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[Preprint]. 2025 Nov 13:2025.11.12.688078.
doi: 10.1101/2025.11.12.688078.

Functional Activity of HIV-1 bNAbs Across Diverse Strains is Driven by Binding Site and Can be Enhanced Through Fc Engineering

Chia Jung Li  1 , Eunice Lim  1 , Meredith Phelps  1 , Jacqueline M Brady  1 , Vintus Okonkwo  1 , Caitlin McCarthy  1 , Caroline Alexander  1 , Margaret E Ackerman  2 , Daniel Lingwood  1 , Alejandro B Balazs  1
Affiliations

Functional Activity of HIV-1 bNAbs Across Diverse Strains is Driven by Binding Site and Can be Enhanced Through Fc Engineering

Chia Jung Li et al. bioRxiv. .

Abstract

Broadly neutralizing antibodies (bNAbs) are promising tools for HIV-1 treatment and prevention, due to their ability to mediate both Fab-dependent neutralization and Fc-dependent effector functions. While antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) have been implicated in antiviral activity, the extent to which these functions vary across bNAb epitopes, viral strains, and Fc region remains unclear. Here, we systematically evaluated twenty bNAbs targeting five distinct Env epitopes against a diverse panel of nine HIV-1 strains. We found that epitope specificity and Env sequence both critically influenced bNAb effector functions. CD4 binding site (CD4bs)- and V3 glycan-targeting bNAbs mediated the broadest and most potent ADCC and ADCP, while V1/V2 apex-directed bNAbs preferentially induced ADCP. In contrast, MPER-targeting bNAbs triggered ADCC more selectively, and gp120/gp41 interface-targeting bNAbs showed limited activity. Irrespective of binding epitope, different strains exhibited a broad range of effector function sensitivities. To investigate the impact of Fc modifications on this variability, we subclass-switched and introduced previously identified Fc mutations known to change Fcγ receptor affinity. Some of these mutations significantly boosted ADCC, while IgG3 subclass switching dramatically enhanced ADCP, even against highly resistant strains. Collectively, these results demonstrate that effector function is shaped by both antibody specificity and viral Env context and that rational Fc modification has the potential to improve antibody-based therapeutics against HIV.

Keywords: ADCC; ADCP; Env sequence; Fc engineering; HIV bNAb.

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Conflict of interest statement

Competing interests: A.B.B. is a founder of Cure Systems LLC. The other authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.. Effector functions of bNAbs vary by HIVREJO.c Env epitope.
A panel of twenty HIV broadly neutralizing antibodies (bNAbs), targeting five distinct epitopes, was selected for functional analysis against HIVREJO.c. (A) Neutralization potency of the bNAb panel, measured by IC50 against replication-competent HIVREJO.c. Data were pooled by epitope, with each dot representing an individual bNAb. (B-D) Functional characterization of the bNAb panel against HIVREJO.c Env-expressing CEM.NKr cells are colored by epitope specificity. Env binding (B), ADCC (C), and ADCP (D) are represented by scatter plots. Horizontal bars represent the mean value for each epitope. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test (*p < 0.0322, **p < 0.0021, ***p < 0.0002, ****p < 0.0001). (E) Heatmap depicting the functional profiles of bNAbs against HIVREJO.c. Data is presented as −log(IC50) for neutralization, log-transformed AUC for binding, and AUC for ADCC and ADCP, normalized within each function. A value of 1 represents the highest activity, and 0 represents the lowest.
Figure 2.
Figure 2.. Effector functions across diverse HIV strains are dependent on Env epitope.
(A) Phylogenetic tree of HIV-1 strains based on Env amino acid sequences, constructed using the neighbor-joining method. The scale bar indicates 0.04 amino acid substitutions per site. (B) Pairwise percentage amino acid identity matrix of HIV Env sequences. (C) Neutralization potency (IC50) of bNAbs against diverse viruses. Scatter plots display pooled data from multiple bNAbs targeting the same epitope. (D-F) Functional activity of the bNAb panel against CEM.NKr cells expressing Env from nine HIV-1 strains are colored by epitope specificity. Env binding (D), ADCC (E), and ADCP (F) are represented by scatter plots. Horizontal bars indicate mean values. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test (*p < 0.0322, **p < 0.0021, ***p < 0.0002, ****p < 0.0001).
Figure 3.
Figure 3.. Antibody-mediated effector functions vary by strain.
Heatmap showing the functional profiles of bNAbs against the HIV-1 panel. Data are displayed as −log10(IC50) for neutralization, log10-transformed AUC for Env binding, and AUC for ADCC and ADCP. All values were normalized on a 0 to 1 scale within each function. bNAbs were grouped and color-coded by epitope specificity. HIV clades are indicated on the heatmap. Viral Envs included HIV398F1 (clade A), HIV246F3 (clade AC), HIVREJO.c (clade B), HIVJR-CSF (clade B), HIVCNE55 (clade CRF01_AE), HIVCH119 (clade CRF07_BC), HIV25710 (clade C), HIVCE1176 (clade C), and HIVX1632 (clade G).
Figure 4.
Figure 4.. Functional activity correlates with Env binding.
(A and B) Scatter plot correlations between bNAb-mediated ADCC (A) or ADCP (B) and Env binding were evaluated across multiple HIV-1 strains. Each data point represents a single bNAb-strain pair and is color-coded by epitope specificity. (C) Correlation between Env binding and neutralization. (D) Correlation between ADCP and ADCC. Pearson correlations were performed and r and P values are indicated. Nonlinear fit of each correlation is represented by a black line.
Figure 5.
Figure 5.. Fc engineering enhances effector functions of N49P7 across diverse strains.
Functional characterization of wild-type, Fc-engineered, or IgG3-switched forms of N49P7 against diverse HIV-1 Envs. (A and B) Normalized ADCC (A) and ADCP (B) activities of Fc-engineered variants relative to wild-type N49P7. Values greater than 1 indicate enhanced activity; values less than 1 indicate reduced activity. (C and D) Heatmaps showing normalized ADCC (C) and ADCP (D) activities, scaled from 0 to 1 within each effector function across diverse Envs. Fc variants tested include ALE (G236A, A330L, I332E), ADE (G236A, S239D, I332E), LPLIL (F243L, R292P, Y300L, V305I, P396L), DLE (S239D, A330L, I332E), YTE (M252Y, S254T, T256E), and IgG3 (constant region replaced with IgG3).
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