Potency testing of bacterial vaccines for human use.

The potency tests for bacterial vaccines are quite diverse. For some products (pertussis, cholera, anthrax, typhoid and BCG vaccines) these are specified as Additional Standards in the Code of Federal Regulations. For other products (tetanus and diphtheria toxoids, plague vaccine) the testing is done according to so-called Minimum Requirements, which have less regulatory authority than Additional Standards. Still other products (e.g., polysaccharide conjugate vaccines, acellular pertussis vaccine, live oral typhoid) are tested according to individualized criteria that are contained in their specific Product License Applications. For some products there is inadequate knowledge of the pathogenic mechanisms and/or protective factors to design valid in vitro potency tests. In these cases, animal testing with subsequent serologic evaluation or challenge testing is often necessary. Examples would include vaccines such as cholera and plague vaccines. The FDA supports the elimination of animal testing when suitable alternatives are available. Thus, many of the potency tests, especially for newer products, rely on in vitro characterization. For example, the immunogenicity of conventional polysaccharide vaccines is largely proportional to their molecular weight. Potency testing therefore relies heavily on physical characterization in terms of composition, molecular weight, and quantity.

GT1b ganglioside prevents tetanus toxin-induced protein kinase C activation and down-regulation in the neonatal brain in vivo.

A single dose of 0.25 ng of tetanus toxin (TeTx), equivalent to approximately 5 minimal lethal doses, injected intracerebrally to 1-day-old rats, caused translocation, i.e., activation, of Ca(2+)-phosphatidylserine-dependent protein kinase C (PKC) from the cytosolic to the membrane compartment within 1 h. Six hours after treatment with the toxin, a 40-50% reduction in the total brain PKC (cytosolic plus membrane) activity was noticed. GT1b (2 micrograms per brain) ganglioside, a putative receptor for TeTx, completely prevented enzyme translocation when injected intracerebrally 30 min before toxin administration and abolished down-regulation after 6 h from the time of toxin injection. GM1 (2 micrograms per brain), a ganglioside of lesser affinity for TeTx, produced by itself a 20-30% reduction of the total PKC activity and did not reverse TeTx-induced PKC down-regulation after 6 h. 12-O-Tetradecanoylphorbol 13-acetate (TPA) phorbol ester, administered at a concentration of 5 x 10(-5) M, caused activation and down-regulation of the enzyme, although with several orders of magnitude lesser potency. GT1b prevented the TPA-induced down-regulation.

The role of proline 345 in diphtheria toxin translocation.

Diphtheria toxin (DT) can translocate across endosomal membranes in response to low pH. Buried hydrophobic domains localized in the 37-kDa toxin B chain become exposed in response to acidic conditions and are thought to participate in the membrane translocation process. The crystal structure of DT has revealed a structurally distinct translocation domain composed of nine alpha-helices with their interconnecting loops (Choe, S., Bennett, M., Fujii, G., Curmi, P., Kantardjieff, K., Collier, R., and Eisenberg, D. (1992) Nature 357, 216-222). Two of these alpha-helices, TH8 and TH9, are unusually apolar and constitute the central core of the translocation domain. It has been proposed that these domains and the highly charged interconnecting loop undergo a conformation change under acidic conditions producing a dagger-like structure capable of inserting into the membrane thus initiating the translocation process. Proline 345 occupies a strategic location at the end of the TH8 alpha-helix. Proline residues have the ability to undergo a cis-trans isomerization reaction and because of this have been proposed to play a role in the conformational change that is a prerequisite for toxin translocation. The role of the proline at position 345 in membrane translocation was investigated. Pro was mutagenized to Glu and to Gly using a two-step recombinant polymerase chain reaction procedure, and the mutant proteins were expressed in vitro. Glu, an alpha-helix former, and Gly, an alpha-helix breaker, were selected for mutagenesis to distinguish between a structural role for Pro as an alpha-helix breaker and alternative roles, perhaps involving cis-trans isomerization-related conformational changes. Replacing Pro at position 345 with Glu or Gly resulted in a 99% reduction in toxicity to Vero cells. The enzymatic and binding activity of the toxin were not altered by the mutations. Instead, the reduction in toxicity is due to decreased translocation ability, suggesting that the Pro at position 345 plays a specific role in toxin membrane translocation.

