A fungal metabolite which inhibits the interaction of CD4 with major histocompatibility complex-encoded class II molecules

CD4, a cell-surface glycoprotein expressed on a subpopulation of T cells, is the receptor for class II molecules of the major histocompatibility complex (MHC II) and a receptor for the envelope glycoprotein (gp 120) of human immunodeficiency virus-1 (HIV-1). Screening of microbial metabolites for CD4-binding activity using an enzyme-linked immunosorbent assay based on the binding of the CD4-specific monoclonal antibody (mAb), anti-Leu3a, identified a family of compounds comprising several novel polyketides. The parent compound (411F, Vinaxanthone) is a C₂₈ molecule probably arising from a dimerization of two C₁₄ polyketide units. It strongly inhibited the interaction of anti-Leu 3a and that of several other D1/D2 epitope-specific mAb with CD4, but only weakly inhibited the binding of HIV-1 gp120. Binding of a representative MHC class II molecule, HLA-DRB*0401, was also inhibited by 411F with a comparable inhibitory concentration (IC₅₀ = 1 μM ). In functional assays 411F inhibited antigen-induced CD4-dependent T cell proliferative responses of peripheral blood mononuclear cells. At the clonal level 411F exhibited selectivity in that the compound inhibited peptide-induced CD4⁺ T cell proliferative responses but not alloantigen-induced CD8⁺ T cell proliferation. It is hypothesized that 411F, a polyanionic compound in aqueous solution at neutral pH, inhibits CD4-dependent functions by binding over a broad area of the positively charged amino-terminal D1 and D2 domains implicated in the interaction with MHC II molecules. 411F has the potential for development as an immunosuppressive agent with a novel mechanism of action.     

HIV-induced deletion of antigen-specific T cell function is MHC restricted.

When antigen-specific T cells are pulsed by antigen-presenting cells (APC) in the presence of HIV they are functionally deleted following subsequent exposure to syngeneic APC in the absence of HIV. Recombinant soluble HIV envelope (gp120) is able to induce a similar effect which, unlike that induced by HIV, is reversible. Neither HIV nor gp120 affect the ability to respond to IL-2. Thus it is only antigen-specific responses involving the T cell receptor pathways and CD4/MHC class II interaction that appear to be inhibited by HIV-1 and gp120. Furthermore, the functional impairment caused by HIV-1 is specific to the T cells that respond to the antigen in co-culture with HIV, as there is no apparent effect on 'bystander'-activated T cells specific for another antigen. Antigen-specific T cell lines may be deleted by a signalling mechanism which involves molecules other than gp120/CD4 but still requires MHC class II restriction.     

AIDS pathogenesis: HIV envelope and its interaction with cell proteins

The immune deficiency induced by HIV has its origin in the interaction of the outer envelope glycoprotein gp120/gp41 with receptors present on human immunocytes. Virus binding to cells, virus entry and subsequent compartmentalization resulting in productive infection depends on the interaction of gp120/gp41 with CD4 and other accessory molecules. Gp120 and HIV are markedly immunosuppressive of T-cell responses and, in addition, HIV can functionally delete antigen responsiveness of T cells. Abolition of CD4 binding, by denaturation of gp120, allows study of T-cell epitopes in gp120 and shows the denatured molecule is highly immunogenic even in naive subjects (F. Manca, unpublished). The gp120-binding site of CD4 is shared with MHC class II molecules and the reaction of antibodies within this region of CD4 induces conformational changes that may be significant for virus entry into cells or for syncytial formation. The HIV envelope contains sites of sequence homology with monomorphic human MHC class II sites that do not appear to be naturally immunogenic in humans. In addition to the properties of gp120, it is hypothesized that HIV envelope may also represent an 'alloepitope' of class II to the human T-cell repertoire, and is therefore able to induce a chronic allogeneic response not dissimilar to experimentally induced GVHD. These features are of potential importance both for primary vaccination against HIV, and for the long-term treatment of HIV seropositive patients. Induction of effective T-cell responses to gp120 require use of a denatured or otherwise modified product lacking CD4-binding capacity. The potential distortion of the TCR repertoire by the class-II-homologous and CD4-interactive sequences must be assessed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of antibodies that inhibit HIV gp120 antigen processing and presentation

