The alpha(1,2)-mannosidase I inhibitor 1-deoxymannojirimycin potentiates the antiviral activity of carbohydrate-binding agents against wild-type and mutant HIV-1 strains containing glycan deletions in gp120

Exposure of carbohydrate-binding agents (CBAs) (i.e. the mannose-specific plant lectins Hippeastrum hybrid agglutinin and Galanthus nivalis agglutinin) to HIV-1 progressively select for mutant HIV-1 strains that contain N-glycan deletions in their envelope gp120. This results in resistance of the mutant virus strains to the CBAs. Exposure of such mutant virus strains to the alpha(1,2)-mannosidase I inhibitor 1-deoxymannojirimycin (DMJ) results in an enhanced suppression of mutant virus-induced cytopathicity in CEM cell cultures. Moreover, when combined with CBAs at concentrations that showed poor if any suppression of mutant virus replication as single drugs, a synergistic antiviral activity of DMJ was observed. These observations argue for a combined exposure of CBAs and glycosylation inhibitors such as DMJ to HIV to afford a more pronounced suppression of virus replication, prior to, or during, CBA resistance development.     

Mutational pathways, resistance profile, and side effects of cyanovirin relative to human immunodeficiency virus type 1 strains with N-glycan deletions in their gp120 envelopes

Limited data are available on the genotypic and phenotypic resistance profile of the alpha-(1-2)mannose oligomer-specific prokaryotic lectin cyanovirin (CV-N). Therefore, a more systematic investigation was carried out to obtain a better view of the interaction between CV-N and human immunodeficiency virus type 1 (HIV-1) gp120. When HIV-1-infected CEM cell cultures were exposed to CV-N in a dose-escalating manner, a total of eight different amino acid mutations exclusively located at N-glycosylation sites in the envelope surface gp120 were observed. Six of the eight mutations resulted in the deletion of high-mannose type N-glycans (i.e., at amino acid positions 230, 332, 339, 386, 392, and 448). Two mutations (i.e., at position 136 and 160) deleted a complex type N-glycan in the variable V1/V2 domain of gp120. The level of phenotypic resistance of the mutated virus strains against CV-N generally correlated with the number of glycan deletions in gp120, although deletion of the glycans at N-230, N-392, and N-448 generally afforded a more pronounced CV-N resistance than other N-glycan deletions. However, the extent of the decrease of antiviral activity of CV-N against the mutated virus strains was markedly less pronounced than observed for alpha(1-3)- and alpha(1-6)-mannose-specific plant lectins Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA), which points to the existence of a higher genetic barrier for CV-N. This is in agreement with a more consistent suppression of a wider variety of HIV-1 clades by CV-N than by HHA and GNA. Whereas the antiviral and in vitro antiproliferative activity of CV-N can be efficiently reversed by mannan, the pronounced mitogenic activity of CV-N on peripheral blood mononuclear cells was unaffected by mannan, indicating that some of the observed side effects of CV-N are unrelated to its carbohydrate specificity/activity.     

The role of <Emphasis Type="Italic">N</Emphasis>-glycans of HIV-1 gp41 in virus infectivity and susceptibility to the suppressive effects of carbohydrate-binding agents

Background

Carbohydrate-binding agents (CBAs) are potent antiretroviral compounds that target the N-glycans on the HIV-1 envelope glycoproteins. The development of phenotypic resistance to CBAs by the virus is accompanied by the deletion of multiple N-linked glycans of the surface envelope glycoprotein gp120. Recently, also an N-glycan on the transmembrane envelope glycoprotein gp41 was shown to be deleted during CBA resistance development.

Results

We generated HIV-1 mutants lacking gp41 N-glycans and determined the influence of these glycan deletions on the viral phenotype (infectivity, CD4 binding, envelope glycoprotein incorporation in the viral particle and on the transfected cell, virus capture by DC-SIGN⁺ cells and transmission of DC-SIGN-captured virions to CD4⁺ T-lymphocytes) and on the phenotypic susceptibility of HIV-1 to a selection of CBAs. It was shown that some gp41 N-glycans are crucial for the infectivity of the virus. In particular, lack of an intact N616 glycosylation site was shown to result in the loss of viral infectivity of several (i.e. the X4-tropic III_B and NL4.3 strains, and the X4/R5-tropic HE strain), but not all (i.e. the R5-tropic ADA strain) studied HIV-1 strains. In accordance, we found that the gp120 levels in the envelope of N616Q mutant gp41 strains NL4.3, III_B and HE were severely decreased. In contrast, N616Q gp41 mutant HIV-1_ADA contained gp120 levels similar to the gp120 levels in WT HIV-1_ADA virus. Concomitantly deleting multiple gp41 N-glycans was often highly detrimental for viral infectivity. Using surface plasmon resonance technology we showed that CBAs have a pronounced affinity for both gp120 and gp41. However, the antiviral activity of CBAs is not dependent on the concomitant presence of all gp41 glycans. Single gp41 glycan deletions had no marked effects on CBA susceptibility, whereas some combinations of two to three gp41 glycan-deletions had a minor effect on CBA activity.

