Administration of anti-HIV-1 broadly neutralizing monoclonal antibodies with increased affinity to Fcγ receptors during acute SHIVAD8-EO infection

Abstract

Anti-HIV-1 broadly neutralizing antibodies (bNAbs) have the dual potential of mediating virus neutralization and antiviral effector functions through their Fab and Fc domains, respectively. So far, bNAbs with enhanced Fc effector functions in vitro have only been tested in NHPs during chronic simian-HIV (SHIV) infection. Here, we investigate the effects of administering in acute SHIV_AD8-EO infection either wild-type (WT) bNAbs or bNAbs carrying the S239D/I332E/A330L (DEL) mutation, which increases binding to FcγRs. Emergence of virus in plasma and lymph nodes (LNs) was delayed by bNAb treatment and occurred earlier in monkeys given DEL bNAbs than in those given WT bNAbs, consistent with faster clearance of DEL bNAbs from plasma. DEL bNAb-treated monkeys had higher levels of circulating virus-specific IFNγ single-producing CD8⁺ CD69⁺ T cells than the other groups. In LNs, WT bNAbs were evenly distributed between follicular and extrafollicular areas, but DEL bNAbs predominated in the latter. At week 8 post-challenge, LN monocytes and NK cells from DEL bNAb-treated monkeys upregulated proinflammatory signaling pathways and LN T cells downregulated TNF signaling via NF-κB. Overall, bNAbs with increased affinity to FcγRs shape innate and adaptive cellular immunity, which may be important to consider in future strategies of passive bNAb therapy.

Fig. 1. Study design and therapeutic efficacy of anti-HIV-1 bNAb combinations.

A Thirty rhesus macaques were inoculated intrarectally with SHIV_AD8-EO and either left untreated (n = 10) (left) or treated at days 3, 10, and 17 post-challenge with a combination of either VRC07-523-LS and PGT121 (WT bNAbs, n = 10) (middle) or VRC07-523-LS/DEL and PGT121/DEL (DEL bNAbs, n = 10) (right). Each bNAb was infused intravenously at 10 mg/kg. B Plasma viral load up to 72 weeks post-challenge in untreated (left), WT bNAb-treated (middle), and DEL bNAb-treated (right) monkeys. Gray and black lines represent individual and median plasma viral loads, respectively. Vertical dotted lines indicate the timings of bNAb infusions. C Time to first detectable plasma virus leading to persistent infection (left) and to peak plasma viremia (right) in untreated and bNAb-treated monkeys. N = 9, 8, and 7 untreated, WT bNAb-treated, and DEL bNAb-treated monkeys, respectively. P < 0.0001 (left) and p = 0.0007 (right). D Peak plasma viral load (left) and plasma viral load at week 36 post-challenge (right) in untreated and bNAb-treated monkeys. N = 9, 8, and 7 (left) and n = 10, 10, and 10 (right) untreated, WT bNAb-treated, and DEL bNAb-treated monkeys, respectively. E Plasma viral load throughout the first 72 weeks post-challenge in untreated and bNAb-treated monkeys as determined by AUC analysis. N = 9, 8, and 7 untreated, WT bNAb-treated, and DEL bNAb-treated monkeys, respectively. Individual data from the initial 6 monkeys per group are indicated with gray solid lines (B) and circles (C–E), whereas data pertaining to the later 4 monkeys per group are denoted with gray dashed lines (B) and squares (C–E) (see text for more details on the two monkey batches). Bar graphs show the mean and individual datapoints (C–E). The Kruskal–Wallis test followed by Dunn’s multiple comparison test was used to detect significant differences between monkey groups. N indicates the number of biological replicates (monkeys). Source data are provided in the Source Data file. AUC area under the curve, IR intrarectal, IV intravenous; pc post-challenge, TCID₅₀ median tissue culture infective doses, Tx treatment, UnTx untreated, wk week.

Fig. 2. PK of infused bNAbs and ADA responses.

