Biology subjects

Brumme, C. J.

Publications and source records attributed to Brumme, C. J..

8 recordsLinked to original sources

bioRxiv2026

HIV superinfection reveals sequential reservoir reactivation and immune-driven rebound dynamics

Superinfection, where a person with HIV acquires a second phylogenetically distinct strain, provides an opportunity to study reservoir evolutionary dynamics because the two strains can be tracked over time. We here characterize such a case that originally went undetected by clinical monitoring. The participant, who expressed the protective HLA-B*57:03 allele, initially controlled his subtype B infection but lost control after superinfection with a unique recombinant form (URF), which came to dominate in plasma without displacing the original B strain. Five years after initiating therapy, replication-competent proviruses from both strains persisted in blood, though URF proviruses dominated (80%). Despite their unequal reservoir representation, both strains rapidly rebounded upon treatment interruption, though most rebounding sequences belonged to a URF subclade that predated ART initiation. During the treatment interruption, genetically distinct viral lineages continually appeared in plasma, consistent with sequential "waves" of reactivation of diverse reservoir clones. Notably, immune escape variants emerged from the reservoir in a later "wave", displacing initial susceptible variants and underscoring the important role of immune responses in shaping rebound dynamics. Sequence analysis revealed that the URF transmitted/founder virus already harbored key HLA-B*57:03-associated escape mutations, likely explaining the loss of viral control after superinfection. Our results reveal that superinfection can remain undetected despite routine HIV clinical monitoring, that pre-adaptation of the superinfecting strain to host HLA can undermine immune control, that rebound virus can originate from reservoir clones predating ART initiation, and that immune responses can actively shape rebound dynamics by driving escape variant outgrowth during treatment interruption. IMPORTANCEThis case demonstrates that HIV superinfection can go undetected by routine clinical monitoring, underscoring the importance of assays that capture HIV diversity -- particularly those used to screen participants for cure trials. It also illustrates how pre-adaptation of a superinfecting strain to host HLA-restricted immune responses can evade existing immunity, with implications for understanding natural immune control, and underscoring the challenge of viral diversity for vaccine and immunotherapy design. The sequential emergence of genetically diverse HIV lineages during treatment interruption, including minority immune escape variants that emerged from the reservoir to displace the initial susceptible population, highlights the difficulty of predicting rebound virus composition and underscores the key role of immune responses in shaping rebound dynamics. Critically, escaped rebound variants can then reseed the reservoir, thereby enriching it in escaped forms, highlighting a specific vulnerability for cure approaches that harness natural immune responses to eliminate reservoir cells.

molecular biology↗

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↗

bioRxiv2023

HIV reservoirs are dominated by genetically younger and clonally enriched proviruses

In order to cure HIV, we need to better understand the within-host evolutionary origins of the small reservoir of genome-intact proviruses that persists within infected cells during antiretroviral therapy (ART). Most prior studies on reservoir evolutionary dynamics however did not discriminate genome-intact proviruses from the vast background of defective ones. We reconstructed within-host pre-ART HIV evolutionary histories in six individuals and leveraged this information to infer the ages of intact and defective proviruses sampled after an average >9 years on ART, along with the ages of rebound and low-level/isolated viremia occurring during this time. We observed that the longest-lived proviruses persisting on ART were exclusively defective, usually due to large deletions. In contrast, intact proviruses and rebound HIV exclusively dated to the years immediately preceding ART. These observations are consistent with genome-intact proviruses having shorter lifespans, likely due to the cumulative risk of elimination following viral reactivation and protein production. Consistent with this, intact proviruses (and those with packaging signal defects) were three times more likely to be genetically identical compared to other proviral types, highlighting clonal expansion as particularly important in ensuring their survival. By contrast, low-level/isolated viremia sequences were genetically heterogeneous and sometimes ancestral, where viremia may have originated from defective proviruses. Results reveal that the HIV reservoir is dominated by clonally-enriched and genetically younger sequences that date to the untreated infection period when viral populations had been under within-host selection pressures for the longest duration. Knowledge of these qualities may help focus strategies for reservoir elimination. ImportanceCharacterizing the HIV reservoir that endures despite antiretroviral therapy (ART) is critical to cure efforts. Our observation that the oldest proviruses persisting during ART were exclusively defective, while intact proviruses (and rebound HIV) all dated to the years immediately pre- ART, explains why prior studies that sampled sub-genomic proviruses on-ART (which are largely defective) routinely found sequences dating to early infection, whereas those that sampled viral outgrowth sequences found essentially none. Together with our findings that intact proviruses were also more likely to be clonal, and that on-ART low-level/isolated viremia originated from proviruses of varying ages (including possibly defective ones), our observations indicate that: 1) on-ART and rebound viremia can have distinct within-host origins, 2) intact proviruses have shorter lifespans than grossly-defective ones, and therefore depend on clonal expansion for persistence, and 3) the HIV reservoir, being overall genetically younger, will be substantially adapted to within-host pressures, complicating immune-based cure strategies.