Conclusions

We revealed the importance of some gp41 N-linked glycans, in particular the N616 glycan which was shown to be absolutely indispensable for the infectivity potential of several virus strains. In addition, we demonstrated that the deletion of up to three gp41 N-linked glycans only slightly affected CBA susceptibility.

     

Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4⁺ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential.     

Glycosylation is necessary for the correct folding of human immunodeficiency virus gp120 in CD4 binding.

Conflicting results have been reported regarding the role of carbohydrate on human immunodeficiency virus (HIV) envelope glycoprotein gp120 in CD4 receptor binding. Glycosylated, deglycosylated, and nonglycosylated forms of HIV type 1 (HIV-1) and HIV-2 gp120s were used to examine CD4 receptor-binding activity. Nonglycosylated forms of gp120 generated either by deletion of the signal sequence of HIV-1 gp120 or by synthesis in the presence of tunicamycin failed to bind to CD4. In contrast, highly mannosylated gp120 bound to soluble CD4 molecules well. Enzymatic removal of carbohydrate chains from glycosylated gp120 by endoglycosidase H or an endoglycosidase F/N glycanase mixture had no effect on the ability of gp120 to bind CD4. An experiment which measured the ability of gp120 to bind to CD4 as an assay of the proper conformation of gp120 showed that carbohydrate chains on gp120 are not required for the interaction between gp120 and CD4 but that N-linked glycosylation is essential for generation of the proper conformation of gp120 to provide a CD4-binding site.     

Profile of resistance of human immunodeficiency virus to mannose-specific plant lectins

The mannose-specific plant lectins from the Amaryllidaceae family (e.g., Hippeastrum sp. hybrid and Galanthus nivalis) inhibit human immunodeficiency virus (HIV) infection of human lymphocytic cells in the higher nanogram per milliliter range and suppress syncytium formation between persistently HIV type 1 (HIV-1)-infected cells and uninfected CD4(+) T cells. These lectins inhibit virus entry. When exposed to escalating concentrations of G. nivalis and Hippeastrum sp. hybrid agglutinin, a variety of HIV-1(III(B)) strains were isolated after 20 to 40 subcultivations which showed a decreased sensitivity to the plant lectins. Several amino acid changes in the envelope glycoprotein gp120, but not in gp41, of the mutant virus isolates were observed. The vast majority of the amino acid changes occurred at the N glycosylation sites and at the S or T residues that are part of the N glycosylation motif. The degree of resistance to the plant lectins was invariably correlated with an increasing number of mutated glycosylation sites in gp120. The nature of these mutations was entirely different from that of mutations that are known to appear in HIV-1 gp120 under the pressure of other viral entry inhibitors such as dextran sulfate, bicyclams (i.e., AMD3100), and chicoric acid, which also explains the lack of cross-resistance of plant lectin-resistant viruses to any other HIV inhibitor including T-20 and the blue-green algae (cyanobacteria)-derived mannose-specific cyanovirin. The plant lectins represent a well-defined class of anti-HIV (microbicidal) drugs with a novel HIV drug resistance profile different from those of other existing anti-HIV drugs.     

The N-linked glycan of the V3 region of HIV-1 gp120 and CXCR4-dependent multiplication of a human immunodeficiency virus type 1 lymphocyte-tropic variant.

We have previously shown that an N-glycosylation site of N306 of HIV-1 gp120 is not necessary for the HIV-1 infectivity but protects HIV-1 from neutralising antibodies. In contrast Nakayama et al. [FEBS Lett. (1998) 426, 367-372], using a virus with an identical V3 region, suggested that elimination of this particular glycan reduced the ability of T-tropic HIV to bind to CXCR4 and hence its ability to infect T cell lines. We therefore re-examined the ability of a mutant virus, lacking the N306 glycan, to replicate in various types of cells and found no change in co-receptor usage for mutant virus. The ability of mutant virus to replicate or to induce syncytia in infected cells was similar to that of wild type virus. These results corroborate our original observation, confirming that the induced mutation in the N306 glycosylation site neither impairs nor improves the ability of mutant virus to replicate in permissive cells.     

