Role of curdlan sulfate in the binding of HIV-1 gp120 to CD4 molecules and the production of gp120-mediated TNF-alpha

In this paper the costs and benefits associated with DNA diagnosis of subjects who are at risk of having a child with a monogenic disease and who seek genetic counselling because of their reproductive plans are predicted under various assumptions using a mathematical model. Four monogenic diseases have been considered: cystic fibrosis, Duchenne muscular dystrophy, myotonic dystrophy, and fragile X syndrome. Counselling (triggered by previous information) on the basis of DNA diagnosis is compared to the situation that only risk evaluation based on pedigree analysis is possible. The results show for each disease that with DNA diagnosis, couples can be more confident in choosing (further) offspring leading to the birth of more healthy children while the number of affected children is reduced. The costs minus savings within the health care sector depend on the prior risks and on the future burden of the monogenic illness under consideration. DNA diagnosis of relative "low" prior risks of a child with CF (for example, 1:180, 1:240 and 1:480) leads to costs instead of savings. For higher prior risks of CF and for the three other diseases, DNA diagnosis produces considerable savings. This result remains valid when assumptions regarding behaviour, reproduction, and receiving DNA diagnosis under different circumstances are varied.     

HIV-1 gp120: a novel viral B cell superantigen

The envelope glycoprotein of the human immunodeficiency virus (HIV-1), gp120, has recently been characterized as a novel immunoglobulin superantigen (Ig-SAg) [1,2]. Analogous to the interaction of SAgs with T cells, gp120 binds to an unusually large proportion of immunoglobulins (lgs) from HIV-uninfected individuals; most, if not all of these Igs are members of the VH3 family [3]. Functionally, gp120 preferentially stimulates VH3 B cells in vitro. This stimulation correlates with an in vivo VH3 activation during HIV infection. Curiously, this initial activation is followed by a subsequent depletion of VH3-expressing B cells as individuals progress to AIDS. In this article we will review our current understanding of the superantigenic properties of HIV gp120. Specifically we will focus on structural aspects of the binding interaction. on the ontological development of these superantigen-binding antibodies, and on potential roles that this unconventional Ig-pathogen interaction might play in the pathogenesis of HIV-induced disease.     

Identification of the B cell superantigen-binding site of HIV-1 gp120

Previous studies showed that the gp120 envelope protein of HIV-1 is able to crosslink membrane IgM on normal human B cells and to induce their activation in a V(H)3 immunoglobulin gene-family-specific manner. Because this V(H) gene family is the largest in the human repertoire, this superantigen (SAg) property is thought to have deleterious consequences for the host, including a progressive decline of B cells with progression of the HIV-1-induced disease. Here, we have identified the sequence motifs on gp120 involved in SAg binding to normal Igs. We show that this SAg-binding activity is present in gp120s from highly divergent isolates of HIV-1 belonging to clades derived from various geographical origins, and that carbohydrate residues are not essential for its expression. The SAg-binding site is formed by protein sequences from two regions of the gp120 molecule. The core motif is a discontinuous epitope spanning the V4 variable domain and the amino-terminal region flanking the C4 constant domain. The most critical residues appear to be Leu395-Asp397 and Ile425-Gln427. Residues from the C2 constant domain (positions 252-272) also seem to play an accessory role in SAg binding of gp120 to normal human Igs. These findings are important in the design of a successful gp120-based vaccine against HIV-1.     

The recognition of chimeras of rat and human CD4 by HIV-1 gp120 and by monoclonal antibodies

The use of chimeras of rat and human CD4 to probe the HIV-1 gp120 and antibody binding properties of CD4 is reviewed. Short segments of human CD4 sequence were substituted for the equivalent regions of rat CD4 which does not bind gp120, and analysis of the properties of these chimeras established: (i) that residues 33-58 of the NH2-terminal domain of human CD4 encompass the high-affinity gp120 binding site; and (ii) that chimeras containing residues 33-62 mediate HIV-1 infection. The chimera-binding specificities of gp120 and a large panel of anti-CD4 antibodies were also determined. This allowed a critical test of the popular notion that receptor mimics appear at high frequency among antibodies elicited by immunization with receptor ligands and that anti-idiotypic antibodies can be used to identify novel receptors. The data suggest that such mimics appear infrequently, if at all, a result which is consistent with the failure of the anti-idiotype approach to identify new genes encoding receptors with prescribed functions.     

