Crude Toxoid Research Articles

Production, immunogenicity, stability, and safety of a vaccine against Clostridium perfringens beta toxins.

Background and Aim:The beta toxin is causing the most severe Clostridium perfringens-related diseases. This work was dedicated to developing a vaccine against beta toxin using C. perfringens type C (NCTC 3180).Materials and Methods:The crude toxoid harvest contained 710 limits of flocculation (Lf)/mL. The vaccine was formulated. Each 1 mL of the final vaccine product contained at least 50 Lf/mL of beta toxoids, 0.2 mL 3% aluminum hydroxide gel (equivalent to 5.18 mg of aluminum), <0.001% W/V thiomersal, formaldehyde <0.05% W/V, and ~0.7 mL phosphate-buffered saline (pH 7.2). The efficacy of the vaccine was evaluated by potency, stability, and safety tests.Results:The vaccine demonstrated 24.36 IU/mL (standard deviation, ±0.56) and 14.74 IU/mL (±0.36) of neutralizing antibodies in rabbits and cattle, respectively. Indeed, these levels were above the minimum recommended by international protocols since the obtained antibody levels had 2.43- and 1.47-fold increase in both rabbits and cattle, respectively, over the minimum antitoxin level suggested by the United States Department of Agriculture. Interestingly, our formulation was capable of inducing 1.65-fold higher immune responses in rabbits than that stimulated in cattle (65% increase) with a significant difference (p<0.0001). The vaccine was stable up to 30 months. The vaccinated rabbits were suffered from a temporarily slight increase in temperatures in the first 10 h without any significant difference (p>0.05).Conclusion:The research showed a procedure for the manufacturing process of the vaccine against C. perfringens beta toxins with a feasible quantity and the vaccine described here showed to be effective in eliciting levels of neutralizing antibodies higher than required by international standards. In addition, The vaccine was stable up to 30 months. Thus, it may represent an effective and safe for preventing C. perfringens-related diseases in rabbits and cattle, although further studies to prove its efficacy in the field on other farm animals are still needed.

Serum neutralizing antibody response and protection against experimentally induced liver abscesses in steers vaccinated with Fusobacterium necrophorum

Abstract Objective To determine the efficacy of leukotoxin-based Fusobacterium necrophorum vaccines and dietary tylosin in providing protection against experimentally induced hepatic abscesses in steers. Design 30 steers assigned randomly to 6 treatment groups of 5 steers each: 1, phosphate-buffered saline solution (PBSS; control); 2, PBSS control, fed tylosin (100 mg/steer) daily; 3, inactivated whole-cell culture with oil emulsion adjuvant; 4, culture supernatant (crude toxoid) with oil emulsion adjuvant; 5, semipurified leukotoxoid with oil emulsion adjuvant; and 6, semipurified leukotoxoid with saponin adjuvant. Procedure Steers were inoculated SC with emulsified antigen or PBSS on days 0 and 21. Blood samples were collected at weekly intervals to monitor serum antileukotoxin antibody titer. On day 42, all steers were challenge exposed intraportally with F necrophorum culture. Three weeks later (day 63), steers were euthanatized and necropsied to examine liver and assess protection. Results Antileukotoxin antibody titers of all vaccinated groups markedly increased from baseline values, and mean titers of vaccinated groups were higher than those of the control and tylosin-treated groups. Steers vaccinated with culture supernatant with oil emulsion adjuvant or semipurified leukotoxoid with saponin adjuvant had the highest mean antibody titers. All 5 steers in the control group developed liver abscesses. Tylosin feeding did not protect steers challenge exposed with F necrophorum intraportally. Conclusions Culture supernatant was more protective than whole-cell culture or semipurified leukotoxin against experimentally induced hepatic abscesses. Partial purification of leukotoxin appeared to reduce its protective immunity. (Am J Vet Res 1996;57:483–488)

Characterization of the immunogenicity of formaldehyde detoxified Pasteurella multocida toxin

The immunogenicity of the Pasteurella multocida toxin (PMT) was studied in murine model systems. Mice were vaccinated with either formaldehyde treated pure PMT (pure toxoid) or formaldehyde treated crude extract of toxigenic P. moltocida (crude toxoid). The corresponding mean anti-PMT titres,sero-conversion rates and survival rates after challenge with affinity purified PMT were compared. When assessed both by anti-PMT titres and seroconversion and challenge, pure toxoid was a more potent immunogen than crude toxoid. This greater immunogenic potency was unaffected by the addition of killed cell preparations of Bordetella bronchiseptica, non-toxigenic P. multocida and B. pertussis. Increasing anti-PMT titres and seroconversion rates were induced by increasing doses of formaldehyde treated PMT (fPMT) in the pure toxoid vaccines, but not in the vaccines containing crude toxoid. However, improved survival rates were observed for both types of vaccine, when the fPMT content was raised. Immunization of pregnant mice with vaccines containing fPMT induced protection of the offspring against challenge with PMT; the protection of the offspring corresponded to that of the mother.