Experimental evaluation of antitoxic protective effect of new cholera vaccines in mice.

Intraperitoneal immunization of mice and subsequent challenge with purified cholera toxin (CT) were employed to evaluate the anti-cholera toxin protective effect of two new oral cholera vaccines, live CVD 103-HgR and killed B subunit-whole cell (BS-WC). CVD 103-HgR vaccine demonstrated 100% protection of mice against 2.25 LD50 and 70% against 3 LD50 of CT. Mice immunized with BS-WC vaccine were protected against 2.25 and 3 LD50 of CT in 88 and 62% of cases, respectively. All three killed parenteral vaccines failed to protect against CT. We suggest this mouse system for preliminary evaluation of the antitoxic protective activity of cholera vaccines.

The effects of interleukin 2 and alpha-interferon administration on hepatic drug metabolism in mice.

We have administered the cytokines interleukin 2 (IL-2), alpha-interferon (IFN-alpha), and gamma-interferon (IFN-gamma) to mice and measured the alterations in hepatic drug-metabolizing enzyme activities. For comparative purposes and to understand the mechanism of diphtheria and tetanus toxoids and pertussis (DTP) vaccine-induced inhibition of drug metabolism, we also studied the effects of vaccine administration in mice. The administration of IL-2 alone or in combination with IFN-alpha or IFN-gamma causes dose-dependent increases in hexobarbital-induced sleep times. These increases correlate well with the inhibition of specific microsomal mixed-function oxidase activities. Sublethally irradiated mice and athymic nude mice receiving injections of IL-2 or IL-2 plus IFN-alpha do not show the inhibition of drug metabolism seen in normal mice. However, the inhibition of drug metabolism in DTP vaccine-treated mice was similar in all three groups. These observations indicate a possible role for immune cells (probably T-lymphocytes) in the inhibition of drug metabolism caused by administration of these cytokines, which is different from the inhibition of drug metabolism caused by DTP vaccine.

Effect of tetanus toxin on oxytocin and vasopressin release from nerve endings of the neurohypophysis.

The effect of tetanus toxin on neuropeptide hormone release from isolated nerve endings of the neural lobe of rat pituitaries (neurosecretosomes) was measured in a perfusion system. Tetanus toxin inhibited depolarization-evoked release of oxytocin and vasopressin in a time- and dose-dependent manner. At 1 microgram/ml, tetanus toxin blocked stimulated release by 85%. Tetanus toxin that was preincubated with a neutralizing monoclonal antibody or heated to 100 degrees C had no effect on hormone release. The ionophores A23187 and ionomycin were potent stimulators of hormone release in control nerve endings, but were not able to overcome the effect of tetanus toxin in intoxicated nerve endings. 8-Bromo-cyclic GMP, which has been reported to reverse the action of tetanus toxin in PC12 cells, had no effect on the action of tetanus toxin in neurosecretosomes. Neurosecretosomes are the first system in which tetanus toxin has been shown to block release from peptidergic nerve terminals. They appear to be a valuable in vitro system for studying the biochemical mechanism of tetanus toxin action.

Cloning and expression of functional fragment C of tetanus toxin.

A segment of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by using the polymerase chain reaction. This fragment was cloned into expression vector pTTQ8, under the control of the tac promoter. Expression of this plasmid in Escherichia coli resulted in the production of a protein consisting of 8 amino acids of the vector fused to the C-terminal 460 amino acids of tetanus toxin. This protein (rFragment C) was recognized by an antipeptide antibody specific for fragment C in an enzyme-linked immunosorbent assay and on immunoblots. rFragment C could be purified significantly in one step by immunoaffinity chromatography. Immunization of mice with rFragment C resulted in the production of antibodies that were able to protect the mice against a challenge with tetanus toxin. rFragment C bound to ganglioside GT1b and to neuronal cells in a manner indistinguishable from that of fragment C obtained by papain cleavage of tetanus toxin. For many applications, rFragment C appears to be a suitable alternative to tetanus toxin or toxin-derived fragment C.

