Covalent modifications of antitetanus F(ab')2 fragments with natural and synthetic polyamines and their effects on the antibody endocytosis in cultured HL60 cells.

For antibody therapeutics to succeed when intracellular target molecules are involved, a strategy must be applied to increase the delivery of antibodies into cells to reach their targets. Antibody cationization by chemical conjugation of a polyamine could be one such strategy. Both natural polyamines with increasing net charge valencies (putrescine, PUT; spermidine, SPD; and spermine, SPM) and a synthetic polyamine (hexamethylenediamine, HMD) can be used to cationize antibodies, but no comparison of the respective effects of these polyamines on intracellular delivery of antibodies has been performed yet. This study describes the covalent modification of antitetanus F(ab') 2 with these four polyamines using different reaction conditions, and compares the effects of these modifications on antibody interaction with cultured HL60 cells. The cationized antibodies retained > or =80% of the binding activity of the unmodified F(ab') 2 with regard to tetanus toxin, as measured by an antigen-binding capture enzyme immunoassay. This same method was used to quantify the amount of cell-associated F(ab') 2 following incubation with HL60 cells. Cationization was shown to enhance cell interaction of the F(ab') 2 : the higher the number of coupled polyamine molecules, the greater the amount of antibody associated with the cells. Moreover, coupling the F(ab') 2 to the SPD and SPM polyamines had greater effect on cell interaction than coupling the F(ab') 2 to the PUT and HMD diamines. Internalization of the cationized antibodies by the HL60 cells was demonstrated by confocal microscopy. This technique also showed that SPD and SPM were more effective than PUT and HMD in terms of intracellular delivery of the F(ab') 2 . It follows from all these results that electrostatic interaction involving charge density plays a predominant role in the endocytic transport mechanism of the F(ab') 2 modified with these polyamines. However, coupling the F(ab') 2 to SPM and SPD yielded the same maximum effects in terms of cell interaction, although coupling SPM was expected to increase the antibody net charge valency more than coupling SPD. This finding suggests that the effective global charge for the cell interaction and uptake of polyamine-modified antibodies does not simply correspond to the addition of the ionizable amine functions on the coupled polyamines, and that other factors may come into play.

Maternal HIV infection and placental malaria reduce transplacental antibody transfer and tetanus antibody levels in newborns in Kenya.

BACKGROUND: In clinical trials, maternal tetanus toxoid (TT) vaccination is effective in protecting newborns against tetanus infection, but inadequate placental transfer of tetanus antibodies may contribute to lower-than-expected rates of protection in routine practice. We studied the effect of placental malaria and maternal human immunodeficiency virus (HIV) infection on placental transfer of antibodies to tetanus. METHODS: A total of 704 maternal-cord paired serum samples were tested by ELISA for antibodies to tetanus. The HIV status of all women was determined by an immunoglobulin G antibody-capture particle-adherence test, and placental malaria was determined by placental biopsy. Maternal history of TT vaccination was recorded. RESULTS: Tetanus antibody levels were reduced by 52% (95% confidence interval [CI], 30%-67%) in newborns of HIV-infected women and by 48% (95% CI, 26%-62%) in newborns whose mothers had active-chronic or past placental malaria. Thirty-seven mothers (5.3%) and 55 newborns (7.8%) had tetanus antibody levels <0.1 IU/mL (i.e., were seronegative). Mothers' self-reported history of lack of tetanus immunization was the strongest predictor of seronegativity and of tetanus antibody levels in maternal and cord serum. CONCLUSION: Malarial and HIV infections may hinder efforts to eliminate maternal and neonatal tetanus, making implementation of the current policy for mass vaccination of women of childbearing age an urgent priority.

Lectins from Triticum vulgaris and Limax flavus are universal antagonists of botulinum neurotoxin and tetanus toxin.

