Comprehensive epitope mapping using polyclonally expanded human CD8 T cells and a two-step ELISpot assay for testing large peptide libraries.
J Immunol Methods
; 491: 112970, 2021 04.
Article
en En
| MEDLINE
| ID: mdl-33529681
ABSTRACT
The genetic diversity of circulating HIV-1 strains poses a major barrier to the design, development and evaluation of HIV-1 vaccines. The assessment of both vaccine- and natural infection-elicited T cell responses is commonly done with multivalent peptides that are designed to maximally capture the diversity of potential T cell epitopes (PTEs) observed in natural circulating sequences. However, depending on the sequence diversity of viral subtypes and number of the HIV immunogens under investigation, PTE estimates, including HLA-guided computational methods, can easily generate enormous peptide libraries. Evaluation of T cell epitope specificity using such extensive peptide libraries is usually limited by sample availability, even for high-throughput and robust epitope mapping techniques like ELISpot assays. Here we describe a novel, two-step protocol for in-vitro polyclonal expansion of CD8 T cells from a single vial of frozen PBMC, which facilitated the screening 441 HIV-1 Gag peptides for immune responses among 32 HIV-1 positive subjects and 40 HIV-1 negative subjects for peptide qualification. Using a pooled-peptide mapping strategy, epitopes were mapped in two sequential ELISpot assays; the first ELISpot screened 33 large peptide pools using CD8 T cells expanded for 7 days, while the second step tested pool-matrix peptides to identify individual peptides using CD8 T cells expanded for 10 days. This comprehensive epitope screening established the breadth and magnitude of HIV-1 Gag-specific CD8 T cells and further revealed the extent of immune responses to variable/polymorphic epitopes.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Mapeo Epitopo
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Linfocitos T CD8-positivos
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Biblioteca de Péptidos
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Ensayo de Immunospot Ligado a Enzimas
Tipo de estudio:
Prognostic_studies
Límite:
Adult
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Female
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Humans
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Male
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Middle aged
Idioma:
En
Año:
2021
Tipo del documento:
Article