A Novel High-Potency Tetanus Vaccine.

Chemically inactivated tetanus toxoid (CITT) is clinically effective and widely used. However, CITT is a crude nonmalleable vaccine that contains hundreds of Clostridium tetani proteins, and the active component is present in variable and sometimes minor percentages of vaccine mass. Recombinant production of a genetically inactivated tetanus vaccine offers an opportunity to replace and improve the current tetanus vaccine. Previous studies showed the feasibility of engineering full-length tetanus toxin (TT) in Escherichia coli In the present study, full-length TT was engineered with eight individual amino acid mutations (8MTT) to inactivate catalysis, translocation, and host receptor-binding functions, retaining 99.4% amino acid identity to native tetanus toxin. 8MTT purified as a 150-kDa single-chain protein, which trypsin nicked to a 100-kDa heavy chain and 50-kDa light chain. The 8MTT was not toxic for outbred mice and was >50 million-fold less toxic than native TT. Relative to CITT, 8MTT vaccination elicited a strong immune response and showed good vaccine potency against TT challenge. The strength of the immune response to both vaccines varied among individual outbred mice. These data support 8MTT as a candidate vaccine against tetanus and a malleable candidate conjugate vaccine platform to enhance the immune response to polysaccharides and other macromolecular molecules to facilitate a rapid response to emerging microbial pathogens.IMPORTANCE Chemical inactivation is a clinically effective mechanism to detoxify protein toxins to produce vaccines against microbial infections and to serve as a platform for production of conjugate polysaccharide vaccines. This method is widely used for the production of protein toxin vaccines, including tetanus toxoid. However, chemical modification alters the protein structure with unknown effects on antigenicity. Here, a recombinant full-length tetanus toxin (TT) is engineered with 8 mutations (8MTT) that inactivate three toxin functions: catalysis, translocation, and receptor binding. 8MTT is nontoxic and elicits a potent immune response in outbred mice. 8MTT also represents a malleable platform for the production of conjugate vaccines, which can facilitate a rapid vaccine response against emerging microbial pathogens.

IL-36-Induced Toxicity in Neonatal Mice Involves TNF-α Production by Liver Myeloid Cells.

Human and mouse neonates exhibit limited vaccine responses characterized by predominant Th2 and limited Th1 responses. Because IL-36 exerts a synergic adjuvant effect with IL-12, enhancing Th1 polarization in adult (AD) mice, we administered IL-36ß to neonatal (1-wk old) and AD control mice at the time of immunization with tetanus toxoid adsorbed to aluminum hydroxide (TT/Alum). Unexpectedly, the combination of IL-36ß with TT/Alum, which was well tolerated in AD mice, proved toxic and even lethal in neonates. This neonatal toxicity was associated with high Il36r mRNA expression in neonatal liver, resulting in increased cytokine production. Liver Il36r mRNA expression decreased with the termination of fetal liver hematopoiesis, and this decrease correlated with a complete protection from TT/Alum/IL-36ß-induced mortality. The combination of IL-36ß and TT/Alum induced the rapid production of TNF-α and IFN-γ by liver myeloid and lymphoid cells, respectively. These responses were less marked when IL-36ß was used alone, with no adverse effect. The toxicity of IL-36ß + TT/Alum was abrogated by the administration of a neutralizing anti-TNF-α Ab, confirming causality. In conclusion, liver myeloid cells in neonatal mice are an important source of proinflammatory cytokines that may lead to TNF-α-mediated toxicity and even lethality.

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.

Non-invasive, epicutaneous immunisation with toxoid in deformable vesicles protects mice against tetanus, chiefly owing to a Th2 response.

A non-invasive, intra/transcutaneous immunisation of mice with a suitable combination of tetanus toxoid, ultradeformable vesicle (Transfersome®) carrier, and monophosphoryl lipid A adjuvant targets immuno-competent cells in a body and can protect 100% of the tested mice against an otherwise lethal (50×LD50) parenteral tetanus toxin challenge. The late immune response to the epicutaneously applied tetanus toxoid in such vesicles consists chiefly of circulating IgG1 and IgG2b antibody isotypes, indicative of a specific Th2 cellular response bias. Immunisations by subcutaneous injections moreover protect 100% of mice against a similar, otherwise lethal, dose of tetanus toxin. However, the immune response to transcutaneous and invasive immunisation differs. The latter elicits mainly IgG1 and IgG2b as well as IgG2a antibody isotypes, indicative of a mixed Th1/Th2 response. The cytokine response of the intra/transcutaneously and subcutaneously immunised mice reflects the difference in the organ-specific manner. IFN-γ concentration is appreciably increased in the draining lymph nodes and IL-10 in spleen. Since tetanus is a neutral antigen, both the Th1-specific IFN-γ and the Th-2 specific-IL-10 are observable.

