The Ly-3 antigens on mouse thymocytes: immune precipitation and molecular weight characterization.

Specific anti-Ly sera were employed to precipitate Ly antigens from Nonidet P-40 extracts of mouse thymocytes labeled with 125I using lactoperoxidase and with NaB3H4 using galactose oxidase. Thymocytes from mice of the congenic strains C57BL/6J (Ly-2.2, Ly-3.2 positive), C57BL/6Ly-2a, Ly-3a (Ly-2.1, Ly-3.1 positive) and C57BL/6-Ly-2a (Ly-2.1, Ly-3.2-positive) were used as sources of labeled antigens and as immune adsorbants to permit evaluation of the specificity of each anti-Ly serum employed. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions are consistent with the Ly-3.1 antigen containing a glycoprotein subunit with an apparent mol wt of 35,000 daltons. Specific precipitates obtained using anti-Ly-2.1 serum yielded SDS-PAGE profiles identical to that obtained with anti-Ly-3.1 serum, suggesting that the Ly-2 and Ly-3 antigens have the same molecular weight distribution. The relationships of these results to the observed close genetic and topological linkage of Ly-2 and Ly-3 and to the genetic linkage of these loci with the IB-peptide marker, a mouse Bk-region polymorphism, are discussed.

Tissue distribution, immunochemical characterization, and biosynthesis of 47D10, a tumor-associated surface glycoprotein.

This report describes a new monoclonal antibody (MAb) designated 47D10 which was produced by immunizing mice against a human lung adenocarcinoma line, A549. The MAb 47D10 reacts with a surface antigen found in 95% of adenocarcinomas of the pancreas as well as on high percentages of adenocarcinomas from colon, breast, lung, and bile duct. The antigen was not detected in normal pancreas, in pancreatitis, or in a variety of normal tissues with the exception of colon and mature granulocytes. Lymphocytes and erythrocytes were also negative. The binding of 47D10 to tumor cells was unaffected by treatment of cells with neuraminidase. Immunoprecipitation followed by polyacrylamide gel electrophoresis showed that 47D10 MAb recognized a group of glycoproteins ranging in molecular weight from 67,000-98,000 on A549 lung carcinoma cells. Pulse-chase labeling showed two precursor proteins with molecular weights of 69,000 and 67,000 which were processed to the larger polypeptides in 1.5 h. At least part of the carbohydrates associated with the 47D10 antigen was asparagine linked because the antigen was sensitive to endoglycosidases, and tunicamycin inhibited the biosynthesis of 47D10 antigen. The 47D10 antigen was expressed on the cell surface because it could be detected on live A549 cells by enzyme-linked immunosorbant assays as well as by immunofluorescent staining. Furthermore, 47D10 antigens on tumor cell lines and granulocytes were vectorially labeled with 125I. The antigen found on granulocytes showed a higher molecular weight of 150,000-180,000, which was digested by endoglycosidase F to polypeptides with molecular weights ranging from 23,000-27,000. In contrast, the degradation product of the A549 antigen was a Mr 39,000 polypeptide after treatment with endoglycosidase F. The immunochemical characteristics of 47D10 antigen suggest that it is distinct from other antigens associated with pancreatic tumors, such as carcinoembryonic antigen, 19-9, and Du-PAN-2. By virtue of its broad range of tumor cell reactivity and low activity on normal cells, the 47D10 MAb may represent an important immunological reagent for differential diagnosis, especially of pancreatic carcinoma.

Characterization of murine monoclonal antibodies to HIV-1 induced by synthetic peptides.

