Montaner, J. S. G. | bioRxiv Science

bioRxiv2025

Prolonged non-suppressible viremia sustained by a clonally expanded, genomically defective provirus with an immune-evasive HIV protein expression profile

Antiretroviral therapy (ART) potently inhibits HIV replication but does not eliminate HIV proviruses integrated within the genomes of infected cells. Though ART normally suppresses HIV to clinically undetectable levels in blood, some individuals experience non-suppressible viremia (NSV) that is not attributable to suboptimal drug exposure (e.g. due to incomplete medication adherence or pharmacological issues) nor emerging HIV drug resistance, and that does not resolve following regimen modification. We now understand that NSV can originate from expanded cell clones harboring genetically identical proviruses that reactivate en masse to produce clinically detectable viremia. NSV can even originate from proviruses with genomic defects, particularly in HIVs major splice donor (MSD) site, that render the viremia non-infectious. But, because only a limited number of such cases have been described, all in HIV subtype B, the mechanisms that allow cells harboring such proviruses to produce prolonged viremia without being eliminated remain incompletely understood. We characterized a case of MSD-defective, replication-impaired NSV that lasted >4 years in an individual with non-B HIV. Our results reveal that proviruses with MSD deletions can persist by integrating into minimally differentiated CD4+ T-cell subsets that then give rise to the full spectrum of memory and effector subpopulations, and by exhibiting an HIV protein expression profile that would allow these cells to evade cellular and humoral immunity. Our results highlight the need to better understand the biological implications of persistent HIV protein and virion production by genomically defective, clonally expanded proviruses, and for clinical guidelines to acknowledge this type of viremia. ImportanceHIV cure efforts focus on eliminating the viral reservoir, strictly defined as cells harboring proviruses that can produce infectious HIV. But this definition excludes proviruses with defects in HIVs major splice donor site (MSD), which persist readily. Our results confirm that clonally expanded, MSD-defective proviruses can produce HIV transcripts, proteins and clinically-detectable viremia over long periods, underscoring the need to investigate the potential long-term biological consequences of these activities. Our findings also have clinical implications. Most HIV treatment guidelines dont acknowledge that persistent viremia during ART can originate from clonally-expanded proviruses, and all recommend that viremia >200 HIV RNA copies/mL be managed as virologic failure. Clear clinical frameworks should be developed to discriminate persistent viremia that is due to drug adherence, pharmacokinetics or emerging drug resistance (that is clinically actionable), from NSV (that is not). Genotyping of HIVs MSD could also help assess potential transmission risk during NSV.

microbiology↗

bioRxiv2020

SARS-CoV-2 RNA quantification using droplet digital RT-PCR

Quantitative viral load assays have transformed our understanding of - and ability to manage - viral diseases. They hold similar potential to advance COVID-19 control and prevention, but SARS-CoV-2 viral load tests are not yet widely available. SARS-CoV-2 molecular diagnostic tests, which typically employ real-time reverse transcriptase-polymerase chain reaction (RT-PCR), yield semi-quantitative results only. Reverse transcriptase droplet digital PCR (RT-ddPCR), a technology that partitions each reaction into 20,000 nanolitre-sized droplets prior to amplification, offers an attractive platform for SARS-CoV-2 RNA quantification. We evaluated eight primer/probe sets originally developed for real-time RT-PCR-based SARS-CoV-2 diagnostic tests for use in RT-ddPCR, and identified three (Charite-Berlin E-Sarbeco and Pasteur Institute IP2 and IP4) as the most efficient, precise and sensitive for RT-ddPCR-based SARS-CoV-2 RNA quantification. Analytical efficiency of the E-Sarbeco primer/probe set, for example, was ~83%, while assay precision, as measured by the coefficient of variation, was ~2% at 1000 input copies/reaction. Lower limits of quantification and detection for this primer/probe set were 18.6 and 4.4 input SARS-CoV-2 RNA copies/reaction, respectively. SARS-CoV-2 RNA viral loads in a convenience panel of 48 COVID-19-positive diagnostic specimens spanned a 6.2log10 range, confirming substantial viral load variation in vivo. We further calibrated RT-ddPCR-derived SARS-CoV-2 E gene copy numbers against cycle threshold (Ct) values from a commercial real-time RT-PCR diagnostic platform. The resulting log-linear relationship can be used to mathematically derive SARS-CoV-2 RNA copy numbers from Ct values, allowing the wealth of available diagnostic test data to be harnessed to address foundational questions in SARS-CoV-2 biology.

molecular biology↗