Two-phase approach for the expression of high-affinity human anti-human immunodeficiency virus immunoglobulin Fab domains in Escherichia coli.

We describe here a two-phase approach for the development of high-affinity human anti-HIV immunoglobulin Fab domains in a bacterial expression system. The first phase of this technique involves the generation of human hybridoma cell lines producing high-affinity antibodies (MAbs). Anti-HIV-1 human MAbs from peripheral blood lymphocytes (PBLs) were prepared from an HIV-1-seropositive patient and from an HIV-1-seronegative volunteer immunized with HIV-1 rgp160. One MAb (T15G1), derived from the blood of the seropositive donor, was specific for HIV-1 gp41, recognized gp41 on the surface of HIV-1-infected cells and bound this antigen with an apparent dissociation constant of 4 x 10(-10) M. A second MAb (M7B5), developed from the immunized volunteer, was specific for HIV-1 gp120 with a dissociation constant on the order of 8 x 10(-10) M, but was unable to recognize cell surface antigen. In the second phase of this technique the Fab domains of these two MAbs were molecularly cloned into a bacterial expression vector. mRNA was isolated from the M7B5 and T15G1 hybridoma cell lines and used as a template for the production of cDNA. The cDNA was amplified using the polymerase chain reaction (PCR) technique, and then fused, in frame, into a bacterial expression vector. The recombinant Fabs (rFabM7B5 and rFabT15G1) were expressed as dicistronic messages in bacteria using the IPTG-inducible lactose promoter (LacZ). DNA sequencing was used to define the gamma chain isotypes and the VH and VL chain gene usage. The binding specificities of rFabM7B5 and rFabT15G1 were indistinguishable from their respective intact MAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of human monoclonal antibodies to rabies.

A total of nine human monoclonal antibodies (MAbs) to rabies virus were generated from peripheral B lymphocytes of subjects immunized with human diploid cell rabies vaccine by somatic cell hybridization. The MAbs were analyzed for their antigen-binding specificities using ELISA, Western blot, and immunoprecipitation assays. The different assays made it possible to identify MAbs directed to the surface glycoprotein, nucleoprotein, nominal phosphoprotein, and matrix protein. One of the MAbs that recognized the surface glycoprotein neutralized rabies virus.

Molecular mimicry accompanying HIV-1 infection: human monoclonal antibodies that bind to gp41 and to astrocytes.

Monoclonal antibodies that bound to HIV gp41 and cross-reacted with astrocytes were recovered from the blood of three patients infected with HIV-1. Mapping of the specificity of these monoclonal antibodies, using synthetic gp41 peptides, located their epitope to amino acids 644-663 and established their conformation dependence. Six other human monoclonal anti-HIV antibodies were found to bind to HIV gp41 or gp120 but not to reactive astrocytes in brain tissue. Sharing of linear or conformational protein determinants between disparate viral and host proteins is termed molecular mimicry. The consequences of such mimicry by anti-viral antibodies interacting with astrocytes may play a role in the dementia of AIDS patients since a major function of astrocytes is to maintain the appropriate milieu for neuronal function. The finding of such cross-reactive antibodies adds to the evidence for a possible autoimmune pathogenesis in some of the disease manifestations accompanying HIV infection.

Cutaneous disorders and viral gene expression in HIV-1 transgenic mice.

Patients infected with HIV-1 experience several hyperproliferative skin disorders, including seborrheic dermatitis, ichthyosis, and psoriasis. Transgenic mice carrying a subgenomic HIV-1 proviral construct lacking the gag and pol genes were found to develop proliferative epidermal lesions, manifested as diffuse epidermal hyperplasia in homozygous transgenic mice and benign papillomas in heterozygous transgenic mice. Nonpapillomatous skin from both homozygotes and heterozygotes expressed viral RNA, and the viral envelope protein gp120 was localized to the suprabasal keratinocyte. Papillomas contained increased amounts of both viral mRNA and envelope glycoprotein. Exposure of transgenic mice to doses of ultraviolet B (UV-B) irradiation that induced cutaneous injury increased viral gene expression and resulted in the development of papillomas within 14-21 days. Cutaneous injury induced by phenol and liquid nitrogen had similar effects. These data support a role for HIV-1 gene products in the pathogenesis of proliferative epidermal disorders associated with HIV-1 infection. Further, they suggest that the process of wound repair increases HIV-1 gene expression in this transgenic mouse model.