Antibodies to the CD4-binding site (CD4bs) of HIV-1 envelope gp120 have been shown to inhibit MHC class II presentation of this antigen, but the mechanism is not fully understood. To define the key determinants contributing to the inhibitory activity of these antibodies, a panel of anti-CD4bs monoclonal antibodies with different affinities was studied and compared to antibodies specific for the chemokine receptor-binding site or other gp120 regions. Anti-CD4bs antibodies that completely obstruct gp120 presentation exhibit three common properties: relatively high affinity for gp120, acid-stable interaction with gp120, and the capacity to slow the kinetics of gp120 proteolytic processing. None of these antibodies prevents gp120 internalization into APC. Notably, the broadly virus-neutralizing anti-CD4bs IgG1b12 does not block gp120 presentation as strongly, because although IgG1b12 has a relatively high affinity, it dissociates from gp120 more readily at acidic pH and only moderately retards gp120 proteolysis. Other anti-gp120 antibodies, regardless of their affinities, do not affect gp120 presentation. Hence, high-affinity anti-CD4bs antibodies that do not dissociate from gp120 at endolysosomal pH obstruct gp120 processing and prevent MHC class II presentation of this antigen. The presence of such antibodies could contribute to the dearth of anti-gp120 T helper responses in chronically HIV-1-infected patients.     

Ligation of human CD4 interferes with antigen-induced activation of primary T cells

The CD4 molecule functions to enhance T cell activation when it is co-aggregated with the T cell receptor for antigen (TCR) by MHC class II antigenic peptide complexes. However, independent ligation of CD4 has been shown to negatively effect signaling through the TCR in vitro. The interaction between the HIV-1 envelope glycoprotein gp120 and CD4 is a central event in the pathogenesis of AIDS and may contribute to immune deficiency via both direct and indirect mechanisms, including lytic infection of T cells and induction of CD4 signaling events resulting in apoptosis and anergy. Analysis of the consequences of interactions between CD4 and gp120 have yielded contradictory results presumably because most of these studies have focused on T cell clones of questionable relevance to the in vivo target of the virus. Here, we analyzed the effects of CD4 ligation on freshly isolated cells of human CD4 transgenic mice, and show that huCD4 preligation, in the absence of human CXCR4, has an inhibitory effect on both early and late T cell activation events. CD4 signaling negatively regulates the response to antigen, as well as to anti-TCR mAb. In addition, we show here that this negative signal requires the cytoplasmic tail of CD4. These results suggest that in HIV infected patients the interaction of gp120 with CD4 induces unresponsiveness of CD4+ T cells to subsequent activation by antigen.     

Inhibitory activity of HIV envelope gp120 dominates over its antigenicity for human T cells.

HIV-1 envelope glycoprotein (gp120), as a CD4-binding reactant, has been shown to inhibit in its native form human T cell responses to several antigens. Here we show that gp120 in soluble form also inhibits activation of a specific human T cell line that responds to gp120-pulsed autologous antigen-presenting cells. In addition the inhibitory property of gp120 for antigen-driven T cell proliferation depends upon its ability to bind CD4 and is lost when CD4-binding capacity is abolished by denaturation, or blocked by complexing with soluble CD4 or with polyclonal antibodies. In contrast, antigenicity of denatured or complexed gp120 for specific human T cells is preserved. Similar effects are also observed with another CD4-binding reactant (i.e. anti-Leu 3a MoAb), which stimulates and/or inhibits human T cells specific for mouse immunoglobulins depending on native or denatured conformation.     

