Covalently crosslinked complexes of human immunodeficiency virus type 1 (HIV-1) gp120 and CD4 receptor elicit a neutralizing immune response that includes antibodies selective for primary virus isolates.

Specific conformational changes in the envelope glycoprotein gp120 of the human immunodeficiency virus type-1 (HIV-1) may be critical for eliciting a broadly neutralizing immune response against primary virus isolates. Since the interaction of gp120 with its receptor, CD4, induces conformational perturbations in both molecules, gp120-CD4 complexes should present unique immunogenic features that may include novel epitopes for broadly neutralizing antibodies. To test this hypothesis, we raised polyclonal antiserum against covalently crosslinked gp120-CD4 complexes in a goat and examined the ability of the anti-complex antibodies to neutralize primary and laboratory-adapted HIV-1 isolates. In cell-free neutralization assays with HIV-1MN, the antiserum demonstrated the ability to neutralize primary virus more effectively than the laboratory-adapted isolate. The neutralizing capacity of the anti-complex serum extended to primary isolates from distant genetic clades A, D, and E, although the degree of neutralization was found to vary among the clades. The neutralizing activity of the serum was composed of two components. The first component included anti-CD4 antibodies that recognized epitopes outside the gp120 binding site; the second was independent of CD4 reactivity and was retained after removal of cell surface anti-CD4 reactivity by repeated absorption with CD4-positive cells. These results demonstrate that gp120-CD4 complexes can elicit a unique polyclonal antibody response that is relevant to the neutralization of primary isolates of HIV-1.

Detection of HIV-1 infection in vitro using NASBA: an isothermal RNA amplification technique.

Establishment of a sensitive infection assay for HIV-1 is essential for successful screening of antiviral agents and neutralizing antibodies. In this report, an infection assay is described which measures the expression of viral genomic RNA and spliced mRNA intermediates in infected cells by an amplification-based technique called NASBA. The extreme sensitivity of this method permits the detection of viral RNA in peripheral blood mononuclear cells (PBMC) within 48 h of infection by a low dose of virus. Similarly, spliced HIV-1 mRNA could be detected within 24 h of infection of CEM cells by HIV-1IIIB. This NASBA-based infection assay was shown to titer the neutralization of the HIV-1IIIB isolate by serum from an infected human and by a monoclonal antibody to gp120. Furthermore, the inhibitory effects of azidothymidine (AZT) and soluble CD4 on HIV-1IIIB infection were quantitated by this assay. The early detection of virus by NASBA minimizes the contribution of secondary infection, thereby permitting more accurate evaluation of antiviral agents and neutralizing antibodies. This assay may be useful for the study of infection of phenotypically distinct HIV-1 isolates, which differ in terms of their replication kinetics.

Monoclonal antibodies raised against covalently crosslinked complexes of human immunodeficiency virus type 1 gp120 and CD4 receptor identify a novel complex-dependent epitope on gp 120.

The binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120, to its cell surface receptor, CD4, represents a molecular interaction involving distinct alterations in protein structure. Consequently, the pattern of epitopes presented on the gp120-CD4 complex should differ from those on free gp120. To investigate this concept, mice were immunized with covalently crosslinked complexes of viral HIV-1IIIBgp120 and soluble CD4. Two monoclonal antibodies (MoAbs) obtained from the immunized mice exhibited a novel epitope specificity. The MoAbs were marginally reactive with HIV-1IIIBgp120, highly reactive with gp120-CD4 complexes, and unreactive with soluble CD4. The same pattern of reactivity was seen in solid-phase assays using HIV-1(451)gp120. A similar specificity for complexes was evident in flow cytometry experiments, in which MoAb reactivity was dependent upon the attachment of gp120 to CD4-positive cells. In addition, MoAb reactivity was detected upon the interaction of CD4 receptors with purified HIV-1IIIB virions. Notably, seroantibodies from HIV-positive individuals competed for MoAb binding, indicating that the epitope is immunogenic in humans. The results demonstrated that crosslinked gp120-CD4 complexes elicit antibodies to cryptic gp120 epitopes that are exposed during infection in response to receptor binding. These findings may have important implications for the consideration of HIV envelope-receptor complexes as targets for virus neutralization.

