The mechanism of action of MF59 - an innately attractive adjuvant formulation.

MF59 is a safe and effective vaccine adjuvant which was originally approved to be included in a licensed influenza vaccine to be used in the elderly in Europe in 1997. The MF59 adjuvanted influenza vaccine (Fluad™) is now licensed in more than 20 countries worldwide and more than 85 million doses have been administered. More recently the vaccine adjuvant has also been shown to be safe and effective in young children and resulted in a significant increase in influenza vaccine efficacy in a controlled clinical trial in Europe. Since the early days of its discovery we have explored the mechanism of action of MF59, using a variety of available techniques. In recent years we have explored more thoroughly the mechanism of action using new and more sophisticated techniques. It is remarkable how consistent the data has been, using a variety of different approaches both in several small animal models and also using human immune cells in vitro. Here we present a summary of all the work performed to date on the mechanism of action of MF59 and we present a unified theory based on the accumulated data of how it exerts its adjuvant effects. A key element of the mechanism of action appears to be the creation of a transient 'immunocompetent' local environment at the injection site, resulting in the recruitment of key immune cells, which are able to take up antigen and adjuvant and transport them to the local lymph nodes, where the immune response is induced. This recruitment appears to be triggered by the induction of a chemokine driven gradient by the impact of MF59 on local cells, which are activated to secrete further chemokines, which are recruitment factors for more immune cells.

Intranasal immunization of mice with herpes simplex virus type 2 recombinant gD2: the effect of adjuvants on mucosal and serum antibody responses.

Mucosal immunization offers the potential for inducing IgA antibody responses in the vagina, the site of infection for many viruses, including herpes simplex type 2 (HSV-2). To investigate this possibility, mice were immunized intranasally with 10 micrograms glycoprotein D2 (gD2) from HSV combined with a series of adjuvants of proven efficacy; the oil in water emulsion MF59, poly(D,L-lactide-co-glycolide) microparticles (PLG) (encapsulated or co-administered), immune-stimulating complexes (iscoms) (incorporated or co-administered with iscomatrix) and the genetically detoxified enterotoxin from Escherichia coli, LT-K63. Encapsulation of gD2 into PLG microparticles, incorporation of gD2 into iscoms and co-administration of gD2 with LT-K63 induced mucosal IgA antibody responses (nasal wash, saliva and vaginal wash) which were greater than those induced by intramuscular administration of gD2 with MF59. Intranasal immunization with these formulations also induced substantial levels of serum IgG and neutralizing antibodies. These studies demonstrated that intranasal immunization with potent adjuvants is an effective means to induce mucosal antibody responses, even in the lower genital tract.

Recent advances in vaccine adjuvants: the development of MF59 emulsion and polymeric microparticles.

Vaccines produced by recombinant DNA technology are safer than 'traditional' vaccines but they are often poorly immunogenic, requiring adjuvants to enhance their immunogenicity. Particulate adjuvants of defined dimensions (< 5 microns) have been shown to be effective in enhancing the immunogenicity of 'weak' antigens in animal models. Two novel adjuvants that possess significant potential for the development of new vaccines are the MF59 sub-microemulsion and polymeric microparticles. MF59 is an oil-in-water emulsion and has been shown to be both potent and safe in human subjects with several vaccines. Microparticles prepared from the biodegradable polymer poly(lactide-co-glycolide) have been shown to enhance immunogenicity when administered by mucosal routes, such as oral and intranasal, and they also possess considerable potential for the development of single-dose vaccines through the use of controlled-release technology.

Rapid cation-exchange chromatography of hemoglobins and other proteins.

A new, weak cation exchanger (MA7C) is useful for the very rapid resolution of protein mixtures. MA7C columns (30 X 4.6 mm I.D.) are packed with non-porous spherical particles (7-micron in diameter) having carboxylic acid ion-exchange groups. These columns can readily separate human hemoglobin A1c from other hemoglobin species in 7 min. Quantitative results agree well with those of accepted techniques. Other separations are possible with flow-rates up to 5 ml/min. Chromatography on MA7C columns is characterized by very narrow bandwidths, even at high flow-rates.

Rapid protein profiling with a novel anion-exchange material.

