Monoclonal antibodies to the extracellular domain of HIV-1IIIB gp160 that neutralize infectivity, block binding to CD4, and react with diverse isolates.

Ten monoclonal antibodies prepared against a soluble, recombinant form of gp160, derived from the IIIB isolate of HIV-1, were characterized. Four of the antibodies neutralized HIV-1IIIB infectivity in vitro, three blocked the binding of recombinant gp120 to CD4, three were reactive with gp41, and one preferentially reacted with an epitope on gp120 within the gp160 precursor. All three CD4 blocking antibodies bound to distinct epitopes, with one mapping to the C1 domain, one mapping to the C4 domain, and one reactive with a conformation-dependent, discontinuous epitope. Of these, the antibody reactive with the discontinuous epitope exhibited neutralizing activity against homologous and heterologous strains of HIV-1. The binding of these monoclonal antibodies to a panel of seven recombinant gp120s prepared from diverse isolates of HIV-1 was measured, and monoclonal antibodies with broad cross reactivity were identified. The epitopes recognized by 7 of the 10 monoclonal antibodies studied were localized by their reactivity with synthetic peptides and with fragments of gp120 expressed as fusion proteins in a lambda gt-11 gp160 epitope library.     

Characterization of the secreted, native gp120 and gp160 of the human immunodeficiency virus type 1

We have previously shown that the cell line 6D5(451) chronically infected with the HIV-1 isolate HTLV-III(451), secretes the HIV-1 envelope glycoproteins gp120 and gp160 in the extracellular medium. The HTLV-III(451) gp120 and gp160 were purified by sequential affinity chromatographic steps using a monoclonal antibody to HIV-1 gp41 and an anti-HIV-1-positive human serum. Amino acid sequence analysis of gp120 and gp160 showed the loss of the signal peptide. Digestion of the purified gp120 and gp160 with endoglycosidases revealed that both proteins are heavily glycosylated and contain complex carbohydrates, in contrast to the intracellular form of gp160 which has been shown to contain mannose-rich immature sugars. Competitive binding analysis showed that while both gp120 and gp160 bind CD4, the affinity of gp160 was five times lower than that of gp120. Both gp120 and gp160 inhibited syncytia formation by HIV-1-infected cells when mixed with CD4+ cells. Furthermore, both gp120 and gp160 had strong mitogenic effects on the T cells from HIV-1-infected gibbons but not on cells from uninfected gibbons.     

HIV-1 neutralizing monoclonal antibodies induced by a synthetic peptide.

We have developed a series of murine monoclonal antibodies to a region of the 120 kD envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1). This region has previously been implicated as a site for virus neutralization by antisera raised to recombinant proteins and by antibodies made to full-length gp120 purified from virus. The antigen employed was a synthetic peptide containing 15 amino acids, representing amino acid residues 308-322, RIQRGPGRAFVTIGK, of env gp120 (HTLV-IIIB isolate). Five of the monoclonal antibodies raised to this antigen have reactivity with gp120 from divergent strains of HIV-1 in Western blot assays. The two of these five which were tested with live cells infected with the divergent HIV-1 isolates IIIB, MN, and RF were specifically reactive by fluorescence analyses with cells infected with the MN and IIIB isolates. Four of the five monoclonal antibodies blocked the fusion of IIIB-infected cells with uninfected MOLT-4 target cells. The monoclonal antibody most reactive with MN-infected cells by fluorescence, #5025A, blocked the fusion of MN-infected cells with uninfected MOLT-4 cells. Four of the five monoclonal antibodies neutralized the IIIB isolate of HIV-1 in vitro, but none neutralized the MN or RF isolates at the levels of antibody tested (less than or equal to 50 micrograms/ml). Taken together these data indicate that monoclonal antibodies to the immunodominant neutralizing domain of HIV-1 gp120 display different levels of group reactivity depending on the assay system being examined.     

