Synergistic neutralization of human immunodeficiency virus type 1 by combinations of human monoclonal antibodies.

The ability of antibodies to the V3 region and the CD4-binding domain (CD4bd) of human immunodeficiency virus type 1 (HIV-1) to act in synergy to neutralize HIV has been demonstrated previously. However, synergy between antibodies to other HIV-1 epitopes has not been studied. We have used 21 combinations of human monoclonal antibodies (MAbs) directed against different epitopes of the gp120 and gp41 proteins of HIV-1 to evaluate their ability to act in synergy to neutralize HIV-1. Combinations of anti-V3 and anti-CD4bd antibodies, anti-V3 and anti-gp120 C-terminus antibodies, anti-CD4bd and anti-C-terminus antibodies, anti-V3 and anti-gp41 antibodies, and anti-CD4bd and anti-gp41 antibodies were tested. Our results show that some, but not all anti-V3 antibodies can act in synergy with anti-CD4bd antibodies. In addition, for the first time, antibodies to the C-terminus region have been found to act in synergy with the anti-CD4bd antibodies. Various anti-CD4bd MAbs also act in synergy when used together. The use of such cocktails of human MAbs for passive immunization against HIV-1 may prove to be important for therapy in postexposure settings and for prevention of maternal-fetal transmission of the virus. The results also provide information on the types of antibodies that should be elicited by an effective vaccine.     

The role of antibody polyspecificity and lipid reactivity in binding of broadly neutralizing anti-HIV-1 envelope human monoclonal antibodies 2F5 and 4E10 to glycoprotein 41 membrane proximal envelope epitopes

Two neutralizing human mAbs, 2F5 and 4E10, that react with the HIV-1 envelope gp41 membrane proximal region are also polyspecific autoantibodies that bind to anionic phospholipids. To determine the autoantibody nature of these Abs, we have compared their reactivities with human anti-cardiolipin mAbs derived from a primary antiphospholipid syndrome patient. To define the role of lipid polyreactivity in binding of 2F5 and 4E10 mAbs to HIV-1 envelope membrane proximal epitopes, we determined the kinetics of binding of mAbs 2F5 and 4E10 to their nominal gp41 epitopes vs liposome-gp41 peptide conjugates. Both anti-HIV-1 mAbs 2F5 and 4E10 bound to cardiolipin with K(d) values similar to those of autoimmune anti-cardiolipin Abs, IS4 and IS6. Binding kinetics studies revealed that mAb 2F5 and 4E10 binding to their respective gp41 peptide-lipid conjugates could best be defined by a two-step (encounter-docking) conformational change model. In contrast, binding of 2F5 and 4E10 mAbs to linear peptide epitopes followed a simple Langmuir model. A mouse mAb, 13H11, that cross-blocks mAb 2F5 binding to the gp41 epitope did not cross-react with lipids nor did it neutralize HIV-1 viruses. Taken together, these data demonstrate the similarity of 2F5 and 4E10 mAbs to known anti-cardiolipin Abs and support the model that mAb 2F5 and 4E10 binding to HIV-1 involves both viral lipid membrane and gp41 membrane proximal epitopes.     

Specificity of antibodies produced against native or desialylated human immunodeficiency virus type 1 recombinant gp160.

