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Cited by 4HIV Research and TreatmentHIV/AIDS drug development and treatmentVirus-based gene therapy research

Revealing viral and cellular dynamics of HIV-1 at the single-cell level during early treatment periods

Open access
Otte, Fabian, Zhang, Yuepeng, Spagnuolo, Julian, Thielen, Alexander, Däumer, Martin, Wiethe, Carsten, Stoeckle, Marcel, Kusejko, Katharina +58 more·2023·Cell Reports Methods
DOI 10.1016/j.crmeth.2023.100485PMC PMC10326345
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Abstract

While combination therapy completely suppresses HIV-1 replication in blood, functional virus persists in CD4+ T cell subsets in non-peripheral compartments that are not easily accessible. To fill this gap, we investigated tissue-homing properties of cells that transiently appear in the circulating blood. Through cell separation and in vitro stimulation, the HIV-1 “Gag and Envelope reactivation co-detection assay” (GERDA) enables sensitive detection of Gag+/Env+ protein-expressing cells down to about one cell per million using flow cytometry. By associating GERDA with proviral DNA and polyA-RNA transcripts, we corroborate the presence and functionality of HIV-1 in critical body compartments utilizing t-distributed stochastic neighbor embedding (tSNE) and density-based spatial clustering of applications with noise (DBSCAN) clustering with low viral activity in circulating cells early after diagnosis. We demonstrate transcriptional HIV-1 reactivation at any time, potentially giving rise to intact, infectious particles. With single-cell level resolution, GERDA attributes virus production to lymph-node-homing cells with central memory T cells (TCMs) as main players, critical for HIV-1 reservoir eradication.

AI snapshot

Written from the paper’s full text.

TL;DR

Otte et al. present GERDA, a flow-cytometry assay that co-detects HIV-1 Gag and Env after stimulation to identify translation-competent cells in blood, with sensitivity down to about one cell per million. Applying it to early-treated HIV-1 infection, they find rare but detectable viral activity, mainly in lymph-node-homing central memory and naive T cells, even during suppressive ART.

Key findings

  • GERDA detects Gag+/Env+ cells down to about 1 per 10^6 cells, with high linearity in titration experiments (Gag+ R² = 0.9956, p < 0.0001; Gag+/Env+ R² = 0.9936, p < 0.0001) and requires only ~40 mL whole blood with ~9 h hands-on time.
  • In clinical samples, Gag+/Env+ events were rare (median 13.5 per 10^6 CD3+ viable CD4 T cells); 4/7 cases with viral loads between 90 and 51,000 copies/mL had no detectable intracellular viral activity by HIV-1 poly-A transcripts or GERDA.
  • HIV-1 remained transcriptionally active during suppression: P04_56 showed reappearance of viral poly-A transcripts under suppressive ART, and P01_46 had the highest proviral load (4,412 copies/10^6 cells) and poly-A transcripts (85,033 copies/10^6 cells) at TP1.
  • The functional reservoir was attributed mainly to lymph-node-homing cells, with central memory T cells (TCMs) and naive T cells as key populations; for P01_46, TCMs showed 4.01 infectious units per million by QVOA and the highest Gag+/Env+ signals, with CCR7+ and little gut-homing potential.

Methods

  • Design: establishment and benchmarking of GERDA, followed by application to fresh blood from 8 recently diagnosed HIV-1 individuals and pooled cryopreserved PBMCs from 4 Swiss HIV Cohort Study individuals before ART initiation; cell lines included HUT4-3, SupT1, Ramos, SXR5, and TZM-bl.
  • GERDA workflow: CD4-untouched preselection, PHA stimulation for 36–40 h, surface Env staining with a biotinylated broadly neutralizing antibody mix, intracellular Gag staining, and flow cytometry; titration used HUT4-3 cells spiked into Ramos cells at 100,000 to 0.3 presumed infectious cells per 10^6.
  • Analyses: HIV-1 DNA qPCR, poly-A RT-qPCR, quantitative viral outgrowth assay with LTR-lacZ X-Gal readout, single-genome amplification, next-generation sequencing, and tSNE/DBSCAN clustering of FACS data.

