Covalent Linkage of HIV-1 Trimers to Synthetic Liposomes Elicits Improved B Cell and Antibody Responses

Abstract

We have demonstrated that a liposomal array of well-ordered trimers enhances B cell activation, germinal center formation, and the elicitation of tier-2 autologous neutralizing antibodies. Previously, we coupled well-ordered cleavage-independent NFL trimers via their C-terminal polyhistidine tails to nickel lipids integrated into the lipid bilayer. Despite favorable in vivo effects, concern remained over the potentially longer-term in vivo instability of noncovalent linkage of the trimers to the liposomes. Accordingly, we tested both cobalt coupling and covalent linkage of the trimers to the liposomes by reengineering the polyhistidine tail to include a free cysteine on each protomer of model BG505 NFL trimers to allow covalent linkage. Both cobalt and cysteine coupling resulted in a high-density array of NFL trimers that was stable in both 20% mouse serum and 100 mM EDTA, whereas the nickel-conjugated trimers were not stable under these conditions. Binding analysis and calcium flux with anti-Env-specific B cells confirmed that the trimers maintained conformational integrity following coupling. Following immunization of mice, serologic analysis demonstrated that the covalently coupled trimers elicited Env-directed antibodies in a manner statistically significantly improved compared to soluble trimers and nickel-conjugated trimers. Importantly, the covalent coupling not only enhanced gp120-directed responses compared to soluble trimers, it also completely eliminated antibodies directed to the C-terminal His tag located at the "bottom" of the spike. In contrast, soluble and noncovalent formats efficiently elicited anti-His tag antibodies. These data indicate that covalent linkage of well-ordered trimers to liposomes in high-density array displays multiple advantages in vitro and in vivoIMPORTANCE Enveloped viruses typically encode a surface-bound glycoprotein that mediates viral entry into host cells and is a primary target for vaccine design. Liposomes with modified lipid head groups have a unique feature of capturing and displaying antigens on their surfaces, mimicking the native pathogens. Our first-generation nickel-based liposomes captured HIV-1 Env glycoprotein trimers via a noncovalent linkage with improved efficacy over soluble glycoprotein in activating germinal center B cells and eliciting tier-2 autologous neutralizing antibodies. In this study, we report the development of second-generation cobalt- and maleimide-based liposomes that have improved in vitro stability over nickel-based liposomes. In particular, the maleimide liposomes captured HIV-1 Env trimers via a more stable covalent bond, resulting in enhanced germinal center B cell responses that generated higher antibody titers than the soluble trimers and liposome-bearing trimers via noncovalent linkages. We further demonstrate that covalent coupling prevents release of the trimers prior to recognition by B cells and masks a nonneutralizing determinant located at the bottom of the trimer.

Keywords: HIV-1; antibody repertoire; human immunodeficiency virus; immunization; immunology; liposomes; nanoparticles; pathogens; vaccines.

FIG 1

Synthetic nanoparticles as a platform for displaying HIV-1 Env trimers. The schematic shows that liposomes incorporate lipids with modified head groups that mediate coupling of Env trimer to the outer surface. (A) First-generation liposomes contain DGS-NTA in the head group that incorporates nickel or cobalt atoms in the NTA cage, which in turn mediates a noncovalent coupling to a C-terminal His tag of Env trimers. (B) Second-generation liposomes, described here, contain maleimide in the head group that mediates a covalent coupling to a free cysteine group located at the C terminus of each protomeric subunit of each HIV Env trimer.

FIG 2

Conjugation of BG505 NFL to second-generation liposomes. (A) Negative-stain EM images of liposomes with nickel in the head group (left) and cobalt in the head group (right) after coupling to BG505 NFL trimers. Bars, ∼100 nm. (B) Nanoparticle tracking analysis of liposomes of varying compositions and containing various modified head groups. The diameters of the liposomes ranged from 100 to 220 nm, indicating little variation in particle size distribution. (C) BG505 NFL trimers with a terminal cysteine were reduced with 1 mM TCEP (pH 6.7) prior to coupling. (Left) SDS gel showing that TCEP effectively reduces intertrimer disulfide bonds and results in homogeneous trimers for covalent coupling to liposomes. (Middle) Negative-stain EM image of TCEP-reduced trimers confirming native-like individual trimers. (Right) The covalent-coupling reaction is shown as a schematic. (D) Coupling of BG505 NFL trimers with selected amino acid linker lengths between the C terminus of the gp140 sequence and the terminal cysteine to maleimide liposomes. All the liposomes contained 16% maleimide lipid for conjugating the terminal cysteine residue. The loading of trimers to the liposomes improved with the increasing linker length; left, 5 amino acids [aa]; middle, 13 aa; right, 16 aa. The amino acid sequence of the linker is shown below each image.

