Fig 1.
LALA-PG mutation and Fc functions of IgG.
A) Diagram of Fc-engineered HCA variant. Red dots on the Fc region indicate approximate locations of the three amino acid mutations and their respective residue substitutions. B) Fc functions inhibited by the Fc LALA-PG mutation. Created with BioRender.com.
Fig 2.
Sperm agglutination by HCA and HCA-LALAPG.
A) Agglutination kinetics assay shows similar dose response curves for HCA and HCA-LALAPG. B) Sperm escape assay shows similar percent of progressive sperm relative to the medium-only control for HCA and HCA-LALAPG at all observed antibody concentrations. Data are expressed as means ± SEM of three independently performed experiments. Statistical analyses were conducted using repeated measures two-way ANOVA of log-transformed data followed by Sidak multiple comparisons test. No significant differences were found between the two groups for either assay (p > 0.6). SEM, standard error of the mean.
Fig 3.
Sperm immobilization test with HCA and HCA-LALAPG.
HCA significantly immobilized sperm in the presence of complement (C) in a concentration-dependent manner (p = <0.0001, 0.0005, 0.0001 for 12.5, 6.25, and 3.12 μg/mL, respectively). Heat-inactivated complement (HiC) was used as a negative control and did not cause immobilization. HCA-LALAPG did not significantly affect sperm motility with either C or HiC (p = 0.9105). Data are shown as mean ± SEM and is representative of three independently performed experiments. Statistical analyses were conducted using a two-way ANOVA followed by a Tukey multiple comparisons test. Results were significant when p ≤ 0.05. (*** = p ≤ 0.001, **** = p ≤ 0.0001).
Fig 4.
Cervical mucus penetration test with HCA and HCA-LALAPG.
The number of progressively motile sperm at 90 minutes in ovulatory cervical mucus was significantly lower in the HCA treatment group than with HCA-LALAPG at all capillary tube depths (1–4 cm). Both antibodies were used at a concentration of 12.5μg/mL. Data are shown as mean ± SEM and is representative of four independent experiments, each with a different cervical mucus donor. Statistical analyses were conducted using a repeated measures ANOVA of log transformed data followed by a Tukey multiple comparisons test. Results were significant when p ≤ 0.05. (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001).
Table 1.
Cervical mucus penetration at 90 minutes with HCA and HCA-LALAPG.
Fig 5.
Antibody-dependent sperm phagocytosis.
A) Significantly more sperm were associated with U937 macrophages in HCA-treated cultures than in cultures treated with HCA-LALAPG or medium-only control (p = 0.0128, p = 0.0131, respectively). “Associated sperm” were defined as those either attached to the surface of the macrophage (i.e., at the beginning of the phagocytosis process), or inside the cell. Campath, an anti-CD52 monoclonal antibody known to mediate ADCP effector function, was used as a positive control. Final antibody concentrations were 50 μg/mL. B) The number of internalized sperm per macrophage was quantified following a 2.5-hour incubation with antibody-treated sperm. Final antibody concentrations were 50 μg/mL. The number of internalized sperm was significantly higher following treatment with HCA than with HCA-LALAPG, medium-only control, or VRC01 isotype control (p = 0.037; negative control run twice, isotype control run once). (C) Images of macrophages incubated with sperm exposed to either medium-only (top left), HCA-LALAPG (top right), HCA (bottom left), or Campath (bottom right). Images were taken at 200x magnification. (D) Sperm internalization post-HCA treatment (red arrows) by U937 macrophages. Images were taken at 400X magnification. Data are expressed as mean ± SEM of three independently performed experiments. Statistical analyses were conducted using either repeated measures one-way ANOVA or mixed-effects analysis of log-transformed data followed by Tukey multiple comparisons tests.