Improved targeting and elicitation of VRC01-class precursors by an optimized glycan-masking mutant of OD-GT8

VRC01-class broadly neutralizing antibodies (bnAbs) target the conserved CD4-binding site (CD4bs) on the HIV-1 envelope. An engineered envelope outer domain eOD-GT8 60mer nanoparticle elicits VRC01-class precursors but also significant off-target responses in transgenic mice and humans, limiting on-target bnAb development. To mask off-target epitopes, N-linked glycans were engineered into eOD-GT8 to further focus immunogenicity to the CD4bs. Here, we developed an improved, highly glycosylated eOD-GT8 variant (eOD-GT8.Mut49) that retains affinity to all tested VRC01-class precursors but does not bind off-target antibodies. Mut49 more efficiently labels VRC01-class B cell precursors in naïve human PBMCs than eOD-GT8. Moreover, Mut49 60mer elicits higher levels of CD4bs-specific responses and VRC01-class precursors than eOD-GT8 60mer and its other derivatives in multiple transgenic mouse models expressing physiologically low frequencies of VRC01-class precursors. These studies support the further development of Mut49 as a priming immunogen in germline-targeting HIV-1 vaccine regimens.