Enhancing the Magnitude of Antibody Responses through Biomaterial Stereochemistry Rajagopal Appavu, † Charles B. Chesson, ‡,§ Alexey Y. Koyfman, † Joshua D. Snook, † Frederick J. Kohlhapp, ∥ Andrew Zloza, ∥ and Jai S. Rudra* ,†,§ † Department of Pharmacology & Toxicology, ‡ Institute for Translational Sciences, and § Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas 77555, United States ∥ Departments of Microbiology/Immunology and Internal Medicine, Rush University Medical Center, Chicago, Illinois 60612, United States ABSTRACT: D-Amino acid analogs of peptides and proteins are attractive for applications in biotechnology and medicine due to their reduced proteolytic sensitivity. Here, we report that self-assembling peptide nanofibers composed of D-amino acids act as immune adjuvants, and investigate their ability to induce antibody responses in comparison to their L-amino acid counterparts. The model antigenic peptide OVA (chicken egg ovalbumin aa 323−339) from chicken egg ovalbumin, known to elicit antibody responses in mice, was linked to an L- or D- amino acid self-assembling peptide domain to generate enantiomeric nanofibers displaying the same epitope. The chiral nature of the fusion peptides was confirmed by circular dichrosim spectroscopy and transmission electron microscopy studies indicated that OVA-bearing enantiomers self-assembled into nanofibers with similar morphologies. In mice, D-amino acid peptide nanofibers displaying OVA elicited stronger antibody responses, equivalent levels of CD4+ T cell responses, and long-term antigen-presentation in vivo compared to L-amino acid nanofibers. Our findings indicate that self-assembling peptides composed of D-amino acids are strong immune adjuvants and that biomaterial stereochemistry can be used as a design tool to program adaptive immune responses for vaccine development. KEYWORDS: adjuvant, enantiomer, nanofiber, peptide, stereochemistry, self-assembly 1. INTRODUCTION Biomaterials constructed from self-assembling peptides have attracted considerable interest for applications in biotechnology and medicine because of their ease of synthesis, biocompati- bility, and biodegradation properties. 1−7 Recently, self-assem- bling peptides that form β-sheet rich nanofibers have been reported to act as effective immune adjuvants and elicit strong antibody and cellular immune responses in mice. 8−12 When a peptide or protein antigen is covalently linked to a self- assembling peptide domain the resulting nanofibers display the antigen on their surface in a multivalent fashion. 8 Immunization with antigen-bearing peptide nanofibers has been shown to be protective in murine models of malaria, 10 cancer, 11 and influenza. 12 Moreover, this phenomenon was not limited to a single self-assembling peptide domain, route of administration, disease model, or mouse strain. 9,11 Also, no immune responses have been detected against self-assembling peptides themselves even when administered with strong exogenous adjuvants. 8 This is of immense interest for applications in vaccine development and immunotherapy because of the toxicity and compositional heterogeneity of currently used immune adjuvants. 13 Because naturally occurring proteins utilize only L-form amino acids, most self-assembling peptides designed to date have utilized naturally occurring L-amino acids. 14 Although D- amino acids very rarely participate in protein synthesis, they are vital to all living organisms including bacteria (D-alanine is a component of the cell wall) and mammals (D-serine is involved in glutamatergic neurotransmission in the central nervous system). 15,16 In humans, physiological fluids such as plasma, cerebrospinal fluid, and amniotic fluid have been reported to contain high levels of D-amino acids. 17 In an effort to control the rate of protease-mediated degradation and enhance stability of peptide therapeutics in vivo, one logical strategy that has been used is the full or partial replacement of L-amino acids with their D-enantiomers. D-Amino acid peptides are resistant to proteolysis and have numerous applications in microbiology, physiology, and medicine. 18−20 In the course of applying this strategy to self-assembling peptides, Zhang and co-workers first reported the effects of stereochemistry on the assembly and behavior of self- assembling peptide biomaterials using the enantiomers L- EAK16 and D-EAK16. 14,21 Although both peptides assembled into well-ordered nanofibers with mirror image secondary structures, significant differences were observed in responses to external stimuli like pH, temperature, and the presence of Received: March 19, 2015 Accepted: June 5, 2015 Article pubs.acs.org/journal/abseba © XXXX American Chemical Society A DOI: 10.1021/acsbiomaterials.5b00139 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX