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Biomedical subjects

L O Arthur

Publications and source records attributed to L O Arthur.

At least 73 records · Page 4Linked to original sources
PubMed1989

Challenge of chimpanzees (Pan troglodytes) immunized with human immunodeficiency virus envelope glycoprotein gp120.

The human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, infects humans and chimpanzees. To determine the efficacy of immunization for preventing infection, chimpanzees were immunized with gp120 purified from human T-cell lymphotrophic virus type IIIB (HTLV-IIIB)-infected cell membranes and challenged with the homologous virus, HTLV-IIIB. A challenge stock of HTLV-IIIB was prepared by using unconcentrated HTLV-IIIB produced in H9 cells. The titer of the virus from this stock on human and chimpanzee peripheral blood mononuclear cells and in human lymphoid cell lines was determined; a cell culture infectivity of 10(4) was assigned. All chimpanzees inoculated intravenously with 40 cell culture infectious units or more became infected, as demonstrated by virus isolation and seroconversion. One of two chimpanzees inoculated with 4 cell culture infectious units became infected. Chimpanzees immunized with gp120 formulated in alum developed antibodies which precipitated gp120 and neutralized HTLV-IIIB. Peripheral blood mononuclear cells from gp120-vaccinated and HIV-infected animals showed a significantly greater response in proliferation assays with HIV proteins than did peripheral blood mononuclear cells from nonvaccinated and non-HIV-infected chimpanzees. Two of the gp120-alum-immunized chimpanzees were challenged with virus from the HTLV-IIIB stock. One animal received 400 cell culture infectious units, and one received 40 infectious units. Both animals became infected with HIV, indicating that the immune response elicited by immunization with gp120 formulated in alum was not effective in preventing infection with HIV-1.

Animals↗
PubMed1989

Molecular characterization and comparison of simian immunodeficiency virus isolates from macaques, mangabeys, and African green monkeys.

Simian immunodeficiency virus (SIV)/Mne has been inoculated into three species of macaques and into baboons. Virus was isolated from all the macaques who subsequently died at 15 to 120 weeks (mean 80 weeks) with various manifestations of immune deficiency. Individual animals varied in their viral antibody profile as a function of time after infection. Independent SIV isolates obtained from African green monkeys and magabeys were compared to SIV/Mne for their ability to replicate in lymphocytes and macrophages and with respect to the immunological relatedness of their viral proteins. Antibodies present in human immunodeficiency virus-2 (HIV-2)-infected individuals were readily detected by the virus produced by a single-cell clone of SIV/Mne.

Animals↗
PubMed1989

Proliferative T-cell response to HIV envelope glycoprotein in immunized and infected primates and human beings.

Human immunodeficiency virus (HIV)-specific helper T-cell response was studied in human subjects and nonhuman primates either infected with HIV or immunized with different HIV protein preparations. A strong group-specific T-cell response involving T-cell proliferation and lymphokine secretion was observed in immunized chimpanzees and rhesus monkeys as well as HIV-infected chimpanzees and gibbons. HIV-infected people demonstrated a low or no HIV-specific T-cell response. In contrast, five of 14 HIV antibody-negative sexual partners of HIV-infected men recognized one or more T-cell epitopes in the envelope glycoprotein of HIV.

Acquired Immunodeficiency Syndrome↗
PubMed1988

HLA-DR is involved in the HIV-1 binding site on cells expressing MHC class II antigens.

The primary interaction of HIV-1 with the target cell involves the viral large envelope protein (gp120) and the cellular CD4 molecule. mAb reacting with portions of CD4 have been shown to block HIV-1 attachment and infection. In one of the early reports describing HIV-1 cell interaction, some mAb reacting with MHC class II Ag were also found to block infection. To investigate further a possible role for MHC class II in HIV-1 binding, a cultured T lymphocyte cell line (H-9) that expresses MHC class II molecules and PHA-stimulated PBL was exposed for various time periods to concentrated viral particles and individual HIV-1 proteins. A decrease in the ability to detect the CD4a epitope and HLA-DR was observed after the cells were exposed to virus for 15, 30, and 60 min whereas HLA-DP and HLA-DQ Ag increased or remained unchanged. After 120 min of virus exposure, the CD4a epitope remained diminished whereas HLA-DR was detected at levels found on cells not exposed to virus. mAb detecting the CD4a epitope and HLA-DR, as well as alloantisera detecting the specific HLA-DR Ag on the target cell, blocked HIV-1 binding. When immunopurified gp120 was added to PHA-stimulated and unstimulated PBL, the CD4a epitope decreased in the same manner as was observed with whole virus preparations. In contrast to exposure to the intact virus, HLA-DR expression appeared to increase. Other viral proteins, p17, p24, and a portion of the small envelope protein, gp41, had no effect on the ability to detect cell surface Ag. Thus, although CD4 is the primary receptor for HIV-1 binding, HLA-DR appears to be involved in the binding site, probably by virtue of its close proximity to the CD4 molecule on the cell surface.

