Abstract
HIV-1 infection cannot be cured due to the presence of the latent reservoir (LR). Novel cure or treatment strategies, such as “shock and kill” or therapeutic vaccination, aim to reduce or eradicate the LR. Cure strategies utilise robust DNA quantification assays to measure the change in the LR in low copy scenarios. No standard assay exists, which impedes the reliable comparison of results from different therapy and vaccine trials and HIV-1 total DNA quantification methods have not been previously compared. The HIV-1 long terminal repeat (LTR) has been shown to be the best target for DNA quantification. We have analysed two HIV-1 quantification assays, both able to differentiate between the variant HIV-1 DNA forms via the use of pre-amplification and primers targeting LTR. We identify a strong correlation (r=0.9759, P<0.0001) between assays which is conserved in low copy samples (r=0.8220, P<0.0001) indicating that these assays may be used interchangeably. The RvS assay performed significantly (P=0.0021) better than the CV assay when quantifying HIV-1 total DNA in patient CD4+ T lymphocytes. Sequence analysis demonstrated that viral diversity can limit DNA quantification, however in silico analysis of the primers indicated that within the target region nucleotide miss-matches appear infrequently. Further in silico analysis using up to-date sequence information led to the improvement of primers and enabled us to establish a more broadly specific assay with significantly higher HIV-1 DNA quantification capacity in patient samples (p=0.0057, n=17).
Highlights
The development of antiretroviral therapy (ART) has been a major breakthrough in the treatment of human immunodeficiency virus type 1 (HIV-1) infection, effectively preventing the progression to acquired immunodeficiency syndrome (AIDS) (Brechtl et al, 2001)
Our aim was to examine the performance of the two assays Claire Vandergeeten assay (CV) and Rene van der Sluis assay (RvS) and using the vast amount of sequence information available to date develop a new assay that will perform most optimally with the highest specificity and sensitivity. The incentive for this consideration was that both HIV-1 DNA quantification assays target the long terminal repeat (LTR) of the HIV-1 genome, well established as the most conserved region of the genome, and both utilise a pre-amplification step allowing for the separate quantification of different viral life-cycle stages
The lack of a standard quantification assay to measure total HIV-1 DNA has led to the development of a number of ‘in-house’ assays targeting different genomic regions for quantification, but this variation may render the results of different clinical trials incomparable
Summary
The development of antiretroviral therapy (ART) has been a major breakthrough in the treatment of human immunodeficiency virus type 1 (HIV-1) infection, effectively preventing the progression to acquired immunodeficiency syndrome (AIDS) (Brechtl et al, 2001). A number of studies have explored the therapeutic potential of vaccination in both simian immunodeficiency virus (SIV) models (De Rose et al, 2008; Fuller et al, 2012, 2006; Hel et al, 2002, 2000; Lu et al, 2003) and in human trials (Barouch et al, 2013; Garcia et al, 2013; Lévy et al, 2014; Lu et al, 2004) with vaccine agents including DNA based vaccines expressing antigen, viral vectors expressing antigen, passive transfer immunotherapy, dendritic cells (DC) primed for HIV-1 antigen presentation or combinations of these (Mylvaganam et al, 2015) These studies have demonstrated that therapeutic vaccination can achieve reduced viral loads, increased time to viral rebound, reduction in size of the LR and in inducing stronger and more sustained immune response against HIV-1 (Mylvaganam et al, 2015). Strategies which aim to completely eradicate the HIV-1 LR are popular in current research and Abbreviations: RvS, Rene van der Sluis assay; CV, Claire Vandergeeten assay; CRx, Christine Rouzioux ⁎ Corresponding author at: Ronald Ross Building, 8 West Derby Street, Liverpool, L687BE, United Kingdom
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