A response to P.H. Duesberg with reference to an idiotypic network model of AIDS immunopathogenesis

Professors in British Columbia, Canada have devised an autoimmunity model of AIDS (MIAMI model) based on an atypical network theory of regulation of the immune system. It presents different stimuli as cofactors for AIDS: allogenic stimuli in some risk groups and MHC mimicking antigenic stimuli in other risk groups. V regions are on helper T cells that are somewhat anti-self class II MCH. Helper T cell idiotypes interact with both class II MHC and particular suppressor T cell idiotypes, therefore both may be similar. In fact, foreign lymphocytes also can induce an immune response similar to that of MHC image (MI). Hence the MI response is against the anti-self MHC, i.e., foreign idiotypes identify self, particularly self MHC. Further, the HIV envelope protein (gp120) binds to CD4 at a site that overlaps the site where CD4 interacts with class II MHC. Thus recombinant gp120 well as antibodies that recognize the gp120 undying site of CD4 can prevent the interaction of CD4 with class II MHC. Some mutations of CD4 effect the gp120 binding site but not the class II binding site and vice versa and others effect both. This similarity and others HIV can be considered an image of class II MHC, and the anti HIV immune response may be anti MHC image (AMI). MI and AMI responses are against each other and against idiotypic determinants expressed on helper and suppressor T cells respectively. A dual attack on the idiotypes of helper and suppressor T cells accompany these responses thereby causing an imminent collapse of the entire immune system. The model's significant predictive power thereby suggests that we may be able to prevent HIV from causing AIDS by inducing immunological tolerance to HIV components that resemble MHC molecules. This model rejects the 11 paradoxes identified by Duesberg who surmises that HIV is not a cofactor or cause of AIDS.     

Human tonsillar dendritic cell-induced T cell responses: analysis of molecular mechanisms using monoclonal antibodies

Dendritic cells, isolated from human tonsillar tissue, were found to be potent stimulators of the sodium periodate T cell oxidative mitogenesis reaction. Monoclonal antibodies against CD2, CD4, CD11a, CD18, LFA-3, ICAM-1, class I and class II major histocompatibility complex (MHC) inhibited T cell proliferation in this response, whereas antibodies against CD8, CD11b, CD11c and CD16 had no effect. Further, antibodies against CD2, CD11a, CD18, LFA-3 and ICAM-1 inhibited the early dendritic cell-T cell clustering event which occurs in this cell interaction. In contrast, antibodies against CD4, class I and class II MHC did not inhibit clustering. Studies examining the expression of the respective molecules upon isolated dendritic cells and T cells suggest that anti-LFA-3 and anti-class II MHC antibodies inhibit at the level of the dendritic cell, whereas anti-CD2 and anti-CD4 antibodies inhibit at the level of the T cell. However, antibodies against CD11a, CD18, ICAM-1 and class I MHC may inhibit at either or both cell levels. These findings have enabled us to propose a molecular mechanism for dendritic cell-T cell interaction in oxidative mitogenesis. Dendritic cell-T cell clustering is mediated by bidirectional binding of LFA-1 (CD11a and CD18) and ICAM-1 (involving both molecules on both cell types) and unidirectional binding of CD2 and LFA-3 (involving T cell CD2 and dendritic cell LFA-3). This initial event permits a second interaction of dendritic cell and T cell molecules, involving T cell CD4, class I MHC (possibly at both cellular levels) and dendritic cell class II MHC, which deliver the signal for proliferation.     

Suppression of major histocompatibility complex class II-associated invariant chain enhances the potency of an HIV gp120 DNA vaccine

Summary One function of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) is to prevent MHC class II molecules from binding endogenously generated antigenic epitopes. Ii inhibition leads to MHC class II presentation of endogenous antigens by APC without interrupting MHC class I presentation. We present data that in vivo immunization of BALB/c mice with HIV gp120 cDNA plus an Ii suppressive construct significantly enhances the activation of both gp120-specific T helper (Th) cells and cytotoxic T lymphocytes (CTL). Our results support the concept that MHC class II-positive/Ii-negative (class II(+)/Ii(-)) antigen-presenting cells (APC) present endogenously synthesized vaccine antigens simultaneously by MHC class II and class I molecules, activating both CD4(+) and CD8(+) T cells. Activated CD4(+) T cells locally strengthen the response of CD8(+) CTL, thus enhancing the potency of a DNA vaccine.     

