Lessons Learned from Monitoring Adeno-Associated Virus-9 Neutralizing Antibody (AAV9-NAb) Seroconversion in a Cohort of Cynomolgus Macaques (Macaca fascicularis)
The purpose of this study was to track seroconversion in a cohort of 12, pair-housed, macaques that were previously screened negative for adeno-associated virus-9 neutralizing antibody (AAV9-NAb). Over a 6-month period, specific biosecurity strategies were implemented with the intention of understanding if following defined protocols could play a role in preservation of AAV9-NAb negative status. AAV9-NAb-negative animals were selected for shipment to the facility approximately 2 months after the initial screening. After arrival, animals were paired and housed in a single room with a dedicated housing corridor, cage wash, and equipment. They were then screened for AAV9-NAb status monthly by 2 different labs to confirm results and ascertain potential for variation of results. Upon initial screening at the facility (within one week of arrival), 2 of the 12 NHPs that were seronegative before shipment had seroconverted to AAV9-NAb positive status. The positive animals and their negative partners were moved to a different room but remained within the same isolated corridor with the same biosecurity practices. Serum that was taken on a monthly basis was also used to screen other AAVs. At the end of the 6-month period, AAV9 NAb status did not change from the time the animals were initially screened on site until the end of the study. Paired animals that were cohoused in the same cage with a positive partner did not seroconvert. Although a control group was not used to validate that biosecurity practices played a role in mitigating seroconversion, unpublished data from a facility employing less restricted biosecurity strategies suggest that the seroconversion process involves a more intricate process.
Schematic Showing the Process Involved With Screening, Procuring, Shipment, and Quarantine of Animals Before Study Start
Neutralizing and Binding Antibody Profiles in Cynomolgus Macaques. Serum from 12 cynomolgus macaques was evaluated for neutralizing (NAb) and binding antibodies (IgG and IgM) against AAV9 every month over a period of 6 months. (A) NAb titer changes plotted as a reciprocal of serum dilution. (B and C) Absorbance values at OD450 for IgG and IgM antibodies that bind to immobilized AAV9 capsids using a modified ELISA assay. The x-axes are time in months.
Neutralizing Antibody Titer Changes in Cynomolgus Macaques Against a Panel of AAV Capsids. Serum from 12 cynomolgus macaques was evaluated for neutralizing (NAb) every month over a period of 6 months against 6 different capsids ([A] AAV1, [B] AAV2, [C] AAV3b, [D] AAV5, [E] AAV6, and [F] AAV8). NAb titers are plotted as a reciprocal serum dilution. The x-axes are time in months.
Anti-AAV IgG Binding Antibody Levels in Cynomolgus Macaques. Serum from 12 cynomolgus macaques was evaluated for IgG binding antibodies over a period of 6 months against 6 different capsids ([A] AAV1, [B] AAV2, [C] AAV3b, [D] AAV5, [E] AAV6, and [F] AAV8). IgG levels are plotted as absorbance values at OD450 for antibodies that bound immobilized AAV capsids using a modified ELISA assay. The x-axes are time in months.
Anti-AAV IgM Binding Antibody Levels in Cynomolgus Macaques. Serum from 12 cynomolgus macaques was evaluated for IgG binding antibodies over a period of 6 months against 6 different capsids ([A] AAV1, [B] AAV2, [C] AAV3b, [D] AAV5, [E] AAV6, and [F] AAV8). IgM levels are plotted as absorbance values at OD450 for antibodies that bound immobilized AAV capsids using a modified ELISA assay. The x-axes are time in months.
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