February 15, 2008 (Vol. 28, No. 4)

Reveal and ProVE Technologies Help Accelerate the Development Process

The development of modern vaccines to prevent or treat conditions that have failed to respond to more traditional treatments or early vaccination approaches is becoming more widespread. Many diseases with major unmet medical needs today including HIV, viral hepatitis, malaria, tuberculosis, and most forms of cancer are candidates for this approach.

Identification of the antigens or epitopes that play a role in these conditions and which the immune system can effectively target is critical to design optimized vaccination systems and to monitor the immunological effects of vaccination throughout the product-development process.

Epitope discovery methods that make use of algorithms to predict peptide binding to MHC molecules can be relatively unreliable, and functional assays, though more successful, are usually time consuming and require large quantities of sample, which may be unavailable.

New systems are now on the market that can accelerate the identification of epitopes and hence, speed up the development of these potentially revolutionary treatments. These include the Reveal and ProVE™ Rapid Epitope Discovery System, which is a unique modular system offered as a discovery service by ProImmune.

Epitope Identification

Using algorithms to predict peptide binding to MHC molecules does not always reveal which epitopes are the most naturally immunogenic and thus the most appropriate for inclusion in vaccines. Functional in vitro assays such as ELISPOT and intracellular cytokine staining that test whether pools of peptides can induce T cells to produce cytokines are better predictors of this, as are functional cellular assays like the T2 assay that monitors the ability of a peptide to stabilize an MHC class I molecule. Unfortunately, these approaches usually require several rounds of screening and are thus labor intensive.

Even using these approaches, it is difficult to assess the true MHC restriction of the epitope involved in generating the immune response. Carrying out exhaustive screening of peptide libraries using these functional assays with samples from infected patients is also strongly limited by the quantity of sample that can be obtained from each patient. Results can also be confounded by restricted sample numbers and the heterogeneity of patients’ tissue types.

ProImmune’s Reveal and ProVE Rapid Epitope Discovery System can overcome these problems and accelerate the epitope-discovery process. Designed as a modular process, it combines in vitro Reveal MHC-peptide binding and rate assays with the synthesis of ProVE MHC Class I Pentamers for conclusive confirmation of epitopes in patient samples.
This step-by-step approach allows the narrowing down of many candidate epitopes to identify those that actually cause relevant immune responses in a matter of weeks rather than months. The technology is widely applicable across many disease areas including all areas of cancer and infectious disease. It is also ideally suited to accelerate research programs with particular time pressures such as vaccine development for emerging diseases.


Any starting set of synthetic peptides can be screened using the Reveal binding assay, provided the length of these matches the restriction of the alleles to be investigated. Candidate sequences may be generated initially as an overlapping peptide library from a known protein. Alternatively, epitopes can be chosen from a more limited selection of known sequences that have been prescreened using an algorithm of choice to identify those that are most likely to bind to MHC molecules.

The high-throughput Reveal binding assay quickly determines the ability of each candidate peptide to bind to one or more MHC alleles and stabilize the ternary MHC-peptide complex. By comparing this ability to that of a pass/fail control peptide that has borderline affinity to the MHC allele of interest, the most likely immunogenic peptides in a protein sequence can be identified. Unlike traditional functional assay approaches, the Reveal binding assay determines the MHC restriction of peptides at the outset.

This information can be used to identify those epitopes that could play a key role as targets in the development of novel immunotherapies, or alternatively, when applied to therapeutic proteins, the information can be used to ensure proteins are deimmunized to avoid producing adverse reactions and/or neutralizing antibodies.

We performed a pilot study to explore which epitopes within the protein sequence of the key H5 protein in the avian influenza virus H5N1 could bind to MHC proteins and thus be used as effective epitopes in vaccine development. From the N-terminus of the H5N1 HA subunit, 94 overlapping peptides of nine amino acids in length were synthesized.

These peptides were screened for binding against seven MHC alleles using the Reveal MHC-peptide binding assay. Figure 1 shows the results of the assay for peptide binding to one of these MHC alleles. Eleven peptides out of the 94 screened were found to bind to the MHC molecule at a strength greater than that of a pass/fail control peptide and so could be considered as candidates for further development.

Figure 1

To help decide which peptides to take further, ProImmune can carry out additional assays to give detailed kinetic information about the binding properties of individual peptides. Assessing the on- and off-rates for peptide binding to MHC molecules can help to identify which candidate epitopes will be appropriate for use in vaccine development by indicating how long individual epitopes could be presented to T cells.


Following the identification of candidate epitopes using the Reveal assay, ProImmune can generate ProVE MHC Class I Pentamers with specificity for these peptides. These Pentamers comprise five MHC-peptide complexes assembled through a coiled-coil domain that binds to epitope-specific T cells (Figure 2). They are suitable for use in flow cytometry, enabling the customer to rapidly identify and quantify different populations of these cells.

With this new system, the use of precious patient samples is shifted to the final validation step at the end of the discovery process, which ensures that the use of such samples is optimized. Since the MHC restriction of the candidate epitopes is known when the patient samples are tested with the Pentamers, the true specificities of the target protein can be fully confirmed when T cells specific to an epitope are detected.

Figure 2

Scientists at Oxford BioMedica used the ProVE Pentamer Library module in conjunction with flow cytometry to detect single antigen-specific T cells in patient samples (Figure 3). These patients had received Oxford BioMedica’s cancer vaccine, TroVax®, which targets the tumor antigen 5T4. Using the Pentamers, they confirmed the MHC restriction of the T-cell epitopes they had previously identified, demonstrated that the T cells present in the samples were truly antigen-specific, and therefore conclusively validated the tested peptide sequences from 5T4 as relevant T-cell epitopes.

The frequency of specific T cells detected using the Pentamers was approximately twofold greater than what was detected using an IFN-g ELISPOT assay against the same peptide antigen, which demonstrated a good correlation between these two assays. Overall, this analysis with ProVE Pentamers showed that cytotoxic, CD8+ T-lymphocyte responses to single peptide sequences can be determined clearly.

Figure 3


ProImmune’s Reveal and ProVE have been developed as a modular system that can significantly reduce the time for T-cell epitope discovery as well as reduce the need for large patient sample volumes. The system can be customized to include peptide synthesis and on-/off-rate binding assays alongside epitope discovery and confirmation.

Nikolai Schwabe, Ph.D., is CEO,
and Amanda Turner is marketing
operations manager at ProImmune.
Web: www.proimmune.com. Phone: 44 870 042 7279. E-mail: [email protected].

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