Transmembrane diffusion channels in Mycoplasma gallisepticum induced by tetanolysin.

The permeability properties of Mycoplasma gallisepticum cells treated with a purified preparation of tetanolysin were investigated by determining the initial swelling rates of cells suspended in an isoosmotic solution of electrolytes or nonelectrolytes. The swelling, initiated by the tetanolysin, depended on the tetanolysin concentration and was markedly affected by the molecular size of the various osmotic stabilizers utilized. Thus, the initial swelling rates in an isoosmotic solution of monosaccharides were much higher than those in isoosmotic solutions of di-, tri-, or tetrasaccharides. Cell swelling induced by tetanolysin was much lower with energy-depleted M. gallisepticum cells, with arsenate-treated cells, or when the membrane potential (delta psi) was collapsed by valinomycin (10 microM) plus KCl (100 mM). Swelling was not affected by the proton-conducting ionophore carbonyl cyanide-m-chlorophenylhydrazone (1 to 10 microM) or by nigericin (5 microM). These results support the concept that the damage induced by tetanolysin is due to the formation of water-filled pores within the membranes of energized M. gallisepticum cells. Such pores allow the diffusion of hydrophilic molecules into the cells and may vary in size, depending on the tetanolysin concentration utilized.

Isolated light chain of tetanus toxin inhibits exocytosis: studies in digitonin-permeabilized cells.

Previous work indicates that the heavy chain of tetanus toxin is responsible for the binding of the toxin to the neuronal membrane and its subsequent internalization. In the present study, the light chain of tetanus toxin mimicked the holotoxin in inhibiting Ca2+-dependent secretion of [3H]norepinephrine from digitonin-permeabilized adrenal chromaffin cells. Preincubation of tetanus toxin with monoclonal antibodies to the light chain prevented the inhibition by tetanus toxin. Preincubation of tetanus toxin with nonimmune ascites fluid or with monoclonal antibodies directed against the C fragment (the C-terminal of the heavy chain) or the heavy-chain portion of the B fragment did not prevent inhibition by tetanus toxin. The data indicate that the light chain is responsible for the intracellular blockade of exocytosis.

Immune response of the llama (Lama glama) to tetanus toxoid vaccination.

An ELISA was developed to measure serum concentrations of tetanus toxoid-specific immunoglobulins. The titers obtained with this assay were compatible with those obtained by the standard mouse toxin-neutralization test. Serum samples from 123 llamas were analyzed for ELISA titers to tetanus toxoid. Of the 82 vaccinated adults, 75 (91%) had titers greater than or equal to 1:50. The vaccination status and titers of weanlings and juveniles (3 to 12 months old) varied; of the 21 vaccinated, 17 (81%) had titers greater than or equal to 1:50 and 7 of 9 (78%) unvaccinated llamas had titers less than 1:50. The ELISA titers of unvaccinated llamas less than 8 weeks old (crias) were matched with the maternal titers. All crias with titers less than 1:50 had dams with titers greater than or equal to 1:50.

Proteolytic cleavage of tetanus toxin increases activity.

Tetanus toxin is initially synthesized in the form of a single polypeptide chain and then proteolytically "nicked" by the bacteria to produce a two-chain structure joined by a disulfide bond. This two-chain form of the toxin is the form known to be biologically active. Whether such nicking is necessary for activity, as it is for certain other bacterial toxins, has not been demonstrated previously. Single-chain toxin preparations produced by salt extraction from the bacteria are characterized and compared with pure two-chain toxin obtained from extracellular filtrates. The ability of these various toxin preparations to produce paroxysmal activity in mouse spinal cord neurons grown in dissociated cell culture is described. The pure two-chain toxin is demonstrated to have greater activity than the single-chain toxin preparations. Indeed the activity of the single-chain toxin preparations can be explained by the small amounts of residual two-chain toxin present in these extracts. Using a protease from a toxin-minus strain of Clostridium tetani to convert a single-chain toxin preparation to two-chain toxin increases toxin activity. In vivo the single-chain toxin preparation is also less toxic. These findings indicate that proteolytic nicking of tetanus toxin increases activity. The unnicked, single-chain form of tetanus toxin may be a relatively nontoxic protoxin form of the toxin; this is a structure-function relationship similar to that of other bacterial protein toxins.