New insights into the mechanisms whereby low molecular weight CCR5 ligands inhibit HIV-1 infection

CC chemokine receptor 5 (CCR5) is a G-protein-coupled receptor for the chemokines CCL3, -4, and -5 and a coreceptor for entry of R5-tropic strains of human immunodeficiency virus type 1 (HIV-1) into CD4(+) T-cells. We investigated the mechanisms whereby nonpeptidic, low molecular weight CCR5 ligands block HIV-1 entry and infection. Displacement binding assays and dissociation kinetics demonstrated that two of these molecules, i.e. TAK779 and maraviroc (MVC), inhibit CCL3 and the HIV-1 envelope glycoprotein gp120 binding to CCR5 by a noncompetitive and allosteric mechanism, supporting the view that they bind to regions of CCR5 distinct from the gp120- and CCL3-binding sites. We observed that TAK779 and MVC are full and weak inverse agonists for CCR5, respectively, indicating that they stabilize distinct CCR5 conformations with impaired abilities to activate G-proteins. Dissociation of [(125)I]CCL3 from CCR5 was accelerated by TAK779, to a lesser extent by MVC, and by GTP analogs, suggesting that inverse agonism contributes to allosteric inhibition of the chemokine binding to CCR5. TAK779 and MVC also promote dissociation of [(35)S]gp120 from CCR5 with an efficiency that correlates with their ability to act as inverse agonists. Displacement experiments revealed that affinities of MVC and TAK779 for the [(35)S]gp120-binding receptors are in the same range (IC(50) ～6.4 versus 22 nm), although we found that MVC is 100-fold more potent than TAK779 for inhibiting HIV infection. This suggests that allosteric CCR5 inhibitors not only act by blocking gp120 binding but also alter distinct steps of CCR5 usage in the course of HIV infection.     

Human immunodeficiency virus type 1 envelope gp120 is cleaved after incubation with recombinant soluble CD4.

Human immunodeficiency virus type 1 (HIV-1) infects human CD4+ cells by a high-affinity interaction between its envelope glycoprotein gp120 and the CD4 molecule on the cell surface. Subsequent virus entry into the cells involves other steps, one of which could be cleavage of the gp120 followed by virus-cell fusion. The envelope gp120 is highly variable among different HIV-1 isolates, but conserved amino acid sequence motifs that contain potential proteolytic cleavage sites can be found. Following incubation with a soluble form of CD4, we demonstrate that gp120 of highly purified HIV-1 preparations is, without addition of exogenous proteinase, cleaved most likely in the V3 loop, yielding two proteins of 50 and 70 kDa. The extent of gp120 proteolysis is HIV-1 strain dependent and correlates with the recombinant soluble CD4 sensitivity to neutralization of the particular strain. The origin of the proteolytic activity in the virus preparations remains unclear. The results support the hypothesis that cleavage of gp120 is required for HIV infection of cells.     

Molecular Characterization of HIV-1 Subtype C gp-120 Regions Potentially Involved in Virus Adaptive Mechanisms

The role of variable regions of HIV-1 gp120 in immune escape of HIV has been investigated. However, there is scant information on how conserved gp120 regions contribute to virus escaping. Here we have studied how molecular sequence characteristics of conserved C3, C4 and V3 regions of clade C HIV-1 gp120 that are involved in HIV entry and are target of the immune response, are modulated during the disease course. We found an increase of “shifting” putative N-glycosylation sites (PNGSs) in the α2 helix (in C3) and in C4 and an increase of sites under positive selection pressure in the α2 helix during the chronic stage of disease. These sites are close to CD4 and to co-receptor binding sites. We also found a negative correlation between electric charges of C3 and V4 during the late stage of disease counteracted by a positive correlation of electric charges of α2 helix and V5 during the same stage. These data allow us to hypothesize possible mechanisms of virus escape involving constant and variable regions of gp120. In particular, new mutations, including new PNGSs occurring near the CD4 and CCR5 binding sites could potentially affect receptor binding affinity and shield the virus from the immune response.     

Role of CD4 epitopes outside the gp120-binding site during entry of human immunodeficiency virus type 1.

