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 HIV Env trimer immunogens; however, the optimal implementation to different immunization strategies remains to be determined.

Fig 1. Evaluation of the antigen-presenting components.

(a) Antigen-bearing components were generated by fusing the N-termini of trimeric nanoparticle building blocks to BG505-SOSIP.v5.2(7S) (left). The SDS-PAGE gel of the purified antigen-bearing components denatured in the presence (+) and absence (-) of reducing agent (right). (b) NS-EM analysis of the purified antigen-bearing components (representative raw micrograph and 2D class averages). Red arrows indicate the location of the fused nanoparticle component. Analysis of the resulting 2D classes suggests that the Env antigen is in a native-like, trimeric form in all three antigen-bearing components. A small percentage of monomer/dimer-like particles were detected in the BG505-SOSIP-T33_dn10A sample (2^nd class from the left in the bottom row). (c) BLI analysis of the antigenicity of three antigen-bearing components compared to BG505-SOSIP.v5.2(7S). (d) Glycan composition analysis for the three antigen-bearing components and BG505-SOSIP.v5.2(7S). Peaks sensitive to endoglycosidase H digestion correspond to oligomannose-type glycans and are colored green.

Fig 2. Nanoparticle assembly and characterization.

(a) Schematic representation of individual components and assembled nanoparticle (left). Nanoparticle assembly tests were performed at different temperatures and the assembly efficacy was assessed using Native PAGE. NP, nanoparticle; ABC, antigen-bearing component; AC, assembly component; (right). (b) Negative stain EM analysis of purified nanoparticles. Representative raw micrographs, 2D class averages and 3D reconstructions are shown for BG505-SOSIP-T33_dn2 (left), -T33_dn10 (middle) and -I53_dn5 (right) nanoparticles. 3D maps are segmented and color-coded (BG505 SOSIP, gray; antigen-bearing component, orange; assembly component, blue). Particle diameters and the average apex-apex distance between the two closest neighboring Env trimers are shown for each nanoparticle. These data are also described in Ueda, Antanasijevic et al., 2020 [38]. (c) BLI analysis of antigenicity of assembled nanoparticles compared to the BG505-SOSIP.v5.2(7S) trimer.

Fig 3. Cryo-EM analysis of tetrahedral and icosahedral nanoparticles.

(a) Tetrahedral BG505-SOSIP-T33_dn10 nanoparticle; (b) Icosahedral BG505-SOSIP-I53_dn5 nanoparticle. Sample micrograph, 2D class averages and initial 3D reconstructions of the full nanoparticles are displayed in the top left part of the corresponding panels. Focused refinement was applied to generate a 3D reconstruction of the nanoparticle core (top and bottom right, maps are in light gray). The refined model of T33_dn10 and the Rosetta_design model of I53_dn5 are docked into the corresponding maps (antigen-bearing component, orange; assembly component, blue). Localized reconstruction approach was used for analysis of the presented antigen (bottom left). Refined BG505-SOSIP models are shown in black.

Fig 4. B-cell activation by trimeric components and nanoparticles.

Ca²⁺ flux (Indo I fluorescence) was used to assess the activation of B cells expressing HIV Env-specific IgM receptors (PGT145, VRC01 and PGT121) by equimolar amounts of BG505-SOSIP in the form of free trimers, fused to the antigen-bearing components (left column) and on the surface of assembled nanoparticles (right column). The antigen was introduced 60 s after the start of each measurement. Ionomycin was added after 240 s. Anti-IgM antibody was used as a positive control.

Fig 5. Immunogenicity of nanoparticle-presented ConM-SOSIP.

Two groups of 5 rabbits were immunized with 30 μg of soluble ConM-SOSIP.v7 trimer (Group 1, open circles) or the equivalent amount presented on the T33_dn2 tetrahedral nanoparticle (Group 2, black squares). The immunization (large arrows) and bleed (small arrows) schedules are shown on the top left, and depictions of the immunogens on the top right. The serum anti-trimer binding and neutralizing antibody titers in each animal at weeks 4, 8 and 22 are presented as scatter plots with the mean titers indicated by lines. The mean titers for each group are plotted longitudinally on the right with the bars corresponding to the standard error of the mean. An AUC statistical analysis (based on two-tailed Mann-Whitney U-test) of titer values in Group 1 versus Group 2 as a function of time results in p values of 0.69 and 0.056 for the binding and neutralization titers, respectively. The data for Group 1 (ConM-SOSIP.v7) were adapted from Brouwer et al., 2019 ([37]; see Methods section for details).

Fig 6. EMPEM analysis of antibody responses in immunized animals.

Composite figures generated from EMPEM analysis performed using sera collected from the two animals in each group that have the highest ConM NAb titers at week 22. Data are shown for the week 4 (post-prime) and week 22 (post-final boost) time points. For simplicity, only a single antibody is shown for each epitope cluster. Epitope definitions are summarized in the text and color coded as indicated at the foot of the figure. The anti-trimer binding antibody and neutralizing antibody titers for each serum sample are listed below the images.