Glycoform and Net Charge Heterogeneity in gp120 Immunogens Used in HIV Vaccine Trials

Abstract

BackgroundThe RV144 clinical trial showed for the first time that vaccination could provide modest but significant protection from HIV-1 infection. To understand the protective response, and to improve upon the vaccine's efficacy, it is important to define the structure of the immunogens used in the prime/boost regimen. Here we examined the heterogeneity in net charge, attributable to glycoform variation, of the gp120 immunogens contained in the AIDSVAX B/E vaccine.Methodology/Principal FindingsIsoelectric focusing and glycosidase digestion were used to assess variation in net charge of the gp120s contained in the AIDSVAX B/E vaccine used in the RV144 trial. We observed 16 variants of MN-rgp120 and 24 variants of A244-rgp120. Glycoform variation in gp120 produced in Chinese hamster ovary cells was compared to glycoform variation in gp120 produced in the 293F human embryonic kidney cell line, often used for neutralization assays. We found that gp120 variants produced in CHO cells were distinctly more acidic than gp120 variants produced in 293 cells. The effect of glycoform heterogeneity on antigenicity was assessed using monoclonal antibodies. The broadly neutralizing PG9 MAb bound to A244-rgp120, but not to MN-rgp120, whether produced in CHO or in 293. However, PG9 was able to bind with high affinity to MN-rgp120 and A244-rgp120 produced in 293 cells deficient in N-acetylglucosaminyltransferase I.Conclusions/SignificanceMN- and A244-rgp120 used in the RV144 trial exhibited extensive heterogeneity in net charge due to variation in sialic acid-containing glycoforms. These differences were cell line-dependent, affected the antigenicity of recombinant envelope proteins, and may affect assays used to measure neutralization. These studies, together with recent reports documenting broadly neutralizing antibodies directed against carbohydrate epitopes of gp120, suggest that glycoform variation is a key variable to be considered in the production and evaluation of subunit vaccines designed to prevent HIV infection.

HIV-1 Envelope Proteins and V1/V2 Domain Scaffolds with Mannose-5 to Improve the Magnitude and Quality of Protective Antibody Responses to HIV-1

“…Transfections were carried out with polyethyleneimine, and the supernatant was collected on day 3 or day 4 (55). For gD-tagged constructs, immunoaffinity chromatography was used to purify the proteins as described previously (47). To purify His 6 -tagged constructs, HisTrap HP columns (GE Healthcare) were used.…”

“…Proteins were digested as described previously (47). Samples were run on NuPAGE (Invitrogen) precast gels (4 -12% BisTris) with MES running buffer.…”

“…In previous studies, we showed that the two gp120 vaccine antigens (MN-rgp120 and A244-rgp120 produced in CHO cells) incorporated in the AIDSVAX B/E vaccine (42,46) and used in the RV144 trial possess high levels of sialic acid-containing glycans and lacked the high mannose glycans required for the binding of some PG9-like bNAbs (PG9, CH01, and CH03) (47). Because PG9 binding requires mannose-5 at Asn-156 and Asn-160, we decided to investigate the immunogenicity of the gp120 and V1/V2 scaffolds produced in GnTI Ϫ cells.…”

Structural Determinants for the Selective Anti-HIV-1 Activity of the All-β Alternative Conformer of XCL1

“…Negatively charged surfaces have been characterized in the early crystal structures of HIV-1 gp120 in complex with CD4 (26), and more recently changes in the net negative charge of the fourth variable (V4) domain of gp120 were shown to influence HIV-1 coreceptor usage (7). Furthermore, analysis of gp120 proteins contained in the AIDSVAX B/E vaccine indicated that the producer cell can influence the net surface negative charge of gp120, and consequently the antigenicity of the proteins, which could in turn influence susceptibility to neutralization (27). Preliminary data from our laboratory indeed demonstrated a dramatic reduction of XCL1 activity against HIV-1 pseudoviruses produced in continuous cell lines, compared to virus produced in primary cells which displays a more physiological glycosylation pattern (C. Guzzo et al, unpublished data).…”

Conference Report: “Functional Glycomics in HIV Type 1 Vaccine Design” Workshop Report, Bethesda, Maryland, April 30–May 1, 2012

“…39 Both the VAX003 study and the RV144 study used the same vaccine design platform: bivalent AIDSVAX B/E vaccine prepared from gp120s from the MN and A244 strains of HIV-1. Both proteins were made in CHO cells and purified by immunoaffinity chromatography (no selection for a particular type of carbohydrate).…”