A hybrid toxin containing fragment A from diphtheria toxin linked to the B protomer of cholera toxin.

We have constructed and characterized a hybrid toxin containing the A chain of diphtheria toxin linked via a disulfide bridge to the B protomer of cholera toxin. Cholera toxin B protomer, previously derivatized with 4-5 cystaminyl groups per pentameric protomer, was reacted with reduced diphtheria toxin chain A to give the desired hybrid, containing an average of 2 molecules of diphtheria toxin chain A per cholera toxin B protomer. A concentration of 0.3 nM hybrid inhibited protein synthesis by 50% in 24 h in several cultured cell lines; thus the hybrid was about 10-fold more toxic than of a (diphtheria toxin chain A)-SS-(concanavalin A) conjugate described previously. Evidence was obtained that toxicity of the hybrid was dependent on the functional contributions of both the diphtheria toxin chain A and cholera toxin B protomer moieties.

Recombinant gp160 as a therapeutic vaccine for HIV-infection: results of a large randomized, controlled trial. European Multinational IMMUNO AIDS Vaccine Study Group.

OBJECTIVES: The primary objective of this study was to expand the safety and immunogenicity database of recombinant gp160 as a therapeutic vaccine in the treatment of HIV-infection. Preliminary efficacy data was also sought. DESIGN: This trial was a randomized, double-blind, placebo-controlled study. Two-hundred and eight volunteers, 96 therapy-naive with CD4 cell count >500x10(6)/l (group A) and 112 with CD4 cell count of 200-500x10(6)/l (group B, 51 out of 112 on treatment with one or two nucleoside analogues), received monthly injections of rgp160 IIIB vaccine or placebo for the first 6 months of the study; booster immunizations with rgp160 MN or placebo were given at times 15, 18, and 21 months. METHODS: Safety and immunogenicity data were obtained and measurements of CD4 cell count, plasma viral RNA, and proviral DNA were performed. Clinical outcome was recorded for the 24 months of study. RESULTS: The vaccine was safe and well tolerated. Despite the induction of new rgp160-specific lymphoproliferative responses and the presence of positive delayed type hypersensitivity skin tests to rgp160 at the end of the 24 month study, no effect on the natural history of HIV infection was detected. Within 24 months, AIDS-defining illnesses had occurred in 19 of the vaccinated volunteers and in 18 of the placebo recipients. Persons with higher plasma viral RNA levels and higher proviral DNA had a more rapid decline in CD4 cell count when compared to persons with lower values. Vaccine did not alter viral RNA or proviral DNA levels. CONCLUSION: There was no clinical benefit to therapeutic immunizations with rgp160, despite the induction of new lymphoproliferative responses.

Quantitative multiple competitive PCR of HIV-1 DNA in a single reaction tube.

A quantitative multiple competitive PCR (QMC-PCR) for determination of DNA copy numbers is described. Four competitive DNA templates for the env region of HIV-1 were constructed with sizes longer (187 and 163 bp) or shorter (122 and 105 bp) than the 142 bp of the wild-type PCR product. Varying amounts of each of these competitors are introduced together with the sample into a single reaction tube. Since competitors and wild-type fragments share the same primer recognition sequence (SK68/SK69), amplification occurs according to the rate of the introduced copy numbers. The PCR products are run on an agarose gel, and the copy number of the sample is determined by analyzing the bands with a video densitometer and calculating the equivalence point in a linear regression plot.

Detection of false-negative results in nested primer PCR of proviral HIV-1 DNA.

A control template for a competitive nested primer PCR of the HIV-1 gag region was constructed. This construct shares the primer recognition sequences with the wild-type template and yields a 97-bp fragment after amplification (wild-type: 115 bp). To provide an internal control for the individual PCR runs, six copies of this nested primer control plasmid were introduced into a reaction tube containing the specific sample (under the PCR conditions used, this copy number reproducibly gave a positive PCR signal). The results of our study show the feasibility of this concept by analyzing a plasmid (pBH10) containing HIV-1 wild-type sequences, and examination of samples from a cohort of HIV-1-seropositive subjects demonstrated the clinical usefulness of this test. The control plasmid was detectable in all of the samples but one, which without the use of the control template would have yielded a false-negative result.

Down regulation of Fc receptors by IVIgG.