In a previous report we have shown that, in contrast to antibodies produced against native or fully deglycosylated human immunodeficiency virus type 1 (HIV-1) gp160 in rabbits, antibodies raised against desialylated HIV-1 gp160 also recognize gp140 from HIV-2 at high titers. Here, we characterize the fine specificity of these cross-reactive antibodies. Inhibition assays with a panel of synthetic peptides as competitors showed that cross-reactivity to gp140 was due to antibodies that were specific for the region encompassing HIV-1 gp41 immunodominant epitope, mimicked by peptide P39 (residues 583 to 609), the latter being able to totally inhibit the formation of complexes between radiolabeled HIV-2 gp140 and antibodies elicited by desialylated HIV-1 gp160. In addition, anti-desialylated gp160 antibodies retained on a P39 affinity column still bound HIV-2 gp140. Fine mapping has enabled us to localize the cross-reactive epitope within the N-terminal extremity of the gp41 immunodominant region. Interestingly, this cross-reactive antibody population did not recognize glycosylated or totally deglycosylated simian immunodeficiency virus gp140 despite an amino acid homology with HIV-1 within this region that is comparable to that of HIV-2. This cross-reactivity between HIV-1 and HIV-2 did not correlate with cross-neutralization. These results illustrate the influence of carbohydrate moieties on the specificity of the antibodies produced and clearly indicate that such procedures may be an efficient way to raise specific immune responses that are not type specific. Moreover, this cross-reactivity might explain the double-positive reactivity observed, in some human sera, against both HIV-1 and HIV-2 envelope antigens.     

Folding, assembly, and intracellular trafficking of the human immunodeficiency virus type 1 envelope glycoprotein analyzed with monoclonal antibodies recognizing maturational intermediates.

Monoclonal antibodies (MAbs) that bind linear or conformational epitopes on monomeric or oligomeric human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins were screened for their recognition of maturational intermediates. On the basis of reactivities with gp160 at different times after pulse-labeling, the MAbs were sorted into groups that exhibited binding which was immediate and constant, immediate but transient, delayed, late, or very late. This grouping was consistent with the selectivity of the MAbs for structural features of gp160. Thus, a MAb to the V3 loop reacted with envelope proteins at all times, in accord with the relative conformational independence and accessibility of the epitope. Several MAbs that preferentially react with monomeric gp160 exhibited diminished binding after the pulse. A 10-min tag occurred before gp160 reacted with conformational MAbs that inhibited CD4 binding. The availability of epitopes for other conformational MAbs, including some that react equally with monomeric and oligomeric gp160 and some that react better with oligomeric forms, was half-maximal in 30 min and closely followed the kinetics of gp160 oligomerization. Remarkably, there was a 1- to 2-h delay before gp160 reacted with stringent oligomer-specific MAbs. After 4 h, approximately 20% of the gp160 was recognized by these MAbs. Epitopes recognized by monomerspecific or CD4-blocking MAbs but not by oligomer-dependent MAbs were present on gp160 molecules associated with the molecular chaperone BiP/GRP78. MAbs with a preference for monomers reacted with recombinant or HIV-1 envelope proteins in the endoplasmic reticulum, whereas the oligomer-specific MAbs recognized them in the Golgi complex. Additional information regarding gp160 maturation and intracellular trafficking was obtained by using brefeldin A, dithiothreitol, and a low temperature.     

Efficient isolation of novel human monoclonal antibodies with neutralizing activity against HIV-1 from transgenic mice expressing human Ig loci

Despite considerable interest in the isolation of mAbs with potent neutralization activity against primary HIV-1 isolates, both for identifying useful targets for vaccine development and for the development of therapeutically useful reagents against HIV-1 infection, a relatively limited number of such reagents have been isolated to date. Human mAbs (hu-mAbs) are preferable to rodent mAbs for treatment of humans, but isolation of hu-mAbs from HIV-infected subjects by standard methods of EBV transformation of B cells or phage display of Ig libraries is inefficient and limited by the inability to control or define the original immunogen. An alternative approach for the isolation of hu-mAbs has been provided by the development of transgenic mice that produce fully hu-mAbs. In this report, we show that immunizing the XenoMouse G2 strain with native recombinant gp120 derived from HIV(SF162) resulted in robust humoral Ab responses against gp120 and allowed the efficient isolation of hybridomas producing specific hu-mAbs directed against multiple regions and epitopes of gp120. hu-mAbs possessing strong neutralizing activity against the autologous HIV(SF162) strain were obtained. The epitopes recognized were located in three previously described neutralization domains, the V2-, V3- and CD4-binding domains, and in a novel neutralization domain, the highly variable C-terminal region of the V1 loop. This is the first report of neutralizing mAbs directed at targets in the V1 region. Furthermore, the V2 and V3 epitopes recognized by neutralizing hu-mAbs were distinct from those of previously described human and rodent mAbs and included an epitope requiring a full length V3 loop peptide for effective presentation. These results further our understanding of neutralization targets for primary, R5 HIV-1 viruses and demonstrate the utility of the XenoMouse system for identifying new and interesting epitopes on HIV-1.     

