Identification Of HIV-1Env-specific Monoclonal Antibodies And Research Of Heteroligation Antibodies

The neutralizing antibody is an important component of protective immuneresponses against HIV-1. So identification of broadly neutralizing antibodies isimportant for AIDS therapy and prevention.In recent years, several kinds of high throughput and sensitive monoclonalantibody screening methods were developed. Monoclonal antibodies identified by Bcell sorting and single cell RT-PCR methods can reflect the natural form ofantibodies better. However, dependence on the expensive flow cytometry haslimited its application. Therefore, we established a method using magnetic beadsinstead of flow cytometry to obtain B cells specifically binding HIV-1Gp120antigen. Combined with single-cell RT-PCR and expression cloning, severalmonoclonal antibodies against Env were obtained.In brief, PBMCs were isolated from peripheral venous blood by Ficoll-Paquedensity gradient centrifugation. Human memory B cells were purified by negativesorting from PBMCs of HIV-1infected individuals, and memory B cells werefurther enriched using anti-CD27microbeads. Gp120antigen labeled with biotinwas incubated with memory B cells to specifically bind IgG on cells membrane. Thememory B cells expressing the Env-specific antibody were harvested by anti-biotinmagnetic beads separating, counted and diluted to the level of a single cell in eachPCR well loading with catch buffer containing RNase inhibitor to get RNAs. Theantibody genes were amplified by single cell RT-PCR and nested PCR, cloned intoeukaryotic expression vectors and transfected into293T cells. The binding activityof antibodies to Env was tested by ELISA. The neutralizing activity of antibodies toEnv was tested by micro-neutralization assay. Three Env-specific antibodies wereidentified from one HIV-1infected individual. Two of them could bind Gp140andFLSC, the other one binds Gp120, FLSC and Gp140with similar binding activity.Studies showed that modified heteroligation bivalent anti-Env antibody bindingdifferent epitopes could neutralize HIV-1more effectively compared with wildantibodies. Whether the antibodies with no or very weak neutralizing activity hadsynergy effect has not been investigated yet. Hence, to investigate the interaction ofdifferent antibodies is our next goal. Firstly, we test the interaction of different antibodies which were identifiedpreviously in our lab by micro-neutralizing assay. Then, the heteroligationantibodies were constructed as follows. Two heavy chain expression vectorIgG-T270Y and IgG-Y311T were constructed by site-directed mutation to form the"knob-in-hole" structure which can promote the formation of heteroligation antibody.To easy detection, the FLAG label was introduced at C terminal of CH3domain ofIgG-T270Y, and the6×His label was introduced at the C terminal of CH3domain ofIgG-Y311T. The heavy chain variable region genes and light chain variable regiongenes were linked together to form the scFv by the overlap PCR. scFv was clonedinto modified expression vectors. Heteroligation antibodies were produced bycotransfection. The transfection supernatant was collected, and the heteroligationantibodies were purified by protein A and Ni Sepharose high performance affinitychromatography. The neutralizing activity of heteroligation antibodies were testedby neutralization. Two heteroligation antibodies CD4-FLAG/1F4-His and1F4-His/3H2-FLAG were expressed successfully, and the neutralizing activity ofheteroligated antibodies were significantly improved compared with wild antibodies.In summary, Env-specific antibodies were obtained using biotin labeled HIVGp120antigen, magnetic beads separating, single cell RT-PCR and expressioncloning. Monoclonal antibodies with no or very weak neutralizing activity usedtogether maybe have synergy effect. The heteroligation antibodies were successfullyconstructed and their neutralizing activity was better than the wild antibodies.