TY - JOUR
T1 - High-throughput high-resolution class I HLA genotyping in East Africa
AU - Koehler, Rebecca N.
AU - Walsh, Anne M.
AU - Sanders-Buell, Eric E.
AU - Eller, Leigh Anne
AU - Eller, Michael
AU - Currier, Jeffrey R.
AU - Bautista, Christian T.
AU - Wabwire-Mangen, Fred
AU - Hoelscher, Michael
AU - Maboko, Leonard
AU - Kim, Jerome
AU - Michael, Nelson L.
AU - Robb, Merlin L.
AU - McCutchan, Francine E.
AU - Kijak, Gustavo H.
N1 - Funding Information:
A sample set from Tanzania was used to field test the real-time PCR-SSP platform. Between September 2002 to April 2003, 3096 volunteers from Mbeya (southwestern Tanzania, latitude 8°54′53″S and longitude 33°27′43″E) were enrolled in a prospective community cohort study, with the objective of assessing the suitability of different population groups for HIV vaccine cohort development. The composition of this cohort was described in detail elsewhere . The study was conducted jointly by the Mbeya Regional AIDS Control Programme (Tanzanian Ministry of Health), the Department of Infectious Diseases & Tropical Medicine, Ludwigs-Maximillians University (Munich, Germany), the Walter Reed Army Institute of Research (Rockville, MD, USA), and the Henry M. Jackson Foundation for the Advancement of Military Medicine (Rockville, MD, USA). Blood samples collected from 174 randomly selected individuals, out of the 2479 participants who remained HIV sero-negative for the 42-month duration of the study, were available for HLA typing. This sample set included 110 female (63.2%), and the median age was 26 years (inter-quartile interval: 21–35 years). All of the tested participants were Black Africans residing in the Mbeya Region, and were recruited from the urban Ghana ward in Mbeya Town, and from the small rural village of Itende. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) separated from whole blood (MagNA pure total nucleic acid extraction, Roche Diagnostics Corp., Indianapolis, IN).
PY - 2010
Y1 - 2010
N2 - HLA, the most genetically diverse loci in the human genome, play a crucial role in host-pathogen interaction by mediating innate and adaptive cellular immune responses. A vast number of infectious diseases affect East Africa, including HIV/AIDS, malaria, and tuberculosis, but the HLA genetic diversity in this region remains incompletely described. This is a major obstacle for the design and evaluation of preventive vaccines. Available HLA typing techniques, that provide the 4-digit level resolution needed to interpret immune responses, lack sufficient throughput for large immunoepidemiological studies. Here we present a novel HLA typing assay bridging the gap between high resolution and high throughput. The assay is based on real-time PCR using sequence-specific primers (SSP) and can genotype carriers of the 49 most common East African class I HLA-A, -B, and -C alleles, at the 4-digit level. Using a validation panel of 175 samples from Kampala, Uganda, previously defined by sequence-based typing, the new assay performed with 100% sensitivity and specificity. The assay was also implemented to define the HLA genetic complexity of a previously uncharacterized Tanzanian population, demonstrating its inclusion in the major East African genetic cluster. The availability of genotyping tools with this capacity will be extremely useful in the identification of correlates of immune protection and the evaluation of candidate vaccine efficacy.
AB - HLA, the most genetically diverse loci in the human genome, play a crucial role in host-pathogen interaction by mediating innate and adaptive cellular immune responses. A vast number of infectious diseases affect East Africa, including HIV/AIDS, malaria, and tuberculosis, but the HLA genetic diversity in this region remains incompletely described. This is a major obstacle for the design and evaluation of preventive vaccines. Available HLA typing techniques, that provide the 4-digit level resolution needed to interpret immune responses, lack sufficient throughput for large immunoepidemiological studies. Here we present a novel HLA typing assay bridging the gap between high resolution and high throughput. The assay is based on real-time PCR using sequence-specific primers (SSP) and can genotype carriers of the 49 most common East African class I HLA-A, -B, and -C alleles, at the 4-digit level. Using a validation panel of 175 samples from Kampala, Uganda, previously defined by sequence-based typing, the new assay performed with 100% sensitivity and specificity. The assay was also implemented to define the HLA genetic complexity of a previously uncharacterized Tanzanian population, demonstrating its inclusion in the major East African genetic cluster. The availability of genotyping tools with this capacity will be extremely useful in the identification of correlates of immune protection and the evaluation of candidate vaccine efficacy.
UR - http://www.scopus.com/inward/record.url?scp=77956274161&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0010751
DO - 10.1371/journal.pone.0010751
M3 - Article
C2 - 20505773
AN - SCOPUS:77956274161
SN - 1932-6203
VL - 5
JO - PLoS ONE
JF - PLoS ONE
IS - 5
M1 - e10751
ER -