Mechanism of enzyme inhibition mediated by anti-reverse transcriptase antibodies from HIV type 1-infected individuals.

We have examined the mechanisms of reverse transcriptase (RT) inhibition mediated by anti-RT antibodies, isolated by affinity chromatography, from four HIV-1-positive individuals. In kinetics assays, anti-RT immunoglobulin (Ig) obtained from three of the sera mediated a noncompetitive type of inhibition against template primer; two of these three also mediated noncompetitive inhibition with respect to deoxyribonucleoside triphosphate. Such inhibition did not require that the Ig be preincubated with RT prior to the addition of reaction components. In contrast, a more complicated pattern of inhibition was exhibited by anti-RT Ig from the fourth serum. Preincubation of this Ig with enzyme markedly enhanced the inhibition. The results demonstrate that the specificities of RT-inhibiting antibodies vary among HIV-1-infected individuals, but that one prevalent mechanism of inhibition involves interactions with epitopes outside of the enzyme active site.

Enzyme immunoassay using native envelope glycoprotein (gp160) for detection of human immunodeficiency virus type 1 antibodies.

An enzyme immunoassay using the purified native gp160 for the detection of human immunodeficiency virus type 1 (HIV-1) antibody was developed. This assay was determined to be highly specific, since (i) 157 serum samples that were confirmed negative by Western blot (immunoblot) (WB) were negative, (ii) 41 serum samples from populations with medical conditions that might cause nonspecific assay reactivity were all negative, and (iii) all 15 serum samples that showed false-positive reactions in one or more commercial HIV-1 screening tests were negative. The assay gave 100% specificity with a randomly selected and unlinked panel of 1,000 serum samples from healthy blood donors. The sensitivity of the assay was assessed by testing 238 samples confirmed as HIV-1 antibody positive by a standardized WB assay. All 238 serum samples (100%) were reactive in the native gp160 assay. In a dilution panel of 14 weakly WB-positive serum samples, 7 samples reacted two-to fivefold more strongly in the gp160 assay than in a virus lysate-based assay; the remaining 7 samples gave comparable reactivities in the two tests. The reactivities of 13 of these 14 serum samples in the gp160 assay were higher than in a commercial enzyme immunoassay that uses a recombinant envelope protein as the antigen. The native gp160 assay was more sensitive to identify seroconversion. In a well-characterized panel of sequential blood samples from a seroconverter, the new assay detected antibodies at least one sample ahead of the other commercial assays tested.

Glycoprotein of human immunodeficiency virus type 1 synthesized in chronically infected Molt3 cells acquires heterogeneous oligosaccharide structures.

Diversity of oligosaccharide structures on the glycoprotein of HIV-1 was studied in individual clones of Molt3 cells chronically infected with HIV-1IIIB. A glycoprotein of molecular weight 140 kD (gp140) was found to be shed into the medium from one of these clones, which unlike normally processed gp120, contained significant proportions of endo H resistant oligosaccharides. Treatment of infected cells with the inhibitors of oligosaccharide trimming enzymes affected the glycosylation pattern as well as the secretion of the glycoprotein into the medium. The exposure of the principal neutralizing domain (PND) on the surface of gp140, as measured by its accessibility to thrombin cleavage, was comparable to that observed with gp120. Sera obtained from mice inoculated with purified gp140 contained high titered anti-V3 antibodies and blocked HIV-1IIIB-induced syncytium formation. These results demonstrate that although glycosylation of viral glycoproteins is governed by the host cell glycosyl transferases, glycoprotein secreted from biological clones of the same host cells acquires different oligosaccharide structures. Exposure and immunogenicity of the PND in one such glycosylation variant are comparable to the normally processed gp120 molecule.

