CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

It has been demonstrated that CD8⁺ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4⁺ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8⁺ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8⁺ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8⁺ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8⁺ T-cell supernatants; and (iii) the CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8⁺ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8⁺ T-cell-derived HIV-suppressor factors.     

Role of the HIV type 1 glycoprotein 120 V3 loop in determining coreceptor usage.

Macrophage (M)-tropic HIV-1 isolates use the beta-chemokine receptor CCR5 as a coreceptor for entry, while T cell line-adapted (TCLA) strains use CXCR4 and dual-tropic strains can use either CCR5 or CXCR4. To investigate the viral determinants involved in choice of coreceptor, we used a fusion assay based on the infection of CD4+ HeLa cells that express one or both coreceptors with Semliki Forest virus (SFV) recombinants expressing the native HIV-1 gp160 of a primary M-tropic isolate (HIV-1BX08), a TCLA isolate (HIV-1LAI), or a dual-tropic strain (HIV-1MN). We examined whether the V3 region of these glycoproteins interacts directly with the corresponding coreceptors by assaying coreceptor-dependent cell-to-cell fusion mediated by the different recombinants in the presence of various synthetic linear peptides. Synthetic peptides corresponding to different V3 loop sequences blocked syncytium formation in a coreceptor-specific manner. Synthetic V2 peptides were also inhibitory for syncytium formation, but showed no apparent coreceptor specificity. A BX08 V3 peptide with a D320 --> R substitution retained no inhibitory capacity for BX08 Env-mediated cell-to-cell fusion, but inhibited LAI Env-mediated fusion as efficiently as the homologous LAI V3 peptide. The same mutation engineered in the BX08 env gene rendered it able to form syncytia on CD4+CXCR4+CCR5-HeLa cells and susceptible to inhibition by SDF-1alpha and MIP-1beta. Other substitutions tested (D320 --> Q/D324 --> N or S306 --> R) exhibited intermediate effects on coreceptor usage. These results underscore the importance of the V3 loop in modulating coreceptor choice and show that single amino acid modifications in V3 can dramatically modify coreceptor usage. Moreover, they provide evidence that linear V3 loop peptides can compete with intact cell surface-expressed gp120/gp41 for CCR5 or CXCR4 interaction.     

gp120 envelope glycoproteins of human immunodeficiency viruses competitively antagonize signaling by coreceptors CXCR4 and CCR5

Signal transductions by the dual-function CXCR4 and CCR5 chemokine receptors/HIV type 1 (HIV-1) coreceptors were electrophysiologically monitored in Xenopus laevis oocytes that also coexpressed the viral receptor CD4 and a G protein-coupled inward-rectifying K⁺ channel (Kir 3.1). Large Kir 3.1-dependent currents generated in response to the corresponding chemokines (SDF-1α for CXCR4 and MIP-1α; MIP-1β and RANTES for CCR5) were blocked by pertussis toxin, suggesting involvement of inhibitory guanine nucleotide-binding proteins. Prolonged exposures to chemokines caused substantial but incomplete desensitization of responses with time constants of 5–7 min and recovery time constants of 12–19 min. CXCR4 and CCR5 exhibited heterologous desensitization in this oocyte system, suggesting possible inhibition of a common downstream step in their signaling pathways. In contrast to chemokines, perfusion with monomeric or oligomeric preparations of the glycoprotein of M_r 120,000 (gp120) derived from several isolates of HIV-1 did not activate signaling by CXCR4 or CCR5 regardless of CD4 coexpression. However, adsorption of the gp120 from a T-cell-tropic virus resulted in CD4-dependent antagonism of CXCR4 response to SDF-1α, whereas gp120 from macrophage-tropic viruses caused CD4-dependent antagonism of CCR5 response to MIP-1α. These antagonisms could be partially overcome by high concentrations of chemokines and were specific for coreceptors of the corresponding HIV-1 isolates, suggesting that they resulted from direct interactions of gp120–CD4 complexes with coreceptors and that they did not involve the desensitization pathway. These results indicate that monomeric or oligomeric gp120s specifically antagonize CXCR4 and CCR5 signaling in response to chemokines, but they do not exclude the possibility that gp120s might also function as weak agonists in some cells. The gp120-mediated disruption of CXCR4 and CCR5 signaling may contribute to AIDS pathogenesis.     

CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4+ T cells

AIDS is characterized by a progressive decrease of CD4⁺ helper T lymphocytes. Destruction of these cells may involve programmed cell death, apoptosis. It has previously been reported that apoptosis can be induced even in noninfected cells by HIV-1 gp120 and anti-gp120 antibodies. HIV-1 gp120 binds to T cells via CD4 and the chemokine coreceptor CXCR4 (fusin/LESTR). Therefore, we investigated whether CD4 and CXCR4 mediate gp120-induced apoptosis. We used human peripheral blood lymphocytes, malignant T cells, and CD4/CXCR4 transfectants, and found cell death induced by both cell surface receptors, CD4 and CXCR4. The induced cell death was rapid, independent of known caspases, and lacking oligonucleosomal DNA fragmentation. In addition, the death signals were not propagated via p56^lck and G_iα. However, the cells showed chromatin condensation, morphological shrinkage, membrane inversion, and reduced mitochondrial transmembrane potential indicative of apoptosis. Significantly, apoptosis was exclusively observed in CD4⁺ but not in CD8⁺ T cells, and apoptosis triggered via CXCR4 was inhibited by stromal cell-derived factor-1, the natural CXCR4 ligand. Thus, this mechanism of apoptosis might contribute to T cell depletion in AIDS and might have major implications for therapeutic intervention.     

Primary CD8+ cells from HIV-infected individuals can suppress productive infection of macrophages independent of β-chemokines

The productive infection of human monocyte-derived macrophages (M) by HIV was suppressed by primary CD8⁺ cells from asymptomatic HIV-infected individuals. This anti-HIV response was noncytotoxic; removal of the CD8⁺ cells from the infected M leads to virus production. CD8⁺ cells inhibited HIV replication when separated from the infected M by a transwell filter insert, indicating a diffusible factor made by the CD8⁺ cells suppressed productive infection of M. Three β-chemokines, which can be secreted by activated CD8⁺ cells, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1α and MIP-1β prevented HIV replication in the M cultures. In addition, incubation of acutely infected M with a mixture of neutralizing antibodies to RANTES, MIP-1α, and MIP-1β enhanced virus replication. Nevertheless, neutralization of β-chemokines with specific antibodies did not abolish the suppression by CD8⁺ cells of HIV replication in M. Thus, even though β-chemokines decrease HIV replication in M, these cytokines are not responsible for the ability of CD8⁺ cells to inhibit HIV production in these cells.     

CD40 ligand (CD154) stimulation of macrophages to produce HIV-1-suppressive β-chemokines

β-chemokines play an important role in the development of immunologic reactions. Macrophages are major β-chemokine-producing cells during T-cell directed, delayed-type hypersensitivity reactions in tissues, and have been reported to be important producers of β-chemokines in the lymph nodes of HIV-1-infected individuals. However, the physiological signals responsible for inducing macrophages to produce β-chemokines have not been established. Two soluble T cell products, interferon-γ and granulocyte-macrophage colony stimulating factor, were added to cultured macrophages, but failed to stimulate the production of macrophage inflammatory protein-1α and -1β; regulated upon activation, normal T cell expressed and secreted (RANTES); or monocyte chemoattractant protein-1. Instead, direct cell–cell contact between macrophages and cells engineered to express CD40L (also known as CD154) resulted in the production of large amounts of macrophage inflammatory protein-1α and -1β, and RANTES (all ligands for CCR5), and monocyte chemoattractant protein-1 (a ligand for CCR2). Supernatants from CD40L-stimulated macrophages protected CD4⁺ T cells from infection by a nonsyncytium-inducing strain of HIV-1 (which uses CCR5 as a coreceptor). These results have implications for granulomatous diseases, and conditions such as atherosclerosis and multiple sclerosis, where CD40L-bearing cells have been found in the macrophage-rich lesions where β-chemokines are being produced. Overall, these findings define a pathway linking the specific recognition of antigen by T cells to the production of β-chemokines by macrophages. This pathway may play a role in anti-HIV-1 immunity and the development of immunologic reactions or lesions.     

