Structure-guided Screening Of Protective Epitopes And Development Of Self-assembling Protein Nanoparticle Delivery System For Epitope-based Vaccines

Background:Currently,most vaccines mainly consist of attenuated or inactivated microorganisms and their toxins,which can induce effective and durable adaptive immune responses.However,these conventional vaccines are associated with problems such as the risk of infection and difficulties in producing pathogens in vitro,which may contain impurities.The specific response stimulated by antigens is the aggregation of epitope-based specific responses.Only a portion of these epitopes,known as immune-dominant epitopes,can exert protective effects in immune responses.An epitope-based vaccine is composed of one or several protective epitopes that can accurately and efficiently induce a specific immune response.Epitope-based vaccines have been proven to be safer and more potent,with the properties of flexibility in design and convenience in modification.However,there are some difficulties that limit the application of epitope-based vaccines,including the identification of protective epitopes and the lack of delivery systems.The structure of vaccine or antigen is a determining factor in immune responses.Therefore,screening epitopes based on structure and optimizing delivery system are effective strategies to overcome the difficulties.To solve these problems above,we carried out the following two researches:Part I:A novel structurally identified epitope delivered by macrophage membrane-coated PLGA nanoparticles elicits protection against Pseudomonas aeruginosaObjectives:Pseudomonas aeruginosa（PA）is a prevalent pathogen responsible for hospital-acquired infections,and a primary bacterial pathogen associated with burns and trauma.In recent years,the antibiotic resistance of PA has gradually increased.The emergence of multi-drug and pan-drug resistant PA strains has led to the need for effective PA vaccines to prevent infectious diseases and reduce antibiotic use to ultimately combat drug resistance.However,no marketed PA vaccine exists,largely due to the variation in virulence gene expression during the infection process,which may compromise vaccine efficacy.Our study focuses on identifying upregulated proteins serving as ideal vaccine targets and screening protective epitopes to construct a PA vaccine.Bacterial membrane proteins are the primary vaccine antigens against bacteria,as antibodies against these antigens not only mediate antibody-dependent cell-mediated cytotoxicity（ADCC）effects but also block their pathogenic effects.However,the hydrophobicity of these outer membrane proteins poses a challenge when used as vaccine candidates.Advances in structural vaccinology provide potential solutions to this challenge,with extracellular loops of outer membrane proteins being the central functional and immunogenic regions.In this study,we aim to select outer membrane proteins with the most significantly upregulated expression according to transcriptome data and screen for immune dominant epitopes.To enhance immunogenicity,we will construct a macrophage membrane-encapsulated PLGA nanoparticle to deliver the identified epitopes.The immune response and protective effect of the nanovaccine will be evaluated in a mouse model.Methods:1.After the transcriptome analysis,transmembrane proteins exhibiting significant changes in m RNA levels were chosen as potential antigens.Then,PRED-TMBB was used to predict the extracellular loops,transmembrane domains and intercellular loops.Subsequently,Luminex-based assay was employed to screen for dominant epitopes.To evaluate the immunogenicity of these epitopes,they were synthesized and conjugated to KLH（keyhole limpet hemocyanin）.The peptide-KLH conjugates formulated with the adjuvant Al（OH）₃ were administered through intramuscular immunization of Balb/c mice.Blood samples were collected via the tail vein seven days after the final immunization,and the reactivity of mouse sera against each peptide was determined by ELISA.2.The PNPs（PLGA nanoparticles）were synthesized via a reported water-in-oil-in-water double emulsion method with slight modifications.The mouse macrophage cells（RAW264.7）were prepared by repeated freeze-thaw and were mixed with PNPs in a weight ratio of 1:1.To obtain PNPs@M carrying Ep_167-193(PNPs@M-Ep_167-193),PNPs@M and Ep_167-193 conjugated to1,2-distearoyl-sn-glycero-3-phosphoethanolaminepoly（ethylene glycol）-2000（DSPE-PEG2000）were employed to prepare PNPs@M-Ep_167-193 based on the interaction between DSPE segments and the cell membrane.The size and morphology of the nanoparticles were determined using a transmission electron microscope（TEM）.The Zeta-potential and size distribution were measured at room temperature using a Nano-ZS.SDS-PAGE was carried out to confirm the membrane camouflage.The toxicity of PNPs@M-Ep_167-193 was determined by the standard Cell Count Kit（CCK-8）assay.Erythrocytes diluted in 0.9%Na Cl solution were incubated with PNPs@M-Ep_167-193 to evaluate the hemolysis effects.The biocompatibility of PNPs@M-Ep_167-193 in vivo was