HIV-1 gp41 shares a common immunologic determinant with normal human blood lymphocytes and monocytes.
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Amino Acid Sequence↗
Biomedical subjects
F Steindl
Publications and source records attributed to F Steindl.
Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS.
Previously we identified the highly conserved amino acids Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ecto-domain of gp41 as the epitope of a neutralizing monoclonal antibody (2F5) directed against human immunodeficiency virus type 1. In the present study, the sequence defining the epitope was introduced into the loop of antigenic site B of the influenza virus hemagglutinin. The resulting chimeric virus was able to elicit ELDKWA-specific immunoglobulins G and A in antisera of mice. Moreover, the distantly related human immunodeficiency virus type 1 isolates MN, RF, and IIIB were neutralized by these antisera. These data suggest that this conserved B-cell epitope is a promising candidate for inclusion in a vaccine against AIDS. The results also show that influenza virus can be used to effectively present the antigenic structure of this B-cell epitope.
Amino Acid Sequence↗
HIV-1 gp41 enhances major histocompatibility complex class I and ICAM-1 expression on H9 and U937 cells.
Based on our findings that HIV-1 (human immunodeficiency virus type 1) soluble gp41 (sgp41; amino acids 539-684) bound to human T, B, and monocyte cells and enhanced major histocompatibility complex (MHC) class I and II antigen expression on Raji cells, we examined the effect of HIV-1 sgp41 on the surface expression of MHC I and II, ICAM-1, and CD4 molecules on human H9 and U937 cells. Flow cytometry (FACS) analysis demonstrated that sgp41 selectively enhanced MHC class I expression by about 75% on H9 cells and by about 85% on U937 cells, while the ICAM-1 expression was increased by about 70% only on H9 cells and remained unaltered on U937 cells; other molecules, such as MHC class II and CD4, remained unaltered. By comparison, alpha-, beta-, and omega-interferons, but not gamma-interferon, showed similar effects as sgp41 on the expression of MHC class I and ICAM-1 on H9 and U937 cells. The results suggest that HIV-1 gp41 may have a biological function that is involved in the regulation of human MHC class I and ICAM-1 expression.
Antigens, CD↗
HIV-1 gp41 binds to several proteins on the human B-cell line, Raji.
Based on our findings that HIV-1 gp41 independently of CD4, can bind to the helper T-cell line H9, we characterized putative binding of HIV-1 gp41 to B-cell lines, Raji, Bjab and Ramos. Using fluorescence-activated cell sorter (FACS) we examined the binding of soluble gp41 (sgp41; Env amino acid 539-684) to these B-cell lines. Using sgp41 attached to sepharose beads Raji cell lysates were absorbed. The sgp41-eluate of Raji cell lysates could inhibit the sgp41-binding to Raji cells. By SDS-PAGE of sgp41-eluate of Raji cell lysates four strong protein band, 37, 45, 49 and 62 kD, and a weak band of 92 kD were stained with Coomassie blue. By Western blot (ligand blot) analysis using sgp41 four protein bands, 37, 45, 49 and 62 kD, were observed in sgp41-eluate of Raji cell lysates. To test the individual proteins the five proteins were isolated from the sgp41-eluate of Raji cell lysates. Three proteins, 45, 49 and 62 kD, each could partially inhibit the sgp41-binding to Raji cells. The results suggest that these three proteins in Raji cell lysates are possible candidates for the putative gp41 receptor(s).
B-Lymphocytes↗
Enhancement of HIV-1 gp41 binding to Raji cells by PWM, LPS, interferon-gamma and interleukin-6.
Based on our finding that HIV-1 gp41 independently of CD4 can bind to several proteins (gp41 binding protein: GBP) on the human T-cell line H9, B-cell line Raji and monocyte-cell line U937, we examined the effect of mitogens and cytokines on binding of gp41 to H9, Raji and U937 cells. Flow cytometry (FACS) analysis demonstrated that PWM and LPS, IFN-gamma and IL-6, but not Con A, IFN-alpha, -beta, -omega and IL-2, could increase gp41 binding to Raji cells. In controls, none of the regulators (IFN-alpha, -beta, -gamma, -omega, IL-2, IL-6, Con A, PWM, LPS) could modify the binding potential of H9 and U937 cells. Our data suggest that the expression of HIV-1 binding proteins is subject to regulation by PWM, LPS, IFN-gamma and IL-6 in the case of B-cells, while on T-cells and macrophages, the binding proteins may be constitutively expressed.
B-Lymphocytes↗
The human monocyte cell line U937 binds HIV-1 gp41 by proteins of 37, 45, 49, 62 and 92 kDa.
