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Development of a minimal saponin vaccine adjuvant based on QS-21.

Fernández-Tejada A, Chea EK, George C, Pillarsetty N, Gardner JR, Livingston PO, Ragupathi G, Lewis JS, Tan DS, Gin DY - Nat Chem (2014)

Bottom Line: We found that the entire branched trisaccharide domain of QS-21 is dispensable for adjuvant activity and that the C4-aldehyde substituent, previously proposed to bind covalently to an unknown cellular target, is also not required.Biodistribution studies revealed that active adjuvants were retained preferentially at the injection site and the nearest draining lymph nodes compared with the attenuated variants.Overall, these studies have yielded critical insights into saponin structure-function relationships, provided practical synthetic access to non-toxic adjuvants, and established a platform for detailed mechanistic studies.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology & Chemistry Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065, USA.

ABSTRACT
Adjuvants are materials added to vaccines to enhance the immunological response to an antigen. QS-21 is a natural product adjuvant under investigation in numerous vaccine clinical trials, but its use is constrained by scarcity, toxicity, instability and an enigmatic molecular mechanism of action. Herein we describe the development of a minimal QS-21 analogue that decouples adjuvant activity from toxicity and provides a powerful platform for mechanistic investigations. We found that the entire branched trisaccharide domain of QS-21 is dispensable for adjuvant activity and that the C4-aldehyde substituent, previously proposed to bind covalently to an unknown cellular target, is also not required. Biodistribution studies revealed that active adjuvants were retained preferentially at the injection site and the nearest draining lymph nodes compared with the attenuated variants. Overall, these studies have yielded critical insights into saponin structure-function relationships, provided practical synthetic access to non-toxic adjuvants, and established a platform for detailed mechanistic studies.

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Radioiodinated saponin [131I]-6 and fluorescent saponin 3 localize to lymph nodes and injection site in mice(a) Biodistribution of active adjuvant [131I]-6 ([131I]-SQS-0-0-5-18) and attenuated adjuvant [131I]-8 ([131I]-SQS-0-3-7-18) with OVA antigen, indicating accumulation of [131I]-6, but not [131I]-8, at injection site and lymph nodes (see Supplementary Fig. 2); error bars indicate standard deviation from mean for five mice; statistical significance indicated graphically only for lymph nodes and injection site for clarity: * = p ≤ 0.05: liver, muscle, lymph node, skin, thyroid; ** = p < 0.01: blood, lungs, spleen, kidneys, bone, injection site; *** = p < 0.001: heart. (b) Imaging at injection site (yellow arrows indicate ink circles) with fluorescein-labeled active adjuvant 3 (SQS-0-0-5-12) or unlabeled inactive adjuvant 2 (SQS-0-0-5-11) and Alexa-647-labeled OVA (OVA-A647), indicating retention of 3 and OVA-A647 at injection site; green crescent in fluorescein image for Mouse 2 is due to software ghosting effect (see Supplementary Fig. 5). (c) Imaging of dissected lymph nodes with active adjuvant 3 (SQS-0-0-5-12) or inactive adjuvant 2 (SQS-0-0-5-11) and OVA-A647, indicating increased accumulation of OVA-A647 with 3 but not 2. Mice were injected in left flank and right lymph node serves as negative control within each animal.
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Figure 2: Radioiodinated saponin [131I]-6 and fluorescent saponin 3 localize to lymph nodes and injection site in mice(a) Biodistribution of active adjuvant [131I]-6 ([131I]-SQS-0-0-5-18) and attenuated adjuvant [131I]-8 ([131I]-SQS-0-3-7-18) with OVA antigen, indicating accumulation of [131I]-6, but not [131I]-8, at injection site and lymph nodes (see Supplementary Fig. 2); error bars indicate standard deviation from mean for five mice; statistical significance indicated graphically only for lymph nodes and injection site for clarity: * = p ≤ 0.05: liver, muscle, lymph node, skin, thyroid; ** = p < 0.01: blood, lungs, spleen, kidneys, bone, injection site; *** = p < 0.001: heart. (b) Imaging at injection site (yellow arrows indicate ink circles) with fluorescein-labeled active adjuvant 3 (SQS-0-0-5-12) or unlabeled inactive adjuvant 2 (SQS-0-0-5-11) and Alexa-647-labeled OVA (OVA-A647), indicating retention of 3 and OVA-A647 at injection site; green crescent in fluorescein image for Mouse 2 is due to software ghosting effect (see Supplementary Fig. 5). (c) Imaging of dissected lymph nodes with active adjuvant 3 (SQS-0-0-5-12) or inactive adjuvant 2 (SQS-0-0-5-11) and OVA-A647, indicating increased accumulation of OVA-A647 with 3 but not 2. Mice were injected in left flank and right lymph node serves as negative control within each animal.

