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Feb 1, 2011 (Vol. 31, No. 3)

Rapid Evaluation of Protein Antigenicity

New Platform Designed to Accelerate Subunit Vaccine Development for Infectious Diseases

  • Identification in HIV-1

    Click Image To Enlarge +
    Figure 1. Results of the Reveal MHC-peptide binding assay for controls and the 12 peptides that were considered potential epitopes; binding signal is shown as a percentage relative to the pass/fail control; peptide 8, HPVHAGPIA (red) was further validated by MHC Class I Pentamer staining.

    In HIV immunity, factors that potentially distinguish between protective and ineffective anti-HIV-1 immune responses and control disease progression relate to epitope-MHC binding as well as stability and affinity. Researchers at ProImmune recently supported a study carried out by Samantha Westrop and her colleagues at Imperial College to examine the details of HIV-1 CD8+ T-cell epitope recognition. This study used rapid peptide synthesis, Reveal binding and affinity assays, and ProVE Pentamer synthesis in conjunction with flow cytometry, as well as ELISpot analyses to discover new T-cell epitopes for HIV-1.

    The rate of HIV-1 disease progression is also influenced by the genetic profile of an individual. For example, individuals with HLA-B35 and HLA-B8 profiles have been associated with an increased rate of disease progression in HIV-1+ infections. To discover possible reasons for this rapid disease progression, ProImmune’s methods were used to investigate the HIV-1 Gag peptide-specific CD8+ T-cell immune response mounted by HLA-B35+ individuals. Initially, forty-four 9-mer HIV-1 Gag peptides were synthesized as a PEPscreen® Custom Peptide Library, and were assessed for binding to B*35:01 using the Reveal MHC-peptide binding assay.

    The results implicated 12 peptides as potential epitopes (Figure 1), and these were further analyzed with the Reveal rate assay to determine their rates of dissociation from the MHC complex. Two of the 9-mer peptides, YPLTSLRSL (peptide 1) and HPVHAGPIA (peptide 8) were found to form a stable complex with B*35:01, indicating that these may be potential novel epitopes.

  • Click Image To Enlarge +
    Figure 2. Pro5 Pentamer staining of live lymphocytes gated on CD3+ cells; the left plot shows staining with an allele mismatched negative control Pentamer, the right plot shows staining with the HA9 (B*35:01/HPVHAGPIA) Pentamer. 0.39% of CD3+ live lymphocytes are CD8+/ B*35:01/HPVHAGPIA Pentamer+ with a background stain of 0.03%.

    Pro5® MHC Class I Pentamer staining was then carried out on patient samples, which confirmed the presence of a well-defined Pentamer+/CD8+ population for the B*35:01/HPVHAGPIA epitope (Figure 2). Additionally, no naïve or central memory T cells specific for the HPVHAGPIA peptide were found, indicating a lack of proliferative potential in infected HIV-1+ individuals. Further validation with ELISpot assays showed that the HPVHAGPIA peptide elicited the highest ex vivo response of the peptides studied.

    Additionally, using the Reveal and ProVE system, a previously unknown epitope was identified, which may be involved in an immune response in B*35:01-positive individuals. The results of this study advance our understanding of HIV-1 immunopathogenesis and disease progression and contribute toward the ultimate goal of vaccine development.

  • Summary

    ProImmune’s rapid antigen-characterization platform encompasses complementary B- and T-cell epitope discovery systems and functional cellular assays such as T-cell proliferation and ELISpot. Not only do these technologies characterize antigenicity of proteins in terms of T- and B-cell responses and allow ranking and selection as part of the overall epitope identification effort, but they also establish the key biomarkers for measuring responses in downstream clinical work.


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