Limitations

  • Stated in the paper: all patients recruited for GERDA evaluation were White, male, and of Caucasian ethnicity and contained only subtype B viruses; only PBMC preparations from peripheral blood were used, with no biopsy material, so formal proof of assay predictiveness for organs requires side-by-side tissue biopsy comparison.
  • Stated in the paper: most individuals did not present with high viral activity at or shortly after diagnosis, and mitogen stimulation is nonspecific for virus or cell type.
  • Evident from the text: the clinical sample is small (8 fresh-blood individuals and 4 pooled cryopreserved samples), follow-up is limited for some individuals, and no clinical event information was available to explain the poly-A activity spike in P04_56.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.crmeth.2023.100485

Cites

67 of 68 references verified0 citation errors · 1 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

23 of 36 cited sources checked · 5 supported · 1 flagged · accuracy not yet measured

  • Interpretationcitation
    § Simultaneous detection of HIV-1 Gag and Env proteins on HIV-1-infected cells

    The combination of Gag and Env detection covers the functionality of most of the viral genome length, avoiding a 5' or 3' constrained bias.

    “Thereby, the combination covers the functionality of most of the viral genome length, thus avoiding a 5 0 or 3 0 constrained bias”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [36] source

      The Methods (Bioinformatic analysis; page unavailable) place the assay’s two amplicons at 5′ Ψ and 3′ env, and the Abstract says they jointly identify more than 90% of deleted proviruses as defective. This supports using targets at opposite ends to detect defects, but the paper uses Ψ—not gag—and does not establish that the assay tests the functionality of most of the genome or eliminates 5′/3′ bias. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [37] source

      The Results section on FLIPS versus sub-genomic sequencing (page unavailable) examines env and gag-pol regions but reports that sequences appearing intact in those regions were defective when the entire genome was examined. It also says those regions missed defects in the 5′ packaging signal and major splice donor site, contrary to the claim that Gag and Env detection avoids a 5′-constrained bias. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • Rests on an assumption: Gag serves as a proxy for the production of viral particle components, while Env indicates the presence of the essential viral receptor-binding function for the target cell.
  • Premisecitation
    § Methods

    Infected cells are found mainly in various distant organs, including lymph nodes, gut, brain, and others.

    “Therefore, infected cells are found mainly in various distant organs, including lymph nodes, gut, brain, and others. 13,14”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [13] 13. Marie-Ange ´ lique De Scheerder, A., Vrancken, B., Dellicour, S., Schlub, T., Lee, E., Shao, W., Rutsaert, S., Verhofstede, C., Kerre, T., Malfait, T., et al.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [14] source

      The Discussion reports HIV-infected cells in the CNS and several other organs, and the study found intact HIV sequences across 28 sampled anatomical compartments, including lymph nodes and gut. It does not establish that infected cells are found mainly in distant organs; the Results identify blood and lymphoid tissues as the main sources of viral dissemination. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • Premisecitation
    § Methods

    Multiple approaches to quantitatively reactivate latent proviruses during suppressive ART have failed or were only marginally successful.

    “Multiple approaches to quantitatively reactivate latent proviruses during suppressive ART have failed or were only marginally successful. 15”
    Quote in the paperSubstantial claim1 of 1 sources checked
  • Premisecitation
    § Methods

    During therapy, approximately 95% of all detected integrated proviral genomes are defective.

    “during therapy R 95% of all detected integrated proviral genomes are defective. 31,32”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [31] source
    • cites [32] source

      The Results section, “Hypermutation and large internal deletions render most non-induced proviruses defective” (page unavailable), reports that 88.3% of non-induced proviruses had obvious defects precluding replication. The Discussion says defective genomes can integrate and be detected by PCR. This supports the general point, but the paper does not establish the claimed approximately 95% figure for all detected integrated proviral genomes during therapy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Premisecitation
    § Methods

    Organ-specific non-peripheral cells with tissue-homing markers can transiently appear in blood cell preparations.

    “In these blood cell preparations, organ-specific non-peripheral cells with tissue-homing markers can also transiently appear. 16-20”
    Quote in the paperSubstantial claim2 of 5 sources checked
    • cites [16] source
    • cites [17] source
    • cites [18] source

      The Results report that peripheral blood CD4+ T cells expressed integrin β7, a marker involved in homing to the intestinal mucosa. They do not establish that these cells originated in an organ or appeared only transiently in blood cell preparations; the proposed trafficking direction is from the periphery to the gut. Page numbers are unavailable in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [19] source
    • cites [20] source

      The Introduction (page unavailable) identifies α4β7 as an intestinal-homing marker, and the Results section on pre-HIV frequencies finds α4β7-expressing CD4+ T cells in blood. The paper does not establish that these are organ-resident, non-peripheral cells appearing transiently in blood preparations; it reports relatively stable frequencies across blood samples. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • May combine two claims. 36
  • Classified as probably not a substantial claim. 16
  • The quote only partly states this claim. 3

  • May combine two claims.

