HIV/HBV coinfection remodels the immune landscape and natural killer cell ADCC functional responses.

BACKGROUND AND AIMS: HBV and HIV coinfection is a common occurrence globally, with significant morbidity and mortality. Both viruses lead to immune dysregulation including changes in natural killer (NK) cells, a key component of antiviral defense and a promising target for HBV cure strategies. Here we used high-throughput single-cell analysis to explore the immune cell landscape in people with HBV mono-infection and HIV/HBV coinfection, on antiviral therapy, with emphasis on identifying the distinctive characteristics of NK cell subsets that can be therapeutically harnessed. APPROACH AND RESULTS: Our data show striking differences in the transcriptional programs of NK cells. HIV/HBV coinfection was characterized by an over-representation of adaptive, KLRC2 -expressing NK cells, including a higher abundance of a chemokine-enriched ( CCL3/CCL4 ) adaptive cluster. The NK cell remodeling in HIV/HBV coinfection was reflected in enriched activation pathways, including CD3ζ phosphorylation and ZAP-70 translocation that can mediate stronger antibody-dependent cellular cytotoxicity responses and a bias toward chemokine/cytokine signaling. By contrast, HBV mono-infection imposed a stronger cytotoxic profile on NK cells and a more prominent signature of "exhaustion" with higher circulating levels of HBsAg. Phenotypic alterations in the NK cell pool in coinfection were consistent with increased "adaptiveness" and better capacity for antibody-dependent cellular cytotoxicity compared to HBV mono-infection. Overall, an adaptive NK cell signature correlated inversely with circulating levels of HBsAg and HBV-RNA in our cohort. CONCLUSIONS: This study provides new insights into the differential signature and functional profile of NK cells in HBV and HIV/HBV coinfection, highlighting pathways that can be manipulated to tailor NK cell-focused approaches to advance HBV cure strategies in different patient groups.

Determinants of worse liver-related outcome according to HDV infection among HBsAg positive persons living with HIV: Data from the ICONA cohort.

OBJECTIVES: We aimed to study hepatitis D virus (HDV) prevalence and risk of progression to severe liver-related events (SLRE) in HBsAg positive people living with HIV (PLWH) in Italy; role of HDV-RNA copy levels, HCV coinfection and nadir CD4 counts were also investigated. METHODS: People living with HIV (PLWH) from Italian Foundation cohort Naïve antiretrovirals (ICONA) with available HBsAg and HDV Ab were enrolled. HBsAg, HDV Ab, HDV-RNA and HDV genotypes were tested. PRIMARY END-POINT: time from first HDV screening to Severe Liver Related Events (SLRE: decompensated cirrhosis, liver transplantation, HCC). Fine-grey regression models were used to evaluate the association of HDV Ab, HDV-RNA, HDV/HCV coinfection, CD4 nadir and outcome. Secondary end-points: time to SLRE or death; HDV Ab and HDV-RNA prevalence. RESULTS: A total of 152/809 (18.8%) HBsAg positive PLWH showed HDV Ab reactivity; 63/93 (67.7%) were HDV-RNA positive. Being male, persons who inject drugs (PWID), HCV Ab positive, with FIB-4 > 3.25 were independent factors of HDV Ab positivity. In a median follow-up of 5 years, 37 PLWH (4.1% at 5-year) developed SLRE and 97 (12.0%) reached the SLRE or death end-point. HDV-RNA positive (independently from HDV-RNA copy level) PLWH had a 4.6-fold (95%CI 2.0-10.5) higher risk of SLRE than HDV negatives. PLWH positive for both HCV Ab and HDV Ab showed the highest independent risk of SLRE (ASHR: 11.9, 95%CI: 4.6-30.9 vs. HCV neg/HDV neg). Nadir CD4 < 200/mL was associated with SLRE (ASHR: 3.9, 95% 1.0-14.5). CONCLUSIONS: One-fifth of the HBsAg positive PLWH harbour HDV infection, and are at high risk of progression to advanced liver disease. HCV contributes to worse outcomes. This population needs urgently effective treatments.

Droplet digital PCR assay as an innovative and promising highly sensitive assay to unveil residual and cryptic HBV replication in peripheral compartment.