Mentions: To identify tissues and organs that could play roles in saponin mechanisms of action, we compared the acute in vivo biodistribution of the active adjuvant [131I]-6 and the attenuated adjuvant [131I]-8 in mice co-administered with OVA. Treatment with [131I]-6, compared to [131I]-8, resulted in significantly higher recovery of radioactivity at the site of injection (17-fold higher, 78% ID/g [injected dose per gram]) and in the nearest draining lymph nodes (24-fold higher, 27% ID/g) at 24 h post-injection (Figure 2a), which was retained at 72 h and 96 h post-injection (Supplementary Fig. 2). In contrast, radioactivity in other tissues where large fold-differences were initially observed (muscle, bone, skin) decreased rapidly at the later timepoints. Minimal deiodination of [131I]-6, evidenced by low thyroid uptake (0.21% ID/g), was observed at 24 h (Figure 2a), although deiodination increased at later time-points (Supplementary Fig. 2). In contrast, the attenuated adjuvant [131I]-8 was recovered at significantly lower levels at the site of injection (4.5% ID/g) and nearest draining lymph nodes (1.1% ID/g) at 24 h post-injection (Figure 2a) and was further cleared from both sites at later time-points (Supplementary Fig. 2). Taken together, these data indicate that only the active adjuvant 6 (SQS-0-0-5-18) localizes to and is retained at the injection site and lymph nodes, while the attenuated adjuvant 8 (SQS-0-3-7-18) is not.


Development of a minimal saponin vaccine adjuvant based on QS-21.

Fernández-Tejada A, Chea EK, George C, Pillarsetty N, Gardner JR, Livingston PO, Ragupathi G, Lewis JS, Tan DS, Gin DY - Nat Chem (2014)

