Increased immunogen valency improves the maturation of vaccine-elicited HIV-1 VRC01-like antibodies

Abstract

Antibodies belonging to the VRC01-class display broad and potent neutralizing activities and have been isolated from several people living with HIV (PLWH). A member of that class, monoclonal antibody VRC01, was shown to reduce HIV-acquisition in two phase 2b efficacy trials. VRC01-class antibodies are therefore expected to be one component of an effective HIV-1 vaccine elicited response. In contrast to the VRC01-class antibodies that are highly mutated, their unmutated forms do not engage HIV-1 envelope (Env) and do not display neutralizing activities. Hence, specifically modified Env-derived proteins have been designed to engage the unmutated forms of VRC01-class antibodies, and to activate the corresponding naïve B cells. Selected heterologous Env must then be used as boost immunogens to guide the proper maturation of these elicited VRC01-class antibodies. Here we examined whether and how the valency of the prime and boost immunogens influences VRC01-class antibody-maturation. Our findings indicate that, indeed the valency of the immunogen affects the maturation of elicited antibody responses by preferentially selecting VRC01-like antibodies that have accumulated somatic mutations present in broadly neutralizing VRC01-class antibodies isolated from PLWH. As a result, antibodies isolated from animals immunized with the higher valency immunogens display broader Env cross-binding properties and improved neutralizing potentials than those isolated from animals immunized with the lower valency immunogens. Our results are relevant to current and upcoming phase 1 clinical trials that evaluate the ability of novel immunogens aiming to elicit cross-reactive VRC01-class antibody responses.

Fig 1. Antibody responses 2 weeks following the prime immunization.

Mice (n = 4) were primed with adjuvanted 426c.Mod.Core- Fer or C4b NPs at week 0 and plasma at week 2 was assayed by ELISA. (A and B) Binding against 426.Mod.Core (solid lines), HxB2.WT.Core (solid lines), as well as their corresponding CD4-BS knock-out antigens (KO; dotted lines) for individual animal in both NP groups are shown. (C) Total titers against 426.Mod.Core (red circles) and HxB2.WT.Core (blue circles) for all animals are shown; ‘*’ indicates significant differences using Mann-Whitney test. (D) CD4-BS specific values against the indicated proteins are shown; ‘*’ indicates significant differences using two-sample t-test assuming unequal variances. A pool of pre-bleed samples was used as an internal control in all ELISAs.

Fig 2. Antibody responses during the course of immunization study.

Mice (n = 4) were primed with adjuvanted 426c.Mod.Core- Fer or C4b NPs at week 0, followed by immunization with corresponding NP form of adjuvanted HxB2.WT.Core at week 23. Mice were bled at the indicated time points (x-axis) and plasma was assayed by ELISA for binding. (A) Mean endpoint titers with s.e.m. values against 426.Mod.Core (red solid line), HxB2.WT.Core (blue solid line), as well as their corresponding antigens with CD4-BS knock-out (KO) (dotted lines) are shown. (B) CD4-BS specific percentages against 426.Mod.Core (red circles) and HxB2.WT.Core (blue circles) are shown for indicated time points with ‘*’ indicating significant differences using two-sample t-test assuming unequal variances. A pool of pre-bleed samples was used as an internal control in all ELISAs.

Fig 3. Heavy and Light chain sequence analysis post prime (W2 and W23), and boost (W25) immunizations.

Bar graphs indicate VH (A, B) and VL (C to F) characteristics from individually sorted Env-specific B cells from pooled mouse samples at the indicated time point in each NP group. The number of HC and LC sequences analyzed is shown at the bottom of the bar graph. (A) VH-gene usage, (B) HCs with the H35N mutation, (C) aa length of the CDRL3 domains in the LC, (D) LC-gene usage, where shades of blue slices represent other 5-aa long CDRL3s. (E) LC-gene usage in other 5-aa long CDRL3s and (F), Presence or absence of Glu₉₆ within the LC sequences with 5-aa long CDRL3 domains. See also S1 Fig.

Fig 4. Binding properties of VRC01-like mAbs from both NP groups, evaluated using BLI assay.

33 VRC01-like mAbs were generated between week 2, week 23, and week 25 timepoints, and tested against the indicated soluble monomeric Envs. Heat map shows the maximum signal (values depicted by corresponding colors shown in the legend) obtained in the assay for each mAb against the indicated Env. Crosses indicate no mAb testing. See also S2 Fig and S1 Table.

Fig 5. Summary of binding properties of VRC01-like mAbs from both NP groups.

(A) 33 VRC01-like mAbs were evaluated against the indicated heterologous WT.Core Envs, and variants of 426c SOSIPs. No binding: (−); Up to 0.1: + /-; 0.1 to 0.5: + ; 0.5 to 1: ++; and >1: +++. (B) Binding curves of two mAbs (3M23 and 3M24) against the indicated variants of 426c SOSIPs are shown. mVRC01 (solid pink line) and glVRC01 (solid cyan line) were included as internal controls in all assays. Black dotted lines indicate end of association and dissociation phases. See also S3 and S4 Figs and S1 Table.

Fig 6. Nucleotide changes in the HC and LC of paired sequences from both NP groups.

Each circle represents a paired sequence and ‘*’ indicates significant differences using Kruskal-Wallis test. See also S5 Fig and S1 Table.

Fig 7. Phylogenetic analysis using HC/LC sequences from the Fer NP group.

Scatter plots are shown with two variables that were designed to discriminate between “boosted subtrees” induced by booster vaccination vs a background hypothesis of no boost (see text): the size of single-timepoint subtrees (x-axis) and their length (distance to common ancestor, y-axis). The real data (top left) is compared to three synthetic cases that inform our understanding of observations in data. The real data (top left) shows several large such subtrees (highlighted in S6 Fig), just as we would expect if the data were generated by processes similar to those modeled in the “boosted” simulation (bottom left). In contrast, the top right (where we have destroyed timepoint information by shuffling it), as well as bottom right (showing simulation with no boosting immunogen) show no such large single-timepoint subtrees. In both data and simulation, large single-timepoint subtrees occur in cases where we expect to observe the effects of boosting (left column), but they are absent where we do not (right column). The effect in common ancestor distance (y-axis) is less clear than in size (x-axis). A similar approach was used in [66]. See also S6 Fig.

Fig 8. Sequence alignment of VRC01-like mAbs from the Fer NP group.

Germline VH1-2*02 and κ8-30*01 sequences are used as reference for alignment, and CDRs are highlighted in red. Green shaded regions highlight the residues commonly present in mature VRC01-class antibodies that are only, or more frequently, found in post-boost mAbs. See also S7 Fig.