Results of an international transferability study of the BINACLE (binding and cleavage) assay for in vitro detection of tetanus toxicity

Tetanus vaccines contain detoxified tetanus neurotoxin. In order to check for residual toxicity, the detoxified material (toxoid) has to be tested in guinea pigs. These tests are time-consuming and raise animal welfare issues. In line with the “3R” principles of replacing, reducing and refining animal tests, the “binding and cleavage” (BINACLE) assay for detection of active tetanus neurotoxin has been developed as a potential alternative to toxicity testing in animals. This in vitro test system can discriminate well between toxic and detoxified toxin molecules based on their receptor-binding and proteolytic characteristics.Here we describe an international study to assess the transferability of the BINACLE assay. We show that all participating laboratories were able to successfully perform the assay. Generally, assay variability was within an acceptable range. A toxin concentration-dependent increase of assay signals was observed in all tests. Furthermore, participants were able to detect low tetanus neurotoxin concentrations close to the estimated in vivo detection limit.In conclusion, the data from this study indicate that the methodology of the BINACLE assay seems to be robust, reproducible and easily transferable between laboratories. These findings substantiate our notion that the method can be suitable for the routine testing of tetanus toxoids.     

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.     

Detoxification of diphtheria and tetanus toxin with formaldehyde. Detection of protein conjugates.

In a model system of purified diphtheria and tetanus toxins it was shown that conjugates between the two proteins are formed during detoxification with formaldehyde. Detoxification mixtures were fractionated by HPLC. Two protein conjugates with different molecular weights were detected and quantified by capture ELISA assay. In vivo the existence of the largest diphtheria-tetanus toxoid conjugate was demonstrated by its antibody response in mice vaccinated with a calcium phosphate adjuvated column fraction of detoxification mixture. To eliminate the risk of cross-linking foreign proteins to toxoids in an attempt to reduce the frequency of adverse reactions in vaccination programmes, it is preferable to purify toxins before treatment with formaldehyde.     

Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation

Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor.     

F型肉毒毒素的制备及类毒素免疫原性初探

将F型肉毒梭菌经适宜条件产毒培养后,以硫酸铵盐析和酸沉两种不同工艺制备的F型肉毒毒素,用分段脱毒法脱毒制备类毒素,分别免疫豚鼠、马匹后测定免疫血清抗体效价。结果显示,两种工艺制备的毒素其类毒素都具有较好的免疫原性。     

Murine red blood cells as efficient carriers of three bacterial antigens for the production of specific and neutralizing antibodies.

Three bacterial toxoids, CRM 197 (mutagenized diphtheria toxin), tetanus toxoid (formaldehyde-treated tetanus toxin), and PT-9K/129G (double mutant of pertussin toxin) were encapsulated within red blood cells (RBCs) of B6D2F1 and Balb/C mice according to a mild procedure based on hypotonic dialysis-isotonic resealing that yielded undamaged RBCs. The toxoid-loaded RBCs were injected intravenously in order to immunize animals and their effects were compared to those of identical amounts (30-95 micrograms per mouse subdivided into multiple injections) of the corresponding free toxoids injected intravenously in saline. Sera from treated mice were collected and tested for titers of specific antibodies against each of the three antigens and also for titers of neutralizing antibodies, i.e., affording protection from toxic effects induced by the corresponding native toxins. In all experiments, significant seroconversion was observed with both immunization systems. Titers of both specific and neutralizing antibodies against CRM 197 and tetanus toxoid were several-fold higher upon immunization with the RBC-encapsulated toxoids, than with the free toxoids. These differences were not due to qualitatively different recognition patterns of antigenic determinants by the two types of sera. Conversely, intravenous immunization with pertussis toxoid either as RBC-encapsulated or as free antigen elicited a comparably high production of specific and of neutralizing antibodies. These data demonstrate that properly engineered RBCs behave as natural carriers and possibly adjuvants for antigens of vaccinal interest.     

Sodium periodate-induced suppressor T cells of the human in vitro antibody response

Combinations of T and B lymphocytes from normal individuals booster immunized 14–30 days previously with a combination of diphtheria and tetanus toxoids, synthesized IgG antitetanus toxoid, and IgG antidiphtheria toxoid antibodies when stimulated by pokeweed mitogen in vitro. The addition of 5 μg of soluble tetanus toxoid to the cultures during the first 2 days incubation resulted in greater than 90% suppression of the subsequent production of IgG antitetanus toxoid antibodies. The synthesis of IgM antitetanus toxoid antibodies, total IgG, total IgM, and IgG antidiphtheria toxoid antibodies were unaffected. Similarly, the addition of 5 μg of soluble diphtheria toxoid suppressed the synthesis of IgG antidiphtheria toxoid antibodies with no effect on the synthesis of IgG antitetanus antibodies. Allogeneic combinations of B and T lymphocytes were capable of mediating the suppression, and irradiation of the T cells caused only a partial and variable reversal of the suppression. The antigen-induced specific suppression of antibody synthesis could not be demonstrated in cultures stimulated with soluble T-cell-derived helper factors.     

