Abstract
Background:Identification of HIV-1 genomes responsible for establishing clinical infection in newly infected individuals is fundamental to prevention and pathogenesis research. Processing, storage, and transportation of the clinical samples required to perform these virologic assays in resource-limited settings requires challenging venipuncture and cold chain logistics. Here, we validate the use of dried-blood spots (DBS) as a simple and convenient alternative to collecting and storing frozen plasma.Methods:We performed parallel nucleic acid extraction, single genome amplification (SGA), next generation sequencing (NGS), and phylogenetic analyses on plasma and DBS.Results:We demonstrated the capacity to extract viral RNA from DBS and perform SGA to infer the complete nucleotide sequence of the transmitted/founder (TF) HIV-1 envelope gene and full-length genome in two acutely infected individuals. Using both SGA and NGS methodologies, we showed that sequences generated from DBS and plasma display comparable phylogenetic patterns in both acute and chronic infection. SGA was successful on samples with a range of plasma viremia, including samples as low as 1,700 copies/ml and an estimated ~50 viral copies per blood spot. Further, we demonstrated reproducible efficiency in gp160 env sequencing in DBS stored at ambient temperature for up to three weeks or at -20°C for up to five months.Conclusions:These findings support the use of DBS as a practical and cost-effective alternative to frozen plasma for clinical trials and translational research conducted in resource-limited settings.
Highlights
Accurate identification and characterization of the virus that establishes productive clinical HIV-1 infection, or the transmitted/founder (TF) virus, is fundamentally important to many areas of HIV research
Using both single genome amplification (SGA) and next generation sequencing (NGS) methodologies, we showed that sequences generated from dried-blood spots (DBS) and plasma display comparable phylogenetic patterns in both acute and chronic infection
These findings support the use of DBS as a practical and cost-effective alternative to frozen plasma for clinical trials and translational research conducted in resource-limited settings
Summary
Accurate identification and characterization of the virus that establishes productive clinical HIV-1 infection, or the transmitted/founder (TF) virus, is fundamentally important to many areas of HIV research. SGA and phenotypic characterization of breakthrough viruses emerging during or after passive administration of broadly neutralizing antibodies (bNAbs) allows accurate identification of neutralization escape variants and their pathways of developing resistance [20]. The HIV-1 field is currently testing multiple novel prevention strategies in large clinical trials, including vaccines, passively administered bNAbs, pre-exposure prophylaxis, and microbicides. For each of these trials, identifying and analyzing the breakthrough TF viruses for relevant properties (e.g., drug resistance, neutralization sensitivity, linked transmission networks, etc.) will be of paramount importance to the interpretation of trial results. We validate the use of dried-blood spots (DBS) as a simple and convenient alternative to collecting and storing frozen plasma.
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