Droplet digital PCR is an innovative and promising approach for highly sensitive quantification of nucleic acids that is being increasingly used in the field of clinical virology, including the setting of hepatitis B virus (HBV). Here, we comprehensively report a robust and reproducible ddPCR assay for the highly sensitive quantification of serum HBV-DNA. The assay showed a limit of detection of 4 copies/ml (<1IU/ml) by Probit analysis, showed a good linearity (R2 = 0.94) and a high intra- and inter-run reproducibility with differences between the values obtained in the same run or in two independent runs never exceeding 0.14logcopies/mL and 0.21logcopies/mL, respectively. By analysing serum samples from chronically HBV infected patients (mostly under antiviral treatment), ddPCR successfully quantified serum HBV-DNA in 89.8% of patients with detectable serum HBV-DNA < 20 IU/mL [equivalent to <112copies/ml] by classical Real-Time PCR assay, with a median (IQR) of 8(5-14)IU/mL [45(28-78)copies/ml], and in 66.7% of patients with undetectable serum HBV-DNA, with a median (IQR) of 5(4-9)IU/mL [28(20-50)copies/ml]. Similarly, by analysing serum samples from patients with a serological profile compatible with occult HBV infection (anti-HBc+/HBsAg-), ddPCR successfully quantified serum HBV-DNA in 40% of patients with a median (IQR) value of 1(1-2)IU/mL [5(5-11)copies/ml], in line with the extremely limited viral replication typically observed in occult HBV infection. Overall, the availability of assays for the highly sensitive quantification of serum HBV-DNA can provide an added value in optimizing the diagnosis of occult hepatitis B infection, improving the therapeutic management of chronically HBV infected patients, also in the light of innovative drugs (upcoming in clinical practise) aimed at achieving HBV functional cure.

Novel concepts on mechanisms underlying Hepatitis Delta virus persistence and related pathogenesis.

Hepatitis Delta virus is the smallest known human virus, exploiting the HBV surface proteins (HBsAg) for the release of its progeny and de novo entry into hepatocytes. Ever growing evidence have highlighted the existence of multiple mechanisms underlying HDV persistence including integrated HBV-DNA as a source of HBsAg production and the capability of the HDV genome to propagate through cell proliferation, thus supporting a potential HDV persistence even in the absence of HBV. Chronic HDV-infection causes the most severe form of viral hepatitis, leading to the development of cirrhosis in 15% of cases within 1-2 years and in 50%-60% of cases within 5-10 years. The rates of hepatocellular carcinoma and hepatic decompensation are also 2-3-fold higher than for HBV mono-infection. There is the evidence that persistent viral replication plays a key role in triggering liver injury, suggesting the existence of direct viral cytopathic properties that can modulate, synergistically with immune-responses, the progression towards end-stage liver diseases. All these aspects can be further exacerbated by the extraordinary degree of viral genetic variability that can promote HDV evasion from immune responses and has enabled viral differentiation into genotypes and subgenotypes with potential different pathobiological properties. In this light, this review aims at providing comprehensive insights of mechanisms (with a focus on virological factors) underlying HDV persistence and pathogenesis, critical in shaping the clinical outcome of the infection. Dissecting these mechanisms is pivotal to optimize therapeutic strategies aimed at fully counteracting this fascinating and fearsome virus.

Whole exome HBV DNA integration is independent of the intrahepatic HBV reservoir in HBeAg-negative chronic hepatitis B.

OBJECTIVE: The involvement of HBV DNA integration in promoting hepatocarcinogenesis and the extent to which the intrahepatic HBV reservoir modulates liver disease progression remains poorly understood. We examined the intrahepatic HBV reservoir, the occurrence of HBV DNA integration and its impact on the hepatocyte transcriptome in hepatitis B 'e' antigen (HBeAg)-negative chronic hepatitis B (CHB). DESIGN: Liver tissue from 84 HBeAg-negative patients with CHB with low (n=12), moderate (n=25) and high (n=47) serum HBV DNA was analysed. Covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA) were evaluated by quantitative PCR, whole exome and transcriptome sequencing was performed by Illumina, and the burden of HBV DNA integrations was evaluated by digital droplet PCR. RESULTS: Patients with low and moderate serum HBV DNA displayed comparable intrahepatic cccDNA and pgRNA, significantly lower than in patients with high HBV DNA, while hepatitis B core-related antigen correlated strongly with the intrahepatic HBV reservoir, reflecting cccDNA quantity. Whole exome integration was detected in a significant number of patients (55.6%, 14.3% and 25% in high, moderate and low viraemic patients, respectively), at a frequency ranging from 0.5 to 157 integrations/1000 hepatocytes. Hepatitis B surface antigen >5000 IU/mL predicted integration within the exome and these integrations localised in genes involved in hepatocarcinogenesis, regulation of lipid/drug metabolism and antiviral/inflammatory responses. Transcript levels of specific genes, including the proto-oncogene hRAS, were higher in patients with HBV DNA integration, supporting an underlying oncogenic risk in patients with low-level to moderate-level viraemia. CONCLUSIONS: HBV DNA integration occurs across all HBeAg-negative patients with CHB, including those with a limited HBV reservoir; localising in genes involved in carcinogenesis and altering the hepatocyte transcriptome.

