Abstract
The lipopeptide, R4Pam2Cys, associates electrostatically with soluble protein antigens and significantly enhances their ability to induce protective humoral and cell-mediated responses. We demonstrate that antibody titers elicited by the antigen ovalbumin (OVA) associated with R4Pam2Cys are higher than those elicited by OVA in the presence of alum and comparable to those elicited by OVA formulated with complete Freund’s adjuvant (CFA). The hierarchy of anti-OVA antibody avidities was CFA > R4Pam2Cys = alum. Each of the three adjuvants facilitated IgG class-switching with significantly more IgG1 elicited by OVA when formulated with R4Pam2Cys. The effects of substituting naturally occurring l-stereoisomers of the cationic residues within R4Pam2Cys with d-stereoisomers revealed that substitution did not affect the ability of R4Pam2Cys to stimulate dendritic cell maturation or its ability to elicit antibody production when used as an adjuvant. Minor detrimental effects were, however, observed in the ensuing CD8+ T cell responses suggesting that the use of d-amino acids affects antigen processing and presentation pathways involved in generation of cell-mediated immunity at least when facilitated through TLR2. Both d- and l-forms were found to be resistant to digestion by trypsin, indicating resistance of the branched structure to protease activity.
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Acknowledgements
The authors would like to acknowledge Dr. Weiguang Zeng for advice on the synthesis of the lipopeptides used in this study.
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The manuscript was written with contributions from all authors each of whom have given approval to the final version of the manuscript.
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The study was supported by funds from Innavac Pty. Ltd. and DCJ is chief scientific officer of Innavac Pty. Ltd.
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ARW was in receipt of student support from Bio Farma Perseo, Bandung, Java, Indonesia during the course of this work. DCJ is recipient of research fellowship APP1042018 from the National Health and Medical Research Council of Australia.
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All animal experimentation was performed with approval from The University of Melbourne’s animal ethics committee.
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Handling Editor: J. D. Wade.
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Wijayadikusumah, A.R., Sullivan, L.C., Jackson, D.C. et al. Structure–function relationships of protein–lipopeptide complexes and influence on immunogenicity. Amino Acids 49, 1691–1704 (2017). https://doi.org/10.1007/s00726-017-2466-6
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DOI: https://doi.org/10.1007/s00726-017-2466-6