Combined Active-Passive Immunization Against Tetanus in Man

A schedule for the prevention of tetanus in the injured, which has been in operation in the emergency department of a large hospital for over two years, is proposed. For the majority of nonimmunized persons, it is recommended that a dose of toxoid and 50 units tetanus immune globulin (human) (TIGH) be given, in separate sites, to be followed later by additional doses of toxoid for the completion of active immunization. Combined active-passive immunization with tetanus toxoid and 50 units TIGH gives a low level of passive immunity and stimulates early onset of active immunization. In combined active-passive immunization, adsorbed tetanus toxoid produced a significantly higher response than the fluid toxoid. The injection of 400 units TIGH somewhat suppressed the induction of immunity following the first dose of AlPO₄-tetanus toxoid.     

Changes in the proliferative response of human lymphocytes in vitro on exposure to subcellular components of Bordetella pertussis]

Different components of B. pertussis were found to have a similar inhibitory effect on thymidine-3H incorporation caused by phytohemagglutinin (PHA) in the culture of lymphocytes taken from donors immunized with tetanus toxoid. However, the same fractions of B. pertussis produced differential effects on the reaction of lymphocyte proliferation in response to tetanus toxoid: murein-"ontaining membranes enhanced thymidine-3H incorporation, RNA-containing fractions reduced it and water-soluble components of disintegrated B. pertussis produced no effect on lymphocyte proliferation.     

Diphtheria-tetanus overimmunization in children with no records: can it be prevented?

A pilot study was undertaken to assess the validity of two new tests for predicting the immune response of Toronto schoolchildren with no acceptable evidence of prior administration of diphtheria or tetanus toxoid to a routine booster injection of diphtheria and tetanus (DT) toxoid. The tests, an inexpensive enzyme-linked immunosorbent assay (ELISA) fingerprick test for tetanus antibodies and a modification of the Schick skin test for susceptibility to diphtheria, were administered before the booster injection. One week later the ELISA test was repeated and the result of the modified Schick test read. On both occasions a diphtheria microneutralization assay was done for "gold standard" evidence of prior exposure to diphtheria toxoid or toxin. The results were used to determine the sensitivity and specificity of a single prebooster tetanus ELISA test or a modified Schick test for predicting which children with no records could be safely protected with only one DT booster dose instead of the primary series of three or four doses usually given to such children. Only 6 of the 34 subjects (18%) were totally without prior exposure to tetanus toxoid. Two of the six (6% of 33 subjects) appeared to mount a primary immune response to diphtheria toxoid as well. An initial ELISA titre of 0.01 IU/ml or lower correctly identified all six children needing a full series of tetanus toxoid (sensitivity for a primary immune response 100%) and falsely identified only 3 of 28 immune children as needing the series (specificity for immunity 89.3%). The modified Schick test appeared to have even greater accuracy for identifying children needing a full series of diphtheria toxoid. However, its use, entailing the costs of an extra nurse visit, would have prevented only seven more children from receiving an unnecessary full series of diphtheria toxoid than use of the baseline tetanus ELISA test alone.     

The regulatory roles of T4 and T8 subsets in tetanus toxoid-induced in vitro immunoglobulin production

A new mitogenic system for in vitro immunoglobulin production induced by tetanus toxoid is presented and the role of T4 and T8 subsets in tetanus toxoid-induced in vitro immunoglobulin production is investigated. Purified T, T4, T8, and B cells from normal individuals previously immunized but not boosted with tetanus toxoid were cultured in helper and suppressor assays and the number of immunoglobulin-secreting cells were enumerated after culture using a hemolytic plaque assay. The regulatory roles of T4 and T8 cells in this tetanus toxoid system were compared with the role of these subsets after pokeweed mitogen stimulation. Although most of the immunoglobulin produced in the tetanus toxoid system was polyclonal, there were differences in the time course, the magnitude of the responses, the radiosensitivity of the subsets, and optimal T- to B-cell ratios for immunoglobulin production which distinguish the tetanus toxoid and pokeweed mitogen systems.     

