HIV-1-infected macrophages participate in virus dissemination and establishment of virus reservoirs in host tissues, but the mechanisms for virus cell-to-cell transfer to macrophages remain unknown. Here, we reveal the mechanisms for cell-to-cell transfer from infected T cells to macrophages and virus spreading between macrophages. We show that contacts between infected T lymphocytes and macrophages lead to cell fusion for fast and massive transfer of CCR5-tropic viruses to macrophages. Through the merge of viral material between T cells and macrophages, these newly formed lymphocyte/macrophage fused cells acquire the ability to fuse with neighboring non-infected macrophages. Together, these two-step envelope-dependent cell fusion processes lead to the formation of highly virus-productive multinucleated giant cells reminiscent of the infected multinucleated giant macrophages detected in HIV-1-infected patients and SIV-infected macaques. These mechanisms represent an original mode of virus transmission for viral spreading and a new model for the formation of macrophage virus reservoirs during infection. We reveal a very efficient mechanism involved in cell-to-cell transfer from infected T cells to macrophages and subsequent virus spreading between macrophages by a two-step cell fusion process. Infected T cells first establish contacts and fuse with macrophage targets. The newly formed lymphocyte/macrophage fused cells then acquire the ability to fuse with surrounding uninfected macrophages leading to the formation of infected multinucleated giant cells that can survive for a long time as evidenced in lymphoid organs and the central nervous system. This route of infection may be a major determinant for virus dissemination and the formation of macrophage virus reservoirs in host tissues during HIV-1 infection.

h Few broadly neutralizing antibodies targeting determinants of the HIV-1 surface envelope glycoprotein (gp120) involved in sequential binding to host CD4 and chemokine receptors have been characterized. While these epitopes show low diversity among various isolates, HIV-1 employs many strategies to evade humoral immune response toward these sensitive sites, including a carbohydrate shield, low accessibility to these buried cavities, and conformational masking. Using trimeric gp140, free or bound to a CD4 mimic, as immunogens in llamas, we selected a panel of broadly neutralizing single-domain antibodies (sdAbs) that bind to either the CD4 or the coreceptor binding site (CD4BS and CoRBS, respectively). When analyzed as monomers or as homo-or heteromultimers, the best sdAb candidates could not only neutralize viruses carrying subtype B envelopes, corresponding to the Env molecule used for immunization and selection, but were also efficient in neutralizing a broad panel of envelopes from subtypes A, C, G, CRF01_AE, and CRF02_AG, including tier 3 viruses. Interestingly, sdAb multimers exhibited a broader neutralizing activity spectrum than the parental sdAb monomers. The extreme stability and high recombinant production yield combined with their broad neutralization capacity make these sdAbs new potential microbicide candidates for HIV-1 transmission prevention.

The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and ␤19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor-and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy.

The targeting of HIV-1 using antibodies is of high interest as molecular tools to better understand the biology of the virus or as a first step toward the design of new inhibitors targeting critical viral intracellular proteins. Small and highly stable llama-derived single-domain antibodies can often be functionally expressed as intracellular antibodies in the cytoplasm of eukaryotic cells. Using a selection method based on the Sos Recruitment System, a cytoplasmic yeast two-hybrid approach, we have isolated single-domain antibodies able to bind HIV-1 Vpr and Capside proteins in the yeast cytoplasm. One anti-Vpr single domain antibody was able to bind the HIV-1 regulatory Vpr protein in the cytoplasm of eukaryotic cells, leading to its delocalization from the nucleus to the cytoplasm. To our knowledge, this is the first description of a functional single-domain intrabody targeting HIV-1 Vpr, isolated using an in vivo cytoplasmic selection method that alleviates some limitations of the conventional yeast two-hybrid system.

BackgroundThe HIV-1 Nef protein is essential for AIDS pathogenesis by its interaction with host cell surface receptors and signaling factors. Despite its critical role as a virulence factor Nef is not targeted by current antiviral strategies.ResultsWe have determined the crystal structure of the complex formed by a camelid single-domain antibody fragment, termed sdAb19, bound to HIV-1 Nef together with a stabilizing SH3 domain. sdAb19 forms a stoichiometric 1:1 complex with Nef and binds to a conformationally conserved surface at the C-terminus of Nef that overlaps with functionally important interaction sites involved in Nef-induced perturbations of signaling and trafficking pathways. The antibody fragment binds Nef with low nanomolar affinity, which could be attenuated to micromolar affinity range by site-directed mutagenesis of key interaction residues in sdAb19. Fusion of the SH3 domain to sdAb19, termed Neffin, leads to a significantly increased affinity for Nef and formation of a stoichiometric 2:2 Nef–Neffin complex. The 19 kDa Neffin protein inhibits all functions of Nef as CD4 and MHC-I downregulation, association with Pak2, and the increase in virus infectivity and replication.ConclusionsTogether, sdAb19 and Neffin thus represent efficient tools for the rational development of antiviral strategies against HIV-1 Nef.

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...

The isolation of antibody fragments targeting proteins implicated in cancers and other diseases remains a crucial issue on targeted therapy or diagnostic tool development (Hoogenboom HR, Henderikx P, de Haard H. Adv Drug Deliv Rev 31 (1-2):5-31, 1998). In many case, the protein of interest, or a relevant portion of this protein such as its extracellular domain is available as purified protein. In such cases, phage display on purified antigen is an easy and fast way to select antibody fragment able to efficiently bind this antigen. However, the output of phage selection can vary significantly depending on the way to immobilize the purified antigen during selection. The following protocols describe the selection of phage antibody on purified antigen adsorbed on plastic, i.e. panning, or a selection in solution, using a biotinylated antigen as well as the corresponding screening produces, and gives hints on the advantage and drawbacks of each approach.

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