Antibodies, Monoclonal↗
PubMed1988

Antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24-amino acid sequence of the viral envelope, gp120.

Antisera to recombinant human immunodeficiency virus (HIV) proteins containing the entire envelope, gp160, or the central portion of the envelope, PB1, can inhibit fusion of virally infected cells in culture. This fusion inhibition is HIV-variant specific--that is, anti-gp160-IIIB inhibits fusion of isolate HTLV-IIIB-infected cells but not of isolate HTLV-IIIRF-infected cells. Both anti-gp160 and anti-PB1 are completely blocked in fusion inhibition activity by the addition of PB1 protein. A 24-amino acid peptide (RP135, amino acids 307-330) completely blocks fusion inhibition activity of both antisera and also blocks the activity of serum from a chimpanzee infected with HTLV-IIIB. Thus, the principal epitope that elicits fusion-inhibiting antibodies is located in the central portion of gp120.

Amino Acid Sequence↗
PubMed1988

A method for predicting individual HIV infection status in the absence of clinical information.

Latent structure analysis can be used to determine sensitivity and specificity rates of human immunodeficiency virus antibody assays in the absence of previous clinical or laboratory results. The technique was applied to the analysis of data obtained when a panel of serum samples, collected as part of a large-scale screening project, were subjected to four conventional bioassays (ag121, p24, gp120, and an enzyme-linked immunosorbent assay). To determine the accuracy of this statistical approach, the results of latent structure analysis were compared with the known clinical diagnoses of patients from whom the samples were taken, and nearly 100% agreement was obtained. Although a two-class latent structure model had some predictive value, a three-class model more adequately explained assay patterns. The use of the four standard assays in conjunction with the statistical methods described here would largely reduce the need for confirmatory Western blot assays in analyses of large panels of samples.

AIDS Serodiagnosis↗
PubMed1988

Partial purification of native HIV transmembrane protein gp41: generation of polyclonal and monoclonal antibodies.

We partially purified the human immunodeficiency virus (HIV) glycoprotein gp41 from infected H9 cells by immunoaffinity chromatography using a column containing the M25 monoclonal antibody (diMarzo-Veronese et al., 1985). A pH 11.5 buffer worked best for eluting the glycoprotein from this column. The eluted gp41 was used in a sensitive slot blot immunoassay to detect antibodies to HIV in human sera and to prepare rabbit polyclonal antibodies and the 41-1S mouse monoclonal antibody. These antibodies reacted with gp41 in immunoprecipitation and in Western blot assays, but did not neutralize HIV in a syncytium-forming microassay. A pH 2.5 buffer was found to be the most effective solution for eluting gp41 from a 41-1S monoclonal antibody column.

Antibodies, Monoclonal↗
PubMed1988

Purification and characterization of the external envelope glycoprotein from two human immunodeficiency virus type 1 variants, HTLV-IIIB and HTLV-IIIRF.