Conformational perturbation of the envelope glycoprotein gp120 of human immunodeficiency virus type 1 by soluble CD4 and the lectin succinyl Con A.

We have studied perturbation of the gp120/gp41 envelope complex of HIV-1 in the presence of the mannose-specific lectin succinyl Con A (SC) and compared the effect with that observed in the presence of soluble CD4 (sCD4). SC did not inhibit the binding of gp120 to CD4. Both sCD4 and SC inhibited syncytium formation induced by HIV-1-infected Molt3/HIV-1IIIB cells. The infectivity of HIV-1 was markedly reduced when the virions were preincubated with SC or when SC was mixed simultaneously with virus and cells. The conformation of gp120 was altered in the presence of SC as evidenced by an increased susceptibility of the principal neutralizing epitope (V3 loop) to thrombin digestion. SC treatment of [35S]-methionine-labeled virions derived from Molt3/HIV-1IIIB cells resulted in the dissociation of gp120 from the viral membrane. The effect was less pronounced than that observed with sCD4. These results suggest that although interacting with different regions of gp120, the mannose-specific lectin alters the conformation of the glycoprotein in a manner similar to that induced by sCD4, causing destabilization of the gp120/gp41 complex.

High prevalence of serum antibodies to equine infectious anemia virus reverse transcriptase.

The immunogenicity of the equine infectious anemia virus (EIAV) reverse transcriptase (RT) was examined by immunoblot assay with recombinant EIAV RT. All of the 19 sera from EIAV-infected horses tested contained antibodies that recognized EIAV RT and directly inhibited the polymerase activity of the enzyme. An examination of sera obtained sequentially from two experimentally infected animals revealed that anti-RT antibodies arise early in infection and increase in level. The appearance of the antibodies correlated with progression toward the asymptomatic period of infection.

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.

Identification of a major growth factor for AIDS-Kaposi's sarcoma cells as oncostatin M.

Conditioned medium from human T cell leukemia virus type 2 (HTLV-II)-infected T cells supports the growth and long-term culture of cells derived from acquired immunodeficiency syndrome (AIDS)-associated Kaposi's sarcoma lesions (AIDS-KS cells). A protein of 30 kilodaltons was purified from conditioned medium that supports the growth of AIDS-KS cells. The amino-terminal sequence of this protein was identical to the amino-terminal sequence of Oncostatin M, a glycoprotein that inhibits the growth of a variety of cancer cells. Oncostatin M from conditioned medium stimulated a twofold increase in the growth of AIDS-KS cells at a concentration of less than 1 nanogram of the protein per milliliter of medium.

Characterization of a neutralizing monoclonal antibody to the external glycoprotein of HIV-1.

The major neutralizing epitope on the external glycoprotein of HIV-1 was studied with an envelope-specific monoclonal antibody and with a human serum positive for antibodies to HIV-1 proteins, both of which were able to neutralize virus infectivity. The monoclonal antibody reacted specifically with gp120 from HIV-1IIIB, and was shown to neutralize infection of CEM cells by cell-free virions, and inhibited the formation of syncytia normally observed when uninfected cells are cocultured with HIV-1-infected cells. Similar neutralization of viral infection and inhibition of syncytia formation was also demonstrated by the HIV-1-antibody-positive human serum. By examining a number of overlapping peptides from a region of HIV-1 gp120 known to contain a neutralizing epitope, this epitope was localized between amino acids 307 and 320 (V3 loop) in the external glycoprotein molecule. The monoclonal antibody did not interfere with the binding of gp120 to CD4, or with the subsequent step of CD4-induced shedding of gp120 from the viral envelope. However, it blocked the proteolytic cleavage of the V3 loop by thrombin, suggesting that the antibody may be inhibiting the interaction of the loop with other membrane-bound proteins.

Purification and partial characterization of equine infectious anemia virus reverse transcriptase.

