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Background:  The frequency of latent reservoirs and integrated viral DNA within resting CD4⁺ T cells appear to be relatively constant in patients in the presence and absence of effective HAART. The limiting dilution co-culture assay is considered the “gold standard” to measure the frequency of reservoir cells, but it is technically cumbersome and time consuming and has inherent high error rates. End-point PCR assays that measure integrated HIV-1 DNA have similar shortcomings. We set out to develop a more robust assay for HIV-1 integration that would allow us to reliably detect even small differences between integration frequencies.

Methods:  The amount of integrated DNA present within purified DNA samples from HIV-1 infected individuals (n = 8, all on effective HAART) was assessed using a modified version of a kinetic DNA PCR assay that we developed based on primers that bind to the repetitive genomic element Alu and the HIV-1 gag region. To measure integration in patient DNA, repetitive sampling techniques were required as only a fraction of samples gave positive signals due to the low frequency of integration. Each patient sample was assayed 40 times using primers to Alu and gag. To enumerate the number of integration events, DNA from a control integration standard cell line was also assayed repetitively after diluting it in uninfected DNA to mimic low frequency integration. This integration standard is polyclonal and reproduces the diversity of integration that occurs with acute infection, but does not contain unintegrated DNA. 

Results:  On average a 12-fold excess of unintegrated DNA relative to integrated DNA was present in the patient samples. A mathematical model was developed to estimate the confidence intervals for the frequency of integration for our patient samples using data from our integration standard assayed at low copy numbers. This mathematical model enabled us to obtain sufficiently tight confidence intervals to detect 2-fold differences. We found the integration frequency varied from 1.1-3.3 per 10,000 PBMC between patient samples. The 10% lower bound and 90% upper bound integration frequency were 0.8 to 5.0. in 10,000 PBMC.

Conclusion:  We have developed a quantitative assay for HIV-1 integration based on kinetic PCR with repetitive sampling and a statistical model which provides point estimates and tight confidence intervals. This assay could be used to monitor therapies designed to reduce viral reservoirs.

Keywords: latency; integration; reservoirs