CD4 is the primary receptor for human immunodeficiency virus (HIV). The binding site for the surface glycoprotein of HIV type 1 (HIV-1), gp120, has been mapped to the C'-C" region of domain 1 of CD4. Previously, we have shown that a mutant of rat CD4, in which this region was exchanged for that of human CD4, is able to mediate infection of human cells by HIV-1, suggesting that essential interactions between HIV and CD4 are confined to this region. Our observations appeared to conflict with mutagenesis and antibody studies which implicate regions of CD4 outside the gp120-binding site in postbinding events during viral entry. In order to resolve this issue, we have utilized a panel of anti-rat CD4 monoclonal antibodies in conjunction with the rat-human chimeric CD4 to distinguish sequence-specific from steric effects. We find that several antibodies to rat CD4 inhibit HIV infection in cells expressing the chimeric CD4 and that this is probably due to steric hinderance. In addition, we demonstrate that replacement of the rat CDR3-like region with its human homolog does not increase the affinity of the rat-human chimeric CD4 for gp120 or affect the exposure of gp41 following binding to CD4, providing further evidence that this region does not play a crucial role during entry of virus.     

Use of a gp120 binding assay to dissect the requirements and kinetics of human immunodeficiency virus fusion events

Binding of the extracellular subunit of human immunodeficiency type 1 (HIV-1) envelope (Env) glycoprotein (gp120) to CD4 triggers the induction or exposure of a highly conserved coreceptor binding site in gp120 that helps mediate membrane fusion. Characterizing the structural features involved in gp120-coreceptor binding and the conditions under which binding occurs is important for understanding the fusion process, the evolution of pathogenic strains in vivo, the identification of novel anti-HIV compounds, and the development of HIV vaccines that utilize triggered structures of Env. Here we use the kinetics of interaction between CCR5 and gp120 to understand temporal and structural changes that occur during viral fusion. Using saturation binding and homologous competition analysis, we estimated the K(d) of interaction between CCR5 and gp120 from the macrophage tropic HIV-1 strain JRFL to be 4 nM. Unlike Env-mediated fusion, gp120 binding to CCR5 did not require divalent cations or elevated temperatures. Binding was not significantly affected by the pH of binding, G-protein coupling of CCR5, or partial gp120 deglycosylation. Oligomeric, uncleaved JRFL gp140 failed to bind CCR5 despite its ability to bind CD4 and monoclonal antibody 17b, suggesting that the uncleaved ectodomain of gp41 interferes with full exposure of the chemokine receptor binding site. Exposure of the chemokine receptor binding site on gp120 could be induced rapidly by CD4, but exposure of this site was lost upon CD4 dissociation from gp120, indicating that the conformational changes in gp120 induced by CD4 binding are fully reversible. The functional gp120-soluble CD4 complex was remarkably stable over time and temperature ranges, offering the possibility that complexes in which the highly conserved coreceptor binding site in gp120 is exposed can be used for vaccine development.     

Sulfotyrosine dipeptide: Synthesis and evaluation as HIV-entry inhibitor

Human immunodeficiency virus type 1 (HIV-1) is responsible for the worldwide AIDS pandemic. Due to the lack of prophylactic HIV-1 vaccine, drug treatment of the infected patients becomes essential to reduce the viral load and to slow down progression of the disease. Because of drug resistance, finding new antiviral agents is necessary for AIDS drug therapies. The interaction of gp120 and co-receptor (CCR5/CXCR4) mediates the entry of HIV-1 into host cells, which has been increasingly exploited in recent years as the target for new antiviral agents. A conserved co-receptor binding site on gp120 that recognizes sulfotyrosine (sTyr) residues represents a structural target to design novel HIV entry inhibitors. In this work, we developed an efficient synthesis of sulfotyrosine dipeptide and evaluated it as an HIV-1 entry inhibitor.     

