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.

Improvement of the systemic prime/oral boost strategy for systemic and local responses.

This paper describes oral boost immunisations of primed animals as an alternative oral vaccination strategy. Mice were primed orally (PO), intranasally (IN), subcutaneously (SC), or intraperitoneally (IP) with ovalbumin (OVA) with or without adjuvant. Boost immunisations were given orally with or without cholera toxin (CT) as adjuvant. Prime immunisations induced variable IgA and IgG(1) titres in serum depending on the route. A subsequent oral boost increased these titres. Use of an adjuvant in the priming significantly increased serum IgA and, to a lesser extend, IgG(1). Oral boost immunisation induced significantly higher serum IgA titres in animals primed via the SC, IP and the IN route compared to the PO route. This was independent of the use of CT. Three oral boosts with OVA plus 5 microg CT given in 5 days to primed mice revealed higher IgA titres compared to single oral boosts and anti-OVA IgA titres in faeces were also detected. Finally, we put together our findings and propose a systemic priming/oral boost strategy in which mice were primed via the SC route with 100 microg OVA plus 50 microg Butyl16-p(AA), and subsequently orally boosted with three doses of 300 microg OVA plus 5 microg CT each. We concluded that oral immunisation is more effective in IN, SC, or IP primed mice than in PO primed mice, and that the IgA antibody response in serum and faeces can be improved by increasing the immunisation frequency and the use of appropriate adjuvants in primary and boost immunisation. The here-formulated strategy improves the probability of success of oral vaccination. The results are discussed in the light of the development of edible vaccines.

Efficacy of oral administration and oral intake of edible vaccines.

To evaluate whether vaccine administration via intragastric gavage is indicative for the outcome of edible vaccines, mice were orally immunised with ovalbumin (OVA) mixed with or without Vibrio cholerae toxin (CT) in various compositions via various routes: (1) OVA dissolved in saline and intragastrically (IG) administered ('IG'); (2) OVA mixed with food extract and administered IG ('food IG'); (3) food chow absorbed with OVA dissolved in saline and fed to the animals ('food'); and (4) OVA dissolved in saline and administered via drinking bottles ('drinking'). When given to naive mice, 'IG' and 'food IG' but not 'food' or 'drinking' induced anti-OVA IgG1 responses in serum, but oral boost immunisations were necessary. Serum IgA was not induced. Oral boosting of subcutaneously (SC) primed mice enhanced the IgG1 and IgA response in serum regardless of the route of immunisation or the vaccine composition. CT did not dramatically enhance the immune response. All immunisation routes except 'drinking' induced antigen-specific IgA antibody secreting cells (ASC) in the lamina propria of naive mice. But antigen-specific antibody responses in faeces were not observed. We concluded that oral (i.e. IG) administration is distinct from oral intake. The composition of the vaccine (food or saline) did not influence oral administration. We thus suggested that the route of administration greatly influenced the outcome of oral immunisation. Although oral administration is a well-accepted route to test the potentials of oral vaccines, our study demonstrated that it is merely indicative for the effectiveness of edible vaccines. Studies on the feasibility of edible vaccines should thus be performed by eating the vaccine.

Protective antiviral immune responses to pseudorabies virus induced by DNA vaccination using dimethyldioctadecylammonium bromide as an adjuvant.

To enhance the efficacy of a DNA vaccine against pseudorabies virus (PRV), we evaluated the adjuvant properties of plasmids coding for gamma interferon or interleukin-12, of CpG immunostimulatory motifs, and of the conventional adjuvants dimethyldioctadecylammonium bromide in water (DDA) and sulfolipo-cyclodextrin in squalene in water. We demonstrate that a DNA vaccine combined with DDA, but not with the other adjuvants, induced significantly stronger immune responses than plasmid vaccination alone. Moreover, pigs vaccinated in the presence of DDA were protected against clinical disease and shed significantly less PRV after challenge infection. This is the first study to demonstrate that DDA, a conventional adjuvant, enhances DNA vaccine-induced antiviral immunity.

Assessment of protective efficacy of live and killed vaccines based on a non-encapsulated mutant of Streptococcus suis serotype 2.

The protective efficacy of a live and killed non-encapsulated isogenic mutant of Streptococcus suis serotype 2 was determined in pigs, and compared with the efficacy of the capsulated wild-type strain. SPF pigs were vaccinated twice intramuscularly at 4 and 7 weeks of age with a dose of 1 x 10(9) formalin-killed CFU of the wild-type (WT-BAC), formalin-killed non-encapsulated mutant (CM-BAC) or live non-encapsulated mutant (CM-LIVE) strain. After 2 weeks, vaccinated pigs and non-vaccinated controls were challenged intravenously with 1 x 10(7) CFU of the homologous, wild-type S. suis serotype 2 strain. Protection was evaluated by clinical, bacteriological, serological and post-mortem examinations. All pigs vaccinated with WT-BAC were completely protected against challenge with the homologous serotype. Pigs vaccinated with CM-BAC were partially protected. Although all pigs vaccinated with CM-BAC survived the challenge, four out of five pigs developed clinical signs of disease for several days. Compared to the WT-BAC and CM-BAC, the CM-LIVE vaccine was less protective. Two out of five pigs vaccinated with CM-LIVE died in the course of the experiment and all of them developed specific clinical signs of disease for several days. The protective efficacy of the vaccines could be associated with serum antibody titers. Antibody titers against cells of wild-type and non-encapsulated mutant strains as well as against muramidase-released proteins (MRP) were high in pigs vaccinated with WT-BAC and CM-BAC. Pigs vaccinated with CM-LIVE showed lower antibody titers. Antibody titers against purified capsular polysaccharides (CPS) of S. suis serotype 2 were only found in pigs vaccinated with WT-BAC. These findings indicate that CPS and other bacterial components of WT-BAC are probably essential for full protection against homologous challenge.