HIV-1 gp41 binding proteins and antibodies to gp41 could inhibit enhancement of human Raji cell MHC class I and II expression by gp41.

Based on our findings, that HIV-1 soluble gp41 could bind to several proteins on the human, T, B and monocyte cells independently of CD4, we examined the effect of HIV-1 soluble gp41 (sgp41;Env amino acids 539-684) on surface expression of MHC I and II, ICAM-1 and CD21 molecules on human Raji cells. Flow cytometry (FACS) analysis demonstrated that sgp41 could selectively enhance MHC class I and II expression on Raji cells, but did not increase expression of other cell surface antigens, such as, CD21 and CD54 (ICAM-1). Soluble gp41 could also enhance MHC class I and II expression on another human B cell line, Bjab. The sgp41-dependent enhancement of the MHC class I and II expression on Raji cells is time- and dose-dependent. The sgp41 enhancement effect on the MHC antigen expression could be inhibited by the sgp41-binding proteins of 45, 49 and 62 kD (isolated from Raji-lysate) which could inhibit the spg41-binding to Raji cells. Interestingly, this sgp41-dependent enhancement of the MHC class I and II expression could also be inhibited by two mAbs to HIV-1 gp41, but not by a third mAb binding to a different site on gp41. These results demonstrate that HIV-1 sgp41 can selectively enhance the human Raji cell MHC class I and II antigen expression and this enhancement effect could be inhibited by the sgp41-binding proteins and anti-gp41 antibodies, and suggest that the sgp41-dependent enhancement is mediated by its binding to Raji membrane proteins of 45, 49 and 62 kD.

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.

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).

Acquisition of host cell-surface-derived molecules by HIV-1.

OBJECTIVE: To determine the acquisition of host cell-membrane-derived molecules by HIV-1 during the budding process, and to investigate whether the uptake of these molecules is cell-type-specific and selective. DESIGN: Virions, propagated by four different cell types were analysed for the presence of adhesion molecules, glycosylphosphatidylinositol (GPI)-anchored proteins and various cell-surface markers. The pattern was compared with the phenotype of the HIV-1-infected cell. METHODS: For phenotypic analysis of virions a two-step assay was used. In the first step, virus was captured with monoclonal antibodies (in some cases polyclonal sera) against different cell-membrane proteins. In a second step, the presence of virus was measured by determining the concentration of the virus-specific p24 core antigen. The expression of surface molecules on uninfected and HIV-1IIIB-infected cells was analysed by FACS. RESULTS: Depending on the cell type used for virus propagation, different cell-membrane molecules were found on the virus surface reflecting the corresponding cell type. The uptake of these molecules was selective to a certain degree. No CD4 and CD87 molecules were detectable on HIV-1, although both molecules were present on uninfected and HIV-1-infected cells. CR3 and CDw108 could not be seen on uninfected cells, but wre detectable on infected cells and virions. CONCLUSIONS: During the budding process HIV-1 acquires a variety of cell-type-specific cell-surface molecules. Certain cell-membrane molecules become upregulated during HIV-1-infection and are then found on virions, whereas other molecules remain on the cell surface and do not become incorporated.

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.

A simple method to quantify staphylococcal protein A in the presence of human or animal IgG in various samples.

Immunoassays designed to measure low concentrations of staphylococcal protein A (SPA) that have been leached into antibody preparations intended for therapeutic use are subject to differing degrees of interference. Methods established to quantify SPA in murine antibody preparations are not accurate in the presence of human or humanized IgG. We report the development of an enzyme-linked immunosorbent assay (ELISA) for SPA with a detection limit of 7 pg/ml and the optimization of a method that permits complete dissociation of SPA-immunoglobulin-complexes. This assay is a modification of our heat-mediated dissociation (HD-SD) treatment with sodium dodecyl sulfate (SDS) and diethylenetriaminepentacetic acid (DTPA) for total immune-complex dissociation, in which the heat treatment has been prolonged and the diluent is characterized by increased protein content and buffering capacity. The diluent developed contains SDS, DTPA and bovine serum albumin dissolved in a 0.1 M phosphate buffer (pH 7.2). To validate the efficiency of this novel method, a series of samples have been assayed, including samples reconstituted in vitro, samples of purified antibodies, and plasma from patients. The described method has been shown to be generally efficient in quantitating all native and recombinant SPA in samples containing up to 50 mg/ml of human IgG. These data demonstrate the utility of this technique in determining SPA contamination of recombinant immunoglobulin therapeutic products.

A simple and robust method for the complete dissociation of HIV-1 p24 and other antigens from immune complexes in serum and plasma samples.