External envelope glycoprotein from cell membranes and culture media of H9 cells infected with human immunodeficiency virus type 1 (HIV-1) isolate HTLV-IIIRF was isolated by immunoaffinity chromatography and compared with similar materials isolated from another variant, HTLV-IIIB. Envelope glycoprotein from IIIB and IIIRF appears to be identical, whether isolated from infected cell membranes or culture media. The molecular size of the IIIRF external envelope glycoprotein was 110 kilodaltons, whereas the relative size of IIIB gp120 was 123 kilodaltons. Amino-terminal sequence analysis of purified external envelope glycoprotein isolated from infected cell membranes or culture fluids revealed identical single sequences for the first 20 amino acids for each variant. The sequences obtained for IIIB gp120 were identical to those reported for the BH10 clone of the IIIB isolate, and the sequences determined for IIIRF gp110 matched the amino acid sequence predicted for the HAT3 clone of the Haitian HIV isolate. The amino-terminal sequences of external envelope glycoproteins isolated from either HIV-1 variant corresponded to the sequence starting at the proposed proteolytic cleavage site for the processing of the signal peptide of gp160. Immunization with external envelope glycoprotein isolated from either of the two HIV-1 variants yielded goat antibodies that primarily precipitated the homologous antigen. Sequential immunization of a single goat with gp120 and then gp110 resulted in the generation of antibodies that precipitated external envelope glycoprotein from both variants.

Amino Acid Sequence↗
PubMed1988

Purified envelope glycoproteins from human immunodeficiency virus type 1 variants induce individual, type-specific neutralizing antibodies.

Repeated immunizations of goats, horses, or chimpanzees with envelope glycoprotein gp120 isolated from human immunodeficiency virus type 1 (HIV-1) resulted in type-specific neutralizing-antibody responses, which began to decay approximately 20 days following the administration of antigen. This was true repeatedly for serum samples from animals hyperimmunized with gp120s from either the HTLV-IIIB (IIIB) or the envelope-divergent HTLV-IIIRF (RF) HIV-1 isolates. Animals previously immunized with the IIIB gp120 were then inoculated with purified RF gp120. The first response in these animals was an anamnestic resurgence of neutralizing antibody to IIIB without detectable neutralizing antibody for RF. However, with later RF gp120 boosts, the IIIB neutralizing-antibody titers fell and an RF type-specific neutralizing-antibody response developed. When assessed with other HIV-1 variants, no group-specific neutralizing antibody was seen in any of the vaccination protocols evaluated. These results will pose real obstacles in the development of an effective vaccine for HIV.

Animals↗
PubMed1987

HTLV-III large envelope protein (gp120) suppresses PHA-induced lymphocyte blastogenesis.

The addition of inactivated preparations of purified human T cell lymphotropic virus (HTLV-III) was found to inhibit normal human lymphocyte phytohemagglutinin (PHA)-induced blastogenesis but had no effect on concanavalin A (Con A), pokeweek mitogen, or allogeneic stimulation. The inhibition was concentration-dependent and also dependent on adding the virus preparation before or at the same time as PHA. The CD4 molecule is the receptor for HTLV-III binding. Immunopurified large envelope protein (gp120) from HTLV-III was found to bind to the CD4 molecule and also inhibited PHA-induced lymphocyte blastogenesis. These results suggest that the gp120 viral protein may alter immune function by binding to the CD4 molecule, which in turn serves as an "off" signal to lymphocyte response to PHA stimulation. Alternatively, by binding to the CD4 molecule, gp120 may interfere with the interaction of this molecule with class II histocompatibility antigens on accessory cells, thus selectively suppressing PHA response.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

A specific assay measuring binding of 125I-Gp 120 from HIV to T4+/CD4+ cells.

The HIV (HTLV-III) envelope glycoprotein, Gp120, was isolated from virus-infected tissue culture cells using affinity chromatography. A radioimmunoassay was developed to determine the degree of iodinated Gp120 to target CD4+ (T4+) cells. 125I-Gp120 could be shown to selectively bind to CD4+ cells only. The Gp120 remained bound to these cells after repeated washes. Monoclonal anti-CD4 antibodies block the binding of Gp120 to CD4+ cells. Monoclonal antibodies to other cell surface components do not interfere with 125I-Gp120 binding. All IgG antibodies from HIV seropositive donors tested block 125I-Gp120 binding, though with variable titers. We believe that this assay provides further proof for the use of CD4 (T4) as a component of the receptor for HIV. It represents a safe, objective and sensitive method for the analysis of Gp120-CD4 interactions, as well as the potential of antibodies to interfere with this binding.

Antibodies, Monoclonal↗
PubMed1987

Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp120) subunit vaccine.

