TBA225, a fusion toxoid vaccine for protection and broad neutralization of staphylococcal superantigens.

Superantigens (SAgs) play a major role in the pathogenesis of Staphylococcus aureus and are associated with several diseases, including food poisoning, bacterial arthritis, and toxic shock syndrome. Monoclonal antibodies to these SAgs, primarily TSST-1, SEB and SEA have been shown to provide protection in animal studies and to reduce clinical severity in bacteremic patients. Here we quantify the pre-existing antibodies against SAgs in many human plasma and IVIG samples and demonstrate that in a major portion of the population these antibody titers are suboptimal and IVIG therapy only incrementally elevates the anti-SAg titers. Our in vitro neutralization studies show that a combination of antibodies against SEA, SEB,and TSST-1 can provide broad neutralization of staphylococcal SAgs. We report a single fusion protein (TBA225) consisting of the toxoid versions of TSST-1, SEB and SEA and demonstrate its immunogenicity and protective efficacy in a mouse model of toxic shock. Antibodies raised against this fusion vaccine provide broad neutralization of purified SAgs and culture supernatants of multiple clinically relevant S. aureus strains. Our data strongly supports the use of this fusion protein as a component of an anti-virulence based multivalent toxoid vaccine against S. aureus disease.

[Cytokine profile in mice with enhanced or depressed immune response to sheep erythrocytes induced by immunomodulator of bacterial origin--purified staphylococcal toxoid].

Influence of immunomodulator of bacterial origin - purified staphylococcal toxoid (PST) - on the synthesisof proinlammatory (IL-1beta, IL-6, TNFalpha, IFN-gamma) and anti-inflammatory (IL- 10) cytokines, as well as cytokines directing the immune response to Th1 (IL-12) or Th2 (IL-4) type was studied in mice. Serum cytokines levels as well as levels of cytokines produced by splenocytes spontaneously or after stimulation by phytohemagglutinin were measured 4 and 24 hours after inoculation of PST. It was shown that PST in wide spectrum of doses (15; 1.5; 0.15 BU per mouse) was able to enhance or suppress synthesis of cytokines. Effect was nonlinear and its direction was depended from cytokine, time interval passed before obtaining the sample and dose of PST. For example, 15 BU of PST enhanced whereas 0.15 BU of PST suppressed the IL-6 production 4 hours after inoculation. Decrease of IL-6 level in serum 24 hours after inoculation of PST was detected. Synthesis of several serum interleukins (IL-2, IL-10) did not changed 4 and 24 hours after inoculation irrespective from dose of PST. It was demonstrated that modulation of humoral immune response in vivo induced by PST did not associated with modulation of cytokine profile. For example, increase of number of cells secreting antibodies to sheep erythrocytes was registered both during increased synthesis of cytokines (4 hours, IL-1beta, IL-6, IL-12) and during period of its depression (IL-6, TNF-alpha, IFN-gamma), as well as during stable production of cytokines (IL-1beta, IL-6, IFN-gamma).

Death by a B cell superantigen: In vivo VH-targeted apoptotic supraclonal B cell deletion by a Staphylococcal Toxin.

Amongst the many ploys used by microbial pathogens to interfere with host immune responses is the production of proteins with the properties of superantigens. These properties enable superantigens to interact with conserved variable region framework subdomains of the antigen receptors of lymphocytes rather than the complementarity determining region involved in the binding of conventional antigens. To understand how a B cell superantigen affects the host immune system, we infused protein A of Staphylococcus aureus (SpA) and followed the fate of peripheral B cells expressing B cell receptors (BCRs) with VH regions capable of binding SpA. Within hours, a sequence of events was initiated in SpA-binding splenic B cells, with rapid down-regulation of BCRs and coreceptors, CD19 and CD21, the induction of an activation phenotype, and limited rounds of proliferation. Apoptosis followed through a process heralded by the dissipation of mitochondrial membrane potential, the induction of the caspase pathway, and DNA fragmentation. After exposure, B cell apoptotic bodies were deposited in the spleen, lymph nodes, and Peyer's patches. Although in vivo apoptosis did not require the Fas death receptor, B cells were protected by interleukin (IL)-4 or CD40L, or overexpression of Bcl-2. These studies define a pathway for BCR-mediated programmed cell death that is VH region targeted by a superantigen.

[Effect of purified staphylococcal toxoid preparation on the in vitro production of interferon by leukocytes from patients with atopic dermatitis]. / Vliianie preparata stafilokokkovogo ochishchennogo anatoksina na produktsiiu interferona leikotsitami in vitro u bol'nykh atopicheskim dermatitom.