genetics↗

bioRxiv2022

HIV proviral genetic diversity, compartmentalization and inferred dynamics in lung and blood during long-term suppressive antiretroviral therapy

The lung is an understudied site of HIV persistence. We isolated 882 subgenomic proviral sequences by single-genome approaches from blood and lung from nine individuals on long-term suppressive antiretroviral therapy (ART), and characterized genetic diversity and compartmentalization using formal tests. Consistent with clonal expansion as a driver of HIV persistence, identical sequences comprised between 9% to 86% of within-host datasets, though their location (blood vs. lung) followed no consistent pattern. The majority (77%) of participants harbored at least one sequence shared across blood and lung, supporting the migration of clonally-expanded cells between sites. No participant exhibited genetic compartmentalization so obvious that it was visually apparent in a phylogeny. When formal tests were applied however, two (22%) participants showed modest yet significant support for compartmentalization when analysis was restricted to distinct proviruses per site. This increased to four participants (44%) when considering all within-host sequences. Thus, while a minority of individuals harbor somewhat distinctive proviral populations in blood and lung, these can simply be due to unequal distributions of clonally-expanded sequences. Importantly, the extent of lung proviral diversity on ART strongly reflected that in blood (Spearman {rho} = 0.98, p < 0.0001), confirming blood as a strong indicator of total body proviral diversity. Analysis of on-ART proviral diversity in context of pre-therapy viral diversity in two participants revealed marked differences in proviral longevity and dynamics. Whereas one participants proviral pool was rich in ancestral sequences that recapitulated more of HIVs within-host evolutionary history, the others largely comprised more contemporary sequences, including ones that re-seeded the reservoir during a three-year treatment interruption. Results highlight the genetic complexity of proviruses persisting in lung and blood during ART, and the uniqueness of each individuals proviral composition. Individualized HIV remission and cure strategies may be needed to overcome these challenges. Author SummaryHIV persists in the body despite long-term suppressive antiretroviral therapy. Much of our knowledge about the HIV reservoir comes from studying proviruses in blood, but a fundamental question is whether these are distinct from those in tissues. The lung could theoretically engender genetically distinctive HIV populations, but this remains understudied. Our analysis of nearly 900 proviral sequences from blood and lung of nine individuals with HIV receiving long-term therapy revealed substantial within-host heterogeneity, yet some common patterns. Identical sequences (consistent with clonal expansion of infected cells) were observed in everyone, though at different (9-86%) frequencies. Recovery of shared sequences across blood and lung was common (77% of participants). Fewer than half of participants exhibited blood-lung genetic compartmentalization, and only modestly so. Moreover, when present, compartmentalization was often attributable to differential distribution of identical sequences, not the presence of genetically distinctive populations, across sites. Results also revealed marked inter-individual differences in proviral dynamics, as evidenced by the proportion of persisting proviruses that represented ancestral versus more recently-circulating viral sequences. Critically, the extent of within-host proviral diversity in blood correlated strongly with that in lung, indicating that despite inter-individual heterogeneity, blood is a strong indicator of proviral diversity elsewhere in the body.

molecular biology↗

bioRxiv2021

HIV proviral burden, genetic diversity and dynamics in viremic controllers who subsequently initiated suppressive antiretroviral therapy

