Current antiviral therapies, such as pegylated interferon-α and nucleos(t)ide analogues, effectively improve the quality of life of patients with chronic hepatitis B. However, they can only control ...the infection rather than curing infected hepatocytes. Complete HBV cure is hampered by the lack of therapies that can directly affect the viral minichromosome (in the form of covalently closed circular DNA cccDNA). Approaches currently under investigation in early clinical trials are aimed at achieving a functional cure, defined as the loss of HBsAg and undetectable HBV DNA levels in serum. However, achieving a complete HBV cure requires therapies that can directly target the cccDNA pool, either via degradation, lethal mutations or functional silencing. In this review, we discuss cutting-edge technologies that could lead to non-cytolytic direct cccDNA targeting and cure of infected hepatocytes.
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In the presence of highly-potent antivirals, persistence of hepatitis B virus (HBV) is most well-characterized by covalently-closed circular DNA (cccDNA) and total intrahepatic DNA ...(IH-DNA). We sought to determine how antiviral therapy could affect their levels during human immunodeficiency virus (HIV)-HBV co-infection.
Sixty co-infected patients from a well-defined cohort with ⩾1 liver biopsy were studied. HBV cccDNA and total IH-DNA were extracted from biopsies and quantified by real-time PCR. Factors associated with intrahepatic viral load were determined using mixed-effect linear regression and half-life viral kinetics during reconstructed follow-up using non-linear exponential decay models.
At biopsy, 35 (58.3%) patients were hepatitis B “e” antigen (HBeAg)-positive and 33 (55.0%) had detectable plasma HBV-DNA (median=4.58log10IU/ml, IQR=2.95–7.43). Overall, median cccDNA was −0.95log10copies/cell (IQR=−1.70, −0.17) and total IH-DNA was 0.27log10copies/cell (IQR=−0.39, 2.00). In multivariable analysis, significantly lower levels of cccDNA and total IH-DNA were observed in patients with HBeAg-negative serology, nadir CD4+ cell counts >250/mm3, and longer cumulative TDF-duration, but not lamivudine- or adefovir-duration. In post-hoc analysis using reconstructed TDF-duration (median 29.6months, IQR=15.0–36.1, n=31), average half-life of cccDNA was estimated at 9.2months (HBeAg-positive=8.6, HBeAg-negative=26.2) and total IH DNA at 5.8months (HBeAg-positive=1.3, HBeAg-negative=13.6). Intrahepatic viral loads remained detectable for all patients, even with prolonged TDF-exposure.
In co-infection, TDF-use is associated with lower levels of HBV replication intermediates and cccDNA. Slow decay of intrahepatic viral loads underscores that TDF is unable to completely block intracellular viral DNA synthesis, which possibly accounts for continuous replenishment of the cccDNA pool.
Chronic hepatitis B virus (HBV) is a persistent infection, while the only real way of knowing the extent of this persistence is through measuring levels of virus in the liver. In this study, we examine levels of HBV in the liver among patients with both HBV and human immunodeficiency virus, or HIV, infection. It would appear that the currently available medication, namely “tenofovir”, works well to decrease virus levels in the liver, but it remains at low levels despite long periods of treatment.
Cerebral white matter hyperintensities (WMH) persist in children and adults living with HIV, despite effective combination antiretroviral therapy (cART). As age and principal routes of transmission ...differ between children (perinatally) and adults (behaviorally), comparing the characteristics and determinants of WMH between these populations may increase our understanding of the pathophysiology of WMH. From separate cohorts of 31 children (NOVICE) and 74 adults (AGEhIV), we cross-sectionally assessed total WMH volume and number of WMH per location (periventricular vs. deep) using fluid-attenuated inversion recovery (FLAIR) MRI images. WMH were either periventricular when within 10mm of the lateral ventricles, or deep otherwise. We assessed patient- or HIV-related determinants of total WMH volume (adjusted for intracranial volume) and location of WMH using logistic regression, while stratifying on children and adults. At enrollment, median age of participants was 13.8 years (IQR 11.4-15.9) for children and 53.4 years (IQR 48.3-60.8) for adults and 27/31 children (87%) and 74/74 adults (100%) had an HIV RNA viral load <200 copies/mL. WMH were present in 16/27 (52%) children and 74/74 adults (100%). The prevalence of deep WMH was not different between groups, (16/16 100% in children vs. 71/74 96% in adults, p = 0,999), yet periventricular WMH were more prevalent in adults (74/74 100%) compared to children (9/16; 56%) (p<0.001). Median WMH volume was higher in adults compared to children (1182 mm3 425-2617 vs. 109 mm3 61.7-625, p<0.001). In children, boys were more likely to have deep WMH compared to girls. In adults, older age was associated with higher total WMH volume, and age, hypertension and lower CD4+ T-lymphocyte nadir with a higher number of periventricular WMH. Our findings suggest that the location of WMH differs between children and adults living with HIV, hinting at a different underlying pathogenesis.
Biofilm production in extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae provides a favourable environment for the exchange of antibiotic-resistance genes and could facilitate ...widespread dissemination. We aimed to assess biofilm development in ESBL-producing E. coli and K. pneumoniae isolates and determine how development relates to microbiological characteristics and clinical outcomes.
