Production and purification of ovine anti-tetanus antibody.

We used the ovine as bioreactor for the production and optimization of anti-tetanus toxin antibody. Four female sheep were immunized with human tetanus vaccine (TT-alum) every two weeks for 16 weeks, after which serum was collected and its titer was estimated by ELISA. The highest titer obtained was 39,000 IU ml-1. To optimize a purification protocol for ovine anti-tetanus toxin, we used four procedures; weak anion (DEAE-Sephadex), weak cation (CM-Sephadex), ammonium sulfate precipitation alone or in combination with caprylic acid. Fifty percent saturation with ammonium sulfate combined with caprylic acid gave us the highest yield of protein with specific activity and the purest Fab product.

Pluronic F127-based systemic vaccine delivery systems.

We have developed a vaccine delivery system based on the non-ionic block copolymer, Pluronic F127 (F127), combined with selected immunomodulators. F127-based matrices are characterized by a phenomenon known as reverse thermogelation, whereby the formulation undergoes a phase transition from liquid to gel upon reaching physiological temperatures. Protein antigens (tetanus toxoid (TT), diphtheria toxoid (DT) and anthrax recombinant protective antigen (rPA)) were formulated with F127 in combination with CpG motifs or chitosan, as examples of immunomodulators, and were compared to more traditional adjuvants in mice. IgG antibody responses were significantly enhanced by the F127/CpG and F127/chitosan combinations compared to antigens mixed with CpGs or chitosan alone. In addition, the responses were significantly greater than those elicited by aluminum salts. Furthermore, the functional activity of these antibodies was demonstrated using either in vivo tetanus toxin challenge or an anthrax lethal toxin neutralization assay. These studies suggest that a block-copolymer approach could enhance the delivery of a variety of clinically useful antigens in vaccination schemes.

Immunogenicity of a combined diphtheria-tetanus-acellular pertussis vaccine in adults.

Two clinical studies were undertaken to evaluate the immunogenicity of an adult-type dTpa booster vaccine (Boostrix by GlaxoSmithKline Biologicals). Blood samples taken prior to vaccination showed that 24.4 and 13.0% of subjects were seronegative for diphtheria and tetanus antibodies, respectively. Moreover, about one-third of the vaccinees had no detectable levels of antibodies to pertussis toxoid (PT) or pertactin (PRN). One month post-vaccination, more than 93% of all individuals, regardless of age or type of vaccine received, had seroprotective antibody levels for diphtheria and tetanus (> or = 0.1IU/ml). In those individuals vaccinated with the adult-type dTpa vaccine (Boostrix), more than 98% were found to be seropositive for antibodies to all three pertussis antigens (PT, filamentous haemogluttin (FHA), and PRN). These data suggest that immunity to diphtheria, tetanus and pertussis (DTP) in adults wanes and that booster vaccination with an adult-type combined dTpa vaccine would boost the serological response to diphtheria antitoxin, tetanus antitoxin and antibodies to Bordetella pertussis PT, FHA and PRN.

Age- and sex-determined differences in the establishment of tetanus antitoxin production in guinea-pigs.

For lack of relevant data of the literature, the tetanus immunisation results obtained in the two sexes were compared in an animal model. Complete immunisation series of weaned, adult and aged guinea-pigs (20-25 animals/group) were performed with aluminium phosphate (AlPO4) adsorbed purified tetanus toxoid (PTAP) as well as with typhoid-tetanus vaccine (TY-TE) containing lipopolysaccharide (LPS). Both vaccines contained 5.0 Lf (limes flocculans, Ramon) per single dose of tetanus toxoid, purity degree: 1500 Lf/mg protein nitrogen (PN). Tetanus antitoxin titres (TAT) were measured after the first shot, and subsequently before and after booster. Compared to TAT of male animals, significantly lower titres were found in female animals after basic immunisation with PTAP in all the three age groups: 1.03 vs. 0.57, 8.75 vs. 5.64, and 0.27 vs. 0.15 IU (international units, related to the Copenhagen International Standard) per ml (sex-chromosome-dependent differences?), as well as in adult animals immunised with TY-TE, before booster: 0.07 vs. 0.02 IU/ml (hormone-dependent differences?). In the latter case the TAT results after booster were 14.49 vs. 12.89 IU/ml. Thus, the lower female prebooster titres were counterbalanced by a quick and effective increase of titres following booster. These results are in accordance with our previous observations in humans (Réthy and Réthy, 1986). From our observations with tetanus immunisation series on guinea-pigs it can be concluded that TAT may be influenced by the effects of sex chromosomes as well as of sexual hormones. During active anti-tetanus immunisation with LPS-containing vaccine (TY-TE) the lower adult female prebooster titres are presumably counterbalanced by the better functionality of the female immune memory.

