Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens

Abstract

Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Computational design of protein nanoparticles with differing sizes and geometries enables combination with antigens of choice to test novel multimerization concepts in immunization strategies where the goal is to improve the induction and maturation of neutralizing antibody lineages. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to an underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. When the immunogenicity of ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle or as soluble proteins were compared in rabbits, the two immunogens elicited similar serum antibody binding titers against the trimer component. Neutralizing antibody titers were slightly elevated in the animals given the nanoparticle immunogen and were initially more focused to the trimer apex. Altogether, our findings indicate that tetrahedral nanoparticles can be successfully applied for presentation of

Disassembly of HIV envelope glycoprotein trimer immunogens is driven by antibodies elicited via immunization

“…While antibody-induced trimer degradation appears to be a problem for many of the current Env trimers being used for immunogenicity studies, there are versions of the trimer resistant to this phenotype. We recently demonstrated that an advanced version of the ConM SOSIP trimer (ConM SOSIP.664v9) that includes an engineered interprotomer disulfide bond between residues 72 and 564 resists serum-induced degradation that was observed in the parental ConM SOSIP.664v7 ( 21 ). An interprotomer disulfide between residues 501 and 663 of the 16055 Env was also recently reported to prevent native flexibility linked (NFL) trimer degradation induced by bnAbs 3BC315 or 1C2 ( 4 , 30 ).…”

“…Encouragingly, Env trimer constructs that contain interprotomer disulfide bonds that are resistant to antibody-dependent degradation already exist ( 21 ). Additional disulfides may provide further stability, although these will need to be tested in immunogenicity studies.…”

“…Attempts to mask the base of the trimer via attachment to nanoparticles or alum have only had limited impact on reducing base-directed antibodies ( 20 , 21 ). Thus, further development of these strategies will be required, likely to include interprotomer disulfide bonds.…”

“…These data suggest that blunting of the base response early on increased the diversity of epitopes targeted, including neutralizing epitopes ( 20 ). Other attempts to mask the base via attachment of Env trimers to nanoparticles have only had limited success ( 21 ).…”

Immunofocusing and enhancing autologous Tier-2 HIV-1 neutralization by displaying Env trimers on two-component protein nanoparticles

“…Negative-stain electron microscopy (nsEM) experiments were performed as previously described 27 , 28 . All samples (free trimers or assembled nanoparticles) were diluted to 20–50 µg/mL and loaded onto the carbon-coated Cu grids (400-mesh, glow-discharged at 15 mA for 30 s).…”