In vitro antigen ELISA for quality control of tetanus vaccines

Consistency of production is recognised as an important aspect of vaccine manufacture and suitably validated in vitro assays are required for quality control testing of these products. For the manufacture and batch release of tetanus vaccines, antigen content and integrity, and degree of adsorption of antigen to the adjuvant are critical parameters that should be monitored for consistency. Here we describe the development and use of an Enzyme Linked Immunosorbent Assay (ELISA) to quantify tetanus antigen in combined vaccine products and to measure the degree of adsorption of antigen to adjuvant. Whilst the antigen assay cannot be assumed to predict potency for different products, it can be used as part of a panel of in vitro methods to provide a more informative product profile and to monitor trends in production. The antigen assay is particularly valuable for providing quantitative information on every final lot when modifications of in vivo potency tests, such as single dilution assays, are used.     

构象稳定的破伤风毒素Hc 片段突变体 (HcM) 的制备及其免疫原性分析

破伤风是由破伤风杆菌侵入人体伤口、生长繁殖、产生毒素而引起的一种急性特异性感染,其死亡率高,严重危害人民生命健康。研究证实破伤风毒素重链C端 (Hc) 具有与毒素受体结合的活性,完全保留了全分子的免疫原性,有望开发成为新的基因工程破伤风亚单位疫苗以替换传统的甲醛灭活类毒素疫苗。由于野生型Hc蛋白 (HcW) 易形成分子间及分子内二硫键,且各构象分子之间易发生不稳定的转换,为疫苗的生产工艺带来困难,因此,通过将破伤风HcW蛋白的869位半胱氨酸突变为丙氨酸,构建构象稳定的破伤风亚单位疫苗突变体HcM,对Hc     

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).     

Evaluation of a guinea pig model to assess interference in the immunogenicity of different components of a combination vaccine comprising diphtheria, tetanus and acellular pertussis (DTaP) vaccine and haemophilus influenzae type b capsular polysaccharide conjugate vaccine.

A guinea pig model to assess the immunogenicity of a combination vaccine containing diphtheria, tetanus and acellular pertussis (DTaP) vaccine and Haemophilus influenzae type b (Hib) capsular polysaccharide conjugated to tetanus toxoid (HibT) was evaluated comparatively with the mouse immunogenicity test to study the effect of combining these antigens on the immunogenicity of various components. The immunogenicity test in mice was performed by subcutaneous injection of groups of 10 animals twice at an interval of four weeks with 1/10 of a single human dose of various formulations of combination vaccines, DTaP or HibT vaccine. The animals were bled at 4 and 6 weeks and IgG or total antibodies to various components were determined by ELISA or RIA. The guinea pig immunogenicity model included groups of animals injected subcutaneously twice at an interval of six weeks with 1.5 times the single human dose of various formulations. The animals were bled at 4, 6 and 8 weeks and serum samples were tested for antibodies to various components by ELISA, RIA and/or neutralization tests. Additionally, potency of tetanus and diphtheria components was assessed as per the US Food and Drug Administration's regulations. Aluminium phosphate (AIPO(4)) adsorbed HibT vaccine or HibT as a combination with AIPO(4)adsorbed DTaP vaccine showed significant increases in IgG antibodies to tetanus toxin in mice as well increased tetanus antitoxin levels in guinea pigs as compared to soluble HibT vaccine. In general, combining DTaP and HibT vaccines did not affect the antibody levels to tetanus and diphtheria toxoids whereas DTaP-HibT combination vaccine elicited significantly lower IgG antibodies to pertussis toxin and filamentous haemagglutinin than DTaP vaccine alone, particularly after first injection. Mice showed similar Hib antibody responses for the combination and HibT alone whereas guinea pigs consistently showed lower anamnestic responses to Hib for combination formulations than for HibT alone. Reducing the amount of HibT and/or tetanus toxoid in the combination formulations reduced this suppression of Hib antibody response in guinea pigs. Suppression of Hib antibody response in combination vaccines has also been reported from recent clinical trials. Based on the results from this study, it appears that the guinea pig model may be able to predict the human response to various components of combination vaccines.     