Previously we raised a rabbit monospecific antibody (C2003) against a synthetic peptide derived from a sequence within the C-terminal portion of the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1). This sequence is found to be conserved in the predicted amino acid sequence of a related lentivirus, the equine infectious anemia virus (EIAV). It was previously determined that the C2003 antibody could cross-react with native EIAV RT and directly inhibit the DNA polymerase activity of the enzyme. We have now fractionated EIAV RT by immunoaffinity chromatography with immobilized C2003 antibody. The procedure yielded an equimolar mixture of two proteins of 66 and 51 kDa associated with both DNA polymerase and RNase H activities. When the EIAV RT proteins were examined by in situ activity gel assays, polymerase activity was found to be principally associated with the 66-kDa component. The fidelity of DNA synthesis by EIAV RT was found to be equivalent to that of HIV-1 RT and lower than that of AMV RT. These observations indicate that the RTs of EIAV and HIV-1 share similar structural and functional properties.

Brefeldin A inhibits the processing and secretion of envelope glycoproteins of human immunodeficiency virus type 1.

The processing and secretion of the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) were studied in chronically infected T cells and in primary macrophages treated with an antiviral antibiotic brefeldin A (BFA). BFA blocks the egress of proteins from the endoplasmic reticulum and has a profound effect on the structure of cis/medial Golgi. In MOLT-3 cells infected with the IIIB strain of HIV-1 (MOLT-3/IIIB), BFA inhibited the intracellular processing of gp160. The secretion of envelope proteins from these cells was significantly inhibited in the presence of BFA. The gag proteins, on the other hand, were processed and secreted normally. BFA also inhibited the proteolytic processing of gp160 in primary macrophages infected with HIV-1. The infectivity of virus pelleted from the medium of MOLT-3/IIIB cells treated with BFA was markedly lower than that obtained from untreated cells. These results demonstrate that the proteolytic processing of gp160 in HIV-1-infected cells takes place after the glycoprotein exists the endoplasmic reticulum and that the transport of glycoprotein to the cell surface is required for assembly of complete HIV-1 particles.

Effect of Evans blue and trypan blue on syncytia formation and infectivity of human immunodeficiency virus type I and type II in vitro.

Polyanionic compounds were used to inhibit infectivity of human immunodeficiency virus in vitro. Suramin, Evans blue, and Trypan blue were shown to inhibit syncytia formation normally observed when HIV-1-infected cells are cocultured with CD4+ cells. The inhibition was more pronounced with Evans blue than with any of the other polyanions studied. The inhibitory effect was significantly weaker in HIV-2 systems. However, the reverse transcriptase activities of both types of viruses were inhibited by Evans blue. Another polyanionic compound, phosphorothioate 28-mer cytidine homopolymer (SdC28) was shown to inhibit syncytium formation induced by HIV-1-and HIV-2-infected cells in an identical manner. Evans blue showed partial blocking of gp120 binding to CD4 in a solid-phase enzyme-linked immunosorbent assay (ELISA). These results suggest that the polyanionic dyes may exert their antiviral effects, at least in part, by interfering with the binding and fusion of HIV with susceptible T cells.

Lateral diffusion of CD4 on the surface of a human neoplastic T-cell line probed with a fluorescent derivative of the envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1).

The envelope glycoprotein (gp120) of HIV-1 was labeled with fluorescein by using 6-[4,6-dichlorotriazinyl]aminofluorescein. The labeled glycoprotein was found to bind to CD4-positive CEM cells. Monoclonal antibody OKT4a but not OKT4 blocked this binding. Similar specific binding of fluorescein-labeled gp120 with CD4 was observed in a solid-phase ELISA where sCD4 was attached to a polystyrene plate. The syncytium formation induced by HIV-1-infected cells on CEM cells was significantly inhibited in the presence of fluorescein-labeled gp120. Fluorescence photobleaching recovery measurements showed that the diffusion coefficient (D) of CD4 molecules complexed with fluorescein-labeled gp120 was approximately 5 x 10(-10) cm2sec-1, with nearly 61% of the receptor molecules being mobile. Binding of anti-gp120 monoclonal antibody to the CD4-gp120 complex reduced the mobile fraction significantly. Diffusion of CD4 labeled with OKT4 IgG was markedly inhibited with reductions in both D and the mobile fraction, but such inhibition was not observed with OKT4 Fab. It appears that crosslinking of multiple molecules of CD4 by OKT4 antibody is required to reduce CD4 mobility. This suggests that the receptor might be present on the membrane plane as molecular clusters containing at least two molecules of CD4.

