Characterization of production processes for tetanus and diphtheria anatoxins.

Tetanus and diphtheria are diseases that still cause significant morbidity and mortality. Clostridium tetani produces the tetanus toxin, a 150-kDa protein. The diphtheria toxin is synthesized by Corynebacterium diphtheriae as a protein of 58 kDa. The objective of this study was to carry out a chemical characterization of the tetanus and diphtheria toxin forms in the several production process stages, and thus to establish an affordable alternative in vitro quality control to aggregate to the classical tests. The 150 kDa band of the tetanus toxin and approximately 58 kDa band of the diphtheria toxin were observed by electrophoresis similar as that described in the literature. The same band of 58 KDa was detected in Western blotting reactions. The results obtained for diphtheria toxin showed very similar protein profiles between distinct lots. For the tetanus toxin, the profiles of the initial stage showed some variability, but the ones of the following stages were similar. The similarity of the electrophoresis results indicated reproduction and consistency of the production processes in Butantan Institute and correlated with the yield and antigenic purity classical data. The establishment of alternative in vitro quality control tests can significantly contribute to achieve the consistency approach supported by WHO.

Production of the first effective hyperimmune equine serum antivenom against Africanized bees.

Victims of massive bee attacks become extremely ill, presenting symptoms ranging from dizziness and headache to acute renal failure and multiple organ failure that can lead to death. Previous attempts to develop specific antivenom to treat these victims have been unsuccessful. We herein report a F(ab)(´)(2)-based antivenom raised in horse as a potential new treatment for victims of multiple bee stings. The final product contains high specific IgG titers and is effective in neutralizing toxic effects, such as hemolysis, cytotoxicity and myotoxicity. The assessment of neutralization was revised and hemolysis, the primary toxic effect of these stings, was fully neutralized in vivo for the first time.

Fed-batch production of tetanus toxin by Clostridium tetani.

This study deals with the effects of the initial nitrogen source (NZ Case TT) level and the protocol of glucose addition during the fed-batch production of tetanus toxin by Clostridium tetani. An increase in the initial concentration of NZ Case TT (NZ(0)) accelerated cell growth, increased the consumption of the nitrogen source as well as the final yield of tetanus toxin, which achieved the highest values (50-60 L(f)/mL) for NZ(0) > or = 50 g/L. The addition of glucose at fixed times (16, 56, and 88 h) ensured a toxin yield ( approximately 60 L(f)/mL) about 33% higher than those of fed-batch runs with addition at fixed concentration ( approximately 45 L(f)/mL) and about 300% higher than those obtained in reference batch runs nowadays used at industrial scale. The results of this work promise to substantially improve the present production of tetanus toxin and may be adopted for human vaccine production after detoxification and purification.

Immunogenicity of a whole-cell pertussis vaccine with low lipopolysaccharide content in infants.

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease. This phase I comparative trial had the aim of comparing the in vitro cellular immune response and anti-pertussis toxin (anti-PT) immunoglobulin G (IgG) titers induced by a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wP(low) vaccine) with those induced by the conventional whole-cell pertussis (wP) vaccine. A total of 234 infants were vaccinated at 2, 4, and 6 months with the conventional wP vaccine or the wP(low) vaccine. Proliferation of CD3(+) T cells was evaluated by flow cytometry after 6 days of peripheral blood mononuclear cell culture with stimulation with heat-killed Bordetella pertussis or phytohemagglutinin (PHA). CD3(+), CD4(+), CD8(+), and T-cell receptor gammadelta-positive (gammadelta(+)) cells were identified in the gate of blast lymphocytes. Gamma interferon, tumor necrosis factor alpha, interleukin-4 (IL-4), and IL-10 levels in supernatants and serum anti-PT IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). The net percentage of CD3(+) blasts in cultures with B. pertussis in the group vaccinated with wP was higher than that in the group vaccinated with the wP(low) vaccine (medians of 6.2% for the wP vaccine and 3.9% for the wP(low) vaccine; P = 0.029). The frequencies of proliferating CD4(+), CD8(+), and gammadelta(+) cells, cytokine concentrations in supernatants, and the geometric mean titers of anti-PT IgG were similar for the two vaccination groups. There was a significant difference between the T-cell subpopulations for B. pertussis and PHA cultures, with a higher percentage of gammadelta(+) cells in the B. pertussis cultures (P < 0.001). The overall data did suggest that wP vaccination resulted in modestly better specific CD3(+) cell proliferation, and gammadelta(+) cell expansions were similar with the two vaccines.

Immunogenicity of a Whole-Cell Pertussis Vaccine with Low Lipopolysaccharide Content in Infants

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease...

Strengthening antivenom production in Central and South American public laboratories: report of a workshop.