An enzyme-linked immunosorbent assay for antibodies to the envelope glycoproteins of divergent strains of HIV-1

We have developed an enzyme-linked immunosorbent assay (ELISA) specific for antibodies to the envelope glycoproteins gp120 and gp160 of HIV-1. An antibody to a conserved epitope on gp120 is adsorbed to a solid phase and used to capture gp120 and/or gp160 from solution. This may be purified recombinant protein or in simple, non-denaturing detergent extracts of different strains of HIV-1. Human serum antibodies bound to the captured antigen are subsequently detected with an anti-human antibody conjugated to alkaline phosphatase, and the AMPAK ELISA amplification system (Novo BioLabs, Cambridge, UK). With this procedure, antibodies can be detected that recognize gp120 from a wide range of divergent HIV-1 strains. The ELISA is sufficiently sensitive to detect env antibodies in sera from HIV-positive individuals at dilutions of 1:300,000. No repeatable false-positives were detected in a screen of 250 normal serum samples. Env antibodies were detected in all 37 strongly HIV-positive sera tested, and in four sera that were borderline or weakly positive in commercial ELISA. However, 55 sera positive in commercial ELISA but unconfirmable by Western blot ('ambiguously' positive) did not contain detectable env antibodies.     

Induction of antibodies to the envelope protein of the human immunodeficiency virus by immunization with monoclonal anti-idiotypes.

Anti-idiotypes that possess the internal image of antigen can induce protective humoral immunity toward microbes. Herein we demonstrate antigen mimicry by monoclonal anti-idiotypes of a distinct epitope of the human immunodeficiency virus (HIV) envelope protein that is defined by a synthetic peptide. This peptide, corresponding to amino acid residues 503-535 (peptide 503-535) of HIV-1 IIIB gp160, induced antibodies in three mammalian species that interacted with HIV-1 gp120 and inhibited in vitro syncytium formation caused by HIV-1, IIIB and MN isolates. Three monoclonal anti-idiotypes were generated against rabbit anti-gp120 antibodies specific for peptide 503-535. These anti-idiotypes recognize an interspecies cross-reactive idiotype expressed on mouse, chimpanzee, baboon, rabbit, and human anti-gp120 antibodies specific for peptide 503-535. The interaction with the cross-reactive idiotype is inhibited by synthetic peptide and HIV-1 gp160. Furthermore, rabbits immunized with the monoclonal anti-idiotypes produced antibodies that also bind HIV-1 gp120 and gp160 and recognized the epitope defined by peptide 503-535.     

Polyclonal antibodies to mannan from yeast also recognize the carbohydrate structure of gp120 of the AIDS virus: an approach to raise neutralizing antibodies to HIV-1 infection in vitro

This study initiates a new method of developing an antigen which might be useful in the prevention of HIV-1 infection. Using a mannan preparation from Saccharomyces cerevisiae neutralizing antiserum was raised in rabbits which prevents HIV-1 infection in vitro up to a titre of 1:128. The corresponding antibody preparation neutralized the in vitro infectivity down to a concentration of 5 micrograms/ml. Analytical studies suggest that the antibodies are directed against the mannose residues of the HIV-1 glycoprotein (gp) 120 and its precursor gp 160.     

Localization of B-cell stimulatory activity of HIV-1 to the carboxyl terminus of gp41

Patients with AIDS are known to have B-cell hyperactivity. We have previously demonstrated that an extract of HIV-1 could induce differentiation of peripheral blood B lymphocytes of healthy volunteers into immunoglobulin-secreting cells. In an attempt to delineate the B-cell stimulatory subregion in HIV-1, we have investigated the influences of native glycoproteins and recombinant proteins of the envelope. The complete envelope glycoprotein, gp160 and a recombinant protein in the carboxyl terminal region of gp41 termed PE-8 were effective in inducing terminal differentiation of normal peripheral blood B lymphocytes and did so in a T-lymphocyte-dependent manner. The activity was not present in the native exterior envelope glycoprotein, gp120 and several other recombinant proteins, viz PE-2 an PE-3, which are in the amino terminal region of gp120 or in env-9, a protein in the junctional region of gp120 and gp41. Polyclonal and monoclonal antibodies directed to diverse regions of the envelope abrogated the influence of gp160. The PE-8-induced B-cell differentiation was abrogated by goat anti-gp160 antibody but not by goat anti-gp120 antibody or monoclonal antibody to the amino terminal of gp41. These studies suggest that a putative polyclonal B-cell stimulatory epitope of HIV-1 is located in the carboxyl end of the envelope glycoprotein.     