A Concentration of VRC07-523-LS (dark pink), PGT121 (dark blue), VRC07-523-LS/DEL (light pink), and PGT121/DEL (light blue), as measured by ELISA in the plasma of monkeys that received either the WT or DEL bNAbs at days 3, 10, and 17 post-SHIV_AD8-EO challenge. Vertical dotted lines indicate the timings of bNAb infusions and horizontal dotted lines indicate the in vitro neutralization IC₈₀ values against SHIV_AD8-EO averaged for VRC07-523-LS and VRC07-523-LS/DEL (pink), and PGT121 and PGT121/DEL (blue). N = 10 for each bNAb and each timepoint except for VRC07-523-LS and PGT121 at week 0 and VRC07-523-LS/DEL and PGT121/DEL at weeks 10–14: n = 4; PGT121 at week 9: n = 9. B Plasma concentration of each infused bNAb up to 14 weeks post-challenge as determined by AUC analysis. N = 10 for each bNAb. Each P < 0.0001, two-sided p-values. C ADA response against VRC07-523-LS (dark pink), PGT121 (dark blue), VRC07-523-LS/DEL (light pink), and PGT121/DEL (light blue), as measured by ELISA in the plasma of monkeys that received either the WT or DEL bNAbs at days 3, 10, and 17 post-SHIV_AD8-EO challenge. Vertical dotted lines indicate the timings of bNAb infusions. N = 10 for each bNAb and each timepoint except for α-VRC07-523-LS at weeks 0 and 1, α-PGT121 at week 0, and α-VRC07-523-LS/DEL at week 6: n = 9; α-VRC07-523-LS/DEL at week 4: n = 8; α-VRC07-523-LS at days 10 and 17 and α-PGT121 at days 3, 10, and 17: n = 4, and α-VRC07-523-LS at day 3: n = 3. D ADA response against each infused bNAb up to 14 weeks post-challenge as determined by AUC analysis. N = 10 for each bNAb. P = 0.0089 for α-VRC07-523-LS vs. α-VRC07-523-LS/DEL and p = 0.0029 for α-PGT121 vs. α-PGT121/DEL, two-sided p-values. Graphs show the mean±SEM (A, C) and the mean and individual datapoints (B, D). The Mann-Whitney test was used to detect significant differences between the WT bNAbs, the DEL bNAbs, or the WT and DEL mutant of the same bNAb. N indicates the number of biological replicates (monkeys). Source data are provided in the Source Data file. ADA anti-drug antibody; AUC area under the curve.

Fig. 3. Detection of SHIV_AD8-EO RNA in LN sections.

A Representative RNAscope staining of LN sections from monkeys that were left untreated or were treated with bNAbs early after SHIV_AD8-EO challenge. Staining was performed in one LN section per timepoint per animal. Big images (scale bars: 1000 µm) show SHIV_AD8-EO RNA (green), CD20 (yellow), and cellular nucleic acids (SYTO, blue) in untreated (left), WT bNAb-treated (middle), and DEL bNAb-treated (right) monkeys at weeks 2 (top) and 8 (bottom) post-challenge. Small images show a LN SHIV_AD8-EO RNA⁺ cell and virions in the DEL bNAb-treated monkey at week 8 post-challenge. Top image (scale bar: 4 µm) shows green spheres denoting the SHIV_AD8-EO RNA⁺ cell and virions; top middle image (scale bar: 4 µm) shows the real signal for the SHIV_AD8-EO RNA⁺ cell; and the middle bottom and bottom images (scale bars: 2 µm) show the spheres and real signal for virions, respectively. Viral events are shown in association with cellular nucleic acids (blue) alone or in combination with either CD20 (yellow) or CD3 (red) and CD4 (turquoise). B, C RNAscope quantification of SHIV_AD8-EO RNA⁺ cells (left) and virions (right) in whole LN sections and in follicular (circles) and extrafollicular (triangles) areas from untreated (n = 2), WT bNAb-treated (n = 5), and DEL bNAb-treated (n = 3) monkeys. P = 0.0357 for WT vs. DEL group at week 8 (B, left and right) and p = 0.0238 for week 2 vs. 8 in WT group (B, right), two-sided p-values. D Percentage of follicular SHIV_AD8-EO RNA⁺ cells (squares) and virions (triangles) in untreated (n = 2) and DEL bNAb-treated (n = 3) monkeys. Bar graphs show the mean and individual datapoints (B–D). The Mann-Whitney test was used to detect significant differences in viral parameters between WT and DEL bNAb-treated monkeys at each timepoint (B), between timepoints for each bNAb-treated group in whole LN sections (B) and in follicular or extrafollicular areas (C), and between follicular and extrafollicular areas at each timepoint in DEL bNAb-treated monkeys (C). N indicates the number of biological replicates (monkeys). Source data are provided in the Source Data file. Tx treatment, UnTx untreated, vRNA viral RNA.

Fig. 4. Detection of infused bNAbs in LN sections.

A Representative confocal microscopy staining to detect infused bNAbs in LN sections from monkeys that were treated with bNAbs early after SHIV_AD8-EO challenge. bNAb staining was performed in one LN section per timepoint per animal. Inset (scale bar: 500 µm) shows CD20 (yellow) and cellular nucleic acids (JOPRO, blue) in the whole LN section; big image (scale bar: 50 µm) shows human Ig kappa light chain⁺ cells (pink), human Ig lambda light chain⁺ cells (turquoise), and cellular nucleic acids (blue); and small images (scale bars: 5 µm) show cellular nucleic acids (blue) with only human Ig kappa light chain⁺ cells (pink) (top left), only human Ig lambda light chain⁺ cells (turquoise) (top right), or an overlay of both (bottom). The big image and small image on the bottom right show pink and turquoise Surface objects assigned by Imaris for easier visualization of human Ig kappa light chain⁺ and lambda light chain⁺ cells. All other images show real staining signals. Quantification of total WT bNAb⁺ and total DEL bNAb⁺ cells (B) or VRC07-523-LS⁺, PGT121⁺, VRC07-523-LS/DEL⁺, and PGT121/DEL⁺ cells (C) in whole LN sections at weeks 2 (circles) and 8 (squares) post-challenge. N = 6 per group per timepoint. Each p = 0.0087, two-sided p-values. D Percentage of follicular VRC07-523-LS⁺, PGT121⁺, VRC07-523-LS/DEL⁺, and PGT121/DEL⁺ cells in LN sections at weeks 2 (circles) and 8 (squares) post-challenge. N = 6 per group per timepoint. P = 0.0260, two-sided p-value. Bar graphs show the mean and individual datapoints (B–D). The Mann-Whitney test was used to detect significant differences in bNAb⁺ cell levels at each timepoint or between timepoints for each type of bNAb (WT or DEL) (B). The Mann–Whitney test was also used to detect significant differences between the levels of VRC07-523-LS⁺ and PGT121⁺ cells, VRC07-523-LS/DEL⁺ and PGT121/DEL⁺ cells, VRC07-523-LS⁺ and VRC07-523-LS/DEL⁺ cells, or PGT121⁺ and PGT121/DEL⁺ cells at each timepoint, or between timepoints for cells bound to each bNAb (C, D). N indicates the number of biological replicates (monkeys). Source data are provided in the Source Data file. kappa, human Ig kappa light chain; lambda, human Ig lambda light chain.

Fig. 5. SIV Gag-specific CD8⁺ T cell responses.

