HIV-1 IPDA® Services

The IPDA® requires cryopreserved or fresh PBMCs from which CD4+ T cells are enriched. Sufficient input – typically hundreds of thousands to millions of CD4+ T cells per sample – is needed to achieve robust statistics at low intact provirus frequencies. Sample shipping by cryoshipper/dry ice with temperature monitoring and chain-of-custody documentation is standard. Accelevir’s PBMC processing services can also support upstream sample preparation if your sites require centralized processing. Contact us for detailed sample volume and shipping and storage requirements tailored to your protocol.

For studies enrolling predominantly HIV-1 subtype B participants – typical of North American and European cohorts – the standard IPDA® Subtype B assay is appropriate. For international clinical trials, studies enrolling participants from sub-Saharan Africa or Asia, or any program with a diverse cohort that may include non-B subtypes (C, A, D, CRFs), the cross-subtype IPDA® version 2 assay significantly reduces detection failures from sequence polymorphisms. In silico validation showed the cross-subtype version correctly identified approximately 100% of intact subtype C sequences versus only ~12% with the original subtype B design. Accelevir’s scientific team can review your study population and recommend the optimal configuration during a technical consultation. HIV reproducibly establishes latent infection across all subtypes, but the viral diversity between clades makes assay selection a critical determinant of data quality.

The quantitative viral outgrowth assay (QVOA), long considered the gold standard for measuring the replication competent HIV reservoir, measures the replication competent HIV reservoir by culturing infected cells under T cell activation conditions and detecting virus production. While QVOA is highly specific, it detects only approximately 1 infectious unit per million CD4+ T cells and misses the large population of replication competent noninduced proviruses that remain latent under standard culture conditions. HIV reservoir size as measured by QVOA is highly correlated to the result by IPDA® in paired analyses. The IPDA® uses digital PCR to detect intact proviruses based on genomic structure – targeting two conserved regions frequently deleted or mutated in defective proviruses – and consistently identifies more intact proviruses than QVOA. The IPDA® also differentiates different subclasses of defective proviruses, providing a quantitative approach to understanding full reservoir composition. While the strength of the QVOA is the direct measurement of viral infection and replication, the IPDA® offers a more sample-conservative assay with increased sensitivity, accuracy, and reproducibility compared to viral outgrowth. Lower cell requirement and increased speed are additional benefits of using the IPDA® compared to older assays like QVOA. However, IPDA® measures proviral DNA integrity rather than demonstrated replication competence; some proviruses classified as intact may harbor defects outside the amplicon regions. For programs requiring functional competence data, Accelevir offers complementary HIV-1 persistence assays including outgrowth-based approaches.

The IPDA® is used in clinical trials to monitor patient responses to therapeutic interventions designed to target the latent proviral reservoir, including immune activation strategies and different immune activation strategies designed to reverse latent infection. IPDA® provides insights into HIV-1 reservoir composition and dynamics – tracking how intact provirus reservoir levels, defective proviruses, and total proviral frequency overall reservoir size change in response to intervention. Intact proviruses can reactivate and cause viral rebound if therapy is interrupted, making accurate quantification of the persistent proviral DNA critical for evaluating whether interventions achieve meaningful reservoir reduction. The assay also helps assess hidden viral reservoirs in tissues beyond circulating blood CD4+ T cells, and Accelevir has supported investigator-initiated trials in both pediatric and adult populations.

After an unprecedented sequencing effort covering all major Group M HIV subtypes, data comparing the frequency of intact HIV provirus by NFL-SGS and IPDA® showed a robust correlation between the two methods. These data show that by building the IPDA® on NFL-SGS analysis of the integrated proviral landscape in subjects on ART, similar accuracy of intact proviral frequency can be achieved with ddPCR, at a fraction of the cost and with increased scalability and accuracy compared to proviral sequencing.

Traditional total HIV DNA assays using single-target PCR amplification measure total and integrated DNA without distinguishing intactness. Because most integrated proviruses in treated individuals are defective and cannot replicate, these assays dramatically overestimate the infectious reservoir. Quantifying intact proviral DNA provides a more relevant estimate of the viral reservoir – only genomic DNA from infected cells that is intact can produce infectious virus. The IPDA® addresses these limitations directly and is recommended for clinical trials evaluating HIV-1 remission strategies.

Given the sequence diversity inherent in HIV biology, assay non-detects due to polymorphisms are rare but inevitable. Every IPDA® dataset undergoes quality control evaluation for potential signal failure attributable to HIV-1 sequence polymorphisms; when one amplicon shows unexpected loss of signal, the event is flagged and investigated. Extensive NFL-SGS and analysis of public HIV genome sequence datasets have minimized the risk of polymorphisms in assay target sites, and the use of optimized ddPCR confers a high degree of tolerance to efficiency-reducing polymorphisms. Accelevir maintains secondary probe sequences and can deploy variant-matched primer/probe sets to rescue affected samples. The availability of alternate primer and probe sets through the cross-subtype IPDA® and triage primer sets ensures the lowest possible data censor rates and maximum data retention on precious clinical samples. For programs anticipating high viral diversity, pre-study pilot testing or in silico screening of participant HIV sequences is available.