HIV-1 gp120- and gp160-induced apoptosis in cultured endothelial cells is mediated by caspases

The immune dysfunction and cell destruction that occur in the human immunodeficiency virus (HIV)-infected host appear to result from the direct cytopathic effects of viral infection and the effects of viral proteins on uninfected bystander cells. Recently, the alpha-chemokine receptor CXCR4 has been reported to mediate apoptosis in neuronal cells and in CD4(+) and CD8(+) T cells after its binding to HIV-1 envelope proteins. In the current study, it was observed that human umbilical vein endothelial cells (HUVEC) undergo apoptosis after their treatment with the HIV-1 envelope proteins gp120/160. Anti-CXCR4 monoclonal antibody decreased HIV-1 gp120/160-induced apoptosis, suggesting that the CXCR4 chemokine receptor mediates the apoptotic effects of these HIV envelope glycoproteins. Further studies revealed that caspases play an important role in this process because the pretreatment of cells with a general caspase enzyme inhibitor decreased the extent of HUVEC apoptosis induced by gp120/160. In addition, it was found that caspase-3 was activated on HIV-1 gp120/160 treatment of these cells. It was also observed that gp120/160 treatment slightly increased the expression of the pro-apoptotic molecule Bax. These results suggest that HIV-1 envelope glycoproteins can disrupt endothelial integrity through the interaction with CXCR4, thereby facilitating virus transit out of the bloodstream and contributing to the vascular injury syndromes seen in acquired immunodeficiency syndrome. (Blood. 2000;96:1438-1442)     

Evidence of HIV type 1 glycoprotein 120 binding to recombinant N-methyl-D-aspartate receptor subunits expressed in a baculovirus system.

Activation of the N-methyl-D-aspartate (NMDA) receptor by HIV-1 envelope glycoprotein 120 (gp120) is thought to represent at least one of the pathways causing neuronal damage in AIDS patients. In the present study, recombinant gp120 binding to NMDA receptor subunits expressed in a baculovirus system was examined by immunocytochemistry and a binding assay, using horseradish peroxidase (HRP)-conjugated and 125I-labeled recombinant gp120, respectively. We found that recombinant gp120 binds to Sf21 cells expressing epsilon1/zeta1 or epsilon2/zeta1 combined NMDA receptor subunits, but not to Sf21 cells infected with mock virus or Sf21 cells expressing a single epsilon1, epsilon2, or zeta1 NMDA receptor subunit. The binding was strongly blocked by unlabeled recombinant gp120, monoclonal anti-HIV-1 gp160 antibody, and a mixture of anti-epsilon1/epsilon2 and anti-zeta1 antibodies. The same results were obtained by flow cytometric analysis. These data suggest that HIV-1 gp120 may directly bind to the NMDA receptor. This evidence enhances our understanding of the mechanism of HIV-1-induced neuronal damage in AIDS patients.     

Role of nitric oxide in the promoting effect of HIV type 1 infection and of gp120 envelope glycoprotein on interleukin 4-induced IgE production by normal human mononuclear cells