The impact of purified staphylococcal toxoid (PST) on the in vitro production of interferon (IFN) by blood leukocytes was evaluated. PST was found to produce a stimulating effect on the production of alpha-IFN in patients with atopic dermatitis (AD): in 88% of cases a two-fivefold increase in IFN production was observed in AD patients in comparison with healthy donors. In addition, the incubation of leukocytes obtained from atopic and nonatopic patients with PST was shown to stimulate the production of gamma-IFN by these leukocytes fivefold, on the average. These results give theoretical basis for use of PST (as an inducer of endogenic IFN) for AD patients treatment.

[The mast cell reaction to a staphylococcal infection occurring against a background of immunodeficiency after cyclophosphane administration]. / Reaktsiia tuchnykh kletok na stafilokokkovuiu infektsiiu, protekaiushchuiu na fone immunodefitsita posle vvedeniia tsiklofosfana.

Materials on the study of the morphofunctional state of mast cells in mouse in experimental staphylococcal infection under the conditions of cyclophosphamide-induced immunodeficiency are presented. As revealed in this study, the infectious process developing in the presence of immunodeficiency is accompanied by the profound and prolonged suppression of the morphofunctional status of mast cells and natural immunity factors at the peak of the disease.

Summing up, weak immunomodulating signals of Tahyna virus and immunomodulating drugs induce immunosuppression in mice.

The successive injection of non-immunomodulating doses of Tahyna virus (100 LD50) and non-immunomodulating doses of immunomodulating drugs, such as purified staphylococcal toxoid or glucosaminylmuramyldipeptide (Likopid), to mice were accompanied by a decrease in the IgM plaque-forming cell response to sheep red blood cells.

[The effect of immunomodulating doses of purified staphylococcal anatoxin on the spontaneous and mitogen-induced proliferation of mouse splenocytes]. / Vliianie immunomoduliruiushchikh doz anatoksina stafilokokkovogo ochishchennogo na spontannuiu i mitogenindutsirovannuiu proliferatsiiu splenotsitov myshei.

Purified staphylococcal toxoid (PST) was shown to alter the spontaneous and mitogen-induced proliferation of mouse spleen cells. In vitro, PST inhibited spontaneous proliferation, as well as proliferation induced by the optimal dose of Con A (2 micrograms/ml) and the optimal and suboptimal doses of lipopolysaccharide (LPS) (100 and 50 micrograms/ml). At the same time the dose of 1.5 binding units (BU) of PST, inhibiting spontaneous proliferation in vitro, induced strong proliferative response in combination with the suboptimal dose of Con A (1 microgram/ml). Our experiments demonstrated that the spontaneous proliferation of mouse spleen cells, immunized with the toxoid, remained unchanged (0, 15 or 15 BU/mouse) or increased (1.5 BU/mouse) the response of spleen cells of immune animals to ConA and LPS also changed in comparison with the control, depending on the conditions of the experiment. After the use of the combination of 2 micrograms of Con A and 1.5 BU of PST or 100 (50) micrograms of LPS and 1.5 BU of PST the inhibition of proliferative response was observed. The summation of the signals of the suboptimal dose of Con A (1 microgram) 1.5 BU of PST was demonstrated.

[The modulation of cellular immunity in vivo and in vitro under the action of purified staphylococcal anatoxin]. / Moduliatsiia kletochnogo immuniteta in vivo i in vitro pod deistviem anatoksina stafilokokkovogo ochishchennogo.

Purified staphylococcal toxoid modulates (mainly suppresses) cell-mediated immune response to heterogeneous antigens of animal origin (to sheep red blood cells as shown by the delayed hypersensitivity reaction) or bacterial origin (to BCG as shown by the splenocyte migration test). The direction and manifestation of modulation depend on the strain of mice, dose of the toxoid, dose and nature of the test antigen and the immunization schedule (intervals between the injections of the antigen).

Staphylococcus aureus alpha-toxin permeabilizes the basolateral membrane of a Cl(-)-secreting epithelium.

Apical membrane ion channels control the rate of transepithelial electrolyte transport in many epithelia. One way to study such channels in their native location, the apical membrane, is to eliminate the resistance of the basolateral membrane to ion flow. Then the opening and closing of apical channels can be measured as a transepithelial current, free from the influence of basolateral membrane transport processes. To develop a method that would permeabilize an epithelial basolateral membrane to ions and nucleotides, we examined the effect of Staphylococcus aureus alpha-toxin on the Cl(-)-secreting T84 epithelial cell line. alpha-Toxin permeabilized the basolateral, but not the apical membrane to Cl-, adenosine 3',5'-cyclic monophosphate (cAMP), and GTP. However, the integrity of signal-transduction pathways, the regulation of apical membrane Cl- channels, and the transepithelial resistance remained intact. In the course of examining the effect of ATP, we found that the basolateral membrane contained purinergic receptors that both stimulated Cl- secretion on their own and, at high concentrations, inhibited cAMP-induced Cl- secretion. These effects of extracellular ATP were eliminated after prolonged exposure to ATP, suggesting receptor downregulation. In addition, depletion of intracellular ATP following permeabilization prevented cAMP-dependent regulation of apical Cl- channels. We conclude that alpha-toxin may prove to be a useful tool for studying the regulation and properties of apical membrane ion channels.