Additive effects characterize the interaction of antibodies involved in neutralization of the primary dualtropic human immunodeficiency virus type 1 isolate 89.6

Human immunodeficiency virus-type I (HIV-1) infection elicits antibodies (Abs) directed against several regions of the gp120 and gp41 envelope glycoproteins. Many of these Abs are able to neutralize T-cell-line-adapted strains (TCLA) of HIV-1, but only a few effectively neutralize primary HIV-1 isolates. The nature of HIV-1 neutralization has been carefully studied using human monoclonal Abs (MAbs), and the ability of such MAbs to act in synergy to neutralize HIV-1 has also been extensively studied. However, most synergy studies have been conducted using TCLA strains. To determine the nature of Ab interaction in HIV-1 primary isolate neutralization, a panel of 12 anti-HIV-1 human immunoglobulin G (IgG) MAbs, specific for epitopes in gp120 and gp41, were used. Initial tests showed that six of these MAbs, as well as sCD4, used individually, were able to neutralize the dualtropic primary isolate HIV-1(89.6); MAbs giving significant neutralization at 2 to 10 microg/ml included 2F5 (anti-gp41), 50-69 (anti-gp41), IgG1b12 (anti-gp120(CD4bd)), 447-52D (anti-gp120(V3)), 2G12 (anti-gp120), and 670-D (anti-gp120(C5)). For studies of reagent interaction, 16 binary combinations of reagents were tested for their ability to neutralize HIV-1(89.6). Reagent combinations tested included one neutralizing MAb with sCD4, six pairs consisting of two neutralizing MAbs, and nine pairs consisting of one neutralizing MAb with another non-neutralizing MAb. To assess the interaction of the latter type of combination, a new mathematical treatment of reagent interaction was developed since previously used methods could be used only when both reagents neutralize. Synergy was noted between sCD4 and a neutralizing anti-gp120(V3) MAb. Antagonism was noted between two pairs of anti-gp41 MAbs (one neutralizing and one non-neutralizing). All of the other 13 pairs of MAbs tested displayed only additive effects. These studies suggest that Abs rarely act in synergy to neutralize primary isolate HIV-1(89.6); many anti-HIV-1 Abs act additively to mediate this biological function.     

Influence of carbohydrate moieties on the immunogenicity of human immunodeficiency virus type 1 recombinant gp160.

The role of carbohydrates in the immunogenicity of human immunodeficiency virus type 1 (HIV-1) glycoproteins (gp160 and gp120) remains poorly understood. We have analyzed the specificity and neutralizing capacity of antibodies raised against native gp160 or against gp160 deglycosylated by either endo F-N glycanase, neuraminidase, or alpha-mannosidase. Rabbits immunized with these immunogens produced antibodies that recognized recombinant gp160 (rgp160) from HIV-1 in a radioimmunoassay and in an enzyme-linked immunosorbent assay. Antibodies elicited by the different forms of deglycosylated gp160 were analyzed for their reactivity against a panel of synthetic peptides. Compared with anti-native gp160 antisera, serum reactivity to most peptides remained unchanged, or it could increase (peptide P41) or decrease. Only antibodies raised against mannosidase-treated gp160 failed to react with a synthetic peptide (peptide P29) within the V3 loop of gp120. Rabbits immunized with desialylated rgp160 generated antibodies which recognized not only rgp160 from HIV-1 but also rgp140 from HIV-2 at high titers. Although all antisera produced against glycosylated or deglycosylated rgp160 could prevent HIV-1 binding to CD4-positive cells in vitro, only antibodies raised against native or desialylated gp160 neutralized HIV-1 infectivity and inhibited syncytium formation between HIV-1-infected cells and noninfected CD4-positive cells, whereas antibodies raised against alpha-mannosidase-treated gp160 inhibited neither virus replication nor syncytium formation. These findings indicate that the carbohydrate moieties of gp160 can modulate the specificity and the protective efficiency of the antibody response to the molecule.     