Key genetic elements, single and in clusters, underlying geographically dependent SARS-CoV-2 genetic adaptation and their impact on binding affinity for drugs and immune control.

OBJECTIVES: To define key genetic elements, single or in clusters, underlying SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) evolutionary diversification across continents, and their impact on drug-binding affinity and viral antigenicity. METHODS: A total of 12 150 SARS-CoV-2 sequences (publicly available) from 69 countries were analysed. Mutational clusters were assessed by hierarchical clustering. Structure-based virtual screening (SBVS) was used to select the best inhibitors of 3-chymotrypsin-like protease (3CL-Pr) and RNA-dependent RNA polymerase (RdRp) among the FDA-approved drugs and to evaluate the impact of mutations on binding affinity of these drugs. The impact of mutations on epitope recognition was predicted following Grifoni et al. (Cell Host Microbe 2020. 27: 671-80.). RESULTS: Thirty-five key mutations were identified (prevalence: ≥0.5%), residing in different viral proteins. Sixteen out of 35 formed tight clusters involving multiple SARS-CoV-2 proteins, highlighting intergenic co-evolution. Some clusters (including D614GSpike + P323LRdRp + R203KN + G204RN) occurred in all continents, while others showed a geographically restricted circulation (T1198KPL-Pr + P13LN + A97VRdRp in Asia, L84SORF-8 + S197LN in Europe, Y541CHel + H504CHel + L84SORF-8 in America and Oceania). SBVS identified 20 best RdRp inhibitors and 21 best 3CL-Pr inhibitors belonging to different drug classes. Notably, mutations in RdRp or 3CL-Pr modulate, positively or negatively, the binding affinity of these drugs. Among them, P323LRdRp (prevalence: 61.9%) reduced the binding affinity of specific compounds including remdesivir while it increased the binding affinity of the purine analogues penciclovir and tenofovir, suggesting potential hypersusceptibility. Finally, specific mutations (including Y541CHel + H504CHel) strongly hampered recognition of Class I/II epitopes, while D614GSpike profoundly altered the structural stability of a recently identified B cell epitope target of neutralizing antibodies (amino acids 592-620). CONCLUSIONS: Key genetic elements reflect geographically dependent SARS-CoV-2 genetic adaptation, and may play a potential role in modulating drug susceptibility and hampering viral antigenicity. Thus, a close monitoring of SARS-CoV-2 mutational patterns is crucial to ensure the effectiveness of treatments and vaccines worldwide.

Establishment of a Seronegative Occult Infection With an Active Hepatitis B Virus Reservoir Enriched of Vaccine Escape Mutations in a Vaccinated Infant After Liver Transplantation.

We describe the establishment of a seronegative occult hepatitis B virus (HBV) infection (OBI) in a successfully vaccinated infant who underwent liver transplantation from an donor positive for antibody to hepatitis B core antigen (anti-HBc). The use of highly sensitive droplet digital polymerase chain reaction assays revealed a not negligible and transcriptionally active intrahepatic HBV reservoir (circular covalently closed DNA, relaxed circular DNA, and pregenomic RNA: 5.6, 2.4, and 1.1 copies/1000 cells, respectively), capable to sustain ongoing viral production and initial liver damage. Next-generation sequencing revealed a peculiar enrichment of hepatitis B surface antigen vaccine-escape mutations that could have played a crucial role in OBI transmission. This clinical case highlights the pathobiological complexity and the diagnostic challenges underlying OBI.

A snapshot of virological presentation and outcome of immunosuppression-driven HBV reactivation from real clinical practice: Evidence of a relevant risk of death and evolution from silent to chronic infection.

