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A major involvement of IFNα in the etiopathogenesis of systemic lupus erythematosus has been suggested by clinical observations, including the increase of serum levels of this cytokine in patients with active disease. Supporting this hypothesis, we have shown that expression of IFNα from a recombinant adenovirus (IFNα Adv) precipitates lupus manifestations in genetically susceptible New Zealand Black (NZB) × New Zealand White (NZW)F1mice (NZB/W) but not in BALB/c mice. In the present investigation, we have prepared an IFNα immunogen, termed IFNα kinoid, which, appropriately adjuvanted, induces transient neutralizing antibodies (Abs) but no cellular immune response to the cytokine and without apparent side effects. Using this preparation, we also showed that, in kinoid-vaccinated NZB/W mice, lupus manifestations, including proteinuria, histological renal lesions, and death triggered by IFNα Adv challenge were delayed/prevented as long as an effective threshold of anti-IFNα inhibitory capacity was present in the serum.

The abnormal cytokine release in the stromal microenvironment of pathologic tissues, contributes to the pathogenesis of viral infections such as AIDS, cancer and auto-immune diseases. Neovacs developed therapeutic vaccines, named Kinoids, which induce anti-cytokine Antibodies. Kinoids are non toxic but immunogenic cytokine derivatives. Kinoid immunizations induce high titre of neutralizing Abs to the corresponding cytokine, is well tolerated and experimentally effective. In transgenic mice expressing huTNFalpha, the TNFalpha kinoid decreases clinical signs of Rheumatoid Arthritis and in mice challenged with syngenic CT26 tumor cell line huVEGF kinoid inhibits lung metastases. After validation by clinical trials, kinoid vaccines could represent a second generation of specific immune therapy to be used to combat ectopic cytokines.

Objective: To evaluate the effect in mice with arthritis of active anti-TNFα immunotherapy based on a keyhole limpet hemocyanin-human TNFα heterocomplex (hTNFα kinoid or TNFK) adjuvanted in incomplete Freund adjuvant. Immunotherapy was evaluated also with methotrexate. Methods: Human TNFα-transgenic mice received hTNFα kinoid with or without methotrexate. Follow-up ranged from 6 weeks (short-term) to 17 weeks (long-term). Arthritis was evaluated clinically and histologically. Monitoring included titration of anti-hTNFα antibodies by ELISA and neutralization assay. Results: Vaccination with TNFK was associated with rapid-onset, long-lasting protection. Long-term results showed significantly milder arthritis in vaccinated animals than in control animals at the peak of the disease. Vaccination was followed by resolution of the clinical evidence of arthritis, contrasting with severe progressive arthritis in the control group. Histological improvements with decreased inflammation and destruction were noted in all immunized groups, even after the shortest follow-up (6 weeks). High titers of neutralizing anti-hTNFα antibodies were detected as early as the fifth post-immunization week and persisted over time. Methotrexate given concomitantly with the vaccine did not influence either the effect on arthritis or the anti-hTNFα antibody titers. Conclusion: Anti-cytokine induction of autoimmune protection against chronic hTNFα overproduction is an efficient alternative to TNFα blockade in experimental arthritis and can be achieved using a TNFK vaccine.

Summary The need to modify tumor cells in order to render them more “immunogenic” was based on the assumption that normal, nonmodified tumor cells are non or weakly immunogenic and as such are unable to raise an efficient protective immune response. Various methods for “xenogenization” (modification of tumor cells) were suggested: induction of new foreign antigens, treatment with either chemicals or enzymes and use of mutagens. Xenogenized tumor cells by their coupling to proteins, and use of chemicals like DTIC (5-[3,3-dimethyl-1-triazeno]-imidazole-4-carboxamide), TZC (8-carbamoyl-3-methyl-imidazo[5,1-d]-1,2,3,5-tetrazin-4 [3H]-one 8-carbamoyl-3-[2-chloroethyl]imidazole [5,1 -d]-1,2,3,5-tetrazin-4[3H]-one) and antiemetic drugs, were tested in experimental models of murine leukemia. Non-tumorigenic clones, xenogenization with DNA hypomethylating agents, aryl-triazine derivatives and DTIC were evaluated for their induction of protective immune response in murine lymphoma. Murine plasmacytoma cells were used for immunization after treatment with glutaraldehyde. Viral modifications of tumor cells were evaluated for their ability to induce a protective tumor response in model systems of rat fibrosarcoma, liver metastatic rat tumor cells, lymphoid tumor cells and hamster tumor cells. In the case of human cancer, attempts were reported to use DNP-conjugated melanoma cells, mutagenic triazine compounds, an autologous colon tumor cell bacillus Calmette-Guerin (BCG) vaccine and genetically engineered vaccines for immunization. The general conclusion drawn from experimental tumor models and for human cancer is, that although modified tumor cells were found to be partially effective in experimental models, it is still necessary to provide more data in order to determine the effective use of xenogenized human tumor cells for immunotherapy.

