Approximately 80 percent of all human sera that react with antigens of HTLV-III, the etiologic agent of the acquired immune deficiency syndrome (AIDS), recognize protein bands at 66 and 51 kilodaltons. A mouse hybridoma was produced that was specific to these proteins. Repeated cloning of the hybridoma did not separate the two reactivities. The p66/p51 was purified from HTLV-III lysates by immunoaffinity chromatography and subjected to NH2-terminal Edman degradation. Single amino acid residues were obtained in 17 successive degradation cycles. The sequence determined was a perfect translation of the nucleotide sequence of a portion of the HTLV-III pol gene. The purified p66/51 had reverse transcriptase activity and the monoclonal immunoglobulin G specifically removed the enzyme activity from crude viral extract as well as purified enzyme.

We have previously shown that the cell line 6D5(451) chronically infected with the HIV-1 isolate HTLV-III(451), secretes the HIV-1 envelope glycoproteins gp120 and gp160 in the extracellular medium. The HTLV-III(451) gp120 and gp160 were purified by sequential affinity chromatographic steps using a monoclonal antibody to HIV-1 gp41 and an anti-HIV-1-positive human serum. Amino acid sequence analysis of gp120 and gp160 showed the loss of the signal peptide. Digestion of the purified gp120 and gp160 with endoglycosidases revealed that both proteins are heavily glycosylated and contain complex carbohydrates, in contrast to the intracellular form of gp160 which has been shown to contain mannose-rich immature sugars. Competitive binding analysis showed that while both gp120 and gp160 bind CD4, the affinity of gp160 was five times lower than that of gp120. Both gp120 and gp160 inhibited syncytia formation by HIV-1-infected cells when mixed with CD4+ cells. Furthermore, both gp120 and gp160 had strong mitogenic effects on the T cells from HIV-1-infected gibbons but not on cells from uninfected gibbons.

The binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120, to its cell surface receptor, CD4, represents a molecular interaction involving distinct alterations in protein structure. Consequently, the pattern of epitopes presented on the gp120-CD4 complex should differ from those on free gp120. To investigate this concept, mice were immunized with covalently crosslinked complexes of viral HIV-1IIIBgp120 and soluble CD4. Two monoclonal antibodies (MoAbs) obtained from the immunized mice exhibited a novel epitope specificity. The MoAbs were marginally reactive with HIV-1IIIBgp120, highly reactive with gp120-CD4 complexes, and unreactive with soluble CD4. The same pattern of reactivity was seen in solid-phase assays using HIV-1(451)gp120. A similar specificity for complexes was evident in flow cytometry experiments, in which MoAb reactivity was dependent upon the attachment of gp120 to CD4-positive cells. In addition, MoAb reactivity was detected upon the interaction of CD4 receptors with purified HIV-1IIIB virions. Notably, seroantibodies from HIV-positive individuals competed for MoAb binding, indicating that the epitope is immunogenic in humans. The results demonstrated that crosslinked gp120-CD4 complexes elicit antibodies to cryptic gp120 epitopes that are exposed during infection in response to receptor binding. These findings may have important implications for the consideration of HIV envelope-receptor complexes as targets for virus neutralization.

the presence of antimicrobial substances on human skin was investigated. Staphyloccus aureus (10-4 colony-forming units) was applied on the forearm of 50 subjects and covered with a semiocclusive device for 24 hr. In 54% of the subjects the organisms persisted, and in 34% S. aureus was inhibited on the skin. Subjects with persistent S. aureus also had persistent Candida albicans, and vice persa. This correlation was not noted with Streptococcus pyogenes. Skin lipids from the two groups of subjects were extracted with acetone and assayed against S. aureus, S. pyogenes, and C. albicans. The percentage of S. aureus or C. albicans recovered was higher (79% and 55%, respectively) in subjects with persistent microrgamisms on their skin than in those without (47% and 28%, respectively). Subjects with persistent S. aureus and C. albicans had higher counts of normal flora (average, 9.2 times 10-3) than those on whose skin these organisms did not persist (average, 7.4 times 10-2). Coagulase-negative Staphylococcus and Micrococcus were found in higher proportions in subjects with persistent test organisms than in those without. Subjects with lower counts of their normal flora had a higher proportion of diphtheroids (34%) than the high-count group (12%).

The first open reading frame of the HIV genome has been identified as the gag gene. The proteins encoded by this gene are p17 as the amino-terminal protein, p24 as the middle peptide, and p15 as the carboxyl-terminal end. A monoclonal antibody recognizing an antigenic determinant on a fragment of p15 has been developed and designated M35/2F8. This monoclonal has been instrumental in radiosequencing the carboxyl-terminal product of p15, p6, and in determining the cleavage site between this protein and the amino-terminal product, p7. By immunoaffinity chromatography it was also possible to purify p6 from HIV lysates and all p6 containing polyproteins from HIV-infected cells. These results gave more insight into the composition and processing of the HIV gag gene.

