High‐Level Expression System for Production of Recombinant HIV‐1 gp120 with Elevated Content of High‐Mannose Glycans
Authors: Knoppova Barbora , Wei Qing , Hargett Audra , Regal Kellie , Brown Rhubell , Hall Stacy , Moldoveanu Zina , Yother Janet , Patel Rakesh , Raska Milan , Renfrow Matthew , Novak Jan 
Published: June 2021

HIV‐1 envelope glycoprotein (Env), a trimer of gp120 and gp41, has N‐glycans that form a “glycan shield” that covers most of the Env surface. It serves as the primary interface with the host and is a target of broadly neutralizing antibodies (BnAbs). Thus, glycosylation should be considered in the design of HIV‐1 Env vaccines. Here we report the comparative glycosylation study of recombinant trimeric gp120 (rgp120) from an immune‐escape variant of HIV‐1 WEAU#4 transiently expressed in the high‐protein yield Expi293F system vs. the commonly used FreeStyle 293‐F cells. To evaluate whether rgp120 expression level affects glycosylation in general and/or in a site‐specific manner, we introduced point mutations that removed specific N‐glycosylation site(s), creating variants WEAU#4 S264N and WEAU#4 N276D, N463D.Normalized amounts of purified proteins were used for gel‐shift and lectin‐binding assays and for monosaccharide compositional analysis by gas‐liquid chromatography. Profiles of N‐glycans were obtained by HPLC with a CarboPac PA100 column. Site‐specific glycosylation heterogeneity was assessed by high‐resolution liquid chromatography‐mass spectrometry (hrLC‐MS). Real‐time PCR array was used for expression profiling of genes encoding key glycosyltransferases/glycosidases. α‐mannosidase activity was measured in cell lysates. Binding of BnAbsVRC01 and PGT122 was assessed by ELISA.Expi293F cells produced ~10‐times more rgp120 protein than FreeStyle 293‐F cells, and the secreted rgp120 exhibited differences in glycosylation. Monosaccharide compositional analysis revealed that Expi293F cells secreted rgp120 with a higher content of mannose compared to FreeStyle 293‐F cells. The ongoing N‐glycan profiling determines the impact on overall glycan composition whereas hrLC‐MS documents site‐specific changes. Our results indicated a higher content of high‐mannose glycans on rgp120s from Expi293F cells, indicating reduced glycan processing. Real‐time PCR confirmed reduced expression of genes encoding several α‐mannosidases in Expi293F vs. FreeStyle 293‐F cells, consistent with the results of α‐mannosidase activity assays. We seek to determine whether α‐mannosidases type I or II (or both) are involved. Although BnAb VRC01 (specific for CD4‐binding site) bound similarly to WEAU#4 rgp120 proteins from Expi293F vs. FreeStyle 293‐F cells, BnAb VRC01 exhibited improved binding to the WEAU#4 N276D, N463D variant compared to WEAU#4 and WEAU#4 S264N, irrespective of the producing system. Binding of BnAb PGT122 that targets V3‐loop outer glycan shield was not altered. These results suggest that site‐specific changes improving recognition by BnAb VRC01 have not impacted the recognition by BnAb PGT122.In summary, the Expi293F expression system produced high amounts of rgp120 glycoprotein with elevated proportions of high‐mannose glycans, thus providing a useful resource for Env vaccine‐antigen testing.Support or Funding InformationUAB CFAR developmental grant, UAB School of Medicine pilot grantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.