HPV-16 E6 and E7 oncoproteins impair the cell cycle in human uterine cervix carcinoma cells (HUCC) by acting on p53 and retinoblastoma proteins, respectively. We recently reported that E7 related into the extracellular compartment by HUCC SiHa cells could inhibit immune T-cell response to recall and alloantigens by a mechanism involving an overproduction of the immunosuppressive IFN alpha by antigen presenting cells (APCs). In this study, we found that besides E7, E6 protein and the vascular endothelium growth factor (VEGF) were released into the SiHa cell supernatants, and we further showed that extracellular E7 but not E6 oncoprotein 1) inhibits the immune cell response to recall and alloantigens, and 2) enhances the release of angiogenic cytokines, including TNF alpha, IL-1 beta and IL-6 by macrophages and/or dendritic cells. VEGF unexpectedly released by cancer cells could also contribute to angiogenesis. Thus in HUCC the same E7 oncoprotein which contributes to controlling the cancer cell cycle has the means in its extracellular configuration to contribute to microenvironmental immunosuppressive and angiogenic processes. Neutralizing anti-E7 antibodies either passively administered or induced by active immunization could represent a new immunotherapeutic endeavour to combat the immunosuppression and/or neoangiogenesis effects of extracellular E7 protein.

This randomized, double-blind, placebo-controlled, phase II/III study was designed to evaluate safety, immunogenicity, and efficacy of an active anti-interferon-alpha (anti-IFN-alpha) vaccine in asymptomatic HIV-1-infected patients. The active immunization was aimed at inducing anti-IFN-alpha antibodies to counteract IFN-alpha overproduction. In all, 242 patients, recruited between December 1995 and July 1996 in eight centers in Europe and Israel, with CD4+ counts from 100 to 634 cells/mm3 who were receiving or not receiving antiretroviral therapy (including protease inhibitors) were randomized to receive either anti-IFN-alpha vaccine or placebo. The anti-IFN-alpha immunization regimen consisted of three priming injections delivered intramuscularly at 1-month intervals in a water-in-oil emulsion of inactivated recombinant IFN-alpha-2b (i-IFN-alpha) followed by intramuscular booster injections of i-IFN-alpha adsorbed onto calcium phosphate every 3 months. Immunogenicity to vaccine was defined as an increase of anti-IFN-alpha antibody level of more than twofold the preimmunization value. Clinical progression, changes in antiretroviral treatment, and decrease of CD4+ counts to200 cells/mm3 were considered endpoints for efficacy evaluation. Contrary to our previous experience, in which six to seven oil priming injections induced a90% response rate, the three oil-adjuvanted injections in this trial were suboptimal because only 40 of 122 vaccinees (33%) had raised anti-IFN-alpha antibody following immunization. In vaccinees, both antibody responders (AbRV) and nonresponders (AbNRV), the tolerance to the vaccine was good and was without evidence of significant safety concerns. During the course of the trial, 62% of vaccine responders, 64% of nonresponders, and 63% of placebo patients elected to add protease inhibitor-containing regimens as new treatment guidelines were established, resulting in a marked decrease in clinical and laboratory progression such that the expected endpoints of the study could not be achieved and further follow-up was halted. Despite the unexpectedly low immunogenicity and fewer than expected endpoints, anti-IFN-alpha vaccine recipients, in comparison with placebo recipients, showed a lower rate of disease progression, nonelective treatment changes, and/or CD4+ count decrease to200 cells/mm3, but the difference was not statistically significant. Nevertheless, the subgroup of patients immunized to IFN-alpha who experienced a rise in anti-IFN-alpha antibodies had a significantly lower rate of occurrence of HIV-1-related events and of any combination of the endpoints compared with those of either placebo patients or vaccinees who failed to develop anti-IFN-alpha antibodies, the latter two groups behaving similarly. Further studies of this approach are warranted because these data suggest a beneficial effect of this adjuvant approach.

The Duffy Antigen Receptor for Chemokines (DARC) belongs to a family of erythrocyte chemokine receptors that bind C-X-C and C-C chemokines such as interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1) and regulated-on-activation, normal T cell-expressed and -secreted (RANTES), but not macrophage inflammatory protein 1 alpha (MIP-1 alpha) or MIP-1 beta. DARC has also been identified to a receptor for malaria parasites Plasmodium vivax and Plasmodium knowlesi. In the present study, we show that HIV-1 binds to RBCs from Caucasian individuals via DARC making RBCs able to transmit HIV to peripheral blood mononuclear cells (PBMCs). Furthermore, binding of HIV-1 particles to RBCs is inhibited by treating these cells with recombinant RANTES, but not with recombinant MIP-1 alpha prior to their incubation with HIV-1. This finding suggests that RBCs may function as a reservoir for HIV-1 or as a receptor for the entry of HIV-1 into CD4-cell subsets as well as neurons or endothelial cells.

Summary Extracellular Tat can act as a viral toxin on uninfected cells of different tissues, including the CNS and the immune system, thus in order to immunize humans against Tat we have prepared a biologically inactivated but immunogenic Tat (Tat Toxoid). Tat Toxoid is not toxic in mice even at high doses. It triggers high levels of specific Tat Abs in the mouse and rabbit. Furthermore, in humans Tat Toxoid immunization was safe and induced in seronegatives persistent high levels of Tat Abs and in immunodeficient patients a significant rise of these specific Abs. Facing acute HIV-1 infection, the presence of high level of circulating Tat Abs promoted by Tat Toxoid vaccine should prevent Tatinduced immunosuppression and allow anti-HIV-1 cellular response to develop. As a consequence, early release of β-chemokines could enhance host resistance towards HIV-1, and, in infected people, inhibit viral replication and evolution towards AIDS.