A workshop on antivenom production in Central and South American public laboratories was performed at Instituto Butantan, Sao Paulo, Brazil, from June 12 to June 16, 2006, under the auspices of the program CYTED. The activity was attended by representatives of public laboratories from Costa Rica, Colombia, Perú, Bolivia, Brazil, and Uruguay. Representatives of the World Health Organization (WHO) and the Brazilian Ministry of Health also participated in the workshop. The political, institutional and technological issues related with antivenom manufacture and distribution in these countries were discussed. Current problems on antivenom production and distribution were analyzed, and the need to strengthen collaborative links between countries was stressed, particularly regarding training programs, transfer of technology, and distribution of antivenoms, with the involvement of the Pan American Health Organization and the WHO. Future workshops on other relevant issues, such as quality control and regulatory policies, are being planned, together with collaborative research efforts between the laboratories.

Role of IgG(T) and IgGa isotypes obtained from arachnidic antivenom to neutralize toxic activities of Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus venoms

The ability of IgG(T) and IgGa subclasses—isolated by liquid chromatography from equine arachnidic antivenom (AAV)—to neutralize toxic activities of Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus venoms as well as to remove venom toxins from circulation was investigated. These subclasses showed similar antibody titers against L. gaucho, P. nigriventer and T. serrulatus venoms, and by immunoblotting few differences were observed in the recognition pattern of venom antigens. IgG(T) and IgGa neutralized 100% lethality induced by L. gaucho and 50% of P. nigriventer venom, but IgGa failed to neutralize T. serrulatus venom, in contrast to IgG(T). Both subclasses neutralized local reactions and dermonecrosis induced by L. gaucho venom in rabbits. In mice, IgG(T) and IgGa partially neutralized the edematogenic activity induced by P. nigriventer and T. serrulatus venoms, but only IgG(T) neutralized (ca. 81%) the nociceptive activity induced by T. serrulatus venom. Both subclasses failed to neutralize nociceptive activity induced by P. nigriventer venom. IgG(T) reduced the serum venom levels of animals injected with L. gaucho, P. nigriventer or T. serrulatus venoms, while IgGa solely reduced L. gaucho and P. nigriventer venoms levels. Our results demostrate that IgG(T) and IgGa subclasses neutralize toxic activities induced by P. nigriventer, T. serrulatus and L. gaucho venoms with different efficacies, as well as depurate these venoms from circulation.

Effect of medium composition on the production of tetanus toxin by Clostridium tetani.

The tetanus toxin is a neurotoxin synthesized by the bacillus Clostridium tetani that, after detoxification with formaldehyde, still exhibits antigenic and immunologic properties, hence its denomination of tetanus toxoid. Such a neurotoxin is produced by cultivation of the microorganism in vegetative form on a relatively complex specific medium containing glucose and peptone. The simultaneous effects of the starting levels of glucose (G0) and N-Z Case TT (NZ0) as carbon and nitrogen sources, respectively, on the production of tetanus toxin have been investigated in this work in static cultivations by means of a five-level star-shaped experimental design and evaluated by response surface methodology (RSM) for optimization purposes. The highest final average yield of tetanus toxin (72 Lf/mL), achieved at G0= 9.7 g/L and NZ0= 43.5 g/L, was 80% higher than that obtained with standard cultivations (G0= 8.0 g/L and NZ0= 25.0 g/L).

Tetanus toxoid: 1. purification in 50 kD molecular weight cut-off membrane

The tetanus anatoxin (M.W.=150,000 dALTONS) is the fundamental component for Tetanus Toxoid (TT) combined vaccines production and must follow the parameters established by World Health Organization (WHO). Among other qualifications the purity degree must be equal or greater than 1,000 Lf/mg PN and the residual formaldehyde must be at maximum level of 0,02 per cent. The Tangential Flow Filtration technique with 50,000 Nominal Molecular Weight Limit (NMWL) membranes and gel filtration in Sephadex G-50 were associated to purify 18 consecutive lots of tetanus anatoxin

Effect of beta-propiolactone treatment on the complement activation mediated by equine antisera

A reducao da ativacao do complemento atraves de uma alteracao do fragmento Fc das imunoglobulinas pela beta-propiolactona foi obtida em soros hiperimunes equinos antivirus rabico, venenos Bothrops e toxina difterica. Os resultados foram avaliados por teste de anafilaxia em cobaias, e comparados com aqueles obtidos com os mesmos soros purificados por precipitacao salina (sulfato de amonio), seguidos ou nao por digestao enzimatica com pepsina. Os niveis de pureza proteica foram para o soro antibotropico de 184,5 mg/g e 488,5 mg/g tratado pela beta-propiolactona e digeridos pela pepsina, respectivamente...(au)

Diphtheria anatoxin industrial purification by gel filtration

Diphtheria toxoid is prepared from the toxin produced by fermentative process, of the Park-Williams N§8 strain of Corynebacterium diphteriae grown on F-20 medium. The toxin separeted from the organisms is detoxified and purified. Diphtheria toxoid is the immunizing antigen protecting against the adverse effects of infection by diphtheria organisms and rarely is used alone. It is used in associated vaccines with tetanus toxoid and pertussis vacine. This paper describes the separation of diphtheria anatoxin proteins on Sephadex G-50, Sephacryl S-100 HR and Superdex 75 prep grade