The highly conserved glycan at asparagine 260 of HIV-1 gp120 is indispensable for viral entry

Carbohydrate-binding agents bind to the N-glycans of HIV-1 envelope gp120 and prevent viral entry. Carbohydrate-binding agents can select for mutant viruses with deleted envelope glycans. Not all glycosylation motifs are mutated to the same extent. Site-directed mutagenesis revealed that deletions destroying the highly conserved (260)NGS(262) glycosylation motif resulted in non-infectious virus particles. We observed a significant lower CD4 binding in the case of the N260Q mutant gp120 virus strains, caused by a strikingly lower expression of gp120 and gp41 in the virus particle. In addition, the mutant N260Q HIV-1 envelope expressed in 293T cells was unable to form syncytia in co-cultures with U87.CD4.CXCR4.CCR5 cells, due to the lower expression of envelope protein on the surface of the transfected 293T cells. The detrimental consequence of this N-glycan deletion on virus infectivity could not be compensated for by the creation of novel glycosylation sites near this amino acid, leaving this uncovered envelope epitope susceptible to neutralizing antibody binding. Thus, the Asn-260 glycan in the gp120 envelope of HIV-1 represents a hot spot for targeting suicidal drugs or antibodies in a therapeutic effort to efficiently neutralize a broad array of virus strains.     

Actinohivin, a novel anti-human immunodeficiency virus protein from an actinomycete, inhibits viral entry to cells by binding high-mannose type sugar chains of gp120

We searched human immunodeficiency virus (HIV) entry inhibitors and found a novel anti-HIV protein, actinohivin (AH), in a culture filtrate of the newly discovered genus actinomycete Longispora albida gen. nov., sp. nov. This paper deals with the mechanism of action of the anti-HIV activity of AH. AH exhibited potent anti-HIV activities against various strains of HIV-1 and HIV-2. AH bound to the glycoprotein gp120 of various strains of HIV-1 and gp130 of simian immunodeficiency virus (SIV), but did not bind to non-glycosylated gp120 nor to cells having CD4 and coreceptors, suggesting that AH inhibits viral entry to cells by binding to the envelope glycoprotein. The investigation of the effects of various sugars on AH-gp120 binding by ELISA revealed that yeast mannan alone strongly inhibited the binding (IC50 = 3.0 microg/ml). Experiments investigating the binding of AH to other glycoproteins revealed that AH binds to ribonuclease B and thyroglobulin that have a high-mannose type saccharide chain, but not to other glycoproteins having a N-glycoside type saccharide chain. The above results indicate that high-mannose type saccharide chains of gp120 are molecular targets of AH in its anti-HIV activity.     

Sensitivity to a nonpeptidic compound (RPR103611) blocking human immunodeficiency virus type 1 Env-mediated fusion depends on sequence and accessibility of the gp41 loop region

The triterpene RPR103611 is an efficient inhibitor of membrane fusion mediated by the envelope proteins (Env, gp120-gp41) of CXCR4-dependent (X4) human immunodeficiency virus type 1 (HIV-1) strains, such as HIV-1(LAI) (LAI). Other X4 strains, such as HIV-1(NDK) (NDK), and CCR5-dependent (R5) HIV-1 strains, such as HIV-1(ADA) (ADA), were totally resistant to RPR103611. Analysis of chimeric LAI-NDK Env proteins identified a fragment of the NDK gp41 ectodomain determining drug resistance. A single difference at position 91, leucine in LAI and histidine in NDK, apparently accounted for their sensitivity or resistance to RPR103611. We had previously identified a mutation of isoleucine 84 to serine in a drug escape LAI variant. Both I84 and L91 are located in the "loop region" of gp41 separating the proximal and distal helix domains. Nonpolar residues in this region therefore appear to be important for the antiviral activity of RPR103611 and are possibly part of its target. However, another mechanism had to be envisaged to explain the drug resistance of ADA, since its gp41 loop region was almost identical to that of LAI. Fusion mediated by chimeric Env consisting of LAI gp120 and ADA gp41, or the reciprocal construct, was fully blocked by RPR103611. The gp120-gp41 complex of R5 strains is stable, relative to that of X4 strains, and this stability could play a role in their drug resistance. Indeed, when the postbinding steps of ADA infection were performed under mildly acidic conditions (pH 6.5 or 6.0), a treatment expected to favor dissociation of gp120, we achieved almost complete neutralization by RPR103611. The drug resistance of NDK was partially overcome by preincubating virus with soluble CD4, a gp120 ligand inducing conformational changes in the Env complex. The antiviral efficacy of RPR103611 therefore depends on the sequence of the gp41 loop and the stability of the gp120-gp41 complex, which could limit the accessibility of this target.     