Neutralization synergy of human immunodeficiency virus type 1 primary isolates by cocktails of broadly neutralizing antibodies.

Several reports have described the existence of synergy between neutralizing monoclonal antibodies (MAbs) against human immunodeficiency virus type 1 (HIV-1). Synergy between human MAbs b12, 2G12, 2F5, and 4E10 in neutralization of primary isolates is of particular interest. Neutralization synergy of these MAbs, however, has not been studied extensively, and the mechanism of synergy remains unclear. We investigated neutralization synergy among this human antibody set by using the classical approach of titrating antibodies mixed at a fixed ratio as well as by an alternative, variable ratio approach in which the neutralization curve of one MAb is assessed in the presence and absence of a fixed, weakly neutralizing concentration of a second antibody. The advantage of this second approach is that it does not require mathematical analysis to establish synergy. No neutralization enhancement of any of the MAb combinations tested was detected for the T-cell-line-adapted molecular HIV-1 clone HxB2 using both assay formats. Studies of primary isolates (89.6, SF162, and JR-CSF) showed neutralization synergy which was relatively weak, with a maximum of two- to fourfold enhancement between antibody pairs, thereby increasing neutralization titers about 10-fold in triple and quadruple antibody combinations. Analysis of b12 and 2G12 binding to oligomeric envelope glycoprotein by using flow cytometry failed to demonstrate cooperativity in binding between these two antibodies. The mechanism by which these antibodies synergize is, therefore, not yet understood. The results lend some support to the notion that an HIV-1 vaccine that elicits moderate neutralizing antibodies to multiple epitopes may be more effective than hereto supposed, although considerable caution in extrapolating to a vaccine situation is required.     

Conserved structures exposed in HIV-1 envelope glycoproteins stabilized by flexible linkers as potent entry inhibitors and potential immunogens

The HIV-1 envelope glycoprotein (Env) undergoes conformational changes while driving entry. We hypothesized that some of the intermediate Env conformations could be represented in tethered constructs where gp120 and the ectodomain of gp41 are joined by flexible linkers. Tethered Envs with long linkers (gp140-14 with 15 aa and gp140-24 with 26 aa) were stable and recognized by conformationally dependent anti-gp120 and anti-gp41 monoclonal antibodies (mAbs). Surprisingly, these proteins potently inhibited membrane fusion mediated by R5, X4, and R5X4 Envs with 5-100-fold lower IC50 than a tethered Env with short linker (gp140-4 with 4 aa), gp120, gp140, soluble CD4, or DP178 (T20). Compared to gp140, gp140-14,24 exhibited increased binding to anti-gp41 cluster II mAbs but not to cluster I mAbs. Cluster II mAbs but not cluster I, IV, or V mAbs reversed the inhibitory effect of gp140-14,24 suggesting a role of exposed conserved gp41 structures for the mechanism of inhibition. These findings suggest the existence of conserved gp41 structures that are important for HIV-1 entry and can be stably exposed in the native environment of the Env even in the absence of receptor-mediated activation. Thus, tethered Envs with long linkers may not only be important as HIV-1 inhibitors but also for elucidation of viral entry mechanisms and development of novel vaccine immunogens.     

Sulfation of the human immunodeficiency virus envelope glycoprotein.

