Exploiting structure-activity relationships of QS-21 in the design and synthesis of streamlined saponin vaccine adjuvants

Article abstract of DOI:10.1039/c9cc07781b

We report the design, synthesis, immunological evaluation, and conformational analysis of new saponin variants as promising vaccine adjuvants. These studies have provided expedient synthetic access to streamlined adjuvant-active saponins and yielded molecular-level insights into saponin conformation that correlated with their in vivo adjuvant activities.

Full text of DOI:10.1039/c9cc07781b

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Exploiting structure–activity relationships of QS-21
in the design and synthesis of streamlined
saponin vaccine adjuvants†
Cite this:DOI: 10.1039/c9cc07781b
Received 4th October 2019,
Accepted 12th November 2019
a
a
Mattia Ghirardello,
Diego Barriales,
Francisco Corzana,
Ane Ruiz-de-Angulo,
Jesus Jimenez-Barbero,
Nagore Sacristan,a
b
cd
c
Ana Poveda,
and Alberto Fernandez-Tejada
´
´
e
bd
ad
DOI: 10.1039/c9cc07781b
Juan Anguita
*
*
´
rsc.li/chemcomm
We report the design, synthesis, immunological evaluation, and backbone8 followed by truncation of the right-hand carbohy-
conformational analysis of new saponin variants as promising drate provided stable, adjuvant-active variants having a linear
vaccine adjuvants. These studies have provided expedient synthetic trisaccharide on the ‘‘eastern’’ domain.9 Conservative modifica-
access to streamlined adjuvant-active saponins and yielded molecular- tions of the central b-glycosyl ester linkage identified a potent
level insights into saponin conformation that correlated with their b-thioester variant albeit with dose-limiting toxicity.10 Truncation of
in vivo adjuvant activities.
the left-hand trisaccharide and modification of the triterpene
provided saponin variants with an aryl iodide moiety at the acyl
Adjuvants are essential components in modern vaccines that chain terminus that exhibited adjuvant activity and reduced
boost the immunogenicity of the target antigens potentiating toxicity.11 However, the presence of a hydrophobic and potentially
the immune response. Despite important advances in the field, immunologically active substituent (aryl iodide) limited saponin
only few vaccine adjuvants have been licensed for human use.1 aqueous solubility and could have further immunological effects
QS-21 is a purified saponin fraction extracted from the bark of that might confound SAR information.
the Quillaja saponaria tree2 that consists of a E2 : 1 mixture of
Herein we report rationally designed, streamlined synthetic
isomers sharing the four main structural domains but differing variants (3, 4, 6, 7) that feature optimal structural modifications for
at the terminal apiose (QS-21-Api, 1), or xylose (QS-21-Xyl, 2) sugar potent adjuvant activity and non-toxicity (Fig. 1b). These include a
(Fig. 1a). With the exception of its recent approval as part of the
malaria and shingles vaccines,3 the scarcity, heterogeneity and
dose-limiting toxicity of QS-21 have hampered its further use in
human vaccines.4 Therefore, practical access to new, structurally
simpler and improved saponin-based adjuvants via shortened
synthetic strategies, and the search for alternative, sustainable
sources of adjuvant-active saponins constitute an important focus
in chemical immunology.5 The total synthesis of QS-216 and
subsequent structure–activity relationship (SAR) studies7 by Gin
and co-workers enabled the development of a number of saponin
variants with various structural modifications. Ester-to-amide
replacement of the acyl chain linkages and simplification of its
a Chemical Immunology Lab, Centre for Cooperative Research in Biosciences,
CIC-bioGUNE, 48160 Derio, Biscay, Spain. E-mail: afernandeztejada@cicbiogune.es
b Inflammation and Macrophage Plasticity Lab, Centre for Cooperative Research in
Biosciences, CIC-bioGUNE, 48160 Derio, Biscay, Spain.
E-mail: janguita@cicbiogune.es
c Chemical Glycobiology Lab, Centre for Cooperative Research in Biosciences,
CIC-bioGUNE, 48160 Derio, Biscay, Spain
d Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Spain
e
˜
Department of Chemistry, University of La Rioja, Logrono, 26006, Spain
Fig. 1 (a) Chemical structure of natural QS-21 Api (1) and Xyl (2), and
(b) streamlined saponin variants 3–8 studied in this work.
