Neutralizing antibodies against Mayaro virus require Fc effector functions for protective activity

Abstract

Despite causing outbreaks of fever and arthritis in multiple countries, no countermeasures exist against Mayaro virus (MAYV), an emerging mosquito-transmitted alphavirus. We generated 18 neutralizing mAbs against MAYV, 11 of which had "elite" activity that inhibited infection with EC₅₀ values of <10 ng/ml. Antibodies with the greatest inhibitory capacity in cell culture mapped to epitopes near the fusion peptide of E1 and in domain B of the E2 glycoproteins. Unexpectedly, many of the elite neutralizing mAbs failed to prevent MAYV infection and disease in vivo. Instead, the most protective mAbs bound viral antigen on the cell surface with high avidity and promoted specific Fc effector functions, including phagocytosis by neutrophils and monocytes. In subclass switching studies, murine IgG2a and humanized IgG1 mAb variants controlled infection better than murine IgG1 and humanized IgG1-N297Q variants. An optimally protective antibody response to MAYV and possibly other alphaviruses may require tandem virus neutralization by the Fab moiety and effector functions of the Fc region.

Graphical abstract

Figure 1.

Anti-MAYV mAbs neutralize strains from both D and L genotypes. (A) Viral strains were subjected to next-generation sequencing. A phylogenetic tree was generated using a Jukes–Cantor genetic distance model by aligning structural gene nucleotide sequences for each MAYV strain. (B) Serial dilutions of mAbs were incubated with 10² FFU of the indicated MAYV strain, representing either genotypes D (top) or L (bottom), before inoculation of Vero cells. Cells were overlaid with methylcellulose and incubated for 18 h. Viral foci were stained, counted, and plotted relative to a no-antibody control. Data are representative of two experiments performed in triplicate. Error bars represent SD within one experiment.

Figure 2.

Cross-reactivity and cross-neutralization of related arthritogenic alphaviruses. (A) Dendrogram showing the phylogenetic relationship of the structural genes of related alphaviruses used for cross-neutralization testing. (B–E) Neutralization assays were performed with anti-MAYV mAbs that bound to cells infected by UNAV (B), CHIKV (C), RRV (D), or ONNV (E). Serial dilutions of the indicated mAbs were incubated with 10² FFU of the indicated alphavirus before inoculation of Vero cells as described in Fig. 1. Data are representative of two experiments performed in triplicate. Error bars represent SD within one experiment.

Figure 3.

Neutralizing mAbs block MAYV infection at postattachment steps. (A and B) Attachment inhibition assay. MAYV-CH was incubated with soluble heparin (0.5 to 2 mg/ml; A), BSA (0.5–2 mg/ml; A), or anti-MAYV mAbs (10 µg/ml; B) for 1 h before addition to Vero cells at 4°C. After unbound virus was removed by extensive rinsing, cell-adsorbed viral RNA was quantified by qRT-PCR, standardized to GAPDH levels, and plotted relative to an untreated (A) or isotype mAb-treated (B) control. Data are the mean and SD of three experiments performed in triplicate (one-way ANOVA with Dunnett’s post-test compared with the isotype control mAb). (C) Pre/postattachment neutralization assays. Serial dilutions of anti-MAYV mAbs were incubated with 10² FFU of MAYV and added to Vero cells. Infection proceeded for 18 h before foci were stained, counted, and plotted relative to a no-antibody control. Data are the mean and SD of two experiments performed in triplicate. (D) Postattachment neutralization assay. 10² FFU of MAYV was adsorbed to Vero cells at 4°C. Unbound virus was removed by extensive washing, and serial dilutions of anti-MAYV mAbs were added. Infection proceeded for 18 h at 37°C. Viral foci were stained, counted, and plotted relative to a no-mAb control. (E and F) FFWO assay. Cells were incubated with virus at 4°C. After removing unbound virus by rinsing, cells were treated with 1 µg/ml of the indicated mAbs and then pulsed for 2 min with medium at pH 7.6 (E) or pH 5.5 (F) at 37°C. After pH neutralization, cells were cultured in medium supplemented with 20 mM NH₄Cl to prevent viral fusion via canonical endosomal pathways. Fusion inhibition was measured by flow cytometry by staining cells for MAYV E2 antigen 18 h later. Data are the mean and SD of three experiments performed in duplicate (one-way ANOVA with Dunnett’s post-test compared with the isotype mAb control). (G and H) Egress inhibition assay. RNA isolated from MAYV-infected cells was transfected into Vero cells. Medium was added with the indicated concentrations of anti-MAYV mAbs. RNase A–resistant encapsidated viral RNA in the supernatant was quantified by qRT-PCR at 1 h (G) or 6 h (H) after transfection. Data are the mean and SD of three experiments performed in duplicate (one-way ANOVA with Dunnett’s post-test compared with the isotype mAb control). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 4.