New prospects for the development of a vaccine against human immunodeficiency virus type 1. An overview

During the past few years, definite progress has been made in the field of human immunodeficiency virus type 1 (HIV-1) vaccines. Initial attempts using envelope gp120 or gp140 from T-cell line-adapted (TCLA) HIV-1 strains to vaccinate chimpanzees showed that neutralizing antibody-based immune responses were protective against challenge with homologous TCLA virus strains or strains with low replicative capacity, but these neutralizing antibodies remained inactive when tested on primary HIV-1 isolates, casting doubts on the efficacy of gp120-based vaccines in the natural setting. Development of a live attenuated simian immunodeficiency virus (SIV) vaccine was undertaken in the macaque model using whole live SIV bearing multiple deletions in the nef, vpr and vpx genes. This vaccine provided remarkable protective efficacy against wild-type SIV challenge, but the deletion mutants remain pathogenic, notably in neonate monkeys. Study of the mechanisms of protection in the SIV model unravelled the importance of the T-cell responses, whether in the form of cytotoxic T-lymphocyte (CTL) killing activity, or in that of antiviral factor secretion of cytokines, beta-chemokines and other unidentified antiviral factors by CD8+ T-cells. Induction of such a response is being sought at this time using various live recombinant virus vaccines, either poxvirus or alphavirus vectors or DNA vectors, which can be combined together or with a gp120/gp140 boost in various prime-boost combination strategies. New vectors include attenuated vaccinia virus NYVAC, modified vaccinia strain Ankara (MVA), Semliki Forest virus, Venezuelan equine encephalitis virus, and Salmonellas. Recent DNA prime-poxvirus boost combination regimens have generated promising protection results against SIV or SIV/HIV (SHIV) challenge in macaque models. Emphasis is also put on the induction of a mucosal immune response, involving both a secretory IgA response and a mucosal CTL response which could constitute a 'first line of defence' in the vaccinated host. Finally, a totally novel vaccine approach based on the use of Tat or Tat and Rev antigens has been shown to induce efficient protection from challenge with pathogenic SIV or SHIV in vaccinated macaques. The only vaccine in phase 3 clinical trials in human volunteers is a gp120-based vaccine, AIDSVAX. A prime-boost combination of a recombinant canarypoxvirus and a subunit gp120 vaccine is in phase 2. Emphasis has been put recently on the necessity of testing prototype vaccines in developing countries using immunogens derived from local virus strains. Trial sites have thus been identified in Kenya, Uganda, Thailand and South Africa where phase I trials have begun or are expected to start presently.     

Expression and characterization of a single-chain polypeptide analogue of the human immunodeficiency virus type 1 gp120-CD4 receptor complex

The infection of CD4(+) host cells by human immunodeficiency virus type 1 (HIV-1) is initiated by a temporal progression of interactions between specific cell surface receptors and the viral envelope protein, gp120. These interactions produce a number of intermediate structures with distinct conformational, functional, and antigenic features that may provide important targets for therapeutic and vaccination strategies against HIV infection. One such intermediate, the gp120-CD4 complex, arises from the interaction of gp120 with the CD4 receptor and enables interactions with specific coreceptors needed for viral entry. gp120-CD4 complexes are thus promising targets for anti-HIV vaccines and therapies. The development of such strategies would be greatly facilitated by a means to produce the gp120-CD4 complexes in a wide variety of contexts. Accordingly, we have developed single-chain polypeptide analogues that accurately replicate structural, functional, and antigenic features of the gp120-CD4 complex. One analogue (FLSC) consists of full-length HIV-1BaL gp120 and the D1D2 domains of CD4 joined by a 20-amino-acid linker. The second analogue (TcSC) contains a truncated form of the gp120 lacking portions of the C1, C5, V1, and V2 domains. Both molecules exhibited increased exposure of epitopes in the gp120 coreceptor-binding site but did not present epitopes of either gp120 or CD4 responsible for complex formation. Further, the FLSC and TcSC analogues bound specifically to CCR5 (R5) and blocked R5 virus infection. Thus, these single-chain chimeric molecules represent the first generation of soluble recombinant proteins that mimic the gp120-CD4 complex intermediate that arises during HIV replication.     

Formation of HIV-1 envelope-hepatitis B core antigen hybrids with high affinity for CD4.

We have identified an acceptor site on HIV gp120, where foreign protein sequences can be inserted while retaining the native conformation of gp120. The resulting hybrids showed dual antigenicity, normal glycosylation, and high affinity binding of the CD4 receptor. This site allows insertion of highly immunogenic proteins such as core antigen of hepatitis B virus. By combining the immunogenicity of the carrier protein with the antigenicity of gp120, these hybrids may lead to modified HIV-1 antigens with enhanced immunogenicity.