Efficacy of a therapeutic cocaine vaccine in rodent models.

Cocaine abuse is a major medical and public health concern in the United States, with approximately 2.1 million people dependent on cocaine. Pharmacological approaches to the treatment of cocaine addiction have thus far been disappointing, and new therapies are urgently needed. This paper describes an immunological approach to cocaine addiction. Antibody therapy for neutralization of abused drugs has been described previously, including a recent paper demonstrating the induction of anti-cocaine antibodies. However, both the rapidity of entry of cocaine into the brain and the high doses of cocaine frequently encountered have created challenges for an antibody-based therapy. Here we demonstrate that antibodies are efficacious in an animal model of addiction. Intravenous cocaine self-administration in rats was inhibited by passive transfer of an anti-cocaine monoclonal antibody. To actively induce anti-cocaine antibodies, a cocaine vaccine was developed that generated a high-titer, long-lasting antibody response in mice. Immunized mice displayed a significant change in cocaine pharmacokinetics, with decreased levels of cocaine measured in the brain of immunized mice only 30 seconds after intravenous (i.v.) administration of cocaine. These data establish the feasibility of a therapeutic cocaine vaccine for the treatment of cocaine addiction.

Influence of environmental factors on the infectivity of Echinococcus multilocularis eggs.

The sensitivity of eggs of Echinococcus multilocularis to environmental and controlled laboratory conditions was tested. Egg material was exposed and the infectivity was subsequently monitored by in vitro activation and by oral infection of the natural host, Microtus arvalis. To study the impact of environmental conditions in an endemic area of south-western Germany, eggs were sealed into bags of nylon mesh and exposed to the natural climate during various seasons. The maximal survival time of eggs was 240 days in an experiment performed in autumn and winter and 78 days in summer. A study of the tenacity of eggs under laboratory conditions revealed a high sensitivity to elevated temperatures and to desiccation. At 45 degrees C and 85-95% relative humidity the infectivity was lost after 3 h as well as after 4 h exposure to 43 degrees C suspended in water. Exposure to 27% relative humidity at 25 degrees C as well as exposure to 15% relative humidity at 43 degrees C resulted in a total loss of infectivity within 48 and 2 h, respectively. Temperatures of 4 degrees C and of -18 degrees C were well tolerated (478 days and 240 days survival, respectively), whereas exposure to -83 degrees C and to -196 degrees C quickly killed off the eggs (within 48 h and 20 h, respectively). Eggs of E. multilocularis were not killed off by exposure to various commercially available disinfectants applied according to the manufacturers' instructions and by exposure for 24 h to low concentrations of ethanol. Irradiation with 40 krad. from a 137Caesium source prevented the development of metacestodes but allowed seroconversion of infected rodents.

A universal T cell epitope-containing peptide from hepatitis B surface antigen can enhance antibody specific for HIV gp120.

Peptide-based vaccines that directly target T cell or B cell epitopes may have significant advantages over conventional vaccines. Further, synthetic chimeric peptides that combine strong T cell epitopes with poorly immunogenic, but immunodominant, B cell epitopes or strain-conserved B cell epitopes may be useful in eliciting antibody to such important regions. Here we characterize a human T cell epitope analyzed in 54 individuals immunized with a hepatitis B virus surface Ag vaccine. Primary cultures from a total of 59 immunized donors were assessed for their ability to respond to hepatitis B virus surface Ag and peptides, and five were non-responders (8.5%). T cell lines were established from the remaining 54 responders. Of the responders, it was found that the peptide representing amino acids 19 through 33 (19-33) elicited significant proliferation in lines derived from 50 donors. This "universal" T cell epitope, which was recognized in donors of many different HLA-DR and -DQ haplotypes, was then used to construct a chimeric peptide containing 19-33 and the third V region loop structure (V3 loop) of HIV-1 envelope gp 120, in an attempt to augment the immune response to the V3 loop peptide. The V3 loop is the region to which significant neutralizing antibody is directed. Thus, a strong immune response to a synthetic peptide that contains the strain-conserved V3 loop region could have significant therapeutic implications. The V3 loop/19-33 peptide was then used to prime mice, to determine whether V3 loop-specific antibody could be induced. The peptide elicited potent 19-33-specific proliferation in T cells isolated from draining lymph nodes, and in six of six mice anti-V3 loop antibody was elicited. Further, V3 loop/19-33-primed animals made significant levels of antibody that bound rgp120. These data suggest that, when a major T cell epitope is synthesized in tandem with the V3 loop, a significant immune response against the loop can be elicited. Thus, given the finding that neutralizing antibody may play a role in the control and/or prevention of HIV infection, an HIV vaccine composed of a T cell epitope-containing peptide may prove effective. In addition, this type of approach can be generalized to the design of peptide-based vaccines.

The potential use of T cell epitopes to alter the immune response.