Mapping of neutralizing anti-MAYV mAbs to sites within E1 and E2 proteins. (A–C) Solvent-exposed residues on the MAYV E2 B domain were changed to the indicated amino acids. WT and mutant MAYV B domain proteins were purified, and binding to anti-MAYV mAbs was tested by ELISA. Regions were divided into groups A (blue), B (red), and C (green) based on patterns of mutations that resulted in loss of binding of mAbs. Representative mAbs from each group are shown (MAY-117, group A [A]; MAY-125, group B [B]; and MAY-139, group C [C]), with the remainder of the data in Fig. S4. (D–F) 293T cells were transfected with a C-E3-E2-6K-E1 plasmid containing alanine mutations in the B domain of E2 and tested for binding with anti-MAYV mAbs by flow cytometry. Additional arginine or glutamic acid changes were made to residues in two loops in the B domain (residues 179–186 and 212–218). Representative mAbs from three binding groups are shown (MAY-117, group A [D]; MAY-125, group B [E]; and MAY-139, group C [F]), with the remainder of the data in Fig. S4. Critical residues were defined as those with ≤25% binding to an individual mAb but ≥75% binding to an oligoclonal pool of anti-MAYV mAbs. Data are from three experiments performed in triplicate. Error bars represent SD within one experiment. (G) Alignment of the B domain of E2 of MAYV, UNAV, CHIKV, RRV, and ONNV with the critical interaction residues identified for each mAb marked. Residues mapped by structure-guided mutagenesis and ELISA (circles), alanine scanning mutagenesis and flow cytometry (triangles), or both (squares) are marked. Boxes around amino acids 179–186 and 212–218 indicate loops in the B domain. Bars above the B domain indicate regions used to define binding groups A (blue), B (red), and C (green). (H–J) Neutralization escape mutants were generated by serial passage of MAYV (strain CH) in the presence of MAY-115 or MAY-131. Sequence-confirmed mutations were introduced into an infectious cDNA clone of the parental MAYV strain and tested for neutralization by MAY-117 (H), MAY-115 (I), or MAY-131 (J). Data are representative of two experiments performed in triplicate. Error bars represent SD within on experiment. (K) Key residues necessary for mAb engagement are highlighted on the surface representation of CHIKV (Basore et al., 2019; left; PDB: 6NK5) and depicted as balls and sticks on a ribbon diagram of the predicted structure of MAYV E2-E1 monomer generated using Phyre2 (right). Inset: Zoomed-in view of a trimeric spike. The E1 glycoprotein is in light gray, with the FL in green and the B-, C-, and D-strands of domain II in cyan. The E2 glycoprotein is in dark gray, with the B domain in yellow. Epitope-mapped residues in E2 domain B and E1 domain II are colored in blue and magenta, respectively.

Figure 5.