Modulation of CD4 lateral interaction with lymphocyte surface molecules induced by HIV-1 gp120

CD4, a lymphocyte surface glycoprotein, serves as co-receptor for antigen with the T cell receptor (TCR). It is also the lymphocyte receptor for HIV by binding the gp120 viral envelope protein. Interaction of gp120 with CD4 is crucial for viral infection, but is not sufficient to allow viral entry into cells. Recombinant gp120 alters CD4⁺ T cell responsiveness to activation stimuli. To express its co-receptor function fully, CD4 must be laterally associated with the TCR and CD45 to form multi-receptor complexes competent to transduce potent activation signals. Here, we examine the possibility that gp120/CD4 binding alters lateral associations of CD4 with other lymphocyte surface molecules, and that assembly of abnormal multi-molecular complexes is involved in the gp120-induced CD4⁺ T cell dysfunction and in viral entry. In the absence of gp120, CD4 displayed high association with CD3, CD5, CD45RC, CD25, CD28, CD44, and CD53; weak association with CD2, CD38, CD45RB, CD62L, and CD26; and no association with CD45RA, CD45RO, CD11b, CD11a, CD54, CD7, CD48, CD98, CD59, CD55, HLA class I and class II molecules. Treatment with gp120 significantly increased CD4 association with CD3, CD45RA, CD45RB, CD59, CD38, CD26, and HLA class I, and decreased that with CD45RC. Specificity of these results were assessed at various levels. First, gp120 did not influence lateral associations displayed by other molecules, such as HLA class II. Second, the Leu3 mAb, which binds CD4 on a site overlapping the gp120 binding site, did not elicit the same CD4 lateral associations as gp120, and finally, a direct gp120/CD4 interaction was needed to induce the lateral associations, as shown by the observation that blocking the gp120/CD4 binding by the Leu3 mAb inhibited the gp120-induced associations. These results can be interpreted in several ways. gp120/CD4 interaction could trigger an inside-out signal responsible for the associations, or gp120 could induce steric modifications of CD4 that increase its affinity for the associating molecules. Alternatively, these molecules may interact directly with gp120, bridging them with CD4. It is also possible that the associations may be mediated by additional components, interacting with both gp120 and the associating surface molecule. The last hypothesis is likely for CD59, whose gp120-induced association with CD4 required the presence of serum in the co-capping assay. Since both CD59 and gp120 bind complement, the observed association could be mediated by complement components.     

Construction of a recombinant bacterial human CD4 expression system producing a bioactive CD4 molecule.

The CD4 protein expressed on helper T lymphocytes is a restriction element for major histocompatibility class II immune responses. This molecule is also used by the human immunodeficiency virus as its specific cellular receptor facilitating binding of virus to cells. As soluble forms of CD4 inhibit HIV infection in tissue culture, attention has focused on this molecule. Bacterially produced CD4 would facilitate studies of the biology of the CD4 molecule. However, bacterially expressed CD4 must be refolded for assumption of its interaction with conformationally dependent anti-CD4 monoclonal antibodies as well as the HIV-1 envelope protein gp120. We report here the engineering of an external domain construct of the CD4 gene into a novel expression vector containing the nucleotide sequence encoding the pelB leader peptide of Erwinia carotovara (pDABL), to facilitate correct folding of CD4 in bacteria. Monoclonal antibodies specific for important conformational epitopes of the CD4 molecule were able to bind bacterial colonies containing the pDABL/CD4 vector but not colonies with vector alone. Importantly, recombinant gp120 produced in baculovirus bound specifically to bacterial colonies expressing the CD4 recombinant molecule. This system presents a simple screening mechanism for molecules that bind to the external domain of the CD4 glycoprotein. Vectors such as pDABL will also facilitate the production of large amounts of biologically active proteins in bacteria.     