Exposure to HIV type 1 (HIV-1) does not usually lead to infection. Although this could be because of insufficient virus titer, there is now abundant evidence that some individuals resist infection even when directly exposed to a high titer of HIV. This protection recently has been correlated with homozygous mutations of an HIV-1 coreceptor, namely CCR5, the receptor for the β-chemokines. Moreover, earlier results already had shown that the same chemokines markedly suppress the nonsyncitial inducing variants of HIV-1, the chief virus type transmitted from person to person. CCR5 mutation, as a unique mechanism of protection, is, however, suspect because HIV-1 variants can use other chemokine receptors as their coreceptor. Moreover, recent results have established that infection can indeed sometimes occur with such mutations. Here, we report on transient natural resistance over time of most of 128 hemophiliacs who were inoculated repeatedly with HIV-1-contaminated Factor VIII concentrate from plasma during 1980–1985 before the development of the HIV blood test. Furthermore, and remarkably, 14 subjects remain uninfected to this date, and in these subjects we found homozygous CCR5 mutations in none but in most of them overproduction of β chemokines. In vitro experiments confirmed the potent anti-HIV suppressive effect of these chemokines.

Circulating interferon (IFN) was investigated in HIV-1 seropositive patients by measuring the IFN alpha antiviral effect in the serum. While serum of healthy seronegative individuals exhibits an antiviral effect, not due to IFNs, considered as background, serum of seropositive patients showed an additional antiviral effect due to the abnormal presence of IFN alpha. Increased titers of IFN alpha were found in the course of the HIV infection and seemed to correlate with the evolution of AIDS disease. Furthermore, patients immunized against IFN alpha had both stabilized CD4 cell count and decreased IFN alpha in their serum. HIV-1-infected patients also exhibited higher titers of natural anti-IFN antibodies than seronegative controls and the level of specific antibodies (Abs) markedly increased in immunized patients. Finally, serum from immunized patients, when compared to seronegative controls, exhibits an interferon neutralizing capacity.

Since the immune system is impaired in the course of HIV-infection, the purpose of any AIDS vaccine therapy should be the restoration in the patient of an adequate immunocompetence to enable him to respond to the antigenic stimulus represented by the virus. In the present investigation we have shown the antiproliferative action on activated T-cells in culture of: sera taken from HIV-infected, but not seronegative individuals; T lymphocytes taken from seronegative subjects and infected in vitro with HIV but not non infected cells; native alpha-IFN and the time-dependent inactivation of this activity by formaldehyde treatment of alpha-IFN. Thus is confirmed the major contribution provided by alpha-IFN to the immunosuppression occurring in the course of HIV-infection. These results also strongly support the new AIDS vaccine therapy strategy based on the administration to HIV-infected patients of inactivated, but still immunogenic alpha-IFN. To the alpha-IFN treatment could also be combined the administration of fixed autologous suppressive cells. The induction of gamma-IFN in addition to alpha-IFN production by stimulation of cells from healthy donors with gp120 should encourage the use of a vaccine combining both inactivated alpha-IFN and gamma-IFN. On the other hand, the IL-12 cytokine with its potential to restore compromised cell-mediated functions associated with HIV infection should also be a valuable adjuvant treatment.

The purification and analysis of IgGs from sera of HIV-1-infected and non infected individuals are reported. The effect of antibodies purified from sera of infected individuals on antigen-induced T cell proliferation was investigated in relation to their possible involvement in an autoimmune reaction in AIDS, in view of the previously unravelled striking peptide similarities between HIV-1 gp120 and the immunoregulatory CD4 and Fas molecules. However, our data do not allow definite conclusions to be drawn. The necessity of purifying antibodies against specific peptides to show their direct effect on T-cell activation is further stressed.

Four cardinal immune disorders interacting with each other may promote the progressive T cell depletion and immunosuppression characterizing AIDS. Immune activation of HIV-1 infected T4 cells leads to virus release and premature cell death. Both virus release with its resulting viral load and dead cells are the source of gp120 stimulus. Anergy of non-infected CD4 cells, resulting in cytokine dysregulation may be promoted by impairing the CD4-MHC interaction during CD4 cell activation either directly through the SLWDQ pentapeptide identity with the CD4 molecule and the CD4 binding region or through a gp120-induced autoimmune reaction to CD4. Overproduction of IFNα, the known antiproliferative and cytolytic cytokine may promote in a paracrine manner to neighbouring cells the immunosuppression generated by the lack of IL2 secretion following CD4 cell anergy. Apoptosis of activated non infected T cells could be induced by effector components of the sutoimmune reaction (CTL, Lymphotoxins or Abs?) directed towards the 2 consensus gp120 sequence identity/similarity (INCTR and FYCNST) shared with the APO/Fas molecule. These two sequences are known as immunodominant sites of the gp120. Furthermore, IFNa overproduction may also render circulating memory T cells competent to apoptosis by upregulating the cascade of metabolic events leading to programmed cell death.

