HIV-gp160 modulates differentially the production in vitro of IgG, IgA and cytokines by blood and tonsil B lymphocytes from HIV-negative individuals.

HIV1-gp160 holds promises in anti-HIV vaccinal strategies. However, this molecule has been described to exhibit superantigenic activities. The present study aimed at examining the effect(s) of HIV1-gp160 on human B cells and in particular on B cells originating from HIV- donors. We purified human B cells of various origins, i.e. from blood and from tonsils (representing a mucosal-type origin), and we tested these cells (stimulated with a polyclonal B cell activator, interleukin (IL)-2 and IL-10 as cytokines, and recombinant HIV1-gp160) for the production of IgG and IgA in an in vitro model. Gp160 induced significantly less total IgG by blood - but not tonsil-originating - B cells and did not affect total IgA production. Further, HIV1-gp160 up-regulated IL-2-, IL-4- and IL-10-mRNA levels in stimulated blood B cells (these cytokines are known to be active on B cell activation and differentiation). Interestingly, HIV1-gp160 also up-regulated IL-1beta-, transforming growth factor (TGF)-beta-, interferon (IFN)-gamma- and IL-12-mRNA levels in stimulated mucosal-type, tonsil-originating, B cells. As these latter cytokines are involved in proinflammatory activities, HIV-gp160 delivery at the mucosal sites would be compatible with an adjuvant activity.

Immunogenicity of a recombinant human immunodeficiency virus (HIV)-canarypox vaccine in HIV-seronegative Ugandan volunteers: results of the HIV Network for Prevention Trials 007 Vaccine Study.

In the first preventative human immunodeficiency virus (HIV) vaccine study to be carried out in Africa, 40 HIV-seronegative Ugandan volunteers were randomly assigned to receive a canarypox vector containing HIV-1 clade B (env and gag-pro) antigens (ALVAC-HIV; n = 20), control vector containing the rabies virus glycoprotein G gene (n = 10), or saline placebo (n = 10). Cytotoxic T lymphocyte activity against target cells expressing clade A, B, and D antigens was assessed using standard chromium-release and confirmatory interferon-gamma enzyme-linked immunospot (ELISPOT) assays. Neutralizing antibody responses to cell line-adapted strains and primary isolates in all 3 clades were also tested. Twenty percent of vaccine recipients generated detectable cytolytic responses to either Gag or Env, and 45% had vaccine-induced HIV-specific CD8(+) T cell responses, as measured by the ELISPOT assay. In contrast, only 5% of the control group had vaccine-specific responses. Neutralizing antibodies against primary and laboratory-adapted HIV-1 clade B strains were seen in 10% and 15% of vaccine recipients, respectively, but responses against clades A and D were not detected. Although the immunogenicity of this clade B-based vaccine was low, ALVAC-HIV elicited CD8(+) T cell responses with detectable cross-activity against clade A and D antigens in a significant proportion of vaccine recipients.

Polymorphisms in HLA class I genes associated with both favorable prognosis of human immunodeficiency virus (HIV) type 1 infection and positive cytotoxic T-lymphocyte responses to ALVAC-HIV recombinant canarypox vaccines.

Carriers of certain human leukocyte antigen class I alleles show favorable prognosis of human immunodeficiency virus type 1 (HIV-1) infection, presumably due to effective CD8(+) cytotoxic T-lymphocyte responses, but close relationships between class I variants mediating such responses to natural and to vaccine HIV-1 antigen have not been established. During 6 to 30 months of administration and follow-up in trials of ALVAC-HIV recombinant canarypox vaccines, cells from 42% of 291 HIV-1-negative vaccinated subjects typed at class I loci responded to an HIV-1 protein in a lytic bulk CD8(+) cytotoxic T-lymphocyte assay. By 2 weeks after the second dose, higher proportions of vaccinees carrying one of two alleles consistently associated with slower progression of natural HIV-1 infection reacted at least once: B*27 carriers reacted to Gag (64%; odds ratio [OR] = 10.3, P = 0.001) and Env (36%; OR = 4.6, P = 0.04), and B*57 carriers reacted to Env (44%; OR = 6.6, P < 0.05). By 2 weeks after the third or fourth dose, B*27 carriers had responded (two or more reactions) to Gag (33%; OR = 4.4, P < 0.05) and B*57 carriers had responded to both Gag (39%; OR = 5.3, P = 0.013) and Env (39%; OR = 9.5, P = 0.002). Homozygosity at class I loci, although conferring an unfavorable prognosis following natural infection, showed no such disadvantage for vaccine response. Individual class I alleles have not previously demonstrated such clear and consistent relationship with both the clinical course of an infection and cellular immunity to a vaccine against the infectious agent. This proof of principle that class I an alleles modulate both processes has implications for development of HIV-1 and presumably other vaccines.

Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MalE protein.

The highly conserved amino acid sequence ELDKWA of HIV-1 gp41 has been inserted into Escherichia coli MalE protein which had been shown to be an adequate carrier to present foreign epitopes to the immune system. We first investigated whether eight different permissive sites of MalE are able to tolerate an insertion of 7-50 residues encoding this epitope. Secondly, antigenicity of the epitope inserted in MalE protein was estimated from monoclonal antibody 2F5 binding analysis using the BIAcore(R) technology and its immunogenicity in mice was measured as the ability of hybrid proteins to elicit antibodies against a synthetic peptide containing this epitope. This study revealed a good correlation between the antigenicity of the inserted epitope and its immunogenicity. Increasing the length of the inserted epitope, as well as inserting multicopies of this epitope increased both its antigenicity and immunogenicity. However, none of the MalE hybrid proteins tested induced anti-HIV-1 neutralizing antibodies. This study strongly suggests that the capacity of the 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented.

Immunization with recombinant canarypox vectors expressing membrane-anchored glycoprotein 120 followed by glycoprotein 160 boosting fails to generate antibodies that neutralize R5 primary isolates of human immunodeficiency virus type 1.

Antibodies generated by candidate HIV-1 vaccines in a phase I clinical trial were assessed for neutralizing activity with a panel of eight well-characterized, genetically diverse clade B primary isolates having an R5 phenotype. The vaccines consisted of one of three different recombinant canarypox vectors expressing membrane-anchored HIV-1(MN)gp120 (ALVAC vCP205, vCP1433, and vCP1452) followed by boosting with a soluble gp160 hybrid consisting of MNgp120 and the majority of gp41 from strain IIIB. Serum samples from a subset of volunteers in each arm of the trial, containing moderate to high titers of neutralizing antibodies to HIV-1 MN, were analyzed. Competition assays with peptides revealed that the majority of neutralizing activity was specific for the MN-V3 loop. Despite MN-specific neutralization titers that sometimes exceeded 1:500, no neutralization of primary isolates was detected and, in some cases, mild infection enhancement was observed. In addition, little or no neutralization of the HIV-1 IIIB heterologous T cell line-adapted strain of virus was detected. These results reinforce the notion that monovalent HIV-1 ENV is a poor immunogen for generating cross-reactive neutralizing antibodies.

Safety and immunogenicity of a live recombinant canarypox virus expressing HIV type 1 gp120 MN MN tm/gag/protease LAI (ALVAC-HIV, vCP205) followed by a p24E-V3 MN synthetic peptide (CLTB-36) administered in healthy volunteers at low risk for HIV infection. AGIS Group and L'Agence Nationale de Recherches sur Le Sida.

