Rational Assembly Of Multi-Component Vaccine To Enhance Immune Responses

Vaccines play an indispensable role in disease prevention and control,making them an important tool for global economies and in human health.With the discovery of vaccine-related immune mechanisms,multi-component vaccines composed of antigens,immune stimulants and delivery systems are an important aspect in vaccine development.However,improving immune responses to these vaccines through the rational design and assembly of each component of a multi-component vaccine is challenging.In response to these challenges,rigid cationic nanoparticles and mannose-targeted flexible Pickering nucleic acid emulsions were constructed for the rational loading and efficient delivery of antigens and immunomodulators.The related mechanisms of the elicited immune response were investigated with in vivo and in vitro experiments.The specific results of our study were as follows:(1)The simultaneous loading of antigen,immunostimulant CpG,and all-trans retinoic acid into rigid cationic nanoparticles PLNP for efficient intracellular delivery.The PLNP used the biodegradable polymer,poly(lactic-co-glycolic acid)(PLGA),as a core compound that was loaded with all-trans retinoic(atRA)through hydrophobic embedding,while lipid DC-cholesterol was used as a positive charge modifier.The positive charge carried on the surface of the nanoparticles could efficiently absorb the negatively charged antigens(76.95%±4.82%)and CpG(80.10%±4.17%).Dynamic light scattering and SEM imaging showed that PLNP had a uniform spherical morphology(particle size:190.4 nm±2.1 nm,PDI:0.143±0.068).In vitro cell experiments showed that PLNP effectively promoted antigen uptake by BMDCs and antigen cross presentation through the lysosomal escape effect.Moreover,PLNP effectively increased the expression of CD80 and CD86 on the surface of BMDCs,thereby promoting BMDC maturation and activation.(2)PLNPs were developed for the co-delivery of atRA,CpG,and antigen for the efficient dual response of systemic and gastrointestinal immunity through the“lymph-node-amplifying effect”.PLNP induced DCs migration and enrichment in the draining lymph nodes by up-regulating the expression of the draining lymph node homing receptor CCR7 on the surface of DCs recruited to the injection site.The abundant immune microenvironment in the draining lymph nodes was used to promote the interaction between the OVA~+DCs and T cells,as well as to promote the expression of the intestinal homing receptor CCR9 on the T-cell surface,which then induced intestinal homing of the T cells.After two immunizations in mice,the PLNP group exhibited an effective promotion of serum antigen-specific IgG and intestinal mucosa IgA antibody production and secretion.Moreover,the proportion of antigen-specific CD8~+T cells in the spleen and mesenteric lymph nodes was significantly increased when compared to those in the control group.Animal experiments using the EV71 recombinant protein antigen VP1 have also shown that PLNP can efficiently elicit a dual response of systemic and gastrointestinal immunity.(3)A mannose-modified Pickering nucleic acid emulsion efficiently targeted APCs recognition and activation.A Pickering emulsion with mannose-based targeting was prepared using nanoparticles assembled by block copolymer PLGA-PEG-mannose and PLGA-PEG-N3as an emulsion stabilizer to further improve its targeting property.With a click chemical reaction(N3-DBCO)between CpG-DBCO and the-N3 group on the surface of the Pickering emulsion,CpG was efficiently added onto the emulsion surface to creat Pickering nucleic acid emulsion Man-PMPE-CpG with a mannose modification on its surface.A Pickering emulsion with a particle size of 1000 nm was prepared by optimizing the nanoparticle concentration,the type and p H of the external aqueous buffer,the ultrasonic power,and ultrasonic time of the emulsion formation process.Additionally,in order to solve the low loading rate of polypeptide antigens into the Pickering emulsions,we encouraged the efficient loading of polypeptide antigens into the Pickering emulsions using palmitic acid modification of polypeptide antigens and the“one-step emulsification method”.In vitro cell experiments showed that Man-PMPE-CpG benefited from the targeting effect of mannose,which increased both the uptake of the emulsion by BMDCs and the immune stimulation effect of CpG to efficiently activate BMDCs.(4)A peptide tumor vaccine was constructed using a Pickering nucleic acid emulsion Man-PMPE-CpG modified with a mannose group for tumor immunotherapy.Man-PMPE-CpG forms a strong antigen reservoir effect at its injection site,and can effectively recruit a large number of immune cells,such as DCs and macrophages that prolong the interaction time between antigens and APC.Man-PMPE-CpG improved the efficiency of antigen presentation;the antigen-specific cellular immune response in mice immunized with Man-PMPE-CpG was significantly enhanced compared to that in the control mice.In an OVA antigen mouse tumor model,Man-PMPE-CpG significantly inhibited tumor growth.In addition,when combined with the immune checkpoint inhibitor PD-1 antibody,Man-PMPE-CpG synergistically enhanced the anti-tumor ability.