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9cc07781b
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simplified, immunologically silent carboxylic acid acyl chain with a
Acylation of amines 17–22 with dodecanedioic acid mono-
terminal negative charge (carboxylate) to increase synthetic effi- benzyl ester 23 to give 24–29, and subsequent global deprotec-
ciency and polarity, and the right-hand linear trisaccharide as tion by hydrogenolysis (H2, Pd/C) and acid hydrolysis (TFA/
optimal carbohydrate moiety within this domain. Notably, despite H2O) afforded saponin variants 3–8 in good to moderate yields
the toxicity showed by the central b-glycosyl ester and thioester (Scheme 2). To minimize undesired reduction of the thioester
variants bearing the left-hand trisaccharide,10 their superior adju- functionality to the corresponding C28-aldehyde, the hydro-
vant activity led us to select these two central linkages to connect genolysis reaction of the b-thioester variants was carefully
the acyl chain-trisaccharide conjugate to the original quillaic acid monitored and the time was reduced to 20 minutes.
(QA) core as well as to the echinocystic acid (EA) triterpene, in
We next evaluated saponin adjuvant activity (50 mg dose) by
which the native C4-aldehyde substituent is replaced with a methyl vaccinating mice in combination with OVA (20 mg) as a model
group. To solve the toxicity issue and based on previous SAR,12 antigen at days 0, 14, and 28. As controls, mice were immunized
these saponins were designed and synthesised devoid of the entire with OVA alone (20 mg) (no-adjuvant) and with OVA (20 mg) plus
branched trisaccharide domain, providing new variants through a QS-21 (20 mg) (positive control), a QS-21 dose known to induce
shortened synthetic route. The corresponding b-glycosyl ester and optimal antibody responses with acceptable toxicity in mice.
thioester congeners incorporating oleanolic acid (OA) (5, 8), in Antibody titres against OVA in mouse sera were determined by
which the triterpene C4-aldehyde substituent and the C16-hydroxyl indirect ELISA before the second boost (day 26) and at the time
group are lacking, were also synthesised and evaluated for com- of sacrifice (day 46). On day 26, anti-OVA IgG titres elicited by
parative purposes (Fig. 1b). Conformational analysis by NMR and echinocystic and quillaic acid variants were significantly higher
molecular dynamics (MD) simulations on the ester variants (3–5) than those of the no-adjuvant control and comparable to (for
identified a key role for the C16-hydroxyl group in saponin con- echinocystic acid variants), or just slightly below (for the
formation that correlated with adjuvant activity.
quillaic acid derivatives) those of the QS-21 group (Fig. 2a). In
The syntheses of ester variants 3–5 started with Schmidt all cases, replacement of the central ester linkage with the
glycosylation (BF3ꢀOEt2) of triterpenes 9–1111 with trisaccharide thioester linker (6–8) did not impair saponin adjuvant activity.
trichloroacetimidate 159 to provide, after benzeneselenol Oleanolic acid variants 5 and 8 elicited lower antibody
(PhSeH) reduction of the azide to the amine, the QA, EA and responses, similar to the no-adjuvant control. The observed
OA b-ester intermediates 17–19 in good overall yields (Scheme 1). trend was maintained on day 46 (Fig. 2b), with increased titres
For the synthesis of the thioester variants 6–8, the triterpene acyl for all three ester variants compared to those on day 26. On day
chlorides 12–14 were first prepared by treatment of acids 9–11 46, however, the anti-OVA IgG titres with quillaic acid thioester
with thionyl chloride. Glycosylation of triterpenes 12–14 with 6 were no longer significantly higher than the no-adjuvant
trisaccharide thiohemiacetal 1610 in the presence of NaH, group. Antibody subtyping of the anti-OVA IgG isotypes (day 46)
followed by azide reduction gave the corresponding b-thioester showed that both echinocystic acid variants 4 and 7 elicited
derivatives 20–22.
the highest IgG1, IgG2b and IgG2c titres among the synthetic
Scheme 1 Reagent and conditions: (a) SOCl2, Py, DCM, 0 1C to r.t., 2 h,
99%; (b) BF3ꢀEt2O, 4 Å MS, DCM, ꢁ78 1C, 6 h (b)0 NaH, THF, 0 1C to r.t., 3 h;
(c) PhSeH, Et3N/THF/toluene 10 : 3 : 3, 40 1C, 3 h, 83% (2 steps) (for 17),
64% (2 steps) (for 18), 66% (2 steps) (for 19); 57% (2 steps) (for 20), 99%
(2 steps) (for 21), 75% (2 steps) (for 22).
