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

Abstract

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 4 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 show 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 IC 50 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 AMP (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-VRC26.25, 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.

Ultrasensitive quantification of HIV-1 cell-to-cell transmission in primary human CD4 + T cells measures viral sensitivity to broadly neutralizing antibodies

“…This process is relatively resistant in vitro to most bnAbs and particularly resistant to VRC01 neutralization ( 6 – 8 , 38 ). Resistance may be related, at least partially, to limited penetration of antibodies to the VS ( 39 ). Thus, we are now in the process of obtaining resistant HIV-1 strains from immunotherapy clinical trials to study their ability to transmit between primary CD4 + T cells and to investigate the relationship between HIV-1 resistance to bnAbs and cell-cell transmission efficiency ex vivo .…”

Viral and Cellular Factors Contributing to the Hematogenous Dissemination of Human Cytomegalovirus via Polymorphonuclear Leukocytes

“…Single chain variable fragment (scFv) antibodies may have the advantage over conventional neutralizing antibodies of being able to achieve viral transmission between PMN and endothelial cell. Such antibodies have recently been increasingly tested against various viruses such as hepatitis C virus or measles virus [ 68 , 69 ] and there are reports that they can inhibit the cell-associated spread of HIV-1 in contrast to conventional neutralizing antibodies [ 70 ]. scFv have also been studied in the context of HCMV, for example against subunits of gB or the pentamer [ 71 , 72 ], and their use may thus also be promising against PMN-mediated transmission.…”

Relationship of human cytomegalovirus‐infected endothelial cells and circulating leukocytes in the pathogenesis of disseminated human cytomegalovirus infection: A narrative review

“…This approach has been already used against different viruses. [92][93][94] In addition, scFv have been used against subunits of pentamer complex, 95 thus promising to be useful in the control of PMNL-mediated HCMV transmission. 96 By using: i) immortalised human endothelial cells 97 and human foetal fibroblasts; ii) PMNL derived from HCMV-seronegative blood donors; iii) the Merlin HCMV reference strain, 98 as a parental BAC clone comprising the entire wild-type HCMV genome; iv) four KO genes [UL128 (pentamer complex), UL74 (gO), UL83 (pp65), and UL82 (pp71)], which were generated by introduction into each gene of two stop codons, according to markerless mutagenesis, 99 the following results were obtained 90 in both directions of PMNL uptake and transmission.…”