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. 2024 May 29;32(3):101273.
doi: 10.1016/j.omtm.2024.101273. eCollection 2024 Sep 12.

Global seroprevalence of neutralizing antibodies against adeno-associated virus serotypes used for human gene therapies

Amit Chhabra  1 , George Bashirians  2 , Christos J Petropoulos  3 , Terri Wrin  3 , Yuvika Paliwal  4 , Peter V Henstock  5 , Suryanarayan Somanathan  2 , Candida da Fonseca Pereira  6 , Ian Winburn  7 , John E J Rasko  8   9   10
Affiliations

Global seroprevalence of neutralizing antibodies against adeno-associated virus serotypes used for human gene therapies

Amit Chhabra et al. Mol Ther Methods Clin Dev. .

Abstract

Adeno-associated virus (AAV) vectors are promising gene therapy candidates, but pre-existing anti-AAV neutralizing antibodies (NAbs) pose a significant challenge to successful gene delivery. Knowledge of NAb seroprevalence remains limited and inconsistent. We measured activity of NAbs against six clinically relevant AAV serotypes across 10 countries in adults (n = 502) and children (n = 50) using a highly sensitive transduction inhibition assay. NAb prevalence was generally highest for AAV1 and lowest for AAV5. There was considerable variability across countries and geographical regions. NAb prevalence increased with age and was higher in females, participants of Asian ethnicity, and participants in cancer trials. Co-prevalence was most frequently observed between AAV1 and AAV6 and less frequently between AAV5 and other AAVs. Machine learning analyses revealed a unique clustering of AAVs that differed from previous phylogenetic classifications. These results offer insights into the biological relationships between the immunogenicity of AAVs in humans beyond that observed previously using standard clades, which are based on linear capsid sequences. Our findings may inform improved vector design and facilitate the development of AAV vector-mediated clinical gene therapies.

Keywords: AAV1; AAV5; AAV6; AAV8; AAV9; AAVRh74var; biotechnology; clinical trials; genetic diseases; rare disease.

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Conflict of interest statement

J.E.J.R. has received supply of material (Material Transfer Agreement), consultancy, or honoraria from bluebird bio, Cynata, Novartis, Pfizer, RareCyte, Roche, and SPARK Therapeutics and has shareholdings with RareCyte and Woke. Y.P., C.d.F.P., and I.W. are employees of, and own stock/options in, Pfizer. G.B., P.V.H., S.S., and A.C. were employed at Pfizer at the time of this study. C.J.P. and T.W. are employees of, and hold equity in, Labcorp.

Figures

None
Graphical abstract
Figure 1
Figure 1
Prevalence of NAb positivity in global adult and pediatric participants by AAV serotype and serum dilution (A) Heatmap of the prevalence of NAb activity for adult participants (n = 502). (B) Heatmap of the prevalence of NAb activity for pediatric participants (n = 50). Heatmaps show the prevalence of NAb activity against six AAV serotypes across all serum dilutions. Numbers inside squares indicate the percentage of serum samples positive for NAb activity against the designated serotype at the designated serum dilution (out of all samples tested for that AAV at that serum dilution). Gray squares indicate that all tested serum samples were negative for NAb activity against the serotype at the specified dilution. AAV, adeno-associated virus; Nab, neutralizing antibody.
Figure 2
Figure 2
Prevalence of NAb positivity in adult participants by AAV serotype and country at 1:1 and 1:4 serum dilutions Heatmaps show the prevalence of NAb activity against six AAV serotypes for all adult participants (left column; n = 502) and the subset of participants from each country for the (A) 1:1 and (B) 1:4 serum dilutions. Numbers inside squares indicate the percentage of serum samples positive for NAb activity against the designated serotype in the designated country (out of all samples tested for that AAV in that country). AAV, adeno-associated virus; Nab, neutralizing antibody; USA, United States; CAN, Canada; UK, United Kingdom; DEU, Germany; ESP, Spain; ITA, Italy; FRA, France; AUS, Australia; JPN, Japan; KOR, South Korea.
Figure 3
Figure 3
Prevalence of NAb positivity in adult participants by AAV serotype and serum dilution across geographical regions Heatmaps show the prevalence of NAb activity against six AAV serotypes for adult participants (n = 502) across all serum dilutions on a per country basis, sorted by geographical region (green, North America; yellow, Europe; pink, Asia-Pacific). Numbers inside squares indicate the percentage of serum samples positive for NAb activity against the designated serotype at the designated serum dilution (out of all samples tested for that AAV at that serum dilution). Gray squares indicate that all tested serum samples were negative for NAb activity against the serotype at the specified dilution. AAV, adeno-associated virus; Nab, neutralizing antibody.
Figure 4
Figure 4
Pairwise co-prevalence of NAb positivity in all participants at the 1:1, 1:2, and 1:4 serum dilutions Circles show the co-prevalence of NAb activity against pairs of the six AAV serotypes across serum dilutions for all participants (n = 552). Circle colors indicate the percentage of serum samples positive for NAb activity against both AAV serotypes in pair. AAV, adeno-associated virus; Nab, neutralizing antibody.
Figure 5
Figure 5
Machine learning analysis of relationship between different AAV serotypes (A) Cladogram showing phylogenetic classifications of AAV serotypes, based on Gao et al. and Mietzsch et al., (B) Results of Ward’s hierarchical clustering of AAV NAb titers showing the relationship between the six analyzed AAV serotypes. AAV1 and AAV6 formed a cluster, indicating that they are closely related. AAV8 and AAVRh74var also formed a cluster, which was found to be related to AAV5. AAV9 was least similar to the other AAVs. (C) Principal-component analysis (PCA) of normalized NAb titers in all participants for the six AAV serotypes. AAVs that are nearer one another in the two-dimensional projection are more closely related. As observed with the clustering analysis (B), AAV1 and AAV6 were closest to one another, followed by AAVRh74var, AAV8, and AAV5, while AAV9 was most distant from all the other AAVs. AAV, adeno-associated virus; Nab, neutralizing antibody; PC, principal component.
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