Curing HIV will require eliminating the reservoir of integrated, replication-competent proviruses that persist despite antiretroviral therapy (ART). Understanding the burden, genetic diversity and longevity of persisting proviruses in diverse individuals with HIV is critical to this goal, but these characteristics remain understudied in some groups. Among them are viremic controllers, individuals who naturally suppress HIV to low levels but for whom therapy is nevertheless recommended. We reconstructed within-host HIV evolutionary histories from longitudinal single-genome amplified viral sequences in four viremic controllers who eventually initiated ART, and used this information to characterize the age and diversity of proviruses persisting on therapy. We further leveraged these within-host proviral age distributions to estimate rates of proviral turnover prior to ART. This is an important yet understudied metric, since pre-ART proviral turnover dictates reservoir composition at ART initiation (and thereafter), which is when curative interventions, once developed, would be administered. Despite natural viremic control, all participants displayed significant within-host HIV evolution pre-therapy, where overall on-ART proviral burden and diversity broadly reflected the extent of viral replication and diversity pre-ART. Consistent with recent studies of non-controllers, the proviral pools of two participants were skewed towards sequences that integrated near ART initiation, suggesting dynamic proviral turnover during untreated infection. In contrast, proviruses recovered from the two other participants dated to time-points that were more evenly spread throughout infection, suggesting slow or negligible proviral decay following deposition. HIV cure strategies will need to overcome within-host proviral diversity, even in individuals who naturally controlled HIV replication before therapy. ImportanceHIV therapy is life-long because integrated, replication-competent viral copies persist within long-lived cells. To cure HIV, we need to understand when these viral reservoirs form, how large and genetically diverse they are, and how long they endure. Elite controllers, individuals who naturally suppress HIV to undetectable levels, are being intensely studied as models of HIV remission, but viremic controllers, individuals who naturally suppress HIV to low levels, remain understudied even though they too may hold valuable insights. We combined phylogenetics and mathematical modeling to reconstruct proviral seeding and decay from infection to therapy-mediated suppression in four viremic controllers. We recovered diverse proviruses persisting during therapy that broadly reflected HIVs within-host evolutionary history, where the estimated half-lives of the persistent proviral pool during untreated infection ranged from <1 year to negligible. Cure strategies will need to contend with proviral diversity and between-host heterogeneity, even in individuals who naturally control HIV.

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↗

bioRxiv2020

HIV Diversity Considerations in the Application of the Intact Proviral DNA Assay (IPDA)

Opening Paragraph (serves as abstract for submission) and BodyThe Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a precise and scalable method for intact HIV reservoir quantification1. This duplexed droplet digital PCR (ddPCR) assay simultaneously targets the HIV Packaging Signal ({Psi}) and the Rev Responsive Element (RRE) within Envelope (env) to distinguish genomically intact proviruses against a large background of defective ones2. The IPDA requires less time, resources, and biological material than the gold standard for replication-competent HIV reservoir measurement, the Quantitative Viral Outgrowth Assay (QVOA)3, and is being adopted in research and clinical studies4-7. In our cohort of HIV-1 subtype B-infected individuals from North America however, the IPDA yielded a 28% failure rate due to HIV polymorphism. We further demonstrate that within-host HIV diversity can lead the IPDA to underestimate intact HIV reservoir size, which could negatively impact clinical trial results interpretation. While the IPDA represents an important methodological advance, HIV diversity should be addressed before its widespread adoption.

genetics↗

bioRxiv2019

Systematic determination of in vitro phenotypic resistance to HIV-1 integrase strand transfer inhibitors from clinical samples

Phenotypic resistance data is relatively sparse for the newest HIV-1 integrase strand transfer inhibitors (INSTIs), dolutegravir (DTG), bictegravir (BIC), and cabotegravir (CAB). In this study, we report the phenotypic susceptibility of a large panel of oligo-clonal patient-derived HIV-1 integrase viruses. Representative clinical samples (N=141) were selected from a large database (N=17,197) of clinically-derived HIV integrase sequences, based on the presence of permutations of substitutions at 27 pre-defined positions in integrase (N=288). HIV-1 RNA was extracted from patient samples and diluted to approximately 500 HIV RNA copies/mL. Using an \"oligo-clonal\" amplification approach to achieve single-copy amplification, these dilutions were subjected to 12 parallel RT-PCR reactions to amplify integrase. Confirmed clonal amplicons were co-transfected with linearized pNL4.3{triangleup}int into CEM-GXR cells. In total, 162 HIV-1 viruses that carried no mixtures and had a unique sequence were harvested, and phenotyped in MT4-LTR-EGFP cells subsequently. Variants with the highest fold change (FC) had G140S and Q148R/H and resistant to all five drugs; R263K was the only single variant conferring >3-FC to DTG, BIC and CAB. There was extensive cross-resistance between DTG, BIC, and CAB and phenotypic resistance values for all the three INSTIs were almost collinear. The greatest exceptions were variants with N155H/G163E or L74I/T97M/F121C/V151I/E157Q/G163K, where both had >70-FC for CAB, while <3-FC for DTG and BIC. While site-directed mutagenesis is invaluable; the systematic selection of representative mutational patterns observed in vivo provides an efficient way to identify clinically relevant drug resistance.

molecular biology↗