Use of sephacryl high resolution (HR) in tetanus anatoxin purification.

The tetanus purified anatoxin is used in the preparation of the tetanus toxoid and multiple vaccines (dT, DT and DTP), all of them strictly following specifications established by the WHO with a minimum antigenic purity equal to 1,000 Lf/mgPN. Aiming to establish more sensitive and accurate methods for purification, samples from four different lots of tetanus anatoxin were submitted to gel filtration in twenty independent trials using the Sephacryl S-100 HR and S-200 HR resins. The Authors were careful to optimize their parameters of performance as to sample volume, elution and selectivity flow for tetanus anatoxin purification, allowing their use in industrial scale. The Sephacryl S-100 HR resin presented the best selectivity, that is, the best separation, allowing a greater linear-flow and, consequently, the best purity index. Satisfactory results were also achieved with the Sephacryl S-200 HR resin after optimization of chromatographic parameters for elution flow and volume of the sample applied. The good results of purification obtained, as well as the high chemical stability, have pointed out both the Sephacryl S-100 HR and S-200 HR resins as equally efficient for industrial production.

Development and validation study for the chromatographic purification process for tetanus anatoxin on Sephacryl S-200 High Resolution.

The tetanus purified anatoxin is used in the preparation of multiple immunoprophylactics. WHO (World Health Organization) specifies that the tetanus anatoxin must exhibit a degree of purity greater than or equal to 1,000 Lf/mg protein nitrogen (PN). Today liquid chromatography is a well established technique for the purification of tetanus anatoxin and several different methods are used in production scale. On a small scale, we purified tetanus anatoxin on Sephacryl S-200 High Resolution (gel filtration) and we obtained a successful high-yield purification. On the basis of these results, by combining conventional tangential flow filtration (TFF) at 50,000 N.M.W.L. (Nominal Molecular Weight Limit) ultrafiltration membrane with gel filtration on Sephacryl S-200 High Resolution, we have been able to purify 14 lots of tetanus anatoxin using the Bioprocess System (Amersham Pharmacia Biotech) to a large scale operation. Using this method, 77,401,332 doses of tetanus toxoid were prepared in 14 consecutive lots, supporting the reproducibility and reliability of the method presented here.

Production of an effective anti-Bothrops-tetanus mixed hyperimmune serum of equine origin

The present investigation reveals the possibility of simultaneous immunization of horses with Bothrops or Crotalus snake venoms and Tetanus antigens for the production of anti-Bothrops-Tetanus or anti-Crotalus-Tetanus mixed serum, with high titers of the respective specific antibodies. Bothrops antivenoms with an average neutralizing titer of 4.16 mg venom/ml were obtained from plasma of horses with titers lower than 0.5 mg venom/ml when Tetanus antigens were not used. This suggests the existence of a synergism between Bothrops venoms and Tetanus antigens in the stimulation of the antibody response. The pooled plasma of the animal had a neutralizing titer of 21.0 mg/ml reference Bothrops venoms and 3,300 IU/ml to Tetanus antigens after purification by enzymatic digestion and ammonium sulphate precipitation. These experiments lead us to conclude that Bothrops envenomation therapy can be successfully performed using Anti-Bothrops-Tetanus serum also serving as Tetanus prophylaxis. anti-Crotalus-Tetanus serum can also be produced, although it is not of medical interest as Crotalus envenomation rarely results in local necrotizing lesions.

Specific peptides of casein pancreatic digestion enhance the production of tetanus toxin.

Casein pancreatic digest is the basic bacterial growth medium used for diphtheria, botulinum and tetanus toxin vaccine production. It is known that the variation in the peptide content of the casein digest directly affects final toxin yields. In this study, the identification and sequences of eight peptides, four to eight amino acids in length, of casein pancreatic digestion, which seem to be involved in the enhancement of tetanus toxin production, are described. They all contain one or two residues of proline/molecule and a predominance of hydrophobic amino acid residues. The most active peptides show a general structure of Pro-aromatic-Pro, and this pattern resembled the motif displayed by bradykinin-potentiating peptides found in snake venoms. By analogy with the mechanism of bradykinin potentiation through inhibition of the proteolytic degradation of bradykinin, it is suggested that the six peptides identified here could protect the tetanus toxin from proteolysis, once secreted by the bacteria.

Neutralization of the effect of Crotalus durissus terrificus venom by gangliosides.

We determined the ability of a mixture of gangliosides (16% GD1b, 19% GT1b, 21% GM1, 40% GD1a) to neutralize the effect of Crotalus durissus terrificus (Cdt) venom in vitro and in vivo. Protection was indicated by the absence of muscular contractions, hind limb paralysis or death of BALB/c mice (16-18 g) after receiving Cdt venom (1 microgram Cdt venom containing 0.6 microgram protein) at the doses indicated. A dose of Cdt venom above 0.9 microgram (ip) or 1 microgram (im) induced muscular contraction and above 1.2 micrograms (ip) or 5.5 micrograms (im) the venom induced muscular contraction and hind limb paralysis. Cdt venom above 2.5 micrograms (ip) or 9 micrograms (im) induced all these symptoms and 95 to 100% death in experimental animals. The lethal dose 50% of the Cdt venom used was 8 micrograms (im) and 1.5 micrograms (ip). In in vitro studies, 4 mg gangliosides neutralized the effect of up to 1.5 micrograms Cdt venom. Quantities as low as 0.2 mg gangliosides were capable of neutralizing 0.9 microgram of Cdt venom in vitro. Intramuscular treatment with 1 mg gangliosides performed 60 min after the intramuscular injection of 5 micrograms Cdt venom protected 100% of the animals. In contrast, no protection was achieved with intraperitoneal treatment with gangliosides. The data show that gangliosides were effective in neutralizing the toxic effects induced by Crotalus durissus terrificus venom both in vitro and in vivo and that post-exposure intramuscular treatment with gangliosides could protect animals experimentally inoculated with the venom.

Neutralization of the effect of Crotalus durissus terrificus venom by gangliosides

We determined the ability of a mixture of gangliosides (16 percent) GDlb, 19 percent GT1b, 21 percent GM1, 40 percent GD1a) to neutralize the effect of Crotalus durissus terrificus (Cdt) venom in vitro and in vivo. Protection was indicated by the absence of muscular contractions, hind limb paralysis or death of BLB/c mice (16-18g) after receiving Cdt venom (1µgCdt venom containing 0.6 µg protein) at the doses indicated. A dose of Cdt venom above 0.9µg (ip) or 1 µg (im) induced muscular contraction and above 1.2 µg (ip) or 5.5 µg (im) the venom induced muscular contraction and hind limb paralysis. Cdt venom BOVE 2.5 µG (IP) OR 9 µg (im) induced all these symptoms and 95 to 100 percent death in experimental animals. The lethal dose 50 percent of the Cdt venom used was 8µ (im) and 1.5 µg (ip). In vitro studies, 4 mg gangliosides neutralized the effect of up to 1.5 µg Cdt venom. Quantities as low as 0.2 mg gangliosides were capable neutralizing 0.9 µg of Cdt venom in vitro. Intramuscular treatment with 1 mg gangliosides performed 60 min after the intramuscular injection of 5 µg Cdt venom protected 100 percent of the animals. In contrast, no protection was achieved with intraperitoneal treatment with gangliosides. The data show that gangliosides were effective in neutralizing the toxic effect induced by Crotalus durissus terrificus venom both in vitro and in vivo and that post-exposure intramuscular treatment with gangliosides could protect animals experimentally inoculated with the venom