Mechanisms of prionSc- and HIV-1 gp120 induced neuronal cell death

In vitro experiments revealed that the scrapie prion protein, PrP(Sc), as well as the PrP fragment PrP106-126, and the HIV-1 coat protein gp120 induce apoptosis of rat cortical neurons. The toxic effect displayed by PrP and gp120 could be blocked by NMDA receptor antagonists. Treatment of neuronal cells with PrP106-126 resulted in a drop of intracellular glutathione level and changes in the level of Bcl-2. Evidence is presented that gp120 causes an activation of phospholipase A2, resulting in the increased release of arachidonic acid, which may in turn sensitize the NMDA receptor.     

Immunogenicity of synthetic HIV-1 gp120 V3-loop peptide-conjugate immunogens

A successful prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine must elicit an immune response that will prevent establishment of the persistent viral infection. The only response shown to be effective in this regard is virus-neutralizing antibody directed against the viral gp120 hypervariable V3-loop region. Conjugate immunogens, containing cyclic peptides representing the V3 determinant covalently bound to a carrier protein, were capable of eliciting virus-neutralizing antibodies. The consistency of the response was related to peptide size. The smaller cyclic peptides, expressing relatively conserved sequences from the V3-loop apex, were poor inducers of neutralizing activity. In contrast, the largest cyclic peptides mediated neutralizing responses that were similar to those observed and previously reported for intact gp120 immunogens. A cyclic synthetic peptide expressing most of the prototypic HIV-1 MN variant V3 determinant warrants further study as a potentially effective vaccine immunogen.     

Dendritic cells from HIV-1-infected patients naturally express HIV-1 gp120 V3 loop-derived peptide ligands

Little is known of the peptide ligands expressed in vivo on antigen-presenting cells (APC) or of the APC lineages involved. In this study we have addressed this question using HLA-DRbeta1*0101-restricted CD4 T cell clones (TLC) specific for a synthetic peptide based on the HIV-1 gp120 V3 loop consensus sequence for the Clade B isolates predominantly found in European and North American patients. These TLC were found to respond, in a dose-dependent manner, to freshly isolated HIV-infected patient APC in the absence of exogenously added peptides. Further APC purification showed that the naturally expressed peptide ligands were present in both the APC lineages shown to be infected with the virus and were most strongly detectable on purified blood dendritic cells. Peptides based on consensus sequences of viruses isolated from one of the patients over the period when naturally expressed peptide ligands could be detected were all found to stimulate TLC proliferation. These studies, therefore, show that peptide ligands derived from natural infection are detectable on APC lineages, particularly on dendritic cells which play an important role in the immune response to viruses. Even small differences in sequence between the vaccine isolate and the natural infection, if they occur in the key residues of protective T cell epitopes, could therefore have a profound effect on the efficacy of vaccines against viruses with high rates of mutation.     

gp 120s derived from four syncytium-inducing HIV-1 strains induce different patterns of CD4 association with lymphocyte surface molecules