Adjuvant properties of thermal component of hyperthermia enhanced transdermal immunization: effect on dendritic cells.

Hyperthermia enhanced transdermal (HET) immunization is a novel needle free immunization strategy employing application of antigen along with mild local hyperthermia (42°C) to intact skin resulting in detectable antigen specific Ig in serum. In the present study, we investigated the adjuvant effect of thermal component of HET immunization in terms of maturation of dendritic cells and its implication on the quality of the immune outcome in terms of antibody production upon HET immunization with tetanus toxoid (TT). We have shown that in vitro hyperthermia exposure at 42°C for 30 minutes up regulates the surface expression of maturation markers on bone marrow derived DCs. This observation correlated in vivo with an increased and accelerated expression of maturation markers on DCs in the draining lymph node upon HET immunization in mice. This effect was found to be independent of the antigen delivered and depends only on the thermal component of HET immunization. In vitro hyperthermia also led to enhanced capacity to stimulate CD4+ T cells in allo MLR and promotes the secretion of IL-10 by BMDCs, suggesting a potential for Th2 skewing of T cell response. HET immunization also induced a systemic T cell response to TT, as suggested by proliferation of splenocytes from immunized animal upon in vitro stimulation by TT. Exposure to heat during primary immunization led to generation of mainly IgG class of antibodies upon boosting, similar to the use of conventional alum adjuvant, thus highlighting the adjuvant potential of heat during HET immunization. Lastly, we have shown that mice immunized by tetanus toxoid using HET route exhibited protection against challenge with a lethal dose of tetanus toxin. Thus, in addition to being a painless, needle free delivery system it also has an immune modulatory potential.

Calibration of the Ph. Eur. Biological Reference Preparation (BRP) for tetanus vaccine (adsorbed) batch 3.

