Adjuvant development is driven by the need to improve immune responses to vaccine antigens without increasing side effects beyond an acceptable level. Adjuvant use in human vaccines has been both limited and cautious and, until the last decade, only aluminum salt adjuvants were approved. In animal vaccines, however, several adjuvants and antigen-presentation systems have been used, including plain saponins, immune-stimulating complexes (ISCOMs), matrix, montanide oils, carbopol formulations, poxvirus constructs and DNA-based formulations. Here, we show that the novel and widely tested saponin based Matrix M™ (Isconova AB, Uppsala, Sweden) adjuvant formulation is ready for application in humans.
Quillaja saponins
The potent adjuvant effects of saponins have been known for decades Citation[1] and their use in foot-and-mouth disease vaccines dates back to the middle of the last century. The adjuvant activities induced by Quillaja saponin are characterized by enhancement of a combination of antibody and cellular immune responses whereas, for example, aluminum salt adjuvants (Al(OH)3 and AlPO3) and most oil adjuvants mainly promote an antibody response. Adverse effects associated with saponin use are associated with their lytic properties, pain and a general molecular instability leading to degradation at physiological pH at ambient temperatures Citation[2].
ISCOMs
Immune-stimulating complexes were conceived by a combination of scientific reasoning and an element of the unexpected. The idea behind the ISCOM was to coformulate antigen and adjuvant in a particle Citation[3]. The resulting spherical ISCOM particle of approximately 40 nm diameter is remarkably stable, held together by the strong affinity between saponin and cholesterol Citation[4]. The particle also provides chemical stability to the fragile saponin molecules under conditions where free saponins degrade quickly.
Immune-stimulating complexes are designed to be the ultimate adjuvant formulation when it comes to efficacy Citation[5], inducing long-lasting biologically active antibodies and cellular immune responses including cytotoxic T cells (CTLs) Citation[6]. ISCOMs were found to be efficient in a variety of animals Citation[5], including man.
A major challenge in ISCOM manufacturing is related to the physical incorporation of the antigen. With membrane-derived antigens, the technology is feasible, however, with other antigens, particularly recombinant DNA proteins, reproducible and efficient incorporation is difficult. Various technical solutions of binding antigens to ISCOM constituents were useful at the laboratory scale, but were less robust for commercial scale vaccine production. Another challenge is the adjustment of the adjuvant or saponin dose, as the saponins are the cornerstones of the ISCOM particle. The amount of saponin required for the actual construction is often considerably higher than that needed to achieve a potent adjuvant effect. High doses of saponin may be reactive, particularly in mice.
Turning challenges into success
As often occurs in research, challenges prompted new developments and successes. Despite poor antigen incorporation, the efficacy of ISCOMs was surprisingly rarely compromised.
Eventually, this led to the understanding that the physical incorporation of the antigen is often not as crucial as initially perceived. This insight developed slowly, since it contradicted the initial perception of the ISCOM mode-of-action, which was to co-deliver antigen and adjuvant in one-and-the-same particle to antigen-presenting cells. In the cell, the antigen and adjuvant are distributed to the endosome and cytosol compartments being a prerequisite for efficient T-helper and CTL induction, respectively Citation[7].
Matrix formulations
The notion that matrix and antigen do not need to be physically linked to achieve potent immune stimulation made it possible to focus on optimization and customization of the matrix product itself.
Just like ISCOMs, matrix formulations are homogenous populations of physically stable nanoparticles consisting of saponin, cholesterol and phospholipid. Likewise, the bonding of saponin to cholesterol in the matrix complex eliminates the hemolytic activity of saponin.
A major source of saponin for adjuvant use is the tree Quillaja saponaria Molina, containing a number of related molecules differing mainly by sugar moieties and the presence or absence of an acyl side chain. Matrix produced from purified but non-fractionated saponins are extensively used in animal vaccines. For companion animals including horse Citation[8], and for humans Citation[9], purified and well-defined fractions of saponin are used. A handful of registered animal vaccines and several human clinical trials rely on matrix-based adjuvants Citation[9,10]. Matrix is currently produced at industrial scale in bulk quantities.
Adjuvant activity of matrix adjuvant
The adjuvant activities of matrix formulations parallel those of ISCOMs, inducing high and long-lasting levels of broadly reacting antibodies supported by a balanced Th1/Th2 response including multifunctional T cells Citation[10,11] and CTLs Citation[5,6]. The adjuvant properties of matrix fulfil the demands of a modern adjuvant: a formulation with dose-sparing capacity, improved quality of immune responses and an acceptable safety profile.
In contrast to most adjuvant formulations, ISCOMs and matrix adjuvant do not exert adjuvant activity through depot or slow-release effects. On the contrary, adjuvant and antigen is rapidly removed from the site of injection Citation[5].
Matrix formulations perform well with most types of antigen, with the exception of low-molecular-weight hydrophilic antigens. Such antigens probably require some ‘physical’ help in terms of size and/or multimericity. The formulation of vaccines with matrix adjuvant is convenient as the vaccine antigen(s) is simply mixed with a suspension of matrix particles in physiological buffers not requiring adsorption or emulsification.
Further improved qualities by formulation: Matrix M
The development of matrix adjuvant technology relates to the purification and characterization of saponin fractions, but above all, to the formulation of the selected saponin fractions. The novel formulation, Matrix M, consists of two individually formed matrix particles, each with a different and well-characterized saponin fraction with complementary properties. The mixture of individually formulated particles significantly improves both adjuvant activity and safety profile. One type of particle consists of highly adjuvant active saponin molecules (Fraction-C saponin), the other of a weaker but very well-tolerated saponin (Fraction A). By mixing the two matrix particles, the dose of the highly active but slightly reactogenic Matrix C™ component may be lowered by a factor of 5–10 while keeping the high-dose adjuvant activity. Hence the safety margin is substantially increased, which is essential for certain sensitive animal species. Matrix M has recently entered into a human clinical trial with promising results in terms of both efficacy and safety Citation[10,11].
The impact of formulation
In the pharmaceutical industry it is well recognized that a new formulation of the same substance is a new drug as the effect and performance might be significantly influenced. Likewise, new formulations of Quillaja saponin resulted in altered and remarkably improved adjuvant properties. A human vaccine dose formulated with Matrix will probably not contain more than a total of 100–200 µg of material, antigen and adjuvant combined. In contrast, conventional adjuvant formulations usually contain >10 mg of these materials combined.
In the vaccine field, hopes are raised that the physical presentation of vaccine antigens in particulate form (nanoparticles), such as micelles, virosomes and virus-like particles would suffice for optimal immune induction. However, it is appreciated that an adjuvant component is essential to achieve optimal efficacy and quality of the immune responses.
It is well appreciated that Matrix M exhibits all qualities of a modern, safe and effective adjuvant. It induces well-balanced and potent cellular and humoral immune responses. These potent adjuvant properties of Matrix M are currently being evaluated in a human influenza clinical trial and will in the near future, be assessed in clinical trials for additional human indications.
Financial & competing interests disclosure
Karin Lövgren Bengtsson is CSO at Isconova AB and a minority shareholder. Bror Morein is a senior advisor to Isconova and is a shareholder of Isconova AB. Albert Osterhaus is CSO of ViroClinics Biosciences B.V. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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