The major envelope glycoprotein of a human immunodeficiency virus (HIV) has been purified and was utilized as a prototype vaccine in chimpanzees. The 120,000-dalton glycoprotein (gp120) was purified from membranes of human T-lymphotropic virus (HTLV)-IIIB-infected cells and the final preparation contained low levels to no detectable HTLV-IIIB core antigen (p24) and low levels of endotoxin. Chimpanzees inoculated with gp120 responded by developing antibodies that precipitated radiolabeled gp120 and neutralized in vitro infection of HTLV-IIIB. Antibodies to HTLV-IIIB p24 were not detected in the gp120-immunized chimpanzees. Peripheral blood leukocytes from the vaccinated animals were examined for T4+ and T8+ cells, and no decrease in the T4/T8 ratio was found, indicating that immunization with a ligand (gp120) that binds to T4 has no detectable adverse effect on the population of T4+ cells. The only current animal model that can be reproducibly infected with HIV is the chimpanzee. Immunization of chimpanzees with HIV proteins will provide an experimental system for testing the effectiveness of prototype vaccines for preventing HIV infection in vivo.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Simple, rapid, quantitative, syncytium-forming microassay for the detection of human immunodeficiency virus neutralizing antibody.

A simple, rapid, quantitative syncytium-forming microassay for the detection of human immunodeficiency virus (HIV-I) isolates is described. A virus-syncytial sensitive clone of CEM cells (CEM-SS) was identified and made adherent to flat bottom 96-well microtiter dishes. Following the addition of virus, these cells develop easily quantifiable, adherent syncytia on a background of confluent, normal CEM-SS monolayer in 4 to 6 days. One-hit kinetics for syncytia formation were obtained at various multiplicities of infection. Syncytia are associated with complete virion production and cytoplasmic localization of the p24 core protein (detected by immunofluorescence). Total infectious virus can be accurately determined in this assay; these results showed a close correlation with p24 and gp120 induction when microtiter well supernatants were passed to fresh cells and evaluated by competitive radioimmunoassay. Studies of p24 antigen induction at and beyond the end point of syncytia formation indicate that there are no detectable nonsyncytial variants in standard HIV-I stocks. Six divergent HIV-I isolates (HTLV-IIIB, -RFII, -MN, -RUTZ, -CC, and LAV-1), as well as HTLV-IIIB and LAV-1 reisolated from persistently infected chimpanzees, produce quantifiable syncytia which vary slightly in their developmental morphology. Accurate neutralization titers are readily obtained from easily constructed multiplicity curves derived from serial dilutions of test sera. Inherent within this system is a flexible method for studying various kinetics of antibody/virus interactions, as well as blocking and interference studies with any candidate antiviral compounds.

Antibodies, Viral↗
PubMed1987

Purification of 120,000 dalton envelope glycoprotein from culture fluids of human immunodeficiency virus (HIV)-infected H9 cells.

The outer envelope glycoprotein, gp120, has been purified from large volumes (greater than 100 liters) of HTLV-IIIB-infected H9 cell culture fluids using immunoaffinity chromatography resins prepared from immunoglobulins of AIDS patients plasma. By using a single-step immunoaffinity purification, between 7 and 28 micrograms of gp120 was recovered from each liter of culture fluid which represented between 60 and 95% of the total envelope glycoprotein present in the fluid. Envelope glycoprotein in culture media was concentrated more than 10,000 times over the starting material. The water-soluble gp120, containing trace contaminating proteins, was purified to apparent homogeneity by preparative polyacrylamide gel electrophoresis (PAGE). Envelope glycoprotein purified from culture fluids was immunogenic in laboratory animals in both native and PAGE-purified forms and was reactive with AIDS patient sera in immunoassays.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Persistent infection of chimpanzees with human immunodeficiency virus: serological responses and properties of reisolated viruses.