Immune suppressive activities exerted by regulatory T-cell subsets have several specific functions, including self-tolerance and regulation of adaptive immune reactions, and their dysfunction can lead to autoimmune diseases and contribute to AIDS and cancer. Two functionally distinct regulatory T-cell subsets are currently identified in peripheral tissues: thymus-developed natural T regulatory cells (nTregs) controlling self-tolerance and antiinflammatory IL-10–secreting type 1 regulatory T cells (Tr1) derived from Ag-stimulated T cells, which regulate inflammation-dependent adaptive immunity and minimize immunopathology. We establish herein that cell contact-mediated nTreg regulatory function is inhibited by inflammation, especially in the presence of the complement C3b receptor (CD46). Instead, as with other T-cell subsets, the latter inflammatory conditions of stimulation skew nTreg differentiation to Tr1 cells secreting IL-10, an effect potentiated by IFN-α. The clinical relevance of these findings was verified in a study of 152 lupus patients, in which we showed that lupus nTreg dysfunction is not due to intrinsic defects but is rather induced by C3b stimulation of CD46 and IFN-α and that these immune components of inflammation are directly associated with active lupus. These results provide a rationale for using anti–IFN-α Ab immunotherapy in lupus patients.

We have designed a computer strategy in order to detect systematically peptidic sites with the potential of interfering with the immune regulatory processes. Applying this software to HIV-1 proteins has led us to unravel a few peptidic sites which could either act directly or be the targets of an auto-immune reaction during HIV-1 infection. We previously reported that the SLWDQ pentapeptide identity with a critical site of CD4 could trigger in HIV-1 infected individuals both an humoral and a cellular autoimmune reaction. In this study, we focused on surprising similitudes unravelled by our software Automat, between HIV-1/2 and another immunoregulatory molecule, the Fas protein which is also called the apoptosis-mediating cell-surface antigen.

Since influenza morbidity and inortalily arc high in the elderly, the usefulness of vaccination has been evaluated. One hundred and twenty-six older persons aged 60–90 yr were given a vaccine consisting of the 3 antigens Singapore, Shanghai and Yamagata, the latter 2 being new antigens. Antibody levels for each of the antigens were determined by ELISA calibrated in HA units prior to and I month after vaccination. Before vaccination, antibodies to Shanghai and Singapore antigens were found in practically all sera tested, and antibodies to Yamagala antigen in only 66% of sera. After vaccination, significant levels of antibodies to all 3 antigens were found in all sera tested. Thus, it was observed that influenza vaccination proved to be effective in the elderly. The elderly should be immunized against influenza, since this is a safe and efficacious preventive measure. In addition, vaccination should limit the spreading of influenza in nursing homes.

Antiviral therapy, including antiprotease treatment, suppresses viral replication, but it does not restore the HIV-1 induced immunopathogenesis which includes IFN alpha overproduction and cellular immunosuppression. To combat HIV-1 induced immunopathogenesis, anti-IFN alpha kinoid immunization in combination with tritherapy may be beneficial to HIV-1 infected immunodeficient patients.

HAART (highly active antiretroviral therapy) suppresses but does not eradicate HIV-1 infection. However, since the antiretroviral agents used in HAART may also be toxic in the long-term, immunotherapies which correct HIV-1 immunosuppression or the cytokine dysregulation associated with it may be beneficial. In this respect, a double blind multicentric placebo-controlled phase II/III anti-IFN alpha vaccine trial has been carried out on 242 HIV-1 patients, the majority of whom were undergoing HAART treatment. In vaccinated patients (vaccinees) who responded to immunization by increased levels of IFN alpha Abs (whether under HAART or not) when compared to placebo or non-responder vaccinees, a strong correlation was found between an increased IFN alpha neutralizing capacity and the reduction of clinical manifestations.

The abnormal cytokine release in the stromal microenvironment of pathologic tissues, contributes to the pathogenesis of viral infections such as AIDS, cancer and auto-immune diseases. Neovacs developed therapeutic vaccines, named Kinoids, which induce anti-cytokine Antibodies. Kinoids are non toxic but immunogenic cytokine derivatives. Kinoid immunizations induce high titre of neutralizing Abs to the corresponding cytokine, is well tolerated and experimentally effective. In transgenic mice expressing huTNFalpha, the TNFalpha kinoid decreases clinical signs of Rheumatoid Arthritis and in mice challenged with syngenic CT26 tumor cell line huVEGF kinoid inhibits lung metastases. After validation by clinical trials, kinoid vaccines could represent a second generation of specific immune therapy to be used to combat ectopic cytokines.

A procedure for the routine and simultaneous laboratory detection of IgG antibodies produced in humans in the course of various infectious diseases is described. The procedure, based on dot-enzyme-linked immunosorbent assay (ELISA), used single nitrocellulose strips onto which several antigens were dotted in close proximity. Optimal conditions were specified that allowed the unequivocal and simultaneous detection of IgG antibodies specifically directed against Toxoplasma gondii, rubella virus, cytomegalovirus, and herpes simplex virus type 1 and type 2 antigens. This technique has proved to be simultaneously specific, sensitive, and reliable, and it has been applied to prenatal screening of sera from pregnant women. It is suggested that this technique should also be used for the screening of large numbers of sera under field trial conditions.

