitope sequences in different orders,with a flexible linker GGGGS.Based on their antigenicity analysis,two peptide molecules with optimized epitope sequence combinations were selected,and named M2e-FP1 and M2e-FP2.?2?In initial study,M2e-FP fusion proteins were constructed in a prokaryotic expression vector,pQE30,for expression of proteins,which were then used to immunize female BALB/c mice for three times with Alum adjuvant,followed by challenge using H1N1 influenza virus(10³ TCID₅₀).The immunization results showed that compared with the PBS control group,fusion proteins M2e-FP1 and M2e-FP2both worked well in inducing high titer of specific Ig G,IgG1 and IgG2a antibodies.Furthermore,every single peptide?H7M2e,H7FP,H5M2e,H5FP,H1M2e?expression of multi-epitope recombinant proteins of M2e-FP1 and M2e-FP2 can play its immune function.The challenge results demonstrated that mice immunized with recombinant M2e-FP1 and M2e-FP2 proteins all survived from H1N1 challenge.Also,these mice had dramatic decrease of lung viral replication and significant alleviation of histopathological damages.These results suggest that the recombinant proteins tested can inhibit H1N1 influenza virus infection and provide immune protection.2.Study of immunogenicity and protective efficacy of B.subtilis spore candidate vaccines?1?Our study constructed recombinant plasmids named pDG1664-CotB-M2e-FP1 and pDG1664-CotB-M2e-FP2,and then recombinant plasmids were integration into the genome of B.subtilis PY-79 by genetic homologous recombination.The M2e-FP1 and M2e-FP2 genes were confirmed for correct insertions by PCR and DNA sequencing analysis.SDS-PAGE and Western Blot assays were utilized for detection of the expression of influenza virus M2e-FP proteins expressed on the spore coat of the recombinant strain,and flow cytometry and immunofluorescence assays were used to determine the expression of M2e-FP proteins on the spore coat.And our study analyzed the growth conditions and biochemical characteristics of recombinant spores.The results showed that M2e-FP proteins were presented successfully on the surface of B.subtilis spores.?2?To further analyze the immunogenicity and immune characteristics of recombinant spores expressing M2e-FP1 and M2e-FP2 proteins,we used these vaccines to immunize mice orally,and challenged mice with 10³ TCID₅₀0 of the H1N1virus.The results revealed that oral immunization of recombinant spores without adjuvants can elicit high titer of specific IgG,IgG1,and IgG2a antibodies.Also,recombinant spore-vaccinated mice demonstrated significant decrease of viral replications and obvious alleviation of histopathological damages in their lungs,as well as significant increase of survival rate.The above results suggest that recombinant M2e-FP1 and M2e-FP2 spores are able to provide effective cross-protection against divergent H1N1 influenza viruses.3.Study of immune response characteristics and mechanisms of action of B.subtilis spore-based influenza candidate vaccines?1?In this study,RAW264.7 cells were used as macrophage models in vitro by measuring the activity of lactic acid dehydrogenase?LDH?and alkaline phosphatase?ACP?in cell lysates.The data illustrated that the activities of intracellular ACP and LDH in recombinant B.subtilis spore M2e-FP1 and PY-79 groups were higher than the PBS group at 12 h.ACP and LDH are the marker enzymes activated by macrophages,which are related to the function of macrophage clearance and phagocytosis.The increase of ACP and LDH activity in macrophages indicated that both recombinant and wild-type spores can activate macrophages and enhance their immune function.?2?This study further investigated the activation and proliferation of lymphocytes in the mesenteric lymph nodes and splenic lymphocytes by analyzing the splenic lymphocyte subsets after immunization,and explored the effect of recombinant spores on the systemic immune response.The results demonstrated that M2e-FP1 and PY-79 group can enhance specific lymphocyte proliferation,among which the percentage of CD3⁺CD4⁺T cells was significantly up-regulated,suggesting that recombinant spores can enhance intestinal mucosal and systemic immune response,with the focus on humoral immunity.?3?To investigate the role of toll-like receptors?TLRs?in the activation of intestinal mucosa after oral immunization,we detected the TLR2,TLR4,and TLR9gene transcription and cytokine secretion by extraction of mRNAs from intestinal tissues and performance of RT-PCR.Immunohistochemistry analysis was used to investigate the expression and distribution of TLR2 and TLR4 in the intestinal mucosa.The results indicated that there were significant up-regulation of TLR2 and TLR4 mRNAs between the M2e-FP1 or PY-79 groups and the PBS group,but no significant differences of TLR9 in all three groups.In addition,significantly increased expression of TLR2 and TLR4 was shown in the intestinal mucosa,and the transcriptional level of mRNA of cytokines also increased significantly in the small intestinal mucosa of recombinant M2e-FP1 spore-immunized mice.These data suggest that recombinant spores may induce antigen-specific immune responses by activation of TLR2 and TLR4.In summary,this study explored B.subtilis as a new form of heat-stable vaccines through oral delivery without the use of adjuvants.By targeting influenza virus with extreme variability,this study designed the candidate vaccines using highly conserved sequences of HA-FP and M2e of H5N1 and H7N9 subtypes of influenza virus.The recombinant B.subtilis spores were prepared successfully,which can stably express heterologous M2e-FP protein on the surface.The recombinant B.subtilis spores induced potent immune responses in the immunized mice,and recombinant spores also can protecte mice against influenza virus infection effectively.Our study not only plays a foundation for the research and studies of universal influenza vaccines,but also provides a new technical approach for the development of novel vaccines against other pathogenic viruses.