Immunogenicity of purified Habu snake-venom toxoid in guinea pigs and monkeys with special reference to HR1 component.

Purity of the parent toxin influenced greatly the immunogenicity in guinea pigs of HR1 component of Habu-venom toxoid. The potency of HR1 toxoid in terms of the immunizing unit (ImU) appeared to be related to the antigen and aluminum contents. The immune response of the animals differed depending on the purity of the toxoid when the dose-response curve was examined over a wide range of the antigen dosage. Anti-HR1 titers of the guinea pigs immunized with crude toxoid (adsorbed) reached a plateau at about 10-20 U/ml ane further rise was not remarkable even when the amout of the antigen was increased several times, while 200 U/ml (maximum 400 U/ml or higher) of annti-HR1 was produced with a large amount of highly purified HR1 toxoid. Purified toxoid showed an excellent immunogenicity also in monkeys. The amounts of antigens necessary for the basic immunization to resist the challenge with a certainly lethal dose of crude venom were 18 and 0.5 mg as protein, for a crude and a highly purified toxoid, respectively. The total amount of aluminum was also reduced from 8.1 (crude toxoid) to 0.5 mg (purified toxoid). Multiple injections for the primary immunization enhanced early production of anti-HR1 in monkeys. However, at later stage of immunization, significant correlation was observed between the amount of HR1 toxoid (in ImU) for the primary immunization and the circulating anti-HR1 titer irrespective of the schedule of immunization. Anti-HR2 higher than 10 U/ml was produced consistently in monkeys with HR2 toxoid of 1.0 ImU for the basic immunization. However, excess HR2 toxoid of more than 10 ImU seemed rather unfavorable for anti-HR2 production.

The role of antigen form and function in the primary and secondary intestinal immune responses to cholera toxin and toxoid in rats.

This report describes studies of the mucosal antitoxic response in rats after enteric administration of several forms of cholera toxin or toxoid, proteins which differ primarily in their ability to bind to cell membranes and activate cellular adenyl cyclase. These two characteristics appeared to markedly enhance the local primary response to these antigens. A single dose of toxoid lacking these features was ineffective in local priming even though it was absorbed and induced a systemic immune response. Single dose mucosal priming occurred only with preparations which bind to cell membranes and was enhanced by those which also activate cellular adenyl cyclase. In contrast, single-dose mucosal boosting was best accomplished by materials with these properties but was also seen with a toxoid lacking both of these functions. The property of membrane binding appears to be most advantageous in mucosal priming, perhaps by increasing effective trapping of absorbed antigen in unprimed mucosal lymphoid tissue, whereas the ability to activate adenyl cyclase appears to enhance primary and secondary type responses about equally. Combinations of crude toxoid and toxin were also more effective in mucosal priming than purified materials, a finding which is unexplained. A single dose of this combination induced mucosal priming which was fully developed in 2 wk, undiminished after 4 too, and only modestly diminished after 8 mo, thus demonstrating relatively prolonged memory in the IgA mucosal immune system. Effective two-dose local immunizing regimens were developed, and it was shown that there was no correlation between the mucosal and systemic secondary antitoxin responses provoked by these regimens.

ジフテリア菌ならびにその他のコリネバクテサウムの血清学的分類とジフテリアトキソイドによるアレルギー反応に関する研究

The antigenic structures of Corynebacterium including C. diphtheriae and others were studied by agglutination reaction and it was confirmed that the strains so far tested contained heat-labile and Oinagglutinable antigen and heatstable antigen. The heat-labile antigen was rather type-specific and strainspecific and heat-stable antigen was cross-reacting to almost all of the tested strains including C. belfanti, C. hofmannii, C. xerosis, and C. ulcerans.In this connection the experimental allergic reaction of guinea pig was investigated and it was confirmed that heat-stable antigen other than toxoid could be responsible for the allergic reaction to the crude toxoid in the market, but it did not sensitize the guinea pig against the purified diphtheria toxoid in the purity of 3300 Lf per mg. P. N.And it was confirmed that the guinea pig could be sensitized against the diphtheria toxoid in the purity of 3300 Lf per mg. P. N.

