Multiple drug resistance to nucleoside analogues and nonnucleoside reverse transcriptase inhibitors in an efficiently replicating human immunodeficiency virus type 1 patient strain.

A human immunodeficiency virus type 1 (HIV-1)-seropositive patient was treated sequentially with the dideoxynucleoside (ddN) analogues zidovudine, didanosine, zalcitabine, stavudine, and lamivudine and the nonnucleoside HIV-1-specific reverse transcriptase inhibitor (NNRTI) loviride (alpha-APA). Accumulation of drug resistance mutations (mainly V75I, F77L, K103N, F116Y, Q151M, and M184V) eventually resulted in a strain that was genotypically and phenotypically resistant to all tested ddNs and the majority of NNRTIs. However, the multidrug-resistant virus retained wild type sensitivities to drugs such as foscarnet, phosphonomethoxyethyl adenine, dextran sulfate, JM3100, saquinavir, and NNRTI TSAO-m3T. Drug-resistant isolates showed replication kinetics and infectivity in an in vitro peripheral blood mononuclear cell system similar to those of the wild type isolate from the same patient. The multi-ddN-resistant isolate was not eliminated in a competition culture with the wild type isolate. Sequential therapy did not prevent the appearance of multidrug-resistant virus with a conserved replication rate.

Syngeneic adoptive transfer of anti-human immunodeficiency virus (HIV-1)-primed lymphocytes from a vaccinated HIV-seronegative individual to his HIV-1-infected identical twin.

Immunotherapy by adoptive transfer of lymphocytes was attempted in identical twins, one who was virus-free and the other who was infected with human immunodeficiency virus-1 (HIV-1), at the stage of acquired immunodeficiency syndrome. The noninfected twin was vaccinated by priming with a recombinant vaccinia virus expressing the envelope glycoprotein of one of his brother's viruses and boosting with the same purified gp160 adsorbed on alum. Vaccination elicited major histocompatibility complex class I-restricted CD8+ cytolytic T lymphocytes specific for HIV-1, but no antibody response. The diseased brother, a 38-year-old homosexual who had developed repeated opportunistic infections since 1990 and had a CD4+ count reduced to practically zero, was treated by infusions of lymphocytes collected from the vaccinated brother by lymphopheresis. After a first transfer of the whole lymphocyte population, no changes were observed in the clinical status and biologic or virologic parameters. A second transfer was then applied with activation of the cells with purified envelope glycoprotein before infusion. The outcome of the treatment was an increase in total lymphocytes, in CD4+ and activated CD8+ DR+ cell counts, and in proliferative responses to HIV antigens. A marked but transient 3-log increase in cellular and plasmatic virus loads was also observed after the second adoptive transfer. These observations will be considered with attention to improve the future adoptive transfer protocols, especially in patients with severe CD4+ depletion.

The convertases furin and PC1 can both cleave the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp160 into gp120 (HIV-1 SU) and gp41 (HIV-I TM).

Intracellular proteolytic processing of human immunodeficiency virus envelope glycoprotein precursor (gp160) is an essential step for virus infectivity. Northern blot analysis provided evidence that furin and PC1, but not PC2, are expressed in the CD4+ human lymphoblastoid H9 cell line, suggesting the possible participation of these convertases in human immunodeficiency virus (HIV) gp160 proteolytic processing. Purified PC1 and furin cleaved specifically in vitro gp160 into gp120 (HIV-I SU) and gp41 (HIV-I TM). NH2-terminal sequence analysis of the produced gp41 (HIV-I TM) demonstrated that the cleavage occurred within the sequence Arg-Glu-Lys-Arg decreases Ala-Val-Gly-Ile, which is identical to the bond cleaved in vivo. Transition state analog peptides were designed and tested in vitro for their ability to inhibit the PC1- or furin-mediated gp160 cleavage. The best inhibitor was decanoyl-Arg-Lys-Arg-Arg-psi [CH2NH]-Phe-Leu-Gly-Phe-NH2.

Influence of genes from the major histocompatibility complex on the antibody repertoire against culture filtrate antigens in mice infected with live Mycobacterium bovis BCG.