A live recombinant canarypox vector expressing HIV-1 gpl20 MN tm/gag/protease LAI (ALVAC-HIV, vCP205) alone or boosted by a p24E-V3 MN synthetic peptide (CLTB-36) was tested in healthy volunteers at low risk for HIV infection for their safety and immunogenicity. Both antigens were well tolerated. ALVAC-HIV (vCP205) induced low levels of neutralizing antibodies against HIV-1 MN in 33% of the volunteers. None of them had detectable neutralizing antibodies against a nonsyncytium-inducing HIV-1 clade B primary isolate (Bx08). After the fourth injection of vCP205, CTL activity was detected in 33% of the volunteers and was directed against Env, Gag, and Pol. This activity was mediated by both CD4+ and CD8+ lymphocytes. On the other hand, the CLTB-36 peptide was poorly immunogenic and induced no neutralizing antibodies or CTLs. Although the ALVAC-HIV (vCP205) and CLTB-36 prime-boost regimen was not optimal, further studies with ALVAC-HIV (vCP205) are warranted because of its clear induction of a cellular immune response and utility as a priming agent for other subunit antigens such as envelope glycoproteins, pseudoparticles, or new peptides.

Correlation between arachidonic acid oxygenation and luminol-induced chemiluminescence in neutrophils: inhibition by diethyldithiocarbamate.

Neutrophils from allergic subjects were hypersensitive to stimulation by low calcium ionophore concentration (0.15 microM), resulting in an increased formation of leukotriene B4 (LTB4), 5S-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5-HETE), and other arachidonic acid metabolites through the 5-lipoxygenase pathway. In parallel, luminol-dependent chemiluminescence was also higher in neutrophils from allergic patients at the basal state and after stimulation by calcium ionophore, revealing an enhancement of radical oxygen species and peroxide production. The activity of glutathione peroxidase, the main enzyme responsible for hydroperoxide reduction, was lowered in these cells. Diethyl-dithiocarbamate (DTC) induced a concentration-dependent decrease in chemiluminescence and arachidonic acid metabolism after neutrophil stimulation. These data show that the elevation of arachidonic acid metabolism in neutrophils from allergic patients is strongly correlated with oxidative status. This elevation may be the consequence of an increased cellular hydroperoxide known to activate 5-lipoxygenase (5-LOX) activity and/or an increased arachidonic acid availability, due either to phospholipase A2 (PLA2) activation or inhibition of arachidonate reesterification into phospholipids. Lowering this oxidative status was associated with a concomitant decrease of this metabolism. Our results suggest that the effect of DTC may be the consequence of an inhibition of peroxyl radical and cellular lipid hydroperoxide production. Thus, DTC may modulate arachidonic acid metabolism in neutrophils by modulating the cellular hydroperoxide level.

Immunological parameters during treatment with ditiocarb (Imuthiol).

OBJECTIVES: To examine the effect of ditiocarb (DTC) treatment on immunological parameters of HIV infection. Immunophenotyping included CD4+ T-cell counting and the analysis of activation markers on CD8+ T cells. Anti-CD3-induced proliferation and anti-CD3-mediated cytotoxicity were monitored as indexes of T-cell function. In addition to the clinical evolution, HIV antigen and anti-p24 levels were monitored during treatment. DESIGN: In this double-blind, placebo-controlled study, 50 HIV-seropositive patients belonging to all clinical disease stages were randomized to treatment with DTC or placebo and followed for 4 months. METHODS: Immunophenotyping on whole blood was performed by flow cytometry, using combinations of anti-CD8 with anti-CD4, anti-HLA-DR, anti-CD38, anti-CD45RO and anti-CD57. Patient lymphocytes were freshly assayed for cytolytic capacity against OKT3-coated targets. T-cell proliferation was measured after 3 days of OKT3-stimulation. RESULTS: No effect was observed on CD4 and CD8+ T-cell counts or on CD8+ T-cell activation markers, except for a selective increase in HLA-DR expressing CD8 cells in the DTC-treated group. Decline in anti-CD3-induced T-cell proliferation and rise in anti-CD3-mediated T-cell cytotoxicity were observed in the DTC and placebo groups. No effect on HIV antigen and anti-p24 antibody titres was observed. The incidence of clinical complications was similar in each group. CONCLUSION: No beneficial immunomodulatory effect of DTC was demonstrated.

Diethyldithiocarbamate (ditiocarb sodium) effect on arachidonic acid metabolism in human mononuclear cells. Glutathione peroxidase-like activity.

