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.

Immunoadjuvants are used to potentiate the activity of modern subunit vaccines that are based on molecular antigens. An emerging approach involves the combination of multiple adjuvants in a single formulation to achieve optimal vaccine efficacy. Herein, to investigate such potential synergies, we synthesized novel adjuvant conjugates based on the saponin natural product QS-21 and the aldehyde tucaresol via chemoselective acylation of an amine at the terminus of the acyl chain domain in QS saponin variants. In a preclinical mouse vaccination model, these QS saponin–tucaresol conjugates induced antibody responses similar to or slightly higher than those generated with related QS saponin variants lacking the tucaresol motif. The conjugates retained potent adjuvant activity, low toxicity, and improved activity–toxicity profiles relative to QS-21 itself and induced IgG subclass profiles similar to those of QS-21, indicative of both Th1 cellular and Th2 humoral immune responses. This study opens the door to installation of other substituents at the terminus of the acyl chain domain to develop additional QS saponin conjugates with desirable immunologic properties.

QS-21 is a potent immunostimulatory saponin that is currently under clinical investigation as an adjuvant in various vaccines to treat infectious diseases, cancers, and cognitive disorders. Herein, we report the design, synthesis, and preclinical evaluation of simplified QS-21 congeners to define key structural features that are critical for adjuvant activity. Truncation of the linear tetrasaccharide domain revealed that a trisaccharide variant is equipotent to QS-21, while the corresponding disaccharide and monosaccharide congeners are more toxic and less potent, respectively. Modification of the acyl chain domain in the trisaccharide series revealed that a terminal carboxylic acid is well-tolerated while a terminal amine results in reduced adjuvant activity. Acylation of the terminal amine can, in some cases, restore adjuvant activity and enables the synthesis of fluorescently labeled QS-21 variants. Cellular studies with these probes revealed that, contrary to conventional wisdom, the most highly adjuvant active of these fluorescently labeled saponins does not simply associate with the plasma membrane, but rather is internalized by dendritic cells.

Aletes acaulis, Cymopterus hendersonii, Cymopterus panamintensis var. acutifolius, Lomatium rigidum, Lomatium scabrum var. tripinnatum, Musineon tenuifolium, Sphenosciadium capitellatum, Tauschia arguta and Tauschia parishii are among the twenty-two species of the Apiaceae family to which female Indra Swallowtail butterflies (Papilio indra: Lepidoptera) are attracted for oviposition. Because plant volatile oils are known to be attractants for female butterflies, the percent composition of the essential oils of each species was studied. Amongst the nine host plants 168 essential oil components were identified representing between 84% and 99% of the oils. Principal Components Analysis and hierarchical cluster analysis on the essential oil compositions of the larval host plants against four non-larval host plants separated the hosts from the non-hosts into distinct clusters. Volatile components of the oils common to the nine species of Apiaceae are correlated with the expression of physiological attraction behavior by the butterfly.

The essential oil compositions of the upper parts of six Lomatium species, Lomatium brandegei, Lomatium east-woodiae, Lomatium graveolens, Lomatium howelii, Lomatium junceum, and Lomatium parryi, were determined by comparing the reported CC, GC/MS data of the oil components as well as by comparing the GC/MS retention times and the mass spectra of several other components with authentic samples. The Indra swallowtail butterfly has been observed to lay eggs oil these Lomatium species. A principal component analysis of the essential oil composition of the six species against the essential oil of Lomatium dissectum var. multifidum, a non-Indra swallowtail butterfly host, was conducted.

BACKGROUND:We examine the accuracy of enzyme catalytic residue predictions from a network representation of protein structure. In this model, amino acid alpha-carbons specify vertices within a graph and edges connect vertices that are proximal in structure. Closeness centrality, which has shown promise in previous investigations, is used to identify important positions within the network. Closeness centrality, a global measure of network centrality, is calculated as the reciprocal of the average distance between vertex i and all other vertices.RESULTS:We benchmark the approach against 283 structurally unique proteins within the Catalytic Site Atlas. Our results, which are inline with previous investigations of smaller datasets, indicate closeness centrality predictions are statistically significant. However, unlike previous approaches, we specifically focus on residues with the very best scores. Over the top five closeness centrality scores, we observe an average true to false positive rate ratio of 6.8 to 1. As demonstrated previously, adding a solvent accessibility filter significantly improves predictive power; the average ratio is increased to 15.3 to 1. We also demonstrate (for the first time) that filtering the predictions by residue identity improves the results even more than accessibility filtering. Here, we simply eliminate residues with physiochemical properties unlikely to be compatible with catalytic requirements from consideration. Residue identity filtering improves the average true to false positive rate ratio to 26.3 to 1. Combining the two filters together has little affect on the results. Calculated p-values for the three prediction schemes range from 2.7E-9 to less than 8.8E-134. Finally, the sensitivity of the predictions to structure choice and slight perturbations is examined.CONCLUSION:Our results resolutely confirm that closeness centrality is a viable prediction scheme whose predictions are statistically significant. Simple filtering schemes substantially improve the method's predicted power. Moreover, no clear effect on performance is observed when comparing ligated and unligated structures. Similarly, the CC prediction results are robust to slight structural perturbations from molecular dynamics simulation.

The variability within calculated protein residue pKa values calculated using Poisson-Boltzmann continuum theory with respect to small conformational fluctuations is investigated. As a general rule, sites buried in the protein core have the largest pKa fluctuations but the least amount of conformational variability; conversely, sites on the protein surface generally have large conformational fluctuations but very small pKa fluctuations. These results occur because of the heterogeneous or uniform nature of the electrostatic microenvironments at the protein core or surface, respectively. Atypical surface sites with large pKa fluctuations occur at the interfaces between significant anionic and cationic potentials.