Immunogenesis and nonspecific factors of natural resistance

A study was made of the change in the blood serum lysozyme content of rabbits immunized with the following vaccine preparations: crude toxoid, chemical divaccine, and microbial tetravaccine against intestinal infections, sorbed diphtheria toxoid, gangrene-tetanus tetratoxoid. It was shown that vaccination caused a distinct reduction of the blood lysozyme content. Restoration of the lysozyme titer to the initial level was observed to occur in most cases not earlier than 20 days after the preparation had been given. There was a direct relationship between the immunizing power of the vaccine (its aount, antigenic activity, and number of administrations) and the extent of the reduction of the lysozyme titer. A definite correlation was noted between the blood content of specific antibodies and lysozyme: the time for the greatest antibody titer rise corresponded to the periods of least lysozyme content.

Factors relating to the virulence of Staphylococci. III. Antibacterial versus antioxic immunity.

Antitoxic and antibacterial immunity have been clearly differentiated in the experimental mouse infection produced by the diffuse colonial variant of the Smith strain of Staphylococcus aureus. Immunization with crude toxoid protected mice from otherwise lethal doses of alpha hemolysin, but did not alter mortality following intraperitoneal infection with living staphylococci. Conversely, animals immunized with heat killed vaccines were readily killed by culture supernates containing alpha hemolysin, but were strikingly protected from otherwise fatal intraperitoneal infection with viable staphylococci. Protection was directly related to the ability of the immunizing substance to promote early intraperitoneal phagocytosis of the infecting inoculum. In these studies with the Smith diffuse variant, rapid intraperitoneal phagocytosis was induced by vaccination with whole cell bacterial vaccines but not by alpha hemolysin toxoid.

Purification of tetanus toxin

The combination of multi-membrane electro-decantation and ammonium sulphate fractionation resulted in an effective purification of tetanus toxin. The most highly purified material obtained in this way contained as much as 4300–4800 Lf per mg protein nitrogen. This toxin is also pure as judged by electrophoretic and sedimentation analyses. The purified toxin yields a single, sharp band in the gel diffusion test and is therefore free of contaminating antigens as far as can be shown by this test. Consequently its use as a prophylactic should produce less side reaction. However, the antigenicity in mice, receiving a single immunising injection of formol toxoid, was shown to be only 35% of that of the crude toxoid. This fact need not detract from the value of the pure toxoid as a prophylactic, since it has other advantages which outweigh its lesser antigenicity under the conditions of these tests.

Protamine tetanus toxoid.

A NUMBER of reports dealing with reactions following immunization against tetanus have appeared in recent years. 1 The elimination of Witte's® peptone from the medium in which the bacillus is grown seemed to reduce the frequency of disturbing reactions. Nevertheless, it appears desirable to improve on the fluid toxoids being used, as well as on the product obtained by the simple addition of relatively large amounts of alum to such crude toxoids. Several methods of purifying crude toxoid or toxin have been described in the literature. Boivin and Izard 2 used trichloroacetic acid to precipitate the toxoid and thereby obtained a considerable removal of inert substances. Eaton and Gronau 3 precipitated tetanus toxin with cadmium chloride, after a previous treatment with calcium chloride. Toxin was then eluted and reprecipitated with ammonium sulfate and the latter removed by dialysis. Pickett, Hoeprich and Germain, 4 following the same procedure and applying it