C57BL/10 and C57BL/6 mice (H-2b); B10 congenic mice with f, k, p, q, r, and s H-2 haplotypes; B10 mice with recombinant g2, o2, a, h2, h4, i5, and bq1 H-2 haplotypes; and B6 mice with major histocompatibility complex (MHC) mutant bm1 and bm13 (class I) and bm12 (class II) haplotypes were infected intravenously with 4 x 10(6) CFU of live Mycobacterium bovis BCG and examined by Western immunoblot analysis for serum antibodies against BCG culture filtrate antigens, following a boost injection with live BCG or with BCG culture filtrate. Parental B10 and B6 mice reacted very intensely with three culture filtrate protein bands with estimated molecular masses of 37, 38, and 40 kDa. Response against the 40-kDa protein was stronger following a boost injection with live BCG than following a boost with culture filtrate. Sera from mice with f, p, i5, bm1, and bm13 haplotypes reacted strongly, with both the 37-38- and 40-kDa antigens, and sera from mice with q and bq1 haplotypes showed a somewhat weaker reaction. Sera from mice with r, s, and bm12 haplotypes reacted against the 37-38-kDa antigen but not against the 40-kDa antigen, and sera from mice with the h2 haplotype reacted only with the 40-kDa antigen but not with the 37-38-kDa antigen. Sera from mice with the k, g2, o2, a, and h4 haplotypes showed, at most, a very weak reaction with the 37-38- and 40-kDa antigens. These results demonstrate that MHC genes profoundly affect the antibody repertoire used against culture filtrate antigens in mice infected with live M. bovis BCG. In particular, as shown in mice with the recombinant H-2 haplotype and in class II mutant bm12 mice, the I-A heterodimer controls the recognition of the immunodominant 40-kDa antigen. By using crossed immunoelectrophoresis, this 40-kDa antigen was identified as antigen 88 according to the reference system of Closs et al. for BCG antigens.

Characterization of monoclonal antibodies against the p17 core protein of the human immunodeficiency virus 1.

Five mouse hybridomas which produce monoclonal antibodies against the p17 core protein of HIV-1 have been isolated. Cross-competition assays and mapping with synthetic peptides demonstrate that two closely related epitopes are identified by these antibodies. Directed against two neighbouring peptides at the carboxy-terminal end of the molecule, they can be used for the selective detection of p17 polypeptide in a viral extract or in an infected cell lysate by a solid-phase sandwich enzyme immunoassay.

Virosomes reconstituted from human immunodeficiency virus proteins and lipids.

Purified Human Immunodeficiency Virus (HIV) was solubilized in octylglucopyranoside. After centrifugation, the supernatant was added to lipid-detergent mixed micelles. Formation of virosomes occurred during overnight dialysis. Centrifugation on a continuous glycerol gradient showed that envelope glycoproteins (gp120 and gp41) and matrix protein p17 but not core protein p25 were associated to virosomes. Proteolytic treatment of virosomes indicates that gp120 is oriented toward the outside as in the virus particles, whereas p17 protein is anchored on both sides of the liposomal membrane. Virosomes are spherical vesicles with approximately the size of the virus as shown by electron microscopy.

A new type of functional VIP receptor has an affinity for helodermin in human SUP-T1 lymphoblasts.

A new type of VIP receptor was characterized in human SUP-T1 lymphoblasts. The order of potency of unlabeled peptides, in the presence of [125I]helodermin, was: helodermin(1-35)-NH2 = helodermin(1-27)-NH2 greater than helospectin greater than VIP = PHI greater than [D-Ser2]VIP greater than [D-Asp3]VIP greater than [D-His1]VIP greater than or equal to [D-Ala4]VIP greater than or equal to secretin = GRF. This specificity was distinct from that of all VIP receptors described so far in that: (i) the affinity for helodermin (Kd = 3 nM) was higher than that of VIP (Kd = 15 nM) and PHI (Kd = 20 nM); and (ii) position 4 played an important role in ligand binding. The labeled sites were likely to be functional receptors as adenylate cyclase in crude lymphoblastic membranes (200-10,000 x g pellets) was stimulated by peptides, in the presence of GTP, with the following order of potency: helodermin(1-35)-NH2 greater than helodermin(1-27)-NH2 greater than helospectin = VIP = PHI.

Glycosylation inhibitors block the expression of LAV/HTLV-III (HIV) glycoproteins.

The glycosylation inhibitors 2-deoxy-D-glucose (2-dGlc) and, to a lesser extent, beta-hydroxynorvaline blocked the formation of syncytia in HIV (LAV/HTLV-III)-infected cells. Using monospecific polyclonal antibodies against recombinant envelope proteins gp110 and gp41 or monoclonal antibodies against env gp110, we could demonstrate a marked reduction in the immunoreactivity of these antigens in HIV-infected cells exposed to the glycosylation inhibitors. There was concomitant accumulation of core proteins p15 and p24, as shown by a solid phase radio-immunoassay, and a decreased oligosaccharide synthesis of env proteins, as monitored by the incorporation of [6-3H]GlcNAc. The reverse transcriptase was not affected by the compounds. Glycosylation inhibitors may be considered for the chemotherapy of AIDS or AIDS-related complex, or chemoprophylaxis of HIV-positive individuals.

Markers of HTLV-III in patients with end stage renal failure treated by haemodialysis.

