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BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) gp41 is a crucial determinant for HIV-1 pathogenicity. We investigated the correlation of enfuvirtide (ENF)-associated gp41 mutational clusters with viroimmunological parameters, as well as the potential underlying mechanisms. METHODS: A total of 172 gp41 sequences and clinical follow-up data from 73 ENF-treated patients were analyzed monthly, from baseline to week 48. RESULTS: There were 7 novel gp41 mutations positively associated with ENF treatment and correlated with classic ENF mutations. The ENF-associated clusters [V38A + N140I ] and [V 38A +T18A ] significantly correlated with an increase in CD4 cell count at week 48 ( an increase from baseline of 112 and 209 cells/microL, respectively), whereas [Q40H + L45M + 268A] significantly correlated with a decrease in CD4 cell count (-53 cells/microL), without a change in the level of viremia. Residues 38 and 18 are located complementarily to each other in the Rev-responsive element, whereas analysis of molecular dynamics showed that the copresence of [V38A + N140 I] abolishes the interaction between residue 38 and 145 important for stabilization of the 6-helix bundle. In contrast, T268A localizes in the gp41 calmodulin-binding domain responsible for gp41-induced CD4(+) T lymphocyte apoptosis. CONCLUSION: Specific gp41 mutational clusters associated with ENF treatment significantly correlate with increases in CD4(+) cell count. Structural analysis suggests that this immunological gain is associated with mechanisms that act at both the protein level and the RNA level (even under conditions of virological failure). This result may help in the selection of patients who can benefit most from ENF treatment and represents a driving force for the design of the next generation of entry inhibitors. Copyright © 2008 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Abstract Background Resistance to the fusion inhibitor enfuvirtide (ENF) is achieved by changes in the gp41 subunit of the HIV envelope glycoprotein (Env). Specific ENF-associated mutational pathways correlate with immunological recovery, even after virological failure, suggesting that the acquisition of ENF resistance alters gp41 pathogenicity. To test this hypothesis, we have characterized the expression, fusion capability, induction of CD4+ T cell loss and single CD4+ T cell death of 48 gp41 proteins derived from three patients displaying different amino acids (N, T or I) at position 140 that developed a V38A mutation after ENF-based treatment. Results In all cases, intra-patient comparison of Env isolated pre- or post-treatment showed comparable values of expression and fusogenic capacity. Furthermore, Env with either N or T at position 140 induced comparable losses of CD4+ T-cells, irrespective of the residue present at position 38. Conversely, Env acquiring the V38A mutation in a 140I background induced a significantly reduced loss of CD4+ T cells and lower single-cell death than did their baseline controls. No altered ability to induce single-cell death was observed in the other clones. Conclusions Overall, primary gp41 proteins with both V38A and N140I changes showed a reduced ability to induce single cell death and deplete CD4+ T cells, despite maintaining fusion activity. The specificity of this phenotype highlights the relevance of the genetic context to the cytopathic capacity of Env and the role of ENF-resistance mutations in modulating viral pathogenicity in vivo, further supporting the hypothesis that gp41 is a critical mediator of HIV pathogenesis.

Abstract Background The third variable loop (V3) of the HIV-1 gp120 surface protein is a major determinant of cellular co-receptor binding. However, HIV-1 can also modulate its tropism through other regions in gp120, such as V1, V2 and C4 regions, as well as in the gp41 protein. Moreover, specific changes in gp41 are likely to be responsible for of damage in gp120-CCR5 interactions, resulting in potential resistance to CCR5 inhibitors. In order to genetically characterize the two envelope viral proteins in terms of co-receptor usage, we have analyzed 526 full-length env sequences derived from HIV-1 subtype-B infected individuals, from our and public (Los Alamos) databases. The co-receptor usage was predicted by the analysis of V3 sequences using Geno2Pheno (G2P) algorithm. The binomial correlation phi coefficient was used to assess covariation among gp120V3 and gp41 mutations; subsequently the average linkage hierarchical agglomerative clustering was performed. Results According to G2P false positive rate (FPR) values, among 526 env-sequences analyzed, we further characterized 196 sequences: 105 with FPR 70%, for X4-using and R5-using viruses, respectively. Beyond the classical signatures at 11/25 V3 positions (S11S and E25D, R5-tropic viruses; S11KR and E25KRQ, X4-tropic viruses), other specific V3 and gp41 mutations were found statistically associated with the co-receptor usage. Almost all of these specific gp41 positions are exposed on the surface of the glycoprotein. By the covariation analysis, we found several statistically significant associations between V3 and gp41 mutations, especially in the context of CXCR4 viruses. The topology of the dendrogram showed the existence of a cluster associated with R5-usage involving E25DV3, S11SV3, T22AV3, S129DQgp41 and A96Ngp41 signatures (bootstrap = 0.88). Conversely, a large cluster was found associated with X4-usage involving T8IV3, S11KRV3, F20IVYV3, G24EKRV3, E25KRV3, Q32KRV3, A30Tgp41, A189Sgp41, N195Kgp41 and L210Pgp41 mutations (bootstrap = 0.84). Conclusions Our results show that gp120V3 and several specific amino acid changes in gp41 are associated together with CXCR4 and/or CCR5 usage. These findings implement previous observations that determinants of tropism may reside outside the V3-loop, even in the gp41. Further studies will be needed to confirm the degree to which these gp41 mutations contribute directly to co-receptor use.

