Fig. 4: Performance of CSC assay in preclinical seroreversion models.

Throughout the figure, Constant refers to the CSC method, where total serum remains fixed at 10%, while Variable refers to the VSC method, where total serum concentration increases with less dilution. A, C, E Transduction curves for serum samples collected from preclinical subjects at various time points post-vaccination or post-injection. Solid curves represent results from the Constant assay, dashed curves represent results from the Variable assay. Vertical error bars indicate standard deviations from the mean. The horizontal gray dashed line at 50% transduction efficiency indicates the neutralization threshold. ND50 dilutions are marked by colored vertical dashed arrows pointing to horizontal bars that denote 95% credible intervals for ND50 estimates. B, D, F Neutralization titers (ND50 estimates) derived from the transduction curves in (A, C, and E). ND50 values are shown for both Constant (filled bars) and Variable (striped bars) assays at corresponding time points. Error bars represent 95% credible intervals. Asterisks denote significant difference (“Methods” section). For samples exhibiting minimal (ND50 < 1/4) or no neutralization, a 1/1 titer is used as a placeholder. A, B Serum samples collected from one cat two weeks and four months after vaccination with a feline CRP vaccine. Constant detected persistent neutralizing activity at four months post-vaccination, while Variable reported seroreversion earlier. C, D Serum samples collected from one cat one, two, and three years after injection with CAP-B22 capsid. Constant detected persistent seropositivity three years post-injection, whereas Variable suggested seroreversion had occurred by year three. E, F Same serum samples as in (C, D) tested against the parental capsid of CAP-B22, AAV9. Seroreversion occurred one year earlier with AAV9 compared to CAP-B22, likely due to limited cross-reactivity between capsids.