October 1, 2006 (Vol. 26, No. 17)

Vaccination as a Strategic Medical Option

Vaccines have arguably saved more lives than any other medical breakthrough. However, despite the long list of diseases that can now be controlled by vaccination, there are still some urgent unmet medical needs, including vaccines for pathogens, such as HIV, malaria, and new strains of influenza.

Successful vaccination enables immunological protection against the target pathogen. Typically, a vaccinated host acquires an immunological memory of a pathogen that will allow a rapid and specific clearing following recognition of pathogen-specific elements. Specialized B and T cells are involved in different arms of the immune response involving antibody and cell-mediated immunity. Pathogen recognition is facilitated by antigen-presenting cells whose primary role is to present antigens to antigen-sensitive lymphoid cells.

While traditional vaccines have used inactivated forms of the pathogen to stimulate a protective immune response, this strategy cannot be employed for all vaccines. A major challenge in the development of vaccines is the difficulty of finding the right immunogenic element that can achieve an efficient and long-lasting immunization effect. Considerable effort has been made to find suitable fragments (protein epitopes) that can be included in vaccines to offer protection against serious life-threatening diseases.

Epitope discovery can be a long process and for protein antigens can include candidate peptide selection and synthesis, binding-characterization assays, assay validation, and data analysis. In the past, the speed of the discovery process has been impeded by issues such as low throughput, lengthy and difficult research efforts, and high costs. Today, new epitope discovery technologies and algorithms for the identification of epitopes have increased the overall efficiency of the entire process.

For example, peptide synthesis was historically an expensive choice in vaccine research and epitope mapping. However, a new synthesis platform from Sigma-Genosys (www.sigma-genosys.com) allows the rapid parallel synthesis of custom libraries at reduced costs, giving access to high-throughput synthetic peptide assays for epitope discovery and vaccine research.

Epitope Mapping with Peptide Libraries

HIV exemplifies the time-consuming and difficult nature of vaccine discovery. HIV has one of the highest mutation rates known, resulting in a virus with significant genetic diversity, a major impediment toward vaccine development. The nine subtypes (clades) within the HIV M group can differ by more than 10% within clades and 30% between clades. Therefore, even if a vaccine is proven effective for a specific clade, it is uncertain that the protection would extend to other strains within the clade and across different clades. This raises the question of whether several vaccines are needed to offer protection against each of the distinct viral variants. As CD8+ T cells have been shown to play an important role in containing HIV infection, vaccine development has focused on the biological mechanism of CD8+ T-cell responses upon HIV infection.

A 2005 study by McKinnon et al., assessed the cross-reactivity across a large population of viral strains and screened for CD8+ T-cell responses to the envelope protein gp160 (Env) in HIV-1 clades A, B, C, and D using ELISPOT analysis with Env-expressing vaccinia virus. The results indicate that responses are rarely restricted to only one clade and sometimes include multiclade recognition.

However, parallel epitope-mapping studies with peptide libraries were required to define the target sequence of these responses with greater precision. Peptide libraries were synthesized using the PEPscreen® platform and comprised 158 15-mer peptides overlapping by 10 amino acids, representing the sequence of the clade A envelope protein. Peptide ELISPOT assays were performed and individual peptide responses were confirmed in separate ELISPOT assays. Statistical analyses were used to examine the relationship between response magnitudes and frequencies of different clades and to compare the magnitude of ELISPOT responses.

Several epitope-specific responses were defined in cross-clade responders. The CD8+ cells recognized relatively conserved regions, providing a generally high amount of cross-clade reactivity. The results indicate that cross-clade CD8+ T-cell responses are common in the population tested, although a number of individuals have Env-specific CD8+ responses limited to clade A, the presumed infecting clade. This might have significant implications in vaccine development because the manner by which each vaccine responds to diversity of the viral strains will determine the success of the vaccine in providing protection against diverse HIV strains.

Identification of Malaria Epitopes

PEPscreen libraries have also facilitated epitope-mapping projects. A study by Ian Cockburn at Johns Hopkins identified epitopes presented by CD8+ T cells during malaria infection. The scientists chose candidate genes that were highly expressed in the sporozoite stage of the protozoan parasite life cycle. Algorithms were employed to predict potential epitope binding to a given MHC molecule. High-scoring peptides were synthesized as 8–10-mer peptides and screened with the special cell line RMA-S. This line is unable to present endogenous peptides, and MHC class I expression on the surface is transient unless tight-binding peptides are provided in the medium. The binding can be measured by flow cytometry using a labeled antibody against MHC class I. Using this method, a novel epitope was identified and data confirmed by an ELISPOT assay.

Future studies will involve the production of T-cell lines for the epitopes identified and studies to verify if such cell lines confer protection against malaria infection. If these studies yield interesting results, the relevant T-cell receptor may be cloned and a transgenic mouse specific for the peptide of interest may be generated.

Figure 1: Principle of the REVEAL binding Assay

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.

Figure 2: Out of 93 peptides, 11 were identified to form the native conformation of the MHC-peptide complex.

Other Applications

The PEPscreen peptide-synthesis platform enables the cost-effective and rapid synthesis of customized peptide libraries for high-throughput ELISPOT assays and epitope mapping studies. It also provides access to innovative tools in vaccine research, such as REVEAL and ProVE from ProImmune. The technology has applications in many other areas of research, including high-throughput protein-protein interaction analysis, customized peptide microarray production, and kinase assays. What was once a cost-prohibitive research tool is now accessible to all labs.

Oscar Monera, Ph.D., is senior technical writer, and Stacey Hoge is product manager for custom peptides at Sigma-Genosys. Web: www.sigma-genosys.com. Phone: (800) 234-5362. E-mail: [email protected]. Jeremy Fry, Ph.D., is director of sales at ProImmune. Web: www.proimmune.com. Phone: +44 870 042 7279

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