We have used short synthetic peptides, 12 and 13 amino acids in length, conjugated to carrier proteins to develop monoclonal antibodies (MAb) to the envelope glycoprotein of 120 (kD) (gp120) and the 3' open reading frame protein (3-orf) of the human immunodeficiency virus type 1 (HIV-1). The peptides employed were chosen because of their strong hydrophilicity and in the case of the gp120 peptide because it represents a highly conserved hydrophilic region in the envelope protein. The MAb developed displayed appropriate specificities with their respective peptides and reacted with appropriate HIV-1 components (i.e., a 120 kD glycoprotein and a 27 kD protein, respectively) as determined by Western blot analysis. In indirect immunofluorescence assays the MAb strongly stained syncytia present in cultures of HTLV-3B-infected H9 cells. The MAb to the envelope component reacted with the RF isolate of HIV-1, as well as with the 3B isolate in immunofluorescence.

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.

Kinetics of soluble interleukin-2 receptor after mechanical and burn trauma.

Extended trauma causes a failure of T-lymphocyte function due to suppressed interleukin-2 synthesis; however, the role of IL-2 receptor, especially its soluble form (sIL-2R), needs to be further evaluated. It was the objective of the study to assess the kinetics of sIL-2R within different settings of trauma and to define its clinical value and possible predictive role. Three groups of patients with trauma were included in the study. Groups 1 and 2 consisted of multiply injured patients (injury severity score 35 +/- 4 and 32 +/- 4, respectively); burned patients formed group 3 (injury severity score 38 +/- 9). Serum samples were collected at the site of the accident (group 1) and during the posttrauma course in the hospital (group 2, daily; group 3, weekly) and sIL-2R was measured in these samples. sIL-2R was within the normal range in groups 1 and 2, but was significantly increased in group 3. There was no correlation between serum concentrations of this mediator and susceptibility to infectious complications or outcome.

Sequential precipitation of mouse thymocyte extracts with anti-Lyt-2 and anti-Lyt-3 sera. I. Lyt-2.1 and Lyt-3.1 antigenic determinants reside on separable molecular species.

Anti-Lyt-2.1 and anti-Lyt-3.1 sera were employed for sequential precipitation of NP-40 extracts of 125I-labeled C57BL/6-Lyt-2a, Lyt-3a thymocytes (Lyt-2.1, Lyt-3.1) to determine whether these alloantigenic determinants are present on the same or different molecular species. Treatment of extracts with anti-Lyt-3.1 serum and SaCI completely precipitated both Lyt-3.1 and Lyt-2.1-specific components, whereas treatment with anti-Lyt-2.1 serum reduced by approximately 37% the quantity of labeled species subsequently precipitable by anti-Lyt-3.1 serum. When 125I-labeled thymocytes were subjected to mild trypsinization before NP-40 extraction, the quantity of radioactive components precipitated by anti-Lyt-2.1 serum was essentially unchanged, but that of anti-Lyt-3.1-precipitable components was greatly reduced. Moreover, sequential precipitation of extracts of trypsinized thymocytes with anti-Lyt-2.1 and anti-Lyt-3.1 sera demonstrated that these molecular species were precipitated independently. Thus 1) Lyt-2.1 and Lyt-3.1 antigenic determinants appear to reside on different molecular species; 2) some Lyt-2.1- and Lyt-3.1-positive molecules appear to be complexed with each other in the NP-40 extract; and 3) this association of Lyt-2.1- and Lyt-3.1-positive species was dependent upon components that were labile to trypsinization of intact thymocytes.

The binding of a glycoprotein 120 V3 loop peptide to HIV-1 neutralizing antibodies. Structural implications.

The structural and antigenic properties of a peptide ("CRK") derived from the V3 loop of HIV-1 gp120 protein were studied using NMR and SPR techniques. The sequence of CRK corresponds to the central portion of the V3 loop containing the highly conserved "GPGR" residue sequence. Although the biological significance of this conserved sequence is unknown, the adoption of conserved secondary structure (type II beta-turn) in this region has been proposed. The tendency of CRK (while free or conjugated to protein), to adopt such structure and the influence of such structure upon CRK antigenicity were investigated by NMR and SPR, respectively. Regardless of conjugation, CRK is conformationally averaged in solution but a weak tendency of the CRK "GPGR" residues to adopt a beta-turn conformation was observed after conjugation. The influence of GPGR structure upon CRK antigenicity was investigated by measuring the affinities of two cognate antibodies: "5023A" and "5025A," for CRK, protein-conjugated CRK and gp120 protein. Each antibody bound to all the antigens with nearly the same affinity. From these data, it appears that: (a) antibody binding most likely involves an induced fit of the peptide and (b) the gp120 V3 loop is probably conformationally heterogeneous. Since 5023A and 5025A are HIV-1 neutralizing antibodies, neutralization in these cases appears to be independent of adopted GPGR beta-turn structure.