A Method for Purifying Diphtheria Toxoid and Combining it with Protamine

Discussion and Summary The use of protamine offers a means of separating bacillary protein and therefore removes a major protein component of crude diphtheria toxoid. No specific and constant formula, in terms of quantity of protamine or with respect to ionic strength can be given which will apply equally well to crude toxoids made in broths of varying composition. It is necessary to determine optimal conditions for each lot. It is desirable to employ toxoids that contain only minimal amounts of protein derivatives precipitated by trichloracetic acid at pH 4.0. Using such preparations, this step precipitates toxoid of high purity, once the bacillary protein is removed. On further treatment with Norite, preparations containing 2000 to 2100 Lf/mgN were obtained. Several additional steps have been employed between the removal of bacillary protein and the addition of trichloracetic acid when the crude toxoid contained larger amounts of protein derivatives originating in the production broth. The chemical steps employed in preparing toxoid of this degree of purity do not appear to have resulted in noticeable changes in the stability of the toxoid, since flocculation has occurred fairly promptly with solutions in phosphate buffer held at 2 C for approximately 2 years and solutions held at 37 C for 4 weeks showed only 10 to 20 per cent loss in Lf. Precipitation of purified toxoid by protamine, and adsorption of the complex on a small quantity of aluminum hydroxide, gave a product which, when suspended in phosphate buffer and warmed at 37 C for 2 weeks, lost only 5 to 10 per cent of its Lf value; when warmed for 4 weeks the loss was 20 per cent. The absence of change in the toxoid molecule as a result of the procedures used is further indicated by the fact that guinea pigs have produced 4 units of antitoxin following injection of the protamine-precipitated product. The high degree of purity of the toxoid obtained is reflected in the absence of disturbing reactions following injection of the protamine-toxoid into human adults. It seems possible that even the occasional individual who is sensitive to toxoid as such will experience less discomfort if given the protamine-precipitated material, since this would be expected to reduce the rate of release of toxoid at the site of the injection. Some data are presented dealing with electrophoretic, and sedimentation-measurements of the purified toxoid.

Detoxification of Crystalline Botulinum Type A Toxin

Summary Botulinum Type A toxoid has been prepared from crystalline toxin by the addition of formaldehyde. The most active preparation consisted of one component electrophoretically, immunized mice in a dose containing 0.01γ of toxoidnitrogen and was 2400 times more active antigenically than crude toxoid on the basis of nitrogen-content.

Purification and Adsorption of Diphtheria Toxoid

THE method for purification of diphtheria toxoid, which h s been used at the State Bacteriological Laboratory of Sweden since 1943, consists of two different procedures: the so-called iso-electric precipitation with trichloracetic acid of the crude toxoid and the aluminium precipitation of the partialy purified material. The method, described brj ny in an earlier publication1, is founded on investigations by Boivin and Yzard2 and by Glenny and Barr3.

The Immunochemistry of Toxins and Toxoids

Summary Methods are given for the accurate and reproducible assay of the biological properties of tetanal toxin and tetanal toxoid. The solubilities of toxin and toxoid, and the optimal points for their precipitation in methanol-water mixtures under controlled conditions of pH, ionic strength and temperature have been determined. Tetanal toxin is quantitatively precipitated at a pH 5.1 ± 0.1 in 40 per cent methanol at ionic strength of 0.09 and at a temperature of −5 C. MLD and Lf concentrations of over 300 times greater than the original crude toxin on a nitrogen basis and of over 5000 times on a volume basis have been obtained. A purified toxin prepared from a special toxin (Mueller) contained 16,000,000 MLD and 2400 Lf units per mg of nitrogen, and further purification of this material has resulted in a crystalline product containing 75,000,000 MLD per mg of nitrogen. Tetanal toxoid is quantitatively precipitated at a pH between 4.8 and 4.9 in 40 per cent methanol at ionic strength 0.09 and at a temperature of −5 C. Lf concentrations of 400 times on a nitrogen basis over the crude toxoid have been obtained. On a volume basis concentrations of over 5,000-fold over the original crude toxoid are possible. The purified toxoid is relatively free of sensitizing material as indicated by anaphylactic studies. I am indebted to Miss Ruth G. Wittler and Mr. Donald B. Grossberg for their help in carrying out assay and analytical procedures. Wyeth Incorporated generously supported this work.

An Alum Toxoid Prepared from Diphtherial Toxin Produced on a Gelatin-Hydrolysate Medium

Abstract The literature on the concentration and purification of diphtherial toxin and toxoid, as reviewed by Smith (1932), and Leonard and Holm (1933), shows a long list of agents that have been used for these purposes. Both publications agreed that the best yields as well as superior immunizing values were obtained with toxoids precipitated with alum. Roux and Yersin (1889) used aluminum salts to precipitate toxin from solution. However, the enhanced antigenicity of alum toxoid was first demonstrated by Glenny, Pope, Waddington and Wallace (1926), who showed that unwashed and undissolved alum-precipitated diphtherial toxoid was superior to crude toxoid in the immunization of guinea-pigs and rabbits. Glenny and Barr (1931), and Wells, Graham and Havens (1932), found that the optimal amount of alum required for complete precipitation, with the least amount of extraneous material in the precipitate, differed with the various types of media used in the production of the toxin.