    Findingown data
    § Methods

    The SXR5 system yielded clearer infection events, while overall sensitivity correlated very well with TZM-bl cells.

    “In our hands, the SXR5 system yielded clearer infection events, while overall, the sensitivity correlated very well with the TZM-bl cells 43 used by others”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [43] source

      The Materials and Methods describe use of JC53BL-13 reporter cells to measure HIV infection, and the Discussion reports their sensitivity. Our copy does not call them TZM-bl cells or compare their sensitivity with the citing authors’ SXR5 system; the claimed correlation and clearer infection events are not established by this source. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    The number of Gag molecules (about 2,000 per nascent particle) exceeds the smaller number of Env trimers (about 10 per virion).

    “even though the number of Gag molecules (about 2,000 per nascent particle 56 ) exceeds the smaller number of Env trimers (about 10 per virion). 57”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [56] source

      The Introduction (page unavailable) reports an estimate of 2,000–5,000 MA monomers per mature HIV-1 particle, supporting the approximate scale of the cited Gag count. It does not give about 2,000 Gag molecules per nascent particle. The separate Env-trimer count is attached to citation 57, not this source. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [57] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Methods

    It is known that especially integrin a4b7-expressing cells are predominantly infected and depleted during the early infection phases, with a rather poor recovery during ART.

    “It is known that especially integrin a 4 b 7 -expressing cells are predominantly infected and depleted during the early infection phases, with a rather poor recovery during ART. 18,20”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [18] source

      The Results (page not specified) show severe intestinal CD4+ T-cell depletion during primary HIV-1 infection and delayed, incomplete recovery with HAART begun during chronic infection. They measure integrin β7 expression during HAART, but do not show that α4β7-expressing cells are preferentially infected or depleted, or that this specific subset recovers poorly. The Results also report near-complete intestinal CD4+ T-cell restoration when HAART began during primary infection. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [20] source

  • May combine two claims.

    Premisecitation
    § DISCUSSION

    Env expression alone is more affected by genetic defects than the gag region, and assay formats exclusively based on free Gag protein as a reservoir marker only insufficiently assess the presence of infectious particles.

    “Moreover, Env expression alone is more affected by genetic defects than the gag region, 36,53 and assay formats exclusively based on free Gag protein as a reservoir marker only insufficiently assess the presence of infectious particles.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [36] source

      The paper reports genetic defects affecting Env and finds that gag PCR excludes only 30% of defective proviruses (main text, page unavailable). It does not directly compare how often defects impair Env versus Gag expression. The citation follows the Env–gag comparison; the separate claim about free Gag protein assays is also not tested in this paper. (LLM, conf=0.80)

      Experimental — accuracy not yet measured
    • cites [53] source

      The Results report that defective proviruses frequently have env deletions and that full-length env proviruses were 21 times less frequent than gag-positive proviruses. This supports a difference in genetic integrity, but the paper measured proviral DNA, not Env expression. The citation follows the first clause; the later claim about free Gag protein is not attributed to this source. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation
    § DISCUSSION

    A multitude of studies showed that defective HIV-1 genomes inside cells may still produce individual viral transcripts or proteins, which could be mistaken as indicators of a functional reservoir of infectious virus.

    “Conversely, a multitude of studies showed that defective HIV-1 genomes inside cells may still produce individual viral transcripts or proteins, 33-35,55 which could be mistaken as indicators of a functional reservoir of infectious virus.”
    Quote in the paperSubstantial claim3 of 4 sources checked
    • cites [33] source
    • cites [34] source
    • cites [35] source

      The Abstract and Discussion report that defective, replication-incompetent HIV-1 proviruses can produce viral transcripts and proteins. Our copy does not address whether those products have been, or could be, mistaken for indicators of an infectious viral reservoir; that part of the claim is an inference rather than a point the paper makes. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [55] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Methods

    Studies have suggested a close genetic relatedness of recovered viruses, suggesting that viral re- and deactivation and organ homing are interconnected for different body compartments.