Collagen minipellet as a controlled release delivery system for tetanus and diphtheria toxoid.

The use of biodegradable polymer matrices as a single-dose vaccine delivery system was investigated using tetanus toxoid (TT) and diphtheria toxoid (DT). BALB/c mice were immunized with TT or DT in different formulations including individual, in minipellet and aluminum hydroxide (alum), and the antibody responses were monitored for 48 weeks. Antigens entrapped in minipellet elicited higher antibody responses compared to those obtained with individual antigens and antigens adsorbed to alum and the antibody levels remained elevated over 48 weeks. In addition, minipellet formulations induced the same subclasses of antibodies induced by alum formulations. These results raise the possibility to obtain optimal and long-lasting immune responses by a single administration of minipellet formulations.

Comparison of in vitro and in vivo methods to study stability of PLGA microencapsulated tetanus toxoid vaccines.

The purpose of this study was to investigate the utility of various in vitro and in vivo methods to assess the stability of experimental vaccines containing tetanus toxoid (TT) within PLGA microspheres. In vitro, the breakdown of the encapsulating polymers into their acid components led to changes in the structure of TT, as determined by the physico-chemical methods, rendering it undetectable by capture ELISA and altering its structural integrity. The changes in TT were directly related to increasing acidity of the vaccine supernate. Purified toxoid (not encapsulated) exposed to low pH (2.5) underwent similar changes but re-neutralisation of buffer containing free toxoid, even after one week at pH 2.5 led to some re-folding of protein as determined by fluorescence spectroscopy and gel filtration chromatography. The microencapsulated vaccines were still able to generate an antibody response in mice even after prolonged pre-incubation at 37 degrees C and the apparent absence of detectable toxoid in the vaccine supernate. Electron microscopy demonstrated differences in the amount of degradation between different formulations of microspheres. Vaccines that had retained their spherical morphology after incubation in vitro for up to 28 days were able to induce protective antibodies response equal to that of freshly prepared vaccines, which indicates that the toxoid within intact microspheres remained immunogenic. Immunochemical and physico-chemical detection methods, performed on antigen released from PLGA vaccines in vitro, are valuable in providing information on product characteristics but may not be able to predict effectiveness and should be used with in vivo methods to evaluate the stability of such formulations.

Leukocyte alkaline phosphatase and serum gamma-glutamil transferase activities in horses used for production of hyper immune sera.

The activities of leukocyte alkaline phosphatase (l-AP) and serum gamma-glutamil transferase (gamma-GT), and total leukocyte counts were determined in horses submitted to the production of hyper immune sera against tetanus (Clostridium tetani). The purpose of this work was to investigate the prospective changes of mentioned parameters in horses under the described circumstances. In addition, the suitability of these parameters in assessing the health condition of the same horses had to be evaluated. The average total leukocyte count increased in one month from the values considered as physiological (8.8 x 10(9)/L, Schalm et al. 1975) to the value of 12 x 10(9)/L and remained at this level up to the end of the trial. The I-AP activities fell after 30 days of the trial for 100 units, and showed permanent slight decreasing tendency thereafter. On the contrary, the serum gamma-GT activity was increasing gradually throughout the whole trial. The results indicate the possibility of reflecting the dynamics of intensified leukocyte metabolism in the course of its function within the chronic inflammation, and development of accompanying pathological changes in the liver. In addition, the initialisation period of pathological changes in the organism of horses in experiment could be between the fourth and sixth month following exposure to antigen.

Oral immunization against diphtheria and tetanus infections by fluid diphtheria and tetanus toxoids.