Supplement-induced cytotoxic cells (SICC) generated from mouse thymus or spleen cells cultured in the presence of interleukin 2 and/or polyinosinic acid.

Mouse thymocytes and spleen cells from unprimed C57BL/6 donors generate broadly reactive cytotoxic cells during 5 days of culture in vitro with polyinosinic acid (5') (poly(I] and/or supernatant from PMA-treated EL4 leukemia cells which contains interleukin 2 (IL-2) activity. We refer here to such cytotoxic cells as "supplement-induced cytotoxic cells" or SICC. Thymocytes are dependent on the supernatant factor(s), whereas spleen cells are usually stimulated by poly(I) alone. Polyinosinic acid acts synergistically with supernatant factor(s) to stimulate generation of SICC by both thymocytes (SICC-T) and spleen cells (SICC-S) when the IL-2 activity of the supernatant is inadequate alone. SICC can be generated by both splenocytes and thymocytes in medium supplemented with fetal calf serum or syngeneic plasma. SICC are active in 4 hr 51Cr-release tests against syngeneic, allogeneic, and xenogeneic tumors but not against lipopolysaccharide-induced lymphoblasts. Embryonic fibroblasts, too, are sensitive to SICC generated by thymocytes. In complement-dependent depletion tests, cytotoxic activity is partially sensitive (SICC-T) or fully sensitive (SICC-S) to anti-Thy-1 and -H-2 but not to anti-Lyt-1, -Lyt-2, or -asialo GM1.

Broadly reactive murine cytotoxic cells induced in vitro under syngeneic conditions.

Mouse spleen cells became cytotoxic in short-term 51Cr-release assays for a wide variety of target cells after 5 days of culture in vitro with polyinosinic acid in a system that was otherwise entirely syngeneic. This study characterizes these effector cells with respect to target specificity, effect of donor age, time course of their appearance, mouse strain differences, and expression of differentiation antigens Thy-1, Lyt-1, Lyt-2, NK-1, and asialo GM1. The combination of properties of this cytotoxic cell response that make it unique are that a) the broadly reactive cytotoxic activity developed from unprimed spleen cells in the absence of either foreign cells or foreign serum; b) the response did not peak until 4 to 5 days of culture in vitro; c) the broad reactivity pattern included freshly dispersed primary syngeneic sarcoma cells and cultured syngeneic fibroblasts but did not include syngeneic lymphoblast target cells; d) the response was largely monoclonal as defined by target cell binding; and e) cytotoxic cell activity was sensitive in complement-mediated treatments to both anti-NK and anti-theta but not to anti-Lyt-2, anti-Lyt-1, or anti-asialo GM1. Both high- and low-responding mouse strains have been identified.

Susceptibility of adherent versus suspension target cells derived from adherent tissue culture lines to cell-mediated cytotoxicity in rapid 51Cr-release assays.

Preparation of target cells from tissue culture lines which grow adherent to tissue culture vessels is often desirable for tests of cell-mediated cytotoxicity (CMC). In the present study we used cells derived from adherent tissue culture lines to compare the merits of suspension vs. adherent target cells in short-term 51Cr-release assays. Cytotoxic activity of murine spleen cells sensitized in vitro against allogeneic spleen cells or syngeneic sarcoma cells was tested with fibroblast or sarcoma target cells. In parallel tests, aliquots of tissue culture lines were detached and used as either suspension or adherent target cells in CMC assays, matching the concentrations of suspension and adherent target cells. In both allogeneic and syngeneic combinations adherent target cells released less 51Cr spontaneously and were more susceptible to CMC than their suspension counterparts.