Abstract Background The transmission of HIV-1 drug-resistant strains in drug naive patients may seriously compromise the efficacy of a first-line antiretroviral treatment. To better define this problem, a study in a cohort of newly diagnosed HIV-1 infected individuals has been conducted. This study is aimed to assess the prevalence and the patterns of the mutations recently associated with transmitted drug resistance in the reverse transcriptase (RT) and in protease (PR) of HIV-1. Methods Prevalence of transmitted drug resistant strains is determined in 255 newly diagnosed HIV-1 infected patients enrolled in different counselling and testing (CT) centres in Central Italy; the Avidity Index (AI) on the first available serum sample is also used to estimate time since infection. Logistic regression models are used to determine factors associated with infection by drug resistant HIV-1 strains. Results The prevalence of HIV-1 strains with at least one major drug resistance mutation is 5.9% (15/255); moreover, 3.9% (10/255) of patients is infected with HIV nucleoside reverse transcriptase inhibitor (NRTI)-resistant viruses, 3.5% (9/255) with HIV non-NRTI-resistant viruses and 0.4% (1/255) with HIV protease inhibitor (PI)-resistant viruses. Most importantly, almost half (60.0%) of patients carries HIV-1 resistant strains with more than one major drug resistance mutation. In addition, patients who had acquired HIV through homosexual intercourses are more likely to harbour a virus with at least one primary resistance mutation (OR 7.7; 95% CI: 1.7–35.0, P = 0.008). Conclusion The prevalence of drug resistant HIV-1 strains among newly diagnosed individuals in Central Italy is consistent with the data from other European countries. Nevertheless, the presence of drug-resistance HIV-1 mutations in complex patterns highlights an additional potential risk for public health and strongly supports the extension of wide genotyping to newly diagnosed HIV-1 infected patients.

Objectives: To unravel the still unexplored HBV-replicative kinetics in antiHBc-positive/HBsAg-negative people-with-HIV (PWH) suspending TDF/TAF., Methods: 101 antiHBc-positive/HBsAg-negative PWH switching to TDF/TAF-sparing therapy were included. Serum HBV-DNA and HBV-RNA were quantified by droplet-digital-PCR at switching (T0), within 12-months (T1) and 12-24 months post-switch (T2)., Results: At T0, 33.7% had cryptic HBV-DNA (undetected by commercial assays, median[IQR]:2[1-5]IU/ml) and 22% were positive to HBV-RNA alone (median[IQR]:4[3-4]IU/ml), indicating an active HBV-reservoir despite HBsAg-negativity and TDF/TAF-pressure. Notably, antiHBs-titer<100mIU/ml independently correlated with cryptic HBV-DNA at T0 (OR[95%CI]:2.6[1.02-6.5], P=0.04). After TDF/TAF-withdrawal, the rate of PWH achieving HBV-DNA>10IU/ml increased from 12.9% at T1 to 42.6% at T2 (P<0.0001). Likewise, a rise from 2% to 11% was observed for HBV-DNA>100IU/ml (P=0.02); median(IQR) HBV-DNA: 579(425-770)IU/ml. Notably, HBV-DNA>10IU/ml at T2 occurred in 70% of PWH with cryptic HBV-DNA, in 38.5% with HBV-RNA alone and in 25% negative to both HBV-markers at T0 (P=0.01). Cryptic HBV-DNA at T0 and lower nadir CD4+T-cell-count independently predicted HBV-DNA>10IU/ml at T2 (OR[95%CI]:8.2[1.7-40.6], P=0.01; OR[95%CI]:8.1[1.3-52.1], P=0.03). Lastly, persistent HBV-DNA positivity was independently associated with a reduced CD4+T-cell recovery at T2 (OR[95%CI]:0.07[0.01-0.77], P=0.03)., Conclusions: This study underlines the importance to regularly monitor antiHBc-positive/HBsAg-negative PWH undergoing TDF/TAF-sparing regimen and the role of highly-sensitive HBV markers in optimizing their management., (Copyright © 2024. Published by Elsevier Ltd.)

