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)

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). 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 Solanesol lipid rafts of the plasma membrane, efficiently neutralizes HIV-1 contamination 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. IMPORTANCE Lipid rafts are specialized dynamic microdomains Solanesol 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 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. INTRODUCTION Llamas 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 chain-only antibodies. Among these antibodies, JM2 binds the CD4-binding site (CD4BS) of gp120 and neutralizes human immunodeficiency virus type 1 (HIV-1) strains from subtypes B, C, and G, and JM4 binds gp120 and neutralizes HIV-1 strains from subtypes A, B, C, A/E, and G in a CD4-dependent manner (2). A recent crystal structure of JM4 in the complex of HIV-1 Yu2 gp120 core and a CD4 mimic shows that JM4 binds to an epitope spanning the gp120 bridge sheet, V3 loop, 19 strand, the CD4-binding loop, and the glycan at residue Asn386 (3). The JM4 epitope overlaps the b12 epitope in the CD4BS and the 17b, 48d, X5, and 412d epitopes in the coreceptor-binding site (CRBS) of gp120 (3). Thus, consistent with what was found with binding and mutagenesis analyses (2), JM4 targets a hybrid epitope on gp120 that combines elements from both the CD4-binding and coreceptor-binding sites. HIV-1 infects cells by both cell-free and cell-cell mechanisms. Viral transmission from infected to uninfected cells occurs via formation of virological and infectious synapses, nanotubes, and filopodia (4, 5). The formation of such structures allows the coordination of viral assembly with viral entry at sites of cell-cell contacts (6). As a result, HIV-1 contamination of T cells by cell-cell transmission has been found to be 100- to 1 1,000-fold more efficient for spreading virus than cell-free transmission (7, 8). While the relative impact of cell-free and cell-cell transmission remains to be defined, in a bone marrow-liver-thymus (BLT) humanized mouse model, HIV-1-infected T cells in lymph nodes were found to be mobile and to form virological synapses and syncytia. Of note, a sphingosine 1-phosphate receptor 1 (S1PR1) antagonist, FTY720, blocks the egress of migratory T cells from the lymph nodes into efferent lymph vessels, thereby interrupting T cell recirculation. When used Solanesol at the onset of HIV-1 contamination, it limited HIV-1 dissemination and reduced plasma viremia (9), indicating that the cell-cell transmission of HIV-1 could be important in the establishment of systemic HIV-1 contamination. Neutralizing antibodies and entry inhibitors effectively block cell-free HIV-1. But with few exceptions, Rabbit polyclonal to ZW10.ZW10 is the human homolog of the Drosophila melanogaster Zw10 protein and is involved inproper chromosome segregation and kinetochore function during cell division. An essentialcomponent of the mitotic checkpoint, ZW10 binds to centromeres during prophase and anaphaseand to kinetochrore microtubules during metaphase, thereby preventing the cell from prematurelyexiting mitosis. ZW10 localization varies throughout the cell cycle, beginning in the cytoplasmduring interphase, then moving to the kinetochore and spindle midzone during metaphase and lateanaphase, respectively. A widely expressed protein, ZW10 is also involved in membrane traffickingbetween the golgi and the endoplasmic reticulum (ER) via interaction with the SNARE complex.Both overexpression and silencing of ZW10 disrupts the ER-golgi transport system, as well as themorphology of the ER-golgi intermediate compartment. This suggests that ZW10 plays a criticalrole in proper inter-compartmental protein transport they are much less capable of blocking cell-cell viral transmission (7, 8, 10,C14). In T cell-T cell coculture, neutralization was exhibited only when virus-infected donor T cells were Solanesol pretreated with antibodies before being added to target T cells (7, 8, 10,C14). In dendritic cell (DC)-CD4 T cell cocultures, due to variations in assay systems used by different laboratories, the results were quite variable, sometimes even controversial (15,C19). For example, Su et al. showed that both anti-gp120 and anti-gp41 antibodies block the and 37C for 2 h to facilitate transduction. After overnight incubation at 37C, 500.