Otimizaçäo do processo de purificaçäo industrial de toxóide tetânico por gel filtraçäo / Improvement in the industrial purification of tetanus toxoid by gel filtration

A metodologia industrial para obtençäo de Toxóide Tetânico(T.T.) atual, consiste na pré-precipitaçäo pelo (NH4)2SO4 seguida de gel filtraçäo em Sephadex G-50, com o qual obtém-se grau médio de pureza de 2.300 Lf/mgNP, sendo que o mínimo exigido pela O.M.S. é 1.000Lf/mgNP. Visando basicamente a diminuiçäo do tempo empregado no processo de purificaçäo do T.T., utilizamos resinas cromatográficas de 2§ e 3§ geraçäo, o Sephacryl S-100HR e o Superdex 75 prepgrade, respectivamente, que possibilitaram a eliminaçäo da etapa de pré-precipitaçäo pelo (NH4)2SO4 obtendo-se resultados com grau de pureza ao redor de 3.000Lf/mgNP. A atividade imunogênica destes produtos quando absorvidos pelo Al (OH)3 em concentraçäo de 15Lf/ml foi de 4,5UI/ml em cobaios. A O.M.S. recomenda que o T.T. induza a formaçäo de no mínimo 2UI/ml nestes animais. O rendimento do processo de purificaçäo foi de 88,46//, que é similar ao obtido no processo atual. A metodologia introduzida permitiu a diminuiçäo do tempo empregado neste processamento assim como a possibilidade de trabalhar-se com purificaçäo em sistema fechado (coluna cromatográfica)

Toxina tetânica: produçäo e purificaçäo em escala industrial / Tetanus toxin: production and purification in large scale

A obtençäo de toxina tetânica purificada em grandes volumes é um passo fundamental para a produçäo de Toxóide Tetânico. A utilizaçäo de vapor para a esterilizaçäo do meio de cultura para a produçäo desta toxina apresentava títulos baixos de toxina tetânica. O emprego da filtraçäo convencional teve como consequência índices insatisfatórios de pureza (Lf/mgNP) e de rendimento (porcentagem) do processo. Com o objetivo de encontrar soluçöes para estas etapas do processo produtivo, foi utilizada a esterilizaçäo por cartuchos para o meio de cultura e a filtraçäo tangencial através de um sistema Prostak, associado à ultrafiltraçäo molecular em 30Kd para a toxina tetânica. A nova metodologia trouxe aumentos significativos em pureza (Lf/mgNP) e em rendimento (porcentagem). A tecnologia empregada e a comparaçäo dos resultados obtidos nos diferentes processos säo apresentadas neste trabalho

Purificaçäo industrial do toxóide tetânico por gel filtraçäo / Industrial purification of tetanus toxoids by gel filtration

Com o fracionamento pelo sulfato de amônio ou com a precipitaçäo alcoólica, säo obtidos toxóides tetânicos com pureza ao redor de 500 Lf/mg N.P. incompatíveis com as exigências da OMS, de 1000 a 1200 Lf/mg N.P. Foram descritas técnicas eficazes com a utilizaçäo da gel filtraçäo em Sephadex G200, G100 e G75 que, no entanto, pela longa duraçäo do processo, dificultam a produçäo em escala industrial. Introduzimos , assim, uma metodologia baseada na combinaçäo da pré-precipitaçäo pelo sulfato de amônio com a gel filtraçäo em Sephadex G50 com a qual obtivemos toxóides tetânicos com grau médio reprodutivo de pureza ao redor de 2.312,75 Lf/mg N.P. e resposta imunogênica satisfatória

Cross - reactivity of horse monovalent antivenoms to venoms of ten bothrops species

Horses were immunized with B. alternatus, B. atrox, B. cotiara, B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B, moojeni, B. neuwiedi and B. pradoi venoms. Antibodies recognizing the venom antigenic components were either immunochemically detected by the precipattion methods or biologically by the assays measuring the venomns indirect hemolytic and lethal toxic activities . Specific and cross - reacting antivenoms.Modifications in the serum electrophoretic patterns characterized by a reduction of the albumin peak and by a correspondent increase of the y-globulins with a pattent or no modification of the α or β globulins were found in these sera.

Cavalos foram imunizados com veneno de B. alternatus, B. atrox, B. cotiara, B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B, moojeni, B. neuwiedi and B. pradoi. Anticorpos específicos para componentes antigênicos dos venenos foram detectados pelos métodos imuno- enzimático, dupla-difusão e precipitação quantitativa enquanto que os anticorpos neutralizantes foram analisados pelos métodos da hemólise indireta em placas e pela neutralização de seus efeitos letais. Anticorpos, tanto específicos como dando reações cruzadas com venenos botrópicos foram encontrados em todos os dez soros monovalentes. Modificações nos padrões eletroforéticos, caracterizadas por uma redução no pico da albumina e por um correspondente aumento das γ. globulinas com modificações ora acentuadas ora pouco perceptíveis nas frações das α e β globolinas, foram detectadas todos esses soros.