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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
Evaluation of a quantitative real-time PCR assay to measure HIV-specific mucosal CD8+ T cell responses in the cervix
PLoS ONE, Volume 5, No. 10, Article e13077, Year 2010
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Description
Several candidate HIV vaccines aim to induce virus-specific cellular immunity particularly in the genital tract, typically the initial site of HIV acquisition. However, standardized and sensitive methods for evaluating HIV-specific immune responses at the genital level are lacking. Therefore we evaluated real-time quantitative PCR (qPCR) as a potential platform to measure these responses. β-Actin and GAPDH were identified as the most stable housekeeping reference genes in peripheral blood mononuclear cells (PBMCs) and cervical mononuclear cells (CMCs) respectively and were used for normalizing transcript mRNA expression. HIV-specific cellular T cell immune responses to a pool of optimized CD8+ HIV epitopes (HIV epitope pool) and Staphylococcal enterotoxin B (SEB) superantigen control were assayed in HIV infected PBMC by qPCR, with parallel assessment of cytokine protein production. Peak HIV-specific mRNA expression of IFNγ, IL-2 and TNFα occurred after 3, 5 and 12 hours respectively. PBMCs were titrated to cervical appropriate cell numbers to determine minimum required assay input cell numbers; qPCR retained sensitivity with input of at least 2.5×104 PBMCs. This optimized qPCR assay was then used to assess HIV-specific cellular T cell responses in cytobrush-derived cervical T cells from HIV positive individuals. SEB induced IFNγ mRNA transcription was detected in CMCs and correlated positively with IFNγ protein production. However, qPCR was unable to detect HIV-induced cytokine mRNA production in the cervix of HIV-infected women despite robust detection of gene induction in PBMCs. In conclusion, although qPCR can be used to measure ex vivo cellular immune responses to HIV in blood, HIV-specific responses in the cervix may fall below the threshold of qPCR detection. Nonetheless, this platform may have a potential role in measuring mitogen-induced immune responses in the genital tract. © 2010 Chege et al.
Authors & Co-Authors
Chege, Duncan
Canada, Toronto
University of Toronto
Chai, Yijie
Canada, Toronto
University of Toronto
Huibner, Sanja
Canada, Toronto
University of Toronto
McKinnon, Lyle R.
Canada, Toronto
University of Toronto
Canada, Winnipeg
University of Manitoba
Kenya, Nairobi
University of Nairobi
Wachihi, Charles
Kenya, Nairobi
University of Nairobi
Kimani, Makobu
Kenya, Nairobi
University of Nairobi
Jaoko, Walter G.
Kenya, Nairobi
University of Nairobi
Kimani, Joshua
Canada, Winnipeg
University of Manitoba
Canada, Ottawa
Public Health Agency of Canada
Kenya, Nairobi
University of Nairobi
Blake Ball, T.
Canada, Winnipeg
University of Manitoba
Canada, Ottawa
Public Health Agency of Canada
Kenya, Nairobi
University of Nairobi
Plummer, Francis Allan
Canada, Winnipeg
University of Manitoba
Canada, Ottawa
Public Health Agency of Canada
Kenya, Nairobi
University of Nairobi
Kaul, Rupert
Canada, Toronto
University of Toronto
Canada, Toronto
University Health Network University of Toronto
Kenya, Nairobi
University of Nairobi
Rebbapragada, Anuradha
Canada, Toronto
Public Health Ontario
Canada, Toronto
University of Toronto
Statistics
Citations: 12
Authors: 12
Affiliations: 6
Identifiers
Doi:
10.1371/journal.pone.0013077
e-ISSN:
19326203
Research Areas
Genetics And Genomics
Infectious Diseases
Sexual And Reproductive Health
Study Approach
Quantitative
Participants Gender
Female