Antibody-antigen kinetics constrain intracellular humoral immunity

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.

TRIM21—From Intracellular Immunity to Therapy

“…This finding is consistent with earlier studies showing that while neutralization is efficient using engineered rh9C12 IgG1 variants with up to 100-fold reduced binding for TRIM21, NF-κB induction is ablated or severely diminished (42,44). Furthermore, design of rh9C12 IgG1 variants with differences in on-and off-rate kinetics demonstrated that while slower off -rates generally correlate with efficient TRIM21 activity, its signaling function was abrogated faster by a reduction in off -rate compared to its neutralization function (61). This may allow for a low level of TRIM21 activity without inducing an anti-viral state, which might be important for clearing low levels of free antibody displaced into the cytosol.…”

“…Whether adenovirus infections in the eye may benefit from TRIM21 and antibody therapy remains to be addressed, however, injection of antibodies into the vitreous is frequently used in the clinic to treat eye diseases such as age-related macular edema (124,125). While activation of a strong inflammatory response may not be beneficial in a treatment setting, using engineered antibodies with reduced affinity for TRIM21 or altered antigen binding kinetics could be attractive to reduce inflammation without compromising virus neutralization (42,61).…”

“…As hexon is the primary immunogen to which antibodies is generated, rh9C12 that binds to the apex of the hexon trimer spike (61), may be particularly well suited for such pre-coating. Indeed, shielding the Ad5 surface with a trivalent 9C12 scFv with high avidity has been used in combination with targeted designed ankyrin repeat proteins (DARPin's) binding to the fiber knob of an Ad5 vector (110).…”

Antibody and DNA sensing pathways converge to activate the inflammasome during primary human macrophage infection

“…Using H433A h9C12, which ablates TRIM21 binding, did not prevent neutralisation. This is in contrast to non‐immune cells such as 293Ts where h9C12 neutralisation of AdV is completely TRIM21 dependent (Bottermann et al , ). Using a LALA h9C12 with reduced FcγR binding also failed to prevent neutralisation.…”

Intracellular Antiviral Immunity

“…This phenotype can be partially rescued by increasing antibody concentration, but this mutant's signaling activity is always significantly lower than WT (McEwan et al, 2012). In line with this observation, it has been shown that TRIM21 effector functions strongly correlate with antibody off-rate, more so than simply affinity for the antigen, a trend that is again more pronounced for TRIM21's signaling output than its neutralization function (Bottermann et al, 2016). It is intuitive that signaling would be regulated more strictly and have thresholds, since TRIM21's neutralization activity cannot be harmful for the host, while the initiation of potent immune signaling can have potentially severe consequences.…”