Signatures of native-like glycosylation in RNA replicon-derived HIV-1 immunogens

Abstract

RNA-based vaccines have emerged as a highly effective delivery platform. However, this approach eliminates the possibility for immunogen purification, common in manufacturing of recombinant immunogens. In HIV-1 vaccine design, this is of particular importance because non-native epitopes can compromise the desired immune response, and native immunogen assembly is important for presentation of glycan-based epitopes targeted by broadly neutralizing antibodies. Here, we investigate the assembly and glycosylation of the archetypal trimeric HIV-1 immunogen, BG505, in the soluble single-chain format (NFL.664) that bypasses the need of maturation by furin cleavage. We have investigated the presence of the trimer-associated mannose-patch as oligomannose-type structures at these N-linked glycosylation sites are indicative of native-like glycoprotein structure. Despite the presence of features of native-like glycosylation, both electron microscopy and glycopeptide analysis indicated the presence of a sub-population of non-native material. We also investigated the glycosylation of material derived from cell-types that likely produce immunogens near the site of intramuscular RNA injection. We show that replicon-transformed dendritic and muscle cell lines generate immunogens displaying similar oligomannose-type glycan content, whereas sites presenting complex-type glycosylation differed substantially in the levels of glycan processing. Overall, the control of the immunogen assembly by protein engineering is sufficient to drive native-like glycosylation at the majority of glycosylation sites independent of producer cells. Furthermore, we explored the engineering of RNA immunogens to improve glycan site occupancy. Controlling immunogen assembly at the nucleotide level offers a route to enhanced RNA-based immunogens.

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