Crude preparations of interferon (IFN)-gamma derived from human peripheral blood leukocyte (PBL) cultures induced with 12-O-tetra-decanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA) were more cytotoxic to HeLa cells than partially purified nautral or highly purified recombinant human IFN-gamma preparations. Conditioned media from PBL cultures contained, in addition to IFN-gamma, a mixture of cytotoxins, including classic lymphocyte-derived lymphotoxin (LT), and a TPA-induced cytotoxic activity produced by the adherent cell population (presumably monocytes). These two types of cytotoxins, indistinguishable in the mouse L929 cell LT assay, could be differentiated by an antiserum prepared against LT derived from the B lymphoblastoid cell line RPMI 1788. This antiserum neutralized lymphocyte-derived classic LT but failed to neutralize the activity of the monocyte-derived cytotoxin. Processing of conditioned media by sequential chromatography on silicic acid, Con A-Sepharose, and DEAE-Sephacel failed to separate IFN-gamma from the LT activity. However, this procedure did remove the monocyte-derived cytotoxic activity present in the original starting material, leaving predominantly classic LT. This LT showed a slightly basic isoelectric point (pI 7.6) which partially overlapped the more basic pI range of IFN-gamma. The two lymphokine activities also could not be completely separated by fast protein liquid chromatography or molecular sieve chromatography. LT in these partially purified preparations was associated with a protein having an apparent molecular weight of 58,000 on gel filtration. This form dissociated partially into a 20,000 mol wt species after denaturation with 0.1% NaDodSO4. IFN-gamma could be selectively removed from preparations containing both IFN-gamma and LT with the aid of monoclonal antibody to IFN-gamma. The addition of purified LT to purified E. coli-derived recombinant human IFN-gamma resulted in a marked synergistic enhancement of cytotoxicity for HeLa cells.

Cultures of human peripheral blood leukocytes (PBL) induced with phytohemagglutinin (PHA) and the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) produced two types of cytotoxic proteins, indistinguishable in the in vitro assay employing murine L 929 cells as targets. One of these proteins had the antigenic and physicochemical properties of lymphotoxin (LT). We have identified the other cytotoxin as tumor necrosis factor (TNF), mainly on the basis of antigenic cross-reactivity demonstrated with antiserum to TNF, and also by its characteristic physicochemical properties and cell source. Unlike LT, PBL-derived TNF did not bind to Concanavalin A-Sepharose or to several other agglutinin-Sepharose columns specific for carbohydrate moieties common in glycoproteins. The molecular weight of native TNF determined by gel filtration was approximately 40,000 while SDS-PAGE revealed a single sharp peak of 16,500 +/- 500. When cultures of monocytes and lymphocytes separated by elutriation were stimulated with PHA and/or TPA, monocytes were the major source of TNF. In contrast, only lymphocytes produced LT. A mixture of antisera to TNF and LF neutralized all cytotoxicity of crude human lymphokine preparations for L 929 cells, suggesting that TNF and LT are either the only, or the major, cytotoxic proteins present in such crude lymphokine preparations demonstrable in this assay.

A comparison of the proliferative responses of lymphocytes to human immunodeficiency virus (HIV) antigens from long-term, seronegative heterosexual partners of HIV-infected subjects, from normal unexposed controls and from healthy seropositive heterosexual partners or seropositive, asymptomatic men, reveals that lymphocytes from healthy seropositive individuals with strong proliferative responses to recall, microbial antigens respond only minimally to HIV proteins or envelope peptides, and that even these low responses do not occur in all individuals. If the frequency of responses to several HIV antigens are analyzed, lymphocytes from both HIV-exposed seropositive and seronegative partners of infected individuals proliferate to HIV antigens to a greater degree than lymphocytes from unexposed, normal control individuals. Although lymphocytes from seropositive partners proliferate to a greater degree than those from seronegative partners, the latter are more similar to seropositive partners than they are to normal controls. This observation suggests that these seronegative partners may have become sensitized to HIV antigens through sexual exposure but without infection, and/or that the presence or development of these small immune responses in some individuals might be associated with a failure to become infected.

BackgroundNovel strategies are needed for the elicitation of broadly neutralizing antibodies to the HIV envelope glycoprotein, gp120. Experimental evidence suggests that combinations of antibodies that are broadly neutralizing in vitro may protect against challenge with HIV in nonhuman primates, and a small number of these antibodies have been selected by repertoire sampling of B cells and by the fractionation of antiserum from some patients with prolonged disease. Yet no additional strategies for identifying conserved epitopes, eliciting antibodies to these epitopes, and determining whether these epitopes are accessible to antibodies have been successful to date. The defining of additional conserved, accessible epitopes against which one can elicit antibodies will increase the probability that some may be the targets of broadly neutralizing antibodies.Methodology/Principal FindingsWe postulate that additional cryptic epitopes of gp120 are present, against which neutralizing antibodies might be elicited even though these antibodies are not elicited by gp120, and that many of these epitopes may be accessible to antibodies should they be formed. We demonstrate a strategy for eliciting antibodies in mice against selected cryptic, conformationally dependent conserved epitopes of gp120 by immunizing with multiple identical copies of covalently linked peptides (MCPs). This has been achieved with MCPs representing 3 different domains of gp120. We show that some cryptic epitopes on gp120 are accessible to the elicited antibodies, and some epitopes in the CD4 binding region are not accessible. The antibodies bind to gp120 with relatively high affinity, and bind to oligomeric gp120 on the surface of infected cells.Conclusions/SignificanceImmunization with MCPs comprised of selected peptides of HIV gp120 is able to elicit antibodies against conserved, conformationally dependent epitopes of gp120 that are not immunogenic when presented as gp120. Some of these cryptic epitopes are accessible to the elicited antibodies.

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