HIV-1 transmitted drug resistance in newly diagnosed individuals in Italy over the period 2015-21.

BACKGROUND: Transmitted drug resistance (TDR) is still a critical aspect for the management of individuals living with HIV-1. Thus, its evaluation is crucial to optimize HIV care. METHODS: Overall, 2386 HIV-1 protease/reverse transcriptase and 1831 integrase sequences from drug-naïve individuals diagnosed in north and central Italy between 2015 and 2021 were analysed. TDR was evaluated over time. Phylogeny was generated by maximum likelihood. Factors associated with TDR were evaluated by logistic regression. RESULTS: Individuals were mainly male (79.1%) and Italian (56.2%), with a median (IQR) age of 38 (30-48). Non-B infected individuals accounted for 44.6% (N = 1065) of the overall population and increased over time (2015-2021, from 42.1% to 51.0%, P = 0.002). TDR prevalence to any class was 8.0% (B subtype 9.5% versus non-B subtypes 6.1%, P = 0.002) and remained almost constant over time. Overall, 300 transmission clusters (TCs) involving 1155 (48.4%) individuals were identified, with a similar proportion in B and non-infected individuals (49.7% versus 46.8%, P = 0.148). A similar prevalence of TDR among individuals in TCs and those out of TCs was found (8.2% versus 7.8%, P = 0.707).By multivariable analysis, subtypes A, F, and CFR02_AG were negatively associated with TDR. No other factors, including being part of TCs, were significantly associated with TDR. CONCLUSIONS: Between 2015 and 2021, TDR prevalence in Italy was 8% and remained almost stable over time. Resistant strains were found circulating regardless of being in TCs, but less likely in non-B subtypes. These results highlight the importance of a continuous surveillance of newly diagnosed individuals for evidence of TDR to inform clinical practice.

Molecular Genotyping of Circulating Enterovirus in the Lazio Region from 2012 to 2023.

Enteroviruses (EVs) are ubiquitous viruses that circulate worldwide, causing sporadic or epidemic infections, typically during the summer and fall. They cause a broad spectrum of illnesses, ranging from an unspecified febrile clinical presentation to a severe illness. EVs are recognized to be the most frequent etiological agents of aseptic meningitis in children. However, as the infection is usually mild and self-limiting, it remains underestimated, and the epidemiology of EVs is poorly understood. To date, no vaccine or effective therapy for all types of enteroviruses is available, and EVs constitute a public health concern. Here, we investigated the molecular epidemiology of EV strains circulating in the Lazio region over a 10-year time span (2012-2023) by using a sequence-typing approach and phylogenetic analysis. The epidemiological trend of EV infection has undergone changes during the SARS-CoV-2 pandemic (2020-2021), which resulted in a modification in terms of the number of diagnosed cases and seasonality. From 2022, the circulation of EVs showed a behavior typical of the pre-pandemic period, although changes in predominantly circulating strains have been noted. Both epidemic and sporadic circulation events have been characterized in the Lazio region. Further analyses are needed to better characterize any strain with higher potential pathogenic power and to identify possible recombinant strains.

Molnupiravir increases SARS-CoV-2 genome diversity and complexity: A case-control cohort study.

Molnupiravir, an oral direct-acting antiviral effective in vitro against SARS-CoV-2, has been largely employed during the COVID-19 pandemic, since December 2021. After marketing and widespread usage, a progressive increase in SARS-CoV-2 lineages characterized by a higher transition/transversion ratio, a characteristic signature of molnupiravir action, appeared in the Global Initiative on Sharing All Influenza Data (GISAID) and International Nucleotide Sequence Database Collaboration (INSDC) databases. Here, we assessed the drug effects by SARS-CoV-2 whole-genome sequencing on 38 molnupiravir-treated persistently positive COVID-19 outpatients tested before and after treatment. Seventeen tixagevimab/cilgavimab-treated outpatients served as controls. Mutational analyses confirmed that SARS-CoV-2 exhibits an increased transition/transversion ratio seven days after initiation of molnupiravir. Moreover we observed an increased G->A ratio compared to controls, which was not related to apolipoprotein B mRNAediting enzyme, catalytic polypeptide-like (APOBEC) activity. In addition, we demonstrated for the first time an increased diversity and complexity of the viral quasispecies.

Genomic and Epidemiologic Surveillance of SARS-CoV-2 in the Pandemic Period: Sequencing Network of the Lazio Region, Italy.