Sulfation is a posttranslational modification of proteins which occurs on either the tyrosine residues or the carbohydrate moieties of some glycoproteins. In the case of secretory proteins, sulfation has been hypothesized to act as a signal for export from the cell. We have shown that the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor (gp160) as well as the surface (gp120) and transmembrane (gp41) subunits can be specifically labelled with 35SO42-. Sulfated HIV-1 envelope glycoproteins were identified in H9 cells infected with the IIIB isolate of HIV-1 and in the cell lysates and culture media of cells infected with vaccinia virus recombinants expressing a full-length or truncated, secreted form of the HIV-1 gp160 gene. N-glycosidase F digestion of 35SO4(2-)-labelled envelope proteins removed virtually all radiolabel from gp160, gp120, and gp41, indicating that sulfate was linked to the carbohydrate chains of the glycoprotein. The 35SO42-label was at least partially resistant to endoglycosidase H digestion, indicating that some sulfate was linked to complex carbohydrates. Brefeldin A, a compound that inhibits the endoplasmic reticulum to Golgi transport of glycoproteins, was found to inhibit the sulfation of the envelope glycoproteins. Envelope glycoproteins synthesized in cells treated with chlorate failed to incorporate 35SO42-. However, HIV glycoproteins were still secreted from cells in the presence of chlorate, indicating that sulfation is not a requirement for secretion of envelope glycoproteins. Sulfation of HIV-2 and simian immunodeficiency virus envelope glycoproteins has also been demonstrated by using vaccinia virus-based expression systems. Sulfation is a major determinant of negative charge and could play a role in biological functions and antigenic properties of HIV glycoproteins.     

Immunization of Rabbits with Highly Purified,Soluble, Trimeric Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Induces a Vigorous B Cell Response and Broadly Cross-Reactive Neutralization

Previously we described induction of cross-reactive HIV-1 neutralizing antibody responses in rabbits using a soluble HIV-1 gp140 envelope glycoprotein (Env) in an adjuvant containing monophosphoryl lipid A (MPL) and QS21 (AS02_A). Here, we compared different forms of the same HIV-1 strain R2 Env for antigenic and biophysical characteristics, and in rabbits characterized the extent of B cell induction for specific antibody expression and secretion and neutralizing responses. The forms of this Env that were produced in and purified from stably transformed 293T cells included a primarily dimeric gp140, a trimeric gp140 appended to a GCN4 trimerization domain (gp140-GCN4), gp140-GCN4 with a 15 amino acid flexible linker between the gp120 and gp41 ectodomain (gp140-GCN4-L), also trimeric, and a gp140 with the flexible linker purified from cell culture supernatants as either dimer (gp140-L(D)) or monomer (gp140-L(M)). Multimeric states of the Env proteins were assessed by native gel electrophoresis and analytical ultracentrifugation. The different forms of gp140 bound broadly cross-reactive neutralizing (BCN) human monoclonal antibodies (mAbs) similarly in ELISA and immunoprecipitation assays. All Envs bound CD4i mAbs in the presence and absence of sCD4, as reported for the R2 Env. Weak neutralization of some strains of HIV-1 was seen after two additional doses in AS02_A. Rabbits that were given a seventh dose of gp140-GCN4-L developed BCN responses that were weak to moderate, similar to our previous report. The specificity of these responses did not appear similar to that of any of the known BCN human mAbs. Induction of spleen B cell and plasma cells producing immunoglobulins that bound trimeric gp140-GCN4-L was vigorous, based on ELISpot and flow cytometry analyses. The results demonstrate that highly purified gp140-GCN4-L trimer in adjuvant elicits BCN responses in rabbits accompanied by vigorous B cell induction.     

Vaccine-induced protection of chimpanzees against infection by a heterologous human immunodeficiency virus type 1.

