Preparation And Evaluation Of Self-Adjuvant Helicobacter Pylori Lipopeptide Vaccine

Background:Helicobacter pylori（H.pylori）is a gram-negative,spiral-shaped bacterium that colonizes the human gastric epithelium and is the major pathogen of the upper gastrointestinal tract.It can cause chronic gastritis,peptic ulcer,and even progress to gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma（MALT）.Given the increasing rate of antibiotic resistance,the search for effective alternatives is a must.The development of prophylactic or therapeutic vaccines may be an effective way to control H.pylori infection.Compared with intact protein antigens,epitope vaccines composed of immunodominant epitopes have the advantages of stronger immune response specificity,fewer side effects and better targeting.However,the first problem to be solved in the development and application of epitope vaccines is that their weak immunogenicity makes it difficult to induce strong effects.Given that resistance to H.pylori infection depends primarily on antigen-specific mucosal and T-cell responses,aluminum adjuvants-commonly used to enhance systemic humoral and Th2-biased responses-are not theoretically preferred.Therefore,conventional adjuvants that are used to enhance systemic humoral and Th2-biased responses are not theoretically preferred.The lack of safe and effective adjuvant that can induce strong cellular and mucosal immune responses is an important bottleneck limiting the development of H.pylori vaccines.Pam₂Cys is a lipid component of Mycoplasma macrophage-activating lipopeptide 2.Lipopeptide molecules synthesized based on Pam₂Cys are promising adjuvants for immune stimulation.For example,Pam₂CSK₄（P2CSK4）,a Toll-like receptor 2（TLR2）agonist,enhances specific cellular and mucosal immune responses when co-delivered with antigens.In our previous study,lipopeptide as an adjuvant enhanced the protective effect of recombinant Hpa A against H.pylori infection.However,a large number of studies have shown that self-adjuvant vaccines produced by the fusion of antigen and adjuvant can produce more effective immune responses than simple mixtures of antigen and adjuvant,because the combination of the two helps to ensure the delivery and uptake of the two components by the same antigen-presenting cells,thereby promoting the best immune response.Objective:In this study,Pam₂Cys was chemically linked to immunodominant epitopes of Ure B or Cag A derived from H.pylori to synthesize lipopeptide vaccines with self-adjuvant activity.Transcriptome sequencing,flow cytometry,confocal microscopy and ELISA were used to study the activation effect of lipopeptide vaccine on dendritic cells（DCs）.Mice were immunized with the lipopeptide vaccine through the nose to evaluate the epitope-specific cellular and humoral immune responses,and to further evaluate the protective effect of the lipopeptide vaccine against H.pylori infection and explore its possible mechanism.Safe and effective self-adjuvant vaccines that elicit comprehensive immune responses,especially specific cellular and mucosal immune responses,are desirable.Methods:1.Six T cell epitopes and four T/B cell mixed epitopes（named P1-P10）were selected from the popular candidate antigens of H.pylori.Four lipopeptides were successfully synthesized by covalently coupling the epitope peptides with Pam₂Cys（named Hp1-Hp10）by solid-phase synthesis.Two lipopeptide vaccines（Hp4 and Hp10）with TLR2 activation activity were screened by HEK-Blue m TLR2 cells.In vitro self-assembly of Hp4 and Hp10 in polar solutions was observed by transmission electron microscopy.The in vitro safety of lipopeptide vaccines Hp4 and Hp10 was evaluated by in vitro hemolysis test and CCK8cytotoxicity test.The safety of Hp4 and Hp10 lipopeptide vaccines in vivo was further evaluated by observing serum biochemical indicators（alanine aminotransferase,aspartate aminotransferase,glutamyl transpeptidase,total bilirubin,albumin,lactate dehydrogenase,alkaline phosphatase and creatinine）and pathological sections of heart,liver,spleen,lung and kidney in immunized mice.2.Lipopeptide vaccines Hp4 and Hp10 were incubated with bone marrow-derived dendritic cells（BMDCs）,respectively,and the cells and culture supernatants were collected.High-throughput sequencing and bioinformatics analysis（principal component analysis,differential gene screening,differential gene cluster analysis,differential gene enrichment analysis and gene set enrichment analysis）were used to explore the mechanism and possible targets of lipopeptide vaccines Hp4 and Hp10 on BMDCs.Laser confocal microscopy was used to observe the endocytosis of Hp4 and Hp10 by BMDCs to investigate the promotion effect of lipopeptide vaccine on BMDCs’antigen uptake.The effect of lipopeptide vaccines on the maturation of BMDCs was evaluated by detecting the expression levels of costimulatory molecules CD40,CD80 and CD86 on BMDCs after stimulation with lipopeptide vaccines Hp4 and Hp10.ELISA was used to detect the secretion of specific cytokines IL-1,IL-2,IL-4,IL-6,IL-12 and IFN-γin the culture supernatant of BMDCs to further investigate the promotion effect of lipopeptide vaccine on BMDCs maturation.3.Mice were immunized with P4,P4+P2CSK4,Hp4,P10,P10+P2CSK4,Hp10,P4+P10,P4+P10+P2CSK4 and Hp4+Hp10 by intranasal injection on days 1,14 and 28.Fourteen days after the last immunization,half of the mice were sacrificed and eyeball blood,splenic lymphocytes,and gastric and small intestinal lavage fluid were collected.The