The HIV-1 gp120 envelope protein has the intrinsic capacity to stimulate monokine secretion.

Results and conclusions concerning the ability of HIV glycoprotein (gp) 120 to stimulate monokine secretion have been equivocal, based on observations using natural gp120 derived from infected human cells and a Chinese hamster ovary (CHO) cell-derived recombinant fusion protein. Current studies were designed to determine whether differences in recombinant gp120 proteins could result in failure to trigger monokine production. We found that natural gp120 could stimulate monocytes to release TNF-alpha, IL-1 beta, IL-6, and granulocyte-macrophage-CSF, and this effect could be blocked with soluble CD4. Full-length rgp120 either expressed from an adenovirus vector and purified from infected human cells, or derived from CHO cells, could function similarly. In contrast, full-length recombinant envelope protein expressed in a baculovirus system and a CHO cell-derived recombinant fusion protein tested previously, consistently failed to stimulate monokine production. The stimulatory capacity of both natural and full-length CHO cell-derived gp120 was eliminated by heating at 100 degrees C, and could be blocked with excess CHO cell-derived gp120 fusion protein. Inasmuch as the baculovirus-expressed gp120 and the CHO cell-derived recombinant fusion protein can bind to CD4, these results suggest that HIV gp120 binding to CD4 on the monocyte surface may of itself be insufficient for stimulation of monokine secretion. Therefore, primary protein structure, as well as posttranslational protein modifications, may determine this activity.

Large-scale purification of gp70 from Moloney murine leukemia virus.

The external envelope glycoprotein, gp70, of the Moloney murine leukemia virus was extracted from NIH 3T3 cells utilizing the detergent n-octyl-beta-D-glycopyranoside. The extracted gp70 was sequentially purified utilizing lectin-affinity, anion-exchange, and molecular-exclusion chromatography techniques. Approximately 10 mg of gp70 was purified by this method and shown to be 95% homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of purified gp70 from Moloney murine leukemia virus was confirmed by amino acid analysis, amino-terminal sequencing, and immunoreactivity with a monoclonal antibody raised against gp70. The procedure is rapid, utilizes commercially available media, and can be used to purify large amounts of retroviral envelope glycoprotein from virus.

Serological investigation of the fish pathogen Edwardsiella ictaluri, cause of enteric septicemia of catfish.

The serological relationships among 32 isolates of Edwardsiella ictaluri obtained from fish were studied. The strains were extremely homogeneous in protein and lipopolysaccharide preparations as observed by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis. Only minor variations were observed in the structural O-side chain subunits in three isolates; however, such variation did not preclude antigenic recognition by two E. ictaluri antisera in either microagglutination or Western blot immunoassays. The antigenic homogeneity of E. ictaluri was further demonstrated by microagglutination assays with both formalin-killed and heat inactivated cellular antigens. The minimal degree of antigenic variability observed suggested that most isolates of E. ictaluri compose a single antigenic serotype.

Biologic activities of HIV-1 envelope glycoprotein: the effects of crosslinking.

We have examined the biologic activities of native and recombinant preparations of human immunodeficiency virus envelope glycoprotein (gp120), both derived from the HIV-1B strain. Antibody to gp120 was used to evaluate the effects of crosslinking gp120 on signalling by the CD4 receptor. Our results indicate that native and recombinant gp120 produce identical effects in our assay systems. Crosslinking gp120 amplified its chemoattractant activity for lymphocytes and monocytes and increased the peak intracellular calcium level, compared with binding of gp120 alone. The induction of inositol trisphosphate (IP3) production, induction of interleukin 2 receptors (IL2R), and inhibition of lymphocyte proliferation following treatment with gp120 were not enhanced by the addition of crosslinking antibody.

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.

Immune response to immunostimulatory complexes (ISCOMs) prepared from human immunodeficiency virus type 1 (HIV-1) or the HIV-1 external envelope glycoprotein (gp120).

In mice, immunostimulatory complexes (ISCOMs) prepared from HIV-1 B external envelope glycoprotein (gp120) induced 10-fold higher antibody titres than gp120 emulsified in depot adjuvant, as measured by enzyme-linked immunosorbent assay (ELISA). Rhesus monkeys immunized with gp120 ISCOMs produced precipitating and virus neutralizing antibody titres equivalent to those seen in HIV-infected chimpanzees and humans. After multiple immunizations with HIV-1 B gp120 ISCOMs, a rhesus monkey developed a neutralizing response to the HIV-1 isolates RF and MN, but not to the CC isolate. Antisera from ISCOM-immunized rhesus monkeys recognized gp120 on the membranes of HIV-1 B-infected H9 cells, indicating the preservation of epitope structure in the ISCOMs matrix.

