Role of tumour necrosis factor-alpha (TNF-α) in the induction of HIV-1 gp120-mediated CD4+ T cell anergy

The HIV-1 envelope glycoprotein (gp120) is known to induce antigen-specific and non-specific CD4⁺ T cell anergy. We found that early T cell activation, as indicated by HLA-DP expression in the early G₁ (G_1A) phase of the cell cycle, and the inhibition of mitogen-mediated IL-2 production induced by gp120, required TNF-α produced by gp120-stimulated macrophages. In the presence of an antibody to TNF-α, these changes induced by gp120 were inhibited, while recombinant TNF-α induced similar abnormalities of CD4⁺ T cells, even in the absence of gp120. On the other hand, inhibition of the mixed lymphocyte reaction (MLR) in CD4⁺ T cells by gp120, which may be related to gp120-mediated down-regulation of CD4 expression on T cells and activation of protein tyrosine kinase p56^lck in CD4⁺ T cells, was observed even in the absence of macrophage-derived TNF-α induced by gp120. These observations indicate that both TNF-α-dependent and independent events contribute to gp120-mediated CD4⁺ T cell anergy, and TNF-α appears to play an important role in inducing CD4⁺ T cell anergy in HIV-1 infection.     

Potency of HIV-1 envelope glycoprotein gp120 antibodies to inhibit the interaction of DC-SIGN with HIV-1 gp120

The interaction of DC-SIGN with gp120 provides an attractive target for intervention of HIV-1 transmission. Here, we have investigated the potency of gp120 antibodies to inhibit the DC-SIGN-gp120 interaction. We demonstrate that although the V3 loop is not essential for DC-SIGN binding, antibodies against the V3 loop partially inhibit DC-SIGN binding, suggesting that these antibodies sterically hinder DC-SIGN binding to gp120. Polyclonal antibodies raised against non-glycosylated gp120 inhibited both low and high avidity DC-SIGN-gp120 interactions in contrast to polyclonal antibodies raised against glycosylated gp120. Thus, glycans present on gp120 may prevent the generation of antibodies that block the DC-SIGN-gp120 interactions. Moreover, the polyclonal antibodies against non-glycosylated gp120 efficiently inhibited HIV-1 capture by both DC-SIGN transfectants and immature dendritic cells. Therefore, non-glycosylated gp120 may be an attractive immunogen to elicit gp120 antibodies that block the binding to DC-SIGN. Furthermore, we demonstrate that DC-SIGN binding to gp120 enhanced CD4 binding, suggesting that DC-SIGN induces conformational changes in gp120, which may provide new targets for neutralizing antibodies.     

In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120

The binding of antibodies to the CD4-binding site (CD4bs) of the HIV-1 envelope glycoprotein gp120 has been shown to induce gp120 to undergo conformational changes that can expose and/or shield specific epitopes on gp120. Here, we study alterations in the antigenicity and immunogenicity of gp120 when complexed with human monoclonal antibodies (mAbs) specific for the CD4bs of gp120. The data showed that gp120 bound by anti-CD4bs mAbs had enhanced reactivity with mAbs to the V3 and N-terminal regions, but not with mAb to the C terminus. Moreover, mice immunized with the gp120/anti-CD4bs mAb complexes produced higher titers of gp120-specific serum IgG and IgA than mice immunized with uncomplexed gp120 or other gp120/mAb complexes. Notably, the enhanced antibody production was directed against V3 and correlated with better exposure of V3 on the gp120/anti-CD4bs mAb complexes. The higher antibody reactivity was evident against the homologous V3(LAI) peptide, but not against heterologous V3 peptides. Potent neutralization activity against HIV-1(LAI) was also observed in the sera from mice immunized with gp120/anti-CD4bs mAb complexes, although the sera exhibited poor neutralizing activities against other viruses tested. These results indicate that the anti-CD4bs antibodies alter the antigenicity and immunogenicity of gp120, leading to enhanced production of anti-gp120 antibodies directed particularly against the V3 region.     