Increased levels of serum IgE have been described in HIV-1 infection; however, mechanisms implicated in this immunoglobulin production remain unknown. In this study, we demonstrate that in vitro infection of human peripheral blood mononuclear cells (PBMCs) by HIV-1 monocytotropic (Ba-L) or lymphocytotropic (LAI) strains promotes IL-4-induced IgE production, indicating that the HIV-1 infectious process may participate in the IgE production observed in vivo. The effect of membrane glycoproteins (gp160, gp120, and gp41) was also evaluated. It was found that gp120 specifically potentiates in a dose-dependent manner IL-4-induced IgE production and does not affect IL-4-induced IgG, IgA, or IgM production. In these experiments, gp160 was also found to upregulate IL-4-induced IgE production, whereas gp41 was ineffective. This effect of gp120, gp160, and HIV-1 infection on IgE synthesis was not observed in the absence of IL-4. In the presence of IL-4, the inducing effect of gp120 appeared to be indirect because gp120 did not modify purified B lymphocyte IgE production after IL-4 and anti-CD40 monoclonal antibody stimulation. As HIV-1 infection is associated with alterations of PBMC redox metabolism, the role of nitric oxide (NO) in this IgE production by human PBMCs was evaluated. In the presence of a specific inhibitor of NO synthase pathways (L-NAME), IgE production induced by IL-4 and gp120 was abolished. Taken together, these data indicate that HIV-1 envelope glycoprotein gp120 (and gp160) specifically enhances IL-4-induced IgE production by normal human PBMCs, probably through the regulation of the nitric oxide pathway.     

Sensitive ELISA for the gp120 and gp160 surface glycoproteins of HIV-1

We have used a panel of polyclonal and monoclonal antibodies against gp120 and gp160, the envelope glycoproteins of human immunodeficiency virus type 1, to create rapid, simple, and sensitive twin-site sandwich ELISA specific for gp120 and gp160 or for gp160 alone. These assays can detect 500 COS cells in a population transiently transfected with a construct encoding gp120 and gp160, or 50 pg of recombinant gp160. We estimate that the mean amount of gp120 + gp160 in the transfected population is equivalent to 2.5 x 10(6) molecules per cell, 40-50% of which can be recovered from the culture medium as gp120 after 24 hours. The ELISA can be adapted to assess whether gp120 is detectable in the sera of HIV-1-infected persons: we show that gp120/gp160 is completely stable in normal human serum for at least 24 hours at 37 degrees C.     

Characterization of recombinant gp120 and gp160 from HIV-1: binding to monoclonal antibodies and soluble CD4

We compared four preparations of recombinant HIV-1 envelope glycoprotein: mammalian (Chinese hamster ovary cells) gp120 (Celltech); baculovirus gp120 from American Biotechnologies Inc. (ABT) and from MicroGeneSys (MGS); and baculovirus gp160 (Institute of Virology, Oxford, UK). Each envelope glycoprotein binds to a neutralizing monoclonal antibody (MAb) directed against the V3 loop, confirming the integrity of this type-specific neutralization epitope. MGS gp120 binds abnormally well to a MAb which recognizes an epitope preferentially exposed on denatured gp120. Consistent with this finding, MGS gp120 binds to soluble CD4 (sCD4) with an affinity 50-100-fold lower than that of Celltech gp120. The affinity of Celltech gp120 from sCD4 is 2.3 nM, indistinguishable from that of gp120 extracted from HIV-1 virions. Baculovirus gp120 (ABT) and gp160 also have a high affinity for sCD4. A significant proportion of anti-gp120 antibodies in HIV-positive human sera recognize epitopes that are dependent on the mammalian glycosylation pattern, and a human HIV-positive serum inhibits the binding of mammalian gp120 to sCD4 five- to 10-fold more potently than it inhibits baculovirus gp120 binding to sCD4.     

Delineation of immunoreactive, conserved regions in the external glycoprotein of the human immunodeficiency virus type 1.

Immunization of mice and rats with purified external glycoprotein gp120 from two divergent human immunodeficiency virus type 1 (HIV-1) isolates resulted in the development of seven hybridomas secreting monoclonal antibodies able to recognize regions of gp120 which are common among divergent strains of HIV-1. These monoclonal antibodies cross-reacted with env glycoproteins from one African (Rutz), one Haitian (RF), and three North American viral isolates, namely IIIB, MN, and 451 by either immunoblot or radioimmunoprecipitation assays. All recognized denatured gp120 in immunoblots with the exception of one which required a conformationally intact glycoprotein for reactivity. The gp120 epitopes identified by these antibodies were mapped by screening of an env gene library in the lambda gt11 expression system. Three out of four epitopes were found to reside in the amino-terminal half of gp120 (Cys9 to Cys35, Thr44 to Glu72 and Val108 to Met130), the other was located in the middle region (Thr221 to Ser255). By virtue of their extent of cross-reactivity these reagents might provide a unique resource for the detection of new viral isolates related to HIV-1.     