Biochemical and biological characteristics of cross-reacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin: Use as a conjugation protein in vaccines and other potential clinical applications

The biochemical and biological characteristics of CRM₁₉₇ are reviewed. Polysaccharide protein conjugate vaccines represent an important technological advancement that allowed for protection against dangerous diseases in vulnerable populations such as infants. The first carrier proteins, diphtheria and tetanus toxoids, were chosen in the context of an extensive body of information describing their immunogenicity and safety profiles in clinical use. These carriers perform well, and they require detoxification. A non-toxic mutant of diphtheria toxin, cross-reacting material 197 (CRM₁₉₇), is a useful carrier protein with several manufacturing and other potential advantages over toxoids. For over a decade, several important and widely used routine childhood glycoconjugate vaccines against serious illnesses, including Haemophilus influenzae type b and pneumococcal disease, have employed CRM₁₉₇ as carrier protein. Additional clinical applications of CRM₁₉₇, as in chemotherapy, also exist.     

Tetanus toxin production from <Emphasis Type="Italic">Clostridium tetani</Emphasis>, using a casein-based medium in a single-use bioreactor

Clostridium tetani, a spore-forming anaerobic bacterium, and a casein-based semisynthetic medium were used to produce tetanus toxin in this study. N-Z-Case TT (casein hydrolysate) solution and glucose stock media were mixed and autoclaved, which resulted in tetanus toxin expression. The toxin was expressed when the N-Z-Case TT solution reacted with the glucose stock at a high temperature, creating an adequate amount of Maillard reaction products (MRPs). After accumulating in C. tetani cells, tetanus toxin was secreted into the medium when cell lysis was induced by surface aeration. C. tetani was cultivated and tetanus toxin was expressed in a single-use bioreactor, which produced 80 Lf/mL of tetanus toxin in a medium with MRPs. While using the correct medium to induce tetanus toxin was important, other factors played a part in achieving the desired concentration of the toxin, including the medium processing and culture methods inside the bioreactor. Tetanus toxoid with a purity level greater than 2,500 Lf/mgPN was obtained by detoxifying and purifying the toxin recovered from the fermenter or single-use bioreactor. A single-use bioreactor could be used in a limited space without the need for constructing a large scale production facility, to produce the tetanus toxoid antigen for clinical trials.     

Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and toxoids

Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG₁ monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse antitoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and toxoids. The detection sensitivity of diphtheria toxin/toxoid equaled 0.0005 Lf/ml; tetanus toxin and toxoid were detected with sensitivities of 20 LD₅₀/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum toxoids (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E toxoid (0.001 UI/ml).     

Immunogenicity test of tetanus component in adsorbed vaccines by toxin binding inhibition test

Samples from 20 lots of diphtheria-tetanus (adult use dT) vaccine and from 20 lots of diphtheria-tetanus-pertussis (DTP) vaccine were used to standardize and validate the in vitro toxin binding inhibition (ToBI) test for the immunogenicity test of the tetanus component. The levels of tetanus antitoxin obtained by ToBI test were compared to those obtained using the toxin neutralization (TN) test in mice routinely employed to perform the quality control of the tetanus component in adsorbed vaccines. The results ranged from 1.8 to 3.5 IU/ml for dT and 2 to 4 IU/ml for DTP by ToBI test and 1.4 to 3 IU/ml for dT and 1.8 to 3.5 IU/ml for DTP by TN in mice. These results were significantly correlated. From this study, it is concluded that the ToBI test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved.     

Single radial immunodiffusion as a method for the identification and quantitation of diphtheria and tetanus toxoids in adsorbed vaccines

The immunochemical techniques of double diffusion and single radial diffusion in agarose gels were compared and each considered as possible alternative methods to the methods stipulated by the European Pharmacopoeia for the identification of diphtheria and tetanus toxoids in adsorbed vaccines. Both methods identified the toxoids but single radial diffusion was found to be preferable as the precipitin bands formed were visible without staining. Single radial diffusion was further investigated for its suitability as a quantitative method and was found to give reproducible estimates of the amount of toxoid present in all vaccines tested. However, in the case of tetanus toxoids these estimates were lower than the amounts stated to have been incorporated in the vaccines by the manufacturers. It was concluded that single radial diffusion would be a suitable replacement in the European Pharmacopoeia as a method for the identification of the diphtheria and tetanus components of adsorbed vaccines provided that elution could also be achieved from vaccines containing calcium phosphate.     

