The Immunogenicity of AAV-Encoded HIV-1 bNAbs in Rhesus Macaques Is Unaffected by a Short Course of the Immunomodulator CTLA4Ig

Adeno-associated virus (AAV)-vectored delivery of HIV-1 broadly neutralizing antibodies (bNAbs) holds promise for achieving durable HIV-1 immunity in a practical and scalable way, yet AAV-encoded bNAbs often elicit antidrug antibody (ADA) responses that limit transgene expression. Engagement of T cell-expressed CD28 with its ligands CD80/CD86 on professional antigen-presenting cells is crucial for initiating adaptive immunity. Because the immunoglobulin-fusion protein CTLA4Ig can outcompete CD28 for binding to CD80/CD86, CTLA4Ig can inhibit T cell activation and prevent immune responses. Hence, we hypothesized that co-delivering CTLA4Ig during AAV/bNAb administration would prevent ADAs in primates. Six rhesus macaques (RMs) were treated intramuscularly with AAV-1 vectors encoding "rhesusized" (rh) versions of the bNAbs 3BNC117 (IgG1) and 10-1074 (IgG2). The experimental monkeys (<i>n</i> = 3) were dosed intravenously with 20 mg/kg of rh-CTLA4Ig on days 0, 2, 7, and 14, while the control animals (<i>n</i> = 3) did not receive any additional intervention. The experimental monkeys mounted ADAs that inhibited bNAb expression, albeit at different rates for rh-3BNC117-IgG1 (66%) and rh-10-1074-IgG2 (33%). In the control group, the incidence of ADAs leading to loss of bNAb expression was 100% for rh-3BNC117-IgG1 and 0% for rh-10-1074-IgG2. There was no significant difference between the groups in their cumulative levels of ADAs or bNAb expression measured over 20 weeks. Despite the development of ADAs against rh-3BNC117-IgG1 in five out of six animals, and in one out of six against rh-10-1074-IgG2, macaques in both groups exhibited minimal T cell responses to both bNAbs. AAV-1 capsid-specific CD4⁺ T cells trended higher in the control animals. In conclusion, a short course rh-CTLA4Ig did not significantly reduce the immunogenicity of AAV-encoded bNAbs in RMs. Although our study was not powered to detect marginal effects, robust improvements in AAV-driven expression of hypermutated HIV-1 bNAbs may require combination approaches, such as multiple co-stimulation blockers, pharmacological immunosuppression, and/or muscle-specific promoters.