Atomic structure of a thermostable subdomain of HIV-1 gp41

Infection by HIV-1 involves the fusion of viral and cellular membranes with subsequent transfer of viral genetic material into the cell. The HIV-1 envelope glycoprotein that mediates fusion consists of the surface subunit gp120 and the transmembrane subunit gp41. gp120 directs virion attachment to the cell–surface receptors, and gp41 then promotes viral–cell membrane fusion. A soluble, α-helical, trimeric complex within gp41 composed of N-terminal and C-terminal extraviral segments has been proposed to represent the core of the fusion-active conformation of the HIV-1 envelope. A thermostable subdomain denoted N34(L6)C28 can be formed by the N-34 and C-28 peptides connected by a flexible linker in place of the disulfide-bonded loop region. Three-dimensional structure of N34(L6)C28 reveals that three molecules fold into a six-stranded helical bundle. Three N-terminal helices within the bundle form a central, parallel, trimeric coiled coil, whereas three C-terminal helices pack in the reverse direction into three hydrophobic grooves on the surface of the N-terminal trimer. This thermostable subdomain displays the salient features of the core structure of the isolated gp41 subunit and thus provides a possible target for therapeutics designed selectively to block HIV-1 entry.     

HIV-1 Tat protein mimicry of chemokines

The HIV-1 Tat protein is a potent chemoattractant for monocytes. We observed that Tat shows conserved amino acids corresponding to critical sequences of the chemokines, a family of molecules known for their potent ability to attract monocytes. Synthetic Tat and a peptide (CysL_24–51) encompassing the “chemokine-like” region of Tat induced a rapid and transient Ca²⁺ influx in monocytes and macrophages, analogous to β-chemokines. Both monocyte migration and Ca²⁺ mobilization were pertussis toxin sensitive and cholera toxin insensitive. Cross-desensitization studies indicated that Tat shares receptors with MCP-1, MCP-3, and eotaxin. Tat was able to displace binding of β-chemokines from the β-chemokine receptors CCR2 and CCR3, but not CCR1, CCR4, and CCR5. Direct receptor binding experiments with the CysL_24–51 peptide confirmed binding to cells transfected with CCR2 and CCR3. HIV-1 Tat appears to mimic β-chemokine features, which may serve to locally recruit chemokine receptor-expressing monocytes/macrophages toward HIV producing cells and facilitate activation and infection.     

β-Chemokines and neutralizing antibody titers correlate with sterilizing immunity generated in HIV-1 vaccinated macaques

One of the obstacles to AIDS vaccine development is the variability of HIV-1 within individuals and within infected populations, enabling viral escape from highly specific vaccine induced immune responses. An understanding of the different immune mechanisms capable of inhibiting HIV infection may be of benefit in the eventual design of vaccines effective against HIV-1 variants. To study this we first compared the immune responses induced in Rhesus monkeys by using two different immunization strategies based on the same vaccine strain of HIV-1. We then utilized a chimeric simian/HIV that expressed the envelope of a dual tropic HIV-1 escape variant isolated from a later time point from the same patient from which the vaccine strain was isolated. Upon challenge, one vaccine group was completely protected from infection, whereas all of the other vaccinees and controls became infected. Protected macaques developed highest titers of heterologous neutralizing antibodies, and consistently elevated HIV-1-specific T helper responses. Furthermore, only protected animals had markedly increased concentrations of RANTES, macrophage inflammatory proteins 1α and 1β produced by circulating CD8⁺ T cells. These results suggest that vaccine strategies that induce multiple effector mechanisms in concert with β-chemokines may be desired in the generation of protective immune responses by HIV-1 vaccines.     