147 ESBL-producing E. coli and 82 K. pneumoniae were genetically characterized. Biofilm formation was measured at 1.5, 4, 6, and 24 h during culture in blood heart infusion using a microbead immobilization assay (BioFilm Ring test®). Results were given as biofilm formation index (BFI) with lower values indicating increased presence of biofilm (range = 0–21).
In total, 57.1% of strains were strong producers of biofilm (BFI < 2), whereas 13.4% lacked biofilm production (BFI > 18). Standard biofilm production (BFI < 7) was common in E. coli isolates (61.9%). For E. coli, biofilm production was less frequently observed in ST131 clones (p = 0.03) but more frequently in strains harbouring toxin (p = 0.008) or adhesin (p = 0.008) virulence factor genes. Despite almost all K. pneumoniae having standard biofilm production (90.2%), there was a 2.4-times higher odds of observing biofilm in ST29/147/323 versus other ST-types (p = 0.13). Patients with standard biofilm producing isolates were not at increased risk of transfer to intensive-care (odds-ratio=2.80, 95%CI=0.59–13.21) or death within 12-months (odds-ratio=1.61, 95%CI=0.75–3.43).
In these ESBL-producing strains, biofilm production is linked to certain virulence factors in E. coli and is common in K. pneumoniae. Further exploration of whether biofilm production increases dissemination and risk of severe clinical outcomes is needed in larger collections of isolates.
To facilitate HCV diagnosis, we developed an HCV-RNA testing service, which involved home-sampled dried blood spots (DBS). The main objective of this study was to evaluate the feasibility of ...self-sampling at home. Furthermore, to optimise the processing of DBS samples for RNA detection, we evaluated two elution buffers: phosphate-buffered saline (PBS) and L6-buffer. 27 HCV-RNA and 12 HIV-1 RNA positive patients were included. Laboratory spotted DBS (LabDBS) were made by a technician from blood samples drawn at inclusion. Patients received a DBS home-sampling kit and were requested to return their self-sampled DBS (ssDBS) by mail. We compared the RNA load of PBS and L6-eluted labDBS, and of L6-eluted ssDBS, L6-eluted labDBS and plasma. LabDBS load measurements were repeated after 7-13 and 14-21 days to evaluate RNA stability. All 39 plasma samples provided quantifiable RNA loads. In 1/39 labDBS sample, RNA could not be detected (plasma HCV load: 2.98 log10 IU/ml). L6-eluted samples gave a 0.7 log10 and 0.6 log10 higher viral load for HCV and HIV-1 respectively, compared to PBS-eluted samples. Strong correlations were found between labDBS and ssDBS HCV RNA (r = 0.833; mean difference 0.3 log10 IU/mL) and HIV-1 RNA results (r = 0.857; mean difference 0.1 log10 copies/mL). Correlations between labDBS and plasma values were high for HCV (r = 0.958) and HIV-1 (r = 0.844). RNA loads in DBS remained stable over 21 days. Our study demonstrates that self-sampling dried blood spots at home is a feasible strategy for the detection of HCV and HIV-1 RNA. This could facilitate one-step diagnostics and treatment monitoring in communities with high HCV prevalence.
This study identified subgroups of sexual behaviors associated with increased STI/HIV risk among those eligible for but not using pre-exposure prophylaxis (PrEP) in order to improve PrEP uptake and ...prioritization in the context of restricted capacity. We used data from sexual health centers (SHCs) in the Netherlands, including all visits of eligible but non-PrEP using men who have sex with men (MSM), men who have sex with men and women (MSMW) and transgender persons between July 2019 (start of the Dutch national PrEP pilot (NPP)) and June 2021. Using latent class analysis (LCA), we identified classes of sexual behaviors (number of partners, chemsex, group sex and sex work) and explored whether these classes were associated with STI diagnosis and sociodemographics. Across 45,582 visits of 14,588 eligible non-PrEP using individuals, the best fitting LCA model contained three classes of sexual behaviors. Classes were distinguished by seldomly reported sexual behaviors (class 1; 53.5%, n = 24,383), the highest proportions of ≥6 partners and group sex (class 2; 29.8%, n = 13,596), and the highest proportions of chemsex and sex work (class 3; 16.7% of visits, n = 7,603). Visits in classes 2 and 3 (vs. class 1) were significantly more often with individuals who were diagnosed with an STI, older (≥36 vs. ≤35 years), MSMW (vs. MSM), and visiting an urban (vs. non-urban) SHC; while these visits were significantly less often with individuals from an STI/HIV endemic area. The percentage of visits at which an STI was diagnosed was 17.07% (n = 4,163) in class 1, 19.53% (n = 2,655) in class 2 and 25.25% (n = 1,920) in class 3. The highest risk of STI, and thereby HIV, was in those engaging in specific subgroups of sexual behavior characterized by frequently reporting multiple partners, group sex, sex work or chemsex. PrEP uptake should be encouraged and prioritized for these individuals.
