A comparative analysis of unintegrated HIV-1 DNA measurement as a potential biomarker of the cellular reservoir in the blood of patients controlling and non-controlling viral replication

Abstract

Background: The persistence of HIV-1 in reservoir cells is one of the major obstacles to eradicating the virus in infected individuals receiving combination antiretroviral therapy (ART). HIV-1 persists in infected cells as a stable integrated genome and more labile unintegrated DNA (uDNA), which includes linear, 1-LTR and 2-LTR circular DNA. 2-LTR circle DNA, although less abundant, is considered a surrogate marker of recent infection events and is currently used instead of the other unintegrated species as a diagnostic tool. This pilot study aimed to investigate how to best achieve the measurement of uDNA. Methods: A comparative analysis of two qPCR-based methods (U-assay and 2-LTR assay) was performed on the blood of 12 ART-naïve, 14 viremic and 29 aviremic On-ART patients and 20 untreated spontaneous controllers (HIC), sampled at a single time point. Results: The U-assay, which quantified all unintegrated DNA species, showed greater sensitivity than the 2-LTR assay (up to 75%, p < 0.0001), especially in viremic subjects, in whom other forms, in addition to 2-LTR circles, may also accumulate due to active viral replication. Indeed, in aviremic On-ART samples, the U-assay unexpectedly measured uDNA in a higher proportion of samples (76%, 22/29) than the 2-LTR assay (41%, 12/29), (p = 0.0164). A trend towards lower uDNA levels was observed in aviremic vs viremic On-ART patients, reaching significance when we combined aviremic On-ART and HIC (controllers) vs Off-ART and viremic On-ART subjects (non-controllers) (p = 0.0003), whereas 2-LTR circle levels remained constant (p ≥ 0.2174). These data were supported by the high correlation found between uDNA and total DNA (r = 0.69, p < 0.001). Conclusions: The great advantage of the U-assay is that, unlike the 2-LTR assay, it allows the accurate evaluation of the totality of uDNA that can still be measured even during successful ART when plasma viremia is below the cutoff of common clinical tests (< 50 copies/mL) and 2-LTR circles are more likely to be under the quantification limit. UDNA measurement in blood cells may be used as a biomarker to reveal a so far hidden or underestimated viral reservoir.

Persistence of Unintegrated HIV DNA Associates With Ongoing NK Cell Activation and CD34+DNAM-1brightCXCR4+ Precursor Turnover in Vertically Infected Patients Despite Successful Antiretroviral Treatment

“…Several studies showed that uDNA can still be measured during successful ART using an improved assay, even when plasma viremia is below the cut-off for common clinical tests, thus signaling recent replication events ( 17 , 20 , 21 ).…”

Unveiling Human Non-Random Genome Editing Mechanisms Activated in Response to Chronic Environmental Changes: I. Where Might These Mechanisms Come from and What Might They Have Led To?

“…On the other hand, this evidence suggests the origin of microRNAs as tools of host defence against complex eukaryotic pathogens (e.g., Plasmodium protists) that were developed by "domestication" of pathogen DNA sequences. Interestingly, phenomena similar to those occurring in HbC and HbS subjects can be observed in long-term non-progressors (subjects with high viral load) and spontaneous controllers (subjects with undetectable or low viral load) of HIV infection [65] and possibly in SARS-CoV-2 infected subjects [157], suggesting that both "pre-symbiotic" intermediates (e.g., in HbC subjects) and forms of domestication and exaptation of pathogen sequences into host defence functions (e.g., in HbS subjects) may be common stages of pathogen-host interplay. Regarding the global pandemic threat of SARS-CoV-2, the chronic engagement of angiotensin-converting enzyme 2 (ACE2) cell surface protein for viral entry is expected to induce ACE2 gene "hyper-transcription" [158] and ACE2 hyper-production was recently documented [159]; consequently, its hypermutation by non-random genome editing mechanisms is also expected, a prediction that should be verified in patients with chronic SARS-CoV-2 infection.…”

“…From this standpoint, even the integration of HIV fragments and defective HIV-1 provirus producing novel HIV protein-coding RNA transcripts [64] could represent not only a "Trojan Horse" reservoir of HIV, but also products of virus domestication and new biotechnological tools to combat HIV itself and/or future viruses. Similarly, plasmid-like unintegrated circular HIV DNAs [65] might represent tools to spread either infective or domesticated HIV DNAs. Hence, it is possible to imagine a new generation of vaccines that aim to insert specific HIV-engineered transposons with an appropriate unique sequence recognising conserved HIV sequences and conserved repeats for APOBEC binding.…”

Quantification of Total HIV DNA as a Marker to Measure Viral Reservoir: Methods and Potential Implications for Clinical Practice

“…Therefore, even though the quantification of integrated and unintegrated forms can provide information in select studies, it does not appear reliable for routine use as an HIV reservoir marker. Instead, the quantification of unintegrated forms has been proposed as indicative of ongoing viral replication [ 42 , 43 ]; in any case, controversies remain.…”

“…When compared to other virologically effective 2DRs, the combination of DTG plus lamivudine (3TC) had several advantages in terms of lower risk of drug interactions and metabolic complications. Furthermore, it has shown excellent viro-immunological properties in both naïve and ART-experienced HIV-infected patients [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , ...…”