[The immunomodulating properties of a diphtheria bacterial vaccine in in vitro experiments]. / Immunomoduliruiushchie svoistva difteriinoi bakterial'noi vaktsiny v opytakh in vitro.

In this investigation lymphocytes sensitized with Corynebacterium diphtheriae antigens obtained from carriers and convalescents were used. The new diphtheria bacterial vaccine Codivac, in contrast to other comparable preparations (diphtheria toxoid, staphylococcal toxoid, staphylococcal vaccine, levamisole), was found to produce a more direct effect by modulating the levels of T-lymphocytes, depending on their initial levels in the patient. Codivac, together with other preparations, can be used for the study of the problems of immunostimulation and immunocorrective therapy.

[Effects of surface-active substances with antistatic properties on several immunity indices]. / Vliianie poverkhnostno-aktivnykh veshchestv s antistaticheskimi svoistvami na nekotorye pokazateli immuniteta.

In experiments performed on rats, it was established that the antibodies' formation was stimulated as a result of a combination of staphylococcal anatoxin or E. coli vaccine immunization with the administration of the non-ionogenic surface-active substances (SAS) Stearox-920 and OC-20. Those SAS proved to have immunostimulating properties, and Stearox-920 was more effective. Another experimental study revealed a growing bactericide activity of the skin, which could be seen from the rate of its self-cleaning from the standard E. coli suspension.

[Effect of staphylococcal toxin and antistaphylococcal gamma-globulin on the electrical and contractile activity of the guinea pig myocardium]. / Vliianie stafilokokkovogo toksina i ego kompleksa s antistafilokokkovym gamma-globulina na élektricheskuiu i sokratitel'nuiu aktivnost' miokarda morskoi svinki.

A study was made of the action of staphylococcal toxin (ST) and its combination with antistaphylococcal gamma-globulin (ASGG) on intracellular potentials (rest potential--RP, and action potential--AP), and isometric contractions of guinea-pig auricle. ST (initial concentration 18.10(-2)Lh) diluted with normal Tyrode's solution at 1:1000, 1:100 and 1:10 (spontaneously active preparations), and Tyrode's solution with 13.5 mM KCl (evoked activity of preparations), significantly increased the duration of AP of myocardial cells. In evoked activity of preparations, RP and the amplitude of AP declined as the concentration of ST was raised. The amplitude of isometric contractions and maximal rates of their growth and fall increased under the effect of ST (1:1000) and decreased at 1:100 and 1:10. ASGG combined with ST (1:100) did not produce any protective effect on the myocardium. On the contrary, it provoked a still greater inhibition of contractility. The inhibitory action of combined ST and ASGG was seen at all ratios of ST to ASGG (use was made of ASGG shortage, equivalent amount and excess as regards ST) and reached 50% for all study characteristics of contractility. Anatoxin (inactivated toxin) combined with ASGG also produced a cardiodepressant action which was manifested in an approximately 50% decrease in the maximal rate of the growth and fall of contractions in the absence of significant changes in the contraction amplitude.

Effects of diphtheria toxin and other exotoxins on oxidant generation by human and murine phagocytes.

Bacterial exotoxins such as diphtheria toxin (D.T.), staphylococcal alpha toxin and Leucocidin can powerfully activate granulocytes and macrophages as detected by production of chemiluminescence in presence of Luminol. Production of superoxide by granulocytes and of prostaglandin E2 in macrophages is also stimulated by D.T. In contrast with the known resistance of rodent parenchymal cells to the diphtheria toxin, human and rodent leucocytes have similar sensitivities to D.T.

The binding of fluorescein-labelled stapbylococcal alpha toxoid to erythrocytes.

Erythrocytes of different animal species have variable hemolytic sensitivity to staphylococcal alpha toxin. Specific and non-specific binding of toxin was measured using fluorescein-labelled toxoid. These studies indicate that toxoid binding to erythrocytes increases with concentration for all species tested. Scatchard plot analyses of 35 animals representing seven species indicate that rabbit, pig, cow, and chicken erythrocytes possess 125 980, 103 920, 82 500, and 41 200 receptors per cell, respectively. The number of receptors remains constant over a period of at least 10 days. No detectable receptors were found for human, rat, and guinea pig erythrocytes. A correlation coefficient of 0.992 exists between receptor number and hemolytic sensitivity for those species having receptors. Variation in hemolytic sensitivity is governed by receptor number and not by variation in the dissociation constant. A threshold sensitivity of 37 000 receptors per cell has been calculated. Since species lacking detectable receptors have considerable sensitivity to hemolysis, it is proposed that two binding mechanisms, specific and non-specific, exist which prepare erythrocytes for destruction.