Sequence homology between tetanus and botulinum toxins detected by an antipeptide antibody.

The extent of immunological similarity between tetanus toxin and botulinum toxins A, B, C1, and E was studied by using 10 antibodies produced against synthetic peptides representing different sequences of tetanus toxin, mouse antitetanus serum, and human Tetanus Immune Globulin. Antibodies produced against the synthetic peptides recognized tetanus toxin in an enzyme-linked immunosorbent assay and on Western blots (immunoblots) but did not appear to recognize the native protein. One of the antitetanus peptide antibodies, which was produced against a peptide from the amino terminal, cross-reacted with three of the four botulinum toxins on immunoblots. This antibody, 1, reacted strongly with botulinum toxins B and C1 and weakly with E but did not recognize type A toxin. None of the other peptide antibodies cross-reacted with the botulinum toxins. Mouse antitetanus serum and human Tetanus Immune Globulin did not recognize any of the botulinum toxins on immunoblots. The amino-terminal region of the light chain of tetanus toxin and botulinum toxin types A, B, C1, and E are known to have sequence homology. Our data demonstrate that for tetanus toxin and botulinum toxin types B, C1, and E this region also has immunological homology. Type A, which has the least amount of homology with tetanus toxin in this region, does not share this immunological homology. These data also suggest that although the native structures of tetanus and botulinum toxins have relatively few common immunological determinants, the two toxins may contain short stretches of identical or very similar amino acid sequences.

Effects of exogenous growth hormone on growth plate cartilage in rats.

Slipped capital femoral epiphysis (SCFE) occurs with greater frequency in children with growth hormone deficiency than in children in the general population. This epidemiologic observation suggests that SCFE may be a complication of growth hormone deficiency, associated pituitary hormone deficiencies, growth hormone therapy, or a combination of these factors. To clarify the relationship of growth hormone treatment to SCFE, homologous growth hormone was administered to rats that were without growth hormone deficiency. This treatment was found to alter the material properties of the growth plate, increasing material stiffness and decreasing specific energy-absorptive capacity. Such material changes render the growth plate more susceptible to injury or separation. The observed effects of growth hormone on the growth plates of intact animals suggest that children without growth hormone deficiency who are treated with growth hormone may be at increased risk for growth plate separation. The experimental data also indicate that growth hormone treatment of children with this deficiency may contribute to the observed increased frequency of SCFE among these patients.

Tetanus toxin in dissociated spinal cord cultures: long-term characterization of form and action.

The clinical course of tetanus is notable, in addition to its often dramatic clinical presentation, by the long duration of the neuromuscular symptoms. Survivors may have tetanic manifestations for several weeks after the onset of the disease. In this article we correlate the duration of specific electrophysiologic effects produced by tetanus toxin with the degradation of cell-associated toxin in primary cultures of mouse spinal cord neurons. From these studies we can conclude that the toxin has a half-life of 5-6 days. Both the heavy and the light chains of tetanus toxin degrade at similar rates. Labeled toxin, visualized by radioautography, is associated with neuronal cell bodies and neurites, and its distribution is not altered during a 1-week period following toxin exposure. Blockade of synaptic activity persists for weeks at the concentration of radiolabeled toxin used in these studies. This blockade of transmission is reversed as the toxin is degraded, suggesting that degradation of toxin may be a sufficient mechanism for recovery from tetanus.

Reevaluation of the role of gangliosides as receptors for tetanus toxin.