Immunization of mice and rats with purified external glycoprotein gp120 from two divergent human immunodeficiency virus type 1 (HIV-1) isolates resulted in the development of seven hybridomas secreting monoclonal antibodies able to recognize regions of gp120 which are common among divergent strains of HIV-1. These monoclonal antibodies cross-reacted with env glycoproteins from one African (Rutz), one Haitian (RF), and three North American viral isolates, namely IIIB, MN, and 451 by either immunoblot or radioimmunoprecipitation assays. All recognized denatured gp120 in immunoblots with the exception of one which required a conformationally intact glycoprotein for reactivity. The gp120 epitopes identified by these antibodies were mapped by screening of an env gene library in the lambda gt11 expression system. Three out of four epitopes were found to reside in the amino-terminal half of gp120 (Cys9 to Cys35, Thr44 to Glu72 and Val108 to Met130), the other was located in the middle region (Thr221 to Ser255). By virtue of their extent of cross-reactivity these reagents might provide a unique resource for the detection of new viral isolates related to HIV-1.

Four mouse hybridomas secreting monoclonal antibodies specific for p24, the major core antigen of the human T-cell leukemia virus type Im (HTLV-III), have been developed, and their specificities have been partially characterized. These antibodies specifically recognized p24 of HTLV-III in extracts of HTLV-III and in HTLV-HI-producing cells. No epitopes cross-reactive with HTLV-I and -II were detected with these antibodies. These hybridomas will be extremely valuable reagents in identifying expression of HTLV-Ill in infected cultures and in cells or tissues from patients with suspected immunodeficiency syndrome.The human T-cell leukemia viruses (HTLVs) are a family of T-cell lymphotropic retroviruses etiologically associated with alteration of T-cell functions. Those (7)(8)(9)(10). AIDS is a newly described disease characterized by severe immune depression with depletion of the OKT4+ subset of T lymphocytes (11-13) and is accompanied by multiple opportunistic infections or neoplasias (9). Common biological features of all members of the HTLV family include their preferred tropism for T cells (5, 14, 15) and morphologic appearance of budding and early forms of the virus under electron microscopy.Even though HTLV-III is antigenically distinct from HTLV-I and -II, it shares a number of determinants (16,17) and some nucleotide sequence homology (18,19). Like the other two isolates, HTLV-III has a major structural protein with a molecular weight of 24,000 (p24) (17) and a reverse transcriptase with a molecular weight of 100,000, but the protein homologous to p19 is somewhat smaller (p17) (ref. 20; unpublished data). Another difference involves the in vitro transmission of the virus to primary human T cells. Whereas HTLV-I and -II can transform some of these cells and be propagated indefinitely in them (14, 15), HTLV-III is only transiently produced in these cells (5). The problem has been overcome by propagating the virus in a human neoplastic cell line (HT) (4).Isolation of the AIDS agent has enormous clinical implications because it allows the possibility of development of specific assays to identify HTLV-III infection, the first step in interrupting its spread. The correlation between the presence of antibodies to HTLV-III proteins and AIDS strongly identifies this virus as the causative agent for AIDS (20-24). The only reported antibody reagent for subtyping HTLV-III to date is a polyclonal antibody to p24 (17). Here we describe the production and characterization of a series of hybridomas secreting monoclonal antibodies to HTLV-III p24, the major core protein. MATERIALS AND METHODSPreparation of Monoclonal Antibody. BALB/c mice (Charles River Breeding Laboratories) were immunized with successive intraperitoneal inoculations of detergent lysates of density gradient-purified HTLV-III (100 ,tg) emulsified in complete Freund's adjuvant for the first inoculation and incomplete adjuvant for the following four boosters given 1 week apart. Three days following a final intraperitoneal booster with disrupted v...

The major internal protein, p30, of rat type C virus (RaLV) was purified and utilized to establish intra-and interspecies radioimmunoassays. Three rat viruses were compared in homologous and heterologous intraspecies assays with no evidence of type specificity. The only heterologous viruses to give inhibition in these species assays were the feline (FeLV) and hamster (HaLV) type C viruses; these reactions were incomplete and required high virus concentrations. An interspecies assay using a goat antiserum prepared after sequentially immunizing with FeLV, RD 114, and woolly monkey virus p30's and labeled RaLV p30 was inhibited by all mammalian type C viruses, although preferentially by RaLV, FeLV, and HaLV. Thus, as in a previously reported assay developed with HaLV p30, rat, hamster, and cat p30's seem more closely related to each other than to mouse type C virus p30. High levels of specific antigen were found in all cell lines producing rat virus, whereas embryonic tissues from several rat strains and cell lines considered virus-free based on other tests were negative for p30. Rats bearing tumors containing Moloney murine sarcoma virus (RaLV) did not contain free circulating antibody to RaLV p30. Fifty-one human tumor extracts (including two tumor cell lines) were tested for activity in the RaLV species and 47 in the interspecies assays after Sephadex gel filtration and pooling of material in the 15,000to 40,000-molecular-weight range. At a sensitivity level of 7 ng/ml (0.7 ng/assay) in the interspecies assay, all human tissues, with one exception, were negative. The one positive result is considered nonspecific based on proteolysis of the labeled antigen. Input tissue protein of the purified tumor extracts averaged 1.9 mg/ml with a range of <0.025 to 22 mg/ml. Tissues from NIH Swiss mice processed in the same manner were positive in the interspecies assay but negative in the intraspecies RaLV assay.