A joint collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) and the World Health Organization (WHO)/National Institute for Biological Standards and Control (NIBSC) to establish replacement batches for the European Pharmacopoeia (Ph. Eur.) Tetanus Vaccine (adsorbed) Biological Reference Preparation (BRP) batch 2 and for the WHO 3rd International Standard (IS) for Tetanus toxoid (adsorbed). Two freeze-dried stabilised tetanus vaccine (adsorbed) candidate preparations (Preparation A, 08/218 and Preparation B, 08/102) were calibrated against the current 3rd IS/BRP batch 2 (Preparation C) using challenge methods in guinea pigs and mice as described in the Ph. Eur. general chapter 2.7.8. Assay of tetanus vaccine (adsorbed). They were also assayed by serology methods. The WHO 2nd IS for Tetanus toxoid adsorbed (TEXA-2) was additionally included in the sample panel as Preparation D. Thirty-four laboratories (regulatory organisations and manufacturers) from 22 countries participated in the collaborative study. The majority of participants performed 2 independent challenge tests. Nine laboratories performed challenge assays in guinea pigs and 30 laboratories performed challenge assays in mice. Eight laboratories performed serology in guinea pigs and 1 laboratory performed serology in mice. For Preparation A, the geometric mean (GM) potency estimate (with 95 % confidence interval (CI)) in guinea pigs for all laboratories that provided valid results (n = 6) was 488.5 (354.2-673.6) IU/ampoule. For valid mouse assays (n = 25) the GM potency (with 95 % CI) was 259.8 (223.5-302.0) IU/ampoule. The inter-laboratory geometric coefficient of variation (GCV) was 36 % for guinea pig assays and 45 % for mouse assays. This compared favourably with the calibration of the 3rd IS/BRP batch 2 where the inter-laboratory GCV was 36 % and 42 % in guinea pigs and mice, respectively. For Preparation B, the GM potency estimate (with 95 % CI) in guinea pigs for all laboratories that provided valid results (n = 6) was 107.9 (64.1-181.7) IU/ampoule. For valid mouse assays (n = 24) the GM potency (with 95 % CI) was 147.9 (126.3-173.1) IU/ampoule. The inter-laboratory GCV was 64.3 % for guinea pig assays and 45.2 % for mouse assays. From the collaborative study, Preparation A appeared more suitable to be the replacement Ph. Eur. BRP as it is similar to the Tetanus vaccine (adsorbed) BRP batch 2, except for nature of the stabiliser. Preparation A was confirmed to have higher potency, readily detectable tetanus toxoid, and confirmed satisfactory stability and performance in challenge assays. Preparation A was adopted in January 2011 by the Ph. Eur. Commission as the Tetanus vaccine (adsorbed) BRP batch 3, with assigned potencies of 490 IU/ampoule in the guinea pig challenge assay and of 260 IU/ampoule in the mouse challenge assay. The same Preparation A was adopted in October 2010 as the WHO 4th IS for Tetanus toxoid (adsorbed), with the assigned activity of 490 IU/ampoule from guinea pig challenge assays. A follow-up study (reporting study) was organised by the EDQM to assess the impact of the potency assigned to the BRP batch 3 for mouse challenge assays on the outcome of batch release testing in Europe. Eight laboratories including official medicines control laboratories (OMCLs) and manufacturers reported the results of their routine testing, using the BRP batch 3 in addition to their regular reference preparation. For each tested product, participants calculated the potency relative to their routine reference and relative to the BRP batch 3. No common sample panel was distributed to participants. In total, data on 40 batches of different marketed tetanus vaccines were reported. Overall, a good concordance was observed between the potencies calculated relative to the BRP batch 2 and relative to the BRP batch 3. On average, the potency estimates were 10 % lower when expressed relative to the BRP batch 3. Cases of discrepant decisions for batch release were very limited and affected mainly batches with specifications close to the pharmacopoeial requirements. The reasons for differences in estimated potencies are discussed. The study showed that the use of the BRP batch 3 with an assigned potency of 260 IU/ampoule does not result in substantial change in the potency of different marketed products. This confirmed that the mouse challenge potency value assigned to the BRP batch 3 is suitable.

In vitro determination of tetanus toxicity by an endopeptidase assay linked to a ganglioside-binding step.

Assays for the detection of tetanus neurotoxin (TeNT) are relevant for research applications as well as for the safety testing of tetanus vaccines. So far, these assays are usually performed as toxicity tests in guinea pigs or mice. The alternative methods described to date were mostly based on the detection of the toxin's proteolytic activity. However, these endopeptidase assays turned out to be unreliable because they only measure the enzymatic activity as sole determinant of tetanus toxicity, while not taking into account other parameters like the toxin's capacity to bind to target cells. In order to better reflect the in vivo situation of a tetanus infection, we have linked an endopeptidase assay to a ganglioside-binding step. The resulting method, which offers a unique combination of two functionally linked assays, detects those TeNT molecules only which possess both a functional binding domain as well as an active enzymatic domain. Our results demonstrate that this assay is able to reliably detect TeNT, and therefore might provide a basis for the replacement of the animal tests for detection of tetanus toxicity. Moreover, the assay concept could also be useful for in vitro toxicity measurements of other toxins with similar subunit structures.

Residual enzymatic activity of the tetanus toxin light chain present in tetanus toxoid batches used for vaccine production.

The light chain of tetanus neurotoxin (TeNT) is a zinc-dependent metalloprotease which specifically cleaves the synaptic vesicle protein synaptobrevin. This crucial mechanism of tetanus toxicity leads to a blockade of inhibitory neurotransmitter release. We recently reported the development of a highly sensitive endopeptidase assay for the specific in vitro detection of active TeNT based on this proteolytic feature. Using this method, we could show that formaldehyde-inactivated TeNT preparations (toxoids), which are used for the production of tetanus vaccines, contain a high residual synaptobrevin-cleaving activity. Such an activity was detected in numerous tetanus toxoid batches obtained from several vaccine manufacturers which did not display any in vivo toxicity in the obligatory animal tests. The enzymatic activity could be attributed to the presence of free TeNT light chains whose function had not been restrained by the formaldehyde treatment, but which lack the functional heavy chain necessary for entering neurons in vivo. To our knowledge, this is the first report describing a residual proteolytic activity in tetanus toxoids.