IVIgG preparations are now widely applied for immune modulatory treatment in various forms of autoimmune and immune complex diseases. Several controlled studies clearly demonstrated the clinical efficacy of this type of treatment; the underlying pathophysiological mechanisms, however, have yet to be elucidated. Among the mechanisms suggested to play a role in this context is the interaction of gamma globulin with Fc gamma receptors (Fc gamma R) expressed in the membrane of immunocompetent cells. Our studies concentrated on these aspects and focused on possible functional consequences of IgG-Fc gamma R interaction. By using the peripheral blood monocyte as a model system for an Fc gamma R-bearing cell, we confirmed previous reports by showing differences in Fc gamma R binding and Fc gamma R modulation induced by IgG in its various forms (monomeric IgG, Polymeric IgG, immune complexes). As biological consequences of Fc gamma R modulation, changes in effector and accessory function of these cells were observed. The results presented in this brief review emphasize especially the difference between ligand-oriented Fc gamma R diffusion (induced by surface-bound IgG) and true long-term down-modulation of Fc gamma R (mediated by fluid-phase IgG polymers) and show that only the down-modulation of Fc gamma R correlated with impaired functions of the affected cell.

CD3, CD8 double-positive cells from HIV-1-infected chimpanzees show group-specific inhibition of HIV-1 replication.

The CD8-positive (CD8+) lymphocyte population in the chimpanzee is composed of two major subsets, a CD3-positive, CD8-positive (CD3+CD8+) and a CD3-negative, CD8-positive (CD3-CD8+) population. The aim of this study was to delineate the function of CD3+CD8+ T cells with respect to inhibition of HIV-1 replication. It could be shown that this cell population had the capacity to control the growth of HIV-1 in exogenously infected CD4-positive (CD4+) lymphocytes. This effect was observed only with cells from HIV-1-infected chimpanzees, was operative only in an autologous and not in a homologous situation, and was not due to cytotoxicity. CD3+CD8+ lymphocyte-mediated inhibition of HIV-1 replication was group-specific in that CD3+CD8+ cells of HIV-1 (IIIB)-infected chimpanzees were capable of inhibiting replication of HIV-1 strains IIIB, MN, and RF. The effect observed was operational during the early stages of HIV-1 replication only; the effector cells had to be added to CD4+ cells within 3 days after HIV-1 infection in order to suppress growth of the virus. The presence of CD3+CD8+ lymphocytes with anti-HIV-1 activity in the circulation of HIV-1-infected chimpanzees might contribute to the lack of progression toward AIDS observed in this species.

An experimental prime-boost regimen leading to HIV type 1-specific mucosal and systemic immunity in BALB/c mice.

Induction of mucosal as well as systemic immunity to HIV-1 is considered to have high priority in current concepts of future AIDS vaccines. Here we show that the desired immune responses can be elicited by an experimental prime-boost regimen consisting of mucosal (intragastric) application of a recombinant vaccinia virus carrying the HIV-1 env gene (vSC25), followed by parenteral (intradermal) immunization with the recombinant HIV-1 glycoprotein 160 (rgp160). Following intragastric immunization of mice with vSC25 in combination with the mucosal adjuvant cholera toxin (CT), HIV-1 env-specific IgA was secreted by B cells of Peyer's patches and the lamina propria. Moreover, mucosal (intragastric and intranasal) application of vSC25 (both in presence or absence of CT) induced a long-lasting, HIV-1 env-specific systemic cytotoxic T cell response. Subsequent intradermal boosters with rgp160 led to HIV-1-specific T cell memory and serum antibodies.

Allostimulated lymphocytes inhibit replication of HIV type 1.

Previous reports demonstrated that alloantigen- or xenoantigen-specific antibodies displayed neutralizing activity toward human or simian immunodeficiency viruses. In the present article we have addressed the question of alloantigen-induced cell-mediated anti-HIV activity. We show that allostimulation resulted in a lymphocyte population (largely of the CD8-positive phenotype) with the capacity to inhibit HIV-1 replication in PHA blasts of homologous and, unexpectedly, also autologous origin. The allostimulated effector cells exerted their activity via a noncytolytic mechanism. Experiments in which direct cell-to-cell contact between allostimulated effectors and HIV-1-infected PHA blasts was prevented by a semipermeable membrane indicated that soluble mediators were involved in inhibition of HIV-1 replication. As such allostimulated effectors not only would have the capacity to prevent viral replication in allogeneic HIV-1-infected cells (known to play an important role in HIV-1 transmission in vivo), but also might inhibit HIV-1 growth in autologous lymphocytes, the concept of an AIDS vaccine containing both HIV-1 antigens and alloantigens warrants further consideration.

Dual tropism of HIV-1 IIIB for chimpanzee lymphocytes and monocytes.

