Engineered Probiotics as a Strategy to Induce Oral Tolerance to AAV-Delivered Broadly Neutralizing Antibodies

About This Grant

Project Summary/Abstract Given the vast assortment of human monoclonal antibodies with potent neutralizing activity, one can envision long-term delivery of a combination of antibodies to achieve sterilizing immunity against most circulating HIV strains. Adeno-associated virus (AAV) vectors are well suited for such sustained antibody delivery. Our group has demonstrated high circulating levels of broadly neutralizing antibodies (bNAbs) following a single intramuscular AAV injection in rhesus macaques, as well as long-term virologic suppression for more than nine years after AAV-mediated delivery of 10-1074 and 3BNC117. These studies have not only demonstrated that long-term expression of AAV-delivered bNAbs can lead to a functional cure but can also eliminate the viral reservoir. In three macaques, intact proviral DNA levels were at or below the limit of detection five years following AAV delivery. Despite these successes, host immune responses against the delivered antibodies have frequently limited long-term transgene expression. To address the crippling effects of anti-drug antibody (ADA) responses, this proposal seeks to induce antigen-specific oral tolerance using engineered probiotics. We aim to determine whether pretreatment with these probiotics can prevent, and potentially reverse, immune responses to AAV-delivered bNAbs, thereby enabling sustained antibody expression in both uninfected and HIV-infected hosts. To test this concept, we will develop and validate designer probiotic strains that can be delivered orally, colonize the gut, and secrete functional bNAb immunoadhesins. Bifidobacterium longum will be engineered to secrete immunoadhesins derived from the bNAbs 3BNC117 and 10-1074, while Lactococcus lactis will be engineered to secrete the cytokine IL-10 to enhance tolerogenic signaling and support oral tolerance induction. Rhesus macaques will be pretreated with weekly oral probiotics prior to intramuscular AAV9 delivery. This approach will be evaluated longitudinally to assess ADA formation, regulatory T cell induction, microbial persistence, and the durability of AAV-mediated bNAb expression. This approach will be directly compared to oral delivery of purified bNAbs, as classically used in oral tolerance studies. In addition, we will assess whether sustained probiotic exposure can reprogram established humoral immunity and restore bNAb expression following AAV delivery. Finally, probiotic pretreatment will be evaluated in SHIV chronically infected rhesus macaques receiving antiretroviral therapy. This model is more clinically relevant and will allow us to determine whether oral tolerance can be established in the setting of chronic immune inflammation and dysregulation. If successful, this project will establish a microbiome-based, non-immunosuppressive strategy for overcoming the last critical barrier to AAV-mediated bNAb delivery. These studies could enable the development of long-term, ART-free HIV treatment and prevention and would provide a broadly applicable approach for improving the durability of AAV-delivered biologics across many diseases.