Evaluación de la toxicidad por dosis única de la vacuna antitetánica vax-TET en ratas Sprague Dawley / Evaluation of toxicity by single dose of the tetanus vaccine vax-TET in Sprague Dawley rats

La vacuna vax-TET® está indicada en la prevención del tétanos y es efectiva si se logra un completo y apropiado esquema de inmunización. Para investigar el potencial tóxico de este producto se realizó una prueba toxicológica con una dosis única, por vía intramuscular, en un volumen de 0,2 mL en ratas Sprague Dawley. La composición de la vacuna de ensayo probada fue la misma de la vacuna comercial. Los animales fueron observados diariamente en buscade síntomas locales y sistémicos de toxicidad. Se realizaron mediciones del consumo de agua y alimento, así como del peso corporal. Dos semanas después de la inoculación las ratas fueron sacrificadas por métodos de eutanasia sin dolor y sometidas a necropsia. No se observaron muertes ni síntomas de toxicidad en los animales estudiados. Tampoco se encontraron diferencias de interés toxicológico entre los grupos experimentales en cuanto a las variables medidas. El estudio anatomopatológico reveló la presencia de formaciones granulomatosas de tipo macrofágico asociadas, fundamentalmente, al hidróxido de aluminio. Estos resultados permitieron concluir que, bajo las condiciones delestudio y según los criterios establecidos, esta vacuna no produce efectos adversos en el modelo animal usado, por lo que se considera potencialmente no tóxica para humanos(AU)

The tetanus vaccine vax-TET® is indicated for the prevention of tetanus and it is effective if a complete and adequate immunization scheme is achieved. In order to investigate the toxic potential of this product, a toxicological test at single dose(0,2 ml), by intramuscular route was developed in Sprague Dawley rats using the same substance concentrations suggested by the manufacturer. The animals were daily observed looking for local and systemic symptoms of toxicity. Water and food consumption and body weight were also monitored during the experiment. Two weeks after inoculation, rats were slaughteredand submitted to necropsy studies. No deaths or toxicity symptoms were observed in the animals studied. No differences of toxicological interest were found among experimental groups regarding the variables measured. The anatomic-pathological study showed the presence of granulomatous formations mainly associated to aluminium hydroxide in the formulation. It wasconcluded that under the study conditions and following the established criteria, this vaccine does not produce any adverse reactions in the animal model used, suggesting a low potential toxicity in humans(AU)

Inbreeding effects on immune response in free-living song sparrows (Melospiza melodia).

The consequences of inbreeding for host immunity to parasitic infection have broad implications for the evolutionary and dynamical impacts of parasites on populations where inbreeding occurs. To rigorously assess the magnitude and the prevalence of inbreeding effects on immunity, multiple components of host immune response should be related to inbreeding coefficient (f) in free-living individuals. We used a pedigreed, free-living population of song sparrows (Melospiza melodia) to test whether individual responses to widely used experimental immune challenges varied consistently with f. The patagial swelling response to phytohaemagglutinin declined markedly with f in both females and males in both 2002 and 2003, although overall inbreeding depression was greater in males. The primary antibody response to tetanus toxoid declined with f in females but not in males in both 2004 and 2005. Primary antibody responses to diphtheria toxoid were low but tended to decline with f in 2004. Overall inbreeding depression did not solely reflect particularly strong immune responses in outbred offspring of immigrant-native pairings or weak responses in highly inbred individuals. These data indicate substantial and apparently sex-specific inbreeding effects on immune response, implying that inbred hosts may be relatively susceptible to parasitic infection to differing degrees in males and females.

Replacement of the in vivo neutralisation test for efficacy demonstration of tetanus vaccines ad us. vet.