Objectives: The aim of this analysis was to investigate the impact of hepatitis B virus (HBV) coinfection on the risk of HIV viral rebound (VR) after achieving suppression for the first time following initiation of antiretroviral therapy (ART) in the real-world setting., Design: Patients living with HIV (PLWH) who were enrolled in the ICONA Foundation Study cohort and achieved viral suppression ≤50 copies/mL for the first time after starting ART were prospectively evaluated and divided in three exposure groups according to serology test results: (a) HIV-monoinfected; (b) HIV-positive/HBcAb-positive/HBsAg-negative; (c) HIV-positive/HBsAg-positive. The occurrence of VR, defined as two consecutive HIV-RNA values >50 copies/mL after achieving viral suppression for the first time (baseline), was investigated., Methods: Standard survival analysis by means of Kaplan-Meier curves and Cox regression analysis with the serology exposure fitted as a time-fixed covariate measured at baseline was employed after controlling for key confounding factors., Results: Of a total of 5657 patients included, 4090 (72%) were HIV-monoinfected, 1342 (23.7%)were HBcAb-positive, and 225 (3.9%) were HbsAg-positive coinfected. Overall, 654 (11.5%) PLWH experienced VR > 50 copies/mL during follow-up. After controlling for all sources of measured confounding, coinfected PLWH showed an increased risk of experiencing VR compared with those who were HIV-monoinfected. In particular, the strongest associations were seen for the HIV/HBsAg-positive participants [adjusted hazard ratio (aHR) = 1.56, 95% confidence interval (CI): 1.03-2.38, p = 0.037] but an excess of risk was also seen in those who were HIV-positive/HBcAb-positive/HBsAg-negative (aHR = 1.25, 95% CI: 1.00-1.55, p = 0.047)., Conclusions: Coinfection with HBV seems to have an impact on the probability of maintaining HIV viral suppression achieved for the first time after ART initiation. Of note, even PLWH positive for HBcAb, a marker of inactive HBV infection, appeared to be at higher risk of VR compared with those who were HIV-monoinfected and their HIV-RNA should be carefully monitored., (© 2024 British HIV Association.)

More than 80 million people worldwide have been infected with the human immunodeficiency virus (HIV). There are now approximately 39 million individuals living with HIV/acquired immunodeficiency syndrome (AIDS). Although treatments against HIV infection are available, AIDS remains a serious disease. Combination antiretroviral therapy (cART), also known as highly active antiretroviral therapy (HAART), consists of treatment with a combination of several antiretroviral drugs that block multiple stages in the virus replication cycle. However, the increasing usage of cART is inevitably associated with the emergence of HIV drug resistance. In addition, the development of persistent cellular reservoirs of latent HIV is a critical obstacle to viral eradication since viral rebound takes place once anti-retroviral therapy (ART) is interrupted. Thus, several efforts are being applied to new generations of drugs, vaccines and new types of cART. In this review, we summarize the antiviral therapies used for the treatment of HIV/AIDS, both as individual agents and as combination therapies, and highlight the role of both macrophages and HIV cellular reservoirs and the most recent clinical studies related to this disease.

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., (Copyright © 2024 American Association for the Study of Liver Diseases.)

Equine hepacivirus (EqHV, Flaviviridae , hepacivirus) is a small, enveloped RNA virus generally causing sub-clinical hepatitis with occasional fatalities. EqHV is reported in equids worldwide, but for Italy data are limited. To address this, a survey study was set up to estimate prevalence at a national level and among different production categories (equestrian; competition; work and meat; reproduction) and national macro-regions (North, Central, South, and Islands). Data obtained testing 1801 horse serum samples by Real-Time RT PCR were compared within the categories and regions. The NS3 fragment of the PCR-positive samples was sequenced by Sanger protocol for phylogenetic and mutational analysis. The tertiary structure of the NS3 protein was also assessed. The estimated national prevalence was 4.27% [1.97-6.59, 95% CI] and no statistical differences were detected among production categories and macro-regions. The phylogenesis confirmed the distribution in Italy of the three known EqHV subtypes, also suggesting a possible fourth sub-type that, however, requires further confirmation. Mutational profiles that could also affect the NS3 binding affinity to the viral RNA were detected. The present paper demonstrates that EqHV should be included in diagnostic protocols when investigating causes of hepatitis, and in quality control protocols for blood derived products due to its parental transmission.