other half was used to establish a H.pylori infection model.The levels of IFN-γ,IL-4 and IL-17A secreted by spleen lymphocytes of mice immunized with lipopeptide vaccines were detected by ELISpot and ELISA to reflect the strength of specific cellular immune responses induced by lipopeptide vaccines Hp4 and Hp10.The ability of lipopeptide vaccine to stimulate memory cellular immune response was evaluated by detecting the proportion of high expression of CD44 and CD62L in T lymphocytes by flow cytometry.Serum Ig G and Ig A antibody levels were detected by ELISA to evaluate the ability of lipopeptide vaccine to induce specific humoral immune response in mice.ELISA was used to detect the levels of gastric mucosal s Ig A,small intestinal mucosal s Ig A,salivary s Ig A and vaginal s Ig A antibodies to evaluate the ability of lipopeptide vaccine to stimulate specific mucosal immune response.The protective effect of lipopeptide vaccine was evaluated by establishing H.pylori infection model and detecting H.pylori colonization in the stomach of mice by real-time fluorescence quantitative PCR.Results:1.Pam₂Cys was chemically linked to immunodominant epitopes of Ure B or Cag A derived from H.pylori to synthesize lipopeptide vaccines Hp4 and Hp10 with self-adjuvant activity.Hp4 and Hp10 can self-assemble into about 50 nm particles in vitro,which is beneficial for antigen presenting cells to recognize and phagocytize.When the concentration of Hp4 and Hp10 was 50-200μg/m L,the hemolysis rate of red blood cells was less than 2%,and the CCK8 results showed that the survival rate of DC2.4 cells was more than 98%,indicating that the lipopeptide vaccines Hp4 and Hp10 had good safety in vitro.There was no significant difference in serum biochemical indicators（alanine aminotransferase,aspartate aminotransferase,glutamyl transpeptidase,total bilirubin,albumin,lactate dehydrogenase,alkaline phosphatase and creatinine）between mice immunized with Hp4 and Hp10 by intranasal administration and PBS control group,indicating that Hp4 and Hp10 had no obvious liver and kidney toxicity.There was no significant difference in the pathological sections of heart,liver,spleen,lung and kidney between the mice immunized with Hp4 and Hp10 by intranasal administration and the PBS control group,indicating that Hp4 and Hp10had no major organ toxicity.The above results indicate that it is safe in vivo.2.KEGG signaling pathway enrichment analysis showed that three PRRs in BMDCs,including Toll-like receptors（TLRs）,NOD-like receptors（NLRs）,and retinoic acid-induced gene（RIG）I-like receptors（RLRs）,were activated by lipopeptide vaccines Hp4 and Hp10.GSEA enrichment analysis focused the activated pathways on TLR2,TLR3,TLR4,NOD2and RIG-I.Quantitative real-time RT-PCR showed that the gene expressions of key components of TLR pathway（Tlr2 and Tlr4）,NLR pathway（Nod2 and Nlrp3）and RLR pathway（Rig-i and Isg15）were significantly up-regulated.Confocal laser scanning microscopy showed that the epitopes P4 and P10 were only adsorbed on the surface of BMDCs,while the lipopeptide vaccines Hp4 and Hp10 were endocytosed by BMDCs.GSEA enrichment analysis showed that BMDCs were in a"mature"state after lipopeptide vaccine treatment.The maturation of BMDCs was further confirmed by the increased expression of co-stimulatory molecules CD40,CD80 and CD86 detected by flow cytometry and the increased secretion of IL-1,IL-2,IL-4,IL-6,IL-12 and IFN-γdetected by ELISA.3.For cellular immunity,the number of IFN-γ,IL-4 and IL-17A secreting cells in mice immunized with lipopeptide vaccines Hp4 and Hp10 was significantly increased,indicating that lipopeptide Hp4 or Hp10 could induce potent Th1,Th2 and Th17 cellular immune responses.Moreover,the increase of IFN-γ,IL-4 and IL-17 in Hp4 group was higher than that in Hp10 group,which may be related to the fact that Hp4 only contains T cell epitopes.For humoral immunity,only Hp10 immunized mice could detect high levels of serum Ig G antibodies,while Hp4 immunized mice could not induce serum Ig G antibodies,indicating that only Hp10 based on T/B mixed epitopes could stimulate specific humoral immune responses in mice.For mucosal immunity,only Hp10 could induce high level of mucosa-specific s Ig A antibody,indicating that Hp10 could induce potent mucosal immune response.The results of challenge test showed that only Hp10 could reduce H.pylori colonization in the stomach of mice.Combined immunization of Hp4 and Hp10 in mice can mediate stronger cellular and mucosal immunity,and reduce the level of H.pylori colonization in the stomach to a greater extent.These results suggest that only vaccines that induce both cellular and mucosal immunity have the ability to resist H.pylori infection.Conclusion:In summary,Pam₂Cys was chemically linked to immunodominant epitopes of Ure B or Cag A derived from H.pylori to synthesize lipopeptide vaccines Hp4 and Hp10 with self-adjuvant activity.Both of them can mediate the recognition,phagocytosis and activation of dendritic cells through self-assembly and activation of PRRs,and further induce potent cellular immune responses.However,only Hp10 based on T/B mixed epitopes can simultaneously induce effective mucosal antibody responses and mediate protection against H.pylori.Co-immunization of Hp4 and Hp10 elicited stronger cellular immunity and mucosal antibody production in mice.These results support the hypothesis that effective cellular and mucosal antibody responses may mediate protection against H.pylori infection.