The inositol 5-phosphatase SHIP-1 and adaptors Dok-1 and 2 play central roles in CD4-mediated inhibitory signaling

CD4 functions to enhance the sensitivity of T cells to antigenic peptide/MHC class II. However, if aggregated in isolation, e.g. in the absence of T cell receptor (TCR), CD4 can transduce yet undefined signals that lead to T cell unresponsiveness to antigen and apoptosis. In Human Immunodeficiency Virus-1 (HIV-1) disease, CD4(+) T cell loss can result from gp120-induced CD4 signaling in uninfected cells. We show here that CD4 aggregation leads to Lck-dependent phosphorylation of the RasGAP adaptors Downstream of kinase-1/2 (Dok-1/2) and the inositol 5-phosphatase-1 (SHIP-1) and association of the two molecules. Studies using SHIP-1 shRNA, knockout mice and decoy inhibitors further indicate that CD4-mediated inhibition of TCR-mediated T cell activation is SHIP-1 and Dok-1/2 dependent, and involves SHIP-1 hydrolysis of Phosphatidylinositol 3,4,5-trisphosophate (PI(3,4,5)P3) needed for TCR signaling. Our studies provide evidence for a novel mechanism by which ill-timed CD4-mediated signals activated by ligands such as HIV-1 gp120 lead to disarmament of the immune system.     

Development and characterization of a whole-cell radioligand binding assay for [125I]gp120 of HIV-1.

The binding of HIV-1 envelope glycoprotein, gp120, to the CD4 receptor is an important step in productive infection. The development of agents which interrupt this binding phenomenon should be of therapeutic interest. The present study characterizes a whole cell gp120/CD4 radioligand binding assay (radioligand binding assay) modified for use in a high volume screening format. Modifications include the use of human CD4 receptor stably expressed in a Chinese hamster ovary cell line and the gentle fixation (paraformaldehyde) of the CD4 receptor just prior to assay. Binding of [125I]gp120 to fixed CD4 was of high affinity (KD = 6 nM), saturable, reversible, and specific. The kinetics of binding were identical to those of viable (non-fixed) CD4 receptor. [125I]gp120 binding was inhibited by unlabeled recombinant gp120, soluble CD4, and the anti-CD4 monoclonals OKT4A and LEU3A. A number of compounds reported to inhibit gp120 binding and/or gp120 induced syncytium formation were also active in this assay. This modified radioligand binding assay was developed to initiate a rational and extensive screening program to assist in the identification of potential chemotherapeutic agents based on their ability to inhibit gp120 binding to host cells.     

Short synthetic peptides derived from viral proteins compete with HIV gp120 for the binding to CD4 receptors

In the complex mechanism of adhesion, internalization, and infection of cells by human immunodeficiency virus (HIV) viral particles, a determinant role is played by the viral envelope glycoprotein gp120, which binds to CD4 receptors of T cells and monocytes. We tested the ability of a panel of 7- to 12-residue synthetic peptides, selected from the region 414-434 of the HIV-1 gp120, to inhibit the binding of the viral protein to CD4 receptors of cultured human lymphoid cells. The assay was based on the observation that the binding of gp120 to the receptors interferes with the binding of a specific anti-CD4 monoclonal antibody, as a result of the masking of the antibody epitope; thus, we tested whether preincubation of cells with the peptides before gp120 addition might restore the recognition of the CD4 molecule by the antibody. High expression of CD4 receptors was thus assumed as indication that the binding of the viral protein had been inhibited. Maximum activity was displayed by a 9-residue peptide located near the amino terminal end of the 414-434 fragment. In addition, several fragments deduced from other viral proteins, possessing partial amino acid sequence homology with the HIV gp120 fragment, exhibited a similar type of interaction with the CD4 receptor. All active peptides contain the Cys residue (position 423 of gp120). This residue is essential, although not sufficient, for inhibiting gp120 binding, as few other amino acid residues within the fragment play a complementary role in increasing or decreasing the inhibitory ability.     

HIV-1 infection: is it time to reconsider our concepts?

