Antibody bivalency improves antiviral efficacy by inhibiting virion release independently of Fc gamma receptors

Abstract

Across the animal kingdom, multivalency discriminates antibodies from all other immunoglobulin superfamily members. The evolutionary forces conserving multivalency above other structural hallmarks of antibodies remain, however, incompletely defined. Here, we engineer monovalent either Fc-competent or -deficient antibody formats to investigate mechanisms of protection of neutralizing antibodies (nAbs) and non-neutralizing antibodies (nnAbs) in virus-infected mice. Antibody bivalency enables the tethering of virions to the infected cell surface, inhibits the release of virions in cell culture, and suppresses viral loads in vivo independently of Fc gamma receptor (FcγR) interactions. In return, monovalent antibody formats either do not inhibit virion release and fail to protect in vivo or their protective efficacy is largely FcγR dependent. Protection in mice correlates with virus-release-inhibiting activity of nAb and nnAb rather than with their neutralizing capacity. These observations provide mechanistic insights into the evolutionary conservation of antibody bivalency and help refining correlates of nnAb protection for vaccine development.

Keywords: Fc gamma receptors; antibody bivalency; antiviral protection; humoral immunity; immunoglobulin superfamily; inhibition of virion release; lymphocytic choriomeningitis virus (LCMV); virus budding; virus neutralization.

Graphical abstract

Figure 1

Passive antibody treatment suppresses viremia independently of antibody PRNT potency and Fc-mediated effector functions (A) PRNT activity of rKL25 against rCl13/WE and rCl13/WE^∗. (B–G) We infected WT mice (B–D, G) and mice of the indicated genotypes (E and F) with rCl13/WE or rCl13/WE^∗ intravenously (i.v.) and treated them 3 days later (arrow) with 300 μg of either antibody format. WT mice in (G) were treated either by a single dose of KL25 IgG2a or with F(ab')₂ fragments in a repeated dosing regimen to mimic the washout of KL25 IgG2a (see Figure S1K). Controls were given no antibody (no Ab). Viremia was monitored over time. (B and C) Comparable efficacy of KL25 against rCl13/WE and rCl13/WE^∗. (D) Efficacy of FcγR- and C1q-blind (D265A mutant) antibody independently of antibody isotype. (E and F) KL25 efficacy in FcγRnullC3KO and TRIM21KO mice. Symbols represent the mean ± SEM of three replicate samples (A), or four mice per group (B, C, F, and G), or three mice per group (D and E). One representative experiment of two is shown.

Figure 2

KL25 efficacy is not due to an indirect immunostimulatory effect but relies on the prevention of viral spread (A–D) (A) Schematic of the experiment in (B) and (C). We co-infected WT mice (n = 4) with a 1:1 mixture of rCl13/WE and rCl13/WE-N119S. KL25-IgG2a was administered 3 days later, serum was collected on d3, d6, and d11 (B), and organs were harvested on d3 and d6 (C). rCl13/WE and rCl13/WE-N119S RNA copy numbers in co-infected mice were individually quantified by RT-qPCR from serum (B) and organs (C). ^∗p < 0.05, ^∗∗p < 0.01 by unpaired Student's t test on log-converted values (B) and by one-way ANOVA with Dunnett's post-test comparing each group with the d6 + no Ab group (C). Arrow in (B) indicates Ab treatment. Symbols in (B, left) show the mean ± SEM of four mice per group, with individual d11 values shown in (B, right) and compared by Student's t tests. Bars in (C) represent the mean ± SEM of four mice. (D) Schematic of the experiments in (F) and (G) (setup A) and (H) and (I) (setup B). In setup A, mice were infected with rCl13/WE, KL25 was administered on d3, and organs were assessed on d6 (F, G). In setup B, KL25 was administered to mice, and replication-deficient rCl13ΔGP(WE) vector was injected i.v. 5 h later. Organs were analyzed on d2 (H, I). Control groups were without antibody treatment (no Ab). (E) FCNT verified that KL25 antibody neutralizes rCl13ΔGP(WE) vectors. Representative fluorescence-activated cell sorting (FACS) panels (left) and dose-dependent neutralization (right). (F and H) Virus-infected cells (LCMV NP-positive) in spleen, liver and kidney of rCl13/WE-infected (F) or rCl13ΔGP(WE) vector-inoculated (H) mice. (G and I) The percentage of LCMV antigen-stained tissue surface (left) and viral RNA copies by RT-qPCR (right). Antibody efficacy calculated as fold reduction of LCMV NP-positive tissue surface and viral RNA copy numbers, respectively, are indicated. Scale bars: 100 μm (F and H). Representative histology images from four individual mice are shown, two or three visual fields were analyzed per organ and animal. Bars in (G) and (I) represent the mean ± SEM of four mice per group. One representative experiment of two is shown for (B), (E), (G), and (I). Data in (C) are independently reproduced in Figure S2. ^∗p < 0.05, ^∗∗p < 0.01; statistical analyses were performed by unpaired Student's t tests on log-converted values.