Persistent infection by human immunodeficiency virus (HIV-1) in the chimpanzee may be valuable for immunopathologic and potential vaccine evaluation. Two HIV strains, the tissue culture-derived human T-cell lymphotropic virus type IIIB (HTLV-IIIB) and in vivo serially passaged lymphadenopathy-associated virus type 1 (LAV-1), were injected intravenously into chimpanzees. Two animals received HTLV-IIIB as either virus-infected H9 cells or cell-free virus. A third animal received chimpanzee-passaged LAV-1. Evaluation of their sera for virus-specific serologic changes, including neutralizations, was done during a 2-year period. During this period all animals had persistently high titers of antibodies to viral core and envelope antigens. All three animals developed a progressively increasing type-specific neutralizing LAV-1 versus HTLV-IIIB antibody titer during the 2-year observation period which broadened in specificity to include HTLV-HIRF, HTLV-IIIMN, and HTLV-IIICC after 6 to 12 months. The antibody titers against both viruses were still increasing by 2 years after experimental virus inoculation. Sera from all animals were capable of neutralizing both homologously and heterologously reisolated virus from chimpanzees. A slightly more rapid type-specific neutralizing response was noted for the animal receiving HTLV-IIIB-infected cells compared with that for cell-free HTLV-IIIB. Sera from all persistently infected chimpanzees were capable of mediating group-specific antibody-mediated complement-dependent cytolysis of HIV-infected cells derived from all isolates tested. Viruses reisolated from all three animals at 20 months after inoculation revealed very similar peptide maps of their respective envelope gp120s, as determined by two-dimensional chymotrypsin oligopeptide analysis. One peptide, however, from the original HTLV-IIIB-inoculated virus was deleted in viruses from all three animals, and in addition, we noted the appearance of a new or modified peptide which was common to LAV-1 as well as to HTLV-IIIB reisolated from infected chimpanzees. It thus appears that a group-specific neutralizing antibody response as well as a group-specific cytotoxic response can develop in chimpanzees after an inoculation of a single HIV variant. This finding suggests that a common, less immunodominant determinant(s) is present on a single HIV strain which could induce group-specific antibodies during viral infection and replication. The identification of this group-specific epitope and the induction of analogous immunity may be relevant to vaccine development against human acquired immunodeficiency syndrome.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Direct identification of class II histocompatibility DR proteins in preparations of human T-cell lymphotropic virus type III.

Class II histocompatibility DR antigen alpha and beta chains were isolated from preparations of human T-cell lymphotropic virus type III grown in human H-9 cells. The proteins were purified by reversed-phase high-pressure liquid chromatography and identified by direct N-terminal amino acid sequence analysis of each chain. The purified DR alpha chain had an N-terminal amino acid sequence identical to the known sequence of human DR alpha chain through the first 37 residues. The N-terminal amino acid sequence of the purified DR beta chain was identical to that of human DR4 beta chain. The DR alpha and beta chains appeared to be identical to the p34-36K and p30-32K proteins, respectively, concentrated in immunostimulatory complexes prepared from unfractionated virus and were the major immunogens in these complexes. These proteins represent a ready source of antigens which can cause false-positive enzyme-linked immunosorbent assay reactions in individuals previously exposed to allogenic histocompatibility antigens. The removal of the DR chains from virus preparations by use of available monoclonal antibodies or other means should result in a lower rate of initial false-positive enzyme-linked immunosorbent assay reactions.

Cell Line↗
PubMed1986

Human retroviral env and gag polypeptides: serologic assays to measure infection.

Humoral antiviral responses to human retrovirus infections identify persistently infected individuals and can be used to characterize virus-host interactions. Antibodies to native viral polypeptides have been reliably measured, although quantitation of env antibodies is difficult due to a lack of purified antigens. To quantitate antibodies to env antigens, bacterially expressed cloned env polypeptides from the transmembrane regions of human T lymphotropic virus types I and III were applied to nitrocellulose filters in an immunodot assay. A combination of the sensitivity of the Western blot procedure and the specificity of peptides from defined viral sequences was used to detect 49/49 HTLV-III/LAV-infected individuals previously defined as seropositive by radioimmunoprecipitation sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of these HTLV-III/LAV envelope seropositive people, 22% lacked antibody to p24 in a radioimmunoassay. In contrast, the sensitivity of antibody detection to HTLV-I env antigens and p24 were comparable. Antibodies to HTLV-I and HTLV-III/LAV env transmembrane peptides were not cross-reactive. Levels of antibody to env antigens of both HTLV-I and HTLV-III/LAV persisted without change for at least 26 mo, suggesting that most infections represent stable virus-host interactions. The use of bacterially expressed env peptides offers a rapid serologic approach for distinguishing human retroviral infections and can be used to define immune responses to specific regions of the viral genome.

Acquired Immunodeficiency Syndrome↗