A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield

The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man(9) at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificity. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.     

HIV-infected cells are killed by rCD4-ricin A chain

The gp120 envelope glycoprotein of the human immunodeficiency virus (HIV), which is expressed on the surface of many HIV-infected cells, binds to the cell surface molecule CD4. Soluble derivatives of recombinant CD4 (rCD4) that bind gp120 with high affinity are attractive vehicles for targeting a cytotoxic reagent to HIV-infected cells. Soluble rCD4 was conjugated to the active subunit of the toxin ricin. This conjugate killed HIV-infected H9 cells but was 1/1000 as toxic to uninfected H9 cells (which do not express gp120) and was not toxic to Daudi cells (which express major histocompatibility class II antigens, the putative natural ligand for cell surface CD4). Specific killing of infected cells can be blocked by rgp120, rCD4, or a monoclonal antibody to the gp120 binding site on CD4.     

Human CD4 binds immunoglobulins

T cell glycoprotein CD4 binds to class II major histocompatibility molecules and to the human immunodeficiency virus (HIV) envelope protein gp120. Recombinant CD4 (rCD4) bound to polyclonal immunoglobulin (Ig) and 39 of 50 (78%) human myeloma proteins. This binding depended on the Fab and not the Fc portion of Ig and was independent of the light chain. Soluble rCD4, HIV gp120, and sulfated dextrans inhibited the CD4-Ig interaction. With the use of a panel of synthetic peptides, the region critical for binding to Ig was localized to amino acids 21 to 38 of the first extracellular domain of CD4. CD4-bound antibody (Ab) complexed with antigen approximately 100 times better than Ab alone. This activity may contribute to the Ab-mediated enhancement of cellular HIV interaction that appears to depend on a trimolecular complex of HIV, antibodies to gp120, and CD4.     

Involvement of a leukocyte adhesion receptor (LFA-1) in HIV-induced syncytium formation

Cell fusion (syncytium formation) is a major cytopathic effect of infection by human immunodeficiency virus (HIV) and may also represent an important mechanism of CD4+ T-cell depletion in individuals infected with HIV. Syncytium formation requires the interaction of CD4 on the surface of uninfected cells with HIV envelope glycoprotein gp120 expressed on HIV-infected cells. However, several observations suggest that molecules other than CD4 play a role in HIV-induced cell fusion. The leukocyte adhesion receptor LFA-1 is involved in a broad range of leukocyte interactions mediated by diverse receptor-ligand systems including CD4-class II major histocompatibility complex (MHC) molecules. Possible mimicry of class II MHC molecules by gp120 in its interaction with CD4 prompted an examination of the role of LFA-1 in HIV-induced cell fusion. A monoclonal antibody against LFA-1 completely inhibited HIV-induced syncytium formation. The antibody did not block binding of gp120 to CD4. This demonstrates that a molecule other than CD4 is also involved in cell fusion mediated by HIV.     

Increasing the potency and breadth of an HIV antibody by using structure-based rational design

Antibodies against the CD4 binding site (CD4bs) on the HIV-1 spike protein gp120 can show exceptional potency and breadth. We determined structures of NIH45-46, a more potent clonal variant of VRC01, alone and bound to gp120. Comparisons with VRC01-gp120 revealed that a four-residue insertion in heavy chain complementarity-determining region 3 (CDRH3) contributed to increased interaction between NIH45-46 and the gp120 inner domain, which correlated with enhanced neutralization. We used structure-based design to create NIH45-46(G54W), a single substitution in CDRH2 that increases contact with the gp120 bridging sheet and improves breadth and potency, critical properties for potential clinical use, by an order of magnitude. Together with the NIH45-46-gp120 structure, these results indicate that gp120 inner domain and bridging sheet residues should be included in immunogens to elicit CD4bs antibodies.     

Type-restricted neutralization of molecular clones of human immunodeficiency virus

In a study of the immunologic significance of the genetic diversity present within single isolates of human immunodeficiency virus type 1 (HIV-1), the neutralization of viruses derived from molecular clones of the HIV-1 strain HTLV-IIIB by an extensive panel of sera was compared. Sera from HIV-1-infected patients and from goats immunized with polyacrylamide gel-purified HIV-1 envelope glycoprotein (gp120), native gp120, or gp120-derived recombinant peptides, showed marked heterogeneity in neutralizing activity against these closely related viruses. The change of a single amino acid residue in gp120 may account for such "clonal restriction" of neutralizing activity.     

