Carbohydrate fatty acid monosulphate ester is a potent adjuvant for low-dose seasonal influenza vaccines.

There is still a need for a better and affordable seasonal influenza vaccine and the use of an adjuvant could solve both issues. Therefore, immunogenicity of a combination of low dose of 1/5TH (3 µg of HA) a licensed seasonal flu vaccine with the novel carbohydrate fatty acid monosulfate ester (CMS)-based adjuvant was investigated in ferrets and safety in rabbits. Without CMS, hemagglutination inhibition (HI) antibody titers ranged from ≤5 to 26 three weeks post immunization 1 (PV-1) and from 7 to 134 post-immunization 2 (PV-2) in ferrets. Virus neutralizing (VN) antibody titers ranged from 20 to 37 PV-1 and from 21 to 148 PV-2. CMS caused 10 to 111- fold increase in HI titers and 3 to 58- fold increase in VN titers PV-1 and PV-2, depending on influenza strain and dose of adjuvant. Eight mg of CMS generated significantly higher antibody titers than 1 or 4 mg, while 1 and 4 mg induced similar responses. Three µg of HA plus 4 mg of CMS was considered the highest human dose and safety of two-fold this dose was determined in acute and repeated-dose toxicity studies in rabbits conducted according to OECD GLP guidelines. The test item did not elicit any clinical signs, local reactions, effect on body weight, effect on urine parameters, effect on blood biochemistry, or gross pathological changes. In blood, increased numbers of neutrophils, lymphocytes and/or monocytes were noted and in iliac lymph nodes, increased cellularity of macrophages of minimal to mild degree were observed. In both ferrets and rabbits, body temperature increased with increasing dose of CMS to a maximum of 1 ˚C during the first day post-immunization, which returned to normal values during the second day. In the local tolerance study, histopathology of the site of injection at 7 days PV-1 revealed minimal, mild or moderate inflammation in 5, 8 and 5 animals, respectively. In the repeated-dose study and 21 days PV-3, minimal, mild or moderate inflammation was observed in 15, 18 and 3 animals, respectively. We concluded that the data show CMS is a potent and safe adjuvant ready for further clinical development of a seasonal influenza vaccine and combines high immunogenicity with possible antigen-sparing capacity.

Carbohydrate fatty acid monosulphate esters are safe and effective adjuvants for humoral responses.

Carbohydrate fatty acid sulphate esters (CFASEs) formulated in a squalane-in-water emulsion are effective adjuvants for humoral responses to a wide range of antigens in various animal species but rise in body temperature and local reactions albeit mild or minimal hampers application in humans. In rabbits, body temperature increased 1°C one day after intramuscular (IM) injection, which returned to normal during the next day. The effect increased with increasing dose of CFASE but not with the number of injections (up to 5). Antigen enhanced the rise in body temperature after booster immunization (P<0.01) but not after priming. Synthetic CFASEs are mixtures of derivatives containing no sulphate, one or multiple sulphate groups and the monosulphate derivatives (CMS) were isolated, incorporated in a squalane in-water emulsion and investigated. In contrast to CFASE, CMS adjuvant did not generate rise in body temperature or local reactions in rabbits immunized with a purified, recombinant malaria chimeric antigen R0.10C. In comparison to alum, CMS adjuvant revealed approximately 30-fold higher antibody titres after the first and >100-fold after the second immunization. In ferrets immunized with 7.5µg of inactivated influenza virus A/H7N9, CMS adjuvant gave 100-fold increase in HAI antibody titres after the first and 25-fold after the second immunisation, which were 10-20-fold higher than with the MF59-like AddaVax adjuvant. In both models, a single immunisation with CMS adjuvant revealed similar or higher titres than two immunisations with either benchmark, without detectable systemic and local adverse effects. Despite striking chemical similarities with monophospholipid A (MPL), CMS adjuvant did not activate human TLR4 expressed on HEK cells. We concluded that the synthetic CMS adjuvant is a promising candidate for poor immunogens and single-shot vaccines and that rise in body temperature, local reactions or activation of TLR4 is not a pre-requisite for high adjuvanticity.