Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses

Abstract

Recombinant adeno-associated viruses (AAVs) have unique gene-transfer properties that speak to their potential as carriers for gene therapy or vaccine applications. However, the presence of neutralizing antibodies to AAV as a result of previous exposure can significantly limit effective gene transfer. In this study, we obtained 888 human serum samples from healthy volunteers in 10 countries around the world. Samples were assayed for neutralizing antibodies to AAV1, AAV2, AAV7, and AAV8, as well as to a novel, structurally distinct AAV vector, rh32.33, in an in vitro transduction inhibition assay. Our data revealed that neutralizing antibodies to AAV2 were the most prevalent antibodies in all regions, followed by antibodies to AAV1. The seroprevalences of antibodies to AAV7 and to AAV8 were lower than that for antibodies to AAV1, and neutralization of AAVrh32.33 was only rarely detected. Our data also indicate a strong linkage of seroreactivity between apparently distinct serotypes that has not been predicted previously in animal models.

Figure 1.

Phylogenetic tree and prevalence of neutralizing antibodies (NAbs) against different adeno-associated virus (AAV) types in 100 serum samples from Australia, 281 from Europe, 407 from Africa, and 100 from the United States. A, Neighbor-joining phylogeny was inferred with Poisson correction for the protein sequences of the AAV VP1 Cap proteins. Scale, evolutionary distance of the number of substitutions per site. B and C, Samples were considered positive if serum dilutions of >1:20 (A) or >1:80 (B) inhibited vector transduction by ≥50%. **Less prevalent than anti-AAV2 Nab (P < .05); ***less prevalent than anti-AAV2 and anti-AAV1 NAbs (P < .05).

Figure 2.

Alignment of amino acid sequences for capsid protein VP1 of adeno-associated virus (AAV) 2, 1, 7, and 8 and rh32.33.

Figure 3.

Prevalence of neutralizing antibodies (NAbs) against different adeno-associated virus (AAV) types in 73 samples from Entebbe, Uganda; 60 from Kakira, Uganda; 60 from Kigali, Rwanda; 112 from Cape Town, South Africa; 51 from Nairobi, Kenya; and 51 from Lusaka, Zambia. Samples were considered positive if serum dilutions of >1:20 (A) or >1:80 (B) inhibited vector transduction by ≥50%. **Less prevalent than anti-AAV2 Nab (P < .05); ***less prevalent than anti-AAV2 and anti-AAV1 NAbs (P < .05); ****less prevalent than anti-AAV2, anti-AAV1, and anti-AAV7 Nabs (P < .05).

Figure 4.

Prevalence of neutralizing antibodies (NAbs) against different adeno-associated virus (AAV) types in 100 serum samples from Belgium, 81 from Greece, and 100 from Italy. Samples were considered positive if serum dilutions of >1:20 (A) or >1:80 (B) inhibited vector transduction by ≥50%. *Less prevalent than anti-AAV1 NAb (P < .05); **less prevalent than anti-AAV2 NAb (P < .05); ***less prevalent than anti-AAV2 and anti-AAV1 Nabs (P < .05).

Figure 5.

Global distribution and magnitude of the neutralizing antibody (NAb) response in serum samples from the United States, Australia, Europe, and Africa in which the NAb titer was ≥1:20. Each box encloses 50% of the data, with the upper and lower limits denoting the interquartile range; the horizontal line denoting the median value; the whiskers denoting minimum and maximum values in the data set that fell within an acceptable range; and the circles denoting outliers.