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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.


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Modulation of h9C12 on and off-rate kinetics have disproportionate affects on antiviral activity.(a) SPR titrations of selected h9C12 variants. (b) Neutralization of Adv5 infection by h9C12 variants in 293 T WT cells or 293 T TRIM21 KO cells. (c) Induction of NFκB in 293 T WT and TRIM21 KO cells by h9C12 variants upon adenovirus infection. Cellular experiments were performed in triplicate.
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f4: Modulation of h9C12 on and off-rate kinetics have disproportionate affects on antiviral activity.(a) SPR titrations of selected h9C12 variants. (b) Neutralization of Adv5 infection by h9C12 variants in 293 T WT cells or 293 T TRIM21 KO cells. (c) Induction of NFκB in 293 T WT and TRIM21 KO cells by h9C12 variants upon adenovirus infection. Cellular experiments were performed in triplicate.

Mentions: In order to investigate how the kinetics of antigen binding influences TRIM21 activity we sought CDR mutations that modulated interaction without abolishing it. Binding of WT h9C12 to hexon is characterized by an on-rate constant of 6.35 × 105 M−1 s−1 and an off-rate constant of 0.00052 s−1 (Fig. 3a). Mutations N100A and T55S had only marginal impact on binding kinetics and maintained ~1 nM Kd to hexon (Fig. 4a). Consistent with the binding data, these mutations had no significant impact on TRIM21-dependent neutralization or NFκB activation (Fig. 4b and c). Mutations G29R/R30S and G98C/S99H both reduced the off-rate constant by 10-fold and the on-rate constant ~10-fold and ~30-fold, respectively. Both mutants also showed an intermediate phenotype for TRIM21 dependent neutralization and NFκB activation (Fig. 4b and c). Q97A represented a threshold variant where only the affinity could be measured as the kinetics were too fast to determine (Fig. 4a and Supplementary Figure 1). With a Kd of 462 nM, Q97A had the weakest affinity of the antibody mutants tested; however, it did not have the least potent TRIM21 dependent neutralization. The mutant that was least protective in neutralization was G53P, despite the fact that it demonstrated higher affinity for hexon than Q97A and its on-rate constant was only 2.6 fold reduced compared to WT (Fig. 4a and b). However, G53P displayed a 152-fold increase in off-rate. This suggested that antigen binding kinetics may be an important determinant of TRIM21 activity. Further comparison of mutant activity and their kinetics provided additional evidence. G53P had significantly reduced activity in both neutralization and NFκB assays with respect to G98C/S99H, even though both mutants had similar affinity for hexon. The most striking difference between these two mutants is their variability in off-rate, which is much faster for G53P (Fig. 4a and Table 2). Conversely, G98C/S99H and G29R/R30S had very similar neutralization and NFκB activities, despite having very different affinities for hexon (Fig. 4b and c). Rather, the similar activities are predicted by their off-rates, which were almost identical (Fig. 4a). Thus, off-rate rather than on-rate is crucial for TRIM21 function. Indeed, if on-rate were the critical factor, then G53P would be one of the most active mutants rather than the weakest.


Antibody-antigen kinetics constrain intracellular humoral immunity
Modulation of h9C12 on and off-rate kinetics have disproportionate affects on antiviral activity.(a) SPR titrations of selected h9C12 variants. (b) Neutralization of Adv5 infection by h9C12 variants in 293 T WT cells or 293 T TRIM21 KO cells. (c) Induction of NFκB in 293 T WT and TRIM21 KO cells by h9C12 variants upon adenovirus infection. Cellular experiments were performed in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Modulation of h9C12 on and off-rate kinetics have disproportionate affects on antiviral activity.(a) SPR titrations of selected h9C12 variants. (b) Neutralization of Adv5 infection by h9C12 variants in 293 T WT cells or 293 T TRIM21 KO cells. (c) Induction of NFκB in 293 T WT and TRIM21 KO cells by h9C12 variants upon adenovirus infection. Cellular experiments were performed in triplicate.
Mentions: In order to investigate how the kinetics of antigen binding influences TRIM21 activity we sought CDR mutations that modulated interaction without abolishing it. Binding of WT h9C12 to hexon is characterized by an on-rate constant of 6.35 × 105 M−1 s−1 and an off-rate constant of 0.00052 s−1 (Fig. 3a). Mutations N100A and T55S had only marginal impact on binding kinetics and maintained ~1 nM Kd to hexon (Fig. 4a). Consistent with the binding data, these mutations had no significant impact on TRIM21-dependent neutralization or NFκB activation (Fig. 4b and c). Mutations G29R/R30S and G98C/S99H both reduced the off-rate constant by 10-fold and the on-rate constant ~10-fold and ~30-fold, respectively. Both mutants also showed an intermediate phenotype for TRIM21 dependent neutralization and NFκB activation (Fig. 4b and c). Q97A represented a threshold variant where only the affinity could be measured as the kinetics were too fast to determine (Fig. 4a and Supplementary Figure 1). With a Kd of 462 nM, Q97A had the weakest affinity of the antibody mutants tested; however, it did not have the least potent TRIM21 dependent neutralization. The mutant that was least protective in neutralization was G53P, despite the fact that it demonstrated higher affinity for hexon than Q97A and its on-rate constant was only 2.6 fold reduced compared to WT (Fig. 4a and b). However, G53P displayed a 152-fold increase in off-rate. This suggested that antigen binding kinetics may be an important determinant of TRIM21 activity. Further comparison of mutant activity and their kinetics provided additional evidence. G53P had significantly reduced activity in both neutralization and NFκB assays with respect to G98C/S99H, even though both mutants had similar affinity for hexon. The most striking difference between these two mutants is their variability in off-rate, which is much faster for G53P (Fig. 4a and Table 2). Conversely, G98C/S99H and G29R/R30S had very similar neutralization and NFκB activities, despite having very different affinities for hexon (Fig. 4b and c). Rather, the similar activities are predicted by their off-rates, which were almost identical (Fig. 4a). Thus, off-rate rather than on-rate is crucial for TRIM21 function. Indeed, if on-rate were the critical factor, then G53P would be one of the most active mutants rather than the weakest.

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.


Related in: MedlinePlus