Rational Design And Immunogenicity Assay On Particulate Capsid Protein Of Human Immunodeficiency Virus Type 1 That Bearing Surface Loop Epitopes Of Viral Envelope Protein

It has been more than 30 years since the discovery of the Human Immunodeficiency Virus(HIV)in 1983,and the development of effective,safe and affordable HIV vaccines and antiviral drugs remains the primary task for the HIV prevention and treatment.Scientists from different countries in the world are trying to investigate different techniques to accelerate the vaccine development.With the development of techniques such as single cell sorting and second-generation sequencing,a series of broadly neutralizing antibodies(bNAbs)have been isolated from HIV-infected individuals.Based on the crystallographic analysis of these bNAbs structures,new immunogenic designs have been developed,including focusing on HIV bNAbs epitopes or conserved epitopes.The main purpose is to use a foreign scaffold or virus particles to display different epitopes,which aims to induce immune system responses and elicit bNAbs.HIV-1 capsid is extensive investigated because of highly conserved sequences and a large number of T,B cell immunodominant epitopes.In the previous research of the mutant capsid protein(CA-N21C/A22C),we found it assembles into particles in vitro.In this study,the mutant capsid protein was used as a scaffold to present different Env epitopes to construct fusion proteins to explore its potentials for an immunogen design of the HIV epitope scaffold.The CypA loop in the capsid was considered as the presented region for foreign epitope.And a series of recombinant clones with different Env epitopes were designed.Recombinant N21C/A22C-V3,N21C/A22C-V1V2 proteins,including NL4-3 and 89.6(clade B)and94UG114(clade D),were expressed in the E.coli prokaryotic expression system.The high purity proteins were purified through ion chromatography and hydrophobic chromatography.Then proteins were characterized by ELISA,and Western blot(WB).The immune serum were analyzed to evaluate the protein immunogenicity by flow cytometry,immunofluorescence,ELISA,and TZM-bl neutralization assay based on the immuno-dot(ELISPOT)platform.The results showed that the recombinant proteins had reactivity with different neutralizing monoclonal antibodies,and the immune serum can neutralize different subtypes of viruses and there is an immune cross-reaction,suggesting good antigenicity and immunogenicity.It also stimulated the Thl and Th2 immune response in the immunized mice.In summary,this study evaluated the feasibility of the N21C/A22C as a scaffold,and provided the proof of concept for the further HIV-1 immunogen designand epitope-focused vaccine studies.