Single-Chain Variable Fragments of Broadly Neutralizing Antibodies Prevent HIV Cell-Cell Transmission

Broadly neutralizing antibodies (bNAbs) are able to prevent HIV infection following passive administration. Single-chain variable fragments (scFv) may have advantages over IgG as their smaller size permits improved diffusion into mucosal tissues. We have previously shown that scFv of bNAbs retain significant breadth and potency against cell-free viral transmission in a TZM-bl assay. However, scFv have not been tested for their ability to block cell-cell transmission, a model in which full-sized bNAbs lose potency. We tested four scFv (CAP256.25, PGT121, 3BNC117, and 10E8v4) compared to IgG, in free-virus and cell-cell neutralization assays in A3.01 cells, against a panel of seven heterologous viruses. We show that free-virus neutralization titers in the TZM-bl and A3.01 assays were not significantly different and confirm that scFv show a 1- to 32-fold reduction in activity in the cell-free model, compared to IgG. However, whereas IgG shows 3.4- to 19-fold geometric mean potency loss in cell-cell neutralization compared to free-virus transmission, scFv had more comparable activity in the two assays, with only a 1.3- to 2.3-fold reduction. Geometric mean 50% inhibitory concentration (IC₅₀) of scFv for cell-cell transmission ranged from 0.65 μg/mL (10E8v4) to 2.3 μg/mL (3BNC117), with IgG and scFv neutralization showing similar potency against cell-associated transmission. Therefore, despite the reduced activity of scFv in cell-free assays, their retention of activity in the cell-cell format may make scFv useful for the prevention of both modes of transmission in HIV prevention studies. IMPORTANCE Broadly neutralizing antibodies (bNAbs) are a major focus for passive immunization against HIV, with the recently concluded HVTN Antibody Mediated Protection trial providing proof of concept. Most studies focus on cell-free HIV; however, cell-associated virus may play a significant role in HIV infection, pathogenesis, and latency. Single-chain variable fragments (scFv) of antibodies may have increased tissue penetration and reduced immunogenicity. We previously demonstrated that scFv of four HIV-directed bNAbs (CAP256.25, PGT121, 3BNC117, and 10E8v4) retain significant potency and breadth against cell-free HIV. As some bNAbs have been shown to lose potency against cell-associated virus, we investigated the ability of bNAb scFv to neutralize this mode of transmission. We demonstrate that unlike IgG, scFv of bNAbs are able to neutralize cell-free and cell-associated virus with similar potency. These scFv, which show functional activity in the therapeutic range, may therefore be suitable for further development as passive immunity for HIV prevention.