Subacute toxicity of adsorbed 250 Lf tetanus toxoid in rats

Since the naturally acquired tetanus antitoxin titre in Indian population is mostly less than protective levels, to minimise the incidence of morbidity, Glaxo Laboratories (India) Ltd., Bombay have prepared a single dose potent tetanus vaccine of 250 Lf as compared to 5 Lf previous tetanus toxoid. Subacute toxicity study revealed that this toxoid (250 Lf) injected at different dose levels (1-2 ml/rat/injection x 4) is non-toxic to rats.     

Neonatal tetanus despite protective serum antitoxin concentration

Using the ELISA technique to estimate serum antibodies against tetanus toxin, seven neonates with clinical tetanus were found to have antibody levels 4-13 times higher than the presumed minimum protective level of 0.01 IU/ml. All but one of their mothers had been vaccinated with tetanus toxoid in pregnancy. In two other neonates, whose mothers had received multiple booster doses of toxoid during pregnancy, the anti-toxin concentrations were 100- and 400-times the presumed protective level. Therefore the toxin dose may overwhelm the pre-existing anti-toxin level and produce disease. Furthermore, multiple booster injections of tetanus toxoid may not only enhance serum anti-toxin titres, but could also lead to an ineffective immune response.     

Influence of Mouse Strain on the Assayed Potency (Unitage) of Tetanus Toxoid

The assayed potency of an adsorbed tetanus toxoid C(2), relative to the international standard for tetanus toxoid (adsorbed), varied significantly with the use of different strains of mice. The unitage was highest with NIH mice, and it was not significantly different from that with CFW mice. With CDF(1) and BALB/c mice, however, the assayed potency was significantly lower than with NIH mice. C3H mice failed to respond to the dose range of the toxoids employed with the other four strains. The significance of the influence of the mouse strain on the designation of a prescribed unit requirement for tetanus toxoid, adsorbed, relative to human efficacy is discussed.     

Human peripheral blood lymphocytes transplanted into SCID mice constitute an in vivo culture system exhibiting several parameters found in a normal humoral immune response and are a source of immunocytes for the production of human monoclonal antibodies.

Human PBL from vaccinated healthy blood donors, which was transplanted i.p. into mice with severe combined immunodeficiency (SCID), exhibited an Ag-dependent humoral Ir against tetanus toxoid. This Ir was dose dependent and was completely abrogated by immunizing with large amounts of Ag, suggesting a high dose tolerization of the B cells. A dose-dependent selection of specific, high affinity B clonotypes was also suggested, since immunization with low concentrations of tetanus toxoid produced antisera with higher avidity than immunizations using a high dose of Ag. The production of human Ig and the clonal outgrowth of normal human B cells in the SCID mouse was strongly down-regulated by human NK cells. Human immune B lymphocytes were also recovered from immunized SCID mice and transformed with EBV, yielding lymphoblastoid cell lines producing high affinity antitetanus human IgG antibodies. These results suggest that SCID mice, repopulated with human PBL, can constitute a functional model of several parameters of a normal human humoral Ir and can provide a source of immune B cells for the production of human mAb.     

Human T-lymphocyte chemotactic activity: nature and production in response to antigen