Structures of the CCR5 N terminus and of a tyrosine-sulfated antibody with HIV-1 gp120 and CD4

The CCR5 co-receptor binds to the HIV-1 gp120 envelope glycoprotein and facilitates HIV-1 entry into cells. Its N terminus is tyrosine-sulfated, as are many antibodies that react with the co-receptor binding site on gp120. We applied nuclear magnetic resonance and crystallographic techniques to analyze the structure of the CCR5 N terminus and that of the tyrosine-sulfated antibody 412d in complex with gp120 and CD4. The conformations of tyrosine-sulfated regions of CCR5 (alpha-helix) and 412d (extended loop) are surprisingly different. Nonetheless, a critical sulfotyrosine on CCR5 and on 412d induces similar structural rearrangements in gp120. These results now provide a framework for understanding HIV-1 interactions with the CCR5 N terminus during viral entry and define a conserved site on gp120, whose recognition of sulfotyrosine engenders posttranslational mimicry by the immune system.     

Structure of a V3-containing HIV-1 gp120 core

The third variable region (V3) of the HIV-1 gp120 envelope glycoprotein is immunodominant and contains features essential for coreceptor binding. We determined the structure of V3 in the context of an HIV-1 gp120 core complexed to the CD4 receptor and to the X5 antibody at 3.5 angstrom resolution. Binding of gp120 to cell-surface CD4 would position V3 so that its coreceptor-binding tip protrudes 30 angstroms from the core toward the target cell membrane. The extended nature and antibody accessibility of V3 explain its immunodominance. Together, the results provide a structural rationale for the role of V3 in HIV entry and neutralization.     

Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design

We present the crystal structure at 2.7 angstrom resolution of the human antibody IgG1 b12. Antibody b12 recognizes the CD4-binding site of human immunodeficiency virus-1 (HIV-1) gp120 and is one of only two known antibodies against gp120 capable of broad and potent neutralization of primary HIV-1 isolates. A key feature of the antibody-combining site is the protruding, finger-like long CDR H3 that can penetrate the recessed CD4-binding site of gp120. A docking model of b12 and gp120 reveals severe structural constraints that explain the extraordinary challenge in eliciting effective neutralizing antibodies similar to b12. The structure, together with mutagenesis studies, provides a rationale for the extensive cross-reactivity of b12 and a valuable framework for the design of HIV-1 vaccines capable of eliciting b12-like activity.     

Computation-guided backbone grafting of a discontinuous motif onto a protein scaffold

The manipulation of protein backbone structure to control interaction and function is a challenge for protein engineering. We integrated computational design with experimental selection for grafting the backbone and side chains of a two-segment HIV gp120 epitope, targeted by the cross-neutralizing antibody b12, onto an unrelated scaffold protein. The final scaffolds bound b12 with high specificity and with affinity similar to that of gp120, and crystallographic analysis of a scaffold bound to b12 revealed high structural mimicry of the gp120-b12 complex structure. The method can be generalized to design other functional proteins through backbone grafting.     

HIV-1 coat protein neurotoxicity prevented by calcium channel antagonists

Coat protein gp120 from the human immunodeficiency virus type-1 (HIV-1) increased intracellular free calcium and injured rodent retinal ganglion cells and hippocampal neurons in culture. Highly purified recombinant gp120 envelope protein produced these effects in a dose-dependent fashion at picomolar concentrations. Immunoprecipitation with antibody to gp120, but not with control immunoglobulin-containing serum, depleted solutions of the viral envelope protein and also prevented both the rise in intracellular calcium and neuronal toxicity. The gp120-induced increase in intracellular calcium was abrogated by transiently lowering extracellular calcium or by adding the dihydropyridine calcium channel antagonist nimodipine (100 nM). Calcium channel antagonists also prevented gp120-induced neuronal injury. In addition, intracellular stores appeared to contribute substantially to the increase in calcium elicited by gp120. Since increases in intracellular calcium have been associated with neurotoxicity, it is possible that an injurious effect of gp120 on neurons might be related to this mechanism and that treatment with calcium channel antagonists may prove useful in mitigating HIV-1-related neuronal injury.     