    “Studies have suggested a close genetic relatedness of recovered viruses, suggesting that viral reand deactivation and organ homing are interconnected for different body compartments. 13,14,44,45”
    Quote in the paperNot a substantial claim3 of 4 sources checked
    • cites [13] 13. Marie-Ange ´ lique De Scheerder, A., Vrancken, B., Dellicour, S., Schlub, T., Lee, E., Shao, W., Rutsaert, S., Verhofstede, C., Kerre, T., Malfait, T., et al.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [14] source

      The discussion (page not specified in the index) reports nearly identical viruses in blood and multiple tissues after treatment interruption and suggests that rebounding virus replenished tissue reservoirs. The “Viral dispersal and repopulation” section also documents movement between anatomical sites. The paper does not establish the claimed link between viral deactivation and organ homing. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [44] source

      The Results (page unavailable) report identical proviral sequences in two CD4+ T-cell subsets, supporting genetic relatedness. The Discussion (page unavailable) says that only peripheral-blood cells were studied. The paper does not establish that viral reactivation, deactivation, and organ homing are interconnected across body compartments. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [45] source

      The Discussion (page unavailable) reports that viral populations in blood and lymphoid tissue may be genetically linked and that latent viral populations appear largely equilibrated between blood and gut tissue. It does not establish the claimed connection between viral reactivation or deactivation and organ homing; the authors also state that they did not examine tissue reservoirs in this study. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • The quote only partly states this claim.

    Findingnone given
    § Highlights

    GERDA enables sensitive detection of Gag+/Env+ protein-expressing cells down to about one cell per million using flow cytometry.

    “Sensitive detection down to 1 cell per million targeting different splice products”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Gag serves as a proxy for the production of viral particle components, while Env indicates the presence of the essential viral receptor-binding function for the target cell.

  • May combine two claims.

    Findingown data
    § Methods

    CD4 expression became downmodulated in all Gag+/Env+ double-positive cells, while it remained high in uninfected control cells.

    “while CD4 expression became downmodulated in all Gag+/Env+ double-positive cells, it remained high in the uninfected control cells”
    Quote in the paperNot a substantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data
    § Methods

    For all individuals, RNA VL was positive at study start (except P07_07) and HIV-1 DNA remained detectable in peripheral blood during the entire observation period, while cell-associated Gag+/Env+ protein double-positive events were much rarer (median 13.5/1×10^6 CD3+ viable CD4 T cells).

    “cell-associated Gag+/Env+ protein double-positive events were much rarer in the samples (median 13.5/1 3 10 6 CD3 + viable CD4 T cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Immunecharacterization of recently diagnosed patients with HIV-1

    Overall infection rates with about 70 infected cells per 10^6 CD3+ viable CD4 T cells in the pooled cell pool were low, with a decent, significant correlation of Gag+/Env+ double-positive expression signals (R2 = 0.34, p = 0.017).

    “the overall infection rates with about 70 infected cells per 10 6 CD3 + viable CD4 T cells in this cell pool were low with a decent, significant correlation of Gag+/Env+ double-positive expression signals (R 2 = 0.34, p = 0.017; Figure S3B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Methods

    No unspecific staining of Ramos cells was observed, supporting high HIV-1 specificity of the GERDA system.

    “No unspecific staining of Ramos cells was observed(FigureS2F),furthersupportingthehighHIV-1specificity of the GERDA system.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Findingnone given
    § Methods

    Included individuals were all suppressed within a short period of time and stayed suppressed.

    “Included individuals were all suppressed within short period of time and stayed suppressed.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Methods

    After cell stimulation, no Gag+/Env+ expression was detected by GERDA, and no cellular LTR induction was observed in the reporter QVOA for P07_07.

    “This was confirmed since, after cell stimulation, no Gag+/Env+ expression was detected by GERDA, and no cellular LTR induction was observed in the reporter QVOA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Limitations of the study

    Most individuals did not present with high viral activity at or shortly after diagnosis and, due to their potent ART, viral activity inside cells had largely declined.

    “Most individuals did not present with high viral activity at or shortly after diagnosis and, due to their potent ART viral activity inside cells, had largely declined as shown in Figures 2B and 2C and Table 1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Methods

    The gold standard QVOA with the LTR-reporter system (via X-Gal) correlates precisely with a detection limit down to about 3 infectious cells per million cells and proves to be accurate.

    “Also, the gold standard QVOA with the LTR-reporter system (via X-Gal), reflecting a reliable functional assay, correlates precisely with a detection limit down to about 3 infectious cells per million cells and proves to be accurate (Figure S3C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Methods

    At 6 months of follow-up for P01_46, high signals for provirus, poly-A transcripts, and poly-A per HIV-1 DNA, the highest double-positive cell counts, and numerous viral outgrowth events were detected in the central memory cell fraction.

    “At 6 months of follow-up, we still detected high signals for provirus, poly-A transcripts, and poly-A per HIV-1 DNA, the highest double-positive cell counts, and numerous viral outgrowth events in the central memory cell fraction in this individual (Figure 4A; Table 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Cell II Reports

    SGA sequences for V3 data were confirmed by NGS and were stable over time.