A new anion-exchange material has been developed which allows very rapid resolution of protein mixtures. The Microanalyzer MA7P matrix consists of small (7 micron), spherical, non-porous, polymethacrylate beads with polyethyleneimine (PEI) covalently coupled to the surface. When packed 30 X 4.6 mm I.D. columns, this matrix is particularly well suited for applications in which 1-300 micrograms of a protein mixture must be resolved in a minimum of time. Recoveries of injected proteins are usually quantitative, even when the amounts of individual proteins are in the submicrogram range. Chromatography on Microanalyzer MA7P columns is characterized by very narrow bandwidths, even at relatively high flow-rates. This is due to the combined effects of short column length, high selectivity, and the lack of velocity-dependent bandbroadening attributable to diffusion into and out of pores. These columns have no discernable gel filtration effects in the molecular weight range from 10(3) to 10(6) daltons. Columns are very rapidly equilibrated with new solvents, further reducing cycle-to-cycle times.

Anion-exchange high-performance liquid chromatography of human serum apolipoproteins.

The rapid separation of seven urea-soluble apolipoprotein species from delipidated human serum very low density lipoproteins (VLDL) and high density lipoproteins (HDL) has been achieved by high-performance liquid chromatography on an anion-exchange column of Syn-Chropak AX 300. Effluent chromatographic peaks were detected by absorbance at 280 nm in a flow-through cell. Peaks corresponding to apolipoproteins AI1, AI2, AII, CI, CII, CIII1, and CIII2 were identified by amino acid analysis, gel electrophoresis, and isoelectric focusing. Maximum efficient loading of semipreparative columns (250 X 9.0 mm) was established to be ca. 20 mg HDL apolipoprotein. Minimum detectable protein was shown to be ca. 1 microgram on an analytical-scale column (300 X 4.5 mm). Chromatographic resolution is comparable to that of conventional DEAE-cellulose column chromatography. The ratio of apoAI1 to apoAI2 was considerably greater in high-performance liquid chromatography, suggesting that the variants seen in conventional chromatography and isoelectric focusing are in part artifactual.

A spectroscopic and electron microscopic examination of the highly condensed DNA structures formed by denaturation in Mg(ClO4)2.

1. Thermal denaturation in 1.5 M Mg(ClO4)2 of the DNA from bacteriophage lambda results in four well-separated subtransitions, as monitored by the accompanying increase in absorbance. The midpoint of the hyperchromic spectrum is significantly lowered compared to either 1.5 M MgCl2 or 3.0 M NaClO4. 2. The first two subtransitions are associated with the melting of the A . T-richest regions of the lambda DNA, as revealed by electron micrographs following fixation with formaldehyde. 3. Commencing with the third subtransition, an unusual DNA structure is observed in electron micrographs. In this structure the A . T-rich half of the molecule appears completely condensed, whereas the G . C-rich half remains native. 4. During the fourth subtransition DNA molecules condense completely and eventually aggregate to form extremely high molecular weight particles containing centers of electron density. Tendrils of DNA, primarily duplex, radiate outward from these centers. 5. The aggregation may be reversed by the removal of magnesium. The intramolecular condensation may be at least partly reversed by increasing the Mg(ClO4)2 concentrations to saturating levels.

The DNA melting transition in aqueous magnesium salt solutions.

The melting transition of the magnesium salt of DNA has been systematically examined in the presence of various types of anions. The addition of ClO4- to a concentration of 3.0 N results in the biphasic optical transition, with the first phase exhibiting rapid reversibility and independence of the DNA concentration. This subtransition, which is interpreted as an intramolecular condensation to a collapsed form of DNA, is followed by a DNA concentration-dependent aggregation reaction. The aggregation can be reversed by increasing the ClO4- concentration to 6.0 N while elevating the temperature to post-transition levels. Alternatively, both the collapse and the aggregation can be prevented by melting in the presence of trichloroacetate, the most strongly chaotropic solvent for DNA which has been reported (K. Hamaguchi and E. P. Geiduschek (1962), J. Am. Chem. Soc. 84, 1329). The forces responsible for mediating both the collapse and the aggregation are superficially similar to those involved in maintaining duplex stability. The collapsed form, in particular, possibly possesses features in common with the condensed structures which can be produced in aqueous solution of certain polymers, such as polyethylene glycol (Lerman, L.S. (1971), Proc. Natl. Acad. Sci. U.S.A. 68, 1886).