Lectins from Anguilla anguilla, Artocarpus integrifolia, Canavalia ensiformis, Datora stramonium, Glycine max, Limax flavus, Ricinus communis and Triticum vulgaris were tested for their abilities to antagonize the binding of botulinum neurotoxin and tetanus toxin to rat brain membranes and to antagonize the ability of these toxins to block neuromuscular transmission in mouse phrenic nerve-hemidiaphragm preparations. Lectins from Limax flavus and Triticum vulgaris, both of which have affinity for sialic acid, were antagonists of the various serotypes of botulinum neurotoxin and tetanus toxin. When tested against the high affinity binding site for botulinum neurotoxin type B, the lectin from Limax flavus had a Ki of 3.1 x 10(-7) M and the lectin from Triticum vulgaris had a Ki of 3.75 x 10(-7) M. When tested against the high affinity binding site for tetanus toxin, the lectins from Limax flavus and Triticum vulgaris had Ki values of 1.5 x 10(-7) and 1 x 10(-6) M, respectively. In all cases the lectins behaved as competitive antagonists. In reverse experiments, neither botulinum toxin nor tetanus toxin was a very effective antagonist of lectin binding to brain membranes. Studies on isolated neuromuscular preparations showed that the lectin from Triticum vulgaris did not affect transmission at concentrations of 10(-6) to 10(-3) M, but at a concentration of 3 x 10(-5) M the lectin produced highly statistically significant antagonism of the neuromuscular blocking properties of botulinum neurotoxin types A, B, C, D, E and F as well as tetanus toxin. The lectin did not antagonize beta-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetanus antitoxin binds to intracellular tetanus toxin in permeabilized chromaffin cells without restoring Ca2(+)-induced exocytosis.

Tetanus toxin blocks Ca2(+)-evoked catecholamine release from permeabilized bovine adrenal chromaffin cells preloaded with gangliosides. Tetanus toxin preincubated with its specific antibodies F(ab')2 is without any effect on exocytosis. Specific antitetanus F(ab')2 presented to chromaffin cells which are pretreated with tetanus toxin and permeabilized by digitonin cannot restore exocytosis. Under the same conditions, however, 125I-labeled F(ab')2 accumulates in chromaffin cells. The accumulation depends on the presence and concentration of tetanus toxin and can be prevented by an excess of unlabeled F(ab')2. Once tetanus toxin has initiated block of exocytosis, it cannot be neutralized by binding to its specific antibody.

[Several properties of low-molecular weight tetanus antitoxin]. / O nekotorykh svoistvakh nizkomolekuliarnogo stolbniachnogo antitoksina

Following intravenous injection of tetanus antitoxin, obtained by tryptic digestion of the horse immunoglobulin "Diaferm-3", purification and concentration of active fragments, the antitoxin was eliminated from the rabbit organism three times more rapidly than after the injection of the original "Diaferm-3" antitoxin. After injection of the split antitoxin its urinary excretion lasted up to 6 days, whereas following injection of the "Diaferm-3" antitoxin it was excreted for up to 19 days; in the first case considerably less antitoxin was excreted than in the second one (2 and 3.5%, respectively). In both cases in the antitoxin excreted with urine represented monovalent. Fab'-fragments, producing a delay in precipitation in the cross reaction in agar gel between the tetanus toxoid and the tetanus antiserum. Fab'-fragment obtained by the mentioned method possessed anaphylactogenic properties.

The effect of co-trimoxazole on antitoxin response to tetanus toxoid.

The effect of a 4-day course of co-trimoxazole (Septrin) on antitoxin response to tetanus vaccine was assessed in a double-blind study involving 22 healthy adults. The tetanus antitoxin levels were measured by in vivo and in vitro methods for up to 8 weeks after the first of two injections of tetanus vaccine, given 4 weeks apart. No sigificant difference was observed in the level of tetanus antitoxin produced in subjects who received co-trimoxazole (Septrin) in recommended therapeutic dosage during the first 4 days of the trial from that in subjects who received placebo tablets. The results indicate that a 4-day course of co-trimoxazole (Septrin) does not affect antibody production.