Khadir E, Burns J, Palermo A, Budrewicz J, Mota J, Horn M, Tzafriri AR. 'Seroprevalence of AAV8 Neutralizing Antibodies in Domestic Swine: Implications for Preclinical Gene Therapy Studies.’ Presented at ASGCT 2026.
Introduction: Adeno-associated virus serotype 8 (AAV8) is widely used in gene therapy for its superior hepatic tropism and efficient muscle transduction. Pre-existing neutralizing antibodies (NAbs) can block vector transduction, reduce transgene expression, and compromise therapeutic efficacy. Human AAV8 seroprevalence ranges from 19-60% depending on geographic population and assay sensitivity, with NAb titers as low as 1:5 sufficient to impair transduction in some contexts. Domestic pigs are increasingly utilized as translational large animal models due to their anatomical organ size, physiological responses, and immunological similarities to humans. However, comprehensive AAV8 NAb data in swine populations remain limited, creating uncertainty in experimental planning. Understanding baseline seroprevalence and titer distribution is critical for appropriate study design, subject selection criteria, and accurate interpretation of vector performance and therapeutic outcomes.
Methods: Serum samples from 14 domestic pigs housed at a regulated research facility under standardized environmental conditions were systematically analyzed for AAV8 NAbs. A validated assay was employed to quantify the functional neutralizing capacity of IgG against AAV8. Briefly, a permissible cell line was incubated with diluted pig serum samples that were pre-incubated with the AAV8 reporter vector (GFP). Each serum sample was two-fold serial diluted from 1:5 to 1:2560 to determine precise titer endpoints by measuring the 50% reduction of the GFP signal for each sample. The assay cutoff for seropositivity was established at 1:5 dilution. Longitudinal blood sampling at multiple timepoints enabled monitoring of seroconversion events and temporal antibody dynamics within the cohort.
Results: Overall seroprevalence was 64.3% (9/14 animals). Among seropositive animals, NAb titers demonstrated marked heterogeneity, ranging from 1:10 to 1:1022 (102-fold range) with a median titer of 1:36. The titer distribution was non-uniform: the majority of seropositive (7/9, 77.8%) exhibited titers ≤1:231, while two animals (14.3% of total cohort) displayed exceptionally high titers of 1:881 and 1:1022. These elevated titers would typically result in exclusion from many clinical gene therapy trials. Notably, one initially seronegative animal underwent seroconversion during the study period, providing direct evidence of ongoing environmental AAV8 exposure within the housing facility. No correlation was observed between housing duration and antibody titer magnitude in this limited cohort.
Conclusion: The observed 64.3% seroprevalence combined with 102-fold titer variability demonstrates the critical necessity of implementing systematic pre-screening protocols for AAV8 NAbs before enrolling pigs in preclinical gene therapy studies. This substantial baseline seropositivity reflects natural exposure to wild- type AAV8 in swine populations and parallels findings reported in other pig strains. The presence of high- titer animals (14.3% of cohort) represents a significant confounding variable that could mask genuine treatment effects or lead to erroneous conclusions about vector efficacy if subjects are not properly screened. The documented seroconversion event underscores the dynamic nature of antibody status and suggests that single-timepoint screening may be insufficient for long-duration studies. It is important to note that NAb titters measured in vitro do not necessarily predict the degree of transduction inhibition in vivo, as the relationship between titer threshold and functional vector blockade depends on multiple factors including vector dose, route of administration, and target tissue. These findings provide strong rationale for mandatory serological screening as standard practice in porcine gene therapy research to ensure experimental reproducibility and minimize inter-subject variability. Further studies with larger sample sizes are needed to establish standardized cutoff criteria for subject selection and to better define the correlation between NAb titters and in vivo vector performance across different experimental conditions.