The main finding of this study was that the triblock peptide-PEG-peptide elicited a moderate antibody response, whereas the native peptide and γKEI did not elicit any detectable response. None of the peptide groups elicited IL-2 or IFN-γ production in cultures of lymph node cells or splenocytes from immunized mice. The circular dichroism data indicated that both γKEI and the triblock appeared to possess similar folding, but the triblock molecule was more highly oligomerized as observed by AUC. Collectively, these results suggested that the more extensive oligomerization of the triblock molecule led to the observed increase in its immunogenicity.
Supporting the interpretation that the triblock's oligomerization into higher molecular weight aggregates was the cause of the increased antibody production, other oligomerized peptide systems have shown similar effects. For example, immune responses to self-assembled β-sheet fibrillizing peptides displaying a known epitope on the fibrillar surface were similar to the immune responses observed for the triblock γKEI-PEG-γKEI molecule, though they appeared to be stronger for the β-sheet peptides [21
]. For both the β-sheet fibril system investigated previously and the coiled coil triblock investigated in the present study, an antibody response was observed in the absence of any associated production of IFN-γ or IL-2 in splenocyte cultures [21
]. In the β-sheet system, this response was not elicited by un-assembled epitope peptides without any adjuvant, nor was it elicited by mixtures of β-sheet peptide and soluble antigen peptides, indicating that immunogenicity was dependent on peptide assembly. Aggregation has also been shown to enhance the immunogenicity of recombinant protein therapeutics, and recent approaches in vaccine design have utilized supramolecular assembly to generate immunogens capable of inducing high antibody titers [20
]. For some time it has been known that high molecular weight polymeric compounds with highly repetitive epitopes can activate T cell-independent (TI) antibody responses by delivering prolonged and persistent signaling to B cells [51
]. Such TI antibody responses can be generated in the absence of any additional adjuvant [20
]. Owing to the structural similarity between previously known TI antigens, the triblock investigated here, and the β-sheet fibrillar system reported previously, a similar type of response may have been elicited for the coiled coil triblock. The T cell independence of this response could be definitively ascertained in future studies using T cell knockout mice or another model animal that cannot raise a specific T cell response.
The present study points to the oligomerization of the triblock being a factor that contributed to its immunogenicity, but other mechanisms cannot be explicitly ruled out. For example, although the folding between γKEI and the triblock appeared to be highly similar by CD, there may have been subtle conformational changes between the two not detectable by this method. It is also not known if the native and γKEI peptide would be immunogenic when delivered with an adjuvant like CFA, or if a native-PEG-native triblock could induce a response similar to γKEI-PEG-γKEI triblock. Investigating the immunogenicity of the peptides conjugated to linear, branched, or dendrimeric macromolecules could shed further light on the antigen valency necessary for producing the observed responses. In addition, the triblock possessed an additional Cys residue and a maleimide-thiol linkage to the PEG chain, possibly producing a novel linear epitope. In other contexts, maleimide linkages have been found to be capable of generating immune responses [52
], and this possibility cannot be ruled out in the present system. Control peptides representing γKEI functionalized with small maleimides would resolve this question. With regards to the ELISA measurements, their accuracy is supported by the fact that none of the peptides investigated had an abnormally high background when incubated with irrelevant sera. However, in almost all ELISAs, it is impossible to completely rule out the possibility that different samples could adsorb or present epitopes with different efficiencies on ELISA plates. Given the uniform background levels and the high-binding plates used, however, this seems unlikely.
The role of biomaterials as immune adjuvants is becoming increasingly appreciated. Pioneering work by Babensee's group has shown that biomaterials such as poly(lactic-co-glycolic acid) (PLGA), chitosan, hyaluronic acid, and agarose induce maturation of dendritic cells (DC) [53
]. Immunizing mice with ovalbumin pre-adsorbed onto these polymeric biomaterials can induce moderate immune responses that can be sustained for as long as 18 weeks [54
]. It has been hypothesized that such polymeric biomaterials induce DC activation in a manner analogous to pathogen associated molecular patterns (PAMPs), through pattern recognition receptors (PRRs) including Toll-like receptors (TLRs), or by contributing to the production of damage associated molecular patterns (DAMPs) at the tissue/biomaterial interface [53
]. Dendritic cell maturation can be dependent on the physical shape and surface area of the implanted biomaterial [53
], and antibody responses elicited by PLGA carriers have been shown to be primarily Th2 helper T cell-dependent, as illustrated by a predominance of IgG1 with respect to other antibody isotypes [54
]. Although antibody isotypes were not investigated in the present study, previously investigated β-sheet fibrillizing peptides also exhibited strong IgG1 responses [21
], indicating that there may be some similarities between the responses elicited by self-assembled peptide materials and those elicited by other biomaterials such as PLGA. Additionally, native ECM proteins including fibrinogen have been shown to activate macrophages and promote the attraction of immune infiltrates [55
]. Although it seems unlikely, it is possible that the native immunogenicity of fibrinogen itself could contribute to the immune responses observed for the fibrin-based coiled coil materials described here.
The results of the experiments reported here have a few implications for the development of self-assembling biomaterials, particularly those that have a high degree of oligomerization or multivalency. Many biomaterials currently in development contain peptides, proteins, or other molecules in highly multivalent arrays. Ligand-functionalized polymer hydrogels and self-assembling biomaterials under development as cell scaffolds for regenerative medicine are particular examples [3
]. The present study is part of a growing number of reports indicating that multivalency can lead to measurable antibody responses for such materials. The questions that remain open are how strong of an antibody response, and what type of an antibody response, can be acceptable in different biomaterials contexts? Many applications of highly multimeric biomaterials with conjugated proteins or peptides have been investigated in vivo with good results [8
], making it possible that some degree of antibody response may be acceptable or even desirable. It is expected that the acceptability of any antibody response would be highly dependent on the specific application and tissue site. Elucidation of the mechanisms of immune responses against multivalent materials will continue to be important considerations as they are developed further towards clinical use.