Based on our findings that HIV-1 gp41 independently of CD4 can bind to the helper T cell line H9 and B cell line Raji, we characterized the putative binding of HIV-1 gp41 to the monocyte cell lines U937 and HL60. Using flow cytometry (FACS) we examined the binding of soluble gp41 (sgp41; env amino acids 539-684) to these monocyte cell lines. Using sgp41 attached to Sepharose beads, U937 cell lysates were absorbed. The sgp41 eluate of U937 cell lysates could inhibit sgp41 binding to U937 cells. With SDS-PAGE of sgp41 eluate of U937 cell lysates, three strong protein bands, (37, 45 and 62 kDa) and two weak bands (49 and 92 kDa) were stained with Coomassie blue. With Western blot (ligand blot) analysis using sgp41, three strong protein bands (37, 49 and 62 kDa) and a very weak band (42-45 kDa) were observed in sgp41 eluate of U937 cell lysates. The results suggest that the four proteins 37, 42-45, 49 and 62 kDa in U937 cell lysates are possible candidates for the putative gp41 receptor(s). We compared the blocking activities of sgp41 eluates from different cell lysates. Not only U937 and Raji lysate-sgp41 eluates, but also H9 and HL60 lysate-sgp41 eluates could block sgp41 binding to U937 and Raji cells. The results indicate that the sgp41-binding proteins on U937, or Raji (H9 and HL60, respectively) probably could have an identical blocking (or binding) specificity; these cell types carry very similar receptor(s) for HIV-1 gp41 binding.
B-Lymphocytes↗
HIV-1 gp41 binding to human peripheral blood mononuclear cells occurs preferentially to B Lymphocytes and monocytes.
Based on our findings that HIV-1 gp41 independently of CD4 can bind to the human helper T lymphoid cell line H9, B cell line Raji and monocyte cell line U937, we characterized putative binding of HIV-1 gp41 to human peripheral blood lymphocytes (PBLs) and monocytes. Using flow cytometry (FACS), we demonstrated that the recombinant soluble HIV-1 gp41 (sgp41; Env amino acid 539-684) can bind to the normal human peripheral blood mononuclear cells (PBMCs), preferentially to B lymphocytes and monocytes independently of gp120-binding sites on CD4 molecules. This binding is dose-dependent. The HIV-1 sgp41 binds to blood B lymphocytes and monocytes more strongly than to T lymphocytes. By two-color flow cytometric analysis, we identified that sgp41 can bind 10% of CD4+ T lymphocytes, 11.9% of CD8+ T lymphocytes, 47% of CD19+ B lymphocytes and 44.2% of CD14+ monocytes.
B-Lymphocytes↗
A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1.
Vaccination against human immunodeficiency virus type 1 (HIV-1) requires an immunogen which will elicit a protective immunity against viruses that show a high degree of genetic polymorphism. Therefore, the identification of neutralizing epitopes which are shared by many strains would be useful. In previous studies, we established a human monoclonal antibody (2F5) that neutralizes a variety of laboratory strains and clinical isolates of HIV-1. In the present report, we define the amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ectodomain of gp41 as the epitope recognized by this antibody. The sequence was found to be conserved in 72% of otherwise highly variable HIV-1 isolates. Escape mutants were not detected in cells infected with HIV-1 isolates MN and RF in the presence of antibody 2F5. Since sequence variability of neutralizing epitopes is considered to be a major obstacle to HIV-1 vaccine development, the conserved B-cell epitope described here is a promising candidate for inclusion in a vaccine against AIDS.
Amino Acid Sequence↗
HIV-1 gp41 contains two sites for interaction with several proteins on the helper T-lymphoid cell line, H9.
OBJECTIVE: To characterize putative binding sites for HIV-1 gp41 to H9 cells. DESIGN: Based on accumulating evidence in the literature that HIV-1 can bind to cell surfaces independent of CD4, we attempted to clarify whether gp41, the transmembrane HIV-1 protein, contributes to CD4-independent binding. We therefore tested binding of gp41 to cells. METHODS: Using fluorescence-activated cell-sorter analysis, we examined the binding of recombinant gp160 (gp160) and soluble gp41 (sgp41; Env amino acids 539-684) to H9 cells, and located the putative binding site(s) of gp41 by inhibition using 16 HIV-1 Env peptides. Putative HIV-1 receptor proteins in H9 cell lysates were Western blotted and precipitated using sgp41. RESULTS: sgp41 bound to the CD4+ H9 cells and rgp160 bound to H9 cells independent of gp120-binding sites on CD4 molecules. Two gp41 peptides (Env amino acids 591-605 and 651-665) inhibited the binding of sgp41 to H9 cells. Four bands, of 37, 40, 55 and 97 kD, were blotted in H9 cell lysates, and three bands, 40, 97 and 108 kD, were observed in the precipitation analysis using lysates of 125I-surface-labelled H9 cells and sgp41 attached to sepharose beads. CONCLUSIONS: HIV-1 gp41 contains two putative binding sites to H9 cells. These sites may be located within Env amino acids 591-605 (ERYLKDQQLLGIWGC) and 651-665 (TLLEESQNQQEKNEQ). Using two different techniques, five proteins (37, 40, 55, 97 and 108 kD) were identified in H9 lysates as possible candidates for gp41 binding.