assessed by a mouse experiment.3.On day 0,14 and 21,the mice in each group were immunized with PNPs@M-Ep_167-193,Ep_167-193,PNPs@M or PBS.Blood was collected via the tail vein,and serum was isolated.The titers of total Ig G and the subtypes against Ep_167-193 in the sera were determined by ELISA.To evaluate if PNPs@M-Ep_167-193-induced immunity could confer effective protection in mice,seven days after the last immunization,the mice in each group were intratracheally injected with a sublethal dose of PA XN-1.Then,the infection was scored according to the breathing,piloerection,movement,nasal secretion and posture of the mice.Meanwhile,body weights were recorded.The number of bacteria colonized and the levels of the proinflammatory cytokines IL-1β,TNF-α,IL-6 and IL-12 in the lung were determined.Histopathological analysis of the lungs was also carried out.Results:1.Eight transmembrane proteins with the most significant changes in m RNA levels were selected and a total of 54 peptides spanning the transmembrane loops of these eight proteins were predicted.Eight epitope peptides with good immunogenicity were identified by Luminex,they were PA2398 Q737-K754,PA4554C167-W193,PA2398 F636-G672,PA0165 R163-A174,PA2398 K694-Q712,PA0958 A200-Q235,PA2398T302-V331,PA4554 D148-T172,PA2398V552-R568 and PA0958 Q295-S319.ELISA indicated PA2398 Q737-K754 and PA4554 C167-W193 were the top 2 immunogenic epitopes in both humans and mice.The protective effect of PA2398 Q737-K754 was assessed previously.As a result,we investigated the epitope PA4554C167-W193(Ep_167-193)in subsequent studies.2.Nanoparticles carrying the epitope PA4554 C167-W193(PNPs@M-Ep_167-193)were successfully produced,and SDS-PAGE confirmed the successful coating of the MM（Macrophage membrane）.The hydrodynamic diameter of PNPs@M-Ep_167-193 was 272.2±4.0nm（PDI=0.281±0.007）and the Zeta potential was-18.2±0.5 m V.TEM observation showed that the size of PNPs@M-Ep_167-193 vaccine particles was about 242.5 nm,with smooth spherical surface and no obvious aggregation.Moreover,PNPs@M-Ep_167-193was safe for in vitro and in vivo evaluation.3.Ep_167-193-specific Ig Gs and Ig G subtypes（Ig G1,Ig G2a and Ig G2b）were significantly increased in the sera of PNPs@M-Ep_167-193 group compared with Ep_167-193,PNPs@M or PBS groups.The effect of PNPs@M-Ep_167-193 on immunity elicited by intratracheal injection of PA XN-1 showed that the onset of infection symptoms in the PNPs@M-Ep_167-193 group was clearly slower than that in the other groups.The number of bacteria and the levels of cytokines IL-1β,TNF-α,IL-6 and IL-12 in the mouse lungs from the PNPs@M-Ep_167-193 group were significantly lower than that in the Ep_167-193,PNPs@M vector and PBS control groups.A similar trend was found after histopathological analysis of the lungs.Congestion and neutrophil infiltration in the lungs of the PNPs@M-Ep_167-193-treated mice were significantly restricted.The alveolar structures were not severely damaged,showing significant differences compared with the other three groups.Conclusions:1.Ep_167-193 from PA4554 is a novel predominant epitope in human and mice.2.Nanoparticles carrying the epitope PA4554 C167-W193(PNPs@M-Ep_167-193)were successfully produced.3.PNPs@M-Ep_167-193 was safe for in vitro and in vivo evaluation and can induce a Th2-biased immune response and confer effective protection in mice.PartⅡ:Novel nanovaccine carrier based on self-assembling ferritin nanoparticles improves the efficacy of cancer vaccinesObjectives:Cancer has become a major public health concern in China,posing a serious threat to both human health and economic development.With an increasing understanding of antitumor immunity,immunotherapy has emerged as a powerful means for oncotherapy.Specifically,personalized vaccines have shown great potential due to their low cost and good safety profile.However,personalized vaccines still face challenges such as poor stability and weak immunogenicity,which limit their clinical efficacy and application.Fortunately,a promising solution to these challenges lies in the development of vaccine delivery systems based on nanoparticles.Epitopes can be encapsulated within nanoparticles or loaded onto their surface following appropriate modifications,allowing them to self-assemble into nanoparticle vaccines.There are many advantages of nanoparticle vaccines.For example,nanoparticles are able to protect antigen against enzymatic degradation and enhance uptake by antigen-presenting cells（APCs）.Moreover,nanoparticle vaccines are able to form a depot effect,retaining the antigen at the injection site and prolonging exposure to immune cells.The ability to mimic the characteristics of the pathogen such as size,shape,and pathogen-associated molecular patterns（PAMPs）is another advantage.Furthermore,nanoparticle vaccines are able to target specific organs or cells through modifications of the nanoparticles.In the study of PA vaccine PNPs@M-Ep_167-193,nanoparticle-based carrier significantly improved the immune response.To further improve the efficacy of personalized cancer vaccines,we aim to develop a novel