NMR study and comparison of the antigenic properties of a peptide recognized by two HIV-1 neutralizing antibodies.

Fab-peptide complexes formed between a 15 residue peptide derived from the HIV-1 gp120 V3 loop and two of its cognate monoclonal antibodies, 5023A and 5025A, were studied using isotope-edited solution nuclear magnetic resonance (NMR) techniques. Since these antibodies neutralize HIV-1 virus with different strain specificities, this study was conducted to better understand the nature of these differences. The amide proton and nitrogen NMR resonances of specific residues were used to monitor the backbone of this peptide in these complexes. Three central residues of this peptide ('RAF') were found to be strongly affected by binding to both antibodies. Several other peptide residues were affected by binding to antibody 5023A but not 5025A. The antibody epitopes mapped by NMR are similar to those obtained previously via PEPSCAN at higher pH. One main difference between the PEPSCAN and NMR determined epitopes for 5023A involved two glycine residues of the peptide. By NMR, one of these glycines was more dramatically affected by antibody binding than predicted by PEPSCAN, while the other was much less so.

T100: a new murine cell surface glycoprotein detected by anti-Lyt-2.1 serum.

A cell surface glycoprotein (designated T100) of apparent m.w. 100,000 by SDS-PAGE under reducing and nonreducing conditions was precipitated from NP-40 extracts of surface radiolabeled thymocytes from a variety of inbred strains of mice by the standard noncongenic Lyt-2.1-typing serum. The inbred stain distribution, trypsin sensitivity on intact cells, and apparent m.w. of T100 suggest that it is different from Lyt-2.1. Inheritance and expression of T100 suggest that it is determined by an allele at a single locus, and testing of CXB recombinant inbred strains and B6.C minor histocompatibility congenic strains suggest that this locus is linked to H-25. Antiserum absorption experiments, two-stage cytotoxicity assays, and results of immunoprecipitations performed after prebinding antibody to radiolabeled thymocytes suggest that some T100 is accessible to antibody on the intact cell surface. However, for unknown reasons the number of cells required to absorb anti-T100 precipitating activity from antiserum was much higher than for removal of anti-Lyt-2.1 activity. A molecule with properties of T100 was also detected on lymph node cells and on the AKTB-1 lymphoma.

An enzyme-linked immunosorbent assay for the measurement of human IgA antibody responses to Epstein-Barr virus membrane antigen.

An enzyme-linked immunosorbent assay (ELISA) which uses a recombinant truncated form of the Epstein-Barr virus (EBV) membrane antigen gp350/250 has been developed and used to measure human IgA antibody responses to that antigen. From comparisons with conventional immunofluorescence assays (IFA) for measuring IgA antibody responses to EBV viral capsid antigens, the ELISA shows comparable specificity and is approximately 4-fold more sensitive. Since IgA antibodies to EBV indicate a high risk of developing nasopharyngeal carcinoma (NPC), the described ELISA, which is more sensitive and objective than IFA, has potential for use in the diagnosis of NPC and for large-scale screening to identify individuals at risk for the development of this disease.

Effects of iodipamide on human C3 and factor B in vitro.

The effects of iodipamide on C3 and factor B in normal human serum and in purified form have been examined by immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Temperature-dependent changes in immunoelectrophoretic profiles have been observed; however, these are not the same as those obtained after treatment of normal human serum (NHS) with cobra venom factor Naja naja. Analyses of iodipamide-treated NHS and purified C3 and factor B by reducing SDS-PAGE indicate that no macromolecular changes have occurred in C3 and factor B that can be ascribed to proteolysis (i.e., activation). The changes observed in C3 and factor B, including loss of hemolytic activity, appear to be due to direct interactions between iodipamide and C3 and factor B. In the case of factor B, iodipamide treatment at 37 degrees C induces aggregation, which is reversible upon reduction with beta-mercaptoethanol.