International audience; Tumor growth depends on blood supply, requiring the development of new vessels, and vascular endothelial growth factor (VEGF) plays a central role in neoangiogenic processes. For this reason, VEGF represents a target for the development of new therapeutic antiangiogenic molecules. Clinical trials using anti-VEGF mAbs such as bevacizumab have validated the efficacy of this therapeutic approach but have also revealed adverse effects. Here we report that a VEGF-derived immunogen, consisting of a heterocomplex of a murine (m)VEGF and keyhole limpet hemocyanin, called "mVEGF kinoid," triggered a strong Ab immune response in mice. The anti-VEGF Abs inhibited both the proliferation of human umbilical vein endothelial cells cultured in the presence of mVEGF and the binding of mVEGF to its receptor-2 Flk-1. In mVEGF kinoid-immunized BALB/c mice challenged with syngeneic CT26 colorectal tumor cells, the number and size of lung metastases were significantly decreased. In human (h)VEGF kinoid-immunized BALB/c mice, high levels of serum Abs to hVEGF were present, and purified IgG from these mice decreased by > or =50% the tumor growth of human A673 rhabdomyosarcoma cells and HT29 colon carcinoma xenografted in Swiss nude and NOD/SCID mice, respectively. Tumor cell growth inhibition was similar to that observed in mice receiving therapeutic doses of bevacizumab. These experiments suggest that a therapeutic vaccine containing VEGF kinoid may represent a strategy for safely combating VEGF-dependent neovascularization and metastases occurring in malignant tumors.

The proinflammatory cytokine TNFalpha is a potent mediator of septic shock and a therapeutic target for chronic inflammatory pathologies including rheumatoid arthritis and Crohn's disease. As an alternative to anti-human TNFalpha (hTNFalpha) mAbs and other hTNFalpha blocker approved drugs, we developed an active anti-hTNFalpha immunotherapy, based on a vaccine comprised of a keyhole limpet hemocyanin-hTNFalpha heterocomplex immunogen (hTNFalpha kinoid) adjuvanted in incomplete Freund's adjuvant. In mice transgenic for hTNFalpha (TTg mice), hTNFalpha kinoid vaccination elicited high titers of Abs that neutralized hTNFalpha bioactivities but did not result in a cellular response to hTNFalpha. The vaccine was safe and effective in two experimental models. Kinoid-immunized but not control TTg mice resisted hTNFalpha-driven shock in one model and were prevented from spontaneous arthritis, inflammatory synovitis, and articular destruction in a second model. These data demonstrate an anti-cytokine induction of autoimmune protection against both acute and chronic hTNFalpha exposure. They show that active vaccination against a human cytokine can be achieved, and that the immune response can be effective and safe.

Anti-Tat vaccination experiments were carried out in mice with a view to inducing systemic in addition to mucosal immunity. For this. three types of immunizing preparations were tested, which consisted of Tat toroid embedded in either an adjuvant oily structure (IMS), or nanoparticles of chitosan, or microparticles of polylactide-co-glycolide (PLG). Administered by either the intranasal or oral route all preparations triggered anti-Tat IgG and IgA antibodies. Sera from mice immunized with either of these preparations could also inhibit significantly the Tat transactivating activity. These results open up a new avenue to the development of an effective anti-AIDS protective vaccine. (C) 2001 Editions scientifiques et medicates Elsevier SAS.

The Tat protein is essential for HIV type 1 (HIV-1) replication and may be an important virulence factor in vivo . We studied the role of Tat in viral pathogenesis by immunizing rhesus macaques with chemically inactivated Tat toxoid and challenging these animals by intrarectal inoculation with the simian/human immunodeficiency virus 89.6PD. Immune animals had significantly attenuated disease with lowered viral RNA, interferon-α, and chemokine receptor expression (CXCR4 and CCR5) on CD4 + T cells; these features of infection have been linked to in vitro effects of Tat and respond similarly to extracellular Tat protein produced during infection. Immunization with Tat toxoid inhibits key steps in viral pathogenesis and should be included in therapeutic or preventive HIV-1 vaccines.

Summary Extracellular Tat can exercise its deleterious effects on cells surrounding HIV-1-infected cells and allow spreading of virus. Extracellular Tat should be neutralized by anti-Tat vaccination using as an immunogen a functionally inactivated but immunogenic Tat preparation (Tat toxoid). In the present paper we show that native Tat can be inactivated without impairment of its immunogenicity by subjecting it to various chemical treatments. Since the carboxyamidation reaction can be easily monitored and the carboxyamidated Tat retained the whole immunogenicity of the native molecule, it should be the toxoid of choice for mass immunization.

The human papillomavirus type 16 (HPV-16) E7 oncogenic protein is found in the culture supernatant of SiHa cells, a cervical carcinoma cell line. Extracellular E7 protein, acting as a viral toxin in human immune cells, induces the overproduction of the immune suppressive IFN alpha cytokine by APCs, and inhibits the T-cell response to recall and allogenic antigens. These effects should be taken into account for the design of anti-human cervical carcinoma vaccines.

Summary Cultured human peripheral blood monocytes (PBMC) and the cell line H9 release a lectin. This lectin is not the previously described sarcolectin, since it does not specifically recognize the sugars lactose and sialic acid. The lectinic T-cell factor reduces the release by APCs of IFNα — a key cytokine known to inhibit the proliferation of activated T-lymphocytes.

Because administration of Tat protein, the HIV-1 toxin that induces immunosuppression and apoptosis, may be deleterious to the host immune system, a chemically inactivated but nonetheless immunogenic Tat preparation, Tat toxoid, was used to immunize seronegative individuals against Tat. In an open, controlled, phase I clinical trial, Tat toxoid turned out to be safe, well tolerated, and able to trigger a specific immune reaction. In particular, a threefold to more than 10-fold increase of circulating antibodies directed against the native Tat was observed after immunization in all of 5 immunized study subjects, together with a positive reaction to delayed-type hypersensitivity (DTH) skin test with Tat toxoid in vivo and increased lymphoproliferative response to native Tat in vitro. Persistent (> or =1 year) high levels of circulating anti-Tat antibodies could prevent the Tat-induced immune suppression and, following HIV-1 exposure, allow the anti-HIV-1 cellular immune response, with its early release of protective beta-chemokines, to occur leading to an increase of host resistance, that is, protection.