This work extends our previous finding that lymphocyte treatment with gp120IIIB specifically induces CD4 association with several surface molecules to other molecules and to three other gp120s from different HIV-1 strains. The ability to induce this association was displayed by the four gp120s employed, i.e. gp120IIIB, gp120SF2, gp120MN and gp120(451), and the association patterns were different, as shown by both co-capping and immunoprecipitation. Co-capping showed that all four gp120s significantly potentiated CD4 association with CD3, CD45RA, CD45RB, CD38, CD26, CD59 and class I MHC molecules. By contrast, CD4 association with CD95 was induced only by gp120(451) and gp120MN; that with CD11a only by gp120SF2 and gp120MN; and that with CD27 and CD45RO only by gp120MN and gp120(451) respectively. All gp120s induced significant CD4 association with CD49d, but gp120SF2 displayed a significantly weaker effect than gp120IIIB. Induction of association was not mediated by inside-out signaling via the CD4-associated tyrosine kinase p58lck, since it was not inhibited by the tyrosine kinase inhibitors herbymicin and genistein, nor by CD45 bridging between CD4 and the associating molecule, since similar patterns of association were detected IN cells expressing different CD45 isoform patterns. Moreover, it was not mediated by chemokine receptors interacting with the gp120 V3 loop, since RANTES did not alter the gp120-induced CD4 association pattern. By contrast, the observation that gp120s from four HIV-1 strains induce different CD4 association patterns suggests that gp120 directly interacts with the associating molecules, possibly via their hypervariable regions.     

Characterization of the MN gp120 HIV-1 vaccine: antigen binding to alum

PURPOSE: The characterization of recombinant MN gp120/alum vaccine requires the study of the gp120-alum interaction for the successful formulation of an alum-based HIV-1 vaccine. METHODS: Several observations suggest that the gp120-alum interaction is weak, wherein buffer counterions such as phosphate, sulfate, bicarbonate may cause the desorption of gp120 from alum. Comparison of gp120 with other proteins using particle mobility measurements shows that the weak binding of gp120 to alum is not an anomaly. Serum and plasma also cause desorption of gp120 from alum with a half-life of only a few minutes, wherein this half-life may be faster than the in-vivo recruitment of antigen presenting cells to the site of immunization. RESULTS: Immunization of guinea pigs, rabbits and baboons with gp120 formulated in alum or saline demonstrated that alum provides adjuvant activity for gp120, particularly after early immunizations, but the adjuvant effect is attenuated after several boosts. CONCLUSIONS: These observations indicate that both the antigen and the adjuvant require optimization together.     

Induction of lymphomonocyte activation by HIV-1 glycoprotein gp120. Possible role in AIDS pathogenesis

Dysfunction of cytokine secretion pattern has been suggested to play a central role in the immunopathogenesis of HIV infection. In fact a shift of T helper cell functions from a Th1-type to TH0- or TH2-type has been observed in HIV-1 infected subjects undergoing disease progression. The inhalance of cytokine network is accompanied by persistent activation of the immune system, impaired ability to mount a proper activation response (anergy), and priming to apoptosis. Extensive investigation during the last decade has been conducted on the influence of HIV-1 gp120 or of its precursor gp160 on several lymphocyte and monocyte functions. Gp120 is able to rise intracellular calcium concentration and to induce the formation of inositol triphosphate, can block mitogen- or antigen-driven T cell activation, can induce altered cytokine production by activated PBMC subpopulations, determines impaired cytotoxicity and chemotactic response to antigens, interferes with the activity of antigen presenting cells, enhances or induces apoptosis, stimulates polyclonal B cell activation and induces or up-modulates a number of cytokines, including IL-6. TNF, IL-1-alpha and -beta, IL-10 and IL-8. Furthermore, both IFN-alpha and -gamma, as well as several markers of IFN activity, such as beta 2-microglobulin and neopterin, are induced in gp120-stimulated PBMC. However, neither IL-4 (Th2-type) nor IL-2 (Th1-type), nor DNA synthesis are activated by gp120. On the other hand gp120-stimulated PBMC express increased IL-2 receptors, and can be induced by exogenous IL-2 to proliferate, suggesting that they are in a state of at least partial activation. According to this hypothesis, other activation markers, both early (such as CD69), and late (such as CD45RO and CD71), are induced by gp120, but this even partial activation does not lead to the ability of PBMC to support productive infection by HIV-1, unless in the presence of exogenous IL-2. The HIV-induced cytokines can influence HIV infection either directly, by up- or down-modulating virus replication, or indirectly, by modulating the expression of cellular molecules. In fact, during the budding process, the HIV envelope captures a number of cell membrane proteins, including cytokine receptors such as IL-2R, adhesion molecules such as LFA-1, ICAM-1, -2, HLA Class I and II, as well as cell lineage markers. Gp120-induced cytokines, particularly IFN-gamma, upmodulate the cellular expression of intercellular adhesion molecules, such as ICAM-1. We have shown that the IFN-gamma-driven increase of the expression of ICAM-1 by cells chronically infected with HIV-1 can be transmitted to the virus progeny, resulting in phenotypic alteration of the virus, and leading to the expansion of its host cell spectrum to CD4-negative cells expressing the appropriate ligands, i.e. LFA-1. Intercellular adhesion molecules are also involved in the cell-mediated transmission of HIV infection, and the increased ICAM-1 expression induced by IFN-gamma determines a stimulation of the transmission of HIV from abortively infected endothelial cells to permissive CD4 lymphocytes. On the whole, these data indicate that HIV, or its soluble products such as gp120, can modify several PBMC functions, by inducing a number of cytokines and a partial state of immune activation. It is possible that the gp120-driven changes of PBMC functions are not only an epiphenomenon of HIV infection, but rather, it is likely that they can participate in the immunopathological events responsible for disease progression.     