Cell-mediated cytotoxic responses of spleen cells from mice bearing primary, chemically induced tumors.

Sarcomas were induced in C57BL/6 mice by using 3-methylcholanthrene. Spleen cells taken from these mice bearing primary, chemically induced tumors and from matched control mice were assessed for the capacity to generate cell-mediated cytotoxic cell activity after in vitro sensitization. Spleen cells from tumor-bearing mice generated strong cell-mediated cytotoxic responses against alloantigens and antigens on syngeneic cells.

Small virus-like particles in leukosis-like syndrome induced by certain antigens and immunostimulators.

A transmissible syndrome which is characterized by splenomegaly and myeloid metaplasia was induced in BALB/c mice by injections of certain homologous and heterologous antigens and complete Freund's adjuvant or dextransulfate. From the plasma of these animals small viruslike particles (30--50 nm) were isolated.

[Preparation of liposomes possessing immunologic specificity]. / Poluchenie liposom, obladaiushchikh immunologicheskoi spetsifichnost'iu

The authors obtained artificial lipid vesicles--liposomes containing immunoglobulins. IgG in the complexes with liposomes proved to retain their immunological activity: the liposomes containing rabbit anti-mouse IgG agglutinated in the presence of donkey anti-rabbit IgG or mouse serum. As shown by the use of liposomes containing H3-inulin and immunoglobulins against the cell surface determinants, these immuno-liposomes selectively bound the target, but not the control cells. Specific binding with the antigenic cell surface determinants was also demonstrated in the case of liposomes bearing the nonimmune globulins besides the immunoglobulins. By the indirect immunofluorescence method it was shown that the nonimmune globulins in complex with the immune liposomes were selectively bound by target cells. A possible use of the immuno-liposomes to deliver various substances selectively to the cells of particular types, and to incorporate new antigens into the cell membrane is discussed.

Active cell edge and movements of concanavalin A receptors of the surface of epithelial and fibroblastic cells.

Movements of the receptors of concanavalin A on various parts of the surfaces of substrate-attached cells were compared. Cultured mouse embryo cells of several types were used: epithelial kidney cells and normal and transformed fibroblasts. Initial distribution of receptors was random on the cells of all types. Binding of concanavalin A induced patching of its receptors on all the cell parts. In contrast, directional centripetal movement of receptors was observed only on the surface of certain cell parts, namely, only the surface of peripheral lamellar cytoplasm was cleared of the receptors. Clearing was always initiated in the zone of lamellar cytoplasm located near active cell edges. In epithelial sheets, clearing was not observed on the surface of central cells that had no lamellar cytoplasm. Concanavalin A receptors on the cleared areas of cell surface were gradually restored after the end of incubation. It is suggested that anchoring of the patches of membrane receptors by cortical microfilaments is possible only on the surface of pseudopods and of lamellar cytoplasm but not on the surface of other cell parts. Besides receptor movements, this hypothesis may be explain differences in the adhesive properties of various parts of the cell surface.

[About the heterogeneity of the murine oncornavirus group-specific antigen gs-1 (author's transl)]. / Uber die Heterogenität des gruppenspezifischen Antigens (gs-1) der murinen Oncornaviren

In isoelectric focusing of twofold gradienpturified and Tween 80-ether disrupted RLV the groupspecific antigen (gs-1) was found in 3 pH-zones (4,45-5,5; 5,65-6,0; 6,45-6,6). For the gs-1 from plasma und spleen cells of leukemic mice was shown heterogeneity by several methods: 1. Gs-1 from plasma showed in isoelectric focusing a scattering along the whole elution profile. 2. Agarimmunoelectrophoresis resulted in a longer precipitationline for gs-1 from plasma in comparison with gs-1 from spleen cells. 3. Polyacrylamidgelelectrophoresis with following immunodiffusion made evident the particularly discrete character of this heterogeneity: gs-1 from both materials were found in two main zones.