Stable,uncleaved HIV-1 envelope glycoprotein gp140 forms a tightly folded trimer with a native-like structure

The HIV-1 envelope spike [trimeric (gp160)₃, cleaved to (gp120/gp41)₃] is the mediator of viral entry and the principal target of humoral immune response to the virus. Production of a recombinant preparation that represents the functional spike poses a challenge for vaccine development, because the (gp120/gp41)₃ complex is prone to dissociation. We have reported previously that stable HIV-1 gp140 trimers, the uncleaved ectodomains of (gp160)₃, have nearly all of the antigenic properties expected for native viral spikes. Because of recent claims that uncleaved gp140 proteins may adopt a nonnative structure with three gp120 moieties “dangling” from a trimeric gp41 ectodomain in its postfusion conformation, we have inserted a long, flexible linker between gp120 and gp41 in our stable gp140 trimers to assess their stability and to analyze their conformation in solution. The modified trimer has biochemical and antigenic properties virtually identical to those of its unmodified counterpart. Both forms bind a single CD4 per trimer, suggesting that the trimeric conformation occludes two of the three CD4 sites even when a flexible linker has relieved the covalent constraint between gp120 and gp41. In contrast, an artificial trimer containing three gp120s flexibly tethered to a trimerization tag binds three CD4s and has antigenicity nearly identical to that of monomeric gp120. Moreover, the gp41 part of both modified and unmodified gp140 trimers has a structure very different from that of postfusion gp41. These results show that uncleaved gp140 trimers from suitable isolates have compact, native-like structures and support their use as candidate vaccine immunogens.The HIV-1 envelope glycoprotein mediates initial steps of virus infection by engaging cellular receptors and facilitating fusion of viral and target-cell membranes (1). Biosynthesis of the virus-encoded envelope glycoprotein yields a precursor, gp160, which following trimerization undergoes cleavage by a furin-like protease into two noncovalently associated fragments: the receptor-binding fragment, gp120, and the fusion fragment, gp41 (1). Three copies each of gp120 and gp41 form the mature envelope spikes (gp120/gp41)₃, the major viral surface antigen. Binding, through a site on gp120, to the host primary receptor, CD4, and then, through a second site, to a coreceptor (e.g., CCR5 or CXCR4) triggers large conformational changes that include reduced interaction between gp120 and gp41 (probably leading to dissociation of the former) and a cascade of ensuing gp41 refolding events (2, 3). Within the precursor gp160, gp41, with its C-terminal transmembrane (TM) segment anchored in the viral membrane, folds into a prefusion conformation. Cleavage of gp160 makes this prefusion conformation metastable with respect to a rearranged, postfusion conformation. Thus, the loss of constraint on gp41 that accompanies coreceptor binding to gp120 triggers a transition in gp41 to an extended, membrane-bridging conformation (sometimes called a “prehairpin” conformation) (4) with a hydrophobic “fusion peptide” at its N terminus inserted into the target-cell membrane and the TM segment in the viral membrane. This relatively long-lived, transient conformation is the target of fusion inhibitors, such as enfuvirtide (5), and of several broadly neutralizing antibodies (bnAbs) (6–8). Folding back of each chain into an α-helical hairpin creates a stable, six-helix bundle—the “postfusion conformation”—placing the fusion peptide and TM segment at the same end of the molecule. This irreversible refolding of gp41 brings the two membranes together, leading to bilayer fusion and viral entry. Thus, during the fusion process, there are at least three distinct conformational states of the envelope protein: the prefusion conformation of (gp120/gp41)₃, the extended intermediate of gp41, and the postfusion conformation of gp41 (with release of free gp120). Moreover, conformational changes of the prefusion form occur upon CD4 and perhaps also coreceptor binding.The envelope glycoprotein is also the primary target of humoral responses in HIV-1–infected individuals. Studies of human monoclonal antibodies (mAbs) have identified a subset members that neutralize a wide range of HIV isolates (see Table S1 for a partial catalog and original references) (9, 10). These bnAbs are of particular interest, because they may guide a search for immunogens to elicit them in vaccinees. Epitopes on gp120 recognized by human bnAbs include the CD4-binding site, a trimer-specific epitope in the relatively invariant parts of the V2 and V3 loops, and