Characterization of Pre-existing Immunity to AAV5

Introduction Seroprevalence studies indicate many prospective gene therapy (GT) patients have been previously exposed to adeno-associated viruses (AAV) and harbor pre-existing AAV antibodies which may impact the administration and efficacy of GT. The objective of this study was to further characterize pre-existing antibody responses to AAV serotype 5 (AAV5) in severe hemophilia A patients (n=540) who are GT naïve.

Method We evaluated AAV5 neutralizing factors (transduction inhibition, TI) using a cell-based assay, AAV5 total binding antibodies (TAb) using a bridging immunoassay, AAV serotype specificity using a bridging immunoassay, AAV5 complement activation, and AAV5 antibody isotypes [1].

Results Most subjects with pre-existing immunity to AAV5 were positive for TAb and TI, with a small subset showing discordant results. Pre-existing AAV5 TAb titers were much lower than AAV5 post-treatment titers. We previously¹ assessed the seroprevalence of pre-existing immunity to AAV5 and four other AAV serotypes (AAV2/6/8/rh10). Using those data, we assigned subjects to AAVx positivity groups (Negative, 1, 2, 3, 4, 5) based on the number of positive serotypes. There was no association between higher AAV5 TAb titers and the presence of antibodies reactive with other serotypes, suggesting that AAV5-specific TAb titers were not broadly a result of previous AAV exposures and subsequent cross-reactivity. In a subset of subjects (n=43), AAV5-induced complement activation was assessed in vitro by measuring complement split products (C3a, C4a, C5a, Bb). In some samples with AAV5 TAb, a>200% increase in activated complement split products was observed; however, this was not correlated with AAV5 TAb titer magnitude. Complement activation was not observed in TAb-negative subjects, and not all TAb-positive subjects exhibited complement activation. Based on AAVx positivity, mean complement activation to AAV5>200% in vitro was measured only in subjects that were positive for all five serotypes (AAV5/2/6/8/rh10). Activation of complement may enhance complement receptor-mediated clearance, alter the biodistribution away from the target tissue, and therefore reduce transduction ([Fig. 1]).

Fig. 1 AAVx Positivity (out of 5 total)

In another subset of subjects (n=78) with pre-existing AAV5 TAb positivity, we characterized the isotype and subtype of the anti-AAV5 response (IgM, IgG1, IgG2, IgG3, IgG4, IgA). Responses were predominantly the IgG isotype. AAV5 TAb titers>100 and TI titers>60 were associated with detection of IgG1, but AAV5 TAb titers>100 that were TI negative/titer<60 did not reveal a similar trend. Consequently, highly neutralizing antibody responses were associated with detection of AAV5-specific IgG1.

Conclusion Our in-depth characterization of pre-existing immunity to AAV5 can help elucidate the most important immunological determinants that limit AAV vectored transduction of target tissues. Understanding these determinants of pre-existing immunity could enable more patients to gain access to GT.

Publikationsverlauf

Artikel online veröffentlicht:
26. Februar 2024

• References

• 1 Klamroth R, Hayes G, Andreeva T, Gregg K. et al. Global Seroprevalence of Pre-existing Immunity Against AAV5 and Other AAV Serotypes in People with Hemophilia A. Hum Gene Ther 2022; 33 (7/8) 432-44