Immunogenicity Assay On Fusion Proteins Harboring Envelope V3 Loop Of Human Immunodeficiency Virus And Subsequent Neutralizing Monoclonal Antibody Screening

Acquired immunodeficiency syndrome(AIDS)is an infectious disease characterized by severe systemic immune system damage.Human immunodeficiency virus type 1(HIV-1)infection is the archcriminal of AIDS.By 2015,the number of HIV global infectious person reached 36.7 million,seriously endangering human health.So far,there is no protective vaccine or drugs that can completely remove the HIV virus.Therefore,the study of safe and effective vaccines and antiviral drugs for the prevention and treatment of HIV is of great significance.Since 2009,a large number of potent and broadly neutralizing antibodies and corresponding epitopes have been reported,animal models and RV144 vaccine protection effects have shown that induction of broadly neutralizing antibodies is the key to vaccine design.HIV-1 envelope glycoprotein gp120 the third variable region(V3)is essential for viral infection.In our previous study,we immunized mice with virus and screened antibody against HIV strain NL4-3 V3 epitope,the V3 neutralizing epitope was shown on CRM197-A and HPV-VLP to assess the immunogenicity of V3 neutralizing epitopes and the potential for research and development of epitope vaccines,and then provide the basis for prophylactic and therapeutic antibody research.In this study,we showed different fusion protein of different length and different types of V3 in CRM 197-A and HPV-VLP,and evaluated the humoral immunity of mice by neutralization experiment and enzyme-linked immunosorbent assay(ELISA)effect.Based on the mouse monoclonal antibody screening platform,the monoclonal antibody was screened using the immunoblot blot(ELISPOT),based HIV neutralizing antibody screening platform.The characterization of the monoclonal antibody were analyzed by neutralization assay and ELISA.Through the analysis of mouse serum and monoclonal antibody characterization analysis,the immunogenicity of V3 neutralizing epitope was comprehensively evaluated,and the possibility of developing epitope vaccine was further explored.In the course of the study,CRM197-A and HPV-VLP were fused with different length and different types of V3 neutralization epitopes.Serum monitoring results showed that CRM197-A-NL4-3-299-328 and HPV-NL4-3-296-311 fusion protein immunized serum has neutralizing effect on HIVNL4-3.CRM197-A-NL4-3-299-328 fusion protein was screened 8 neutralizing monoclonal antibodies with specific neutralizing HIVNL4-3,most of which IC50 was less than 0.02μg/mL,which was potent monoclonal antibody.HPV-NL4-3-296-311 fusion protein was screened 20 strains of neutralizing monoclonal antibody with different capacities.Most of the monoclonal antibodies were able to bind to different types of HIV envelope protein gp120,which wih cross-reactivity.The mAb 4H4 could neutralize the laboratory existing strains HIVNL4-3,HIV89.6,HIVMJ4,HIV94,and IC50 of HIVNL4-3,HIV89.6,HIVMJ4 and HIV94 were 12-15μg/mL.In conclusion,in this study,V3 neutralizing epitope fusion protein stimulated mice to produce potency antibody,and screened for type-specific potent neutralizing antibodies,and also produced cross-neutralizing antibodies,suggesting that V3 neutralizes epitope fusion has strong immunogenicity and has the potential to induce broadly neutralizing antibodies,which providing basis for further studies on the design of broadly neutralizing antibody antigens and epitope vaccines.