Figure 3

Bivalent but not monovalent antibody molecules tether virions to the infected cell surface and inhibit virion release (A) Experimental design of the VRI assay. (B–D) KL25 antibody formats were tested in a VRI assay on RAW264.7 cells. Twelve hours post infection the cells were processed for TEM (B) or immunogold TEM (C) to assess cell-surface-tethered virions. Tethered virions as shown in representative images were found in about three to five cells per 100 KL25-, F(ab')₂- or MonoFab-treated cells. This variability is presumably due to compartmentalized virion release and imperfect coverage of the cell surface compartments by the ultrathin TEM sections. Conversely, not a single tethered virion was found in >200 cells of untreated or Fab-treated samples. (D) Viral RNA copies in cell culture supernatant were monitored. (E) Bivalent KL25-IgG2a and F(ab')₂ but not Fab molecules are active in VRI. (F) Intracellular viral RNA copies in a VRI assay. (G) Junin virus GP-specific mAbs QC03 and OD01 but not LCMV-GP-specific KL25 inhibit the release of Junin-GP-expressing LCMV (rCl13/JUNGP). (H) The GP-specific mAb KL25 but not the NP-specific mAbs KL53 and VL4 are active in VRI assays. (I) VRI assay on rCl13/WE- or rCl13/WE^∗-infected RAW264.7 cells document similar KL25 dose response. (J) VRI activity of polyclonal sera collected at the indicated time points after rCl13/WE infection and pre-diluted 1:50. Each experiment was performed with three technical replicates. VRI activity calculated as fold-change compared with no Ab (D–I) or naive serum (J). (K and L) d30-IgG (K-M) in FCNT (K) and in ELISA (L) against GP1 and GP-C of WE. (M) Mice were infected with rCl13/WE, treated with d30-IgG on d3 (arrow), or were left untreated (no Ab) and viremia was monitored. (B–D) One representative image out of several regions analyzed in at least three TEM images captured. Symbols show the mean ± SEM of three technical replicates in (D), (K), and (I) (red squares) or individual values in (E)–(J) and (L). Bars represent the mean ± SEM of three technical replicates in each group. The mean ± SEM of two mice is shown in (M). One representative experiment of two similar ones (F–M) or one out of three experiments (D and E) is shown. Scale bars in (B) and (C): 500 nm. ^∗p < 0.05, ^∗∗p < 0.01 by one-way ANOVA of log-converted RNA copy numbers.