Since the beginning of the COVID-19 pandemic, large-scale genomic sequencing has immediately pointed out that SARS-CoV-2 has rapidly mutated during the course of the pandemic, resulting in the emergence of variants with a public health impact. In this context, strictly monitoring the circulating strains via NGS has proven to be crucial for the early identification of new emerging variants and the study of the genomic evolution and transmission of SARS-CoV-2. Following national and international guidelines, the Lazio region has created a sequencing laboratory network (WGSnet-Lazio) that works in synergy with the reference center for epidemiological surveillance (SERESMI) to monitor the circulation of SARS-CoV-2. Sequencing was carried out with the aims of characterizing outbreak transmission dynamics, performing the genomic analysis of viruses infecting specific categories of patients (i.e., immune-depressed, travelers, and people with severe symptoms) and randomly monitoring variant circulation. Here we report data emerging from sequencing activities carried out by WGSnet-Lazio (from February 2020 to October 2022) linked with epidemiological data to correlate the circulation of variants with the clinical and demographic characteristics of patients. The model of the sequencing network developed in the Lazio region proved to be a useful tool for SARS-CoV-2 surveillance and to support public health measures for epidemic containment.

Treatment-Emergent Cilgavimab Resistance Was Uncommon in Vaccinated Omicron BA.4/5 Outpatients.

Mutations in the SARS-CoV-2 Spike glycoprotein can affect monoclonal antibody efficacy. Recent findings report the occurrence of resistant mutations in immunocompromised patients after tixagevimab/cilgavimab treatment. More recently, the Food and Drug Agency revoked the authorization for tixagevimab/cilgavimab, while this monoclonal antibody cocktail is currently recommended by the European Medical Agency. We retrospectively reviewed 22 immunocompetent patients at high risk for disease progression who received intramuscular tixagevimab/cilgavimab as early COVID-19 treatment and presented a prolonged high viral load. Complete SARS-CoV-2 genome sequences were obtained for a deep investigation of mutation frequencies in Spike protein before and during treatment. At seven days, only one patient showed evidence of treatment-emergent cilgavimab resistance. Quasispecies analysis revealed two different deletions on the Spike protein (S:del138-144 or S:del141-145) in combination with the resistance S:K444N mutation. The structural and dynamic impact of the two quasispecies was characterized by using molecular dynamics simulations, showing the conservation of the principal functional movements in the mutated systems and their capabilities to alter the structure and dynamics of the RBD, responsible for the interaction with the ACE2 human receptor. Our study underlines the importance of prompting an early virological investigation to prevent drug resistance or clinical failures in immunocompetent patients.

Virological characterization of HIV-1 RNA elements detected exclusively through the LTR region by the dual-target Aptima HIV-1 Quant Dx assay in a subset of positive patients.

BACKGROUND: In a restricted subset of HIV patients with suppressed viral load (i.e., pol-undetected HIV-RNA), the Aptima HIV-1 Quant Dx Assay (Aptima), a dual-target (pol and LTR) and dual-probe test for viral load (VL) monitoring, can detect HIV-RNA exclusively through amplification of the LTR region. OBJECTIVES: To analyze the virological characteristics of the HIV-RNA elements detected only through LTR amplification (LTR-e). STUDY DESIGN: LTR-e isolated from plasma and peripheral blood mononuclear cells (PBMC) were evaluated for their ability to trigger productive infections. Viral pellets morphology and ultrastructural characteristics of PBMC from LTR-e patients were examined by electron microscopy. Plasma LTR-e underwent Sanger sequencing. Exosomes were examined with Aptima for LTR-e content. RESULTS: In-vitro, LTR-e could not infect PBMC, induce cytopathic effects, or cause syncytia, even at high VL (e.g., >10,000 copies/mL). Under the electron microscope, plasma pellets and PBMC from patients with LTR-e showed atypical vesicles. Sanger sequencing of LTR-e yielded no results. Moreover, in plasma samples, LTR-e were associated with cell debris, never with exosomes. CONCLUSIONS: Differently from other dual-target but single-probe assays, Aptima unveils VL based only on LTR amplification in some HIV patients. Here, we show that LTR-e represent partial/incomplete/non-canonical transcripts unable to trigger productive infection or transmit HIV-1 infection. The recognition of VL based only on LTR-e in infected individuals is crucial as it allows to avoid inappropriate decisions in the clinical management of HIV patients, such as retesting of VL and switching of ART. Physicians and HIV-RNA dual-target assay manufacturers should consider the important implications of not recognizing this singular type of VL.

Non-B subtypes account for a large proportion of clustered primary HIV-1 infections in Italy.