Diethyldithiocarbamate (DTC), a thiol delivery agent, has been shown to significantly reduce the frequency of primary opportunistic infections in HIV-infected patients. This therapeutic effect has been related to the capacity of DTC to reverse the deleterious effects of the oxidative stress occurring in HIV infection. The influence of DTC on the oxygenated metabolism of arachidonic acid (AA) was investigated in mitogen-stimulated human peripheral blood mononuclear cells (PBMC). Upon incubation with PBMC previously labelled with [3H]AA, Concanavalin A (Con A) markedly increased cyclooxygenase and lipoxygenase activities, within 30 min, as judged by thromboxane B2 (TxB2) and hydroxyeicosatetraenoic acid (HETE) production. Con A activation of [3H]AA platelets also increased 12-HETE production but did not induce any TxB2 synthesis. Micromolar concentrations of DTC, added simultaneously with the mitogen, significantly enhanced the synthesis of HETEs above the Con A-induced level while TxB2-induced synthesis was inhibited but only at DTC concentrations higher than 50 microM. In the presence of nordihydroguaiaretic acid, a lipoxygenase inhibitor, which inhibited the Con A-induced synthesis of HETEs by 78%, DTC no longer stimulated HETE production above the Con A-induced level. Reverse phase HPLC analysis showed that Con A increased the PBMC production of 5-, 12- and 15-HETEs. In the presence of 5 microM DTC, 5-HETE production was entirely suppressed whereas the 15-HETE level was markedly enhanced, 12-HETE production by the contaminating platelets remained unchanged. In vitro experiments indicated that DTC alone did not significantly influence 15-hydroperoxyeicosatetraenoic (15-HPETE) production by the soybean 15-lipoxygenase but, in the presence of added reduced glutathione, DTC markedly reduced 15-HPETE into 15-HETE. In addition, DTC was able to substitute for cellular extract in the glutathione peroxidase (GPx) assay system. Taken together, these results indicate that DTC, through its "GPx-like" activity is able to modify the lipoxygenase cascade. Its ability to selectively reduce 15-HPETE known to stimulate immunosuppressive T-cells might help to explain its positive regulatory effect upon the immune system.

Intravenous gamma-globulin in myasthenia gravis: interaction with anti-acetylcholine receptor autoantibodies.

Clinical improvement has been observed in myasthenia gravis patients treated by intravenous immunoglobulin (IVIg). In order to investigate the mechanism of action of these IVIg, we looked for an in vitro interaction between IVIg and the anti-acetylcholine receptor autoantibodies. Significant inhibition by IVIg of anti-acetylcholine receptor autoantibody activity from 30 MG sera was observed and binding of anti-acetylcholine receptor autoantibodies on IVIg was found for four of five myasthenia gravis sera. These observations suggest that IVIg contains Ig directly binding to and inhibiting pathogenic autoantibodies.

[Lysosomal enzyme deficiency: in vitro correlation of deficient cells using a conditioned medium or by co-culturing with non-deficient cells]. / Déficience en enzyme lysosomiale: correction in vitro de cellules déficitaires par du milieu conditionné ou par des co-cultures avec des cellules non déficitaires.

We demonstrate that lysosomal enzyme (alpha-L-fucosidase) can enrich deficient fibroblasts, with purified enzyme brought by the medium, or with an enzyme supply by various cell sources. The co-culture systems lead to a deficient cell correction, whatever donor cells are lymphocytes or lymphoblastoid cells. This correction arise only with alive cells, and is strongly inhibited by mannose-6-phosphate. Our results do not support the hypothesis that cell to cell contact independently of mannose-6-phosphate binding site is necessary for transfer of lysosomal enzyme from lymphocytes to fibroblasts. We suggest that the neighbourhood of cells leads to a phosphorylated precursor increase in the pericellular area, which creates an enzyme stabilizing effect favourable at its incorporation.

Prevalence of CMV ELISA antibody: titer and neutralizing activity in intramuscular immune globulin of placental origin.