The bacterium Clostridium (C.) tetani is an ubiquitous pathogen. This anaerobic, gram-positive bacterium can form spores and can be found in the whole environment. It enters the body via injuries of the skin and wounds where it releases the neurotoxin "tetanospasmin" (= tetanus toxin). The animals most susceptible to tetanus infection are horses and sheep. Only active immunisation by tetanus vaccine provides effective protection against tetanus intoxication. The marketing authorisation requirements stipulate that efficacy of tetanus vaccines ad us. vet. must be demonstrated in all target animal species via determination of neutralising tetanus serum antitoxin concentrations. The standard method used for this purpose is still the toxin neutralisation test (TNT), as it quantifies the tetanus toxin-neutralising effect of tetanus serum antibodies in vivo. In this test, tetanus toxin is added to dilutions of serum from vaccinated horse and sheep. The serum dilutions are then administered to mice or guinea pigs, which are observed for toxic symptoms. Against the background of animal protection, the goal of one project of the Paul-Ehrlich-Institut (Bundesministerium fuer Bildung und Forschung (Federal Ministry for Education and Research), 0312636) was to establish an alternative to the toxin neutralisation test, enabling the testing of efficacy of tetanus vaccines with serological in vitro methods. For this purpose, a so-called double antigen ELISA (DAE) was established which enables the testing of sera of different species in one assay. In addition, the sera were tested in an indirect ELISA for horses and sheep separately. Altogether, ten groups of horses and eight groups of sheep were immunised with ten animals per group each. The tetanus vaccines comprised almost all products authorised for the German market at the start of the project. 564 horse sera and 257 sheep sera were tested using the two ELISA methods. Some sera were also tested in vivo. The kinetics of antibody responses were recorded. The in vitro DAE method seems to be suitable to replace the mouse neutralisation test used for the detection of tetanus antitoxin in sera of target animal species. The comparison of some sera in the ELISA and the TNT showed good equivalence of results. Nevertheless, before an ELISA titre in horse and sheep sera indicating unambiguous protection against tetanus can be fixed, further comparative assays of low titre sera in the TNT and the DAE will have to be performed.

In vitro determination of specific toxicity in tetanus vaccines.

Tetanus vaccine is prepared from detoxified tetanus neurotoxin. To ensure the absence of residual toxin activity or to exclude the reversion to toxicity reliable control testing is based on in vivo methods, because no in vitro assay provides the required specificity and sensitivity. Tetanus neurotoxin is a 150 kDa protein produced by Clostridium tetani. The 50 kDa light chain of this neurotoxin belongs to the family of zinc metalloproteases. It cleaves synaptobrevin, a small synaptic vesicle protein, which is involved in neuroexocytosis, at the single Q76-F77 peptide bond. To develop a sensitive in vitro assay capable of quantifying the proteolytic activity of this toxin, we used as substrate a recombinant fragment of synaptobrevin2 (1-97). For detecting the cleavage products a peptide antibody raised against the N-terminal cleavage site was used. In Western Blot analysis only the cleaved substrate was detected while the uncleaved substrate showed no signal. In different approaches, recombinant synaptobrevin was either (i) bound to a microtitre plate, reduced toxin was added and the N-terminal cleavage product was detected by a specific antibody or (ii) the cleavage was performed in test tubes, the samples were transferred to a microtitre plate and immobilised cleavage products were detected. When toxoid or crude toxin is used, non-specific cleavage of synaptobrevin substrate occurs. Depending on the toxoid used different patterns of degradation of substrate are visible in Western Blots. Different protease inhibitors and reaction conditions seem to have an effect on the inhibition of this non-specific cleavage.

Application of an in vitro endopeptidase assay for detection of residual toxin activity in tetanus toxoids.

Tetanus vaccine is composed of chemically denatured tetanus toxin (TeNT), thus safety testing requires confirmation of freedom from residual and reversible toxicity. Currently, TeNT activity is estimated using in vivo assay models. Information that TeNT acts by selectively inactivating protein leading to the blocking of release of neurotransmitters has provided the opportunity to develop in vitro biochemical assay for toxin activity. In this study we describe development and use of an in vitro endopeptidase assay for detection of TeNT activity in toxoid vaccine formulations.

Disturbance of cardiovascular circadian rhythms by pertussis vaccine in freely-moving rats.

Vaccination of young children with diphtheria, tetanus, poliomyelitis and pertussis (DTPoP) vaccine is effective in preventing outbreaks of whooping cough but adverse events sometimes occur. This pilot study shows that in freely-moving rats, multiple treatment with DTPoP (at day 0 and day 5, 6 ml/kg i.v.) increased heart rate (HR) for 5 days after the first treatment and decreased diastolic blood pressure (DBP) for at least 26 days after the first treatment and inhibited the circadian rhythm of HR and DBP for at least 10 days. DTPo vaccine, containing no pertussis vaccine, was free of such effects. Thus, in rats, the pertussis component of DTPoP acts on the cardiovascular system and disturbs its circadian rhythm. The contribution of these findings to clinical adverse effects is as yet unknown and needs further research.