    “Of note, SGA sequences for V3 data were confirmed by NGS and were stable over time (Figure 5A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Revealing viral and cellular dynamics of HIV-1 at the single-cell level during early treatment periods

    The FPR for the outlier branch is 77.76 (R5-tropic), whereas all other branches correspond to a single dominating virus (FPR 49.27, R5-tropic).

    “FPR for outlier branch is 77.76 (R5-tropic, boxed in red), whereas all other branches correspond to single dominating virus (FPR 49.27, R5-tropic).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Methods

    When expanded T cell fractions excluding TCMs were analyzed for viral RNA, small amounts of free genomic RNA indicating viral particles were detected exclusively in supernatants of TNs (in 3/15 wells) and were accompanied by elevated levels of poly-A transcripts.

    “When expanded T cell fractions, excluding fractions of TCMs, were analyzed for the presence of viral RNA, small amounts of free genomic RNA, indicating the presence of viral particles, were detected exclusively in supernatants of TNs (in 3/15 wells) and were accompanied by elevated levels of poly-A transcripts (Table 2).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Study participants

    The median baseline viral load of the study samples was 24,200 copies/mL.

    “with a median baseline viral load of 24,200 copies/mL.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Article Cell Reports

    In P01_46, plasma viral load continuously declined over about 6 months, accompanied by decreasing proviral DNA loads and IC viral RNA expression.

    “with a continuously declining plasma VL over the period of about 6 months that was accompanied by decreasing proviral DNA loads and IC viral RNA expression (Table 1).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Methods

    For most individuals, sampling started with detectable VLs between 7,800 and 556,000, and during the course of the study, most individuals experienced good VL control to below 100 c/mL.

    “For most individuals, sampling started with detectable VLs between 7,800 and 556,000. During the course of the study, most individuals experienced good VL control to below 100 c/mL (Table 1).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Study participants

    The study samples included 2 female and 10 male cohort individuals.

    “For the study samples from 2 female and 10 male cohort individuals were included.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Virus dynamics inside HIV-1 cell reservoirs

    A unique minority of viral genomes appeared in one in-vitro-induced well, and NGS revealed that this virus variant was already present in the earliest clinical sample.

    “In addition, a unique minority of viral genomes (red box in Figure S6A) appeared in one in -vitro -induced well. NGS revealed that this virus variant was already present in the earliest clinical sample (Table S3).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Methods

    HIV-1 DNA and HIV-1 poly-A titration curves show a high correlation with R2 = 1 vs. R2 = 0.9999, respectively.

    “HIV-1 DNA and HIV-1 poly-A titration curves show a high correlation with R 2 = 1 vs. R 2 = 0.9999, respectively.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Virus dynamics inside HIV-1 cell reservoirs

    Geno2pheno called both HIV populations from P04_56 R5 tropic (FPRs of 18.9% or 28.5%), and NGS assigned them the highest abundances in the viral populations.

    “The Geno2pheno [co-receptor] algorithm called both HIV populations R5 tropic (false positive rates [FPRs] of 18.9% or 28.5%), and NGS assigned them the highest abundances in the viral populations.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given
    § Methods

    The HIV-1 DNA detection limit of consistently 10 copies in the background of 1x10^6 cellular DNA molecules is fully in the accepted range for diagnostic PCR assays.

    “The HIV-1 DNA detection limit of consistently 10 copies in the background of 1 3 10 6 cellular DNA molecules is fully in the accepted range for diagnostic PCR assays.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Methods

    Inside the cells of all patients in the study, HIV-1 DNA and poly-A transcripts as well as Gag+/Env+ translation competence were always low.

    “we consistently observed that inside the cells of all patients in the study, HIV-1 DNA and poly-A transcripts as well as Gag+/Env+ translation competence, were always low.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Article Cell Reports

    Other memory compartments showed no or only very low signals for Gag+/Env+ translation-competent cells, with no signals detectable in co-expanded negative controls.

    “Other memory compartments showed no or only very low signals for Gag+/Env+ translation-competent cells (Figures 4A and 4B), with no signals detectable in co-expanded negative controls (Figure S5B).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Study participants

    The median age at infection of the study samples was 45 ± 11.7 years.

    “The median age at infection was 45 ± 11.7 years”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § DISCUSSION

    The high test sensitivity (one infected cell per million), short hands-on time and the power of single-cell characterization of GERDA correlate with IC HIV-1 DNA/RNA levels in PBMC populations or with the gold standard QVOA.

