Construction Of Folic Acid-modified Erythrocyte Membrane-hybrid Liposomal Toxoid Vaccine And Anti Pore-forming Toxin Of Vibrio Vulnificus

The prevalence of drug-resistant bacterial infections,compounded by the extensive clinical application of antibiotics,presents a formidable obstacle to effective drug therapy,resulting in a projected death toll of tens of millions worldwide by the year 2050.Hence,there is a pressing need to devise and advance novel drug dosing approaches to enhance the efficacy of preventing and treating bacterial infections,including those caused by drugresistant strains.Pore-forming toxins(PFTs),a bacterial exotoxin,serve as the primary virulence factor in numerous bacterial infections.By destroying the epithelial barrier and compromising the immune system,PFTs facilitate pathogen invasion and colonization,making them a crucial target in combating bacterial infections.The antivirulence factor strategy entails inhibiting toxins to eliminate pathogens in a straightforward way to avoid drug resistance and is a promising approach for treating bacterial infections.Vaccines serve the purpose of preventing and managing bacterial infections by facilitating the identification of pathogens by the immune system and inducing an immune response that effectively combats bacterial infections.Antitoxin vaccines integrate antitoxin strategies with vaccine therapy to minimize the occurrence of bacterial drug resistance.The conventional antitoxin vaccine,also known as the toxoid vaccine,mitigates the toxicity of toxin antigen through protein degeneration.However,the primary challenge associated with toxoid vaccines is the difficulty in ensuring their safety and immunogenicity simultaneously.Hence,based on the biological attributes and mode of action of pore-forming toxins,it holds immense importance to devise a novel vaccine vector system that integrates biomimetic nanostrategy.Unbound pore-forming toxins have the ability to attach to particular receptors on the cellular membrane surface,undergo folding and polysome assembly,and identify and assemble specific regions that are inserted into the cellular membrane surface.Erythrocytes are a primary target of several pore-forming toxins for inducing toxicity,thereby serving as a potential adsorption carrier for toxins.Following the administration of nanocarriers,the absorption of diverse plasma proteins can facilitate the preservation of specific endogenous protein functions and elicit amplified biological effects.As an illustration,the presence of immunoglobulin IgM in the bloodstream can serve as a natural adjuvant to augment the immune response of nanovine in vivo.Research has demonstrated that upon IgM adsorption by liposomes,there is a notable augmentation in the distribution of splenic B cells,and the production of specific antibody titers in mice following immune stimulation can be sustained at elevated levels for an extended duration.This study presents the construction of a novel toxoid vaccine through the development of a biomimetic nanoparrier that adsorbs and neutralizes the toxicity of bacterial poreforming toxins.Additionally,the vaccine enhances antigen presentation and immune response through the IgM functionalization strategy.The study provides new insights and experimental evidence for the prevention and control of drug-resistant bacterial infections.Part Ⅰ of the study focuses on the construction and characterization of folate-modified erythrocyte membrane-hybrid liposome toxoid vaccines.The Vibrio vulnificus hemolysin A(VvhA)protein was produced using an E.coli expression system and subsequently analyzed and identified through SDS-PAGE and Western Blot techniques.The resulting VvhA protein exhibited a concentration of 2 mg/m L,a molecular weight of 51 k Da,and a purity of 90%.Preparation of folate modified liposomes(FA-Lips)by thin film evaporation method,followed by extrusion fusion with red blood cell membrane,folate modified red blood cell membrane hybrid liposomes(RCM-FA-Lips)were prepared,and folate modified red blood cell membrane hybrid liposomes toxin vaccine(RCM-FA-Lips(VvhA))was prepared by adsorption of VvhA.The fusion of erythrocyte membranes and liposome was confirmed through fluorescent colocalization experiments,fluorescence resonance energy transfer(FRET),and mass spectrometry analysis.The characteristics of RCM-FA-Lips(VvhA)were examined,and transmission electron microscopy(TEM)revealed a sphere-like membrane structure with a mean particle size of122.9 ± 2.3 nm and a Zeta potential of-24.5 ± 1.3 m V.In Part Ⅱ,an investigation was conducted on the ability and safety of folic acidmodified erythrocyte membrane-hybrid liposomal toxin neutralization.The results of the anti-hemolysis experiment demonstrated that RCM-FA-Lips were capable of fully neutralizing VvhA toxin at a specific mass ratio,with an anti-hemolysis rate of 98.78 ± 0.08% when the mass ratio of RCM-FA-Lips to VvhA was 8:1.The present study aimed to investigate the impact of RCM-FA-Lips on the proliferative activity of mouse hepatocytes(AML12),intestinal epithelial cells(NCM460),and umbilical vein endothelial cells(HUVECs)using the CCK-8 method.The results indicated that RCM-FA-Lips exhibited a significant effect on the proliferation of AML12,NCM460,and HUVECs,with values of 97.59 ± 0.92%,94.51 ± 4.14%,and 97.76 ± 1.22%.Similarly,the RCM-Lips(VvhA)group also showed a significant impact on the proliferation of these cells,with values of 97.78 ± 1.29%,94.90 ± 1.33%,and 97.62 ± 0.91%,when compared to the control group(P < 0.0001).The