Radioiodinated saponin [131I]-6 and fluorescent saponin 3 localize to lymph nodes and injection site in mice(a) Biodistribution of active adjuvant [131I]-6 ([131I]-SQS-0-0-5-18) and attenuated adjuvant [131I]-8 ([131I]-SQS-0-3-7-18) with OVA antigen, indicating accumulation of [131I]-6, but not [131I]-8, at injection site and lymph nodes (see Supplementary Fig. 2); error bars indicate standard deviation from mean for five mice; statistical significance indicated graphically only for lymph nodes and injection site for clarity: * = p ≤ 0.05: liver, muscle, lymph node, skin, thyroid; ** = p < 0.01: blood, lungs, spleen, kidneys, bone, injection site; *** = p < 0.001: heart. (b) Imaging at injection site (yellow arrows indicate ink circles) with fluorescein-labeled active adjuvant 3 (SQS-0-0-5-12) or unlabeled inactive adjuvant 2 (SQS-0-0-5-11) and Alexa-647-labeled OVA (OVA-A647), indicating retention of 3 and OVA-A647 at injection site; green crescent in fluorescein image for Mouse 2 is due to software ghosting effect (see Supplementary Fig. 5). (c) Imaging of dissected lymph nodes with active adjuvant 3 (SQS-0-0-5-12) or inactive adjuvant 2 (SQS-0-0-5-11) and OVA-A647, indicating increased accumulation of OVA-A647 with 3 but not 2. Mice were injected in left flank and right lymph node serves as negative control within each animal.
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Figure 2: Radioiodinated saponin [131I]-6 and fluorescent saponin 3 localize to lymph nodes and injection site in mice(a) Biodistribution of active adjuvant [131I]-6 ([131I]-SQS-0-0-5-18) and attenuated adjuvant [131I]-8 ([131I]-SQS-0-3-7-18) with OVA antigen, indicating accumulation of [131I]-6, but not [131I]-8, at injection site and lymph nodes (see Supplementary Fig. 2); error bars indicate standard deviation from mean for five mice; statistical significance indicated graphically only for lymph nodes and injection site for clarity: * = p ≤ 0.05: liver, muscle, lymph node, skin, thyroid; ** = p < 0.01: blood, lungs, spleen, kidneys, bone, injection site; *** = p < 0.001: heart. (b) Imaging at injection site (yellow arrows indicate ink circles) with fluorescein-labeled active adjuvant 3 (SQS-0-0-5-12) or unlabeled inactive adjuvant 2 (SQS-0-0-5-11) and Alexa-647-labeled OVA (OVA-A647), indicating retention of 3 and OVA-A647 at injection site; green crescent in fluorescein image for Mouse 2 is due to software ghosting effect (see Supplementary Fig. 5). (c) Imaging of dissected lymph nodes with active adjuvant 3 (SQS-0-0-5-12) or inactive adjuvant 2 (SQS-0-0-5-11) and OVA-A647, indicating increased accumulation of OVA-A647 with 3 but not 2. Mice were injected in left flank and right lymph node serves as negative control within each animal.
Mentions: To identify tissues and organs that could play roles in saponin mechanisms of action, we compared the acute in vivo biodistribution of the active adjuvant [131I]-6 and the attenuated adjuvant [131I]-8 in mice co-administered with OVA. Treatment with [131I]-6, compared to [131I]-8, resulted in significantly higher recovery of radioactivity at the site of injection (17-fold higher, 78% ID/g [injected dose per gram]) and in the nearest draining lymph nodes (24-fold higher, 27% ID/g) at 24 h post-injection (Figure 2a), which was retained at 72 h and 96 h post-injection (Supplementary Fig. 2). In contrast, radioactivity in other tissues where large fold-differences were initially observed (muscle, bone, skin) decreased rapidly at the later timepoints. Minimal deiodination of [131I]-6, evidenced by low thyroid uptake (0.21% ID/g), was observed at 24 h (Figure 2a), although deiodination increased at later time-points (Supplementary Fig. 2). In contrast, the attenuated adjuvant [131I]-8 was recovered at significantly lower levels at the site of injection (4.5% ID/g) and nearest draining lymph nodes (1.1% ID/g) at 24 h post-injection (Figure 2a) and was further cleared from both sites at later time-points (Supplementary Fig. 2). Taken together, these data indicate that only the active adjuvant 6 (SQS-0-0-5-18) localizes to and is retained at the injection site and lymph nodes, while the attenuated adjuvant 8 (SQS-0-3-7-18) is not.

Bottom Line: We found that the entire branched trisaccharide domain of QS-21 is dispensable for adjuvant activity and that the C4-aldehyde substituent, previously proposed to bind covalently to an unknown cellular target, is also not required.Biodistribution studies revealed that active adjuvants were retained preferentially at the injection site and the nearest draining lymph nodes compared with the attenuated variants.Overall, these studies have yielded critical insights into saponin structure-function relationships, provided practical synthetic access to non-toxic adjuvants, and established a platform for detailed mechanistic studies.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology & Chemistry Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065, USA.

ABSTRACT
Adjuvants are materials added to vaccines to enhance the immunological response to an antigen. QS-21 is a natural product adjuvant under investigation in numerous vaccine clinical trials, but its use is constrained by scarcity, toxicity, instability and an enigmatic molecular mechanism of action. Herein we describe the development of a minimal QS-21 analogue that decouples adjuvant activity from toxicity and provides a powerful platform for mechanistic investigations. We found that the entire branched trisaccharide domain of QS-21 is dispensable for adjuvant activity and that the C4-aldehyde substituent, previously proposed to bind covalently to an unknown cellular target, is also not required. Biodistribution studies revealed that active adjuvants were retained preferentially at the injection site and the nearest draining lymph nodes compared with the attenuated variants. Overall, these studies have yielded critical insights into saponin structure-function relationships, provided practical synthetic access to non-toxic adjuvants, and established a platform for detailed mechanistic studies.

Show MeSH
Related in: MedlinePlus