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A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Published on: March 25, 2014
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Comprehensive epitope mapping using polyclonally expanded human CD8 T cells and a two-step ELISpot assay for testing
Clive M Michelo1, Jama A Dalel2, Peter Hayes2
1Zambia Emory HIV Research Project, B22/737 Mwembelelo, Emmasdale, Lusaka, Zambia.
Journal of Immunological Methods
|February 2, 2021
Summary
Developing effective HIV-1 vaccines is challenging due to viral genetic diversity. This study presents a new two-step method to efficiently screen HIV-1 Gag peptides for T cell responses, overcoming limitations of large peptide libraries.
Area of Science:
- Immunology
- Vaccinology
- Virology
Background:
- The genetic diversity of human immunodeficiency virus type 1 (HIV-1) strains presents a significant obstacle for vaccine development.
- Assessing T cell responses to HIV-1 typically involves large peptide libraries representing potential T cell epitopes (PTEs), which can become unmanageably large.
- Evaluating these extensive libraries is often limited by the availability of biological samples, even with advanced techniques like ELISpot assays.
Purpose of the Study:
- To introduce a novel, efficient two-step protocol for in vitro polyclonal expansion of CD8 T cells.
- To facilitate the screening of a large number of HIV-1 Gag peptides for immune responses.
- To overcome sample limitations in epitope mapping for HIV-1 vaccine research.
Main Methods:
- A two-step protocol involving in vitro polyclonal expansion of CD8 T cells from peripheral blood mononuclear cells (PBMCs).
- Screening of 441 HIV-1 Gag peptides across 32 HIV-1 positive and 40 HIV-1 negative subjects.
- Utilizing a pooled-peptide mapping strategy with two sequential ELISpot assays (7-day and 10-day T cell expansion).
Main Results:
- The protocol enabled the screening of 441 HIV-1 Gag peptides, identifying immune responses in HIV-1 positive subjects.
- The two-step ELISpot approach successfully mapped epitopes using pooled and matrix peptides after sequential T cell expansion.
- The study established the breadth and magnitude of HIV-1 Gag-specific CD8 T cell responses and highlighted responses to variable epitopes.
Conclusions:
- The novel two-step CD8 T cell expansion protocol significantly enhances the efficiency of screening numerous HIV-1 peptides.
- This method effectively addresses sample volume limitations in epitope mapping for HIV-1 vaccine research.
- The findings provide a comprehensive assessment of HIV-1 Gag-specific CD8 T cell immunity, including responses to diverse viral sequences.