Toxoid of Clostridium botulinum type F: purification and immunogenicity studies.

Toxin from Clostridium botulinum type F was recovered from dialysis cultures and partially purifed by: (i) ammonium sulfate and ethanol precipitation; (ii) O-(diethylaminoethyl)-cellulose chromatography; or (iii) diethylaminoethyl-cellulose chromatography followed by O-(carboxymethyl)-cellulose chromatography. Toxin purities as reflected by specific activity were 1.83 X 10(6), 9.8 X 10(6), and 2.0 X 10(7) mouse 50% lethal doses (LD50)/mg of N, respectively, for toxins purified by the three methods. The toxins were converted to toxoids by incubation at 35 C in the presence of 0.3 to 0.45% formalin for 21 to 35 days. Toxoids were immunogenic in guinea pigs, as demonstrated by serum antitoxin response and the immunized animals' resistance to challenge by type F botulinal toxin. The immune response to type F toxoids was lower when toxoids of serotypes A, B, C, D, and E were combined with the type F toxoid than when the type F toxoid only was administered. The toxoid prepared from the most highly purified toxin (method [iii]) conferred the highest immunity in guinea pigs at a given dose level. A relation between serum antitoxin level and resistance to challenge was observed. At least 50% of the groups of guinea pigs with 0.015 antitoxin units or more per ml survived challenge by 10(5) mouse LD50 of type F botulinal toxin. A dose of 3.75 mug of N of the most highly purified type F toxoid in combination with the other five serotypes of botulinal toxoid invoked an immune response in guinea pigs comparable to that considered adequate for the other toxoids.     

Determination of Tetanus Toxin and Toxoid by ELISA Using Monoclonal Antibodies

The procedure for obtaining monoclonal antibodies TT-1, TT-2, and TT-3 against tetanus toxin/toxoid is described. It is shown that the commercial DTP vaccine and tetanus toxoid conjugated with a low-molecular-weight hapten can both be used as immunogens. Monoclonal antibodies TT-1 and TT-2 neutralized tetanus toxin in vivo. The monoclonal antibodies obtained were used to design and compare several schemes of quantitative determination of tetanus toxoid and toxin by ELISA. A more sensitive competitive ELISA allowed the detection of as much as 0.01 EC/ml toxoid and 50 LD₅₀/ml toxin.     

Comparative study of the immunoglobulins and specific antibodies in the blood of rabbits immunized with ADTP vaccine and ADT-anatoxin with normal and reduced antigen contents

Adsorbed DPT vaccine and adsorbed DT toxoids with normal and reduced antigen content were used for the immunization of rabbits. The levels of IgM and IgG and the dynamics of antibodies to diphtheria and tetanus toxins and to Bordetella pertussis in the blood sera of the animals were studied in the postvaccinal period (on days 15 and 34). This study revealed that the reduction of the antigen content of adsorbed DT toxoid to 5 Lf of diphtheria toxoid and 5 binding units of tetanus toxoid did not decrease the capacity of the preparation for increasing the levels of IgG and IgM, antibodies to diphtheria and tetanus toxins in the sera of the rabbits. The reduced content of these toxoids in adsorbed DPT vaccine did not affect its capacity for inducing the enhanced synthesis of IgG, antibodies to diphtheria and tetanus toxins, while the production of IgM and IgA remained unchanged. At the same time an increase in the titers of antibodies to B. pertussis in the animals was less pronounced than that observed after the injection of commercial adsorbed DPT vaccine. Additional investigations are necessary in order to establish the protective potency of the pertussis component in adsorbed DPT vaccine with the reduced content of toxoids and to find out the optimum antigenic composition for this preparation.     

Preparative Procedure for the Purification of Toxoids by Gel Filtration

Sephadex gel filtration can be employed as a preparative procedure for the purification of both tetanus and diphtheria toxoids. A toxoid purification sequence is described in the text. By utilizing the described methods and columns, up to 100,000 human doses of diphtheria toxoid could be processed in a single operation. The method has given an 80% yield of diphtheria toxoid with a purity of 1,900 Lf per mg of N. The analysis of the material by immunodiffusion tests showed that a marked increase in purity was achieved. Antigenicity tests demonstrated that there was no significant difference in antigenic potency between the parent toxoid and its purified fraction. Factors limiting the effective separation of tetanus toxoid by gel filtration are discussed. The construction of the columns used is described in detail, as well as packing procedures and column characteristics such as bed volume, void volume, sample size, and flow rate.