    “The high test sensitivity (one infected cell per million), short hands-on time (compare, e.g., Baxter et al. 24 ) and the power of single-cell characterization of GERDA correlate with IC HIV-1 DNA/RNA levels in PBMC populations or with the gold standard QVOA (Figures 1, 2D, 3A, 3B, 3D-3G, 4A, and S5C; Tables 1, 2, and S1).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Baxter et al. 24

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingreasoning
    § Cells

    HUT4-3 cells are HUT78 derivatives that constitutively express infectious HIV-1.

    “HUT4-3cellsareHUT78(M.Martin,NIAID,Bethesda,MD,USA)derivatives,constitutivelyexpressinginfectiousHIV-1.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § Methods

    Our study demonstrates the validity and favorable features of GERDA as being comparable or superior to other sensitive reservoir assays.

    “Our study demonstrates the validity and favorable features of GERDAasbeingcomparableor superior to other sensitive reservoir assays.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Gag serves as a proxy for the production of viral particle components, while Env indicates the presence of the essential viral receptor-binding function for the target cell.

  • May combine two claims.

    Interpretationown data
    § In brief

    GERDA integrates multiple markers to sensitively detect the functional cellular HIV-1 blood reservoir early after diagnosis and to predict the intact viral reservoir in peripheral tissues.

    “Otte et al. present an approach that integrates multiple markers to sensitively detect the functional cellular HIV-1 blood reservoir early after diagnosis and to predict the intact viral reservoir in peripheral tissues.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Gag serves as a proxy for the production of viral particle components, while Env indicates the presence of the essential viral receptor-binding function for the target cell.

  • The quote only partly states this claim.

    Interpretationown data
    § Methods

    The observed low intracellular HIV-1 DNA, poly-A transcripts, and Gag+/Env+ translation competence correlate with the reported only slow establishment of a persistent viral reservoir in distant target tissues.

    “This correlates with the reported only slow establishment of a persistent viral reservoir in distant target tissues.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data
    § Methods

    Cluster Beeplots confirmed that each marker had a clear pattern of positive/negative values for each cluster, demonstrating good cluster assignment by tSNE and DBSCAN.

    “Cluster Beeplots confirmed that each marker had a clear pattern of positive/negative values for each cluster (Figures 3F and 4D), demonstrating good cluster assignment by tSNE and DBSCAN.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning
    § DISCUSSION

    The simultaneous assessment of HIV-1 Gag and Env protein expression pinpoints productively infected T cell classes as dynamic HIV-1 reservoirs and covers the functionality of almost the entire viral genome length, thus avoiding a 5' or 3' constrained bias.

    “The simultaneous assessment of HIV-1 Gag and Env protein expression pinpoints productively infected T cell classes as dynamic HIV-1 reservoirs and covers the functionality of almost the entire viral genome length, thus avoiding a 5 0 or 3 0 constrained bias.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Virus dynamics inside HIV-1 cell reservoirs

    Certain intact virus populations stably co-exist over time and can be readily reactivated, e.g., by antigen stimulation.

    “This indicates that certain intact virus populations stably co-exist over time and can be readily reactivated, e.g., by antigen stimulation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § Study participants

    Ethics approval for SHCS project no 839 was obtained from the local ethics committees of all study sites of the Swiss HIV Cohort Study.

    “SHCS project no 839 approval was obtained from the local ethics committees of all study sites of the Swiss HIV Cohort Study (Kantonale Ethikkommission Bern, Ethikkommission beider Basel, Ethikkommission des Kantons St. Gallen, Kantonale Ethikkommission Zurich, Comitato etico cantonale del Ticino, Commission d'ethique de la recherche clinique de la faculte de biologie et de medicine de l'universite”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data
    § Methods

    The standardized analysis of the multidimensional data combined tSNE and DBSCAN, allowing analysis of all data in an identical manner and verification of final data clusters with classical gating.

    “The standardized analysis of the multidimensional data combined tSNE and DBSCAN, allowing us to analyze all data in an identical manner and verify the final data clusters with classical gating.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning
    § Article Cell II Reports

    This confirms the existence and critical contribution of non-peripheral virus sanctuary sites with TCMs and TNs as key cell pools with mainly lymphoid homing properties.

    “This confirms the existence and critical contribution of non-peripheral virus sanctuary sites with TCMs and TNs as key cell pools with mainly lymphoid homing properties (Tables 1 and 2).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § Limitations of the study

    Another potential limitation of the study is the inevitable use of mitogens, triggering a general cell stimulation with no specificity for virus or cell type.