The study was undertaken in order to provide a snapshot from real clinical practice of virological presentation and outcome of patients developing immunosuppression-driven HBV reactivation. Seventy patients with HBV reactivation were included (66.2% treated with rituximab, 10% with corticosteroids and 23.8% with other immunosuppressive drugs). Following HBV reactivation, patients received anti-HBV treatment for a median (IQR) follow-up of 31(13-47) months. At baseline-screening, 72.9% of patients were HBsAg-negative and 27.1% HBsAg-positive. About 71.4% had a diagnosis of biochemical reactivation [median (IQR) HBV DNA and ALT: 6.9 (5.4-7.8) log IU/mL and 359 (102-775) U/L]. Moreover, 10% of patients died from hepatic failure. Antiviral prophylaxis was documented in 57.9% and 15.7% of HBsAg-positive and HBsAg-negative patients at baseline-screening (median [IQR] prophylaxis duration: 24[15-33] and 25[17-36] months, respectively). Notably, HBV reactivation occurred 2-24 months after completing the recommended course of anti-HBV prophylaxis in 35.3% of patients. By analysing treatment outcome, the cumulative probability of ALT normalization and of virological suppression was 97% and 69%, respectively. Nevertheless, in patients negative to HBsAg at baseline-screening, only 27% returned to HBsAg-negative status during prolonged follow-up, suggesting the establishment of chronic infection. In conclusion, most patients received a diagnosis of HBV reactivation accompanied by high ALT and 10% died for hepatic failure, supporting the importance of strict monitoring for an early HBV reactivation diagnosis. Furthermore, HBV reactivation correlates with high risk of HBV chronicity in patients negative for HBsAg at baseline-screening, converting a silent into a chronic infection, requiring long-term antiviral treatment. Finally, a relevant proportion of patients experienced HBV reactivation after completing the recommended course of anti-HBV prophylaxis, suggesting the need to reconsider proper duration of prophylaxis particularly in profound immunosuppression.

Quantification of intrahepatic total HBV DNA in liver biopsies of HBV-infected patients by a modified version of COBAS® Ampliprep/COBAS®TaqMan HBV test v2.0.

Intrahepatic total HBV DNA (it-HBV DNA) level might reflect the size of virus reservoir and correlate with the histological status of the liver. To quantitate it-HBV DNA in a series of 70 liver biopsies obtained from hepatitis B chronic patients, a modified version of the COBAS®Ampliprep/COBAS®TaqMan HBV test v2.0 was used for this purpose. The linearity and reproducibility of the modified protocol was tested by quantifying serial dilutions of a full-length HBV containing plasmid and it-HBV DNA from a reference patient. A good linear trend between the expected values and those generated by the assay was observed at different concentrations of both plasmid and reference patient (R 2 = 0.994 and 0.962, respectively). Differences between the values obtained in two independent runs were ≤0.3 log IU for the plasmid and ≤0.6 log IU/mg for the reference patient, showing a high inter-run reproducibility. In the 70 liver biopsies, it-HBV DNA level ranged from 1.4 to 5.4 log IU/mg, with a good linearity and reproducibility between the values obtained in two runs [R 2 = 0.981; median (IQR) difference of it-HBV DNA 0.05 (0.02-0.09) IU/mg]. The modified COBAS®Ampliprep/COBAS®TaqMan HBV test v2.0 allows an accurate quantitation of it-HBV DNA. Its determination may have prognostic value and may be a useful tool for the new therapeutic strategies aimed at eradicating the HBV infection.

Utility of accessible SARS-CoV-2 specific immunoassays in vaccinated adults with a history of advanced HIV infection.

Accessible SARS-CoV-2-specific immunoassays may inform clinical management in people with HIV, particularly in case of persisting immunodysfunction. We prospectively studied their application in vaccine recipients with HIV, purposely including participants with a history of advanced HIV infection. Participants received one (n = 250), two (n = 249) or three (n = 42) doses of the BNT162b2 vaccine. Adverse events were documented through questionnaires. Sample collection occurred pre-vaccination and a median of 4 weeks post-second dose and 14 weeks post-third dose. Anti-spike and anti-nucleocapsid antibodies were measured with the Roche Elecsys chemiluminescence immunoassays. Neutralising activity was evaluated using the GenScript cPass surrogate virus neutralisation test, following validation against a Plaque Reduction Neutralization Test. T-cell reactivity was assessed with the Roche SARS-CoV-2 IFNγ release assay. Primary vaccination (2 doses) was well tolerated and elicited measurable anti-spike antibodies in 202/206 (98.0%) participants. Anti-spike titres varied widely, influenced by previous SARS-CoV-2 exposure, ethnicity, intravenous drug use, CD4 counts and HIV viremia as independent predictors. A third vaccine dose significantly boosted anti-spike and neutralising responses, reducing variability. Anti-spike titres > 15 U/mL correlated with neutralising activity in 136/144 paired samples (94.4%). Three participants with detectable anti-S antibodies did not develop cPass neutralising responses post-third dose, yet displayed SARS-CoV-2 specific IFNγ responses. SARS-CoV-2 vaccination is well-tolerated and immunogenic in adults with HIV, with responses improving post-third dose. Anti-spike antibodies serve as a reliable indicator of neutralising activity. Discordances between anti-spike and neutralising responses were accompanied by detectable IFN-γ responses, underlining the complexity of the immune response in this population.

