Studies indicate that even in the absence of antiretroviral drugs, approximately 75% of infants that are born to HIV-1 infected mothers remain uninfected despite repeated exposure to HIV-1. Investigation on pediatric immune response and maternal factors in HIV-1 exposed uninfected infants might provide a useful tool in prevention and treatment of HIV-1 infections. Natural killer (NK) cells express cell surface killer immunoglobulin-like receptors (KIR), these receptors regulate activation and inhibition of NK cell responses through recognition of HLA class I molecules on the target cells. Recent studies indicate that NK cells can control HIV-1 replication and is associated with resistance to HIV-1 infection and slower disease progression to AIDS. DNA extraction and KIR genotyping from dried blood spots maybe a useful alternative to the whole blood especially in paediatric studies. DBS filter paper cards can be easily collected and to transported at lower cost.
Research question
Is resistance to HIV-1 infection of HIV-1 exposed, uninfected, infants partially determined by the quality and quantity of Natural Killer (NK) cells expressing pro-inflammatory (i.e. stimulatory) Killer Immunoglobulin-like Receptors (KIR)?
Fit with Africa Centre research portfolio
This research project falls under Objective 1 in the Africa Centre research strategy: HIV dynamics – understanding the HIV epidemic.
Data sources used
This work builds on previous research on mother-to-child transmission at the Africa Centre, and utilises stored blood samples from the Wellcome Trust-funded Vertical Transmission Study (VTS), and the detailed data on perinatally-infected infants and their mothers.
Methods/study design
Using previously stored dried blood spot (DBS) and whole blood samples from the same individuals we have recently established a protocol to obtain genomic DNA of sufficient quantity and quality for reliable KIR genotyping. The KIR genotype results obtained from DBS were consistently comparable to those determined from whole blood; we will therefore use this approach to quantify KIR repertoires among virus-transmitting and non-transmitting mother-infant pairs.
We have recently obtained ethical permission to conduct a retrospective study using DBS from 120 HIV-1 perinatally infected infants and 120 HIV-1 exposed, uninfected, infants from HIV-infected mothers. Using PCR sequence specific primer genotyping, we will assess 12 NK cell KIR genes. In addition HLA-B and HLA-C alleles will be determined by high resolution HLA genotyping. This is a collaborative effort between the Africa Centre, Doris Duke Medical Research Institute, and the Ragon Institute USA.
With 120 virus-transmitting mother-child pairs this study will exceed previous studies of MTCT in the number of transmissions that have found statistically significant differences in HLA associations.
Preliminary work: Use of dried blood spots for determination of killer immunoglobulin-like gene repertoire
To evaluate whether dried blood spots (DBS) can be used to isolate genomic DNA for the analysis of various single nucleotide polymorphism (SNPs) relevant to innate immune responses and to establish a robust method of DNA extraction on DBS, we compared three methods of DNA extraction from DBS. DNA was extracted on 10 samples using a 4.8 mm punch of DBS of the same sample with Qiagen QiaAMP DNA mini kit, Nuclisens Minimag magnetic extraction and prepGEMTM Storage Card Blood kit. DNA quality was quantified with nanodrop, picogreen and HLA-DRB1 PCR. DNA was amplified with REPLI-G midi kit, a whole genome amplification kit (WGA) and KIR typing was done with sequence specific primer (SSP)-PCR. DNA was also extracted on whole blood of the same samples, followed by KIR typing with SSP-PCR. KIR typing results were compared between different methods of DBS extraction and whole blood.
HLA-DRB1 and IL-10 typing were successful in all samples before and after whole genome amplification and the KIR typing results were identical to that of the whole blood. HLA-DRB1 and IL-10 typing was unsuccessful in DNA extracted with Nuclisens minimag, and prep-GEM, however after WGA the KIR typing results were identical to that of whole blood samples. Cleaning up the DNA with Qiagen columns produced better results, however large quantities of DNA were lost and as a result false negative results were produced.
Qiagen QiaAMP DNA mini kit was found to be the best method for DNA extraction from dried blood spots. DNA isolation and whole genome amplification from dried blood spots provide an easy and cost effective way of SNP analysis especially in pediatric studies. The use of DBS for DNA extraction is cheap and does not require expertise to process the PBMC.
Policy implications
This work will add to the literature on the innate immune genetic determinants underlying perinatal MTCT of HIV-1.