HIV type 1 (HIV-1) not only directly kills infected CD4+T cells but also induces immunosuppression of uninfected T cells. Two immunosuppressive proteins, interferon α (IFNα) and extracellular Tat, mediate this process because specific antibodies against these proteins prevent generation of suppressor cells in HIV-1-infected peripheral blood mononuclear cell cultures. Furthermore, the production of C-C chemokines in response to immune cell activation, initially enhanced by IFNα and Tat, ultimately is inhibited by these proteins in parallel with their induction of immunosuppression. The clinical corollary is the immunosuppression of uninfected T cells and the decline in C-C chemokine release found at advanced stages of HIV-1 infection paralleling rising levels of IFNα and extracellular Tat. We, therefore, suggest that IFNα and Tat may be critical targets for anti-AIDS strategies.

Summary Peripheral blood mononuclear cells (PBMCs) are physiologically activated by interleukin (IL)-2. We found that oyster extract (JCOE) currently used as a functional nutrient enhanced in vitro the IL-2 dependent activation as measured by cell count, 3 H-thymidine uptake and up-regulation of a IL-2 receptor. In human immunodeficiency virus (HIV) seropositive individuals, this oyster extract-induced effect was marked in asymptomatic individuals with quasi-normal CD4 cell counts, but was weakly reflected in acquired immunodeficiency syndrome (AIDS) patients.

Twenty-seven HIV-1-infected patients, 16 at early stage of disease and without concomitant antiretroviral therapy and 11 at more advanced stage of disease receiving antiretroviral therapy, have been followed since their enrollment, November 1992 and July 1993, respectively, in phase I/II studies to evaluate safety and immunogenicity of an anti-interferon-alpha (IFN-alpha) vaccine, aimed at modulating the impaired cytokine network in AIDS patients by counteracting IFN-alpha overproduction. We compared clinical, virological, and immunological markers of disease progression, including circulating IFN-alpha levels in a 24- to 30-month follow-up period with those of 62 patients fulfilling the same enrollment criteria and comparable for sex, risk factor, and age, regularly followed at our center. Anti-IFN-alpha immunization consisted of four-six intramuscular injections 1 month apart of a water-in-oil emulsion of 500 micrograms formalin-inactivated recombinant IFN-alpha-2b (iIFN-alpha) followed by intramuscular injections of 250 micrograms iIFN-alpha adsorbed onto calcium phosphate every 3 months. Neither clinical deterioration nor a CD4+ cell count decrease from pretreatment values was observed in IFN-alpha-immunized patients in the follow-up period, whereas clinical and immunological disease progressions were observed among open-comparison patients. Furthermore, statistical analysis showed a strong association between occurrence of clinical manifestations and high circulating IFN-alpha titers, while nonprogression of IFN-alpha-immunized patients was associated with decreased levels of circulating IFN-alpha.

Summary The acute human immunodeficiency virus type 1 (HIV-1) infection of activated peripheral blood mononuclear cells (PBMCs) from normal donors results in inhibition of cell proliferation and generation of functional suppressive T cells. Cultured HIV-1 infected PBMCs but not uninfected PBMCs, following irradiation, can inhibit the proliferation of antigen-activated autologous T cells in a dose-dependent way. CD8+ cell subpopulation is responsible for this inhibition. The presence of anti-alpha interferon (IFNα) and anti-Tat antibodies in the culture medium counteracts the HIV-1-induced immunosuppression and prevents the generation of suppressive T cells by these PBMCs. The reported data should have major implications for strategies of AIDS treatment which, in association with antiviral drugs, aim at targetting immune disorders.

Automat is a novel program which finds exhaustively all the oligopeptide segments shared by a given protein of any size with the proteins of a whole databank. It allows the user to collect statistics on the composition of the sequence studied in reference to the databank used. We present here the rationale and the algorithm underlying this powerful software. Besides its immediate interest for identifying efficiently similarities between proteins (or DNAs), biological applications of this software have already been described in the case of HIV-1 viral proteins, and Automat should prove useful also for studying more generally autoimmune diseases.

A decreased number of macrophages has previously been demonstrated by means of skin-window assays in operable breast cancer patients. However, in this type of cellular inflammatory response, a varying intensity of cellular activation, cellular recruitment and macrophage chemotaxis exists. In this study, we performed skin windows after stimulation by a chemo-attractant (FMLP) and a recall antigen (Candidin-latex). Cellular responses were classified in 19 breast cancer patients according to the presence (A+) or absence (A0) of macrophage activation by comparison to healthy controls. In 13 A0 patients, the admixture of a cytokine (IFN alpha) and an immunodulator (P40) to one agent or the other or both resulted in the restoration of macrophage functions. Our study shows that uniform cellular responses to chemo-attractants and antigens are observed in healthy and not immunocompromised individuals as assessed by skin-window tests. However, the cellular response recorded in immunodeficient cancer patients is altered. It also shows that the admixture to chemoattractants and antigens of particular cytokines or better still of an immunomodulator displaying a wide spectrum of activity offers a way of restoring macrophage functions. Individual responses to immunotherapy in clinical oncology may be related to such results.