•Vaccination did not reduce symptoms of post-acute sequelae (PASC).•Antibody kinetics did not differ between participants with and without PASC.•Early antibody titers were comparable between ...participants with and without PASC.•Therapeutic potential of COVID-19 vaccination on PASC seems unlikely.
Symptoms of post-acute sequelae of COVID-19 (PASC) may improve following SARS-CoV-2 vaccination. However few prospective data that also explore the underlying biological mechanism are available. We assessed the effect of vaccination on symptomatology of participants with PASC, and compared antibody dynamics between those with and without PASC.
RECoVERED is a prospective cohort study of adult patients with mild to critical COVID-19, enrolled from illness onset. Among participants with PASC, vaccinated participants were exact-matched 1:1 on age, sex, obesity status and time since illness onset to unvaccinated participants. Between matched pairs, we compared the monthly mean numbers of symptoms over a 3-month follow-up period, and, using exact logistic regression, the proportion of participants who fully recovered from PASC. Finally, we assessed the association between PACS status and rate of decay of spike- and RBD-binding IgG titers up to 9 months after illness onset using Bayesian hierarchical linear regression.
Of 349 enrolled participants, 316 (90.5%) had ≥3 months of follow-up, of whom 186 (58.9%) developed PASC. Among 36 matched pairs with PASC, the mean number of symptoms reported each month during 3 months of follow-up were comparable between vaccinated and unvaccinated groups. Odds of full recovery from PASC also did not differ between matched pairs (OR 1.57 95%CI 0.46–5.84) within 3 months after the matched time-point. The median half-life of spike- and RBD-binding IgG levels were, in days (95%CrI), 233 (183–324) and 181 (147–230) among participants with PASC, and 170 (125–252) and 144 (113–196) among those without PASC, respectively.
Our study found no strong evidence to suggest that vaccination improves symptoms of PASC. This was corroborated by comparable spike- and RBD-binding IgG waning trajectories between those with and without PASC, refuting any immunological basis for a therapeutic effect of vaccination on PASC.
In individuals infected with hepatitis B virus (HBV), the loss of hepatitis B surface antigen (HBsAg) is the ultimate therapeutic goal, which defines "functional cure." For individuals living with ...human immunodeficiency virus (HIV), functional cure occurs roughly 2 per 100 person-years during potent anti-HBV containing antiretroviral therapy. Although this rate may be higher than expected in treated HBV mono-infected individuals, rates of functional cure widely vary between studies (0.6-10.5 per 100 person-years). Similar to HBV mono-infection, the phase of HBV infection, HBV (sub-)genotypes and hepatitis B "e" Ag-negative variants are associated with functional cure in treated HIV-HBV co-infection. In specifically HIV-HBV co-infected individuals, strong increases in CD4+ T cell counts after treatment initiation have also been linked to functional cure, yet this finding is inconsistent across studies. Several markers directly or indirectly reflecting HBV activity are being developed to predict functional cure, such as quantification of HBsAg, hepatitis B core-related antigen, HBsAg protein composition, anti-hepatitis B core antibodies and interferon-gamma-inducible protein 10. Few have been assessed during treatment in HIV-HBV co-infected individuals and none have been validated to predict functional cure. Novel therapeutics for HBV cure are essential for individuals with HIV-HBV co-infection and need to be separately evaluated in this population.
Understanding the factors that delineate the efficacy of T cell responses towards pathogens is crucial for our ability to develop potent therapies against infectious diseases. Multidimensional ...evaluation of T cell functionality at the single-cell level enables exhaustive analysis of combinatorial functional properties, hence polyfunctionality. We have recently invented an algorithm that quantifies polyfunctionality, the Polyfunctionality Index (Larsen et al. PLoS One 2012). Here we demonstrate that quantitative assessment of T cell polyfunctionality correlates with T cell efficacy measured as the capacity to kill target cells in vitro and control infection in vivo.
We employed the polyfunctionality index on two datasets selected for their unique ability to evaluate the polyfunctional imprint on T cell efficacy. 1) HIV-specific CD8+ T cells and 2) Leishmania major-specific CD4+ T cells were analysed for their capacity to secrete multiple effector molecules, kill target cells and control infection. Briefly, employing the Polyfunctionality Index algorithm we determined the parameter estimates resulting in optimal correlation between T cell polyfunctionality and T cell efficacy.
T cell polyfunctionality is correlated with T cell efficacy measured as 1) target killing (r=0.807, P<0.0001) and 2) lesion size upon challenge with Leishmania major (r=-0.50, P=0.004). Contrary to an approach relying on the Polyfunctionality Index algorithm, quantitative evaluation of T cell polyfunctionality traditionally ignores the gradual contribution of more or less polyfunctional T cells. Indeed, comparing both approaches we show that optimal description of T cell efficacy is obtained when gradually integrating all levels of polyfunctionality in accordance with the Polyfunctionality Index.
Our study presents a generalizable methodology to objectively evaluate the impact of polyfunctionality on T cell efficacy. We show that T cell polyfunctionality is a superior correlate of T cell efficacy both in vitro and in vivo as compared with response size. Therefore, future immunotherapies should aim to increase T cell polyfunctionality.
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