Prevalence of hepatitis D virus infection in Central Italy has remained stable across the last 2 decades with dominance of subgenotypes 1 and characterized by elevated viral replication.

OBJECTIVES: Here we investigate Hepatitis D virus (HDV)-prevalence in Italy and its fluctuations over time and we provide an extensive characterization of HDV-infected patients. METHODS: The rate of HDV seroprevalence and HDV chronicity was assessed in 1579 hepatitis B surface antigen (HBsAg)+ patients collected from 2005 to 2022 in Central Italy. RESULTS: In total, 45.3% of HBsAg+ patients received HDV screening with an increasing temporal trend: 15.6% (2005-2010), 45.0% (2011-2014), 49.4% (2015-2018), 71.8% (2019-2022). By multivariable model, factors correlated with the lack of HDV screening were alanine-aminotransferase (ALT) less than two times of upper limit of normality (<2ULN) and previous time windows (P <0.002). Furthermore, 13.4% of HDV-screened patients resulted anti-HDV+ with a stable temporal trend. Among them, 80.8% had detectable HDV-ribonucleic acid (RNA) (median [IQR]:4.6 [3.6-5.6] log copies/ml) with altered ALT in 89.3% (median [IQR]:92 [62-177] U/L). Anti-HDV+ patients from Eastern/South-eastern Europe were younger than Italians (44 [37-54] vs 53 [47-62] years, P <0.0001), less frequently nucleos(t)ide analogs (NUC)-treated (58.5% vs 80%, P = 0.026) with higher HDV-RNA (4.8 [3.6-5.8] vs 3.9 [1.4-4.9] log copies/ml, P = 0.016) and HBsAg (9461 [4159-24,532] vs 4447 [737-13,336] IU/ml, P = 0.032). Phylogenetic analysis revealed the circulation of HDV subgenotype 1e (47.4%) and -1c (52.6%). Notably, subgenotype 1e correlated with higher ALT than 1c (168 [89-190] vs 58 [54-88] U/l, P = 0.015) despite comparable HDV-RNA. CONCLUSIONS: HDV-screening awareness is increasing over time even if some gaps persist to achieve HDV screening in all HBsAg+ patients. HDV prevalence in tertiary care centers tend to scarcely decline in native/non-native patients. Detection of subgenotypes, triggering variable inflammatory stimuli, supports the need to expand HDV molecular characterization.

New insights into hepatitis B virus lymphotropism: Implications for HBV-related lymphomagenesis.

HBV is one of the most widespread hepatitis viruses worldwide, and a correlation between chronic infection and liver cancer has been clearly reported. The carcinogenic capacity of HBV has been reported for other solid tumors, but the largest number of studies focus on its possible lymphomagenic role. To update the correlation between HBV infection and the occurrence of lymphatic or hematologic malignancies, the most recent evidence from epidemiological and in vitro studies has been reported. In the context of hematological malignancies, the strongest epidemiological correlations are with the emergence of lymphomas, in particular non-Hodgkin's lymphoma (NHL) (HR 2.10 [95% CI 1.34-3.31], p=0.001) and, more specifically, all NHL B subtypes (HR 2.14 [95% CI 1.61-2.07], p<0.001). Questionable and unconfirmed associations are reported between HBV and NHL T subtypes (HR 1.11 [95% CI 0.88-1.40], p=0.40) and leukemia. The presence of HBV DNA in peripheral blood mononuclear cells has been reported by numerous studies, and its integration in the exonic regions of some genes is considered a possible source of carcinogenesis. Some in vitro studies have shown the ability of HBV to infect, albeit not productively, both lymphomonocytes and bone marrow stem cells, whose differentiation is halted by the virus. As demonstrated in animal models, HBV infection of blood cells and the persistence of HBV DNA in peripheral lymphomonocytes and bone marrow stem cells suggests that these cellular compartments may act as HBV reservoirs, allowing replication to resume later in the immunocompromised patients (such as liver transplant recipients) or in subjects discontinuing effective antiviral therapy. The pathogenetic mechanisms at the basis of HBV carcinogenic potential are not known, and more in-depth studies are needed, considering that a clear correlation between chronic HBV infection and hematological malignancies could benefit both antiviral drugs and vaccines.

Evaluation of HIV-DNA and residual viremia levels through week 96 in HIV-infected individuals who continue a two-drug or switch to a three-drug integrase strand transfer inhibitor-based regimen.