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., (© 2023 The Authors. Liver International published by John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

Background: We aimed at evaluating the temporal trend of drug-resistance and APOBEC editing from HIV-DNA genotypic resistance tests (GRT) in virologically suppressed individuals., Material and Methods: Major resistance mutations (MRM), genotypic susceptibility score (GSS) for the current regimen and APOBEC-related mutations (APO-M) were evaluated. Potential changes in trends of MRM and APO-M over-time were assessed and predictors of MRM detection or sub-optimal GSS (GSS<2) at HIV-DNA-GRT were estimated through logistic regression analyses., Results: Among the 1126 individuals included, 396 (35.2%) harboured at least one MRM (23.4% to NRTI, 18.8% to NNRTI, 7.7% to PI and 1.4% to INSTI [N=724]); 132 (12.3%) individuals showed a GSS <2. APO-M and stop codons were found in 229 (20.3%) and 105 (9.3%) individuals, respectively. APO-DRMs were found in 16.8% of individuals and were more likely observed in those individuals with stop codons (40.0%) compared to those without (14.4%, P<0.001). From 2010 to 2021 no significant changes of resistance or APO-M were found. Positive predictors of MRM detection at HIV-DNA GRT were drug abuse, subtype B infection, and a prolonged and complex treatment history. Perinatal infection and having at least 2 stop codons were associated with a current suboptimal regimen., Conclusions: In virologically suppressed individuals, resistance in HIV-DNA and the extent of APOBEC editing were generally stable in the last decade. A careful evaluation of APOBEC editing might be helpful to improve the reliability of HIV-DNA GRT. Further investigations are required to understand how to apply the estimation of APOBEC editing in refining genotypic evaluation., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest related to this manuscript., (Copyright © 2023. Published by Elsevier B.V.)

(1) Background: SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is a promising therapeutic target to fight COVID-19, and many RdRp inhibitors nucleotide/nucleoside analogs, such as remdesivir, have been identified or are in clinical studies. However, the appearance of resistant mutations could reduce their efficacy. In the present work, we structurally evaluated the impact of RdRp mutations found at baseline in 39 patients treated with remdesivir and associated with a different degree of antiviral response in vivo. (2) Methods: A refined bioinformatics approach was applied to assign SARS-CoV-2 clade and lineage, and to define RdRp mutational profiles. In line with such a method, the same mutations were built and analyzed by combining docking and thermodynamics evaluations with both molecular dynamics and representative pharmacophore models. (3) Results: Clinical studies revealed that patients bearing the most prevalent triple mutant P323L+671S+M899I, which was present in 41% of patients, or the more complex mutational profile P323L+G671S+L838I+D738Y+K91E, which was found with a prevalence of 2.6%, showed a delayed reduced response to remdesivir, as confirmed by the increase in SARS-CoV-2 viral load and by a reduced theoretical binding affinity versus RdRp (ΔGbind WT = -122.70 kcal/mol; ΔGbind P323L+671S+M899I = -84.78 kcal/mol; ΔGbind P323L+G671S+L838I+D738Y+K91E = -96.74 kcal/mol). Combined computational approaches helped to rationalize such clinical observations, offering a mechanistic understanding of the allosteric effects of mutants on the global motions of the viral RNA synthesis machine and in the changes of the interactions patterns of remdesivir during its binding.

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.

The SARS-CoV-2 main protease (M pro ) is a crucial enzyme for viral replication and has been considered an attractive drug target for the treatment of COVID-19. In this study, virtual screening techniques and in vitro assays were combined to identify novel M pro inhibitors starting from around 8000 FDA-approved drugs. The docking analysis highlighted 17 promising best hits , biologically characterized in terms of their M pro inhibitory activity. Among them, 7 cephalosporins and the oral anticoagulant betrixaban were able to block the enzyme activity in the micromolar range with no cytotoxic effect at the highest concentration tested. After the evaluation of the degree of conservation of M pro residues involved in the binding with the studied ligands, the ligands' activity on SARS-CoV-2 replication was assessed. The ability of betrixaban to affect SARS-CoV-2 replication associated to its antithrombotic effect could pave the way for its possible use in the treatment of hospitalized COVID-19 patients.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Svicher, Salpini, D’Anna, Piermatteo, Iannetta, Malagnino and Sarmati.)

The aim of this study was to evaluate whether the presence of anti-hepatitis B (HBV) c antibodies (HBcAb positivity) could influence the control of HIV viremia in patients living with HIV (PLWH) who switch to two-drug antiretroviral therapy (2DR) containing lamivudine (3TC) (2DR-3TC-based). A retrospective multicentre observational study was conducted on 160 PLWH switching to the 2DR-3TC-based regimen: 51 HBcAb-positive and 109 HBcAb-negative patients. The HBcAb-positive PLWH group demonstrated a significantly lower percentage of subjects with HIV viral suppression with target not detected (TND) at all time points after switching (24th month: 64.7% vs. 87.8%, p < 0.0001; 36th month 62.7% vs. 86.8%, p = 0.011; 48th month 57.2% vs. 86.1%, p = 0.021 of the HBcAb-positive and HBcAb-negative groups, respectively). Logistic regression analysis showed that the presence of HBcAb positivity (OR 7.46 [95% CI 2.35−14.77], p = 0.004) could favour the emergence of HIV viral rebound by nearly 54% during the entire study follow-up after switching to 2DR-3TC.