Quantification of the Latent HIV-1 Reservoir Using Ultra Deep Sequencing and Primer ID in a Viral Outgrowth Assay.
BACKGROUND
In this study, we measured the latent HIV-1 reservoir harboring replication-competent HIV-1 in resting CD4 T cells in participants on highly active antiretroviral therapy, quantitating the frequency of latent infection through the use of a Primer ID-based Ultra Deep Sequencing Assay (UDSA), in comparison to the readout of the quantitative viral outgrowth assay (QVOA).


METHODS
Viral RNA derived from culture wells of QVOA that scored as HIV-1 p24 capsid antigen positive were tagged with a specific barcode during cDNA synthesis, and the sequences within the V1-V3 region of the HIV-1 env gene were analyzed for diversity using the Primer ID-based paired-end MiSeq platform. We analyzed samples from a total of 19 participants, 2 initially treated with highly active antiretroviral therapy in acute infection and 17 treated during chronic infection. Phylogenetic trees were generated with all viral lineages detected from culture wells derived from each participant to determine the number of distinct viral lineages growing out in each well, thus capturing another level of information beyond the well being positive for viral antigen. The infectious units per million (IUPM) cell values estimated using a maximum likelihood approach, based on the number of distinct viral lineages detected (VOA-UDSA), were compared with those obtained from QVOA measured using limiting dilution.


RESULTS
IUPM estimates determined by VOA-UDSA ranged from 0.14 to 3.66 and strongly correlated with the IUPM estimates determined by QVOA (r = 0.94; P < 0.0001).


CONCLUSIONS
VOA-UDSA may be an alternative readout for that currently used for QVOA.
*Department of Biochemistry and Biophysics, UNC Center for AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC; †Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, NC; and Departments of ‡Medicine; §Microbiology and Immunology, UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.
Sook-Kyung
SK
Lee
Shuntai
S
Zhou
Pedro L
PL
Baldoni
Ean
E
Spielvogel
Nancie M
NM
Archin
Michael G
MG
Hudgens
David M
DM
Margolis
Ronald
R
Swanstrom
D044966:Anti-Retroviral Agents / Q000627:therapeutic use; D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D014644:Genetic Variation; D005838:Genotype; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D010802:Phylogeny
D044966:Anti-Retroviral Agents; D017931:DNA Primers
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