A novel method to identify and characterise peptide mimotopes of heat shock protein 70-associated antigens

The heat shock protein, Hsp70, has been shown to play an important role in tumour immunity. Vaccination with Hsp70-peptide complexes (Hsp70-PCs), isolated from autologous tumour cells, can induce protective immune responses. We have developed a novel method to identify synthetic mimic peptides of Hsp70-PCs and to test their ability to activate T-cells. Peptides (referred to as "recognisers") that bind to Hsp70-PCs from the human breast carcinoma cell line, MDA-MB-231, were identified by bio-panning a random peptide M13 phage display library. Synthetic recogniser peptides were subsequently used as bait in a reverse bio-panning experiment to identify potential Hsp70-PC mimic peptides. The ability of the recogniser and mimic peptides to prime human lymphocyte responses against tumour cell antigens was tested by stimulating lymphocytes with autologous peptide-loaded monocyte-derived dendritic cells (DCs). Priming and subsequent stimulation with either the recogniser or mimic peptide resulted in interferon-γ (IFN-γ) secretion by the lymphocytes. Furthermore, DCs loaded with Hsp70, Hsp70-PC or the recogniser or the mimic peptide primed the lymphocytes to respond to soluble extracts from breast cells. These results highlight the potential application of synthetic peptide-mimics of Hsp70-PCs, as modulators of the immune response against tumours

Cross-stimulation of CD14- cells by mimic peptides.

<p>CD14⁻ cells were incubated with DCs loaded with mimic peptides and then re-stimulated with a fresh batch of DCs loaded with same or a different mimic peptide. T-cell stimulation was monitored by quantifying the amount of IFN-γ released into the medium. The IFN-γ levels were normalized to the levels produced following incubation with DCs that had no peptides loaded. Error bars represent the normalized % error from two independent experiments. The combination of peptides are indicated in the figure and are as follows <b>A.</b> MP1 followed by MP1; red solid line (•), MP1 followed by MP5; red dashed line (▪), MP5 followed by MP5; green solid line (▴) and MP5 followed by MP1; green dashed line (⋄). <b>B.</b> MP3 followed by MP3; black solid line (▴), MP4 followed by MP4; green solid line (•), MP4 followed by MP3; red solid line (▪).</p

Physical properties of recogniser peptides.

<p>Physical properties of recogniser peptides.</p

Physical properties of MUC1pep1, MUC1pep2 and their putative mimic peptides MP1-5.

<p>Physical properties of MUC1pep1, MUC1pep2 and their putative mimic peptides MP1-5.</p

MUC1 peptides and their corresponding recogniser peptides.

*<p>From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049728#pone.0049728-Luo1" target="_blank">[35]</a> Sequence similarities between anti-MUC1 CDRs and recognisers are shown in bold and underlined.</p

MUC1 peptides, corresponding recognisers and putative mimic peptides.

<p>Sequence similarities between mimic peptides (MP2 and MP3) and MUC1pep1 are shown in bold and underlined.</p

Structure of the MUC1 protein.

<p>The secretory signal peptide is shown as a shaded box at the N-terminus. The variable number tandem repeat (VNTR) is shown as an open rectangle. The small C-terminal domain, which is proteolytically derived from a common precursor protein and containing the transmembrane domain is also shown. The expanded region shows the amino acid sequences of three VNTR repeats [in brackets]. The amino acid sequences of the 12-mer and 14-mer peptides (MUC1pep1 and MUC1pep2 respectively) used for biopanning are underlined. The sequence of the core peptides used for eluting bound phage, competition ELISA and DC stimulation are shown in red.</p

Immunostimulation of CD14- cells with peptide-loaded DCs.

<p><b>A.</b> CD14⁻ cells were co-cultured with DCs loaded with MUC1pep1 core peptide (dotted line) or MUC1pep2 core peptide (solid line). After 8 days (arrow), the cells were re-stimulated by addition of a fresh batch of autologous DCs. T-cell stimulation was monitored by quantifying the amount of IFN-γ released into the medium. The IFN-γ levels were normalized to the levels produced following incubation with DCs that had no peptides loaded. Error bars represent the normalized % error from two independent experiments. <b>B.</b> As in A except that DCs were loaded with MP1 (solid line), MP2 (dashed line) or MP3 (dotted line). <b>C.</b> As in A except that DCs were loaded with MP4 (dashed line) and MP5 (solid line).</p

Lager yeasts possess dynamic genomes that undergo rearrangements and gene amplification in response to stress.

A long-term goal of the brewing industry is to identify yeast strains with increased tolerance to the stresses experienced during the brewing process. We have characterised the genomes of a number of stress-tolerant mutants, derived from the lager yeast strain CMBS-33, that were selected for tolerance to high temperatures and to growth in high specific gravity wort. Our results indicate that the heat-tolerant strains have undergone a number of gross chromosomal rearrangements when compared to the parental strain. To determine if such rearrangements can spontaneously arise in response to exposure to stress conditions experienced during the brewing process, we examined the chromosome integrity of both the stress-tolerant strains and their parent during a single round of fermentation under a variety of environmental stresses. Our results show that the lager yeast genome shows tremendous plasticity during fermentation, especially when fermentations are carried out in high specific gravity wort and at higher than normal temperatures. Many localised regions of gene amplification were observed especially at the telomeres and at the rRNA gene locus on chromosome XII, and general chromosomal instability was evident. However, gross chromosomal rearrangements were not detected, indicating that continued selection in the stress conditions are required to obtain clonal isolates with stable rearrangements. Taken together, the data suggest that lager yeasts display a high degree of genomic plasticity and undergo genomic changes in response to environmental stress