The long asymptomatic phase of HIV infection is critical in the progression to AIDS. It probably reflects an ancestral relationship with lentiviruses stemming from the primate-simian immunodeficiency virus evolutionary pathway leading to an idiosyncratic immune tolerance, which needs to be understood if effective vaccines are to be rationally designed. The majority of CD4+ T cells that die due to HIV-1 in the asymptomatic phase are not infected with the virus. Transmission of the predominant HIV-1 R5 variants to T cells is mediated by infected monocyte-derived macrophages. The two cell populations come into intimate contact mainly in the lymph nodes during antigen presentation where there is also active viral replication. We propose that HIV exploits antigen presentation to access target T cells and evade immune surveillance. This is achieved at the assembly point of an immunological synapse between an antigen presenting, HIV-1-infected macrophage and a responding effector/memory CD4+ T cell. Viral envelope gp120 glycoproteins proximal to MHC II molecules cross-link with T cell CD4 molecules, thus establishing a supra molecular immuno-viral synapse. The interaction results in conformational changes of gp120 exposing its V3 domain. Ionic interaction of this domain with the synapse-recruited chemokine receptor CCR5 dimerizes the receptor triggering intracellular signals that contribute to T cell receptor transactivation pathways and subsequent enhancement of T cell activation. HIV-downregulated MHC II gives weak immune complexes. Disruption of the immuno-viral synapse before completion of cell entry is a frequent outcome condemning the responding T cell to a premature activation-induced T cell death. Information on the assembly, mechanistic and functional interactions at the immuno-viral synapses may well assist in elucidating new strategies to combat HIV infection.     

Inhibition of human immunodeficiency virus infection by the lectin jacalin and by a derived peptide showing a sequence similarity with gp120

Jacalin is a plant lectin known to specifically induce the proliferation of CD4⁺ T lymphocytes in human. We demonstrate here that jacalin completely blocks human immunodeficiency virus type 1 (HIV-1) in vitro infection of lymphoid cells. Jacalin does not bind the viral envelope glycoprotein gp120. Besides other T cell surface molecules, it interacts with CD4, the high-affinity receptor to HIV. Binding of jacalin to CD4 does not prevent gp120-CD4 interaction and does not inhibit virus binding and syncytia formation. The anti-HIV effect of the native lectin can be reproduced by its separated a-subunits. More importantly, we have defined in the a-chain of jacalin a 14-amino acid sequence which shows high similarities with a peptide of the second conserved domain of gpl20. A synthetic peptide corresponding to this similar stretch also exerts a potent anti-HIV effect. This peptide is not mitogenic for peripheral blood mononuclear cells and does not inhibit anti-CD3-induced lymphocyte proliferation. These results make jacalin a chain-derived peptide a potentially valuable therapeutic agent for acquired immunodeficiency syndrome.     

Role of curdlan sulfate in the production of ,-chemokines and interleukin-16

The blocking effect of curdlan sulfate (CRDS) on human immunodeficiency virus (HIV) infection has been thought to be related to inhibition of the binding of HIV-1 envelope glycoprotein (gp120) and CD4 molecules. However, recent reports have indicated that blocking the binding of gp120 to CD4 by CRDS only makes a small contribution to the inhibition of HIV-1 infection. We report here that the effect of CRDS on the production of β-chemokines and cytokines might be important in the inhibition of HIV-1 infection, in addition to interference with the binding of gp120 to CD4⁺ cells. Received: 5 November 1997     

Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains

Human immunodeficiency virus (HIV-1) enters target cells by binding its gp120 exterior envelope glycoprotein to CD4 and one of the chemokine receptors, CCR5 or CXCR4. CD4-induced (CD4i) antibodies bind gp120 more efficiently after CD4 binding and block the interaction with the chemokine receptor. Examples of CD4i antibodies are limited, and the prototypes of the CD4i antibodies exhibit only weak neutralizing activity against primary, clinical HIV-1 isolates. Here we report the identification of a novel antibody, E51, that exhibits CD4-induced binding to gp120 and neutralizes primary HIV-1 more efficiently than the prototypic CD4i antibodies. The E51 antibody blocks the interaction of gp120-CD4 complexes with CCR5 and binds to a highly conserved, basic gp120 element composed of the beta 19-strand and surrounding structures. Thus, on primary HIV-1 isolates, this gp120 region, which has been previously implicated in chemokine receptor binding, is accessible to a subset of CD4i antibodies.