Large-scale production and purification of a vaccinia recombinant-derived HIV-1 gp160 and analysis of its immunogenicity

The human immunodeficiency virus (HIV-1) envelope gene was expressed in large-scale microcarrier cultures of Vero cells using a system involving coinfection with two recombinant vaccinia viruses. One recombinant contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second contained the HIV-1 gp160 gene flanked by T7 promoter and termination sequences. The protein was expressed on the surface of infected cells, and it was shown to have a molecular weight of 160 kD and to react with gp41 and gp120 specific monoclonal antibodies. After purification by successive affinity and ion-exchange chromatography, the protein was demonstrated to be present in a particulate form with a diameter in the range of 15-30 nm. When injected into goats a high-titer gp160 specific antibody response was elicited and group-specific neutralizing activity could be demonstrated in vitro. The immunogenicity of the protein was also studied in conjunction with a number of adjuvant formulations, and the highest potency in mice was obtained using a preparation with 0.2% Al(OH)3 and 0.25% deoxycholate.     

Quantitative analysis of human immunodeficiency virus type 1 antibody reactivity by western immunoblots: evaluation of relative antibody levels in seropositive individuals and mothers.

A quantitative analysis of antibody responses to human immunodeficiency virus type 1 (HIV-1) proteins using Western immunoblots and 125I-labeled protein A is reproducible and can be validated. The antibody levels obtained by Western immunoblots were compared with stoichiometric p24 radioimmunoassay over a wide range of antibody (correlation coefficient, .94; P less than .001). Antibody levels to gp160 and gp120 were validated using purified antigens. Analysis of antibody levels from 31 seropositive individuals revealed a statistically significant correlation between antibody levels to p24 and the other viral proteins except gp120. Anti-gag p24 antibody was strongly correlated with antibodies to other env products, specifically gp41 and gp160. Using the validated assay, HIV-1-infected mothers of infants were found to have highly variable levels of antibody to all viral proteins. Mothers of infected infants did not differ significantly from mothers of uninfected infants in antibody pattern or levels to any viral protein including gp120.     

Production and characterization of a human monoclonal antibody, reactive with a conserved epitope on gp41 of human immunodeficiency virus type I.

A human Epstein-Barr virus-transformed lymphoblastoid B-cell line was generated from peripheral blood mononuclear cells (PBMC) of an asymptomatic human immunodeficiency virus type I (HIV-1) seropositive donor, which produces a human monoclonal antibody K14 (IgG1), reactive with an epitope on the transmembrane part (gp41) of the envelope glycoprotein of HIV-1. This monoclonal antibody reacts with a lysate of HIV-1-infected H9 cells, gradient purified HIV-1, and a vaccinia recombinant HIV-1 gp160 protein, but not with HIV-2 antigens in an enzyme-linked immunosorbent assay (ELISA). When used as an immobilized ligand in an immune affinity column, K14 selectively purifies gp41 from a HIV-1-infected H9 cell lysate. Although no reactivity was observed in ELISA with a panel of partially overlapping synthetic nonapeptides spanning the whole length of HIV-1 gp41, it was shown to react with recombinant envelope proteins, provided that they did contain amino acids 643-692: deletion of this part resulted in the disappearance of the reactivity. Testing of an extensive panel of the sera from HIV-1 seropositive or seronegative donors from Europe and Africa, including a selected group of donors before and after HIV-1 seroconversion, in a competition ELISA with horseradish peroxidase-conjugated K14, showed that the epitope recognized on gp41 is immunodominant and conserved. K14 does not neutralize HIV-1 infectivity or virus-mediated cell fusion, and does not mediate antibody-dependent cellular cytotoxicity.     

Immunocytochemical determination of antigen and epitope specificity of HIV-1-specific B cells in lymph-node biopsies from HIV-1-infected individuals.