The capacity of the Corynebacterium granulosum-derived P40 immunomodulator to induce in mice the formation of various cytokines IFN, IL-1, IL-2, alpha-TNF as well as to activate the complement system in rats was investigated. The results showed that P40 injected by the intravenous route was capable of inducing the formation of all four cytokines. High levels of IFN were measured 2 h after P40 stimulation and were still present at 24 h. The kinetic study of IL-1, IL-2 and alpha-TNF induction showed that it was a biphasic phenomenon. The patterns of IL-1 and alpha-TNF induction were quite comparable, whereas the release of IL-2 was delayed with respect to that of IL-1 and alpha-TNF. Oral administration of P40 to rats strongly activated the alternative pathway of the complement system. It was concluded that most of the non-specific effects of P40 on the immune system are likely to be mediated by its capacity to induce cytokine formation and to activate the complement system.

It is known that C granulosum -derived P40 immunomodulator displays strong anti-microbial effects in mice by the intravenous route. Since microbial contamination of humans occurs in many instances via the airways, the effect of P40 on infections was investigated when it was given intranasally or by aerosolization. In order to augment its bioavailability, P40 was derivalized by coupling with polylysine chains (P40-PL). The results showed that P40-PL exercised a significant protective effect, both by the intranasal route and by aerosolization on both influenza and K pneumonlae infections produced by aerosolization or intranasal instillation. Stimulation of the phagocytic capacity of alveolar macrophages by these types of treatment is likely to account for the increased resistance of mice toward microbial infections influenza / K pneumoniae infection / immunomodulator

An ELISA for assessing pneumococcal anticapsular antibodies in human sera is described. Sensitization was carried out by adding to avidin-coated polystyrene plates biotinylated capsular polyosides. This ELISA was used for the determination of anticapsular antibody levels in pre- and post-immunization sera from 20 elderly individuals. The anticapsular antibody titres differed significantly in sera taken prior to and after vaccination. Eighteen of the 20 sera tested showed mean fold increases of the antibody level greater than 2 (2.1 to 21.0) as a result of vaccination. Since morbidity and mortality rates from S. pneumoniae infections are high in the elderly, immunization should be systematically performed. However, the elderly subject is frequently immunosuppressed, and it is therefore advisable to control the result of the vaccination, using a simple procedure, such as the one described here.

An enzyme-linked immunosorbent assay using nitrocellulose strips (dot-ELISA) for the routine laboratory detection of IgG antibodies to mumps and varicella viruses is described. The virus antigens are dotted onto nitrocellulose strips, and the dotted strips are incubated with the sera to be tested. The bound antibodies are revealed using enzyme-labeled antihuman IgG antibodies. Reliable results are obtained when the assay is carried out at 37 degrees C. The reported data indicate that the dot-ELISA can reliably be used for the detection of IgG antibodies to mumps and varicella viruses in human sera.

Anti-Tat therapeutic vaccination has been clinically investigated by different groups [1-4], given that 1) extracellular Tat protein induces T cell apoptosis and cellular immune suppression, 2) epidemiological data showed that LTNP exhibit high level of serum anti-Tat Ab, negatively correlated with p24 antigenemia, 3) in Tat immunized macaques, viremia decreased following SHIV challenge. Anti-Tat therapeutic vaccination using Tat Toxoid adjuvanted either with Seppic [1,2] or with alum or DcChol (Aventis Pasteur) proved to be safe. A prospective structured treatment interruption study (STI) monitored according to EU guidelines was conducted at Hospital St-Pierre, Brussels (Pr. N. Clumeck) on 31 vaccinees who received a DcChol adjuvanted Tat Toxoid (n = 12), a DcChol placebo (n = 8) or non adjuvanted Tat toxoid (n = 11). The 2 year study follow-up showed that vaccinees developing high titer of Abs neutralizing Tat bioactivity prolonged HAART-interruption., info:eu-repo/semantics/published, Oral presentation. From 2006 International Meeting of The Institute of Human VirologyBaltimore, USA. 17–21 November, 2006

In the present investigation, mouse infection models with either intracellular or extracellular bacteria were designed in order to assess the effect of nonspecific immunostimulation with the C. granulosum -derived P40 immunomodulator on infection treatment, performed with doses of antibiotics achieving a low percentage of cures. The results obtained showed that nonspecific immunostimulation was able to significantly enhance antibiotic therapy efficacy. In addition, combined treatments of bacterial infections should allow for a reduction in dosages of antibiotics while at the same time, achieving increased efficacy. Possible mechanisms for the potentiating effect of nonspecific immunostimulation on antibiotic therapy effectiveness are discussed. antibiotic therapy/nonspecific immunostimulation/bacterial infections

The tyrosyl residues of tetanus toxin can be specifically nitrated by tetranitromethane. The introduction of NO2 groups into the toxin molecule is followed by a rapid decrease in toxicity. Although tryptophyl residues are differently involved and some aggregation occurs, these two phenomena do not take any significant part in the toxicity loss. The immunological reactivity in vitro is preserved. However, it is modified when a certain amount of NO2 groups have been introduced. The thus-formed derivative no longer gives a reaction of identity with the native toxin. Furthermore, when a sufficiently large number of NO2 groups have been introduced, two lines of precipitation are seen. Antisera raised in rabbits with a few nitro-derivatives of the toxin exhibit a low avidity. Precipitating power with pure toxin of these antisera is lower than with a normal antitetanus serum. The capability of tetanus toxin to give rise to neutralizing antibodies is not impaired until 33 NO2 groups have been introduced, then it gradually decreases until it finally disappears. Modification of the toxin by nitration allowed us to discriminate three groups of determinants. In particular, the results presented are further substantiated by those given by 3-aminotyrosyl derivatives of tetanus toxin.