OBJECTIVES: To investigate HIV-DNA and residual viremia (RV) levels over 96 weeks (W96) in virologically-suppressed HIV-1-infected individuals enrolled in the Be-OnE Study. Individuals were randomised to continue a two-drug regimen with dolutegravir (DTG) plus one reverse transcriptase inhibitor (RTI) or to switch to elvitegravir/cobicistat/emtricitabine/tenofovir-alafenamide (E/C/F/TAF). STUDY DESIGN: Total HIV-DNA and RV were evaluated at baseline, W48 and W96 using droplet digital polymerase chain reaction (ddPCR) technique. Potential relationships between viro-immunological parameters and between/within arms were also assessed. RESULTS: Median (interquartile range [IQR]) HIV-DNA was 2247 (767-4268), 1587 (556-3543) and 1076 (512-2345) copies/106 CD4+T-cells at baseline, W48 and at W96, respectively; RV was 3 (1-5), 4 (1-9) and 2 (2-4) copies/mL, respectively, with no significant differences between arms. A significant reduction in HIV-DNA and RV from baseline to W96 was observed in the E/C/F/TAF arm (HIV-DNA: -285 [-2257; -45], P=0.010; RV: -1 [-3;0], P=0.007). In the DTG + 1 RTI arm, HIV-DNA and RV levels remained stable (HIV-DNA: -549 [-2269;+307], P=0.182; RV: -1 [-3;+1], P=0.280). For both HIV-DNA and RV, there were no significant changes over time between the arms. A positive correlation was found between baseline HIV-DNA and HIV-DNA at W96 (E/C/F/TAF: Spearman correlation coefficient (rs)=0.726, P=0.0004; DTG + 1 RTI: rs=0.589, P=0.010). In general, no significant correlations were found between HIV-DNA, RV and immunological parameters over time. CONCLUSIONS: In virologically-suppressed individuals, there was a small reduction in HIV-DNA and HIV-RNA levels from baseline to W96 in individuals who switched to the E/C/F/TAF arm compared with those who remained on DTG + 1 RTI. However, there were no significant differences between the two arms in the changes in HIV-DNA and HIV-RNA over time.

Frequency of Atypical Mutations in the Spike Glycoprotein in SARS-CoV-2 Circulating from July 2020 to July 2022 in Central Italy: A Refined Analysis by Next Generation Sequencing.

In this study, we provided a retrospective overview in order to better define SARS-CoV-2 variants circulating in Italy during the first two years of the pandemic, by characterizing the spike mutational profiles and their association with viral load (expressed as ct values), N-glycosylation pattern, hospitalization and vaccination. Next-generation sequencing (NGS) data were obtained from 607 individuals (among them, 298 vaccinated and/or 199 hospitalized). Different rates of hospitalization were observed over time and among variants of concern (VOCs), both in the overall population and in vaccinated individuals (Alpha: 40.7% and 31.3%, Beta: 0%, Gamma: 36.5% and 44.4%, Delta: 37.8% and 40.2% and Omicron: 11.2% and 7.1%, respectively, both p-values < 0.001). Approximately 32% of VOC-infected individuals showed at least one atypical major spike mutation (intra-prevalence > 90%), with a distribution differing among the strains (22.9% in Alpha, 14.3% in Beta, 41.8% in Gamma, 46.5% in Delta and 15.4% in Omicron, p-value < 0.001). Overall, significantly less atypical variability was observed in vaccinated individuals than unvaccinated individuals; nevertheless, vaccinated people who needed hospitalization showed an increase in atypical variability compared to vaccinated people that did not need hospitalization. Only 5/607 samples showed a different putative N-glycosylation pattern, four within the Delta VOC and one within the Omicron BA.2.52 sublineage. Interestingly, atypical minor mutations (intra-prevalence < 20%) were associated with higher Ct values and a longer duration of infection. Our study reports updated information on the temporal circulation of SARS-CoV-2 variants circulating in Central Italy and their association with hospitalization and vaccination. The results underline how SARS-CoV-2 has changed over time and how the vaccination strategy has contributed to reducing severity and hospitalization for this infection in Italy.

Unexpected rise in the circulation of complex HBV variants enriched of HBsAg vaccine-escape mutations in HBV genotype-D: potential impact on HBsAg detection/quantification and vaccination strategies.

