Protective and other biological properties of Bacillus anthracis soluble antigens.

The soluble antigens were explored of the culture filtrate (CF) derived during static growth of B. anthracis vaccine strain 34F2 on a medium containing casein hydrolysate. Electrofocusing of CF preparations revealed that the protective activity was distributed over a wide range of pH 3-7. The most pronounced and stable protective activity was observed at pH 4.6-4.8. Following toxin factors were isolated and identified: protective antigen (87 kD), oedema factor (87 kD) and lethal factor (78-81 kD). The greatest protective activity was associated with antigens characterized by a molecular weight of 78-87 kD and toxic activity. Preparations of the oedema and lethal factors had the same protective activity as protective antigen (PA) preparations. Other CF soluble antigens protected about 30% of immunized guinea pigs. A protein was isolated with a molecular weight of 80 kD and isoelectric point at pH 5.3-5.7 which was not toxic and did not form toxic mixtures in association with other toxin factors; this protein featured a high immunogenic activity, however, it protected only 31% of immunized animals. Factors are analyzed which determine differences in the protective effects of live and chemical vaccines.

[The assessment of antitoxic anti-anthrax immunity]. / Otsenka antitoksicheskogo protivosibireiazvennogo immuniteta.

In experiments on guinea pigs immunized with avirulent noncapsular strains STI, Sterne (34F2) and the avirulent mutant of Bacillus anthracis strain 228/8 the relationship between the titers of serum antibodies to the preparations of purified protective antigens (PA) and purified lethal factor (LF) of B. anthracis toxin and the level of the antitoxic activity (ATA) of blood sera, as well as acquired resistance, was analyzed. The ATA of sera was evaluated in the primary culture of peritoneal macrophages affected by the mixture of PA and LF. The level of relationship (r) between individual ATA values and the titers of antibodies to PA and LF was shown to vary over a wide range, depending on the group of the animals and did not exceed, on the average, 0.19-0.37. At the same time the mean values of these characteristics, followed in their dynamics depending on the immunogenic properties of vaccine strains or the time elapsed after vaccination, were highly correlated (r = 0.76-0.87). The possibility of using these characteristics for the evaluation of acquired resistance are discussed.

[Postinfection and postvaccinal antianthrax immunity in human subjects]. / Postinfektsionnyi i postvaktsinal'nyi protivosibireiazvennyi immunitet u liudei.

Antibodies to Bacillus anthracis protective antigen (PA) and to the lethal factor (LF) of B. anthracis exotoxin in the blood sera of anthrax patients and of subjects with a history of the disease, as well as of persons immunized with STI live vaccine, were studied by the heterogeneous enzyme immunoassay. In 1-6 years after convalescence the levels of anti-PA and anti-LF antibodies (at 75% and 96% detection rates respectively) were higher than on weeks 1-4 from the onset of the disease. In persons having had anthrax antibodies belonged mainly to IgG, and the anti-LF antibody level was higher than the anti-PA antibody level. In persons immunized with STI vaccine the detection rate of antibodies somewhat increased in 2-7 months after immunization, reaching, on the average, 72%, the antibody levels after primary immunization and regular annual booster immunization being similar. In 1-2 years after primary (booster) immunization the isolation rate of antibodies decreases to 21%. Specific features of postinfectious and postvaccinal immunity to anthrax and problems of retrospective diagnosis of this disease are discussed.

[The effect of the protective antigen of Bacillus anthracis on the formation of immunity under the action of live anthrax vaccines]. / Vliianie protektivnogo antigena Bacillus anthracis na formirovanie immuniteta pod deistviem sibireiazvennykh zhivykh vaktsin.

The preparation of low-molecular protective antigen (PA) isolated from strain 34F2 (Sterne) and having a molecular weight of 34 and 51 kD, unlike the preparation of high-molecular PA with a molecular weight of 87 kD, suppressed the formation of acquired resistance to anthrax when introduced into guinea pigs in mixture with live spores of strains of STI, 34F2 and new vaccine strain 228/8; this phenomenon was mainly accompanied by a decrease in the level of antibodies to lethal factor (LF) nad in the antitoxic activity of blood serum. The immunosuppressing action of low-molecular PA depended on the kind of vaccine strain introduced together with this antigen, which suggested the existence of differences in the ligand determinants of strains 34F2 and STI. In contrast to high-molecular PA, low-molecular PA blocked the action of the lethal mixture of PA and LF on the culture of peritoneal mononuclear phagocytes of CBA mice. The competitive relationships between low-molecular PA and high-molecular PA are discussed.