The amino acid analysis of tetanus toxin as purified from bacterial extracts or from culture filtrates of Plectridium tetani, was carried out. The results that have been obtained support the assumption of the identity of both types of toxin. Tetanus toxoid amino acid composition was comparatively determined. Significant decreases in the amounts of some amino acids were found. These deficits are accounted for within the reactions with formaldehyde which lead to the formation of Mannich-like derivatives. The presence of 6 free SH-groups on the molecule of toxin could be substantiated. Two of these groups are difficult to reveal. The existence of two disulfide bridges was established. These results are discussed, in particular, with regard to the expression of the toxicity. The presence of only one N-terminal amino acid residue could be revealed.

O tratamento de infecções crônicas recidivantes (I.C.R.) encontra muitas dificuldades. No presente estudo, o uso do imunoadjuvante P40, quer isolado, quer associado a vacinoterapia no tratamento de I.C.R., demonstrou ser muito eficaz, ao passo que a vacinoterapia isoladamente não o foi. Foi aventada a hipótese de que a imunidade mediada por células pode desempenhar papel importante no contrôle das I.C.R., uma vez que a melhora clínica dos pacientes foi acompanhada de positivação de testes cutâneos anteriormente negativos, testes estes realizados quer com o agente infeccioso específico, quer com antígeno inespecífico de refôrço. The treatment of relapsing chronic infections (RCI) encounters many difficulties. In the present study, the use of the immuno adjuvant P40 either alone or in association with vaccinotherapy for the treatment of RCI turned out to be very effective, whereas vaccinotherapy alone was not. It is hypothesized that cell-mediated immunity may play a major role in controlling RCI, since the clinical improvement of the patients kept up with the positivation of previously negative skin tests carried out with either the specific infecting agent or with recall-anti gens.

Tetanus toxin (TeTx) is a neurotransmission impairing toxin that acts on several neurotransmitter systems. TeTx also inhibits the K+-induced release of acetylcholine (ACh) from synaptosomes isolated from the electric organ of Torpedo. Neither the membrane potential and depolarization, nor the depolarization-induced calcium uptake into cholinergic nerve terminals is modified after TeTx poisoning. On the other hand, it is known that, when cholinergic nerve terminals are stimulated, there is a release of ATP associated with the release of ACh. We have explored the action of TeTx on this co-release, and have found that there is no action of TeTx on the nucleotide release. Thus, TeTx blocks ACh release without modifying ATP release.

We describe a Dot-ELISA system for the rapid, specific and reliable assessment of a subject's antitetanus immune status. The conditions for Dot-ELISA were selected to give positive reactions only for sera with antitetanus antibody titers equal to or higher than 0.06 I.U./ml, considered to represent protection when using "in vitro" methods. This Dot-ELISA method could advantageously be applied to the monitoring of vaccination campaigns, as well as for assessing the antitetanus immune status of the wounded.

The role of gangliosides in rabies virus infection of chick embryo-related (CER) cells was investigated. Cultured cells were pretreated with neuraminidase to render the cells transiently non-susceptible to viral infection. Incubation of these desialylated cells with gangliosides allowed them to incorporate exogenous gangliosides and they recovered their susceptibility to rabies virus infection. Infection of CER cells was monitored by specific fluorescence 24 h after virus inoculation. The use of individual purified gangliosides or mixtures of two gangliosides to restore cellular susceptibility to viral infection showed that GT1b and GQ1b were the most effective. The disialogangliosides were also active, principally GD1b, whereas GM1, GM3 were poorly active and GD3 inactive. Incubation of rabies virus with gangliosides prior to virus infection reduced the percentage of infected cells. The results indicate that highly sialylated gangliosides are part of the cellular membrane receptor structure for the attachment of infective rabies virus. However, it is possible that other glycoconjugates such as glycoproteins or glycolipids also participate as components of a receptor structure for rabies virus.

In the course of our studies on oxidative stress as a component of pathological processes in humans, we showed that microintrusion into cells with microcapillary and ultramicroelectrochemical detection could mimic many types of mechanical intrusion leading to an instant (0.1 s) and high (some femtomoles) burst release of H 2 O 2 . Specific inhibitors of NADPH enzymes seem to support the assumption that this enzyme is one of the main targets of our experiments. Also, human immunodeficiency virus type 1 (HIV-1) gp160 inhibits the cooperative response of uninfected T cells as well as Tat protein release by infected cells does. In this study, we analyzed in real time, lymphocyte per lymphocyte, the T-cell response following activation in relation to the redox state. We showed that the immunosuppressive effects of HIV-1 Tat and gp160 proteins and oxidative stress are correlated, since the native but not the inactivated Tat and gp160 proteins inhibit the cellular immune response and enhance oxidative stress. These results are consistent with a role of the membrane NADPH oxidase in the cellular response to immune activation.