Human immunodeficiency virus glycoprotein (gp120) induction of monocyte arachidonic acid metabolites and interleukin 1.

This study reports on the direct effect of the envelope glycoprotein (gp120) of the human immunodeficiency virus type 1 (HIV-1) on human monocyte function. Addition of preparations of purified gp120 from the HIV-1 to human monocytes resulted in the production of interleukin 1 (IL-1) and arachidonic acid metabolites from the cyclooxygenase and lipoxygenase pathways. Quantification of prostaglandin E2 (PGE2) and IL-1 revealed an increase in both mediators with 50 ng of gp120 per ml and an increase of 12- and 30- to 40-fold with 200-400 ng of gp120 per ml, respectively. Unlike native gp120, the recombinant nonglycosylated gp120 fragments PB1-RF and PB1-IIIB, as well as one of the core structural proteins of HIV-1, p24, did not increase arachidonic acid metabolism or IL-1 activity. Cytofluorometric analysis revealed that gp120 blocked the binding of OKT4A to the CD4 on monocytes, whereas OKT4 binding was unaffected. Involvement of the CD4 in signal transduction was further demonstrated by the ability of OKT4 and OKT4A monoclonal antibodies to increase monocyte PGE2, IL-1 activity, and nanogram amounts of IL-1 beta.

Lymphocyte activation by HIV-1 envelope glycoprotein.

Cell activation by phytohaemagglutinin, phorbol ester and by the supernatant of phytohaemagglutinin-stimulated peripheral blood mononuclear cells induces the expression and cytopathic effects of latent human immunodeficiency virus type-1 (HIV-1) in vitro. The lymphocyte surface protein CD4 has been identified as a receptor for HIV-1 and binds the viral envelope glycoprotein (gp120). In the light of evidence indicating that one natural function of CD4 is as a growth factor receptor, we examined the ability of native gp120 to activate resting CD4-bearing lymphocytes. Our results indicate that gp120 has innate biological activity as a result of a specific interaction with CD4, inducing increases in intracellular levels of inositol trisphosphate and of calcium, and in interleukin-2 receptor expression and cell motility.

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.

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.

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.

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.

An epizootic in chinook salmon (Oncorhynchus tshawytscha) caused by a sorbitol-positive serovar 2 strain of Yersinia ruckeri.

Enteric redmouth disease is described in chinook salmon (Oncorhynchus tshawytscha) at a state hatchery in Sand Ridge, Illinois. Biochemical, isoenzyme, and serological data indicated that the epizootic was caused by a sorbitol-fermenting Serovar 2 strain of Yersinia ruckeri. In laboratory experiments the isolate was pathogenic for both brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar).

Rapid serological analysis of bacterial lipopolysaccharides by electrotransfer to nitrocellulose.

Techniques are described for the rapid screening of proteinase K-treated bacterial lysates by electroblot and immunoenzymatic detection to assess O-specificity of antigens and antisera. Conditions are outlined which permit the use of a single polyacrylamide gel for both electrotransfer to nitrocellulose and silver staining. Immunodetection of transferred LPS bands was equally sensitive to silver stain when whole cell or O-specific antisera were used. The techniques were utilized to identify at least 4 O-serotypes among sorbitol fermenting isolates of the fish pathogen, Yersinia ruckeri. Observed variations in the electrophoretic mobilities of lipopolysaccharides from 17 field isolates of Y. ruckeri were used to accurately predict the O-serotype.

Detection of Vibrio anguillarum antigen by the dot blot assay.

The dot blot assay, modified and adapted for detection of antigens from Vibrio anguillarum in fish tissues, was specific for V. anguillarum and did not react with antigens of V. ordalii, Pseudomonas sp., or Yersinia ruckeri. The blot assay enabled detection of as little as 2.3 ng of a mixture of protein antigens obtained from cell-free extracts of V. anguillarum; it was about 100 times more sensitive than either the indirect fluorescent antibody technique or bacterial isolation for detecting V. anguillarum in fish tissues.

Multilocus Electrophoretic Assessment of the Genetic Structure and Diversity of Yersinia ruckeri.

Multilocus isoenzyme electrophoresis was used to screen 47 field isolates of Yersinia ruckeri for electrophoretic variation at 15 enzyme loci. Only four electrophoretic types were observed, thus indicating that the genetic structure of Y. ruckeri is clonal. Forty-two isolates were of one electrophoretic type, a reflection of the low amount of genetic diversity extant in this species. Although sorbitol fermentation has been considered to be indicative of a second biotype, no significant gene frequency differences were found between the group of 20 isolates that readily used sorbitol as the sole carbon source and the group of 27 that did not.