    “Another potential limitation of our study could lie in the inevitable use of mitogens, triggering a general cell stimulation with no specificity for virus or cell type.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data
    § Methods

    The reliability of our cell homing assessment was reflected in the consistent detection of HIV Gag+/Env+ double-positive cells in TCMs early after diagnosis (with stimulation) but also 6 months after therapy initiation in sorted and thereby separated cell populations.

    “The reliability of our cell homing assessment was also reflected in the consistent detection of HIV Gag+/Env+ double-positive cells in TCMs early after diagnosis (with stimulation; Figures 2B, 2F, and 2G) but also 6 months after therapy initiation in sorted and thereby separated cell populations.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given
    § Limitations of the study

    All patients recruited for GERDA evaluation were White, male, and of Caucasian ethnicity and contained only subtype B viruses.

    “For this study, all our patients recruited for GERDA evaluation were White, male, and of Caucasian ethnicity and contained only subtype B viruses.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § Article Cell II Reports

    Viral evolution occurs even during periods of suppressive ART in vivo, which could contribute to the continuous change and reshaping pool of reactivatable HIV-1 proviruses.

    “This also suggests that viral evolution occurs even during periods of suppressive ART in vivo , which could contribute to the continuous change and reshaping pool of reactivatable HIV-1 proviruses.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given
    § Limitations of the study

    Only PBMC preparations from peripheral blood, but no biopsy materials, were used in this study.

    “Only PBMC preparations from peripheral blood, but no biopsy materials, were used in this study.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Methods

    GERDA was suitable to assign the viral reservoir to TCMs early after infection, which was confirmed 6 months later as the main reservoir by all tested molecular detection formats.

    “In conclusion, GERDA was suitable to assign the viral reservoir to TCMs early after infection, which was confirmed 6 months later as the main reservoir by all tested molecular detection formats.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data
    § Revealing viral and cellular dynamics of HIV-1 at the single-cell level during early treatment periods

    The sorted T cell memory fractions had purities of 97.0 ± 2.9% (TN), 93.1 ± 0.14% (TCM), 93.1 ± 0.42% (TTM), and 95.3 ± 2.19% (TEM).

    “| Cell fraction | Purity (%) |”
    • Also stated in: § Revealing viral and cellular dynamics of HIV-1 at the single-cell level during early treatment periods · plain
      “| 97.0 ± 2.9 | T N |”
    • Also stated in: § Revealing viral and cellular dynamics of HIV-1 at the single-cell level during early treatment periods · plain
      “| 93.1 ± 0.14 | T CM |”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § Study participants

    Written consent from all participating patients is available.

    “written consent from all participating patients is available.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § Cell II Reports

    TTM and TEM fractions were number-wise routinely underrepresented in the QVOA samples compared with TN and TCM fractions.

    “Of note, TTM and TEM fractions were number-wise routinely underrepresented in the QVOA samples compared with TN and TCM fractions.”
    Quote in the paperNot a substantial claim
50 of 50

  • The authors state this as an assumption.

    A high viral DNA content is suggestive of high viral activity in the respective samples.

    “We assumed that a high viral DNA content would be suggestive of high viral activity in the respective samples.”

  • The paper relies on this without support.

    ART completely suppresses viral replication in peripheral blood.

    “ART completely suppresses viral replication in peripheral blood.”

  • The paper relies on this without support.

    Specialized methods for characterizing tissue reservoirs rely on biopsy material and often rely on cell-associated viral transcripts or the Gag p24 protein, and many lack the ability to exclude defective proviruses.

    “Specialized methods for characterizing tissue reservoirs of HIV-1 rely on biopsy material from the organ of interest and often rely on cell-associated viral transcripts or the Gag p24 protein. Many of these assays lack the ability to exclude defective proviruses.”

  • The paper relies on this without support.

    Gag serves as a proxy for the production of viral particle components, while Env indicates the presence of the essential viral receptor-binding function for the target cell.

    “Gag serves a proxy for the production of viral particle components, while Env indicates the presence of the essential viral receptor-binding function for the target cell.”

  • The paper relies on this without support.

    Various T cell subsets are the main contributors to the persisting HIV-1 pool during therapy.

    “As various T cell subsets are the main contributors to the persisting HIV-1 pool during therapy”

  • The paper relies on this without support.

    Cells circulating in peripheral blood are not a homogeneous population of PBMCs but can also stem from distant compartments and immunological organs and only transiently appear in circulating blood.

    “It is known that cells circulating in the peripheral blood are not at all a homogeneous population of PBMCs but that they can also stem from distant compartments and immunological organs and only transiently appear in circulating blood.”