Scheme 2 Reagent and conditions: (a) EtOCOCl, Et3N, THF, 0 1C to r.t.,
4 h, 61% (for 24), 65% (for 25), 90% (for 26); 65% (for 27), 71% (for 28), and
77% (for 29); (b) 1: Pd/C, H2 (1 atm), EtOH/THF 1 : 1, r.t., 16 h (for 24–26);
20 min (for 27–29); 2: TFA/H2O 3 : 1, 0 1C, 1 h, 84% (for 3), 49% (for 4), 63%
(for 5); 46% (for 6), 36% (for 7), and 23% (for 8).
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with no significant differences compared to the no adjuvant
control, confirming the non-toxicity of these saponin variants.
Taken together, these results have identified echinocystic
acid variants 4 and 7 as the most potent saponin variants,
eliciting higher antibody responses than their quillaic acid counter-
parts, at levels closer to those of QS-21. These data highlight the
dispensability of the C4-aldehyde substituent and the importance
of the C16-hydroxyl group of the triterpene for elicitation of potent
humoral responses by these synthetic saponins.
Next, to seek a potential role of the triterpene C16-hydroxyl
in saponin conformational preferences that could explain its
importance in adjuvant activity, we performed a conforma-
tional analysis of the synthetic saponins by combining experi-
Fig. 2 Antibody responses in mice elicited by saponins 3–8 with OVA
antigen (a) on day 26 and (b) on day 46 after first immunization. Horizontal
bars indicate median titres. Statistical significance versus no-adjuvant group
using unpaired Student’s t-test: * p r 0.05, ** p r 0.01, *** p r 0.001.
variants (Fig. S1, ESI†). In general, the mouse IgG1 subclass is mental NMR data with molecular dynamics (MD) simulations.
associated with Th2 cell responses (humoral immunity), Given the similar adjuvant activity showed by the ester and
whereas the IgG2 is related to Th1 responses (cellular thioester congeners, we carried out 0.5 ms MD simulations on
immunity).13 For the quillaic acid variants, the ester analogue the three ester variants 3–5 using AMBER 18 implemented with
3 showed IgG1 and IgG2b titres significantly higher than the GAFF and GLYCAM 06 force-fields.14 In parallel, to validate the
no-adjuvant group, albeit lower than its echinocystic acid MD simulations, we performed 2D-NOESY experiments on the
counterpart 4. In contrast, the thioester analogue 6 elicited three variants in methanol, as their solubility in water was not
IgG1 and IgG2b titres similar to the inactive oleanolic acid sufficient for these NMR studies. Whereas NOE cross-peaks
variants 5 and 8 and the negative control (Fig. S1, ESI†).
between the carbohydrate and the triterpene were negative,
As a standard initial overall toxicity assessment, no mouse died positive NOEs for the distal acyl chain methylene protons were
due to immunization with any of the saponin variants (3–8) and observed (Fig. S3, ESI†), implying that the acyl chain displays
no weight loss was observed among the analysed groups. This is different local motion and higher flexibility than the rest of the
notable for quillaic acid variants 3 and 6, since their parent molecule, in accordance with the MD simulations (Fig. S6,
compounds incorporating the branched trisaccharide were toxic ESI†). Moreover, the observed NOE cross-peaks were compati-
at 20 mg (1/5 mouse dead for the ester variant9 and 2/5 mice dead ble with the main conformations derived from the MD in
for the thioester variant10). To assess potential toxic side effects methanol (Fig. S10, ESI†). These conformations differ in the
beyond mortality and weight loss, we analysed hepatic alanine orientation of the ester linkage and thus in the orientation of
transaminase (ALT) levels in blood serum on day 46 (Fig. S2, the Gal residue (and the trisaccharide) relative to the triterpene.
ESI†). The levels of this liver enzyme remained low for all groups, Fittingly, the folded conformation that is predicted by MD in
Fig. 3 Structural ensembles derived from unrestrained 0.5 ms MD simulations of ester variants 3–5 in explicit water, showing rmsd (Å) for heavy atoms
relative to the average structure, key distances (Å), and 3D plots of torsion angle distributions around the central glycosidic linkage.