a site near the base of the V3 loop involving an N-linked glycan at position 332. A glycan-dependent epitope spans both gp120 and gp41. The membrane-proximal external region (MPER) of gp41 binds a set of bnAbs that were among the earliest broad neutralizers discovered. The domain-swapped, dimeric antibody, 2G12, recognizes only glycans at defined positions, and its reactivity therefore depends on specific glycosylation patterns but not on many other aspects of the gp120 amino acid sequence.Interesting groups of nonneutralizing antibodies, or with a very narrow range of isolates neutralized, include those that bind the so-called CD4-induced (CD4i) epitope, which overlaps the coreceptor site, on the bridging sheet of gp120, when the epitope becomes exposed by the conformational changes that accompany CD4 binding (Table S1). Nonneutralizing antibodies that interact with gp41 fall into two “clusters”: those in cluster I, which recognize the “immunodominant” C-C loop of gp41, and those in cluster II, which bind strongly with a segment just preceding the MPER in the gp41 polypeptide chain, but only when gp41 is in the postfusion conformation. Most of the antibodies listed in Table S1 recognize conformation-dependent epitopes and thus are excellent molecular probes for defining the conformational state of the envelope trimer.A form of gp140, stabilized by a disulfide crosslink between gp120 and gp41 (perhaps related to the disulfide between surface and TM subunits in many oncoretroviruses) was introduced over a decade ago (11) and subsequently modified by introducing an Ile-to-Pro mutation in gp41, to retard formation of the six-helix bundle (12). The product, known as SOSIP (SOS to designate the double cysteine mutations and IP to denote the isoleucine to proline change), is possible only with certain isolates, and the most widely studied has been BG505 SOSIP.664 (13–16). This modified gp140 trimer, which can be cleaved with furin without compromising stability, has greatly facilitated structural analysis, probably by eliminating large-scale conformational fluctuations (13, 14). In this variant, the MPER has been deleted, and the furin site has been replaced with a string of six arginines. Its structure, determined by both electron cryomicroscopy and X-ray crystallography, shows that (as expected from other fusion proteins) the conformation of gp41 in the prefusion state is distinct from the postfusion six-helix bundle (3, 17). The SOSIP.664 structure, which is an extremely important contribution to our understanding of envelope trimer molecular architecture, has some puzzling features. Docking CD4 onto the model suggests that it can bind all three gp120 sites with no clashes. This observation is at odds with a large body of evidence, including a recent biophysical study of the same trimer, showing that CD4 binding induces a substantial structural rearrangement (18–22). Moreover, uncleaved BG505 gp140 without the SOSIP modifications is unstable and heterogeneous. Can one really conclude, as suggested (14), that all HIV-1 uncleaved gp140 trimers are misfolded products resembling three gp120 moieties flexibly linked to a trimer gp41 in the postfusion, six-helix bundle conformation? The answer to this question is of considerable consequence, as it relates directly to design and production of candidate HIV-1 vaccine immunogens.In the work reported here, we have inserted a flexible, 20-residue linker between gp120 and gp41 in the context of a previously characterized, stable gp140. The linker releases the tight covalent constraint between gp120 and gp41, and it should therefore mimic to some extent the furin cleavage (which is effectively a linker of infinite length). If an uncleaved gp140 trimer truly resembles “three balls on a string,” as asserted (14), addition of a linker that can extend as much as 70 Å should exaggerate this property, causing the trimer with the inserted linker to have antigenic properties resembling three monomeric gp120s and a very large hydrodynamic radius. We find, to the contrary, that gp140 with the flexible linker, 20-residue insert (gp140–FL20) has antigenic properties essentially identical to those of the unmodified trimer and very different from those of free gp120 or of a construct with three gp120s held together by a heterologous trimerization tag. Moreover, the linker barely affects the hydrodynamic radius of the trimer, which is even slightly smaller than that of SOSIP. These results show that the stable, uncleaved gp140 trimers we have characterized previously are compact and native-like, and they support our suggestion that they are promising, envelope-based immunogens for clinical testing in vaccine development.     