Amino Acid Sequence↗
Expression of a human monoclonal anti-HIV-1 antibody in CHO cells.
The cDNA coding for the light and heavy chains, respectively, of the human monoclonal antibody 3D6 (IgG1, kappa), which binds specifically to human immunodeficiency virus-1 (HIV-1) gp41, was inserted into three different mammalian expression vectors and transfected into Chinese hamster ovary (CHO) cells. Transcription was under the control of Rous sarcoma virus long terminal repeat (RSV LTR), human cytomegalovirus major immediate early (CMV IE) promoter, and mouse mammary tumor virus long terminal repeat (MMTV LTR), respectively. Antibody productivity was monitored in the supernatants of selected clones. The binding characteristics of the CHO-derived antibody to HIV-1 gp41 were found to be identical to that of the original antibody produced by hybridoma cells.
Animals↗
Isolation of isoproteins from monoclonal antibodies and recombinant proteins by chromatofocusing.
A fast protein liquid chromatographic method for the preparative separation of the various isoproteins is described. Highly purified human monoclonal antibodies, recombinant human superoxide dismutase and human superoxide dismutase from erythrocytes were used as starting material. The isoproteins were separated by chromatofocusing on Mono P columns. A very narrow pH gradient was applied to achieve complete separation of the isoproteins. The prepurification steps and the pretreatment of the samples to achieve optimum resolution are described in detail. The method is also applicable to extremely basic monoclonal antibodies (pI = 9). The successful separation was checked by isoelectric focusing in immobilized pH gradients (Immobilines). The future of these methods is discussed, because for many different biochemical and biophysical investigations pure and homogeneous isoproteins are necessary.
Antibodies, Monoclonal↗
Shifts of isoelectric points between cellular and secreted antibodies as revealed by isoelectric focusing and immobilized pH gradients.
Charge microheterogeneity of monoclonal antibodies, as revealed by isoelectric focusing in carrier ampholytes, has been known for a long time. Here we demonstrate, in the case of monoclonals against the gp-41 of the HIV-1 virus, that this heterogeneity is already present within the cell sap of hybridoma cells during antibody synthesis. When the monoclonals are secreted extracellularly, the same isoelectric point (pI) spectrum is maintained, but there is marked redistribution of the relative isoform abundance towards the lower pI components. This suggests in vivo processing of such forms, possibly via glycosylation or deamidation. The secreted antibodies are also analyzed by immobilized pH gradients (IPG), where they demonstrate an even more extensive heterogeneity, due to the marked increment in resolving power. Single bands are purified by preparative IPGs in a multicompartment electrolyzer and are shown to be stable with time. Thus, artefactual heterogeneity produced by the focusing technique is completely excluded and cellular processing is clearly established.
Animals↗
A continuous multistage roller reactor for animal cell culture: 1. Patterns of growth, production and catabolism of a murine hybridoma.
A Tubular Liquid Film Reactor was designed as a model system to transfer a batch culture kinetic to a continuous cascade. Cell density, product formation and substrate consumption rates were followed during fermentation at two dilution rates. In spite of the high dilution rates effective in each segment by itself high cell densities of up to 10(7) cells/ml were achieved due to cell sedimentation. The model character of the reactor was taken to determine critical values of substrate concentrations that influence production rates and result in an adaptation of metabolism.
Alanine↗
Comparison of protein A, protein G and copolymerized hydroxyapatite for the purification of human monoclonal antibodies.
Protein A Superose, protein G Sepharose fast flow and copolymerized hydroxyapatite were used for the purification of human monoclonal antibodies against HIV 1. Both desalted culture supernatant and a prepurified protein solution were used as starting materials. The different runs were compared with respect to yield and recovery of biological activity. The biological activity (specific reactivity) was checked by antigen enzyme-linked immunosorbent assay with recombinant antigen. The human monoclonal antibodies could not be selectively eluted from the hydroxyapatite but elution could be effected from the protein A Superose at pH 4.0 and from protein G at pH 3.0. The eluted immunoglobulin G was distributed over a broad pH range when protein G Superose was used. Biologically active material could be obtained from protein A Superose and protein G Sepharose fast flow.
