The Glycosylphosphatidylinositol-Anchored Variable Region of Llama Heavy Chain-Only Antibody JM4 Efficiently Blocks both Cell-Free and T Cell-T Cell Transmission of Human Immunodeficiency Virus Type 1

Abstract

The variable regions (VHHs) of two heavy chain-only antibodies, JM2 and JM4, from llamas that have been immunized with a trimeric gp140 bound to a CD4 mimic have been recently isolated (here referred to as VHH JM2 and VHH JM4, respectively). JM2 binds the CD4-binding site of gp120 and neutralizes HIV-1 strains from subtypes B, C, and G. JM4 binds gp120 and neutralizes HIV-1 strains from subtypes A, B, C, A/E, and G in a CD4-dependent manner. In the present study, we constructed glycosylphosphatidylinositol (GPI)-anchored VHH JM2 and JM4 along with an E4 control and transduced them into human CD4؉ cell lines and primary CD4 T cells. We report that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. Expression of GPI-VHH JM4, but not GPI-VHH E4 and JM2, on the surface of transduced TZM.bl cells potently neutralizes multiple subtypes of HIV-1 isolates, including tier 2 or 3 strains, transmitted founders, quasispecies, and soluble single domain antibody (sdAb) JM4-resistant viruses. Moreover, transduction of CEMss-CCR5 cells with GPI-VHH JM4, but not with GPI-VHH E4, confers resistance to both cell-free and T cell-T cell transmission of HIV-1 and HIV-1 envelope-mediated fusion. Finally, GPI-VHH JM4-transduced human primary CD4 T cells efficiently resist both cell-free and T cell-T cell transmission of HIV-1. Thus, we conclude that VHH JM4, when targeted to the lipid rafts of the plasma membrane, efficiently neutralizes HIV-1 infection via both cell-free and T cell-T cell transmission. Our findings should have important implications for GPI-anchored antibody-based therapy against HIV-1. IMPORTANCELipid rafts are specialized dynamic microdomains of the plasma membrane and have been shown to be gateways for HIV-1 budding as well as entry into T cells and macrophages. In nature, many glycosylphosphatidylinositol (GPI)-anchored proteins localize in the lipid rafts. In the present study, we developed GPI-anchored variable regions (VHHs) of two heavy chain-only antibodies, JM2 and JM4, from immunized llamas. We show that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. GPI-VHH JM4, but not GPI-VHH JM2, in transduced CD4؉ cell lines and human primary CD4 T cells not only efficiently blocks diverse HIV-1 strains, including tier 2 or 3 strains, transmitted founders, quasispecies, and soluble sdAb JM4-resistant strains, but also efficiently interferes T cell-T cell transmissions of HIV-1 and HIV-1 envelope-mediated fusion. Our findings should have important implications in GPI-anchored antibody-based therapy against HIV-1. L lamas naturally produce heavy chain-only antibodies. The variable regions (VHHs) of these heavy chain-only antibodies exhibit antigen-specific binding affinity comparable to that of conventional immunoglobulins (1). Previously, using trimeric gp140 bound to a CD4 mimic as immunogens in llamas, we isolated a panel of broadly neutralizing VHHs of heavy cha...

Glycosyl-Phosphatidylinositol-Anchored Anti-HIV Env Single-Chain Variable Fragments Interfere with HIV-1 Env Processing and Viral Infectivity

“…Our past studies of anti-HIV Env GPI-anchor antibody derivatives have shown increased potency and breadth of neutralization of HIV-1 compared to levels of their secreted counterparts (28)(29)(30)(31)33). In those studies, we demonstrated that anti-HIV Env GPI-anchored antibody derivatives severely restrict viral replication at the level of entry.…”

“…It is known that CD4, the receptor for HIV-1 entry (25,26), and the HIV Env protein colocalize with polarized raft markers GM1 and CD59 but not with transferrin receptor, which is excluded from lipid rafts (27). Previously, we showed that binding regions of anti-HIV Env monoclonal antibodies, such as single-chain Fv (scFv) (28), the heavy-chain complementarity-determining region 3 (HCDR3) of PG16, which binds a linear epitope on the HIV-1 Env (29), llama single-chain (30) antibodies, or even the C34 peptide of the HIV Env gp41 protein (31), can be targeted to lipid rafts by genetically linking them to a glycosyl-phosphatidylinositol (GPI) attachment signal from decay-accelerating factor (DAF) (32). Interestingly, when these molecules are targeted to the lipid raft using a GPI anchor, they exhibited potent neutralizing activity, blocking entry of different HIV-1 subtypes and variants in both cell-free infection and cell-cell transmission assays (28)(29)(30)(31)33).…”

Single-Domain Antibodies As Therapeutics against Human Viral Diseases

“…Further, Liu et al developed GPI-anchored variable regions by genetically linking sdAbs with a glycosylphosphatidylinositol (GPI) attachment signal, which targeted lipid rafts of plasma membrane. Transduction of human CD4 + cell lines and primary CD4 T cells with GPI-VHH JM4 conferred broad and potent neutralization of HIV-1 and efficiently interfered with cell-cell transmission of HIV-1 and HIV-1 envelope-mediated fusion ( 76 ). One extremely potent and broad HIV-1-neutralizing sdAb from an immunized llama, J3, targeted the CD4-binding site and neutralized 96% of all strains tested ( 11 ).…”

Tandem bispecific neutralizing antibody eliminates HIV-1 infection in humanized mice

“…In addition, it is possible that the binding of BiIA-SG Hu5A8 domains to CD4 increases the local concentration of PGT128 near the cell membrane for enhanced HIV-1 neutralization. This mechanism is possible because antibody membrane anchoring can increase neutralizing activities of both bnAbs and nonneutralizing anti-HIV antibodies (55,56). Future structural analysis of BiIA-SG is necessary to answer its mode of action.…”