Phenotypic knockout of HIV type 1 chemokine coreceptor CCR-5 by intrakines as potential therapeutic approach for HIV-1 infection

A genetic defect in a CC-chemokine receptor (CCR)-5, the principal coreceptor for the macrophage-tropic HIV type 1 (HIV-1), recently was found to naturally protect CCR-5-defective, but healthy, individuals from HIV-1 infection. In this study, we mimic the natural resistance of the CCR-5-defective individuals by designing a strategy to phenotypically knock out CCR-5. The inactivation of the CCR-5 coreceptor is accomplished by targeting a modified CC-chemokine to the endoplasmic reticulum to block the surface expression of newly synthesized CCR-5. The lymphocytes transduced to express the intracellular chemokine, termed “intrakine,” were found to be viable and resistant to macrophage-tropic HIV-1 infection. Thus, this gene-based intrakine strategy targeted at the conserved cellular receptor for the prevention of HIV-1 entry should have significant advantages over currently described approaches for HIV-1 therapy.     

Anti-HIV-1 and chemotactic activities of human stromal cell-derived factor 1α (SDF-1α) and SDF-1β are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage

CD26 is a leukocyte-activation antigen that is expressed on T lymphocytes and macrophages and possesses dipeptidyl peptidase IV (DPPIV) activity, whose natural substrates have not been identified yet. CXC chemokines, stromal cell-derived factor 1α (SDF-1α) and 1β (SDF-1β), sharing the receptor CXCR-4, are highly efficacious chemoattractants for resting lymphocytes and CD34⁺ progenitor cells, and they efficiently block the CXCR-4-mediated entry into cells of T cell line tropic strains of HIV type 1 (HIV-1). Here we show that both the chemotactic and antiviral activities of these chemokines are abrogated by DPPIV-mediated specific removal of the N-terminal dipeptide, not only when the chemokines are produced in transformed mouse L cell line to express human CD26 but also when they were exposed to a human T cell line (H9) physiologically expressing CD26. Mutagenesis of SDF-1α confirmed the critical requirement of the N-terminal dipeptide for its chemotactic and antiviral activities. These data suggest that CD26-mediated cleavage of SDF-1α and SDF-1β likely occurs in human bodies and promotes HIV-1 replication and disease progression. They may also explain why memory function of CD4⁺ cells is preferentially lost in HIV-1 infection. Furthermore, CD26 would modulate various other biological processes in which SDF-1α and SDF-1β are involved.     

Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells

Increasing evidence suggests that HIV-1-specific cytotoxic T lymphocytes (CTLs) are a key host immune response to HIV-1 infection. Generation of CTL responses for prevention or therapy of HIV-1 infection has several intrinsic technical barriers such as antigen expression and presentation, the varying HLA restrictions between different individuals, and the potential for viral escape by sequence variation or surface molecule alteration on infected cells. A strategy to circumvent these limitations is the construction of a chimeric T cell receptor containing human CD4 or HIV-1-specific Ig sequences linked to the signaling domain of the T cell receptor ζ chain (universal T cell receptor). CD8⁺ CTLs transduced with this universal receptor can then bind and lyse infected cells that express surface HIV-1 gp120. We evaluated the ability of universal-receptor-bearing CD8⁺ cells from a seronegative donor to lyse acutely infected cells and inhibit HIV-1 replication in vitro. The kinetics of lysis and efficiency of inhibition were comparable to that of naturally occurring HIV-1-specific CTL clones isolated from infected individuals. Further study will be required to determine the utility of these cells as a therapeutic strategy in vivo.     

