Research ArticleANTIBODIES

Optimal therapeutic activity of monoclonal antibodies against chikungunya virus requires Fc-FcγR interaction on monocytes

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Science Immunology  22 Feb 2019:
Vol. 4, Issue 32, eaav5062
DOI: 10.1126/sciimmunol.aav5062
  • Fig. 1 Clinical protection after mAb therapy.

    (A) Four-week-old WT C57BL/6J mice were administered mouse anti-CHIKV mAbs [CHK-152 + CHK-166 (250 μg per mAb; 500 μg total)] or an isotype control (WNV E60; 500 μg) on 3 dpi with 103 FFU of CHIKV. Foot swelling was measured before infection and for 10 dpi (n = 8 per group, two experiments). Graphs show means ± SEM (***P < 0.001, ****P < 0.0001, two-way ANOVA with Sidak’s post-test). (B) mAbs (CHK-166 human IgG1, CHK-152 human IgG1, CHK-166 human IgG1 N297Q, and CHK-152 human IgG1 N297Q) were preincubated with 102 FFU of CHIKV and added to Vero cells for 18 hours. Viral foci were measured and compared with a no mAb control to determine relative infection. WNV hE16 is an isotype control mAb. Each graph represents the mean ± SD (two or three experiments). (C to F) Four-week-old mice were inoculated with CHIKV and then administered a (C and D) cocktail [CHK-152 + CHK-166 (250 μg per mAb; 500 μg total)] or (E and F) monotherapy [CHK-152 or CHK-166 (250 μg total)] of intact or N297Q variants of humanized mAbs or an isotype control (WNV hE16; 500 or 250 μg) on 3 dpi. (C, E, and F) Foot swelling was measured [(C) n = 8 to 10 per group, three experiments; (E) n = 7 per group, two experiments; (F) n = 7 per group, two experiments]. Graphs show means ± SEM (*intact versus isotype mAb, ¢intact versus N297Q, N297Q versus isotype mAb; two-way ANOVA with Tukey’s post-test: *P < 0.05, **P < 0.01, ****P < 0.0001, ¢P < 0.05, ¢¢P < 0.01, ¢¢¢P < 0.001, ¢¢¢¢P < 0.0001, P < 0.05). (D) Human IgG levels in the ipsilateral ankle were determined by ELISA at 5 dpi (n = 8 to 9 per group, two experiments). Bars indicate mean values (ns, not significant; Student’s t test).

  • Fig. 2 Intact mAb therapy reduces viral RNA levels.

    WT mice were inoculated with 103 FFU of CHIKV and administered a cocktail of intact or N297Q variants of humanized anti-CHIKV mAbs or an isotype control mAb at 3 dpi. (A) Ipsilateral ankles were harvested at indicated days, and viral RNA was determined by qRT-PCR (5 and 7 dpi, n = 8 to 9 per group; 28 dpi, n = 7 to 9 per group, two experiments; one-way ANOVA with Tukey’s post-test: **P < 0.01, ***P < 0.001). (B) RNA in situ hybridization of ipsilateral feet using CHIKV-specific probes (479501) from tissues at 5 or 7 dpi. Images show low magnification (top; scale bars, 100 μm) and medium magnification (middle and bottom; scale bars, 100 μm) with a high-magnification inset (scale bars, 10 μm) (representative images from n = 6 per group, two experiments).

  • Fig. 3 Fc effector functions of antibody affect infiltration of immune cells.

    WT mice were inoculated with 103 FFU of CHIKV and administered a cocktail of intact or N297Q variants of humanized anti-CHIKV mAbs or an isotype control at 3 dpi. (A to D) Ipsilateral ankles were collected at 4 dpi and analyzed for chemokines (n = 9 to 10 per group, two experiments). Bars indicate median values (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Kruskal-Wallis ANOVA with Dunn’s post-test). (E and F) At 4 dpi, cells from ipsilateral feet were stained for monocytes (CD11b+CD11cLy6GLy6C+), neutrophils (CD11b+CD11cLy6G+), NK cells (CD3NK1.1+), or (G) MHC class II+ monocytes (CD11b+CD11cLy6GLy6C+MHCII+) and analyzed by flow cytometry. (E) Percentage of indicated cell populations out of CD45+ cells and (F and G) number of viable cells of indicated populations [(E to G) n = 8 per group, two experiments]. Bars indicate mean values (*P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Bonferroni’s post-test). (H to K) Ipsilateral ankles were collected at 7 dpi and analyzed for chemokines (n = 8 to 9 per group, two experiments). Bars indicate median values (*P < 0.05, **P < 0.01, Kruskal-Wallis ANOVA with Dunn’s post-test). (L and M) At 7 dpi, cells from ipsilateral feet were stained for monocytes, moDCs (CD11b+CD11c+Ly6GLy6C+MHCII+), neutrophils, NK cells, CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), B cells (CD3CD19+), or (N) MHC class II+ monocytes and analyzed by flow cytometry. (L) Percentage of indicated cell populations out of CD45+ cells and (M and N) number of viable cells of indicated populations [(L to N) n = 6 to 10 per group, two or three experiments]. All comparisons in (L) to (N) were not statistically significant (one-way ANOVA with Bonferroni’s post-test). (O to S) Indicated cell numbers were compared between 4 and 7 dpi. Bars indicate mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test). The color of asterisks denotes significance for matching group (red, intact combo; blue, N297Q combo; black, isotype).

  • Fig. 4 Fc-FcγR interactions mediate clinical and virological protection.