nanovaccine carrier based on self-assembling nanoparticle composed of ferritin.The CD8⁺T cell epitope peptide(OVA_257-264)of model antigen ovalbumin（OVA）is selected as the immunogen.The optimal transplantation site of OVA_257-264 in Fc will be screened based on the level of recombinant protein expression,then the fusion protein Fc-OVA_257-264carrying epitope OVA_257-264 will be obtained.Fusion protein FNP（Ferritin-based nanoparticle）will be constructed with ferritin and 4 tandem repetitions of the B domain of Staphylococcus aureus A protein（Sp A4B）.A novel ferritin-based nanoparticle vaccine FNP-Fc-OVA_257-264 will be prepared to deliver epitope OVA_257-264 through the high affinity between Sp A4B and Fc.This approach holds great promise for enhancing the potency and effectiveness of personalized cancer vaccines.Methods:1.FNP and Fc-OVA_257-264 proteins were expressed by E.coli and 293F cells,respectively.The FNP-Fc-OVA_257-264complex was produced,and its size and morphology were determined using TEM.The Zeta-potential and size distribution were measured at room temperature using a Nano-ZS.The physicochemical properties of FNP-Fc-OVA_257-264were also evaluated using size exclusion chromatography.The optimal adjuvant formulated with FNP-Fc-OVA_257-264was screened using ELISpot assay.The toxicity of FNP-Fc-OVA_257-264 was evaluated by the CCK-8 assay,hemolysis assay and mouse experiment.2.C57BL/6 mice in each group were immunized on day 0,14 and 28.14 days after the last immunization,the amounts of IFN-γSFCs（Spot-forming cells）and OVA_257-264 specific CD8⁺T cells were determined using an ELISpot assay and tetramer staining assay.Four months after the last immunization,the amount of IFN-γSFCs was determined by ELISpot to evaluate long-term immune response of vaccine.3.C57BL/6 mice were immunized and subcutaneously challenged with E.G7-OVA tumor cells to evaluate antitumor activity of the vaccine in both prophylactic and therapeutic grafted tumor model.Tumor volumes were measured with a caliper and MRI was used to observe tumor growth.Tumor-infiltrating lymphocytes were determined using IHC.4.Confocal laser scanning microscopy（CLSM）and flow cytometry were used to determine the uptake of the FNP-Fc-OVA_257-264and Fc-OVA₂₅₇-₂₆₄particles by DC2.4 cells.The in vitro activation of BMDCs,in vivo activation and cross-presentation of DCs in inguinal lymph nodes（LNs）were analysed by flow cytometry.The antigen-specific CD8⁺T cells were isolated from OT-1 TCR-transgenic mice and subsequently stimulated with FNP-Fc-OVA_257-264 particles.Then the proliferation of these cells was assessed by CFSE dilution assay with flow cytometry.Results:1.Protein FNP and Fc-OVA_257-264were successfully produced with high purity and solubility.Additionally,the complex FNP-Fc-OVA_257-264was successfully produced with a hydrodynamic diameter of 182.4±12.9 nm（PDI=0.252±0.035）,which was significantly larger than that of FNP（35.8±1.5 nm,PDI=0.261±0.004）.Importantly,the nanovaccine FNP-Fc-OVA_257-264was safe for in vitro and in vivo toxicity evaluation.2.Poly I:C was screened as the optimal adjuvant formulated with FNP-Fc-OVA_257-264.Fourteen days after the final immunization,mice inoculated with FNP-Fc-OVA_257-264 showed a significant increase in the production of IFN--specific spots（up to 350 per 10⁶ splenocytes）compared to control groups,including free Fc-OVA257-264 and PBS,which only minimally induced IFN--specific spots.This trend was also observed 4 months after the final immunization.3.The tumor volume of FNP-Fc-OVA257-264-immunized mice was much smaller and their survivals were significantly prolonged in both prophylactic and therapeutic lymphoma models.IHC analysis showed a significant increase in tumor-infiltrating lymphocytes in FNP-Fc-OVA_257-264-immunized mice compared with other two groups.4.CLSM imaging and flow cytometry showed that nanovaccine FNP-Fc-OVA_257-264was efficiently taken up by DC2.4 cells,whereas free Fc-OVA_257-264 was not.Additionally,flow cytometry analysis revealed a significant upregulation of costimulatory signals（such as CD80,CD86,CD40,MHC-I,and MHC-II）on the surface of BMDCs in the FNP-Fc-OVA_257-264-treated group.In vivo results showed that vaccination with FNP-Fc-OVA_257-264resulted in the highest level of CD86⁺CD80⁺DCs in the inguinal lymph nodes（LNs）of treated mice compared to Fc-OVA_257-264 or PBS treatment at 72 h after immunization.Moreover,FNP-Fc-OVA_257-264also resulted in the highest level of CD11c⁺SIINFEKL-H-2Kb⁺antigen cross-presented DCs in draining LNs 72 h post-immunization.Conclusions:1.Vaccine carrying epitope OVA_257-264based on ferritin nanoparticles,named FNP-Fc-OVA_257-264,was successfully produced and it was safe for in vitro and in vivo evaluation.2.Nanovaccine FNP-Fc-OVA_257-264 was capable of efficiently inducing antigen-specific CD8⁺T cells in mice amd showed promising results in inhibiting tumor growth in both prophylactic and therapeutic lymphoma models.3.Immunization with FNP-Fc-OVA_257-264significantly enhances immune activation of APCs,leading to enhanced uptake and cross-presentation of antigen by APCs.