Previous studies have shown that supernatants from concanavalin A-stimulated human peripheral blood mononuclear cells and isolated Leu-2 suppressor/cytotoxic T cells are chemotactic for Leu-3 helper/inducer T cells. The current study shows that lymphocyte chemotactic factor (LCF) is also produced following antigen (tetanus toxoid) challenge of mononuclear cells obtained from recently immunized human donors. LCF was detected in 24-hr supernatants from mononuclear cells challenged with tetanus and was produced maximally at 48 hr. Tetanus toxoid challenge of mononuclear cells obtained from individuals whom had not received a tetanus immunization for 7 to 10 years prior to testing showed little or no production of LCF. Serial studies of these individuals following a tetanus booster immunization showed that LCF was produced by antigen-challenged mononuclear cells obtained 1-5 days postimmunization, was produced optimally 5-15 days postimmunization, and was still produced by antigen-challenged mononuclear cells obtained 6 weeks later. Fractionation of mononuclear cells from immunized donors into glass wool nonadherent lymphocytes, T lymphocytes, and non-T lymphocytes showed that tetanus-toxoid-induced LCF was produced by nonadherent lymphocytes and T cells but not non-T cells. Further fractionation of T lymphocytes into Leu-2 and Leu-3 T-cell subpopulations showed that LCF production by antigen-challenged isolated subpopulations was limited to the Leu-2 suppressor/cytotoxic T-cell subset. Characterization of both Con A and tetanus toxoid-induced LCF by gel filtration on Sephadex G-100 demonstrated the presence of two peaks of LCF corresponding to molecular weights of approximately 14,000-17,000 and 40,000-50,000.     

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.     

Tetanus antibody titers and duration of immunity to clinical tetanus infections in free-ranging rhesus monkeys (Macaca mulatta)

Prior to 1985 tetanus was a major cause of mortality in the free-ranging colony of rhesus monkeys on Cayo Santiago, accounting for almost a quarter of annual deaths. In 1985 and 1986 all animals (except infants) received primary and booster doses, respectively, of tetanus toxoid. In subsequent years primary immunizations were given to all yearlings, and boosters were administered to all 2-year-old animals during the annual capture of the colony. The main objectives of the tetanus immunization program were to reduce the pain and suffering caused by tetanus infections and to decrease mortality in the colony. Other objectives were to evaluate the efficacy of the two-dose tetanus toxoid immunization protocol and to determine whether additional boosters might be required to provide adequate long-term protection against tetanus infections. The immediate effect of the mass immunization program was the elimination of clinical tetanus infections in the population and a 42.2% reduction in the overall mortality rate. Since the immunization program began, no cases of tetanus have been observed in the colony, except in two unimmunized infants, and it has not been necessary to give tertiary injections of tetanus toxoid to maintain protection against infection. A sample collected in 2004 of the original cohort of monkeys immunized in 1985 and 1986 showed that 93.3% (14/15) had protective tetanus antibody titers (>0.01 IU/ml) at the ages of 20-23 years, which is close to the life expectancy of the Cayo Santiago rhesus macaques. Two intramuscular doses of tetanus toxoid provided long-term, if not lifelong, protection against tetanus for rhesus monkeys living in a tropical clime where tetanus is enzootic and the risk of infection is great.     

Immunisation of adults during an outbreak of diphtheria.

In an outbreak of infection due to Corynebacterium diphtheriae in a hospital for mentally subnormal adults sera from 211 members of staff were screened for diphtheria antitoxin titres. Of these, 79 (37%) required immunisation, and a low dose preparation (1 LfU of diphtheria and 10 LfU tetanus toxoids) was offered. Of the 64 subjects who accepted a single immunisation and were subsequently retested, seroconversion to diphtheria toxoid occurred in 45 (70%), the rate being highest in younger adults. Seroconversion to tetanus toxoid occurred in 59% of subjects. Local reactions to the single dose were reported by 29 (43%) subjects, and nine (13%) experienced moderately severe local reactions and systemic symptoms. We conclude that adults should not be vaccinated without previous screening for susceptibility to diphtheria; that neither previous immunisation nor age is reliable in predicting the need for vaccination; and that though a single booster dose of diphtheria toxoid is probably effective in adults under 45, two doses should be given to those in the older age group.     