Recent advances in the understanding of T cell specificity and activation have lead to the design of T cell specific immunomodulators. T cell epitope containing peptides have been proposed as agents which may either enhance or dampen the immune response. In this review, we examine two systems which can benefit from the application of this novel technology. Vaccine development is moving toward the use of defined cloned or synthetic molecules. T cell epitope identification and design can be used to augment the ability of a weak antigen to generate an immune response. In contrast, traditional allergy immunotherapy has been shown to alter the immune response to the allergenic antigen. T cell epitope approaches to allergy desensitization offer a new therapeutic modality.

Structure of the T-cell antigen receptor: evidence for two CD3 epsilon subunits in the T-cell receptor-CD3 complex.

The T-cell antigen receptor (TCR) consists of heterodimeric glycoproteins (TCR alpha beta or gamma delta) that demonstrate homology with immunoglobulins. Noncovalently associated with the alpha beta (or gamma delta) heterodimer are at least five nonvariant proteins (CD3-gamma, -delta, -epsilon, -zeta, and -eta), which together comprise the TCR-CD3 complex. The stoichiometry of the antigen receptor has been assumed to be either alpha beta gamma delta epsilon zeta zeta or alpha beta gamma delta epsilon zeta eta. In this paper we provide several lines of evidence that support the notion that the mature TCR-CD3 complex on the cell surface contains two CD3-epsilon polypeptide chains. Transfection of two murine T cell-T cell hybridomas with the human DNA encoding CD3-epsilon protein demonstrated that both murine and human CD3-epsilon chains were present within the same TCR-CD3 complex. Analysis of thymocytes isolated from transgenic mice that expressed high copy numbers of the human CD3-epsilon gene showed that the heterologous human CD3-epsilon subunits were coexpressed with murine CD3-epsilon in the same TCR-CD3 complex. Since CD3-epsilon was shown to form disulfide-linked homodimers both in human and murine T cells, the two CD3-epsilon subunits present in the TCR-CD3 complex were in direct contact with one another. The presence of two CD3-epsilon polypeptide chains in close proximity to one another in the TCR-CD3 complex may have important implications for its assembly and its signal transduction mechanisms.

Prostaglandin E2-dependent induction of B cell unresponsiveness. Role of surface Ig and Fc receptors.

Previously, we demonstrated that two signals were required for accessory cells to induce B cell unresponsiveness: tolerogenic Ig and PG. The purpose of this study was to investigate whether PGE2, in an accessory cellfree system, promoted fluorescein-specific B cell unresponsiveness in conjunction with ligands which bound to surface Ig (sIg) and/or FcR. Several conditions were found whereby PGE2 was obligatory for unresponsiveness. In the presence of aggregated, but not monomeric non-Ig fluorescein-Ag, direct plaque-forming cell responses were reduced by 60%. In contrast, engagement of the B cell FcR by aggregated IgG2b or by the 2.4G2 anti-FcR mAb failed to induce unresponsiveness, even when PGE2 was present. These data suggested that PGE2 could promote sIg-mediated negative signaling. A second condition where PGE2 promoted unresponsiveness occurred when sIg and FcR were simultaneously engaged by monomeric ligands. However, when sIg and FcR were cross-linked, PGE2-independent B cell unresponsiveness occurred. Interestingly, when subinhibitory doses of cross-linking agents were used, PGE2 dependent negative signaling resulted. PGE2 can thereby promote B cell unresponsiveness in three different situations. First, when sIg is extensively cross-linked by aggregated antigens or those with repeating determinants. Second, when sIg is engaged by monomeric antigen and when the B cell FcR is also occupied. Third, under conditions where B cell sIg and FcR are inadequately cross-linked. These situations can occur in vivo when macrophages in the B cell microenvironment (i.e., follicles) secrete PGE2 and when Ag with repeating epitopes, or immune complexes capable of binding B cell sIg and FcR are present. Thus, PGE2 can serve as an important regulatory element in limiting antibody formation.

E-series prostaglandins are potent growth inhibitors for some B lymphomas.

The ability of prostaglandins (PG) to inhibit the growth of B cell lymphomas was investigated. Macrophage-secreted PGE2 was previously shown to promote unresponsiveness to antigen in normal B lymphocytes. This observation suggested that B lymphomas might also be regulated by prostanoids. Five non-PG-secreting Ly-1+ B lymphomas (CH12, CH31, CH33, NBL and WEHI-231) were incubated for 24-72 h with PGE2, PGE1 or PGF2 alpha. The level of lymphoma growth at the end of culture was determined using a colorimetric assay which detects only viable cells. A marked heterogeneity was observed with respect to the sensitivity of these lymphomas to PGE2 and PGE1. CH31 was very sensitive, being growth inhibited by as little as 10(-8) M PGE. In contrast, CH12, a more mature lymphoma, was highly resistant, whereas CH33, NBL and WEHI-231 were of intermediate resistance. All five lymphomas demonstrated little or no growth inhibition when cultured with PGF2 alpha. Moreover, unlike PGE2, PGF2 alpha failed to elevate intracellular cAMP levels. It was previously shown that CH31, CH33 and WEHI-231 could be growth inhibited by anti-immunoglobulin antibodies which cross-link surface immunoglobulin. Interestingly, these three lymphomas were rendered more sensitive to this treatment if PGE2 was present. For example, 10(-8) M PGE2 alone had little effect on CH33, but significantly augmented growth inhibition induced by suboptimal quantities of anti-immunoglobulin antibody. Cholera toxin, another agent which was found to rapidly elevate intracellular cAMP levels, also synergized with suboptimal doses of anti-immunoglobulin to induce growth inhibition. Overall these data suggest that, in vivo, macrophage-secreted prostanoids may slow the growth of some lymphomas and that anti-immunoglobulin or anti-idiotype treatment may be more effective in the presence of agents which elevate cAMP such as E-series PG.