The HIV-1 gp120 causes ultrastructural changes typical of apoptosis in the rat cerebral cortex

We used transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labelling (TUNEL) techniques to study the neuropathological effects of intracerebroventricular (i.c.v.) injection of recombinant HIV-1 gp 120 in rats. In brain cortical tissue sections from rats treated with a single daily dose of gp120 (100 ng day-1 for 7 or 14 consecutive days) TEM analysis showed chromatin compaction and marginalization along the inner surface of the nuclear envelope followed by masses of condensed chromatin, ultrastructural signs demonstrating the occurrence of apoptotic cell death. These effects were paralleled by in situ DNA fragmentation, as revealed by application of TUNEL technique to cryostat brain tissue sections from rats treated likewise with the viral coat protein. In no instance was apoptosis seen in the brain cortex of control rats. The present data demonstrate that gp120 given i.c.v. produces apoptosis in the neocortex of rats.     

HIV-1 gp120 modulates hypothalamic cytokine mRNAs in vivo: implications to cytokine feedback systems

HIV-1-derived envelope glycoprotein 120 (gp120) may play an important role in HIV-1 neuropathology. Gp120 may act through mediators including proinflammatory cytokines. Here, we investigated the regulation of the IL-1 beta system [IL-1 beta, IL-1 receptor type I (IL-1RI), IL-1 receptor antagonist (IL-1Ra), IL-1 receptor accessory proteins (IL-1R AcP I and II)], TNF-alpha, TGF-alpha, and TGF-beta 1 mRNAs in the hypothalamus of Wistar rats in response to the chronic intracerebroventricular (ICV) microinfusion (via osmotic minipumps) of HIV-1 gp120 (100, 500, and 1000 ng/24 h for 72 h). Gp120 increased IL-1 beta, IL-1Ra, TNF-alpha, and TGF-beta 1 mRNAs. Gp120-induced cytokine mRNA profiles were highly intercorrelated in the same samples. Levels of IL-1RI, IL-1R AcP I and II, and TGF-alpha did not change significantly, and levels of GAPDH mRNA were constant. The data suggest potential cytokine-cytokine interactions with positive (IL-1 beta<-->TNF-alpha) and negative (IL-1Ra-->IL-1 beta; TGF-beta 1-->IL-1 beta/TNF-alpha) feedback in gp120 action. A dysregulation of the balance between stimulatory and inhibitory cytokine mechanisms may participate in the initiation, propagation, and/or aggravation of HIV-1 neuropathology.     