HIV-1 gp120 chemokine receptor-mediated signaling in human macrophages

The chemokine receptors CCR5 and CXCR4 serve as the cellular receptors in conjunction with CD4 for HIV-1 entry and infection of target cells. Although the virus has subverted these molecules for its own use, their natural function is to respond to activation and migration signals delivered by extracellular chemokines. A principal research objective of our laboratory is to understand the consequences of virus-chemokine receptor interactions for cellular function, as well as for entry and infection. We hypothesized that CXCR4-using (X4) and CCR5-using (R5) HIV-1 strains might elicit signals through the chemokine receptors that result in aberrant function and/or regulate virus entry or postentry steps of infection. We have focused on primary human macrophages, which express both CXCR4 and CCR5, because macrophages are a principal target for HIV-1 in vivo, in appropriate macrophage activation appears to play a major role in the pathogenesis of certain sequelae of AIDS, such as HIV encephalopathy, and macrophage infection is regulated at several steps subsequent to entry in ways that are linked to envelope-receptor interactions. This review summarizes our recent findings regarding the mechanisms of chemokine-receptor signaling in macrophages, the role of viral envelope glycoproteins in eliciting macrophage signals, and how these activation pathways may participate in macrophage infection and affect cell functions apart from infection.     

HIV-1 induces tumour necrosis factor and IL-1 gene expression in primary human macrophages independent of productive infection.

Collagen-induced arthritis has been widely used as an animal model of rheumatoid arthritis. We have used this model with a view to determining potential therapeutic targets for the treatment of human disease. To do this we have attempted to modulate the progression of established arthritis over a 10-day time period following the first appearance of disease, by i.p. injection of one of three different MoAbs. These consist of a rat IgG2a specific for the CD5 antigen expressed on all T cells and a subpopulation of B cells, a mouse IgG2b recognizing the CD72 antigen, and a rat IgM specific for the B220 molecule, CD72 and B220 both being expressed on all B cells. None of the three MoAbs had depleting activity in vivo. The progression of arthritis was monitored both clinically, and histologically. The effects of treatment with anti-CD5 and anti-CD72 antibodies were compared with control antibodies of the same species class and subclass. In the case of anti-B220 antibodies, the effects of treatment were compared with administration of PBS. Of these MoAbs, only treatment with anti-CD5 resulted in disease amelioration with significant decrease in disease severity in 60% of the animals. These changes became apparent 6 days after initiation of treatment. There were no significant differences in serum levels of IgG antibodies to native bovine collagen type II between the groups of treated and control mice. Possible mechanisms underlying the modification of disease expression following treatment with anti-CD5 MoAb are discussed.     

Structural requirements of glycosaminoglycans for their interaction with HIV-1 envelope glycoprotein gp120

Heparan sulfate proteoglycans are known to assist HIV-1 entry into host cells, mediated by the viral envelope glycoprotein gp120. We aimed to determine the general structural features of glycosaminoglycans that enable their binding to gp120, by surface plasmon resonance. Binding was found to be dependent on sequence type, size and sulfation patterns. HIV-1 gp120 prefers heparin and heparan sulfate (with at least 16 monomers in length) over chondroitin and dermatan. Sulfate groups were essential to promote this interaction. These results advance the understanding of the molecular-level requirements for virus attachment and cell entry.     

Increased Mycobacterium tuberculosis growth in HIV-1-infected human macrophages: role of tumour necrosis factor-alpha

Synergism between Mycobacterium tuberculosis (M. tuberculosis) and HIV-1 infections was demonstrated in several in vitro models and clinical studies. Here, we investigated their reciprocal effects on growth in chronically HIV-1-infected promonocytic U1 cells and in acutely infected monocyte-derived macrophages (MDM). Phagocytosis of M. tuberculosis induced HIV-1 expression in U1 cells, together with increased TNF-alpha production. M. tuberculosis growth, evaluated by competitive PCR, was greater in HIV-1-infected MDM compared to uninfected cells. M. tuberculosis phagocytosis induced greater TNF-alpha and IL-10 production in HIV-1-infected MDM than in uninfected cells. In uninfected MDM, addition of TNF-alpha and IFN-gamma decreased, whereas IL-10 increased M. tuberculosis growth. On the contrary, in HIV-1-infected MDM, addition of TNF-alpha and IFN-gamma increased, whereas IL-10 has no effect on M. tuberculosis growth. TNF-alpha seems to play a pivotal role in the enhanced M. tuberculosis growth observed in HIV-1-infected MDM, being unable to exert its physiological antimycobacterial activity. Here, for the first time we demonstrated an enhanced M. tuberculosis growth in HIV-1-infected MDM, in line with the observed clinical synergism between the two infections.     