Antibodies, Monoclonal↗
Isolation of human monoclonal antibody isoproteins by preparative isoelectric focusing in immobilized pH gradients.
A method for preparative isolation of human monoclonal antibody isoproteins is described in the present paper. A human monoclonal antibody directed against the transmembrane protein gp 41 from the human immunodeficiency virus (HIV-1) was used in this study. The antibody belongs to the IgG1 subtype and exhibits antibody dependent cellular cytotoxicity. The resolving power of conventional preparative protein separation techniques such as ion-exchange chromatography, chromatofocusing and lectin affinity chromatography is too poor for a complete separation of isoproteins. The more sophisticated technique of chromatofocusing on FPLC-based material (Mono P, Pharmacia) did not satisfy our expectation. With semipreparative IEF in immobilized pH gradients we were able to prepare the different isoproteins of a human monoclonal antibody in milligram amounts. No significant difference between the single isoproteins with respect to specificity and avidity to the recombinant antigen (rec gp 160) was detected. Therefore, we assume that the separation conditions did not influence the immunochemical nature of the antibody and significant denaturation and/or precipitation of the IgG did not occur. Furthermore the method affords preparative separation with resolution equivalent to analytical runs. Experiments for scale up and further characterization of isoproteins (carbohydrate composition, amino acid analysis, half life times etc.) are in progress.
Animals↗
The high efficiency, human B cell immortalizing heteromyeloma CB-F7. Production of human monoclonal antibodies to human immunodeficiency virus.
This paper describes the construction of a new heteromyeloma cell line designated CB-F7. The cell line was derived from xenogeneic somatic cell hybridization between normal human B lymphocytes and the murine HAT-sensitive P3X63Ag8/653 cell line. CB-F7 cells were characterized by rapid cell growth (doubling time about 16 h) and high cloning efficiencies in culture medium supplemented with 10% or 5% fetal calf serum, respectively. The karyotype of the cells consists of about 75-78 chromosomes as well as two chromosomal fragments. Fusions of the cells with human peripheral blood cells resulted in approximately 2-6 clones per 10(5) seeded lymphocytes. Furthermore, the cells are ouabain resistant and therefore suitable for fusions with EBV-transformed lymphoblastoid cell lines. Using CB-F7 as the parental cell line a number of specific human mAb producing hybrids were established. For the first time, we describe here the generation of hybrids secreting human monoclonal antibodies to human immunodeficiency virus (HIV). Two monoclonal antibodies of IgG type and one of IgM type reacted with the major core protein p25 and one IgG antibody reacted with the transmembrane protein gp41.
Antibodies, Monoclonal↗
Combination of Zetaprep mass ion-exchange media and high-performance cation-exchange chromatography for the purification of high-purity monoclonal antibodies.
A procedure involving diafiltration, mass ion exchange on a QAE Zetaprep disk, gel chromatography and cation-exchange chromatography was used for the purification of mouse monoclonal antibodies from hybridoma culture supernatant. Prior to the separation steps, the starting solution was adjusted to the desired pH and conductivity. Diafiltration was used for this purpose in order to keep the volume constant or even to reduce the volume of sample. A QAE Zetaprep disk was used to remove the main protein contaminants from the culture supernatant. After washing unbound proteins out of the Zetaprep disk, slightly bound protein was eluted with a buffer solution containing 50 mM sodium chloride. The monoclonal antibody was eluted with a solution containing 150 mM sodium chloride. The purity of the eluted antibody was 50%, and was increased to 99% by subsequent high-performance cat-ion-exchange chromatography. The purity was determined by means of sodium-dodecyl sulphate polyacrylamide gel electrophoresis and silver staining. The advantage of the two high-performance techniques, mass ion exchange and high-performance cation-exchange chromatography, are the high-flow-rates and the high resolution that can be obtained. These techniques are suitable for the production of injectable therapeutic preparations.
Animals↗
Screening for monoclonal antibodies with covalently linked antigen.
Microtitre plates prepared for ELISA are treated for the maximum adsorption of gamma-globulins (at pH 9). The adsorption of other soluble proteins (e.g. antigens) is sometimes less effective and therefore requires a higher amount of protein for the primary coat of wells. In order to reduce the amount of pure antigen required for the screening of mAb-producing hybridomas, we improved the sensitivity of our screening ELISA technique by coupling the antigen covalently to the surface of the microtiter wells. The antigen (urokinase) was coupled by the glutaraldehyde or carbodiimide procedures respectively using "aminoplates" (Nissho Iwai, Japan). The glutaraldehyde method led to at least a five fold increase of the sensitivity compared to coventional adsorption. Reduced requirement of antigen for the assay is thus achieved by a simple procedure.
Animals↗