双佐剂包被的破伤风类毒素马匹免疫试验

为提高破伤风免疫马匹的血浆抗体效价,应用不同佐剂配制TT抗原,进行马匹超免疫比较研究;采用FIA和植物油双佐剂包被与单佐剂包被的TT抗原,注射马匹进行超免疫,比较三组血浆的效价;结果显示,双佐剂抗原较单佐剂的免疫效果好,但可能对马匹刺激较强,有待调整注射剂量和免疫程序。     

Physicochemical and immunochemical assays for monitoring consistent production of tetanus toxoid

The detoxification of tetanus toxin by formaldehyde is a crucial step in the production of tetanus toxoid. The inactivation results in chemically modified proteins and it determines largely the ultimate efficacy and safety of the vaccine. Currently, the quality of tetanus toxoid lots is evaluated in potency and safety tests performed in animals. As a possible alternative, this article describes a panel of in vitro methods, which provides detailed information about the quality of tetanus toxoid. Ten experimental lots of tetanus toxoid were prepared using increasing concentrations of formaldehyde and glycine to obtain tetanus toxoids having differences in antigenicity, immunogenicity, residual toxicity and protein structure. The structural properties of each individual toxoid were determined using immunochemical and physicochemical methods, including biosensor analysis, ELISA, circular dichroism, TNBS assay, differential scanning calorimetry, fluorescence and SDS-PAGE. The quality of a tetanus toxoid lot can be assessed by these set of analytical techniques. Based on antigenicity, immunogenicity and residual toxicity data, criteria are formulated that tetanus toxoids lot have to meet in order to have a high quality. The in vitro methods are a valuable selection of techniques for monitoring consistency of production of tetanus toxoid, especially for the detoxification process of tetanus toxin.     

Experimental imutiol stimulation of the primary immunological response to tetanus anatoxin]

The data on the influence of imuthiol on humoral and cell-mediated immunity developing after immunization against tetanus are presented. Experiments were made on 166 guinea pigs with the use of imuthiol in doses of 25 and 125 mg/kg and tetanus toxoid in doses of 1.5 and 6.0 BU/kg. The dynamic observation of the animals lasted 30 days. The data of the passive hemagglutination test and the neutralization test indicated that imuthiol had considerable influence on the formation of humoral immunity. The results of skin reactions with tetanin, in vitro cell reactions, as well as resistance tests, showed that imuthiol exerted influence on delayed hypersensitivity, the mechanisms of cell-mediated resistance and survival after challenge. The data on the influence of imuthiol could be registered even at early stages after immunization, the immunostimulating action of the preparation reaching its maximum with tetanus toxoid used in a dose of 6.0 BU/kg and imuthiol, in a dose of 125 mg/kg.     

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.     

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.     

Determination of tetanus toxin and toxoid by ELISA using monoclonal antibodies

The procedure of obtaining monoclonal antibodies TT-1, TT-2, and TT-3 against tetanus toxin/toxoid is described. It is shown that both commercial DTP vaccine and tetanus toxoid conjugated with a low-molecular-weight hapten can be used an 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 detecting as much as 0.01 EC/ml toxoid and 50 LD50/ml toxin.     

The immunization of children with combined diphtheria and tetanus vaccine in Sweden

Two groups derived from 97 children three-four months of age were vaccinated with diphtheria and tetanus vaccines containing either a routinely prepared diphtheria toxoid or a more purified preparation. Two injections were given with an interval of one month and a third injection was given one year after the first. Prior to the third injection no child was without protection against diphtheria, i.e. had an antitoxin titre less than 0.01 IU ml-1. After the third injection 95 and 94% of the children vaccinated with the routinely and more purified diphtheria toxoids, respectively, had diphtheria antitoxin titres greater than 1 IU ml-1 (estimated to provide protection for at least ten years). Systemic reactions such as fever and malaise occurred in five children. Local reactions greater than 10 cm were observed in three children and reactions greater than 5 but less than or equal to 10 cm were seen in 14% of the children. The routinely prepared combined diphtheria and tetanus vaccine, DT, produced very good immunity against diphtheria with moderate side effects. The use of a more purified diphtheria toxoid in the combined vaccine produced the same immunity and side effects.     

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.