Neutralizing activity of anti-peptide antibodies against the principal neutralization domain of human immunodeficiency virus type 1.

Monoclonal antibodies (MAbs) raised against a 15-mer peptide representing the centre of the principal neutralization domain of human immunodeficiency virus type 1 (strain BH10) showed wide variations in neutralizing activity against the homologous strain. The nature of this difference in neutralizing activity was studied by measuring antibody concentration, their affinity for peptide and specificity, by reaction with peptides which differed in the extent of sequence overlap, length and the presence of single amino acid replacements. All MAbs bound to approximately the same region in the principal neutralization domain, within the sequence RIQRGPGRAFV. The peptides with which each antibody was able to react differed by only a few amino acids. The neutralizing activity of each MAb preparation was related to its affinity and concentration; the affinity is related in part to the fine structure of the epitope recognized. MAbs with high affinity for the peptide tended to react only with peptides in which amino acid replacements did not affect the beta-turn potential of the peptide, whereas the reactivity of MABs with low affinity was relatively insensitive to amino acid replacements affecting the beta-turn potential.

In vitro efficacy of anti-HIV immunotoxins targeted by various antibodies to the envelope protein.

Six different anti-HIV envelope antibodies and one irrelevant control antibody were coupled to ricin A chain and tested for their efficacy in inhibiting HIV tissue culture infections. The anti-HIV antibodies consisted of five monoclonals, three of murine and two of human origin, and one polyclonal preparation prepared by affinity purifying pooled serum antibodies from HIV-infected humans on rgp160. The binding specificity of the antibodies was defined by ELISA by using recombinant envelope proteins and synthetic peptides, and by flow cytometry on HIV-infected cells. The in vitro efficacy of the antibodies was tested by the abilities of the immunotoxins to inhibit protein synthesis in persistently infected cell lines and by their abilities to inhibit HIV production during both acute and persistent infection as measured with an HIV-specific focal immunoassay. The immunotoxins were tested against a panel of distinctly different HIV isolates. The results indicate the following: 1) A mAb to the immunodominant neutralizing loop was highly effective against homologous strains of HIV, but had no activity against heterologous HIV. 2) The efficacy of anti-gp41 mAb varied depending upon the epitope recognized and possibly the affinity of binding to gp41. 3) The polyclonal human anti-gp160 antibodies produced the immunotoxin with the broadest specificity for different HIV strains and the greatest specific activity. This is related to the polyclonal nature of the preparation rather than an increase in relative avidity of the antibody. 4) Activity of an immunotoxin is not a direct function of the binding of the antibody to the surface of infected cells. 5) The ability of an immunotoxin to halt the spread of infection through a tissue culture cell population is dependent upon the ability of the antibody to neutralize the virus as well as the activity of the toxin. Our data suggest that efficacious immunotoxins for the treatment of AIDS may be made with polyclonal anti-envelope antibodies derived from the serum of patients who have been infected with HIV or with appropriately chosen anti-gp41 antibodies.

Specific lysis of human tumor cells by T cells coated with anti-T3 cross-linked to anti-tumor antibody.

Heteroaggregates containing anti-T3 cross-linked to anti-target cell antibodies have been shown to cause human T cells to lyse target cells that express antigens recognized by the anti-target cell antibody. In this study, we test targeted human T cells for the ability to lyse human tumor cells as a first step toward the application of this phenomenon to tumor immunotherapy. Several monoclonal anti-human tumor antibodies were assayed for binding to a number of human tumor lines and for the ability to promote specific tumor cell lysis when cross-linked with anti-T3. We found that anti-T3 cross-linked to anti-tumor monoclonal antibodies caused cloned human T cells and fresh peripheral blood T cells to lyse the tumor cells with the same specificity as predicted by the binding studies. Peripheral blood T cells were then tested in the presence of various heteroaggregates for the ability to lyse single cell suspensions prepared from fresh tumor or fresh normal tissue. These studies showed that heteroaggregates containing anti-T3 cross-linked to anti-tumor antibody cause fresh human T cells to specifically lyse fresh tumor cells, but not (with one exception) fresh normal cells.