Patients and members of staff from a haemodialysis unit were tested for markers of infection with human T cell lymphotropic virus type III (HTLV-III), the virus associated with the acquired immune deficiency syndrome (AIDS). An enzyme linked immunosorbent assay showed eight of 100 patients to have antibodies to HTLV-III. In five of these patients past or present infection with HTLV-III was confirmed by Western blot analysis or detection of HTLV-III antigens in lymphocyte cultures, or both. Investigation of other risk factors for AIDS showed that the putative source of HTLV-III was unrelated to dialysis in two patients whereas blood transfusion was the most likely cause of contamination in the others. No member of staff gave a positive result in the enzyme linked immunosorbent assay. Nosocomial transmission of HTLV-III seems unlikely if precautions similar to those recommended for the control of hepatitis B infection are applied.

Bovine leukemia virus-related antigens in lymphocyte cultures infected with AIDS-associated viruses.

An earlier finding that lymphocytes from African patients with the acquired immune deficiency syndrome (AIDS) react with rabbit antiserum to purified antigens of bovine leukemia virus (BLV) prompted a study of the possible cross-reactions between a BLV-infected ovine cell line and human lymphocytes inoculated with a strain of lymphadenopathy syndrome-associated virus (LAV). A solid-phase radioimmunoassay was used to detect antigenic markers of the retroviruses. Crude extracts from short-term cultures of lymphocytes infected with LAV bound rabbit antisera to the LAV glycoprotein gp13 (molecular weight 13,000) and the BLV proteins p24 and gp51, but did not bind antibodies to the p24 of human T-cell leukemia virus type I (HTLV-I). Antiserum to LAV gp13 reacted with an ovine cell line producing BLV but also weakly with virus-free ovine cells. Lymphocyte cultures from four African patients with AIDS expressed BLV-related antigens within 6 to 10 days of culture, at the moment when particle-bound reverse transcriptase was produced. BLV-related antigens were induced in lymphocyte cultures from healthy individuals by addition of filtered supernatant or irradiated cells of the original culture. The antisera to BLV used in this study may prove useful for the detection of AIDS-associated viruses in short-term cultures of lymphocytes from AIDS patients or their contacts.

Production of monoclonal antibodies against HTLV-I proteins recognizing surface epitopes of live infected cells.

The proteins of HTLV-I virus, the only human retrovirus implicated in the etiopathogenesis of the T-cell leukemia, were previously studied with the use of monoclonal antibodies. Different groups have produced specific monoclonal antibodies that recognized the core proteins of the virus p19 and p24 and in one case a monoclonal specific of a gp21 protein. All these antibodies were shown to react with the virus-producing fixed cells. We also developed a battery of antibodies against p24 and p19 antigens from HTLV-I virus but the anti-p19 monoclonal antibodies happened to recognize epitopes exposed on the surface of live HTLV-I infected cells. One of the monoclonal antibody that bound to the surface of HTLV infected cells recognized a protein of an approximate mol. wt of 33 kilodalton (KD). These antibodies that bound to the live cells should be precious tools to study leukemic patients with T-cell leukemia and the evolution of the live cell populations during the course of the disease.

Monoclonal antibodies against baboon endogenous virus and against host cell antigens.

Monoclonal antibodies were produced by murine hybridomas after immunization with semipurified baboon endogenous virus. In a solid-phase radioimmunoassay, two antibodies (F12-9 and B9-18) reacted with viral antigen only. The antibodies A6-8 and C9-12 also reacted with virus-producing cells but not with control cells, whereas antibodies E4-6 and D12-2 bound to virus-free cells as well. The cytofluorometry technique confirmed these results and showed a competition between antibodies A6-8 and C9-12 for binding to virus-producing cells as well as a competition between antibodies D12-2 and E4-6 for binding to virus-free human cells. An immune precipitation assay with disrupted virions indicated that antibodies A6-8, B9-18, and C9-12 were directed against the gp70 glycoprotein, and that antibody F12-9 reacted with a viral antigen with a molecular weight of 18,000. The syncytia induced in RSa cells by baboon molecular weight of 18,000. The syncytia induced in RSa cells by baboon endogenous virus could be inhibited either when antibody A6-8 or C9-12 was combined to the virus or when the RSa cells were treated with the anticellular antibody D12-2 or E4-6. These two effects were not observed with Mason-Pfizer virus. Thus, of three antibodies with specificities for viral gp70, two (A6-8 and C9-12) were directed at viral sites responsible for syncytium formation. Another antiviral antibody (F12-9) reacted with a protein of unknown function with a molecular weight of 18,000. The two anticellular antibodies were directed at similar or neighboring epitopes, which may be situated within the receptor to the virus.