Accuracy of antigen determination in human plasma samples is often adversely affected by immune complex formation between antigens (e.g., HIV-1 p24 protein) and specific antibodies. In this study we describe an optimized method for complete immune complex dissociation (ICD) in plasma. This method is based on heat denaturation of antibodies and utilizes a defined solution of sodium dodecyl sulfate (SDS) and diethylenetriaminepentaacetic acid (DTPA) as diluent. The efficiency of this procedure for ICD was compared with those of published methods, employing heat denaturation alone and acidification. Plasma samples from patients participating in anti-retroviral treatments and samples reconstituted in vitro were treated and analyzed in parallel. HIV-1 p24 antigen was determined by quantitative enzyme-linked immunosorbent assay (ELISA). In 312 samples from 97 patients, antigenemia was found in 44.9% when measured directly and in 87.2% after this treatment. In a subset of 56 samples, 21.4% tested positive prior to treatment, while after either novel treatment, heat denaturation or acidification, these samples tested positive in 80.4%, 62.5% and 60.7%, respectively. In 94% of cases viral RNA was detected. This improved procedure for ICD provides a reliable and convenient method for complete and accurate p24 antigen detection in human plasma and is applicable to commercially available test kits.

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.

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.

HIV-1 gp41 shares a common immunologic determinant with human T, B and monocyte cell lines.

Several examples of molecular mimicry between HIV-1 and human proteins are reported in the literature. Here we report on yet another example. The monoclonal antibody 3D6 recognized a 17-amino-acid region in HIV-1 gp41 (amino acids 602-618) and could bind to the human T-cell lines H9 and Molt4, B cell lines Raji and Bjab, and monocyte cell lines U937 and HL60. By Western blot using 3D6, a strong band of 43 kDa and a very weak band of 80 kDa were detected in lysates of H9, Molt4, Raji and Bjab cell lines, but only a strong band of 43 kDa was observed in case of U937 and HL60 cells, under reducing or non-reducing conditions. The results indicate the presence of a common immunologic determinant between HIV-1 gp41 and membrane proteins of these human T, B and monocyte cells.

A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1.

We have established a panel of human monoclonal antibodies against human immunodeficiency virus type 1 (HIV-1). The antibodies 2F5 and 2G12 have been identified to be two of the most potently in vitro neutralizing antibodies against HIV-1. Here we report on a further antibody, 4E10, of similar in vitro neutralizing potency. 4E10 binds to a novel epitope C terminal of the ELDKWA sequence recognized by 2F5, which has been so far the only described broadly neutralizing anti-gp41 antibody. Both 4E10 and 2F5 bind only weakly to infected cells compared with gp120-specific 2G12 and polyclonal anti-HIV-1 immunoglobulin (HIVIG), but show potent in vitro neutralizing properties. 4E10 neutralizes potently not only tissue culture-adapted strains but also primary isolates of different clades, including A, B, C, D, and E. Viruses that were found to be resistant to 2F5 were neutralized by 4E10 and vice versa; none of the tested isolates was resistant to both anti-gp41 antibodies. This confirms that the region recognized by 2F5 and 4E10 is essential for viral infectivity and may be important for vaccine design. Moreover, our results suggest that 4E10 should be further investigated for passive anti-HIV immunotherapy.

Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization.

Electrofusion and EBV transformation were studied by immortalizing human PBLs from blood of HIV-1-positive volunteers. A panel of 33 cell lines producing human monoclonal antibodies (Hu-MAbs) against HIV-1 was established by cell fusion or EBV transformation. For the first fusion experiments the source of B lymphocytes was peripheral blood of HIV-1-infected donors in CDC stages II or III with CD4 cell counts higher than 500/mm3. Later on, from these patients only, those with high anti-HIV titers were chosen as blood donors. By that means the yield of stable specific hybridomas was increased twofold. In our experiments electrofusion turned out to be a more efficient immortalization method than EBV transformation, due to a high and constant immortalization rate. The hybridomas were stable after intensive subcloning and could be cultivated over a period of 8 months without loss in monoclonal antibody production. Immunoglobulin class, subtype, reactivity against HIV-1 proteins, Western blot patterns, immunofluorescence, and epitopes were characterized. The subtype of all antibodies was IgG1 or IgG3. The light chain was predominantly kappa. All antibodies showed reactivity against HIV-1 envelope or core protein. All hybridomas were stable and suited for mass production. Several Hu-MAbs are becoming an important tool in the field of diagnosis, research, and immunotherapy.

A broadly neutralizing human monoclonal antibody against gp41 of human immunodeficiency virus type 1.