The treatment of human CMV infection in transplant recipients by intravenous or intramuscular administration with high doses of hyperimmune or normal Ig showed inconsistent results. In fact, all hyperimmune Ig preparations were produced from high anti-CMV Ab titer plasma selected either by ELISA, complement fixation, or indirect hemagglutination test. These tests may not investigate the neutralizing activity of antibodies (Ab). We, therefore compared the ELISA titers with the neutralizing Ab titers in various normal Ig preparations from plasma or placenta and for intramuscular or intravenous use. Hyperimmune plasmatic Igs were also included. All the preparations were tested at a 5% dilution. We report the following data: (1) There is a poor correlation between ELISA and neutralization test. (2) The Igs of placental origin have significantly higher ELISA anti-CMV Ab titers than plasmatic normal Igs. They show the same neutralizing activity. (3) When compared with the normal intravenous Ig, normal intramuscular Igs show significantly higher titers with both techniques (P less than .0001). (4) Normal intramuscular Igs from placenta offer similar ELISA Ab titers and similar or higher neutralizing activity than hyperimmune Igs purified from plasma. It would be worth using Igs with high neutralizing activity in CMV prophylaxis in order to correlate in vitro tests and in vivo protection. In this respect, normal intramuscular Igs from placenta might be challenged in a clinical trial.

Specific adsorption of circulating antibodies by extracorporeal plasma perfusions over antigen coated collagen flat-membranes: applications to systemic lupus erythematosus.

A new biocompatible collagen membrane, chemically activated in order to bind several antigens such as albumin or DNA, was successfully used to perform extracorporeal immunoadsorptions (IA) in vivo. First, immunoadsorbent devices composed of albumin-coated membranes, in a multilayer arrangement, were able to remove a large amount of anti-albumin antibodies from dogs actively immunized against heterologous albumin, during a single hemoperfusion. Second, DNA-coated membranes, in the same disposition, were used in 5 SLE-patients for serial plasma perfusions, which resulted in a significant decrease of anti-DNA antibodies (DNA-Ab) and immune complexes in serum. As previously observed in dogs, IA were sometimes followed by a rebound of DNA-Ab levels, which occurred either between successive IA (fast rebound) or 2 weeks after the series of IA, only in patients receiving the lowest steroid dosages. The main factors involved in such a rebound are probably a reequilibration of the antibody pool between the intra- and extravascular spaces, for the "fast" component of this rebound, and a de novo antibody synthesis induced by antibody removal only. Indeed, the hypothesis of a direct stimulation by either antigen release or contact between lymphocytes and antigen linked on the membrane, were ruled out by the results of in vitro studies and of plasma perfusion alone. These results support the usefulness of antigen-coated collagen membranes for in vivo IA, which represent a new approach to the treatment of autoantibody mediated diseases and to the investigation of the factors governing antibody synthesis.

DNA immobilized onto an acyl-azide derivative of collagen membranes for use as immunoadsorbent.

Single- and double-stranded DNA were immobilized on films of highly polymerized collagen by a covalent process. The coupling was efficient at an acidic pH with an optimum at pH 5, while preventing the collagen film from any damage. In addition, no leakage occurred, so it was possible to use this DNA-coated collagen film as an immunoadsorbent. Therefore the findings reported here suggest that the acyl-azide procedure is also suitable for DNA binding on a proteinic support. Promising results in specific clearance of DNA antibodies were obtained in vitro.

New method for the specific clearance of antibodies using antigens linked to a collagen film.

A new type of immunoadsorbent, derived from a commercial haemodialysis module, has been designed. The dialysis membranes were replaced by bovine collagen membranes on which a given antigen had been linked by covalent binding. First, these membranes were tested in vitro: they were placed in contact with a given volume and concentration of antiserum to define their capacity to retain antibodies. Second, they were stacked in a modified haemodialyzer for antibody extraction ex vivo in dogs: the blood of immunized animals was passed over the immunoadsorbent and recirculated after either partial or total removal of antibodies. The degree of purification is related to the area of the membranes used and to the volume of blood to be purified.