Temporal evolution of anti-Clostridium antibody responses in sheep after vaccination with polyvalent clostridial vaccines.

Polyvalent clostridial vaccines, composed of a complex mixture of toxoids from up to 9 different species, are highly effective in controlling clostridial diseases in cattle and sheep. Commercially available vaccines usually state that in normal field conditions two doses administered 4 to 6 weeks apart elicit protective antibody levels that will last for one year. However, studies on the development and duration of the antibody response against the different Clostridium species in target animals are scarce and only partial. Evaluating the temporal evolution of the antibody responses upon vaccination in target species is relevant to understand the bases of protective immunity induced by these vaccines and to develop new optimized vaccines. Here, we assessed the antibody response in sheep against each Clostridium component of two different 9-valent Clostridial vaccines over the period of one year. One vaccine was a commercially available vaccine and the other was an experimental vaccine prepared by us with the same antigens that we used to set up a specific ELISA for each Clostridium species. Both vaccines showed similar results, irrespectively of the origin of the antigens used for the ELISAs, with antibody titers that peaked at day 36 after vaccination and large inter individual variations in the magnitude of the response. Antibody titers were maintained up to 90 days and then markedly decreased, becoming even undetectable in some animals 6 months after vaccination. Given that the current scheme of yearly revaccination has largely shown to be effective at controlling the burden of disease, our results strongly suggest that circulating antibody levels cannot completely explain the protective immunity elicited by these vaccines, and prompt for further studies into the correlates of protection of clostridial vaccines.

Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain.

Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (~30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.