present study assessed the in vivo detoxification capacity of RCM-FA-Lips through skin detoxification experiments and the survival of VvhA-infected mice.In the mouse skin toxin contamination model,the findings revealed that there were no significant differences among the RCM-Lips group,RCM-FA-Lips group,and negative control group(Saline).The findings from the H&E staining analysis indicated that the epidermal layer architecture of both the RCM-Lips group and RCM-FA-Lips group was intact,with normal and tightly packed epithelial cells,and a high abundance of collagen fibers in the dermis,without any discernible signs of inflammation.In the experiment assessing the survival of VvhA-infected mice,the RCM-Lips and RCM-FA-Lips groups demonstrated a 60% survival rate,indicating a significant therapeutic effect on VvhA-infected mice.Conversely,the RCMVs and FALips control mice exhibited a 0% survival rate.To investigate the tissue toxicity of RCMFA-Lips(VvhA),CD68 immunohistochemistry was employed,revealing a positive number of macrophages in the heart,liver,spleen,lung,and kidney of BALB/c mice 24 hours after7 days of administration at a dose of 78 mg/kg.The findings indicate that the experimental group did not exhibit any discernible positive cells in their respective tissues,and there was no significant difference observed when compared to the saline group.Furthermore,the experimental group demonstrated good tissue safety.The third aspect pertains to the immune activation and immunoprotective effects of the folate-modified red membrane-hybrid liposomal toxoid vaccine.Upon incubation of RCM-FA-Lips(VvhA)with serum and subsequent analysis using SDS-PAGE and ELISA detection,it was observed that RCM-FA-Lips(VvhA)exhibited a greater capacity to adsorb IgM in serum as compared to the RCM-Lips(VvhA)group.The findings indicate a lack of discernible positive cells in the various tissues examined.Laser confocal microscopy was utilized to observe the uptake of RCM-FA-Lips(VvhA)by dendritic cells,revealing a significantly higher uptake in BMDCs compared to the RCMLips(VvhA)group.To investigate the immune activation and protective effects of RCM-FALips(VvhA)in mice.The mice were segregated into subcutaneous and intravenous immunization groups,receiving a total of three administrations at seven-day intervals.The four groups included subcutaneous Saline,VvhA,RCM-Lips(VvhA),and RCM-FALips(VvhA),while the remaining two groups received RCM-Lips(VvhA)and RCM-FALips(VvhA)at a dose of 100 μg(2 mg/m L).The ELISA assay revealed that the RCM-FALips(VvhA)group exhibited significantly higher titers of VvhA-specific antibodies in their serum.Specifically,the serum antibody titers in the RCM-FA-Lips(VvhA)mice were 16 times greater than those in the formaldehyde detoxification VvhA group(P < 0.0001)and 2times higher than in the RCM-Lips(VvhA)group(P < 0.0001).The results of the study demonstrate that the RCM-FA-Lips(VvhA)immune mice exhibited serum antibody titers that were 1.6 times higher than those observed in the RCM-Lips(VvhA)group(P < 0.001).These findings suggest that the administration of RCM-FA-Lips(VvhA)can effectively stimulate higher serum antibody titers,regardless of the method of administration(i.e.,subcutaneous or intravenous).Formation of germinal center in lymph nodes was observed using laser confocal microscopy and quantitatively analyzed by flow cytometry.RCM-FA-Lips(VvhA)significantly increased the percentage of GC B cells in mouse lymph nodes.The proportion of GC B cells in the RCM-FA-Lips(VvhA)group was 1.2 times higher than that in the RCM-Lips(VvhA)group for subcutaneous immunization(P < 0.01),and 3.06 times higher than that in the formaldehyde detoxification VvhA group(P < 0.0001).For tail vein immunization,the proportion of GC B cells in the RCM-FA-Lips(VvhA)group was approximately 1.35 times higher than that in the RCM-Lips(VvhA)group(P < 0.01),and2.13 times higher than that in the formaldehyde detoxification VvhA immunization group(P< 0.0001).After intraperitoneal injection of a lethal dose of VvhA into immunized mice,the survival rate of the RCM-FA-Lips(VvhA)group and the RCM-FA-Lips(VvhA)group for tail vein immunization was 100%,while that of the RCM-Lips(VvhA)group for subcutaneous immunization was 90%,the RCM-Lips(VvhA)group for tail vein immunization was 80%,and the formaldehyde detoxification VvhA immunization group was 60%.This suggests that folate-modified red blood cell membrane-hybrid liposome-like toxin vaccines have better immune protection.In summary,this study developed a folate-modified red blood cell membrane-hybrid liposome as a biomimetic carrier.The carrier efficiently loaded the antigen protein Vibrio vulnificus hemolysin A through the pore-forming mechanism of the toxin on the red blood cell membrane,neutralizing its toxicity while preserving its antigenicity.Additionally,folate molecules were modified onto the surface of the cell membrane-hybrid liposome as targeting molecules for IgM binding.In vivo,IgM was adsorbed onto the surface of the carrier,utilizing its natural adjuvant effect to enhance the in vivo immune response of the toxoid vaccine.The results showed that the constructed nanocarrier toxoid vaccine had good antigen presentation and immune activation effects and produced a good immune protective effect in the mouse model.This system can also be applied to the preparation of toxoid vaccines for multiple bacterial toxins,providing new ideas and experimental basis for the prevention and treatment of antibiotic-resistant bacterial infections.