The host adaptive immune response to a pathogen infection comprises both cell mediated and antibody dependent components. Antibody mediated neutralization is a key component of protection against viruses and is the primary focus of this thesis. Antibodies recognize structurally defined epitopes within the context of native proteins. These may be represented by a simple linear sequence of amino acids or a discontinuous sequence of residues brought together by the conformational constraints of the protein. Many protein epitopes recognized by antibodies have been shown to be short α-helices of 3-5 turns. However corresponding synthetic peptides of this length have no structure in water because solvent competes strongly for the hydrogen bonding amides otherwise required to hydrogen bond one another to define an α-helix. This thesis is aimed primarily at (1) synthetically constraining short peptide sequences (9-13 residues) into stable α-helices of 3-4 turns; (2) structurally characterizing such constrained α-helical structures by circular dichroism and 1D and 2D NMR spectroscopy; and (3) evaluating these helix mimetics for serum stability, immunogenicity, antigenicity as well as the biological relevance of the antibodies they induce. The overall aim was to demonstrate that constrained short peptides more effectively structurally and functionally mimic known α-helical B cell epitopes from native proteins than unconstrained short peptides of the same lengths. The primary focus of Chapter 2 was to optimize in vitro ELISA conditions and immunization protocols for potentially assessing antibody responses in mice to short peptides corresponding to segments of important dengue virus proteins (NS1 and the envelope fusion protein, E). The NS1 peptide investigated had been suggested to be an α-helical epitope, but my investigations reveal that it is more likely a turn rather than a helix. While the E protein epitope chosen was not a viable epitope for testing a helix-constraining strategy, it was evaluated as a constrained turn mimic of a viral fusion epitope. Although the constrained peptides from both proteins (NS1 and E) elicited stronger antibody responses in mice than their unconstrained analogues, they still induced relatively poor antibody levels. Interestingly, mouse antibodies raised to the constrained peptide (β-turn analogue) from NS1 protein also reacted with the native protein. To evaluate a helix-constraining strategy for short peptides (less than 15 residues) that have no helix structure in water, an epitope of the HPV E7 protein was selected for mimicry. A short peptide sequence corresponding to this B cell epitope had previously been reported to have α-helical propensity but only in trifluoroethanol-water mixtures, and my initial work showed that it had no detectable helical structure at all in water. Chapter 3 presents an example of a short helical peptide as a B cell epitope, constrained into an α-helix by a side chain to side chain lactam bridge. The constraint involved cyclizing the peptide by specifically linking together side chains of lysine and aspartic acid inserted in the sequence three amino acids apart. CD and NMR structural studies highlighted significant α-helicity in the constrained short peptide, whereas the corresponding unconstrained short peptide had no structure in water. Both unconstrained and constrained short peptide epitopes were injected into mice and antibodies raised were quantified ex vivo by peptide ELISA. The helix-constrained epitope elicited higher antibody titres than the unconstrained peptide which was relatively non-immunogenic. Importantly, antibodies raised to the constrained synthetic α-helical peptide also reacted with the native E7 protein, suggesting that the helical constraint conferred on the peptide a structure analogous to that seen in the protein. In Chapter 4 a constrained α-helical peptide corresponding to a crystallographically defined α-helical sequence in the fusion, F protein of respiratory syncitial virus (RSV) was investigated for its potential to induce an antibody response. Again, while the helix-constrained peptide clearly had α-helicity by CD and NMR studies, the unconstrained short peptide had no detectable helical structure in water. To potentially boost antibody responses, relative to those generated against the dengue virus peptides examined in Chapter 3, both unconstrained and constrained peptides were coupled to the carrier protein KLH before immunizing mice. Significant levels of peptide reactive antibody were generated to both the unconstrained and constrained peptides. However, when investigated in a viral neutralization assay, the antibodies raised to the unconstrained peptide showed a higher neutralization potential than those raised to the constrained peptide. We attribute this unexpected difference to the fact that the region of the F protein corresponding to the epitope chosen, undergoes dramatic conformational changes during the viral fusion process and it is only in its post-fusion form that this helix has been observed. It is possible that the inherent flexibility of the linear, unconstrained counterpart of this epitope may more effectively mimic the conformational intermediates of the native structure on presentation to the immune system. Chapter 5 began an examination of the effects of three different adjuvants on antibody induction by short peptides. They were compared using a candidate peptide vaccine for malaria as a model system. As before, a helix-constrained peptide was compared with its unconstrained peptide sequence in immunization experiments. Higher titres of antibodies were raised to the constrained versus unconstrained peptides. In the second part of this chapter, a putative cancer vaccine peptide was similarly constrained via an ester linkage or a helix-inducing lactam bridge but both methods induced only low T-cell responses compared to their corresponding unconstrained sequences, possibly because the incorrect structure had been stabilized. The focus of this thesis was to evaluate a helix stabilization strategy for its possible application to short peptide vaccines. Using extensive circular dichroism and NMR spectroscopy measurements, we have shown in all cases that helix-constrained peptides were much more α-helical in solution than their corresponding unconstrained short peptide sequences that tended to have no or negligible α-helix structure in water. In some examples, we have compared serum stability and found that constrained peptides have higher serum stability than unconstrained peptides, a difference attributed to their greater stability towards proteolytic degradation – proteases being unable to recognize helices. We have also proven that the helix-constrained peptides induced higher mouse antibody titres than unconstrained peptides. Several attempts were made to boost antibody responses to the peptides by varying either immunization protocols, adjuvant or by attaching a carrier molecule. Further work is needed to optimize this promising new approach to short peptide vaccines.
| Identifer | oai:union.ndltd.org:ADTP/254053 |
| Creators | Dhiraj Hans |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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