The interaction of CD4 with HIV-1 gp120

CD4 is an integral cell surface glycoprotein that is able to enhance T cell specific antigen responses when it interacts with its physiological ligand, class II major histocompatibility (MHC) molecules. In addition, CD4 is a specific cell-surface receptor for the human immunodeficiency virus-1 (HIV-1). Infection by HIV-1 is initiated by the binding of the envelope glycoprotein, gp120, to the first domain of CD4. The binding of CD4 to class II MHC is inhibited by gp120, one possible mechanism for immunosuppression in AIDS patients. In addition, the CD4/gp120 interaction may directly inhibit T cell function. Recently we have synthesized small molecules (CPFs) that specifically inhibit this interaction. CPFs bind to gp120 and prevent the binding of gp120 to CD4, and also inhibit the infectivity of HIV-1.     

HIV-1 envelope glycoprotein 120 increases intercellular adhesion molecule-1 expression by human endothelial cells

Human immunodeficiency virus type 1 (HIV-1) infection is often associated with central nervous system damage and vascular complications. However, the mechanisms of this association are largely unknown. We examined the effect of HIV-1 envelope glycoprotein 120 (gp120) on cell adhesion molecule expression by endothelial cells. We found, for the first time, that both soluble and membrane-bound gp120 could significantly increase the expression of human endothelial intercellular adhesion molecule-1 (ICAM-1) at both mRNA and protein levels, but not vascular cell adhesion molecule-1 and E-selectin. The specificity of gp120-mediated response was demonstrated by blocking experiments using a specific monoclonal antibody against gp120, which successfully abolished the gp120-mediated increase of ICAM-1 expression. Furthermore, there was a significant increase of human monocytic cell line THP-1 adherence onto the gp120-treated endothelial monolayers. This increased cell adhesion was effectively blocked by either anti-gp120 or anti-ICAM antibodies. These findings suggest that HIV-1 gp120-mediated endothelial ICAM-1 expression could be one of the important mechanisms of HIV-1 pathogenesis.     

HIV-1 envelope protein gp120 expression by secretion in E. coli: assessment of CD4 binding and use in epitope mapping.

A non-glycosylated form of the HIV-1 envelope protein gp120 and four truncated derivatives have been expressed as non-fused secreted products in the periplasmic space of E. coli. We show that the full length molecule, whilst folded and soluble, fails to bind to CD4 consistent with other work that suggests an essential role for carbohydrate in gp120 function. In addition, when used in conjunction with the truncated derivatives, rapid epitope mapping of anti-gp120 monoclonal antibodies is achieved using both Western-blot and ELISA formats.     

Human MoAbs produced from normal, HIV-1-negative donors and specific for glycoprotein gp120 of the HIV-1 envelope.

Human MoAbs of IgM class were developed against three regions of the HIV-1 envelope. Uninfected donor lymphocytes were immunized in vitro with recombinant protein pB1. Four out of five antibodies were directed to different parts of the V3 region, which contains a major neutralizing site. Two out of these antibodies were directed to more than one amino acid sequence, indicating reactivity to discontinuous sites. Two of the human MoAbs inhibited viral spread between cells in tissue culture, interpreted as reactivities to conserved amino acid sequences exposed during viral maturation. No MoAb neutralized virus, which may be explained by the relatively low avidity of the antibodies. One MoAb was directed to a region containing amino acids participating in CD4 binding. This technique appears to allow formation of antibodies with fine specificities other than those obtained in infected hosts.     

