Mucosal immunisation with Clostridium botulinum type C 16 S toxoid and its non-toxic component

Abstract

Clostridium botulinum types C and D produce a 16 S (500 kDa) toxin that is formed by conjugation of neurotoxin with a non-toxic component (nonTox). The amino acid sequences of type C and D nonTox components are almost identical. In a previous report it was proposed that nonTox is necessary for the effective absorption of the toxin from the small intestine. This suggested the hypothesis that mucosal immunity against nonTox in the small intestine might prevent the absorption of both C-and D-16 S toxins. The nonTox was purified from a mutant strain, (C)-N71, that does not produce neurotoxin. This nonTox or detoxified C-16 S toxin were mixed with adjuvant (a mutant form of heat-labile toxin of Escherichia coli), and inoculated into mice via the nasal or oral route, or both. The mice inoculated nasally four times with nonTox or toxoid produced high levels of antibodies (including IgA) against the immunogens, both in intestinal fluids and sera. When these nonTox-immunised mice were challenged orally with 2 and 20 oral minimum lethal doses (MLD) of C-or D-16 S toxins, the same results were obtained with both C and D; the mice survived after challenge with 2 MLD of either C or D but were killed by 20 MLD of either toxin although the time to death was significantly longer than in the control non-immunised mice. These results indicate that the local anti-nonTox antibodies reduce absorption of both C-and D-16 S toxins from the small intestine. The C-16 S toxoid-immunised mice showed similar behaviour with type D toxin challenge, probably due to the same mechanism, but were protected against 20 MLD of C-16 S toxin.

Disruption of the epithelial barrier by botulinum haemagglutinin (HA) proteins – differences in cell tropism and the mechanism of action between HA proteins of types A or B, and HA proteins of type C

“…The HA-positive toxin complex rich fraction eluted from this column was collected and dialysed against 0.01 M sodium phosphate buffer (pH 6.0). To obtain the HA-negative toxin complex, fraction 2 was dialysed against 0.05 M sodium acetate buffer (pH 4.2), and applied to an SP-Toyopearl 650M (Tosoh) column as described previously (Mahmut et al, 2002). The HA-negative toxin complex rich fraction eluted from this column was collected and dialysed against 0.01 M sodium phosphate buffer (pH 6.0).…”

“…The HA-positive toxin complex was concentrated in the precipitate which appeared during dialysis (fraction 1), and the HA-negative toxin complex remained in the supernatant (fraction 2). After centrifugation (15 000 g, 30 min, 4 uC), the precipitate, which contained the HA-positive toxin complex, was dialysed against 0.05 M sodium acetate buffer (pH 4.2) and applied to an SP-Toyopearl 650M (Tosoh) column as described previously (Mahmut et al, 2002). The HA-positive toxin complex rich fraction eluted from this column was collected and dialysed against 0.01 M sodium phosphate buffer (pH 6.0).…”

Mucosal vaccine development for botulinum intoxication

“…In this regard, it is essential to develop mucosal vaccines protecting the UR tract from inhalational BoNT intoxication since oral vaccines most likely fail to induce Ag-specific immunity in the respiratory mucosa. It has been shown that nasal immunization with type C progenitor toxoid 16 S (formalin-inactivated type C BoNT with auxiliary proteins, 500 kDa, L toxin) with mutant heat labile toxin from E. coli (deleting tripeptide Arg192-Thr193-Ile194) as nasal adjuvant induced Ag-specific S-IgA Ab responses in the intestinal fluids and plasma [87]. Further, mice given 16 S type C vaccine were protected when orally challenged with 20 times the minimum oral lethal dose of C 16 BoNT [87].…”

Quantitative Analysis of Levels of Serum Immunoglobulin G against Botulinum Neurotoxin Type D and Association with Protection in Natural Outbreaks of Cattle Botulism

“…Since then, ELISA systems have been used for diagnosis of botulism in various species, including wild birds (28), cattle (17,22), and recently a dog (4). ELISA systems, which offer greater flexibility and economy than other immunoassay techniques, were also used for the evaluation of the immune response to various botulinum vaccines in mice (21), primates (19), humans (32), and cattle (3). Previously, the ELISA positive/negative cutoff values used for diagnosis of botulism in cattle were defined as the upper 99% confidence limit of the mean of a population of cattle not exposed to BoNTs plus 3 sample standard deviations (13).…”