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Antibody-antigen kinetics constrain intracellular humoral immunity

View Article: PubMed Central - PubMed

ABSTRACT

During infection with non-enveloped viruses, antibodies stimulate immunity from inside cells by activating the cytosolic Fc receptor TRIM21. This intracellular humoral response relies on opsonized viral particles reaching the cytosol intact but the antigenic and kinetic constraints involved are unknown. We have solved the structure of a potent TRIM21-dependent neutralizing antibody in complex with human adenovirus 5 hexon and show how these properties influence immune activity. Structure-guided mutagenesis was used to generate antibodies with 20,000-fold variation in affinity, on-rates that differ by ~50-fold and off-rates by >175-fold. Characterization of these variants during infection revealed that TRIM21-dependent neutralization and NFκB activation was largely unaffected by on-rate kinetics. In contrast, TRIM21 antiviral activity was exquisitely dependent upon off-rate, with sub-μM affinity antibodies nevertheless unable to stimulate signaling because of fast dissociation kinetics. These results define the antibody properties required to elicit an efficient intracellular immune response during viral infection.

No MeSH data available.


TRIM21-dependent neutralization and signaling is independent from on-rate but proportional to off-rate.Neutralization and signaling efficacy, expressed as percentage of WT activity, and the EC50 of selected h9C12 variants plotted against their off rates (a–c), on rates (d–f) and affinities (g–i) as determined by SPR. The Pearson correlation (Pearson’s correlation coefficient r) and the statistical significance (two-tailed P value) between these kinetic measures are shown.
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f5: TRIM21-dependent neutralization and signaling is independent from on-rate but proportional to off-rate.Neutralization and signaling efficacy, expressed as percentage of WT activity, and the EC50 of selected h9C12 variants plotted against their off rates (a–c), on rates (d–f) and affinities (g–i) as determined by SPR. The Pearson correlation (Pearson’s correlation coefficient r) and the statistical significance (two-tailed P value) between these kinetic measures are shown.

Mentions: To confirm that off-rate of the antibody-antigen complex rather than on-rate determines TRIM21 antiviral activity, we compared the TRIM21-dependent neutralization and signaling of all h9C12 mutants for which we had kinetic data (Fig. 5a–i). For neutralization we examined the correlation between mutant kinetics and both the potency (EC50) and efficacy (maximal neutralization) of the response. When using either measure of neutralization we observed a significant correlation with off-rate (p-values of 0.0054 and 0.0001 respectively) but not with on-rate (p-values of 0.3193 and 0.1208 respectively). Plotting the Kd (kd/ka) also gave a significant correlation with both neutralization measures, albeit the fit to efficacy is substantially poorer than to off-rate. Plotting against signaling responses gave a similar pattern as neutralization, namely that the principle correlate was off-rate, with correlation to affinity also statistically significant but on-rate non-significant. As our kinetic data were obtained using Fab fragments they represent intrinsic rate constants and do not report on any avidity contribution that may result from bivalent binding to virions. To assess whether avidity alters the relative potency of intermediate antibody mutants we compared binding of IgG and Fab fragments to intact adenovirus virions by ELISA. The IgGs had enhanced binding suggesting that bivalent binding is possible, consistent with the arrangement predicted by the crystal structure (Supplementary Figure 2). However, relative binding between mutants was broadly the same between IgG and Fab and a similar p-value was obtained for the correlation with neutralization efficacy.


Antibody-antigen kinetics constrain intracellular humoral immunity
TRIM21-dependent neutralization and signaling is independent from on-rate but proportional to off-rate.Neutralization and signaling efficacy, expressed as percentage of WT activity, and the EC50 of selected h9C12 variants plotted against their off rates (a–c), on rates (d–f) and affinities (g–i) as determined by SPR. The Pearson correlation (Pearson’s correlation coefficient r) and the statistical significance (two-tailed P value) between these kinetic measures are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121590&req=5

f5: TRIM21-dependent neutralization and signaling is independent from on-rate but proportional to off-rate.Neutralization and signaling efficacy, expressed as percentage of WT activity, and the EC50 of selected h9C12 variants plotted against their off rates (a–c), on rates (d–f) and affinities (g–i) as determined by SPR. The Pearson correlation (Pearson’s correlation coefficient r) and the statistical significance (two-tailed P value) between these kinetic measures are shown.
Mentions: To confirm that off-rate of the antibody-antigen complex rather than on-rate determines TRIM21 antiviral activity, we compared the TRIM21-dependent neutralization and signaling of all h9C12 mutants for which we had kinetic data (Fig. 5a–i). For neutralization we examined the correlation between mutant kinetics and both the potency (EC50) and efficacy (maximal neutralization) of the response. When using either measure of neutralization we observed a significant correlation with off-rate (p-values of 0.0054 and 0.0001 respectively) but not with on-rate (p-values of 0.3193 and 0.1208 respectively). Plotting the Kd (kd/ka) also gave a significant correlation with both neutralization measures, albeit the fit to efficacy is substantially poorer than to off-rate. Plotting against signaling responses gave a similar pattern as neutralization, namely that the principle correlate was off-rate, with correlation to affinity also statistically significant but on-rate non-significant. As our kinetic data were obtained using Fab fragments they represent intrinsic rate constants and do not report on any avidity contribution that may result from bivalent binding to virions. To assess whether avidity alters the relative potency of intermediate antibody mutants we compared binding of IgG and Fab fragments to intact adenovirus virions by ELISA. The IgGs had enhanced binding suggesting that bivalent binding is possible, consistent with the arrangement predicted by the crystal structure (Supplementary Figure 2). However, relative binding between mutants was broadly the same between IgG and Fab and a similar p-value was obtained for the correlation with neutralization efficacy.

View Article: PubMed Central - PubMed

ABSTRACT

During infection with non-enveloped viruses, antibodies stimulate immunity from inside cells by activating the cytosolic Fc receptor TRIM21. This intracellular humoral response relies on opsonized viral particles reaching the cytosol intact but the antigenic and kinetic constraints involved are unknown. We have solved the structure of a potent TRIM21-dependent neutralizing antibody in complex with human adenovirus 5 hexon and show how these properties influence immune activity. Structure-guided mutagenesis was used to generate antibodies with 20,000-fold variation in affinity, on-rates that differ by ~50-fold and off-rates by >175-fold. Characterization of these variants during infection revealed that TRIM21-dependent neutralization and NFκB activation was largely unaffected by on-rate kinetics. In contrast, TRIM21 antiviral activity was exquisitely dependent upon off-rate, with sub-μM affinity antibodies nevertheless unable to stimulate signaling because of fast dissociation kinetics. These results define the antibody properties required to elicit an efficient intracellular immune response during viral infection.

No MeSH data available.