Characterization of gp41 as the transmembrane protein coded by the HTLV-III/LAV envelope gene

Radiolabeled amino acid sequencing was used to characterize gp41, an antigen of HTLV-III/LAV, the virus believed to be the etiological agent of the acquired immune deficiency syndrome. This antigen is the one most commonly detected in immunoblot assays by sera of patients with AIDS or AIDS-related complex (ARC) and other individuals infected with HTLV-III/LAV. A mouse monoclonal antibody that was reactive with gp41 precipitated a 160-kilodalton protein (gp160) in addition to gp41, but did not precipitate a 120-kilodalton protein (gp120) from extracts of metabolically labeled cells producing HTLV-III. Extracts of infected cells that had been labeled with tritiated leucine or isoleucine were immunoprecipitated with the monoclonal antibody. The immunoprecipitates were fractionated by polyacrylamide gel electrophoresis and the p41 was eluted from the gel bands and subjected to amino-terminal radiolabeled amino acid sequencing by the semiautomated Edman degradation. Leucine residues occurred in cycles 7, 9, 12, 26, 33, and 34 among 40 cycles and isoleucine occurred in cycle 4 among 24 cycles analyzed. Comparison of the data with the deduced amino acid sequence of the env gene product of HTLV-III precisely placed gp41 in the COOH-terminal region of the env gene product. Gp160 is thus the primary env gene product and it is processed into gp120 and gp41.     

Synergism between HIV gp120 and gp120-specific antibody in blocking human T cell activation

The human immunodeficiency virus (HIV) binds to CD4-positive cells through interaction of its envelope glycoprotein (gp120) with the CD4 molecule. CD4 is a prominent immunoregulatory molecule, and chronic exposure to antibody against CD4 (anti-CD4) has been shown to cause immunodeficiency in mice. T cell-dependent in vitro immune responses can also be inhibited by anti-CD4. Experimental findings reported here indicate that CD4-bound gp120 attracts gp120-specific antibodies derived from the blood of HIV-seropositive individuals to form a trimolecular complex with itself and CD4. Thus targeted to CD4, the gp120-specific antibody functions as an antibody to CD4; it cross-links and modulates the CD4 molecules and suppresses the activation of T cells as measured by mobilization of intracellular calcium (Ca2i+). The synergism between gp120 and anti-gp120 in blocking T cell activation occurs at low concentrations of both components. Neither gp120 nor anti-gp120 inhibits T cell activation by itself in the concentrations tested.     

The MHC-binding and gp120-binding functions of CD4 are separable

CD4 is a cell surface glycoprotein that is thought to interact with nonpolymorphic determinants of class II major histocompatibility (MHC) molecules. CD4 is also the receptor for the human immunodeficiency virus (HIV), binding with high affinity to the HIV-1 envelope glycoprotein, gp120. Homolog-scanning mutagenesis was used to identify CD4 regions that are important in class II MHC binding and to determine whether the gp120 and class II MHC binding sites of CD4 are related. Class II MHC binding was abolished by mutations in each of the first three immunoglobulin-like domains of CD4. The gp120 binding could be abolished without affecting class II MHC binding and vice versa, although at least one mutation examined reduced both functions significantly. These findings indicate that, while there may be overlap between the gp120 and class II MHC binding sites of CD4, these sites are distinct and can be separated. Thus it should be possible to design CD4 analogs that can block HIV infectivity but intrinsically lack the ability to affect the normal immune response by binding to class II MHC molecules.     

Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists

Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation, and development. The G protein-coupled chemokine receptor CXCR4 is specifically implicated in cancer metastasis and HIV-1 infection. Here we report five independent crystal structures of CXCR4 bound to an antagonist small molecule IT1t and a cyclic peptide CVX15 at 2.5 to 3.2 angstrom resolution. All structures reveal a consistent homodimer with an interface including helices V and VI that may be involved in regulating signaling. The location and shape of the ligand-binding sites differ from other G protein-coupled receptors and are closer to the extracellular surface. These structures provide new clues about the interactions between CXCR4 and its natural ligand CXCL12, and with the HIV-1 glycoprotein gp120.     

