| Nucleic acid vaccines,including DNA and mRNA vaccines,have gained attention in infectious disease vaccine development due to their advantages such as easy production and induction of robust and durable humoral and cellular immune responses.Besides vaccine formats,antigen combination and design also play crucial roles in vaccine efficacy.In this study,to overcome the shortcomings of commercially available vaccines,we utilized the nucleic acid vaccine platform to design herpes zoster(HZ)DNA vaccines and rabies virus(RABV)mRNA vaccines with different antigen combinations,respectively.Subsequently,a series of immunological evalutions were performed to reveal immune response elicited by the above vaccines in murine model.The results are shown in the following two parts:1.Preparation and immune efficacy evaluation of Herpes zoster DNA vaccineHZ,caused by the reactivation of latent varicella-zoster virus(VZV)from the sensory ganglia due to aging or immunosuppression,is usually followed by post-herpetic neuralgia(PHN)and other complications.VZV-specific cell-mediated immunity(CMI)elicited by vaccination can effectively prevent the incidence of HZ.Currently,there are two forms of vaccines in the market,live attenuated vaccines(LAV),Zostavax~?and Ganwei~?;and recombinant subunit vaccines,Shingrix~?.However,the efficacy of LAV decreases with age,and they are not recommended to immunocompromised people.Subunit vaccines are limited by the high price and low production of AS01B adjuvant.In contrast,DNA vaccines show the advantages of easy production,convenient transport,and the ability to induce robust cellular immune responses,making them more suitably used in developing countries and regions.Therefore,we utilized the DNA vaccine platform for HZ vaccine development.Glycoprotein E(gE)is the most abundant and immunogenic VZV glycoprotein,which contains B cell and CD4~+T cell epitopes.Therefore,g E is widely used for development of HZ vaccines.Immediate early protein 63(IE63)of VZV is expressed in infected sensory ganglia during the latency period and IE63-specific CMI also plays a key role in preventing HZ reactivation.Therefore,we tried to enhance the VZV-specific CMI and prevent HZ by adding IE63 as antigenic component.In this study,we constructed HZ DNA vaccines expressing truncated g E,IE63,IE63-2A-g E(where 2A is a self-cleaving sequence from porcine teschovirus-1)and IE63-linker-g E.Immunization was carried out in C57BL/6 mice pre-immunized with LAV via intramuscular injection.The immunogenicity of HZ DNA vaccines with different antigen combinations and designs was evaluated in terms of humoral and cellular immune responses.As a result,IE63-2A-g E group induced comparable binding antibody and neutralizing antibody(Nab)titers to that in commercial LAV group and higher levels of IFN-γand IL-2 secretion,inducing a Th1-biased immune response and slight antige-specific Cytotoxic T Lymphocyte(CTL)-mediated immune responses.2.Preparation and immune efficacy evaluation of Rabies virus mRNA vaccineRabies is a lethally zoonotic infection caused by RABV,which causing more than60,000 death every year.The widely used inactivated rabies vaccine requires multiple doses to induce sufficient Nab.In contrast,mRNA vaccines,known for safety and high immunogenicity,can be rapidly produced and low-dose immunization is sufficient to generate robust immune responses.Therefore,we utilized the mRNA vaccine platform for RABV vaccine development.Besides vaccine formats,antigen design is crucial for enhancing vaccine efficacy.To enhance the vaccine immunogenicity,three strategies were explored in this study:(1)designing RABV Glycoprotein(G)with different conformations;(2)forming virus-like particles(VLP);(3)combinating nucleoprotein(N)with RABV VLP.RABV G protein,the only viral surface glycoprotein,is the major antigen for inducing Nab and is widely used in RABV vaccine development.G protein forms trimer on viral surface mediating receptor binding and membrane fusion,and trimeric G protein has been proved with better immunogenicity than monomeric form in several reports.Additionally,G protein,classⅢviral fusion protein,undergoes a reversible conformational change during the fusion process.However,the main epitopes eliciting Nab against RABV exist in prefusion conformation but rarely in postfusion conformation.Therefore,we designed RABV mRNA vaccines expressing wild type G protein,soluble trimeric G protein(t G-MTQ)formed by an artificially trimeric motif(MTQ)and pre-fusion conformation stabilized pre G protein.To further enhance immunogenicity of RABV mRNA vaccines,RABV VLP mRNA vaccine was developed by co-expression of pre G and matrix protein(M)to promote antigen presentation by dendritic cells and activate germinal center(GC).Cellular immunity is equally important for disease control,and N protein can induce humoral and cellular immune responses.Therefore,this study aimed to increase the cellular response by adding N protein as antigenic component in RABV VLP mRNA vaccine.In this study,RABV G,tG-MTQ,preG,M and N mRNAs were produced through plasmid construction,in vitro transcription and capping.RABV G,t G-MTQ,pre G,VLP(pre G+M)and VLP/N(pre G+M+N)mRNA vaccines were encapsulated by microfluidic technique.Subsequently,the immunogenicity,protective efficacy,GC activation,and durability of RABV mRNA vaccines were evaluated in BALB/c mouse model.The results indicated that RABV mRNA vaccines exhibited faster and higher level of hurmoral immune responses compared to that in inactivated rabies vaccine group.And RABV mRNA vaccine groups posed Th1-biased immune responses(Ig G2a/Ig G1and Ig G2b/Ig G1 ratios>1).Moreover,the mice immunized with pre G,VLP and VLP/N induced significantly higher RABV-specific Ig G and Nab titers than those in G and t G-MTQ groups.In terms of cellular immunity,ELISpot and flow cytometry results demonstrated that G and pre G groups induced a Th1-biased cellular immune response,while the VLP and VLP/N groups exhibited a relatively balanced Th1/Th2 immune response.And the challenge experiment results showed that all RABV mRNA vaccines completely protected mice against lethal rabies virus intracranial challenge.Furthermore,the activation of GC after immunization was assessed.The results demonstrated that RABV mRNA vaccines,especially the VLP and VLP/N,effectively induced specific GC B,plasma and T follicular helper cell responses.Moreover,the frequencies of Foxp3~+T regulatory cells and suppressive T follicular regulatory cells in all RABV mRNA groups were significantly lower than those in the inactivated rabies vaccine group.In terms of the persistence of vaccine efficacy,the results demonstrated that VLP and VLP/N groups maintained high level of Nab titer within 4 months after the second immunization and significantly increased the number of RABV-specific long-lived plasma cells and memory B cells.Furthermore,RABV mRNA vaccines sustained memory T cell responses within 4 months after the second immunization.In summary,the IE63-2A-g E DNA vaccine designed on DNA vaccine platform exhibited the ability to induce robust humoral and cellular immune responses against HZ,providing a new idea for antigen design of HZ vaccines.Meanwhile,RABV VLP and VLP/N mRNA vaccines designed on mRNA vaccine platform could induce rapid,high-potency,prolonged Nab production and robust cellular response in mice,demonstrating their potential as rabies vaccine candidates.This research provides novel insights into selection of vaccine platforms and antigen designes for HZ and RABV vaccines. |