In humans, macrophages serve as a major reservoir of human immunodeficiency virus (HIV-1) in the infected host and may play a role in the pathogenesis of the disease. In HIV-1-infected chimpanzees, however, virus could not be recovered from cells of the monocyte/macrophage lineage, leaving the question of macrophage tropism of HIV-1 in this species unresolved. The data reported that HIV-1 IIIB shows dual tropism and is infectious for both chimpanzee monocytes and lymphocytes in vitro. Viral replication in chimpanzee monocytes was clearly demonstrated by infection of allogeneic phytohemagglutinin (PHA) blasts in vitro and by electron microscopy (EM). EM revealed HIV particles associated with 10-15% of the HIV-1 IIIB-infected chimpanzee monocytes. Viral particles budding from the monocyte surface in the typical crescent form were noted as well. This is in contrast to the human situation, where monocytotropic HIV strains preferentially bud into and accumulate in cytoplasmic vacuoles. These results indicate that both lymphocytes and cells of the monocyte/macrophage lineage replicate virus in the chimpanzee; the cell tropism of viral strains, however, is different in chimpanzees and humans.

Immunization of chimpanzees with the HIV-1 glycoprotein gp160 induces long-lasting T-cell memory.

The goal of the present study was to investigate the antigen-specific T-cell response to the recombinant HIV envelope glycoprotein (gp160) and to test the effect of various adjuvant formulations on the efficiency of T-cell priming as well as on magnitude and longevity of the gp160-specific T-cell response. Our studies revealed that, in combination with an appropriate adjuvant (lipid-based adjuvant or mineral carrier complex), immunization with recombinant gp160 led to the appearance of gp160-primed T cells. The T-cell response obtained was substantial (proliferative response of greater than 100,000 delta dpm after one primary and two booster immunizations), gp160-specific (proliferation only in response to gp160, no proliferation after addition of a mock gp160 preparation), and long-lasting (T cell responses of greater than 50,000 delta dpm were observed more than one year after the last booster). The results presented here differ from those of previous studies in that they show the presence of substantial and long-lasting T-cell memory toward the immunogen gp160. Therefore further investigations on the use of these preparations as HIV candidate vaccines appear to be justified.

Effects of cyclosporin A upon humoral and cellular immune parameters in insulin-dependent diabetes mellitus type I: a long-term follow-up study.

Patients who had been included in a randomized double-blind placebo-controlled trial on the efficacy of cyclosporin A (CyA) in producing remissions in insulin-dependent diabetes mellitus (IDDM) type I were investigated for humoral and cellular immunologic parameters. Whereas metabolic derangement before the initiation of insulin treatment led to small but significant decreases in the percentage of CD4-positive lymphocytes as well as of the activity of natural killer (NK) cells and antibody-dependent cellular cytotoxicity (ADCC), the administration of CyA did not influence any of the immunologic parameters tested, which included proliferative lymphocyte responses to mitogens and alloantigens and serum concentrations of immunoglobulins G, A and M. Thus NK cell activity, ADCC as well as the percentage of CD4-positive lymphocytes returned to normal levels in parallel with the normalization of glycosylated haemoglobin (HbAlc), but were not further influenced in their course by the administration of CyA, as compared with patients receiving placebo. Interferon-induced augmentation of NK cell activity did not differ between patients with IDDM on placebo and those under CyA therapy. All other investigated parameters also remained unchanged during the time of CyA therapy. We conclude that metabolic derangement leads to a reversible disturbance of certain cellular immune functions, but their normalization achieved by insulin treatment and their further course remains uninfluenced by the administration of CyA.

Detection of tick-borne encephalitis virus by sample transfer, plaque assay and strand-specific reverse transcriptase polymerase chain reaction: what do we detect?

Experimental inoculation of mice provides a well characterized model for studying infection with tick-borne encephalitis virus (TBEV), a flavivirus pathogenic for humans. Conflicting data on the kinetics of viremia and the development of virus titers in the brain, however, were only recently shown to have resulted from the use of assay systems with different levels of sensitivity in the titration of TBEV, i.e. plaque assay or sample transfer into naive recipient mice. Theoretically, RT-PCR could extend further the detectability to antibody-neutralized virus and when undertaken strand-specifically discriminate active replication from the mere presence of TBEV. We have compared the conventional methods for detection of TBEV with a newly devised RT-PCR method. As expected, RT-PCR, in contrast to the infectivity assays, detected antibody-neutralized virus. Furthermore, the mere presence or active replication of the virus could be differentiated by strand-specific RT-PCR. Plaque assay and sample transfer, in contrast, both detected only infectious virus. However, whereas sample transfer provides higher sensitivity for detection of TBEV from solid organs, the plaque assay is less costly and considering animals welfare more convenient. Thus, the newly devised method may allow the resolution of unanswered questions, while both the traditional infectivity assays retain their benefits in certain situations.