IFN-gamma restores HIV- and non-HIV-specific cell mediated immune response in vitro and its activity is neutralized by antibodies from patients with AIDS.

The addition of IFN-gamma to cultures of peripheral blood mononuclear cells (PBMCs) obtained from asymptomatic HIV-infected patients increased cell proliferation in response to HIV envelope synthetic peptides (Env), influenza A virus (VIRUS), and allogeneic lymphocytes (ALLO) but not to phytohaemagglutinin (PHA) stimulation. F(Ab)2 fragments of IgG purified from the sera of HIV-seropositive patients specifically interfered with IFN-gamma-induced cell proliferation in response to recall antigens. Neutralization of the lymphokine activity was found to be sustained by specific IFN-gamma antibodies. Data obtained demonstrate that IFN-gamma can restore the cell-mediated immunity of a number of asymptomatic HIV+ individuals in vitro, while IFN-gamma antibodies present in sera of patients with AIDS interfere with the activity of the lymphokine.

Aspects of molecular interaction between HIV p17 and human gamma interferon.

We describe the specific interaction between high-purity recombinant human immunodeficiency virus (HIV) type 1 p17 and human gamma interferon (hIFN-gamma) proteins. This interaction was found to be dose dependent and to involve conformational epitopes on both sides. Specificity was confirmed by competition ELISA, using monoclonal antibodies (MAbs) to hIFN-gamma as specific reagents. By competition experiments we also identified the epitope(s) on the hIFN-gamma molecule involved in p17 binding, very close to the receptor binding site. The kinetic constants were determined by surface plasmon resonance (SPR) analysis. The affinity constant (KA) of the complex was 2.78 x 10(8) M-1, that is, the ratio between a low dissociation rate constant (Koff)(1 x 10(-5)sec-1) and a high association rate constant (Kon) (3 x 10(3) M-1sec-1). However, p17 did not displace the binding of hIFN-gamma to its cellular receptor, nor did it interfere with the capability of the lymphokine to induce de novo expression of HLA-DR antigens on human monocytic cells or to inhibit the proliferation of tumor cells.

Purification of LamB proteins using continuous elution electrophoresis: a comparison with immunoaffinity chromatography.

LamB is a membrane protein that allows the exposition of a foreign peptide on the surface of a recombinant E. coli cells. An immunopurified hybrid LamB protein has been used to elicit high-titre antibodies to a foreign epitope. Looking for a simpler purification procedure we have compared the traditional approach, which includes affinity chromatography, to continuous elution electrophoresis, in the purification of two different hybrid LamB proteins as foreign epitopes. The results obtained showed that both methods yielded the same purification, although the electrophoretic procedure had a higher yield. Continuous-elution electrophoresis could be a useful tool for the purification of membrane proteins.

The influence of fetal calf serum on interferon-gamma induced HLA-DR expression on U937 cells.

The induction of HLA-DR antigen expression on U937 cells by interferon-gamma (IFN-gamma) is positively influenced by amount of fetal calf serum (FCS) added to the tissue culture medium. A transient alkalinization of FCS before its addition to the medium, dramatically decreased the immunomodulating activity of IFN-gamma. FCS was also found to be a dose-dependent enhancer of the IFN-gamma-induced 2',5'-oligoadenylate (2-5A) synthetase production. Our findings suggest the need for a serum factor(s), labile at basic pH values, to support at least two of the multiple IFN-gamma activities.

A monoclonal antibody to the NH2-terminal segment of human IFN-gamma selectively interferes with the antiproliferative activity of the lymphokine.

To gain more information about the relationship between the structure of IFN-gamma and its activity, a peptide corresponding to a hydrophilic peak between amino acids 4 and 16 was used to immunize mice and generate mAb. mAb IGMB-15 reacts to both native and rIFN-gamma and neutralizes the antiproliferative activity of IFN-gamma without affecting its antiviral activity or its ability to up-regulate HLA-DR Ag expression. Moreover, we observed that mAb IGMB-15 was unable to inhibit the binding of radiolabeled IFN-gamma to its cellular receptor. These findings show that the NH2-terminal region may somehow be involved in the biologic activity of IFN-gamma. Besides, the capability of mAb IGMB-15 to inhibit the antiproliferative but not the antiviral activity of IFN-gamma in the same cell (HEp-2) suggests the presence of different elements involved in signal transduction, which may account for the multiple activities of the lymphokine.

Expression of activation markers on peripheral-blood lymphocytes following oral administration of Bacillus subtilis spores.

This study was undertaken to assess the capability of Bacillus subtilis spores to modify the peripheral-blood lymphocyte (PBL) subsets or determine the de novo expression of activation markers. The data we obtained show that spores of B. subtilis are able to increase the expression of certain cell activation markers and that such activation is dose-dependent. In fact, doses of 2 x 10(9) spores did not give rise to changes in any of the parameters evaluated, while doses of 6 x 10(9) increased the HLA-DR antigen expression on T-lymphocytes. At the highest dosage used (12 x 10(9), B. subtilis spores caused the appearance of cells bearing the CD25 and CD71 activation markers. Therefore, such cell activation markers may prove useful for monitoring the activity of B. subtilis spores, and possibly of other immunomodulating agents, in the course of clinical research.

High-titre antibodies to a foreign epitope elicited by affinity-purified hybrid LamB proteins.

A monoclonal antibody to the LamB protein, named LBS-1, was developed and characterized. It was then covalently bound to Sepharose and used to purify hybrid LamB proteins from Escherichia coli crude extracts. A peptide of the interferon-gamma (IFN-gamma) NH2-terminal region, inserted within the LamB protein, was used as a model to assess immune response in mice injected with sonicated E. coli extract or with affinity-purified hybrid LamB protein. None of the mice immunized with the whole bacterial extract produced antibodies to IFN-gamma. On the other hand, all the mice immunized with the purified protein developed high-titre anti-IFN-gamma antibodies. These results might be due to the presence of bacterial components capable of masking the LamB protein to the immune system. The use of affinity-purified LamB proteins may constitute in some instances a more effective way of generating an immune response against foreign epitopes as opposed to whole bacterial antigens.

Purification of natural human IFN-gamma antibodies.

Natural antibodies to interferon gamma (IFN-gamma) were found in patients suffering from various viral infections, but also at weak titers in healthy individuals. In the present study we describe a one-step chromatographic procedure for the purification of the anti-IFN-gamma antibodies from human Ig preparations, using a recombinant IFN-gamma-coupled Sepharose CL4B affinity column. The antibodies to IFN-gamma were eluted from the column using 3 different methods without loss of immunological activity. They were found to be Ig, mostly of the IgG1 subclass, and, in the biological assay, to be able to neutralize the de novo expression of Fc receptor sites induced by IFN-gamma on U937 cells.