Binding of tetanus toxin to rat brain membranes was of lower affinity and capacity when binding was determined in 150 mM NaCl, 50 mM Tris-HCl (pH 7.4) than in 25 mM Tris-acetate (pH 6.0). Binding under both conditions was reduced by treating the membranes with neuraminidase. Pronase treatment, however, reduced toxin binding only in the Tris-saline buffer (pH 7.4). In addition, the concentration of gangliosides required to inhibit toxin binding was 100-fold higher in Tris-saline compared to Tris-acetate buffer. The toxin receptors in the membranes were analyzed by ligand blotting techniques. Membrane components were dissolved in sodium dodecyl sulfate, separated by polyacrylamide gel electrophoresis, and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled toxin. Tetanus toxin bound only to material that migrated in the region of the dye front and was extracted with lipid solvents. Gangliosides isolated from the lipid extracts or other sources were separated by TLC on silica gel and the chromatograms were overlaid with labeled tetanus toxin. The toxin bound to areas where the major rat brain gangliosides migrated. When equimolar amounts of different purified gangliosides were applied to the chromatogram, binding of the toxin was in the order GD1b approximately equal to GT1b approximately equal to GQ1b greater than GD2 greater than GD3 much greater than GD1a approximately equal to GM1. Thus, the toxin appears to have the highest affinity for gangliosides with a disialyl group linked to the inner galactosyl residue. When binding of tetanus toxin to transfers and chromatograms was determined in the Tris-saline buffer (pH 7.4), the toxin bound to the same components but the extent of binding was markedly reduced compared with the low-salt and -pH conditions. Our results indicate that the interaction of tetanus toxin with rat brain membranes and gangliosides is greatly reduced under more physiological conditions of salt and pH and raise the possibility that other membrane components such as sialoglycoproteins may be receptors for the toxin under these conditions.

Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor.

Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site.

Differential neurochemical effects of chronic exposure of cerebral cortical cell culture to valproic acid, diazepam, or ethosuximide.

We have assessed the relative neurochemical effects of valproic acid, ethosuximide, and diazepam on dissociated cultures of mouse cerebral cortex. Cultures were exposed chronically (11 days) to each antiepileptic drug and assayed for number of neurons, total protein, tetanus toxin fixation, high-affinity uptake of gamma-aminobutyric acid and beta-alanine, choline acetyltransferase activity, and specific and clonazepam-displaceable benzodiazepine binding. Ethosuximide-exposed cultures did not evidence neuronal toxicity; exposure to valproic acid and diazepam resulted in modest neuronal toxicity. However, exposure to each of these drugs resulted in a marked reduction in benzodiazepine binding. This effect may relate to a common mechanism of action of drugs used to treat absence seizures.

Antibodies against the light chain of tetanus toxin in human sera.

Tetanus toxoid elicits protective antibodies against tetanus toxin in humans and animals. It has been reported that antitoxin from immunized humans contains no anti-light chain antibodies, based on immunodiffusion and quantitative precipitin analyses. We confirmed the absence of precipitating anti-light chain antibodies in tetanus immune globulin. However, the presence of antibodies against the light chain of the toxin was shown by direct binding and inhibition analyses, using enzyme-linked immunosorbent assays. Using a neutralization inhibition test, we also found that about one-fourth of the neutralizing antibodies in tetanus immune globulin are directed against the light chain. These results suggest that the light chain of tetanus toxin contains immunogenic determinants and that antibodies directed against it may have a role in the prevention of tetanus or treatment of tetanus or both.

Differential toxicity of chronic exposure to phenytoin, phenobarbital, or carbamazepine in cerebral cortical cell cultures.

The effects of phenytoin (30 micrograms/ml), phenobarbital (64 micrograms/ml), and carbamazepine (24 micrograms/ml) were assessed in cerebral cortical cell cultures. After antiepileptic drug exposure for eleven days, cultures were assayed for total protein, number of neurons, tetanus toxin fixation, high-affinity uptake of gamma-aminobutyric acid and beta-alanine, activity of choline acetyltransferase, and benzodiazepine binding. Carbamazepine-exposed cultures demonstrated minimal effects, whereas highly significant deficits related to generalized toxicity were observed in cultures exposed to phenytoin or phenobarbital.