[The effect of lethal anthrax toxin on the functional activity of peritoneal mononuclear phagocytes and polymorphonuclear neutrophils in mice]. / Vliianie letal'nogo sibireiazvennogo toksina na funktsional'nuiu aktivnost' peritoneal'nykh mononuklearnykh fagotsitov i polimorfnoiadernykh neitrofilov myshei.

The mixture of purified protective antigen (PA) and the lethal factor (LF) of B. anthracis exotoxin in a dose incapable of causing cell damage inhibited the zymosan-stimulated luminol-dependent chemiluminescence of mononuclear phagocytes (MP) and polymorphonuclear neutrophils (PMN) in CBA mice and enhanced the chemiluminescence of PMN in BALB/c, CC57W and A/Sn mice. The conclusion is made that the capacity of mixture of PA and LF for the in vitro inhibition of stimulated production of active forms of oxygen in MP and PMN is directly related to the in vivo immunosuppressing activity of B. anthracis toxin. But the influence of the mixture of PA and LF on the production of active forms of oxygen in MP and PMN is not the leading factor of natural immunity to B. anthracis infection in mice.

[The detection of specific antigens in experimental anthrax]. / Vyiavlenie spetsificheskikh antigenov pri éksperimental'noi sibirskoi iazve.

The dynamics of various specific antigens was studied in guinea pigs infected with strain 72/12 of Tsenkovskii's second vaccine. The study showed that at the acute stage of the disease toxin antigens prevailed over the levels of somatic antigens and nonprotective protein with a molecular weight of 79 KD. The enzyme immunoassay system for the detection of the lethal toxin factor permitted the detection of the antigen in the blood sera of 100% of infected animals at the prodromal period and the acute stage of the disease. In pathological material obtained from skin lesions the presence of toxin antigens and nonprotective protein was registered in 90-100% of the animals. The diagnostic significance of these assays for the early rapid diagnosis of anthrax during lifetime, as well as for the postmortem rapid diagnosis of the disease.

[Immunosuppressive action of the lethal toxin of Bacillus anthracis in mice]. / Immunosupressivnoe deistvie letal'nogo toksina Bacillus anthracis na myshei.

In experiments on inbred mice infected with B. anthracis capsular strain 71/12 of Tsenkovsky's second vaccine B. anthracis lethal toxin introduced in mixture with spores has been shown to aggravate anthrax infection in CBA mice susceptible to anthrax, while producing a faint effect on the infectious process in BALB mice with hereditary resistance to anthrax. B. anthracis purified edema toxin has been found to produce a weaker aggravating effect with respect to anthrax infection than the lethal toxin. As revealed in these experiments, the capacity of the lethal toxin to suppress the activity of peritoneal macrophages in vitro is the more pronounced, the more resistant to anthrax are the mice used as the donors of these macrophages. The mechanism of hereditary immunity which may ensure resistance to infection in the presence of immunosuppression is discussed.

[Effect of the lethal Bacillus anthracis toxin on phagocytosis and the dynamics of the change in the enzyme activity of the antioxidative system of peritoneal mononuclear phagocytes in mice with differing hereditary immunity to anthrax]. / Vliianie letal'nogo toksina Bacillus anthracis na fagotsitoz i dinamiku izmeneniia aktivnosti fermentov antiokislitel'noi sistemy peritoneal'nykh mononuklearnykh fagotsitov myshei s razlichnym nasledstvennym immunitetom k sibirskoi iazve.