Figure 4

KL25-MonoFab exhibits Fcγ-dependent VRI activity and suppresses viremia in an FcγR-dependent manner (A) Schematic of engineered monovalent antibodies. In AlbuminFab, albumin substitutes for the heavy chain CH2-CH3 domains, with a flexible linker to the VH-CH1 domain. MonoFab is a heterodimer of a modified heavy chain and a light chain derivative, in which the mouse IgG1 CH2 and CH3 domains are fused to the kappa light chain, connected by a part of the hinge domain (Figure S6). (B) KL25 MonoFab and AlbuminFab were purified by affinity chromatography followed by size exclusion chromatography and were reanalyzed by size exclusion chromatography (Figure S6D). A superposition of their elution profiles with elution maxima of KL25 IgG1, Fab2, and Fab molecules is displayed for a comparison of relative molecular weights. (C) Binding of the indicated KL25 constructs to mouse FcγRIIb in ELISA. Symbols indicate the mean ± SD of two independent measurements. (D) WT mice (n = 4) were treated with 300 μg of rKL25, MonoFab, or AlbuminFab 3 days after rCl13/WE^∗ infection. Serum concentrations were determined 24 and 48 h later to calculate the molecules' in vivo half-life under infection conditions. (E) GP1 binding was assessed by ELISA. (F) rCl13/WE PRNT activity of the indicated KL25 constructs. (G and H) We performed VRI assays using rCl13/WE (G) or rCl13/WE^∗ (H) and the indicated antibody constructs. (I–N) WT (I and J) and FcγRnull/C3KO (K and L) mice were infected with rCl13/WE^∗ on d0 and were treated with KL25 IgG1 (300 μg), MonoFab (300 μg), MonoFab-D265A (500 μg), or AlbuminFab (500 μg) on d3 (arrow). MonoFab-D265A and AlbuminFab were dosed higher than KL25 and MonoFab to exclude the possibility that a lack of efficacy was due to borderline dosing. Viremia was determined (I and K). Viral RNA copy numbers in serum were quantified at d10 and d11, respectively (J and L). The fold reduction in viral RNA load compared with untreated animals is indicated. Representative results from two independent experiments are shown. Symbols in (E) and (F) indicate the mean ± SEM of three independent measurements; symbols in (G) and (H) show individual cell culture wells and symbols in (D), (J), and (L) represent individual mice. Bars in (G) and (H) show the mean ± SEM of three technical replicates. Symbols in (I), (K), (M), and (N) represent the mean ± SEM of four mice per group. ^∗p < 0.05, ^∗∗p < 0.01 compared with no antibody controls (no Ab), as determined by one-way ANOVA with Dunnett's post test, conducted on log-converted values.

Figure 5

Additional LCMV-nAb clones corroborate that bivalency-dependent VRI activity correlates with in vivo protection (A–F) GP1 binding of the LCMV-nAbs WEN3 (A, C, and E) and WEN1 (B, D, and F) in IgG1 or MonoFab format (A and B), their PRNT potency (C and D), and VRI activity (E and F). Symbols in (A)–(D) show the mean ± SEM of three technical replicates, (E) and (F) show individual replicates with bars indicating the mean ± SEM. (G and H) WT mice were given 300 μg of WEN3 (G) or WEN1 (H), controls were without antibody treatment (no Ab, same control group reported in G and H). Five hours later, the animals were challenged with rCl13ΔGP(WE), and 2 days after vector administration we determined viral RNA copies in tissues. Antibody efficacy was calculated as viral RNA fold reduction compared with no Ab. (I–L) FcγRnull/C3KO and WT mice were infected with rCl13/WE on d0 and were treated with the indicated antibody constructs (300 μg) on d3 or left untreated (no Ab). (M and N) Viremia was monitored. Viral RNA copy numbers in serum on d6. Fold reduction compared with no-Ab controls is indicated. Representative results from two independent experiments are shown. Symbols and bars in (G)–(L) represent the mean ± SEM of four mice, symbols in (M) and (N) show individual animals. ^∗p < 0.05, ^∗∗p < 0.01 as determined by Student's t tests (G and H) and by one-way ANOVA followed by Dunnett's post test (E, F, M, and N), conducted on log-converted values.