Stereochemical analysis of the antigenic tip of the V3 loop peptide of HIV-1 gp120

A novel multiple turn conformation has been observed for a segment GPGRAFY in the crystal structure of a complex of HIV-1 gp120 V3 loop peptide with the Fab fragment of a neutralizing antibody [Ghiara et al. (1994) Science 264, 82-85]. A structural motif has been defined for the peptide segment, employing idealized backbone conformations characterized by ranges of virtual C alpha torsion angles and bond angles. A search of 122 high-resolution protein crystal structures has permitted identification of 24 examples of similar structural motifs. Two major conformational families have been identified, which differ primarily in the conformation at residue 3. The observed conformation at residue 3 in family 1 is left-handed helical (alpha L) and that in family 2 is right-handed helical (alpha R). Of the 10 examples in family 1, 9 examples have Gly residues at position 3. Of the 12 examples in family 2, 7 examples have Asn/Asp at position 3. Computer modeling of the V3 loop tip sequence using the two backbone conformational families as starting points leads to minimum-energy conformations in which antigenically important side-chains occupy similar spatial arrangements. This stereochemical analysis of the V3 loop tip sequence suggests a rational basis for the design of synthetic analog peptides for use as viral antagonists or synthetic antigens.     

Potent neutralization of primary HIV-1 isolates by antibodies directed against epitopes present in the V1/V2 domain of HIV-1 gp120

Although it is known that some human immune sera possess potent neutralizing activities for primary viruses, the identity of the target epitopes mediating this neutralization is unknown, and currently available immunogens have not been able to induce such activities. Using recombinant fusion glycoproteins expressing native V1/V2 domains of gp120 we have found that sera from a subset of HIV-1-infected humans contain antibodies that recognize broadly conserved V1/V2 epitopes. Such antibodies were isolated from one human serum by affinity chromatography on a column containing a V1/V2 fusion protein, and shown to efficiently neutralize several macrophage-tropic HIV-1 isolates. Rodents immunized with the purified V1/V2 fusion protein produced antibodies reactive with unrelated V1/V2 fusion proteins and with heterologous gp120s. V1/V2-specific immunoglobulins isolated from sera of these animals by affinity chromatography also possessed potent neutralization activity for several primary HIV-1 isolates. These results indicate that the V1/V2 domain of HIV-1 gp120 contains conserved epitopes that mediate potent neutralization of primary viruses, and suggest that subunit vaccines that efficiently induce such antibodies may provide protective humoral immunity against clinically relevant HIV-1 isolates.     

Modification of delivery system enhances MHC nonrestricted immunogenicity of V3 loop region of HIV-1 gp120

A successful peptide vaccine for AIDS is desired to elicit T-helper and cytotoxic T lymphocyte responses besides neutralizing antibodies. The V3 loop peptide of HIV-1 has been shown to contain the principal neutralizing domain, one of the most immunodominant regions, having both B-cell and T-cell determinants. In this study, the tip of the V3 loop region was mutated from GPGR to GPGQ based on the sequence of Indian isolates (CKRKIHIGPGQAFYT). To further enhance the immunogenicity of this epitope, two delivery systems of immune stimulating complexes (ISCOMs) and liposomes were used to incorporate the peptide. Mice of differing haplotypes, H-2b, H-2d, H-2k and H-2s, showed no MHC restriction when immunized with these formulations. The IgG levels as assessed by ELISA were found to be significantly higher (P < 0.05 to P < 0.001) for even five-fold lower doses of the peptide in ISCOMs and liposomes as compared to the conventional alum-based preparation. The major subtype elicited was IgG2a/IgG2b, suggestive of a Th1-like response for all the formulations. Thus, it would appear that the same peptide incorporated in ISCOMs and liposomes selects a Th1 response and may therefore be important not only for neutralization but also for virus clearance.     