It was demonstrated, that the lethal in vitro suppressed the phagocytic activity of peritoneal mononuclear phagocyte (Mph) and enhanced the level activity of glutathione peroxidase to H2O2 (GP-H2O2) in Mph of resistant to anthrax BALB mice. In Mph BALB the authors observed dependent-dose enhancement of GP-H2O2 activity and reduction of the ratio of level glutathione reductase (GR) to GP-H2O2 (GR/GP-H2O2). The enhancement of activity GR-H2O2 in Mph CBA was not dependent on the doses of toxin. The coefficient GR/GP-H2O2 was similar to the control. The mechanisms of hereditary resistance to anthrax were discussed.

[Purification and characteristics of a protective factor from Bacillus anthracis toxin]. / Ochistka i kharakteristika protektivnogo faktora toksina Bacillus anthracis.

It was found that during filtration of a sterile toxic cultural supernatant (TCS) obtained by 24 hour cultivation of the vaccinal strain through a column packed with porous glass or silochrome not only oedematic (OF) and lethal (LF), but also protective (PF) factors of toxin are adsorbed on the column. Elution of adsorbed antigens allowed for rapid concentration and purification of biologically active components of toxin from large volumes of TCS under conditions of limited proteolysis. The experimental results suggest that in 24 hour TCS and PF exists as large (87 kD) molecules as well as low molecular weight fragments whose molecular mass is of the order of 17-18 kD. The PF preparations whose molecular mass is below 68 kD possess a weak biological activity.

[Use of immunological adsorption in heterogeneous immunoenzyme analysis in determining antibodies to the protective determinants of Bacillus anthracis]. / Primenenie immunologicheskoi adsorbtsii v geterogennom immunofermentnom analize pri opredelenii antitel k protektivnym determinantam Bacillus anthracis.

In a heterogeneous enzyme immunoassay system involving the use of polystyrene assay plates, the method of immunological adsorption has been used for studying the spectrum of specific antibodies to individual chromatographically pure fractions of B. anthracis toxin. The relationship between the characteristics of acquired stability and the level of serum antibodies to individual biologically active and biologically inactive toxin antigens in guinea pigs, immunized with live vaccines in a single injection, has been studied. As revealed in this study, the level of serum antibodies to chromatographically pure toxin fractions does not reflect acquired immunity to anthrax.

[Use of immunological adsorption in a heterologous immunoenzyme analytical system for detecting specific antibodies to serogroup B and D1 Salmonella lipopolysaccharides in healthy donors and in persons with a history of salmonellosis]. / Primenenie immunologicheskoi adsorbtsii v sisteme geterogennogo immunofermentnogo analiza pri vyiavlenii spetsificheskikh antitel k sal'monelleznym lipopolisakharidam serogrupp B i D1 u zdorovykh donorov i perebolevshikh sal'monellezami liudei.

The sera obtained from healthy donors and from subjects with a history of Salmonella infections (B, C1, D1 and E1) of medium severity were studied. In the initial testing in the heterologous enzyme immunoassay the levels of antibodies to LPS, groups B and D1, were somewhat higher in the subjects with a history of the infections than in healthy donors. After a single adsorption with homologous LPS the levelling of differences between the two groups was observed. After two adsorptions with heterologous LPS the level of the reaction in both the groups increased, and the differences between these groups augmented. The adsorption of sera with heterologous LPS increased the diagnostic sensitivity of the reaction only to 14-23% in the determination of the serogroup of the causative agent. An increase in the antibody level after adsorption with heterologous LPS was supposedly due to the additional detection of group-specific antibodies with low avidity, which appeared as the result of the elimination of cross-reacting highly avid antibodies, as well as non-immunoglobulin molecules complementary to LPS.

[Hereditary resistance to anthrax and sensitivity to the anthrax toxin in mice]. / Nasledstvennaia ustoichivost' k sibirskoi iazve i chuvstvitel'nost' k sibireiazvennomu toksinu u myshei.

The degree of the hereditary susceptibility of mice to anthrax caused by noncapsular and capsule-forming Bacillus anthracis strains has been found to be directly related to the sensitivity of the animals to the edematogenic and immunosuppressing action of anthrax toxin. The genetic analysis indicates that resistance to anthrax is probably controlled by a dominant gene, not linked with histocompatibility complex H-2 and, probably, unrelated to the presence of hemolytic activity in mouse sera, determined by component C5 of the complement.