AAV3B is a naturally occurring adeno-associated virus serotype that has particular tropism for human hepatocytes after systemic administration. Recombinant AAV vectors use the virus capsid to deliver therapeutic DNA into cells, and AAV3B is especially relevant to inherited metabolic disorders involving the liver. The researchers cite previous mouse work showing that an AAV3B vector carrying a functional ATP7B gene could restore physiological copper metabolism in a model of Wilson’s disease, an inherited disorder in which copper accumulates, particularly in the liver. The current study did not test gene therapy or measure clinical benefit; it instead characterized pre-existing antibody levels in people who might be considered for AAV3B-based interventions.

The biological obstacle is that many people encounter naturally occurring AAV early in life or receive maternal antibodies passively. Antibodies that neutralize the therapeutic vector can bind its capsid and interfere with attachment, cellular entry or transduction. High levels can therefore reduce or completely block delivery of the therapeutic gene. Because of this, prospective participants in AAV gene-therapy trials are commonly screened for neutralizing antibodies, and those with detectable levels may be excluded even when the antibody concentration is low enough that it might potentially be reduced. The investigators argue that a simple positive-or-negative classification can conceal clinically distinct populations and may lead to unnecessary exclusion of people with substantial medical need.

For the study, investigators tested serum using a reporter-based assay developed and validated at Genosafe in France. Human serum was serially diluted and mixed with an AAV3B vector carrying a luciferase reporter gene. HeLa cells were then exposed to the serum–vector mixtures, and reductions in reporter expression were used to estimate neutralizing-antibody titers. A screening threshold of 34.48% transduction was established at a 5% false-positive risk, while a confirmation threshold of 21.74% was used at a 1% false-positive risk. Samples below 1:5 were classified as negative, titers from 1:5 to less than 1:45 were designated intermediate, and titers at or above 1:45 were considered high.

In the first healthy cohort, which included 200 adults aged 18 to 80 from several self-identified ethnic groups, 35.5% were positive for anti-AAV3B neutralizing antibodies, with no difference detected between men and women. Positivity generally increased with age: 21% of donors younger than 30 were positive, compared with approximately 33% at ages 31–40, 36% at ages 41–50, 52% at ages 51–60 and 53% at ages 61–70. Both of the two donors older than 70 were positive, although that group was too small for a firm conclusion. Positivity was 47% among Asian and Black donors, 40% among Hispanic donors and 18% among White donors, with statistically significant differences across the reported ethnic groups. The authors cautioned that the Asian and Black subgroups were small and that the results should not be treated as definitive population estimates.

The second healthy cohort comprised 580 men aged 18 to 56 who were screened for the NAVIgATE Phase I trial of VTX-PID, an IgG-degrading enzyme. Overall, 33% were anti-AAV3B positive, a prevalence the investigators described as consistent with the first cohort’s 35.5%. The age-stratified results were not monotonic: positivity was 31.6% among participants aged 18–30, 66.5% at ages 31–40 and 74.6% at ages 41–50, before falling to 27.4% at ages 51–60. The similar overall prevalence in the two independent healthy cohorts did not mean that their age patterns matched. Most participants in the German cohort were White, and the small numbers in other ethnic categories prevented robust conclusions for those groups.

Titer distributions revealed a second important distinction among seropositive participants. In both healthy cohorts, 19% of positive individuals had intermediate titers between 1:5 and 1:45, while 14% to 16.5% were reported in the high-titer category. A finer analysis of intermediate samples in the German cohort found that 87% had titers below 1:30. The authors defined the intermediate range to identify people who might be excluded from AAV3B trials yet could potentially become eligible after antibody reduction. That threshold was based on the assumption that VTX-PID could produce an eightfold to tenfold proportional reduction in neutralizing antibodies, rather than on evidence that every treated person would receive effective gene therapy.

The Wilson’s disease cohort came from pre-screening for the GATEWAY Phase I/II trial of VTX-801, an AAV3B vector encoding a mini-ATP7B gene. The 23 adults were 18 to 62 years old and included 19 from the United States, three from Denmark and one from the United Kingdom. Nineteen patients, or 82.6%, were antibody negative, while four were positive with titers above 1:45; none had intermediate titers. The positive group included two men aged 60 and 62 and two women aged 24 and 26. The observed prevalence was therefore below 20%, but the small and geographically limited sample means the estimate should not be generalized as a definitive prevalence for all people with Wilson’s disease.

The authors linked the prevalence findings to a potential strategy for widening access to AAV3B-based gene therapy. Existing approaches to lowering antibodies include plasmapheresis and immunosuppression, whose effectiveness can be limited and which can be costly or cause adverse effects. VTX-PID is a recombinant form of IdeS, an enzyme that cleaves IgG. In the NAVIgATE Phase I study cited by the authors, a dose of 0.30 mg/kg reduced total IgG by 72%–81%, undigested IgG by 95%–98% and neutralizing antibodies by 60%–70% in participants with intermediate titers. At 0.6 mg/kg, all intermediate-titer participants reached a non-inhibitory level of 1:5 for six to 10 days, whereas at 0.30 mg/kg five of six reached that level and one reached 1:5.5. These are pharmacodynamic findings from a separate study and do not demonstrate therapeutic efficacy for Wilson’s disease.

Although the reported antibody patterns could help investigators plan trials, the researchers emphasized that neutralizing-antibody assays are not yet standardized across laboratories. Results can vary with the cell line, reporter system, multiplicity of infection, vector preparation and serum factors that disturb transduction. Different positivity thresholds used in earlier studies also limit direct comparisons, which may help explain why this study’s 33%–36% prevalence among healthy adults was lower than estimates reported in some prior AAV3 or AAV3B studies. The defensible conclusion is that anti-AAV3B antibodies affect a substantial minority of healthy adults, their concentrations vary within the seropositive population, and both variables can influence trial eligibility. Establishing whether antibody-lowering treatment can safely improve access to AAV3B gene therapy will require appropriately designed clinical studies rather than inference from prevalence data alone.

Subject of Research: Prevalence and titer distribution of anti-AAV3B neutralizing antibodies in healthy adults and adults with Wilson’s disease

Article Title: Prevalence of adeno-associated virus type 3B neutralizing antibodies in cohorts of healthy donors and Wilson’s disease adults

Article References: Bouquet, C., Tamarit, B., Aseguinolaza, G. G., Ferrer, V., Valero, S., Douar, A., Brossel, A., Fuhr, R., & Combal, J.-P. (2026). Prevalence of adeno-associated virus type 3B neutralizing antibodies in cohorts of healthy donors and Wilson’s disease adults. Gene Therapy. https://doi.org/10.1038/s41434-026-00651-z

Image Credits: AI Generated

DOI: 10.1038/s41434-026-00651-z

Keywords: AAV3B, gene therapy, neutralizing antibodies, Wilson’s disease, seroprevalence, liver-directed therapy, patient eligibility, IgG reduction, AAV vectors, hepatocytes, Prevalence, adeno-associated