人类免疫缺陷病毒I型包膜糖蛋白gp41的基因重组表达

目的 基因重组表达（ＨＩＶ－１ ｇｐ４１）抗原,并研制一种快速,简便,灵敏性高,特异性强的国产ＨＩＶ－１免疫检测试剂。方法 选用ＨＩＶ－１型ＢＨ１０毒株的包膜糖蛋白ｇｐ４１的部分基因（６９７７－７４９７）,重组在ＰＢＶ２２１表达载体上。表达产物通过１５％ＳＤＳ－聚丙烯酰胺凝胶电泳初步分离纯化,根据ＲＦ值,切下含特异蛋白的胶带,以Ｗｅｓｔｅｒｎ ｂｌｏｔ法转移在硝酸纤维素膜上;     

HIV-1 gp120 induces anergy in naive T lymphocytes through CD4-independent protein kinase-A-mediated signaling

The ability of the envelope glycoprotein gp120 [human immunodeficiency virus (HIV) env] to induce intracellular signals is thought to contribute to HIV-1 pathogenesis. In the present study, we found that the exposure of CD4+ CD45RA+ naive T cells to HIVenv results in a long-lasting hyporesponsiveness to antigen stimulation. This phenomenon is not dependent on CD4-mediated signals and also can be generated by the exposure of naive T cell to soluble CD4-HIVenv complexes. The analysis of the proximal signaling reveals that HIVenv does not activate Lck as well as the mitogen-activated protein kinase intermediate cascade. Conversely, the envelope glycoprotein stimulates the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activity and induces the progressive accumulation of the phosphorylated form of the cAMP-responsive element binding. Of note, the ligation of CXCR4 by stromal cell-derived factor-1alpha but not the engagement of CD4 by monoclonal antibody stimulates the PKA activity and induces a long-lasting hyporesponsivity state in naive CD4+ lymphocytes. The pretreatment of lymphocytes with H89, a cell-permeable PKA inhibitor, prevents the induction of anergy. These findings reveal a novel mechanism by which HIVenv may modulate the processes of clonal expansion, homeostatic proliferation, and terminal differentiation of the naive T lymphocyte subset.     

Human immunodeficiency virus type 2 envelope glycoprotein: differential CD4 interactions of soluble gp120 versus the assembled envelope complex.

Utilizing a recombinant vaccinia expression system, we investigated the biological properties and CD4 receptor interactions of the envelope glycoproteins of a noncytopathic human immunodeficiency virus type 2 strain, termed HIV-2/ST, and a highly cytopathic variant derived from it. The efficiency and host cell range of syncytium formation by the recombinant glycoproteins of both viruses were highly restricted compared to those of prototypic strains of HIV (HIV-2/ROD or HIV-1/IIIB). However, the glycoprotein of cytopathic but not wild-type ST generated numerous large syncytia in the human T-cell line Sup T1 from which it was derived. A single cell line (Molt 4 clone 8) was permissive to fusion by both wild-type and cytopathic ST envelopes, but only the glycoprotein of cytopathic ST could be inhibited with a soluble form of the viral receptor CD4 (sCD4). While these results indicated major differences in the envelope glycoprotein-CD4 receptor interactions of wild-type versus cytopathic ST, direct and competition binding assays utilizing soluble external glycoprotein (SU) and sCD4 surprisingly revealed equivalent low binding affinity for both viruses. From these experiments we conclude that relevant biological properties (e.g., CD4 binding, cytopathic potential, and sCD4 neutralization) of HIV viruses which differ in their pathogenic potential are reflected in the sCD4 interactions of the assembled native envelope complex (as on cell or virion surfaces) but not the soluble SU glycoprotein.     

Factors limiting the immunogenicity of HIV-1 gp120 envelope glycoproteins

Efficient immune responses to HIV-1 gene products are essential elements to the development and design of an effective vaccine. Ideally, both humoral and cellular responses will be optimally elicited. It is therefore important to elucidate any factors that might limit the immunogenicity of HIV-1 proteins that are likely to be included in an effective vaccine. Since the HIV-1 exterior envelope glycoprotein gp120 is a major target for neutralizing antibodies, it is a virtual certainty that this gene product will be a component of any vaccine that seeks to elicit neutralizing antibody responses from the host humoral immune system. We report here the testing of several HIV-1 gp120 variants derived from a primary isolate that appears deficient in eliciting immune responses at both the level of CD4+ help and consequently in the generation of high-affinity IgG antibody responses in small animals. Factors limiting an effective immune response include (a) envelope glycoprotein strain variation decreasing functional T-cell help, (b) alteration of the glycosylation patterns of gp120 by expression in different cell types, and (c) the native structure of gp120 itself, which may limit the elicitation of effective T-cell help during natural infection or during parenteral immunization in adjuvant. Such limiting factors and others should be considered in the design and testing of gp120-based immunogens in small animals and possibly in primates as well.     