The extraordinary genetic diversity of human immunodeficiency virus type 1 (HIV-1) is a major problem to overcome in the development of an effective vaccine. In the most reliable animal model of HIV-1 infection, chimpanzees were immunized with various combinations of HIV-1 antigens, which were derived primarily from the surface glycoprotein, gp160, of HIV-1 strains LAI and MN. The immunogens also included a live recombinant canarypox virus expressing a gp160-MN protein. In one experiment, two chimpanzees were immunized multiple times; one animal received antigens derived only from HIV-1LAI, and the second animal received antigens from both HIV-1LAI and HIV-1MN. In another experiment, four chimpanzees were immunized in parallel a total of five times over 18 months; two animals received purified gp160 and V3-MN peptides, whereas the other two animals received the recombinant canarypox virus and gp160. At 3 months after the final booster, all immunized and naive control chimpanzees were challenged by intravenous inoculation of HIV-1SF2; therefore, the study represented an intrasubtype B heterologous virus challenge. Virologic and serologic follow-up showed that the controls and the two chimpanzees immunized with the live recombinant canarypox virus became infected, whereas the other animals that were immunized with gp160 and V3-MN peptides were protected from infection. Evaluation of both cellular and humoral HIV-specific immune responses at the time of infectious HIV-1 challenge identified the following as possible correlates of protection: antibody titers to the V3 loop of MN and neutralizing antibody titers to HIV-1MN or HIV-1LAI, but not to HIV-1SF2. The results of this study indicate that vaccine-mediated protection against intravenous infection with heterologous HIV-1 strains of the same subtype is possible with some immunogens.     

Characterization of the outer domain of the gp120 glycoprotein from human immunodeficiency virus type 1

The core of the gp120 glycoprotein from human immunodeficiency virus type 1 (HIV-1) is comprised of three major structural domains: the outer domain, the inner domain, and the bridging sheet. The outer domain is exposed on the HIV-1 envelope glycoprotein trimer and contains binding surfaces for neutralizing antibodies such as 2G12, immunoglobulin G1b12, and anti-V3 antibodies. We expressed the outer domain of HIV-1(YU2) gp120 as an independent protein, termed OD1. OD1 efficiently bound 2G12 and a large number of anti-V3 antibodies, indicating its structural integrity. Immunochemical studies with OD1 indicated that antibody responses against the outer domain of the HIV-1 gp120 envelope glycoprotein are rare in HIV-1-infected human sera that potently neutralize the virus. Surprisingly, such outer-domain-directed antibody responses are commonly elicited by immunization with recombinant monomeric gp120. Immunization with soluble, stabilized HIV-1 envelope glycoprotein trimers elicited antibody responses that more closely resembled those in the sera of HIV-1-infected individuals. These results underscore the qualitatively different humoral immune responses elicited during natural infection and after gp120 vaccination and help to explain the failure of gp120 as an effective vaccine.     

A mechanism of restricted human immunodeficiency virus type 1 expression in human glial cells.

We characterized in detail the life cycle of human immunodeficiency virus type 1 (HIV-1) in human glioma H4/CD4 cells which stably express transfected CD4 DNA (B. Volsky, K. Sakai, M. Reddy, and D. J. Volsky, Virology 186:303-308, 1992). Infection of cloned H4/CD4 cells with the N1T strain of cell-free HIV-1 (HIV-1/N1T) was rapid and highly productive as measured by the initial expression of viral DNA, RNA, and protein, but all viral products declined to low levels by 14 days after infection. Chronically infected, virus-producing H4/CD4 cells could be obtained by cell cloning, indicating that HIV-1 DNA can integrate and remain expressed in these cells. The HIV-1 produced in H4/CD4 cells was noninfectious to glial cells, but it could be transmitted with low efficiency to CEM cells. Examination of viral protein composition by immunoprecipitation with AIDS serum or anti-gp120 antibody revealed that HIV-1/N1T-infected H4/CD4 cells produced all major viral proteins including gp160, but not gp120. Deglycosylation experiments with three different glycosidases determined that the absence of gp120 was not due to aberrant glycosylation of gp160, indicating a defect in gp160 proteolytic processing. Similar results were obtained in acutely and chronically infected H4/CD4 cells. To determine the generality of this HIV-1 replication phenotype in H4/CD4 cells, nine different viral clones were tested for replication in H4/CD4 cells by transfection. Eight were transiently productive like N1T, but one clone, NL4-3, established a long-lived productive infection in H4/CD4 cells, produced infectious progeny virus, and produced both gp160 and gp120. We conclude that for most HIV-1 strains tested, HIV-1 infection of H4/CD4 is restricted to a single cycle because of the defective processing of gp160, resulting in the absence of gp120 on progeny virus.     

Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1.

Six recombinant human Fab fragments that were derived from the same human immunodeficiency virus type 1 (HIV-1)-infected individual and are directed against the CD4 binding site (CD4bs) of the gp120 envelope glycoprotein were studied. A range of neutralizing activity against the HIV-1 (HXBc2) isolate was observed, with Fab b12 exhibiting the greatest potency among the Fabs tested. The neutralizing potency of Fab b12 was better than that of monoclonal whole antibodies directed against the third variable (V3) region of gp120. To explore the basis for the efficient neutralizing activity of b12, the recognition of a panel of HIV-1 gp120 mutants by the six Fabs was studied. The patterns of sensitivity to particular gp120 amino acid changes were similar for all six Fabs to those seen for anti-CD4bs monoclonal antibodies derived from HIV-1-infected individuals by conventional means. In addition, recognition by Fab b12 demonstrated an atypical sensitivity to changes in the V1 and V2 variable regions. Next, the binding of the Fabs to monomeric gp120 and to the envelope glycoprotein complex was examined. Neither the binding properties of the b12 Fab to monomeric gp120 nor the ability of the Fab to compete with soluble CD4 for monomeric gp120 binding appeared to account for the greater neutralizing potency. However, both quantitative and qualitative differences between the binding of b12 and that of less potent Fabs to the cell surface envelope glycoprotein complex were observed. Relative to less potently neutralizing Fabs, Fab b12 exhibited a higher affinity for a subpopulation of cell surface envelope glycoproteins, the conformation of which was best approximated by the mature gp120 glycoprotein. Apparently, subtle differences in the gp120 epitope recognized allow some members of the group of anti-CD4bs antibodies to bind to the functionally relevant envelope glycoprotein complex and to neutralize virus more efficiently.     

Human immunodeficiency virus type 1 Vpu protein regulates the formation of intracellular gp160-CD4 complexes.

Intracellular transport and processing of the human immunodeficiency virus type 1 (HIV-1) envelope precursor glycoprotein, gp160, proceeds via the endoplasmic reticulum and Golgi complex and involves proteolytic processing of gp160 into the mature virion components, gp120 and gp41. We found that coexpression of gp160 and human CD4 in HeLa cells severely impaired gp120 production due to the formation of intracellular gp160-CD4 complexes. This CD4-mediated inhibition of gp160 processing was alleviated by coexpression of the HIV-1-encoded Vpu protein. The coexpression of Vpu and CD4 in the presence of gp160 resulted in increased degradation of CD4. Although the precise mechanism(s) responsible for the Vpu effect is presently unclear, our findings suggest that Vpu may destabilize intracellular gp160-CD4 complexes.     

Proteolytic Processing of the Human Immunodeficiency Virus Envelope Glycoprotein Precursor Decreases Conformational Flexibility

The mature envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) virions is derived by proteolytic cleavage of a trimeric gp160 glycoprotein precursor. Remarkably, proteolytic processing of the HIV-1 Env precursor results in changes in Env antigenicity that resemble those associated with glutaraldehyde fixation. Apparently, proteolytic processing of the HIV-1 Env precursor decreases conformational flexibility of the Env trimeric complex, differentially affecting the integrity/accessibility of epitopes for neutralizing and nonneutralizing antibodies.     

