Sucrose fatty acid sulphate esters as novel vaccine adjuvant.

In search for novel adjuvants for human and veterinary vaccines, we focus on synthetic carbohydrates because microbial carbohydrates function as important alarming signals to the immune system. Mono- and disaccharides were added chemically with various functional groups and adjuvant activity and reactogenicity were determined in parallel. In our test model, we used poor immunogens to identify the most effective adjuvants and non-rodent mammals to facilitate extrapolation to humans. Disaccharides added with both fatty acid and sulphate esters and immobilized on a vehicle exerted high adjuvanticity. Chemical structure and presentation of the compound were optimized for a maximal in vivo performance. The formulation selected for human therapeutic vaccines (designated as 'CoVaccine HT') consists of a sucrose fatty acid sulphate ester immobilized on the oil droplets of a submicron emulsion of squalane-in-water. Both humoral and cell-mediated responses were enhanced in a dosedependent fashion against a wide range of antigens, e.g. inactivated viruses, bacterial subunits, recombinant proteins, virus-like particles and peptide-protein conjugates. Remarkably high booster reactions indicated strong immunological memory established by the first contact between host and antigen in presence of the adjuvant. In comparison with existing adjuvants, CoVaccine HT revealed similar or even higher adjuvanticity than mineral oil emulsions (O/W, W/O or O/W) but significantly lower reactogenicity. We concluded that CoVaccine HT is a promising adjuvant as it combines the efficacy of strong adjuvants with the safety of mild ones, is effective towards various types of antigens in large non-rodent mammals and is a chemically defined, stable, aqueous formulation.

Sucrose fatty acid sulphate esters as novel vaccine adjuvants: effect of the chemical composition.

Adjuvant activity of novel, synthetic sucrose derivatives towards a recombinant glycoprotein was determined in large, non-rodent animal species. Compared to antigen alone, up to 3000-fold higher virus neutralizing antibody titres (VNTs) and 10-fold higher cellular responses against classical swine fever virus were observed in pigs after two immunizations with the sucrose derivatives combined with a squalane-in-water emulsion. The chemical composition of the derivative was crucial and sucrose esters containing one sulphate and seven dodecanoic (C12) or decanoic (C10) esters exerted the highest adjuvanticity. Derivatives without sulphate, with fewer fatty acid esters or with shorter or longer alkyl esters were less effective. Strong adjuvant activity of these formulations was the result of synergistic collaboration between the sucrose ester and the squalane emulsion, as factor of increase in VNT by the individual components was between 4 and 34. Enhanced humoral and cell-mediated immune responses lasted for at least 24 weeks. We concluded that combinations of hydrophobic, negatively-charged sucrose fatty acid sulphate esters plus submicron emulsions of squalane-in-water are strong adjuvants for humoral and cell-mediated immunity and that these formulations are promising adjuvants for future vaccines containing poor immunogens.

Immunological differences between layer- and broiler-type chickens.

In commercial poultry husbandry, alternatives for the use of antibiotics and vaccines are under investigation, which preferably have to be applicable for both layer- and broiler-type chickens. There are indications that the defense mechanisms vary between layer- and broiler-type chickens. Therefore, the difference in immune response between layer- and broiler-type chickens of the same age was investigated, using TNP-KLH (trinitrophenyl-conjugated keyhole limpet hemocyanin) as antigen without adjuvant. First different routes of immunization (intravenously, intramuscular, subcutaneous and ocular) were examined to find out which immunization route gives the highest antibody titers. The intravenous immunization route resulted in higher TNP-specific antibody responses than the other immunization routes tested and therefore this immunization route was used in both following experiments. In order to investigate the optimal dose of antigen needed for immunization, a dose-response curve in broiler- and layer-type chickens was completed. The humoral immune response was measured in serum by a TNP-specific ELISA and the in vitro cellular immune response by an antigen-specific lymphocyte proliferation assay. The antibody response of layer- and broiler-type chickens appeared to differ, not only in optimal dose and response, but also in kinetics of the response itself. Broiler chickens generated higher IgM anti-TNP titers whereas layer-type chickens generated higher IgG anti-TNP titers. This specific antibody response in broiler-type chickens did not last as long as in layer-type chickens. The TNP-specific cellular immune response was detectable in layer-type chickens, but not in broilers. Both types generate a non-specific cellular immune response, although this response in broilers is lower than in layer-type chickens. From these results, we conclude that broilers primarily respond to TNP-KLH with a high IgM antibody response whereas layer-type chickens respond with a high IgG response. In addition, the cellular response of layer-type chickens is much higher than the response of broilers. The results suggest that broilers are specialized in the production of a strong short-term humoral response and layer-type chickens in a long-term humoral response in combination with a strong cellular response, which is in conformity with their life expectancy.