There is an ever increasing need to develop effective vaccines to combat acute and chronic infections and diseases, in both therapeutic and prophylactic settings. The traditional use of live, attenuated pathogens as immunogens in vaccines has proven successful in addressing a host of maladies, yet there are many pathogens and diseases for which this strategy has proven unsuccessful. As a consequence, many contemporary approaches to vaccine development employ recombinant or synthetic subunit vaccines, which offer improved safety and more precise targeting. However, they are often characterized by poor immunogenicity. As such, subunit antigens must be co-administered with an adjuvant to enhance the immune response.1–8
The discovery of novel adjuvants has emerged as a critical frontline effort in the development of modern vaccine formulations. The adjuvant component enables dose-sparing of rare, expensive, and otherwise impotent antigens. An adjuvant also extends the immunotherapeutic benefits to poor responders, such as elderly or immunocompromised patients. While numerous classes of adjuvants have been explored for vaccine therapies over the past several decades, Alum, a mixture of aluminum salts (hydroxide, phosphate, sulfate),9–12
is still the most popular and is present in approximately half of all human vaccines in the U.S. First introduced for human use in the 1930s, Alum was the only approved adjuvant in man for more than 70 years. It was not until late 2009 that the FDA approved GlaxoSmithKline’s AS04 adjuvant (a proprietary combination of Alum and monophosphoryl lipid A)13
for the Cervarix vaccine to immunize against human papillomavirus (HPV). Thus, there remains a great need for novel adjuvants for use in vaccine therapy.
Currently, one of the most promising new adjuvants undergoing clinical investigation is QS-21, a natural product extract obtained from the bark of the Quillaja saponaria tree, found in the desert regions of Chile, Bolivia, and Southern Peru. This semi-purified extract consists of a mixture of triterpene saponins (), whose principal constituents are characterized by complex bis(desmosides) 1 (QS-21-apiose) and 2 (QS-21-xylose). These immunoactive constituents of QS-21 contain four distinct structural domains: the central triterpene (quillaic acid), a branched trisaccharide at the C3 position of the triterpene, a linear tetrasaccharide attached at C28 of the triterpene, and a branched acyl chain linked to the central fucose moiety of the linear tetrasaccharide.
Structure of the saponin adjuvant QS-21 and four key structural domains.
The importance of QS-21 as an investigational adjuvant is evident in its use in more than 100 clinical trials involving >6,000 total patients. QS-21 has exhibited a remarkable ability to augment clinically significant responses to vaccine antigens targeting a wide landscape of diseases and degenerative disorders (e.g., cancer, Alzheimer’s disease, malaria, HIV, hepatitis). Despite these promising indications, further commercial advancement of QS-21 is hampered by low isolation yields, inconsistent composition, and lack of purity. Moreover, the instability of QS-21, characterized by spontaneous hydrolysis of the acyl domain, limits its efficacy, enhances its local reactogenicity, and complicates formulation and storage protocols. Finally, these hurdles in advancing QS-21 are further exacerbated by the fact that the mechanism by which it potentiates the immune response is unknown. In the absence of insights on this front, there exists no rational path forward to design novel saponin adjuvants with improved vaccine efficacy.
Our previous synthetic studies on QS-21 have permitted access to each specific isomeric saponin within the mixture,14–17
thereby obviating the problems associated with inconsistent purity and composition. While the initial synthetic route was rather lengthy, a significantly streamlined semi-synthetic route was subsequently developed.18
This involved isolation of the entire Quillaja
prosapogenin half of the adjuvant ([QPS]
, ) from the commercial semipurified Quillaja saponaria
extract Quil-A, followed by derivatization into a selectively protected form (protected Quillaja
). Integration of this technology into the synthesis of novel saponins offers a convenient means by which to synthesize and append distinct oligosaccharides and acyl chain variants for evaluation.
Semisynthetic approach to QS-21 analogues 5–7 with variations in the acyl chain domain.
Our initial work in this area involved the preparation and evaluation of three non-natural analogues comprising acyl chain domain variants 5
These syntheses involved modification of the fucose moiety within the linear tetrasaccharide domain of the saponin to reduce hydrolytic instability, allowing the attachment of three distinct lipophilic acyl chains. This initial study revealed that significant structural modification of the central fucosyl pyranose and of the acyl chain of QS-21 causes little or no impairment of adjuvant activity. Moreover, differing toxicity profiles were observed.
Although these initial findings highlighted the exciting prospect of designing improved saponin adjuvants, these three compounds offered a rather narrow insight into the structure–activity profile of these adjuvants. For example, while all three synthetic Quillaja saponins were competent immunostimulators, the complex acyl chain in 5 required an excessively long synthesis, the simplified acyl chain in 6 imparted unacceptable levels of toxicity, and the non-glycosylated acyl chain in 7 limited its aqueous solubility.
Herein, we report efforts that have defined the specific substructures of the saponin adjuvant that are critical for adjuvant activity. Multiple variations of both the acyl chain and linear tetrasaccharide domains have been explored systematically, leading to the development of highly simplified structures that show excellent immunostimulatory properties with encouraging toxicity profiles. In addition, these efforts led to the development of a synthetic Quillaja saponin scaffold that is amenable to attachment of fluorescent reporter groups while still retaining adjuvant activity. The development of this novel class of biochemical tools allows, for the first time, direct investigations of QS-21-derived molecular probes to unravel the cellular mechanisms of saponin immunostimulation.