Purified diphtheria toxoid incorporated in egg yolk and mixed with a medicinal plant seed was used to orally immunize rabbits against diphtheria infection. Animals were partially immunized against a lethal diphtheria toxin challenge. The immunity was complete when gastric enzyme juices were inhibited before oral vaccination by aprotinin, a natural protease inhibitor. Rabbits and monkeys were orally immunized against both diphtheria and tetanus in the same way by pre-treatment with aprotinin. Adult volunteers receiving protease inhibitor before administration of oral toxoids have shown a significant rise in specific circulating antitoxins.

Establishment of stable mouse/human-human hybrid cell lines producing large amounts of anti-tetanus human monoclonal antibodies with high neutralizing activity.

To establish stable hybrid cell lines producing human anti-tetanus antibody with high toxin-neutralizing activity, peripheral lymphocytes from humans hyperimmunized with tetanus toxoid were, after in vitro antigen stimulation, fused with a mouse/human heteromyeloma or human lymphoblastoid cell line and cloned. Unlike the IgM secretors (six clones), the IgG secretors we obtained (six clones) produced anti-tetanus human monoclonal antibodies with high neutralizing activity (the highest one, cell line G2, 4.3 IU/100 micrograms IgG). Appropriate combinations of three or four kinds of monoclonal antibodies of the IgG type resulted in markedly increased neutralizing activity comparable with that of anti-tetanus human polyclonal immunoglobulin preparations currently used clinically on the basis of toxin-specific IgG content. Five of these cell lines produced 10-20 micrograms of antibody per ml for more than 3 months. The cell line G2 produced 6 mg of antibody per day in serum-free medium in a 500-ml bioreactor in perfusion culture and 13-104 mg in a nude mouse. These cell lines satisfied, for the first time, the minimal requirements for applying human monoclonal antibodies to clinical use.

Germinal center kinetics in lymph nodes of primed mice stimulated with complexed as opposed to free antigen.

Primed mice with low titers of circulating tetanus antitoxin (AB) were stimulated via the hind footpads with either fluid tetanus toxoid alone (AG) to create in vivo complexes in AG excess, or the same dose of toxoid complexed at equivalence with isologous antibody (AB-AG CPX), to have in vivo complexes in AB excess. All experimental animals reacted with three topically distinct consecutive waves of enhanced proliferative activity in popliteal lymph nodes, i.e., in the T-zone (peak on day 2), in the medullary area, the main site of plasmocytopoiesis (day 3), and in lymphoid follicles (day 5-6). Maximum serum AB titers following injection of AG-AB CPX were only about 25% of those found in animals boosted with AG alone. This suppressive effect was best reflected in a comparable reduction in plasmocytopoiesis, and to an lesser extent in the proliferative activity within the T-zone, and not at all in the overall magnitude of germinal center formation and/or expansion. However, the patterns of germinal center kinetics differed markedly between the two groups: a high sharp peak of development on day 5, followed by a marked drop on day 6 characterized the response in mice given AG alone, and a broad peak around day 6 that of those receiving AG-AB CPX. These differences could not adequately be accounted for by variations in centroblast/centrocyte proliferation rate vs. pycnotic indices, so that different patterns of lymphoid cell emigration from the centers may be considered. The results suggest that immune complexes, fixed on follicular dendritic cells, with different antigen-to-antibody ratios have divergent effects on the development and kinetics of germinal centers, the principal sites of memory B cell generation.

Autologous red blood cells potentiate antibody synthesis by unfractionated human mononuclear cell cultures.

We have tried to determine the most favourable conditions for the in vitro induction of specific antibody (Ab) responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH). Human peripheral blood mononuclear cells (PBMNC) were obtained from normal volunteers and stimulated with PWM, TT, KLH, and mixtures of PWM and antigens in the presence or absence of autologous red blood cells (RBC) (1:50 ratio of PBMNC/RBC). The cultures were harvested on day 11; immunoglobulins were determined immunonephelometrically and Ab levels by ELISA with human antibodies used for calibration. While anti-TT responses were easy to induce with PBMNC from recently boosted individuals, the production of anti-TT from PBMNC obtained from non-recently boosted individuals was only possible when PBMNC were stimulated with TT and PWM in the presence of autologous RBC. Similarly, anti-KLH responses were easier to induce with PBMNC from an immune donor; maximal response was observed after stimulation with PWM + KLH in the presence of autologous RBC. Stimulation of primary anti-KLH responses with PBMNC from non-immune donors was only successful when the cells were stimulated with KLH + PWM in the presence of autologous RBC. The potentiation of human B-cell responses with autologous RBC can be abrogated by pretreatment of PBMNC with anti-CD2 antibodies and is associated with increased expression of IL-2 receptors and increased production of gamma interferon (IFN-gamma). However, addition of IFN-gamma in different doses and at different times to PWM-stimulated PBMNC cultures was not as effective as addition of RBC in enhancing the production of immunoglobulin and antibody.

