Accelerated Epitope Discovery for Avian Flu Vaccines
The influenza strain H5N1, a fundamental isolate that causes bird flu, is easily transmissible between birds. A major healthcare concern is that the virus might mutate to become transmissible between humans, resulting in a pandemic. A protective vaccine is urgently required, and an effective starting point is to screen for potentially relevant epitopes encoded by the H5N1 genome.
Recent advances in the design of epitope-discovery systems have significantly accelerated the epitope discovery process, giving results in weeks rather than months. Advanced systems, such as ProImmune’s (www.proimmune.com) REVEAL™ and ProVE™, produce results faster than could be expected with traditional methods such as ELISPOT.
These systems were used in a pilot study to explore the sequence of the H5 protein in the avian flu virus for epitopes that could be important in the research to develop a vaccine. As the MHC is highly polymorphic, its binding affinity with target antigens varies greatly. Thus, the MHC-restriction and binding properties of target peptides are crucial factors to be considered in the vaccine development process.
In a study performed by ProImmune, custom peptide libraries were investigated using the REVEAL MHC-peptide binding assay to assess MHC-restriction and binding properties. A total of 93 overlapping PEPscreen 9-mer peptides from the N-terminal region of the Hemagglutinin (HA) subunit of H5N1 were assembled with different MHC alleles. By using the high-throughput REVEAL assay, the individual binding properties for seven MHC alleles with each of the 93 peptides were determined.
Detection of peptide binding is based on the presence or absence of the native conformation of the MHC-peptide complex (Figure 1). Each peptide is given a score relative to the control peptide of a known T-cell epitope. The score is reported as a percentage of the signal generated by the test peptide versus the control peptide, and the peptide is given a pass or fail result (Figure 2). Using this approach, 27 novel epitopes were defined.
The availability of patient material would allow further validation of the novel epitopes using the ProVE module. This step assesses whether the specific T cells are present in response to a pathogen, evaluates the functional status of these populations, and enables immunomonitoring over time. The assay functions by producing recombinant ProVE MHC Pentamers that incorporate the selected synthetic epitopes. The Pentamer-epitope complexes bind specifically to those CD8-positive T-cell populations that detect the peptide antigen. Antigen-specific T cells are detected by flow cytometry using a fluorescent marker associated with the Pentamer. To investigate the binding affinity more thoroughly, kinetic studies of selected synthetic peptides may be performed by using the REVEAL affinity assay technology. This allows a further in-depth analysis and measures on- and off-rates of the MHC-peptide formation. Fast on-rates and slow off-rates indicate a high-binding affinity between the epitope and the MHC molecule.
The use of the PEPscreen synthetic peptide library, coupled with the REVEAL and ProVE assays reduced the complete screening process from several months to less than 30 days. The combined technologies represent a breakthrough in the speed of epitope discovery and facilitation of the vaccine-development process.