A Frequency of SIV Gag-specific CD8⁺ T cells in PBMCs (left) and LN cells (right) from monkeys that were left untreated or were treated with bNAbs early after SHIV_AD8-EO challenge. N = 9 untreated monkeys except for LN cells pre: n = 7, week 2: n = 6, and week 20: n = 4. N = 8 and 7 WT and DEL bNAb-treated monkeys, respectively. P = 0.0474, 0.0480, and 0.0053 for untreated vs. WT group at week 4, 6, and 8, respectively, and p = 0.0485 and 0.0408 for WT vs. DEL group at weeks 6 and 8, respectively (left) (B, C) Polyfunctional profile of SIV Gag-specific CD8⁺ T cell responses in PBMCs (top) and LN cells (bottom) at week 20 post-challenge. N = 9 and 4 untreated monkeys for PBMCs and LN cells, respectively; n = 8 and 7 WT and DEL bNAb-treated monkeys, respectively. B P = 0.04 for WT vs. DEL group in PBMCs; p < 0.001 for PBMCs vs. LN cells in untreated and DEL groups, and p = 0.002 for PBMCs vs. LN cells in WT group; two-sided p-values. C PBMCs, IFNγ⁺MIP-1β⁺TNF^-CD107a^- CD8⁺ T cells: p = 0.0099 and 0.0014 for untreated vs. DEL and WT vs. DEL group, respectively. PBMCs, IFNγ⁺MIP-1β^-TNF^-CD107a^- CD8⁺ T cells: p = 0.0209 and 0.0240 for untreated vs. DEL and WT vs. DEL group, respectively. LN cells, IFNγ^-MIP-1β⁺TNF⁺CD107a⁺ CD8⁺ T cells: p = 0.0333 for untreated vs. WT group. Graphs show the mean (A–C) and individual datapoints. The Kruskal–Wallis test followed by Dunn’s multiple comparison test was used to detect significant differences between monkey groups at each timepoint (A) and for each functional readout combination (C), and between timepoints for each monkey group (A, colored statistics). Statistics in gray, orange, and green, denote, respectively, differences from week 2 for untreated monkeys, week 4 for WT bNAb-treated monkeys, and pre-challenge for DEL bNAb-treated monkeys in PBMCs (A, left), and from week 20 in LN cells (A, right). The permutation test was used to compare pie charts (B). N indicates the number of biological replicates (monkeys). Source data are provided in the Source Data file. LN lymph node, PBMCs peripheral blood mononuclear cells, pre pre-challenge, Tx treatment, UnTx untreated, wk week.

Fig. 6. Transcriptomic programs in LN cells from SHIV_AD8-EO-challenged monkeys on or off early bNAb therapy.

Heatmaps showing the modulation of pathways in sorted LN monocytes (A), NK cells (B), and T cell subsets (C) from untreated and bNAb-treated monkeys at week 2 or 8 post-SHIV_AD8-EO challenge when compared to pre-challenge or week 2 post-challenge, as determined by transcriptomic analysis. Monkeys were either left untreated or were treated at days 3, 10, and 17 post-SHIV_AD8-EO challenge with either VRC07-523-LS and PGT121 or VRC07-523-LS/DEL and PGT121/DEL. Pathways are indicated in rows (database: MSigDB Hallmark) and the comparisons of interest (monkey group & timepoint 1 – monkey group & timepoint 2) in columns. Red cells indicate significant enrichment of a pathway among genes induced in monkey group & timepoint 1 (GSEA nominal p ≤ 0.05 and NES > 0); blue cells indicate significant enrichment of a pathway among genes downregulated in monkey group & timepoint 1 (GSEA nominal p ≤ 0.05 and NES < 0); and gray cells correspond to non-significant enrichment in monkey group & timepoint 1 (GSEA nominal p > 0.05), when compared to monkey group & timepoint 2. A two-sided permutation test was used to calculate the nominal p-value and the Benjamini-Hochberg procedure was used to calculate the adjusted p-value (A–C). Source data are provided in the Source Data file. CM central memory, EM effector memory, fCD8⁺ follicular CD8⁺, GC germinal center, mem. memory, NES normalized enrichment score, pre pre-challenge, Tfh T follicular helper, UnTx untreated, wk week.