Knowledge about B-cell dysfunction and HIV-specific antibody production is necessary for the understanding of both HIV-1-related immunopathology and the (vaccine-induced) humoral immunity involved in protection against AIDS. This paper describes the application of recently developed methods to detect epitope specificity of B cells in lymph-node biopsies with antigen-enzyme conjugates. Cryosections of five lymph-node biopsies from HIV-1-infected individuals and four control tissues were stained with a panel of HIV-1 antigen-enzyme conjugates: recombinant HIV-1 proteins (gp 160, gp 120 and p24), labelled with peroxidase, and synthetic peptides representing neutralizing epitopes from gp120 and gp41, labelled with alkaline phosphatase. Antibody-forming cells (AFCs) were detected in all the HIV-1-infected biopsies with gp160, gp120 and/or p24, in numbers up to 350 per section. AFCs producing specific antibodies against peptide 101 (SP 101), representing the neutralizing epitope 586-608 of gp41, were detected in one patient. These techniques allow correlation of in vivo function of B cells with lymph-node pathology, clinical stage of the disease and serological data. Their potential for the elucidation of HIV-related immunopathogenesis and the development of vaccines is discussed.     

Enhanced HIV-1 neutralization by antibody heteroligation

Passive transfer of broadly neutralizing human antibodies against HIV-1 protects macaques against infection. However, HIV-1 uses several strategies to escape antibody neutralization, including mutation of the gp160 viral surface spike, a glycan shield to block antibody access to the spike, and expression of a limited number of viral surface spikes, which interferes with bivalent antibody binding. The latter is thought to decrease antibody apparent affinity or avidity, thereby interfering with neutralizing activity. To test the idea that increasing apparent affinity might enhance neutralizing activity, we engineered bispecific anti-HIV-1 antibodies (BiAbs) that can bind bivalently by virtue of one scFv arm that binds to gp120 and a second arm to the gp41 subunit of gp160. The individual arms of the BiAbs preserved the binding specificities of the original anti-HIV IgG antibodies and together bound simultaneously to gp120 and gp41. Heterotypic bivalent binding enhanced neutralization compared with the parental antibodies. We conclude that antibody recognition and viral neutralization of HIV can be improved by heteroligation.     

Peptides selected from a phage display library with an HIV-neutralizing antibody elicit antibodies to HIV gp120 in rabbits, but not to the same epitope

Monoclonal antibodies specific for the conserved CD4 binding site region of the HIV envelope protein gp120 were used to select phage from two different random peptide display libraries. Synthetic peptides were made with sequences corresponding to those displayed on the selected phage, and peptide-protein fusions were expressed that contained the selected phage-displayed peptide sequence and either the N-terminal domain of the phage pIII protein or the small heat shock protein of Methanococcus jannaschii or both. For monoclonal antibody 5145A, these constructs containing the selected peptide sequences were all capable of specifically inhibiting the binding of 5145A to HIV-1 gp120. Rabbits immunized with peptide-protein fusions produced antisera that bound to recombinant HIV-1 gp120, but did not bind to HIV-infected cells nor neutralize HIV. The antisera also did not compete with CD4 or antibodies to the CD4 binding site for binding to gp120.     

Accessibility of the highly conserved amino- and carboxy-terminal regions from HIV-1 external envelope glycoproteins

Amino- and carboxy-terminal extremities of the envelope external glycoproteins are regions that have remained highly conserved between human immunodeficiency viruses HIV-1 and HIV-2. The corresponding peptides have been synthesized and their structure and function analyzed. Circular dichroism spectra showed evidence of alpha helical conformation when the peptides were dissolved in the nonpolar solvent trifuoroethanol. These two regions are indeed exposed on the molecule because they were accessible to their respective specific antibodies on the native gp160 precursor or processed gp120 glycoproteins of HIV-1. Neither the peptides nor rabbit or human antibodies directed against the N- and C-terminal peptides interfered with the interaction between HIV-1 external glycoprotein gp120 and its CD4 cellular receptor. Taken together, these results indicate that N- and C-terminal regions of gp120 are accessible on the quaternary structure of the virion as well as on the soluble form of gp120 and that these regions are not directly or indirectly involved in the binding of gp120 to CD4.