FIG 3

Stability and characterization of BG505 NFL trimers on various liposomes. (A) Liposomes were incubated at 37°C in 20% mouse serum and evaluated for retention of trimers by electron microscopy. Samples were collected after 1 h, 4 h, 24 h, and 96 h (shown). (B) Count of trimers visible by EM on various liposomes after incubation in 100 mM EDTA for 30 min. EDTA strips off surface-coupled trimers on nickel liposomes, but not on cobalt- and Cys-linked liposomes. (C) Biolayer light interferometry (Octet) analysis of binding of trimer-specific bNAbs (PGT145 and PG16), CD4bs bNAbs (VRC01), gp120-gp41 interface bNAb (35022), and non-bNAbs (F105 and 447-52D) to BG505 NFL trimers coupled to nickel-, cobalt-, and Cys-linked liposomes, respectively.

FIG 4

B cells are efficiently activated by BG505 NFL trimers when coupled to liposomes. (A) B cells expressing PGV04 were activated with BG505 NFL trimers either in solution or coupled to various liposomes. The resulting Ca²⁺ flux was measured over a period of 120 s. The trimers activated B cells more effectively when coupled to liposomes (all three versions) than soluble trimers. (B and C) Gating strategy (B) and representative flow cytometry plots (C) analyzing B cells from the draining lymph nodes for the expression of the germinal center marker GL-7. The mice were injected with equal amounts of BG505 NFL trimers, either in solution or coupled to liposomes. The control group received blank liposomes, and a naive mouse was included for comparison. (D) Mice were subcutaneously immunized with soluble trimers and different liposome vaccine formulations. Two weeks postimmunization, B cells from the draining lymph nodes were analyzed for the expression of the germinal center marker GL7. The percentage of GL7⁺ B cells detected in each gate is shown for each group. A naive mouse and a group of mice administered blank liposomes were used as negative controls. *, P ≤ 0.05.

FIG 5

BG505 NFL trimers generate early and improved antigen-specific IgG responses when covalently coupled to liposomes. The schedule of immunizations and bleeds is shown. Each animal received 10 μg of BG505 NFL trimer. (A) Longitudinal analysis of BG505 NFL-specific IgG responses in mice to various groups of liposomes. The geometric mean of the binding titers (ED₅₀) is plotted at every bleed time point. The Cys-linked liposomes generated the highest IgG response to either gp120 (left) or trimer (right) on the ELISA plate at every time point analyzed. The gray dashed lines indicate the immunization time points. (B) ED₅₀ binding titers of soluble and various liposome groups after four immunizations (bleed point P4) against BG505 NFL gp120 (left) and BG505 NFL trimer (right). (C) ELISA binding analysis of V3 peptide-specific IgG in mouse serum. ELISA plates were coated with V3 peptide of BG505 Env. Binding was not detected for V3-specific IgG in the sera of all the groups of mice that received BG505 NFL trimers (left) (each symbol represents data for an individual mouse). Control MAbs (at a starting dilution of 10 μg/ml) for V3 peptide binding ELISA are shown on the right.

FIG 6

Comparison of BG505 NFL2 trimer-specific responses in all animals on various liposome platforms. Longitudinal analysis is indicated by ED₅₀ binding titers of soluble and various liposome groups against BG505 NFL gp140 trimer captured by the glycan-directed bNAb, 2G12, on the wells of the ELISA plate. OD₄₅₀, optical density at 450 nm. (A) (Left) Cartoon representation of trimer capture via 2G12 on an ELISA plate. (Right) Representative ELISA binding curves. Each symbol/curve represents data from one mouse. (B) The Cys-linked group has the best responses that are uniform across all animals and show a statistically significant difference (ANOVA analysis) from the soluble group or liposome groups with noncovalently coupled trimers. *, P ≤ 0.05; **, P ≤ 0.01.

FIG 7

Effect of adjuvant on elicitation of BG505 NFL-specific antibodies. Mice were immunized with soluble BG505 NFL trimers or coupled to Cys-linked liposomes in Iscomatrix adjuvant. ELISA analysis indicated a statistically significant difference in BG505 gp120 binding titers (ED₅₀) between the P3 and P4 time points (Student t test). The Cys-linked liposomes generated earlier, better, and more uniform responses to epitopes on gp120 than the soluble trimers. *, P ≤ 0.05; ***, P ≤ 0.001.

FIG 8

Covalent coupling of BG505 NFL trimer to liposomes restricts accessibility to the bottom of the Env spike. Shown is ELISA binding analysis of His tag-specific IgG in mouse serum (analyzed at P3). The viral matrix protein (VP40) of Ebola virus with a His₆ tag was used as the target for binding IgG. The binding curves indicate low His tag-specific IgG in sera of mice administered Cys-linked liposomes. Each symbol and associated curve represents binding data from one mouse from the corresponding group.

FIG 9

Covalent coupling of other clade Env trimers to Cys-linked liposomes. Shown are negative-stain EM images of 16055 NFL (clade C) (A) and SC422 NFL (clade B) (B) trimers coupled to Cys-linked liposomes. Bars, ∼100 nm.