The availability of single-administration vaccines would assist in the control of global mortality caused by infectious diseases where protection can be achieved only upon repeated immunisations with appropriate vaccines. Biodegradable microspheres of poly(lactide-co-glycolide) have been studied pre-clinically for this purpose and shown to be promising for several protein and sub-unit antigens. In view of preparing a microsphere-based tetanus vaccine for clinical trials, final candidate vaccine-formulations were pre-clinically optimised here. Specifically, the importance of particular materials and processing for the induction of neutralising antibodies in guinea pigs were examined. The most efficacious vaccines were small-sized (<5 microm), co-adjuvanted with admixed alum and fabricated from fast-degrading polymers. Interestingly, the immunogenicity was less influenced by the type of antigen-stabilising excipient, the number of microsphere populations mixed together, or the microencapsulation technology, i.e. spray-drying versus coacervation, used. On the basis of these, we plan to prepare clinical samples for safety and immunogenicity testing in man.