Neutralization of HIV-1 by redirection of natural antibodies

The great variability and high glycosylation of gp120 poses a great challenge for the design of a functional immune therapy. The binding region of the CD4 receptor to gp120, however, is well conserved and may constitute a target to limit viral entry and infectivity. Our strategy consists in using a preexisting pool of natural antibodies directed toward the gal(α1,3)gal disaccharide and to redirect it to HIV. We here show that using CD4-derived, gp120-binding, synthetic peptides chemically linked to gal(α1,3)gal can redirect these natural antibodies and improve the HIV-1 neutralizing activity of the CD4-derived peptides in vitro. Importantly, the binding of the CD4-gal(α1,3)gal peptides to HIV-1–infected cells conferred antibody-dependent cellular cytotoxicity after the addition of human sera. Thus, the temporary redirection of naturally occurring antibodies and their biological activities to a new antigen represents a completely new way of targeting a human disease.     

Sensitivity to inhibition by β-chemokines correlates with biological phenotypes of primary HIV-1 isolates

Primary HIV-1 isolates were evaluated for their sensitivity to inhibition by β-chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted), macrophage inflammatory protein 1α (MIP-1α), and MIP-1β. Virus isolates of both nonsyncytium-inducing (NSI) and syncytium-inducing (SI) biological phenotypes recovered from patients at various stages of HIV-1 infection were assessed, and the results indicated that only the isolates with the NSI phenotype were substantially inhibited by the β-chemokines. More important to note, these data demonstrate that resistance to inhibition by β-chemokines RANTES, MIP-1α, and MIP-1β is not restricted to T cell line-adapted SI isolates but is also a consistent property among primary SI isolates. Analysis of isolates obtained sequentially from infected individuals in whom viruses shifted from NSI to SI phenotype during clinical progression exhibited a parallel loss of sensitivity to β-chemokines. Loss of virus sensitivity to inhibition by β-chemokines RANTES, MIP-1α, and MIP-1β was furthermore associated with changes in the third variable (V3) region amino acid residues previously described to correlate with a shift of virus phenotype from NSI to SI. Of interest, an intermediate V3 genotype correlated with a partial inhibition by the β-chemokines. In addition, we also identified viruses sensitive to RANTES, MIP-1α, and MIP-1β of NSI phenotype that were isolated from individuals with AIDS manifestations, indicating that loss of sensitivity to β-chemokine inhibition and shift in viral phenotype are not necessarily prerequisites for the pathogenesis of HIV-1 infection.     

An inducible HIV type 1 gp41 HR-2 peptide-binding site on HIV type 1 envelope gp120

Synthetic peptides of sequences within the HIV-1 gp41 heptad repeat-regions (HR-1 and HR-2) can effectively inhibit cell fusion and viral entry. DP178 (T-20), an HR-2 peptide, acts by inhibiting the association between HR-1 and HR-2, thereby interfering with HIV-1 fusion and viral entry. HR-2 peptide binding is predicted to be an important indicator of the presence of Env gp41 fusion intermediate conformation. A stabilized HR-2/Env conjugate might be an HIV-1 vaccine candidate and have the potential for inducing antibodies against transiently exposed epitopes on HIV-1 Env. To explore the possibility of design of HR-2 stabilized-HIV-1 immunogens, we studied the ability of HIV-1 Env to bind to HR-2 peptides. Using surface plasmon resonance (SPR)-binding assays and precipitation of soluble Env gp120 proteins with HR-2 peptide DP178, we have found that there is an HR-2 peptide-binding site on soluble HIV-1 recombinant gp120. Binding of DP178 was induced by sCD4 and by the anti-gp120 human mAb A32. The induction of DP178 binding was inhibited > 80% by the HIV-1 coreceptor-binding site mAb 17b. Binding of DP178 to gp120 was also inhibited by gp120 C4 peptides with sequences that are centrally located within the HIV-1 coreceptor-binding site. Thus, in addition to interactions with the gp41 HR-1 region, the fusion inhibitor peptide DP178 binds to triggered soluble HIV-1 recombinant gp120 following its interaction with sCD4 or CD4 mimic mAb A32. This may prove to be an important consideration when designing an HIV vaccine that utilizes constrained HIV Env proteins.     