Liposomes as immunological adjuvants: antigen incorporation studies.

Tetanus toxoid was incorporated into liposomes composed of equimolar phospholipid and cholesterol. The toxoid was either passively entrapped into multilamellar vesicles prepared by the dehydration-rehydration procedure (DRV) or covalently coupled by diazotization to the surface of multilamellar vesicles (MLV) prepared by the classical procedure. Up to 82.3% of the antigen used was entrapped in neutral, negatively and positively charged DRV composed of a variety of unsaturated and saturated phospholipids and 63.1% was coupled to MLV composed of egg phosphatidylcholine. After freeze-drying of toxoid-incorporating DRV and MLV and subsequent rehydration, up to 93.5% of the antigen was recovered with liposomes and, in the case of MLV, retained its external localization. Upon freeze-drying in the presence of 0.25 M trehalose, up to 96.1% of the antigen was recovered with the DRV liposomes. In immunization studies using Balb/c mice, DRV composed of equimolar egg phosphatidylcholine and cholesterol were shown to act as immunological adjuvants to the entrapped tetanus toxoid. In addition, there was no difference in immune responses between DRV and MLV of identical composition but bearing the toxoid on their surface. Comparison of immune responses to the toxoid entrapped in DRV made of phospholipids with varying gel to liquid crystalline transition temperature (Tc) revealed a reduction in responses to very low or nil values for DRV made of distearoyl phosphatidylcholine (Tc 54 degrees C).

Are potency tests for tetanus vaccines really necessary?

There is no simple relationship connecting the antigen content to the immunogenic potency of tetanus vaccines and the need for potency tests therefore remains inescapable. However, the quantal response methods of assay presently used for the measurement of potency appear to use unnecessarily large numbers of animals and substantial reductions could be made, particularly in the control of combined vaccines, if toxic challenge were replaced by in vitro titration of antitoxin.

In vitro humoral immune response of human peripheral blood lymphocytes to tetanus toxoid sepharose 4B.

The in vitro immune response of unfractionated human peripheral blood lymphocytes (PBL) from immune donors who had not been re-immunized with tetanus toxoid (TT) prior to donation was investigated. In this study we were able to stimulate PBL with tetanus toxoid coupled to Sepharose 4B (STT) for production of anti-tetanus toxoid antibody (Ab). Soluble tetanus toxoid or STT alone did not stimulate production of specific Ab. Pokeweed mitogen (PWM) and STT were required for optimal production of IgG and IgM antibodies specific to tetanus toxoid. Specific Ab responses were reduced in low and high concentrations of STT. Depletion of monocytes had no effect on either total IgG or specific IgG synthesis, but decreased the synthesis of both total and specific IgM. Depletion of E-rosette-forming cells decreased the production of specific Ab, suggesting T-dependency of the immune response to STT. Simultaneous production of total immunoglobulin and specific Ab by Sepharose 4B was negligible in the absence of PWM. In the presence of PWM, total immunoglobulin production was optimal, and specific anti-TT Ab production was undetectable. The specificity of the anti-TT Ab was studied by absorption of the culture supernates with an STT column which removed all measurable specific Ab.

Cimetidine and the immune response. I. In vivo augmentation of nonspecific and specific immune response.

Cimetidine is a commonly prescribed histamine antagonist useful in the treatment of peptic ulcer disease. Histamine receptors are found on suppressor T cells and therefore we expected to observe enhanced immune responsiveness in animals treated with this drug. Mice given daily subcutaneous injections of cimetidine (25 or 100 mg/kg) were found to produce approximately twice as much specific antibody in response to tetanus toxoid immunization. Furthermore, mitogen-stimulated splenocytes from cimetidine-treated animals proliferated to a greater extent and produced more immunoglobulin in vitro than controls. These observations offer direct in vivo evidence for immunomodulation by cimetidine.