Specific HBsAg mutations are known to hamper HBsAg recognition by neutralizing antibodies thus challenging HBV-vaccination efficacy. Nevertheless, information on their impact and spreading over time is limited. Here, we characterize the circulation of vaccine-escape mutations from 2005 to 2019 and their correlation with virological parameters in a large cohort of patients infected with HBV genotype-D (N = 947), dominant in Europe. Overall, 17.7% of patients harbours ≥1 vaccine-escape mutation with the highest prevalence in subgenotype-D3. Notably, complex profiles (characterized by ≥2 vaccine-escape mutations) are revealed in 3.1% of patients with a prevalence rising from 0.4% in 2005-2009 to 3.0% in 2010-2014 and 5.1% in 2015-2019 (P = 0.007) (OR[95%CI]:11.04[1.42-85.58], P = 0.02, by multivariable-analysis). The presence of complex profiles correlates with lower HBsAg-levels (median[IQR]:40[0-2905]IU/mL for complex profiles vs 2078[115-6037]IU/ml and 1881[410-7622]IU/mL for single or no vaccine-escape mutation [P < 0.02]). Even more, the presence of complex profiles correlates with HBsAg-negativity despite HBV-DNA positivity (HBsAg-negativity in 34.8% with ≥2 vaccine-escape mutations vs 6.7% and 2.3% with a single or no vaccine-escape mutation, P < 0.007). These in-vivo findings are in keeping with our in-vitro results showing the ability of these mutations in hampering HBsAg secretion or HBsAg recognition by diagnostic antibodies. In conclusion, vaccine-escape mutations, single or in complex profiles, circulate in a not negligible fraction of HBV genotype-D infected patients with an increasing temporal trend, suggesting a progressive enrichment in the circulation of variants able to evade humoral responses. This should be considered for a proper clinical interpretation of HBsAg-results and for the development of novel vaccine formulations for prophylactic and therapeutic purposes.

Human Monocyte-Derived Macrophages (MDM): Model 1 (GM-CSF).

Monocytes/macrophages play critical roles in HIV transmission, viral spread (early in infection), and as a reservoir of virus throughout infection. In the current research area in HIV, there has been a recent resurgence of interest in the biology of monocyte subsets and macrophages and their role in HIV pathogenesis, and as long-lived HIV reservoir. Thus, sensitive and specific techniques are needed to measure the impact of these cells in the establishment of the "hard-core" reservoir, and in their capacity to cause a low-level virus production during cART. Here, a protocol is presented for cell culture and HIV-1 infection of granulocyte-macrophage colony-stimulating factor (GM-CSF) differentiated human monocyte-derived macrophages.

Hepatitis B virus DNA integration as a novel biomarker of hepatitis B virus-mediated pathogenetic properties and a barrier to the current strategies for hepatitis B virus cure.

Chronic infection with Hepatitis B Virus (HBV) is a major cause of liver-related morbidity and mortality worldwide. HBV-DNA integration into the human genome is recognized as a frequent event occurring during the early phases of HBV infection and characterizing the entire course of HBV natural history. The development of refined molecular biology technologies sheds new light on the functional implications of HBV-DNA integration into the human genome, including its role in the progression of HBV-related pathogenesis and in triggering the establishment of pro-oncogenic mechanisms, promoting the development of hepatocellular carcinoma. The present review provides an updated and comprehensive overview of the current body of knowledge on HBV-DNA integration, focusing on the molecular mechanisms underlying HBV-DNA integration and its occurrence throughout the different phases characterizing the natural history of HBV infection. Furthermore, here we discuss the main clinical implications of HBV integration as a biomarker of HBV-related pathogenesis, particularly in reference to hepatocarcinogenesis, and how integration may act as a barrier to the achievement of HBV cure with current and novel antiviral therapies. Overall, a more refined insight into the mechanisms and functionality of HBV integration is paramount, since it can potentially inform the design of ad hoc diagnostic tools with the ability to reveal HBV integration events perturbating relevant intracellular pathways and for identifying novel therapeutic strategies targeting alterations directly related to HBV integration.

Update on SARS-CoV-2 Omicron Variant of Concern and Its Peculiar Mutational Profile.