Prevention of HIV-1 infection and preservation of CD4 function by the binding of CPFs to gp120

Infection by human immunodeficiency virus type-1 (HIV-1) is initiated when its envelope protein, gp120, binds to its receptor, the cell surface glycoprotein CD4. Small molecules, termed N-carbomethoxycarbonyl-prolyl-phenylalanyl benzyl esters (CPFs), blocked this binding. CPFs interacted with gp120 and did not interfere with the binding of CD4 to class II major histocompatibility complex molecules. One CPF isomer, CPF(DD), preserved CD4-dependent T cell function while inhibiting HIV-1 infection of H9 tumor cells and human T cells. Although the production of viral proteins in infected T cells is unaltered by CPF(DD), this compound prevents the spread of infection in an in vitro model system.     

Beta 1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis

Neoplastic transformation has been associated with a variety of structural changes in cell surface carbohydrates, most notably increased sialylation and beta 1-6-linked branching of complex-type asparagine (Asn)-linked oligosaccharides (that is, -GlcNAc beta 1-6Man alpha 1-6Man beta 1-). However, little is known about the relevant glycoproteins or how these transformation-related changes in oligosaccharide biosynthesis may affect the malignant phenotype. Here it is reported that a cell surface glycoprotein, gp 130, is a major target of increased beta 1-6-linked branching and that the expression of these oligosaccharide structures is directly related to the metastatic potential of the cells. Glycosylation mutants of a metastatic tumor cell line were selected that are deficient in both beta 1-6 GlcNAc transferase V activity and metastatic potential in situ. Moreover, induction of increased beta 1-6 branching in clones of a nonmetastatic murine mammary carcinoma correlated strongly with acquisition of metastatic potential. The results indicate that increased beta 1-6-linked branching of complex-type oligosaccharides on gp 130 may be an important feature of tumor progression related to increased metastatic potential.     

Dissociation of gp120 from HIV-1 virions induced by soluble CD4

The CD4 antigen is the high affinity cellular receptor for the human immunodeficiency virus type-1 (HIV-1). Binding of recombinant soluble CD4 (sCD4) or the purified V1 domain of sCD4 to the surface glycoprotein gp120 on virions resulted in rapid dissociation of gp120 from its complex with the transmembrane glycoprotein gp41. This may represent the initial stage in virus-cell and cell-cell fusion. Shedding of gp120 from virions induced by sCD4 may also contribute to the mechanism by which these soluble receptor molecules neutralize HIV-1.     

Direct interaction of a ligand for the erbB2 oncogene product with the EGF receptor and p185erbB2

The erbB2 oncogene encodes a 185-kilodalton transmembrane protein whose sequence is similar to the epidermal growth factor receptor (EGFR). A 30-kilodalton factor (gp30) secreted from MDA-MB-231 human breast cancer cells was shown to be a ligand for p185erbB2. An antibody to EGFR abolished the tyrosine phosphorylation induced by EGF and transforming growth factor-alpha (TGF-alpha) but only partially blocked that produced by gp30 in SK-BR-3 breast cancer cells. In two cell lines that overexpress erbB2 but do not expresss EGFR (MDA-MB-453 breast cancer cells and a Chinese hamster ovary cell line that had been transfected with erbB2), phosphorylation of p185erbB2 was induced only by gp30. The gp30 specifically inhibited the growth of cells that overexpressed p185erbB2. An antibody to EGFR had no effect on the inhibition of SK-BR-3 cell colony formation obtained with gp30. Thus, it appeared that gp30 interacted directly with the EGFR and erbB2. Direct binding of gp30 to p185erbB2 was confirmed by binding competition experiments, where gp30 was found to displace the p185erbB2 binding of a specific antibody to p185erbB2. The evidence described here suggests that gp30 is a ligand for p185erbB2.     

Second conserved domain of gp120 is important for HIV infectivity and antibody neutralization

Rabbit antisera were raised against three overlapping synthetic peptides with sequence homology to the second conserved domain of the external envelope glycoprotein (gp120) of the human immunodeficiency virus (HIV). All of the antisera immunoprecipitated the envelope glycoprotein. In particular, an antiserum directed against amino acids 254 to 274 of env was efficient in neutralizing three different isolates of HIV in vitro, without affecting the binding of the virus to CD4-positive cells. Therefore, this conserved region of gp120 appears to be critical in a postbinding event during virus penetration and may represent a target for antibody neutralization of HIV. These findings may be applicable in the design of a vaccine for the acquired immunodeficiency syndrome.