The neutral glycosphingolipid globotriaosylceramide promotes fusion mediated by a CD4-dependent CXCR4-utilizing HIV type 1 envelope glycoprotein

Previously, we showed that the addition of human erythrocyte glycosphingolipids (GSLs) to nonhuman CD4⁺ or GSL-depleted human CD4⁺ cells rendered those cells susceptible to HIV-1 envelope glycoprotein-mediated cell fusion. Individual components in the GSL mixture were isolated by fractionation on a silica-gel column and incorporated into the membranes of CD4⁺ cells. GSL-supplemented target cells were then examined for their ability to fuse with TF228 cells expressing HIV-1_LAI envelope glycoprotein. We found that one GSL fraction, fraction 3, exhibited the highest recovery of fusion after incorporation into CD4⁺ nonhuman and GSL-depleted HeLa-CD4 cells and that fraction 3 contained a single GSL fraction. Fraction 3 was characterized by MS, NMR spectroscopy, enzymatic analysis, and immunostaining with an antiglobotriaosylceramide (Gb3) antibody and was found to be Gal(α1→4)Gal(β1→4)Glc-Cer (Gb3). The addition of fraction 3 or Gb3 to GSL-depleted HeLa-CD4 cells recovered fusion, but the addition of galactosylceramide, glucosylceramide, the monosialoganglioside, GM3, lactosylceramide, globoside, the disialoganglioside, GD3, or α-galactosidase A-digested fraction 3 had no effect. Our findings show that the neutral GSL, Gb3, is required for CD4/CXCR4-dependent HIV-1 fusion.     

Targeted disruption of the Ca2+ channel β3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltagedependent activation of P/Q-type Ca2+ channels in neurons

In comparison to the well characterized role of the principal subunit of voltage-gated Ca²⁺ channels, the pore-forming, antagonist-binding α₁ subunit, considerably less is understood about how β subunits contribute to neuronal Ca²⁺ channel function. We studied the role of the Ca²⁺ channel β₃ subunit, the major Ca²⁺ channel β subunit in neurons, by using a gene-targeting strategy. The β₃ deficient (β₃−/−) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic β₃−/− neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca²⁺ channels was described by two Boltzmann components with different voltage dependence, analogous to the “reluctant” and “willing” states reported for N-type channels. The absence of the β₃ subunit was associated with a hyperpolarizing shift of the “reluctant” component of activation. Norepinephrine inhibited wt and β₃−/− neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca²⁺ channels. The reduction in the expression of N-type Ca²⁺ channels in the β₃−/− mice may be expected to impair Ca²⁺ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.     

Induction of a nonproductive conformational change in gp120 by a small molecule HIV type 1 entry inhibitor

Conformational changes in HIV-1 envelope glycoproteins, gp120 and gp41, is a dynamic process essential for HIV-1 entry. Here we show that a small molecule HIV-1 entry inhibitor, IC9564, induces a conformational change in gp120. The conformational change in gp120 is evidenced by a significant increase in the binding of a conformational monoclonal antibody 17b. As a result of the conformational effect, IC9564 significantly enhances the neutralizing activity of 17b. Unlike CD4, IC9564 does not trigger conformational changes in gp41. In fact, IC9564 inhibits CD4-induced conformational changes in gp41. Thus, IC9564 exploits the dynamic nature of gp120 by inducing a nonproductive gp120 conformation that is not able to trigger a conformational change in gp41 for membrane fusion.