  • The paper relies on this without support.

    Most current HIV-1 quantification assays use HIV-1 DNA, RNA, Gag protein, or progeny virus, often leading to a significant over- or underestimation of the viral reservoir.

    “Most current HIV-1 quantification assays use HIV-1 DNA, RNA, Gag protein, or progeny virus, often leading to a significant over- or underestimation of the viral reservoir.”

  • The paper relies on this without support.

    The clinical treatment files accurately indicate the early virus suppression phase after ART initiation for P01_46.

    “In agreement with the clinical treatment files, this individual represented the early virus suppression phase after ART initiation.”

  • The paper relies on this without support.

    A single HUT4-3 cell contains multiple HIV-1 transcripts.

    “For benchmarking our titration experiments, it is important to note that a single HUT4-3 cell contains multiple HIV-1 transcripts, explaining the high numbers detected even at the”

  • The paper relies on this without support.

    Mitogens trigger a general cell stimulation with no specificity for virus or cell type.

    “Another potential limitation of our study could lie in the inevitable use of mitogens, triggering a general cell stimulation with no specificity for virus or cell type.”
10 of 10

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • P01_46 proviral load differs between prose and Table 1

    Minor

    The prose gives P01_46's highest proviral load as 4,412 copies per million cells; Table 1 gives 4,414 at its first measured time point.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Siliciano, 2005, Enhanced culture assay for detection and quantitation of latently infected, resting virus in HIV-1-Infected individuals, Methods Mol. Biol., 304, 3

Cited by

Sorted by citations

Related

Authors
Otte, F., Zhang, Y., Spagnuolo, J., Thielen, A., Däumer, M., Wiethe, C., Stoeckle, M., Kusejko, K., Klein, F., Metzner, K. J., Klimkait, T., Abela, I., Aebi-Popp, K., Anagnostopoulos, A., Battegay, M., Bernasconi, E., Braun, D. L., Bucher, H. C., Calmy, A., Cavassini, M., Ciuffi, A., Dollenmaier, G., Egger, M., Elzi, L., Fehr, J., Fellay, J., Furrer, H., Fux, C. A., Günthard, H. F., Hachfeld, A., Haerry, D., Hasse, B., Hirsch, H. H., Hoffmann, M., Hösli, I., Huber, M., Jackson-Perry, D., Kahlert, C. R., Kaiser, L., Keiser, O., Klimkait, T., Kouyos, R. D., Kovari, H., Kusejko, K., Labhardt, N., Leuzinger, K., Martinez de Tejada, B., Marzolini, C., Metzner, K. J., Müller, N., Nemeth, J., Nicca, D., Notter, J., Paioni, P., Pantaleo, G., Perreau, M., Rauch, A., Salazar-Vizcaya, L., Schmid, P., Speck, R., Stöckle, M., Tarr, P., Trkola, A., Wandeler, G., Weisser, M., & Yerly, S.
Affiliations
  • University of Basel (CH)
  • University Hospital of Basel (CH)
  • University of Cologne (DE)
  • University of Zurich (CH)
Corresponding author
Otte, Fabian
ORCID
Venue
Cell Reports Methods
Publisher
Elsevier BV
Published
2023-06
Volume
3
Issue
6
Pages
100485
ISSN
2667-2375 (ISSN-L)
DOI
10.1016/j.crmeth.2023.100485
PMC
PMC10326345
OpenAlex
W4377861642
Semantic Scholar
b1fe424e5ed40c3ee2f9f7fa6901c59cbdf442c1
Cited by
4
References
66
Citations by year
2024: 2 · 2023: 2
FWCI
0.48
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports Methods (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Zurich Open Repository and Archive (University of Zurich) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
  • Zurich Open Repository and Archive (University of Zurich) (submittedVersion, cc-by-nc-nd)
  • Zurich Open Repository and Archive (University of Zurich)
Funders
  • China Scholarship Council (CSC201906170031, 201369)
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (839)
  • Swiss HIV Cohort Research Foundation
Topics
  • Life Sciences › Immunology and Microbiology › Virology › HIV Research and Treatment
  • Health Sciences › Medicine › Infectious Diseases › HIV/AIDS drug development and treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
Keywords
HIV-1, viral reservoir, single-cell detection, HIV-1 reactivation, central memory T cells, proviral DNA, early antiretroviral therapy, CD4+ T cells
MeSH terms
HIV Seropositivity, Humans, CD4-Positive T-Lymphocytes, HIV-1, HIV Infections, T-Lymphocyte Subsets

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-14.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-14.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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