We have established a hybridoma clone, designated 2F5, secreting a neutralizing human monoclonal antibody (MAb) specific for gp41 of human immunodeficiency virus type 1 (HIV-1). The epitope of MAb 2F5 was mapped to amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala on the ectodomain of gp41. In this study different in vitro test systems were used to characterize the neutralizing properties of MAb 2F5. In syncytium inhibition assays, fusion inhibition experiments, and neutralization assays on different HIV-susceptible cells (H9, U937, and peripheral blood mononuclear cells) MAb 2F5 showed broad-spectrum neutralizing capacity against HIV-1 laboratory isolates IIIB, MN, RF, and SF2. In addition, primary isolates from AIDS patients were also neutralized.

B cell-mediated infection of stimulated and unstimulated autologous T lymphocytes with HIV-1: role of complement.

In vivo, human immunodeficiency virus type 1 (HIV-1) is opsonized with complement fragments and virus-specific antibodies (Ab). Thus, HIV is able to interact with complement receptor (CR) - and Fc receptor (FcR) - positive cells such as B cells, follicular dendritic cells or macrophages. In this study we demonstrate that the interaction between B cells and HIV has an impact on autologous primary T cell infection in vitro. We confirmed the presence of complement-fragments and virus-specific Ab on serum-treated HIV using a virus-capture assay. In experiments with CR2-specific Ab we showed that the virus/B cell interaction was mainly dependent on CR2. In infection experiments immobilisation of HIV on stimulated tonsil B cells greatly enhanced the infection of interleukin (IL)-2-activated autologous tonsil T cells. Surprisingly, enhancement of T cell infection by B cell-HIV complexes was observed even in the absence of mitogenic stimuli such as PMA and was independent of the addition of exogenous IL-2. Taken together, these results indicate that primary B cells are able to efficiently transmit opsonised HIV to autologous primary T cells and induce a massive enhancement of infection. These in vitro experiments mimic the in vivo situation in the lymphoid tissue and suggest an alternative mechanism for the infection of primary T cells.

HIV-1 gp41 binds to two proteins in cell culture supernatant of human B cell line Raji and monocyte cell line U937.

Based on our findings that HIV-1 gp41 can bind independently of CD4 to the human T cell line H9, B cell line Raji and monocytic cell line U937, as well as human peripheral blood mononuclear cells preferentially to B lymphocytes and monocytes, we examined whether soluble protein for HIV-1 gp41 binding exists in the cell culture supernatant of Raji and U937. Using HIV-1 recombinant soluble gp41 (sgp41; Env amino acid 539-684) attached to sepharose beads, the cell culture supernatants of Raji and U937 were absorbed. By SDS-PAGE of sgp41-eluates of these cell culture supernatants three protein bands of 70, 75 and 92kD were stained with Coomassie blue. By Western blot (ligand blot) analysis using sgp41, two protein bands of 70 and 75kD were observed in sgp41-eluates from Raji and U937 cell culture supernatants. These sgp41-eluates could inhibit the sgp41 binding to Raji and U937 cells. Our data indicate that Raji and U937 cells produce two soluble binding proteins for HIV-1 gp41.

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.

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.

Cytokine activity assay by means of proliferation measured in plane convex microtiter wells.

A new assay to measure cell proliferation by turbidimetric evaluation of cultures in specially designed microtiter plates was set up. The IL-2-dependent proliferation of CTLL-2 cell line was used as a model. The novel microtiter plate detection system, the General Cell Screening System (GCSS) was used for the evaluation of the cell proliferation assay. The test system utilizes the population growth rate (mu) of the cells. The tests were performed in specially designed microtiter plates which were read in a scanning spectrophotometer. These results were compared with colorimetric assays, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) as substrates. In contrast to conventional methods, the presently described system is not an end-point method, this means that cell growth can be observed over a certain period and is not limited to a single moment during the period of cell growth. Another advantage is the reduction of manual manipulations and the avoidance of toxic or radioactive substances and organic solvents. Therefore, a higher number of samples can be analyzed compared to other methods. The range for detection was 0.015-10 units IL-2 per well, and the accuracy is in the range of < 0.005 standard deviation.

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.

Pilot scale production of a human monoclonal antibody against human immunodeficiency virus HIV-1.

Human monoclonal antibodies against the transmembrane protein gp41 of HIV-1 were isolated and purified on a pilot scale. A purification scheme was established for the production of human monoclonal antibodies on the gram scale. 50 1 of culture supernatant can be treated in one purification cycle. The hybridomas were mass cultured in an airlift fermenter. The culture broth was clarified by microfiltration and chromatographed on CM-Sepharose fast flow and protein A Superose. Scale up of the high performance affinity chromatography from 1 ml protein A Superose up to 40 ml is described. All desalting steps were performed by gel filtration on Sephadex G-25 coarse. The yield of the whole purification procedure is in the range of 50-60%. The purity is higher than 99.9%. DNA and reverse transcriptase could not be detected. The whole method is designed as a basis for scale up to industrial scale. Results from quality control assays have proven the validity of this approach.