The process of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversification is still ongoing and has very recently led to the emergence of a new variant of concern (VOC), defined as Omicron or B.1.1.529. Omicron VOC is the most divergent variant identified so far and has generated immediate concern for its potential capability to increase SARS-CoV-2 transmissibility and, more worryingly, to escape therapeutic and vaccine-induced antibodies. Nevertheless, a clear definition of the Omicron VOC mutational spectrum is still missing. Herein, we provide a comprehensive definition and functional characterization (in terms of infectivity and/or antigenicity) of mutations characterizing the Omicron VOC. In particular, 887,475 SARS-CoV-2 Omicron VOC whole-genome sequences were retrieved from the GISAID database and used to precisely define its specific patterns of mutations across the different viral proteins. In addition, the functional characterization of Omicron VOC spike mutations was finely discussed according to published manuscripts. Lastly, residues characterizing the Omicron VOC and the previous four VOCs (Alpha, Beta, Gamma, and Delta) were mapped on the three-dimensional structure of the SARS-CoV-2 spike protein to assess their localization in the different spike domains. Overall, our study will assist with deciphering the Omicron VOC mutational profile and will shed more light on its clinical implications. This is critical considering that Omicron VOC is currently the predominant variant worldwide. IMPORTANCE The Omicron variant of concern (VOC) has a peculiar spectrum of mutations characterized by the acquisition of mutations or deletions rarely detected in previously identified variants, particularly in the spike glycoprotein. Such mutations, mostly residing in the receptor-binding domain, could play a pivotal role in enhancing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity (by increasing binding affinity for ACE2), jeopardizing spike recognition by therapeutic and vaccine-induced antibodies and causing diagnostic assay failure. To our knowledge, this is one of the first exhaustive descriptions of newly emerged mutations underlying the Omicron VOC and its biological and clinical implications.

Impact of pre-existent drug resistance on virological efficacy of single-tablet regimens in people living with HIV.

Despite the wide use of single-tablet regimens (STRs), few real-life data are available regarding the impact of pre-existent drug resistance on virological failure (VF). We aimed to fill this gap by analysing a large cohort of individuals selected from the ARCA database. The impact on VF of pre-existent resistance-associated mutations (RAMs) and cumulative genotypic susceptibility score (cGSS) before STR start was evaluated through survival analysis. Potential emergence of resistance at VF was also evaluated. Overall, 3916 individuals were included, comprising 678 treatment-naïve (G1), 2309 treatment-experienced aviraemic (G2) and 929 viraemic (G3), of whom 65.2% were treated with a STR based on efavirenz (35.2%) or rilpivirine (30.0%). At 2 years after starting a STR, the overall probability of VF was 5.9% in G1, 8.7% in G2 and 20.8% in G3. No impact of pre-existent resistance on VF was found in G1. The probability of VF was higher in patients with cGSS < 3 (reduced susceptibility to at least one drug) than in those with cGSS = 3 (full susceptibility to STR drugs) both in G2 and G3. A higher probability of VF was also found in the presence of pre-existent M184V (alone or in combination with pre-existent thymidine analogue mutations). Among patients who failed STR, a significant emergence of RAMs was found only in those exposed to EFV/FTC/TDF in G3 (specifically K103N and M184V). Our results confirm a high efficacy of STRs in clinical settings. Pre-existent resistance appears to influence virological efficacy of STRs in treatment-experienced individuals (both aviraemic and viraemic).

Highly Sensitive HBsAg, Anti-HBc and Anti HBsAg Titres in Early Diagnosis of HBV Reactivation in Anti-HBc-Positive Onco-Haematological Patients.

The role of novel HBV markers in predicting Hepatitis B virus reactivation (HBV-R) in HBsAg-negative/anti-HBc-positive oncohaematological patients was examined. One hundred and seven HBsAg-negative/anti-HBc-positive oncohaematological patients, receiving anti-HBV prophylaxis for >18 months, were included. At baseline, all patients had undetectable HBV DNA, and 67.3% were anti-HBs positive. HBV-R occurred in 17 (15.9%) patients: 6 during and 11 after the prophylaxis period. At HBV-R, the median (IQR) HBV-DNA was 44 (27-40509) IU/mL, and the alanine aminotransferase upper limit of normal (ULN) was 44% (median (IQR): 81 (49-541) U/L). An anti-HBc > 3 cut-off index (COI) plus anti-HBs persistently/declining to <50 mIU/mL was predictive for HBV-R (OR (95% CI): 9.1 (2.7-30.2); 63% of patients with vs. 15% without this combination experienced HBV-R (p < 0.001)). The detection of highly sensitive (HS) HBsAg and/or HBV-DNA confirmed at >2 time points, also predicts HBV-R (OR (95% CI): 13.8 (3.6-52.6); 50% of positive vs. 7% of negative patients to these markers experienced HBV-R (p = 0.001)). HS-HBs and anti-HBc titration proved to be useful early markers of HBV-R. The use of these markers demonstrated that HBV-R frequently occurs in oncohaematological patients with signs of resolved HBV infection, raising issues of proper HBV-R monitoring.