Simian immunodeficiency virus from African green monkeys.

Simian immunodeficiency virus (SIV) was isolated from the total peripheral blood mononuclear cell population and the monocyte-macrophage adherent cell population of three seropositive green monkeys originating from Kenya. SIV from these African green monkeys (SIVagm) was isolated and continuously produced with the MOLT-4 clone 8 (M4C18) cell line but not with a variety of other cells including HUT-78, H9, CEM, MT-4, U937, and uncloned MOLT-4 cells. Once isolated, these SIVagm isolates were found to replicate efficiently in M4C18, SupT1, MT-4, U937, and Jurkat-T cells but much less efficiently if at all in HUT-78, H9, CEM, and MOLT-4 cells. The range of CD4+ cells fully permissive for replication of these SIVagm isolates thus differs markedly from that of previous SIV isolates from macaques (SIVmac). These SIVagm isolates had a morphogenesis and morphology like that of human immunodeficiency virus (HIV) and other SIV isolates. Antigens of SIVagm and SIVmac cross-reacted by comparative enzyme-linked immunosorbent assay only with reduced efficiency, and optimal results were obtained when homologous antibody and antigen were used. Western blotting (immunoblotting) of purified preparations of SIVagm isolate 385 (SIVagm385) revealed major viral proteins of 120, 27, and 16 kilodaltons (kDa). The presumed major core protein of 27 kDa cross-reacted antigenically with the corresponding proteins of SIVmac (28 kDa) and HIV-1 (24 kDa) by Western blotting. Hirt supernatant replicative-intermediate DNA prepared from cells freshly infected with SIVagm hybridized to SIVmac and HIV-2 DNA probes. Detection of cross-hybridizing DNA sequences, however, required very low stringency, and the restriction endonuclease fragmentation patterns of SIVagm were not similar to those of SIVmac and HIV-2. The nucleotide sequence of a portion of the pol gene of SIVagm385 revealed amino acid identities of 65% with SIVmac142, 64% with HIV-2ROD, and 56% with HIV-1BRU; SIVagm385 is thus related to but distinct from previously described primate lentiviruses SIVmac, HIV-1, and HIV-2. Precise information on the genetic makeup of these and other SIV isolates will possibly lead to better understanding of the history and evolution of these viruses and may provide insight into the origin of viruses that cause acquired immunodeficiency syndrome in humans.

A novel autocrine pathway of tumor escape from immune recognition: melanoma cell lines produce a soluble protein that diminishes expression of the gene encoding the melanocyte lineage melan-A/MART-1 antigen through down-modulation of its promoter.

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as "Ag silencing," is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.

Immunochemical analysis of the gp120 surface glycoprotein of human immunodeficiency virus type 1: probing the structure of the C4 and V4 domains and the interaction of the C4 domain with the V3 loop.

We have probed the structure of the C4 and V3 domains of human immunodeficiency virus type 1 gp120 by immunochemical techniques. Monoclonal antibodies (MAbs) recognizing an exposed gp120 sequence, (E/K)VGKAMYAPP, in C4 were differentially sensitive to denaturation of gp120, implying a conformational component to some of the epitopes. The MAbs recognizing conformation-sensitive C4 structures failed to bind to a gp120 mutant with an alteration in the sequence of the V3 loop, and their binding to gp120 was inhibited by both V3 and C4 MAbs. This implies an interaction between the V3 and C4 regions of gp120, which is supported by the observation that the binding of some MAbs to the V3 loop was often enhanced by amino acid changes in an around the C4 region.