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methanol (Fig. S6, ESI†) was experimentally confirmed by NMR. in reduced steps [i.e. 22 total steps for 3 QA(O) and 4 EA(O)].
Indeed, NOE cross-peaks between protons at the Gal and Rha Overall, replacement of the ester central linker by a thioester
residues and at the triterpene were observed (Fig. S4 and S5, modification did not significantly impair adjuvant activity.
ESI†). Additionally, the existence of weak cross-peaks between Echinocystic acid variants 4 and 7 were found to be superior to
the Gal protons and a methyl group of the triterpene for 3 and 4 their quillaic acid congeners, which makes them attractive leads
(Fig. S4, ESI†) strongly suggests that the extended conformation for further development and provides a more affordable and
is also present, although in a minor proportion. These experi- sustainable triterpene source for potent saponin adjuvants.
mental results validate our computational approach, which was These multidisciplinary studies emphasise the dispensability
also applied to analyse the conformational behaviour of these of the C4-aldehyde and the importance of the C16-hydroxyl in
derivatives in water. Thus, MD simulations of 3 QA(O), 4 EA(O) the adjuvant activity of these truncated variants. Notably, con-
and 5 OA(O) in explicit water were carried out. According to our formational analysis of the ester variants by NMR and molecular
simulations, both adjuvant-active variants, 3 and 4, exhibited a dynamics simulations highlighted a key role of the triterpene
main conformation in water in which the acyl chain (grey) was C16-hydroxyl group in saponin conformation that correlated
mainly folded back over the triterpene (green), while the linear with adjuvant activity, suggesting a mechanism of action
trisaccharide (orange) was oriented away from the centre of the involving interaction with discrete molecular targets. In all, the
triterpene, in a rather extended orientation (Fig. 3). In contrast, superiority of the echinocystic acid variants presented in this
the inactive variant 5 displayed a distinct conformation. The work makes them leading scaffolds for future mechanistic
folded presentation of the acyl chain over the triterpene was studies and synthetic vaccines based on saponin adjuvants.
barely present, instead pointing away from the triterpene.
Funding from the ERC (ERC-2016-STG-716878 to A. F.-T.)
Moreover, its linear trisaccharide showed a more disorganised and Spanish MCIU (CTQ2017-87530-R, RYC-2015-17888 to
conformation, folded over the triterpene. The different geome- A. F.-T.; SAF2015-65327-R and RTI2018-096494-B-100 to J. A.;
tries were quantitatively characterized by measuring the torsion RTI-2018-099592-B-C21 to F. C.; Severo Ochoa Accreditation
angles around the central glycosidic linkage (i.e. triterpene C17 SEV-2016-0644 to CIC bioGUNE) is acknowledged. A. F.-T. thanks
´
to Gal C1) and the distances A and B, as defined in Fig. 3.
Interestingly, the distributions obtained for the central
linkage torsion angle for the active variants 3 QA(O) and 4
EA(O) were basically identical. In these clusters, the Gal residue
displays its b-face approximately perpendicular to the rear edge
of the triterpene, favouring the folding of the acyl chain over
the triterpene. Strikingly, the lack of the C16-hydroxyl group in
the inactive OA(O) variant (5) modifies the three-dimensional
shape of this saponin in comparison to those deduced for 3
and 4. Thus, 5 exhibited an additional population for the
central linkage torsion angle distribution (Fig. 3), and its linear
trisaccharide displayed a different conformational behaviour
(Fig. S7, ESI†), with a more flexible glycosidic linkage between
the xylose and rhamnose units. Moreover, 5 exhibited longer
distances between the triterpene B-ring and the Gal residue
(A, blue) and between the triterpene B-ring and the acyl chain
(B, red), compared to active variants 3 and 4 (Fig. 3). Therefore,
the presentation of the polar and non-polar moieties of these
molecules is markedly different for the active and inactive
saponins, and the different orientation of the carbohydrate
and acyl chain in 3 QA(O) and 4 EA(O) might facilitate the
hydration of the C16-hydroxyl group. The above conformational
Raquel Fernandez for inspiration.
Conflicts of interest
A. F.-T. is co-inventor on patents and patent applications that
include saponin molecules presented in this work.
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In conclusion, we have designed and synthesised streamlined
saponin variants exploiting optimal structural variations at the
acyl chain, triterpene and central linker regions. The synthetic
strategy enabled efficient access to optimized minimal saponins
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