Interaction of C-terminal sequences of human immunodeficiency virus reverse transcriptase with template primer.

We have raised a rabbit monospecific antibody (designated C2003) against a synthetic peptide (CTP66) derived from a conserved sequence in the C-terminal portion of the p66 component of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (DeVico, A.L., Copeland, T.D., Veronese, F.D., Oroszlan, S., Gallo, R. C., and Sarngadharan, M. G. (1989) AIDS Res. Hum. Retroviruses 5, 51-60). This antibody directly inhibits the polymerase activity of HIV-1 RT and of RTs from a variety of retroviruses. HIV-1 RT is protected from this inhibition by preincubation of the enzyme with template primer prior to treatment with the antibody. Such protection is abrogated when the pretreatment is conducted under conditions of high ionic strength. Kinetic studies showed that the antibody-mediated inhibition is competitive with respect to template primer concentration. These results indicate that C2003 antibody acts to interfere with the template binding function of the enzyme and further indicates that conserved residues recognized by the antibody may be directly involved in this function.

Mode of action of 5'-linked cholesteryl phosphorothioate oligodeoxynucleotides in inhibiting syncytia formation and infection by HIV-1 and HIV-2 in vitro.

A phosphorothioate homocytidine 10-mer containing a cholesteryl moiety covalently linked to the 5'-end (Chol-SdC10) inhibited syncytium formation in susceptible T cells induced by HIV-1 and HIV-2. The syncytium inhibition effect was minimal with unmodified cytidine homopolymer of the same net charge. Chol-SdC10 was shown to protect CEM cells against infection by cell-free HIV-1 particles without any apparent toxicity to the growth of CD4+ T cells. The DNA polymerase activity of the purified reverse transcriptase (RT) of HIV-1 was markedly inhibited by Chol-SdC10 but the effect on the RNase H activity of RT was minimal. Analysis of the kinetics of reverse transcriptase inhibition mediated by the drug revealed that the inhibition at a higher concentration was competitive with respect to template primer binding and noncompetitive at lower concentrations. Chol-SdC10 also partially blocked the binding of gp120 to CD4 in a solid-phase ELISA. These results confirm that the anti-HIV activity of phosphorothioate cytidine homopolymers increases markedly by covalent modification with the cholesteryl moiety at the 5'-end and demonstrates that the cytoprotective effect is manifested at multiple steps in the virus life cycle. These steps include inhibition of retroviral replication activity as well as the binding and fusion of HIV with CD4+ T cells.

Differential viral gene expression and its effect on the biological properties of the cell clones of an HIV-1-infected cell line.

We have shown that 6D5 cells infected with the HIV-1 strain HTLV-III451 (6D5(451)) secreted viral envelope proteins gp160 and gp120 into the culture medium. Single cell cloning of 6D5(451) cells separated three distinct phenotypes. All clones secreted unprocessed env protein gp160 along with gp120. Only one phenotype produced infectious virus and contained normally processed gag proteins. The second phenotype was associated with nonproducer cells expressing only the env gene but no extracellular particles. The third phenotype synthesized Pr53gag but no reverse transcriptase, nor did it process the gag precursor. Only immature particles could be seen in the culture. Cells of the first and the third phenotypes produced two sizes of gp160, the normal and one with a small truncation at the C-terminus. Phenotype 2 only produced the smaller gp160. In all cases the gp160 that was secreted into the medium was the truncated molecule.