The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120

We have analyzed the unique epitope for the broadly neutralizing human monoclonal antibody (MAb) 2G12 on the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). Sequence analysis, focusing on the conservation of relevant residues across multiple HIV-1 isolates, refined the epitope that was defined previously by substitutional mutagenesis (A. Trkola, M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan, K. Srinivasan, J. Sodroski, J. P. Moore, and H. Katinger, J. Virol. 70:1100-1108, 1996). In a biochemical study, we digested recombinant gp120 with various glycosidase enzymes of known specificities and showed that the 2G12 epitope is lost when gp120 is treated with mannosidases. Computational analyses were used to position the epitope in the context of the virion-associated envelope glycoprotein complex, to determine the variability of the surrounding surface, and to calculate the surface accessibility of possible glycan- and polypeptide-epitope components. Together, these analyses suggest that the 2G12 epitope is centered on the high-mannose and/or hybrid glycans of residues 295, 332, and 392, with peripheral glycans from 386 and 448 on either flank. The epitope is mannose dependent and composed primarily of carbohydrate, with probably no direct involvement of the gp120 polypeptide surface. It resides on a face orthogonal to the CD4 binding face, on a surface proximal to, but distinct from, that implicated in coreceptor binding. Its conservation amidst an otherwise highly variable gp120 surface suggests a functional role for the 2G12 binding site, perhaps related to the mannose-dependent attachment of HIV-1 to DC-SIGN or related lectins that facilitate virus entry into susceptible target cells.     

Solid-phase proteoliposomes containing human immunodeficiency virus envelope glycoproteins

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein gp120 mediates receptor binding and is the major target for neutralizing antibodies. A broadly neutralizing antibody response is likely to be a critical component of the immune response against HIV-1. Although antibodies against monomeric gp120 are readily elicited in immunized individuals, these antibodies are inefficient in neutralizing primary HIV-1 isolates. As a chronic pathogen, HIV-1 has evolved to avoid an optimal host response by a number of immune escape mechanisms. Monomeric gp120 that has dissociated from the functional trimer presents irrelevant epitopes that are not accessible on functional trimeric envelope glycoproteins. The resulting low level of antigenic cross-reactivity between monomeric gp120 and the functional spike may contribute to the inability of monomeric gp120 to elicit broadly neutralizing antibodies. Attempts to generate native, trimeric envelope glycoproteins as immunogens have been frustrated by both the lability of the gp120-gp41 interaction and the weak association between gp120 subunits. Here, we present solid-phase HIV-1 gp160DeltaCT (cytoplasmic tail-deleted) proteoliposomes (PLs) containing native, trimeric envelope glycoproteins in a physiologic membrane setting. We present data that indicate that the gp160DeltaCT glycoproteins on PLs are trimers and are recognized by several relevant conformational ligands in a manner similar to that for gp160DeltaCT oligomers expressed on the cell surface. The PLs represent a significant advance over present envelope glycoprotein formulations as candidate immunogens for HIV vaccine design and development.     

The role of eukaryotic subtilisin-like endoproteases for the activation of human immunodeficiency virus glycoproteins in natural host cells.

Proteolytic activation of the precursor envelope glycoproteins gp160 of human immunodeficiency virus type 1 (HIV-1) and gp140 of HIV-2, a prerequisite for viral infection, results in the formation of gp120/gp41 and gp125/gp36, respectively. Cleavage is mediated by cellular proteases. Furin, a member of the eukaryotic subtilisin family, has been shown to be an activating protease for HIV. Here, we compared the presence of furin and other mammalian subtilisins in lymphatic cells and tissues. Northern blot analyses revealed that furin and the recently discovered protease LPC/PC7 were the only subtilisin-like enzymes transcribed in such cells. Furin was identified as an enzymatically active endoprotease present in different lymphocytic, as well as monocytic, cell lines. When expressed from vaccinia virus vectors, the proprotein convertases were correctly processed, transported, and secreted into the media and enzymatically active. Coexpression of different subtilisins with the HIV envelope precursors revealed that furin and LPC/PC7 are able to cleave HIV-1 gp160. Moreover, both enzymes proteolytically processed the envelope precursor of HIV-2. gp140 was also cleaved to some extent by PC1, which is not, however, present in lymphatic cells. Furin- and LPC/PC7-catalyzed cleavage of HIV-1 gp160 resulted in biologically active envelope protein. In conclusion, among the known members of the subtilisin family, only furin and LPC/PC7 fulfill the requirements of a protease responsible for in vivo activation of HIV envelope glycoproteins.