    C1q−/− (A, C, D, and F to H) or FcRγ−/− (B, C, E to G, and I) mice were inoculated with 103 FFU of CHIKV and administered a cocktail of humanized (A to C, H, and I) or mouse (D to G) intact anti-CHIKV mAbs or an isotype control mAb at 3 dpi. (A, B, D, and E) Foot swelling was measured before infection and for 10 or 7 dpi [(A) n = 9 to 10 per group; (B) n = 11 to 12 per group; (D) n = 6 to 8 per group; (E) n = 11 to 13 per group, two or three experiments]. Graphs show means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA with Sidak’s post-test). (C and F) The ipsilateral ankle (I. ankle) or (G) spleen was harvested 5 dpi (C) or 7 dpi (F and G), and levels of viral RNA were determined. (C, F, and G) Student’s t test (n = 6 to 13, two or three experiments; **P < 0.01, ***P < 0.001, ****P < 0.0001). (C) Tissue titers from WT mice are from Fig. 2A (5 dpi) and shown for comparison. (H and I) The ipsilateral feet from antibody-treated CHIKV-inoculated C1q−/− (H) or FcRγ−/− (I) mice were harvested at 4 dpi and stained for CD45+ cells, monocytes, neutrophils, and NK cells and analyzed for number of viable cells of indicated populations by flow cytometry [(H) n = 11 to 12 per group, three experiments; (I) n = 9 per group, three experiments]. Bars indicate mean values (*P < 0.05, unpaired t test).

  • Fig. 5 Anti-CHIKV mAbs enhance phagocytosis with mouse monocytes and neutrophils.

    Mouse, humanized intact, and humanized N297Q variants of anti-CHIKV mAbs or an isotype control were evaluated for mouse (A and B) monocyte-directed or (C and D) neutrophil-directed phagocytosis of CHIKV p62-E1–functionalized fluorescent beads (ADCP, antibody-directed cellular phagocytosis; ADNP, antibody-dependent neutrophil phagocytosis; n = 3 donors, two experiments). The dotted line indicates the no antibody control. Graphs show means ± SEM.

  • Fig. 6 Monocytes reduce CHIKV infection in the context of mAb therapy.

    WT mice were inoculated with 103 FFU of CHIKV and administered a cocktail of (A to L) humanized or (M to P) mouse intact anti-CHIKV mAbs or an isotype control mAb at 3 dpi. (A to D) Monocytes and neutrophils, (E to H) neutrophils, or (I to P) monocytes were depleted using anti-Ly6G/Ly6C, anti-Ly6G, or anti-CCR2, respectively. (A and B) Monocyte and neutrophil, (E and F) neutrophil, or (I, J, M, and N) monocyte depletion was confirmed by flow cytometry analysis [(A) anti-Ly6G/Ly6C, (E) anti-Ly6G, or (I or M) anti-CCR2 in each set; the top is the specific cell depletion and the bottom is the nondepleting isotype]. (C, G, K, and O) Foot swelling was measured before infection and for 7 dpi [(C, G, K, and O) n = 6, two experiments]. Bars indicate mean ± SEM (two-way ANOVA with Tukey’s post-test: aanti-CHIKV mAb + depleting mAb versus isotype mAb + depleting mAb (open circle versus open triangle); banti-CHIKV mAb + isotype nondepleting mAb versus isotype mAb + isotype nondepleting mAb (closed circle versus closed triangle); cisotype mAb + depleting mAb versus isotype mAb + isotype nondepleting mAb (open triangle versus closed triangle); danti-CHIKV mAb + depleting mAb versus anti-CHIKV mAb + isotype nondepleting mAb (open circle versus closed circle); a, b, or dP < 0.05, aa, bb, or ddP < 0.01, bbb or cccP < 0.001, bbbb, cccc, or ddddP < 0.0001). (D, H, L, and P) Ipsilateral ankles were collected at 7 dpi, and viral RNA levels were measured. Bars indicate mean values, and significance was determined by Student’s t test between either the depleted or isotype nondepleted ankles [(D, H, L, and P) n = 6, two experiments; **P < 0.01, ***P < 0.001, ****P < 0.0001]. Open symbols denote mice depleted of indicated immune cells, and closed symbols denote mice that receive isotype nondepleting control mAbs.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/32/eaav5062/DC1

    Fig. S1. IM-CKV063 in combination with CHK-166 reduces clinical disease and viral RNA.

    Fig. S2. Viral burden in contralateral ankle with anti-CHIKV mAb therapy.

    Fig. S3. Gating scheme for infiltrating immune cells in WT mice.

    Fig. S4. Similar levels of cellular infiltration in the ipsilateral feet at 7 dpi.

    Fig. S5. Fc effector functions of antibody affect proinflammatory cytokine and chemokine expression.

    Fig. S6. Gating scheme for infiltrating immune cells in FcRγ−/− and C1q−/− mice.

    Fig. S7. NK cell depletion does not affect mAb-mediated protection.

    Table S1. Proinflammatory chemokine and cytokine expression in joint tissue homogenates.

    Table S2. Raw data in Excel file.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. IM-CKV063 in combination with CHK-166 reduces clinical disease and viral RNA.
    • Fig. S2. Viral burden in contralateral ankle with anti-CHIKV mAb therapy.
    • Fig. S3. Gating scheme for infiltrating immune cells in WT mice.
    • Fig. S4. Similar levels of cellular infiltration in the ipsilateral feet at 7 dpi.
    • Fig. S5. Fc effector functions of antibody affect proinflammatory cytokine and chemokine expression.
    • Fig. S6. Gating scheme for infiltrating immune cells in FcRγ−/− and C1q−/− mice.
    • Fig. S7. NK cell depletion does not affect mAb-mediated protection.
    • Table S1. Proinflammatory chemokine and cytokine expression in joint tissue homogenates.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel file). Raw data in Excel file.

    Files in this Data Supplement:

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