Two signals are required for accessory cells to induce B cell unresponsiveness. Tolerogenic Ig and prostaglandin.

We previously demonstrated that a lymphoid dendritic cell-like tumor line (P388AD.2) presented a normally tolerogenic signal, fluoresceinated sheep gamma-globulin (FL-SGG), as an immunogenic one. In contrast, macrophages derived from the peritoneal cavity potentiated the ability of FL-SGG to induce B cell unresponsiveness. In this paper we examined whether two different Ia+ splenic accessory cells differentially presented tolerogen to spleen cells or fluorescein (FL)-binding B cells. Interestingly, lymphoid dendritic cells presented FL-SGG to spleen cells and elicited augmented anti-FL antibody responses, whereas splenic macrophages presented this same moiety and elicited hapten-specific B cell unresponsiveness. The mechanism of splenic macrophage-elicited B cell negative signaling was investigated, and it was found that B cell unresponsiveness was abrogated in the presence of the cyclooxygenase inhibitor indomethacin. This observation suggested a crucial role for PG in B cell negative signaling. The addition of 10 nM PGE2 restored unresponsiveness in cultures treated with indomethacin and tolerogen-pulsed macrophages, even though this dose of PG had no effect on the ability of B cells to be triggered by an immunogenic signal. A role for T cells was excluded, inasmuch as purified hapten-specific B cells were specifically tolerized by FL-SGG-pulsed macrophages. Lymphoid dendritic cells pulsed with FL-SGG did not deliver a tolerogenic or immunogenic signal to FL-specific B cells. However, when PGE2 was supplied, B cell unresponsiveness was induced. Finally, we tested whether "non-tolerogenic" doses of FL-SGG could render hapten-specific B cells unresponsive in the presence of PGE2, but in the absence of accessory cells. Interestingly, the combination of non-tolerogenic amounts (10 to 1000 pg/ml) of FL-SGG in conjunction with PGE2 induced unresponsiveness, whereas neither moiety alone was effective. These results suggest that splenic macrophages and lymphoid dendritic cells exert opposing effects on the immune system as evidenced by the induction of negative or positive B cell signaling. Our observations suggest that one of the key factors in controlling whether an accessory cell delivers a tolerogenic signal is the ability to secrete PG.

Differential presentation of tolerogenic immunoglobulin in vivo by macrophages and by a lymphoid dendritic cell-like tumor line.

Previous work indicated that macrophages and a lymphoid dendritic cell-like tumor line, P388AD.2, possessed a differential ability to present a haptenated immunoglobulin (tolerogen) in vitro. Macrophages presented fluorescein-conjugated sheep gamma globulin (FL-SGG) and elicited B-cell unresponsiveness. In contrast, P388AD.2 presented this normally tolerogenic signal as an immunogenic one and induced augmented anti-hapten antibody responses. The objective of the present study was to determine whether differential tolerogen presentation could occur in vivo using defined accessory cells pulsed with FL-SGG. Interestingly, the intravenous (IV) injection of FL-SGG-pulsed thioglycollate-elicited macrophages, which secreted prostaglandin E2, induced hapten-specific B-cell unresponsiveness in syngeneic recipients. One thousand times as much FL-SGG in soluble form was required to produce the same degree of unresponsiveness. In contrast to macrophage-elicited negative signalling, non-prostaglandin secreting P388AD.2, when pulsed with FL-SGG, induced hapten-specific responses 2-3 times control values. Moreover, as few as 2 x 10(4) FL-SGG-pulsed P388AD.2 induced significant augmentation of the anti-FL antibody response. The presentation of FL-SGG in an immunogenic fashion by P388AD.2 was rapid and long lasting since increased responses were demonstrated as early as 1 day or as long as 21 days after IV injection. P388AD.2 were not simply acting as a passive carrier, nor permitting host presentation of FL-SGG, since there were requirements for P388AD.2 viability, and for syngeneic recipients in order to generate augmented anti-FL antibody responses. Moreover, inappropriate presentation of FL-SGG by P388AD.2 injected into allogeneic recipients did not elicit positive or negative signalling. In order to demonstrate that the ability of P388AD.2 to present FL-SGG in an immunogenic fashion was not simply a property of all tumor cells, the P388D1 cell line was pulsed with FL-SGG and injected. Neither tolerance nor augmentation was induced. Overall these results demonstrate that the type of antigen-presenting cell which introduces the immune system to an immunoglobulin tolerogen is critical to the induction of B-cell unresponsiveness or priming.