IgA nephropathy in mice following repeated administration of conjugated Haemophilus influenzae type B vaccine (PRP-T).

OBJECTIVE: In this study, the risk of IgA nephropathy in Swiss albino mice following the subcutaneous administration of conjugated Haemophilus influenzae type b vaccine (PRP-T), containing capsular polysaccharide of the organism (PRP) conjugated to tetanus protein (T), was evaluated. METHODS: Three treatment and corresponding control groups, each containing mice, were constituted and given 2, 4, 6 injections of 1/4 HD of PRP-T or placebo, respectively, at 2-week intervals. All mice in each treatment group were sacrificed two weeks from the last injection to examine sequential glomerular changes. RESULTS: The niceoscpic examination of renal tissues revealed mesangial proliferation (6/7; 85%) in the first group given 2 doses of vaccine; mesangial proliferation (5/7; 72%) and increase in matrix (7/7; 100%) in the second group given 4 doses; and mesangial proliferation (7/7; 100%), increase in matrix (7/7; 100%), IgA (7/7; 100%) and C3 (3/7; 42%) deposition within mesangium in the third group given 6 doses. No histopathological changes were detected in the renal tissues of any control mouse. When the experimental groups were compared statistically with their respective controls at the light microscopic level, mesangial proliferation in the first group (p: 0.0047), mesangial proliferation (p: 0.021) and increase in matrix (p: 0.001) in the second group, mesengial proliferation (p: 0.001) and increase in matrix (p: 0.001) in the third group were determined to be significantly different. When study and control groups were compared by immunofluorescence microscopy, only the third group revealed a statistically significant difference with respect to IgA deposition (p: 0.001). C3 deposition was also demonstrated in this group, but it was not significantly different (p: 0.192). However, in no instance was a control mouse found to have any form of immune deposition. CONCLUSION: We concluded that conjugated Haemophilus influenzae type b vaccine, given at two-week intervals to a total of six doses, caused secondary IgA nephropathy in mice.

Embryotoxicity and teratogenicity studies of poly (DL-lactide-co-glycolide) microspheres incorporated tetanus toxoid in Wistar rats.

Tetanus Toxoid loaded biodegradable microspheres (MTT) (poly (DL-Lactide-co-Glycolide) were administered intramuscularly to pregnant Wistar rats from Days 6 to 15 of gestation, at 1, 5 and 10-times the human equivalent dose of TT. Developmental defects in relation to soft tissues and skeleton, weight and sex of live pups and early fetal deaths from treated and control rats were analysed. The findings in treatment groups were comparable to those in the controls. These observations show that MTT was safe for pregnant rats and developing pups.

Poly (D,L-lactide) glycolide polymer microsphere entrapped tetanus toxoid: safety evaluation in Wistar rats.

A vaccine preparation consisting of tetanus toxoid (TT) encapsulated in biodegradable poly (D,L-Lactide) glycolide polymer has been developed. The toxicity of the preparation was evaluated in adult male and female Wistar rats injected IM with 20, 100 and 200 Lf tetanus toxoid, in polymer in 0.5 ml vehicle. No adverse effect of the vaccine could be seen, except for a granulomatous reaction at the site of injection. The vaccine was considered safe based on the findings in rats of both sexes.

An effective toxoid from tetanus toxin prepared by stepwise iodination of purified toxin.

Purified samples of tetanus toxin were gradually iodinated by stepwise addition of iodine, up to saturation. The residual capacity of each sample to provoke tetanus was tested by injecting sc 25 Lf (Limit of flocculation) in mice. Toxicity diminished in relation to iodine incorporated, and with iodine-saturated samples, doses of up to 100 Lf in mice, or 500 Lf in guinea-pigs, proved innocuous. Mice immunized with two doses of this toxoid adsorbed on Al(OH)3, and challenged with standard lethal toxin, gained protection against 10 MLD (Minimum Lethal Doses). Guinea-pigs were immunized by a single sc dose of 27.5 Lf with fluid toxoid, and all resisted challenge against 10 MLD applied 30 days later. The mice sera gave strong immunoprecipitation lines against the native toxin. The findings indicated that by controlled iodination of tetanus toxin an effective and inexpensive toxoid can be prepared.