Multiple effects of CD4 CDR3-related peptide derivatives showing anti-HIV-1 activity on HIV-1 gp120 functions

The interaction of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 with CD4 CDR3-related peptide derivatives showing anti-HIV-1 activity has been studied. Conformational changes in gp120, which could affect its interaction with CD4 and its shedding from virions, were detected by fluorescence spectrum analysis of tryptophan residues after addition of peptide representative of the CD4 CDR3-related region, but not the CD4 CDR2-related region. Interestingly, the addition of scrambled peptide, S1 (with altered amino acid sequence compared with the native CDR3-related peptide but unaltered overall composition), which we recently showed to have stronger anti-HIV-1 activity than the original CDR3-related peptide, had no effects on the conformational change in gp120 or on its interaction with CD4 and its shedding from HIV-1 virions. However, all of the CDR3-related peptides, including S1, showed blocking effects on the binding of antibodies against gp120 V3 loop and C-terminus regions. Thus, we concluded that there were at least two separable activities of the CDR3-related peptides in anti-HIV-1 activity, i.e. induction of conformational changes in gp120, which could affect its binding to CD4 and to gp41 (as observed in native CDR3-related peptides), and inactivation of V3 loop and C-terminus regions in gp120 (as observed in all of the CDR3-related peptides, including S1).     

HIV-1 envelope gp120 inhibits the monocyte response to chemokines through CD4 signal-dependent chemokine receptor down-regulation

Since HIV-1 infection results in severe immunosuppression, and the envelope protein gp120 has been reported to interact with some of the chemokine receptors on human T lymphocytes, we postulated that gp120 may also affect monocyte activation by a variety of chemokines. This study shows that human peripheral blood monocytes when preincubated with gp120 either purified from laboratory-adapted strains or as recombinant proteins exhibited markedly reduced binding, calcium mobilization, and chemotactic response to chemokines. The gp-120-pretreated monocytes also showed a decreased response to FMLP. This broad inhibition of monocyte activation by chemoattractants required interaction of gp120 with CD4, since the effect of gp120 was only observed in CD4+ monocytes and in HEK 293 cells only if cotransfected with both chemokine receptors and an intact CD4, but not a CD4 lacking its cytoplasmic domain. Anti-CD4 mAbs mimicked the effect of gp120, and both anti-CD4 Ab and gp120 caused internalization of CXCR4 in HEK 293 cells provided they also expressed CD4. Staurosporine blocked the inhibitory effect of gp120 on monocytes, suggesting that cellular signaling was required for gp120 to inhibit the response of CD4+ cells to chemoattractants. Our study demonstrates a broad suppressive effect of gp120 on monocyte activation by chemoattractants through the down-regulation of cell surface receptors. Thus, gp120 may be used by HIV-1 to disarm the monocyte response to inflammatory stimulation.     

The V3 domain of the HIV-1 gp120 envelope glycoprotein is critical for chemokine-mediated blockade of infection

The ability of CD8 T cells derived from human immunodeficiency virus (HIV)-infected patients to produce soluble HIV-suppressive factor(s) (HIV-SF) has been suggested as an important mechanism of control of HIV infection in vivo. The C-C chemokines RANTES, MIP-1 alpha and MIP-1 beta were recently identified as the major components of the HIV-SF produced by both immortalized and primary patient CD8 T cells. Whereas they potently inhibit infection by primary and macrophage-tropic HIV-1 isolates, T-cell line-adapted viral strains tend to be insensitive to their suppressive effects. Consistent with this discrepancy, two distinct chemokine receptors, namely, CXCR4 (ref. 7) and CCR5 (ref. 8), were recently identified as potential co-receptors for T-cell line-adapted and macrophage-tropic HIV-1 isolates, respectively. Here, we demonstrate that the third hypervariable domain of the gp 120 envelope glycoprotein is a critical determinant of the susceptibility of HIV-1 to chemokines. Moreover, we show that RANTES, MIP-1 alpha and MIP-1 beta block the entry of HIV-1 into cells and that their antiviral activity is independent of pertussis toxin-sensitive signal transduction pathways mediated by chemokine receptors. The ability of the chemokines to block the early steps of HIV infection could be exploited to develop novel therapeutic approaches for AIDS.