Biomimetic Synthesis Of Influenza Vaccines With Virus-Like Particles As "Chassis"

The vaccination of safe and effective vaccines is the most effective way to prevent and control infectious diseases.However,the high antigenic diversity and high mutation rates of viruses impose significant limitations on traditional vaccine manufacturing processes.Influenza caused by influenza virus is one of such infectious diseases that threaten human health seriously thus demands universal vaccines urgently.This work from vaccine synthetic biology point of view proposed a series of nanovaccines against influenza virus by taking apo-ferritin(AFt)and Hepatitis B core antigen(HBc)virus-like particles as "chassis",employing influenza virus hemagglutinin(HA),matrix protein 2 ectodomain(M2e)and nucleoprotein(NP)antigen as "plugins" The presentation of antigens on the VLPs were realized by chemical modification and conjugation,genetic engineering,and a novel temperature-shift based physical encapsulation technologies to simulate their spatial distribution in the natural virus.The spatially programmed VLPs influenza vaccines allowed the presentation of structurally diverse antigens and enhanced their antigen immunogenicity.The results and novelties are as follows:(1)The M2e-AFt and HA-AFt conjugate vaccines were prepared by covalently conjugating the external M2e antigen or full-length HA antigen of influenza virus on the outer surface of AFt nanoparticles through chemical coupling technology.Upon intraperitoneal immunization in mice,the two conjugate vaccines stimulated significantly higher antigen-specific antibody and better protective immune responses than the free antigen,and HA-AFt was superior to M2e-AFt.The(M2e+HA)-AFt externally assembled dual-antigen influenza vaccine was further developed by covalently coupling the external full-length HA and M2e antigens on the AFt surface simultaneously,which could elicit both high levels of M2e and HA antigen-specific antibodies in mice.Compared with the single-antigen vaccine,the dual-antigen vaccine provided stronger broadly protective immune responses for immunized mice,with a 100%protection rate against homologous strain and a 70%protection rate against heterologous strain.(2)From the perspective of particulate structural change,the thermal stability and the variation of pore channel between subunits of AFt nanoparticles with the temperature was investigated.It was found that the intact structures of AFt nanoparticles were retained after heating at 25 to 50℃ for at least 30 min.However,its particle size increased from 9.3 to 12.2 nm,indicating a thermally induced expansion in the nanopores thus resulted in an increase of particle size of AFt nanoparticles.Further increasing the heating temperature led to significant changes in the secondary structure of AFt and even degradation or aggregation of the AFt nanoparticle.Benefited from the discovery of the expansion in the nanopores of AFt nanoparticles at high temperatures,a novel temperature-shift based encapsulation process was proposed for encapsulation of influenza virus internal nucleoprotein(NP)antigens inside AFt nanoparticles.By mixing NP with AFt solution at 50℃ for 45 min,about 20 NP peptides could be encapsulated into each AFt nanoparticle,which was significantly higher than that obtained by traditional pH-dependent disassembly-reassembly encapsulation process(Only 4.5 NP peptides per AFt nanoparticle).Moreover,the new process was easier to operate and control than the pH-dependent strategy.(3)By combining the abovementioned temperature-shift based encapsulation strategy with the chemical conjugation of HA antigens on the AFt surface,an internally-externally assembled biomimetic dual-antigen influenza vaccine HA-AFt+NP with HA chemical conjugated on the surface and NP encapsulated inside the cage was constructed based on the AFt "chassis".Both HA and NP antigen-specific humoral immune responses were elicited by this HA-AFt+NP vaccine,and the IgG2a and Thl immune responses of the surface-coupled HA antigen were synergistically enhanced by the embedded NP antigen.Compared with free HA antigen and the HA-AFt single-antigen vaccine,the biomimetic HA-AFt+NP dual-antigen vaccine conferred 100%protection against a lethal infection of both homologous and heterologous H1N1 strains in the absence of adjuvants,and higher hemagglutination inhibition(HAI)titers against heterologous H5N1 and H3N2 virus strains,which indicated a more potential cross-protective effect of the HA-AFt+NP vaccine against a variety of influenza virus strains.(4)Based on the thermal stability and thermally associated pore-expansion characteristics of HBc VLP nanocages similar to AFt,an internally-externally assembled biomimetic dual-antigen influenza vaccine was further constructed with HBc VLPs as "chassis",by combining genetic engineering with a novel temperature-shift based encapsulation technology.By displaying NP or M2e antigens on the exterior of HBc VLPs through genetic fusion,and further encapsulating M2e or NP antigens inside the VLPs at 60℃ for 30 min,two internally-externally assembled dual-antigen influenza vaccines M2eext-HBc+NPint(A biomimetic vaccine)or NPect-HBc+NPint(A non-biomimetic vaccine)simultaneously targeting M2e and NP antigens was constructed.Both of the dual-antigen vaccines could elicit M2e and NP antigen-specific antibodies simultaneously in mice and synergistically enhanced effects in antibody levels were observed between the loaded two antigens in the dual-antigen vaccines.Most importantly,after a lethal challenge of the heterologous H1N1 virus,the biomimetic vaccine M2eext-HBc+NPint conferred the mice 100%protection without noticeable body weight loss in the absence of any adjuvant.While the protective efficacy conferred by the non-biomimetic vaccine NPext-HBc+M2eint was only 62.5%,accompanying 12.5%body weight loss in the immunized mice.Analysis of the immune mechanisms showed that the biomimetic M2eext-HBc+NPint dual-antigen vaccine was able to stimulate the significantly more efficient formation of germinal center(GC)B cells and a higher level of effector memory CD8+T cell population.All above results demonstrated that with VLPs as the "chassis" for biomimetic assembly of a flexible combination of different antigens as "plugins",the present work not only provides a new strategy for the rapid construction of more effective and universal vaccines against influenza virus but also promising for the production of effective vaccines against other emerging or emergent infectious pathogens.