HIV-1 glycoprotein gp120 disrupts CD4-p56lck/CD3-T cell receptor interactions and inhibits CD3 signaling

Using the CD4⁺ human T cell clone P28, we demonstrated that the HIV-1 glycoprotein gp120 inhibited CD3-induced inositol trisphosphate production, calcium influx and T cell proliferation. Additionally, gp120 was shown to dissociate the tyrosine kinase p56^lck from CD4 in CEM cells, with a concommittant inhibition of CD4-linked kinase activity. We have addressed the question whether disruption of CD4/p56^lck or CD4/CD3-T cell receptor interactions, or both, could account for the inhibitory effect of gp120 in P28 cells. By comparing the effects of various anti-CD4 monoclonal antibodies (mAb) with those of gp120, we show that gp120 and IOT4a modulate CD4 expression, and decrease CD4-associated p56^lck and CD4-linked kinase activity at the plasma membrane. In contrast, OKT4A and OKT4 anti-CD4 mAb have no inhibitory effect. Interestingly, gp120 also inhibits CD3-induced Lck activation and cellular tyrosine phosphorylation, particularly of phosphoinositide-specific phospholipase C-γ-1. Kinetic experiments reveal that the inhibitory effect of gp120 on CD3-induced tyrosine phosphorylation appears as early as 30 min, but culminate when CD4-p56^lck complexes disappear from the cell surface after 4 h. These results suggest that a negative signal is triggered by gp120 that results, after a few hours, in down-modulation of CD4-p56^lck complexes and the impairment of CD3 signaling. Supporting this hypothesis, gp120 inhibits CD3-linked kinase activity as shown by the inhibition of the phosphorylation of CD3 chains, leading to the inhibition of subsequent signal transduction.     

HIV-1糖蛋白gp120激发人小胶质细胞钙内流效应和ERK磷酸化

目的： 探讨HIV-1糖蛋白gp120对人小胶质细胞钙离子内流和ERK磷酸化的作用及其机制。方法: 用钙离子探针Fluo-4标记粘附在盖玻片上的人小胶质细胞，运用共聚焦显微镜以荧光强度为指标实时观察各种条件下的细胞内钙离子水平的变化；用gp120处理并用anti-gp120-FITC进行染色，运用共聚焦显微镜术和流式细胞术分析人小胶质细胞与gp120结合情况；用抗磷酸化ERK 抗体免疫荧光方法进行染色，运用共聚焦显微镜术和流式细胞术进行ERK磷酸化水平分析。结果: 共聚焦显微镜检测结果显示HIV-1 糖蛋白gp120能够激发人小胶质细胞钙离子内流效应；共聚焦显微镜和流式细胞仪分析结果显示gp120可以与人小胶质细胞结合；共聚焦显微镜和流式细胞仪分析结果显示gp120刺激可增加人小胶质细胞ERK磷酸化。结论: HIV-1 糖蛋白gp120能在人小胶质细胞激发钙离子内流并且增加胞内ERK的磷酸化，从而导致了小胶质细胞的活化，这一效应提示，在HIV-1相关性脑炎中，gp120可能参与了某些发病机制。     

HIV-1 gp120 induces NFAT nuclear translocation in resting CD4+ T-cells

The replication of human immunodeficiency virus (HIV) in CD4+ T-cells is strongly dependent upon the state of activation of infected cells. Infection of sub-optimally activated cells is believed to play a critical role in both the transmission of virus and the persistence of CD4+ T-cell reservoirs. There is accumulating evidence that HIV can modulate signal-transduction pathways in a manner that may facilitate replication in such cells. We previously demonstrated that HIV gp120 induces virus replication in resting CD4+ T cells isolated from HIV-infected individuals. Here, we show that in resting CD4+ T-cells, gp120 activates NFATs and induces their translocation into the nucleus. The HIV LTR encodes NFAT recognition sites, and NFATs may play a critical role in promoting viral replication in sub-optimally activated cells. These observations provide insight into a potential mechanism by which HIV is able to establish infection in resting cells, which may have implications for both transmission of HIV and the persistence of viral reservoirs.