Abstract
Over the last decade, multiple broadly neutralizing monoclonal antibodies (bN-mAbs) to the HIV-1 envelope protein (Env) gp120 have been described. Many of these recognize epitopes consisting of both amino acid and glycan residues. Moreover, the glycans required for binding of these bN-mAbs are early intermediates in the N-linked glycosylation pathway. This type of glycosylation substantially alters the mass and net charge of Envs compared to molecules with the same amino acid sequence but possessing mature, complex (sialic acid–containing) carbohydrates. Since cell lines suitable for biopharmaceutical production that limit N-linked glycosylation to mannose-5 (Man5) or earlier intermediates are not readily available, the production of vaccine immunogens displaying these glycan-dependent epitopes has been challenging. Here, we report the development of a stable suspension-adapted Chinese hamster ovary (CHO) cell line that limits glycosylation to Man5 and earlier intermediates. This cell line was created using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system and contains a mutation that inactivates the gene encoding Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase (MGAT1). Monomeric gp120s produced in the MGAT1− CHO cell line exhibit improved binding to prototypic glycan-dependent bN-mAbs directed to the V1/V2 domain (e.g., PG9) and the V3 stem (e.g., PGT128 and 10–1074) while preserving the structure of the important glycan-independent epitopes (e.g., VRC01). The ability of the MGAT1− CHO cell line to limit glycosylation to early intermediates in the N-linked glycosylation pathway without impairing the doubling time or ability to grow at high cell densities suggests that it will be a useful substrate for the biopharmaceutical production of HIV-1 vaccine immunogens.
Highlights
Despite 30 years of research, a vaccine capable of providing protection against human immunodeficiency virus type 1 (HIV-1) has yet to be described
Though there is no HIV-1 vaccine available yet, significant progress has been made in understanding the envelope protein structure and the antibodies that bind to it
Examination of the gp120 subunit vaccines used in the RV144 trial showed that both components (MN-recombinant gp120 [rgp120] and A244-rgp120) were enriched for complex, sialic acid–containing glycans and lacked the high-mannose glycosylation found on the surface of virions and native envelope proteins required to bind the new class of glycan-dependent broadly neutralizing monoclonal antibody (bN-mAb) [16,17,18,19,20]
Summary
Despite 30 years of research, a vaccine capable of providing protection against human immunodeficiency virus type 1 (HIV-1) has yet to be described. Differences in glycosylation between the vaccine immunogens from the RV144 trial and virus particles could, in part, explain the low efficacy of RV144 and other gp120-based vaccines and their inability to elicit bN-mAbs. Previously, we reported that the same gp120s used in the RV144 trial could be modified to bind multiple bN-mAbs when expressed in a cell line (human embryonic kidney 293 [HEK 293] N-acetylglucosaminyltransferase I [GnTI]−) that limited N-linked glycosylation to Man or earlier species (e.g., Man, Man9) [21]. We reported that the same gp120s used in the RV144 trial could be modified to bind multiple bN-mAbs when expressed in a cell line (human embryonic kidney 293 [HEK 293] N-acetylglucosaminyltransferase I [GnTI]−) that limited N-linked glycosylation to Man or earlier species (e.g., Man, Man9) [21] While in theory this cell line could be used to produce a glycan-optimized gp120 vaccine, in reality, this is not practical. The HEK 293 GnTI− system is not suitable for clinical
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