Antibody protection against lethal MAYV challenge. A lethal MAYV challenge model was developed by treating 4 wk-old C57BL/6J male mice with a single 100-µg dose of anti-Ifnar1 mAb 1 d before subcutaneous virus inoculation. (A and B) Prophylaxis studies. A single dose of anti-MAYV mAbs (100 µg/mouse; ∼6/mg/kg) was administered 1 d before inoculation with MAYV-BeH407, and survival was monitored. Antibodies are presented in IgG subclass groups: mouse IgG1 (A) or mouse IgG2a (B). Data are from two experiments. (C and D) Therapeutic studies. Indicated mAbs (100 µg/mouse) were given 1 d after virus inoculation, and survival was monitored. Data are from two experiments. (E–G) Combination mAb therapy. 200 total µg of MAY-115, MAY-134, or a combination (100 µg each) was given to mice beginning at 1, 2, or 3 dpi, and survival was monitored. Data are from two experiments. In this figure, n = 10, log-rank test with Bonferroni correction compared with the isotype mAb control treated group. *, P < 0.05; ***, P < 0.001.

Figure 6.

Antibody protection of MAYV-induced musculoskeletal disease. (A) Swelling observed in the ipsilateral (4 dpi, left) and contralateral (7 dpi, right) ankle following infection with MAYV-BeH407. (B–G) Protective mAbs in the lethal challenge model (Fig. 5) were tested for activity against MAYV-induced musculoskeletal disease. 4-wk-old C57BL/6J male mice were given 100 µg of indicated mAbs via intraperitoneal route 1 d before subcutaneous inoculation of 10³ FFU MAYV-BeH407 in the foot. Swelling was measured in the ipsilateral (B, D, and F) and contralateral (C, E, and G) ankles using digital calipers. Data are the mean and SEM of two experiments (n = 10 mice, two-way ANOVA with Tukey’s post-test). (H–M) Virus titers in the ipsilateral (H and I) or contralateral (J and K) feet or draining inguinal lymph node (L and M) at 1 (H, J, and L) and 7 (I, K, and M) dpi after prophylaxis of mice with 100 µg of the indicated mAbs. Animals were perfused with PBS before tissue collection. Viral titers were determined by qRT-PCR. Data are from two experiments (n = 10 mice, one-way ANOVA with Dunnett’s post-test). *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001. The experiments in B, D, and F or C, E, and G were performed concurrently, and thus a single isotype control mAbs was used and included in each graph.

Figure 7.

Antibody protection against MAYV depends on the IgG subclass and Fc effector functions. (A) 4-wk-old C57BL/6 male and female FcγR^−/− mice were administered 100 µg of the indicated anti-MAYV mAbs and 100 µg of anti-Ifnar1 mAb 1 d before subcutaneous inoculation with 10³ FFU of MAYV-BeH407. Data are from two experiments. (B) Recombinant, isotype-switched mAbs (MAY-115 and MAY-134) were tested by ELISA for binding to soluble mouse and human FcγRs. Binding is presented relative to the mouse IgG2a isotype control of MAY-115 (left) or MAY-134 (right). Data are the mean and SD of two experiments performed in triplicate. (C and D) Neutralizing activity of isotype-switched MAY-115 (C) or MAY-134 (D) was measured by FRNT against MAYV-BeH407. Data are representative of three experiments performed in triplicate. (E) 4-wk-old FcγR^−/− mice were administered 100 µg of the indicated MAY-115 IgG and 100 µg of anti-Ifnar1 mAb 1 d before subcutaneous inoculation with 10³ FFU of MAYV-BeH407. Data are from two experiments. (F–I) Protection by isotype-switched mouse IgG2a mAbs. MAY-115 (F and G) and MAY-134 (H and I) isotype-switched mAbs were tested as prophylaxis against lethal challenge (F and H) or musculoskeletal disease (G and I) as described in Figs. 5 and 6. Survival data are from two experiments. Swelling data are the mean and SD from two experiments (n = 10 mice, two-way ANOVA with Sidak’s post-test). (J–L) Protection by isotype-switched mouse IgG1 mAbs. MAY-117 (J) and MAY-130 (K) isotype-switched mAbs were tested as prophylaxis against lethal challenge (L). Survival data are from two experiments (A, E, F, H, and L; n = 10 mice, log-rank test with Bonferroni correction compared with the isotype control or indicated treatment groups). The experiments in F and H or G and I were performed concurrently, and thus a single isotype control mAbs was used and included in each graph. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.