OBJECTIVES AND DESIGN: Using pol sequences obtained for routine resistance testing, we characterised the molecular patterns of HIV-1 transmission and factors associated with being part of a transmission cluster among individuals who in 2008-2014 presented with primary HIV-1 infection (PHI) at 11 urban centres across Italy. METHODS: Pol sequences were obtained by Sanger sequencing. Transmission clusters were identified by phylogenetic analysis (maximum likelihood method, confirmed by Bayesian analysis). Multivariable logistic regression explored factors associated with a participant being part of a transmission cluster. RESULTS: The PHI cohort comprised 186 participants (159/186, 85.5% males) with median age 44 years, median CD4 count 464 cells/mm3 and median plasma HIV-1 RNA 5.6 log10 copies/mL. Drug resistance associated mutations were found in 16/186 (8.6%). A diversity of non-B subtypes accounted for 60/186 (32.3%) of all infections. A total of 17 transmission clusters were identified, including 44/186 (23.7%) participants. Each cluster comprised 2-6 sequences. Non-B subtypes accounted for seven clusters and 22/44 (50%) of clustered sequences. In multivariable logistic regression analysis, factors associated with being part of a transmission cluster comprised harbouring a non-B subtype (adjusted OR (adjOR) 2.28; 95% CI 1.03 to 5.05; p=0.04) and showing a lower plasma HIV-1 RNA (adjOR 0.80, 95% CI 0.64 to 0.99; p=0.04). CONCLUSIONS: There was a large contribution of diverse non-B subtypes to transmission clusters among people presenting with acute or recent HIV-1 infection in this cohort, illustrating the evolving dynamics of the HIV-1 epidemic in Italy, where subtype B previously dominated.

Intact provirus and integration sites analysis in acute HIV-1 infection and changes after one year of early antiviral therapy.

Background and objectives: HIV-1 provirus integration in host genomes provides a lifelong reservoir of virally infected cells. Although not able to generate viral progeny, the expression of defective proviruses has been associated with activation. Provirus integration may influence host gene transcription and shifts may occur during disease progression or antiretroviral therapy (ART). The study aimed to analyze intact/defective provirus and sites of provirus integration in acute infections: changes after 48 weeks of early therapy were also evaluated. Methods: DNA from peripheral blood lymphomonocytes of 8 acute HIV-1 infections at serodiagnosis (T0) and after 48 weeks of therapy (T1) was used to quantify intact and defective provirus by digital-droplet PCR and to analyze provirus integration sites, by next-generation sequencing of libraries derived from ligation-mediated PCR. Results: A high variability in the amount of intact proviral DNA was observed at both T0 and T1, in the different subjects. Although the ratio of intact/total proviral HIV-1 DNA did not dramatically change between T0 (8.05%) and T1 (9.34%), after early therapy both intact and total HIV-1 DNA declined significantly, p = 0.047 and p = 0.008, respectively. The median number of different (IQR) integration sites in human chromosomes/subject was 5 (2.25-13.00) at T0 and 4 (3.00-6.75) at T1. Of all the integration sites observed at T1, 64% were already present at T0. Provirus integration was observed in introns of transcriptionally active genes. Some sites of integration, among which the most represented was in the neuregulin 2 gene, were shared by different patients, together with the orientation of the insertion. Provirus integration was also observed in intergenic regions, with median (IQR) % of 15.13 (6.81-21.40) at T0 and 18.46 (8.98-22.18) at T1 of all read matches. Conclusions: In acute HIV-1 infection, the amount of intact proviral DNA in peripheral lymphomonocytes did not exceed 10% of total HIV-1 DNA, a percentage that was not substantially changed by early administrated ART. Provirus displayed a relatively small number of recurrent integration sites in introns of transcriptionally active genes, mainly related to cell-cycle control. Consideration should be given to therapeutic strategies able to target the cells harboring defective proviruses, that are not reached by conventional antiviral drugs, these potentially also impacting on replicative competent integrated provirus.

SARS-CoV-2 Variants Identification: Overview of Molecular Existing Methods.

Since the beginning of COVID-19 pandemic the Real Time sharing of genome sequences of circulating virus supported the diagnostics and surveillance of SARS-CoV-2 and its transmission dynamics. SARS-CoV-2 straightaway showed its tendency to mutate and adapt to the host, culminating in the emergence of variants; so it immediately became of crucial importance to be able to detect them quickly but also to be able to monitor in depth the changes on the whole genome to early identify the new possibly emerging variants. In this scenario, this manuscript aims to provide an overview of the existing methods for the identification of SARS-CoV-2 variants (from rapid method based on identification of one or more specific mutations to Whole Genome sequencing approach-WGS), taking into account limitations, advantages and applications of them in the field of diagnosis and surveillance of SARS-CoV-2.

Bictegravir/emtricitabine/tenofovir alafenamide ensures high rates of virological suppression maintenance despite previous resistance in PLWH who optimize treatment in clinical practice.

OBJECTIVES: We evaluated virological response and resistance profiles in individuals who were virologically suppressed who switched to bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) in real life. METHODS: Survival analysis was used to assess probability of virological rebound (VR). Cumulative major resistance mutations (MRM) and cumulative genotypic susceptibility score (cGSS) were evaluated before the switch. RESULTS: Overall, 283 individuals virologically suppressed for a median (interquartile [IQR]) time of 7 (3-9) y were analyzed. Of these, 20.8% were in first-line treatment, 13.1% were highly treatment-experienced (HTE), and 8.5% had experienced previous integrase inhibitor (INI)-failures. Before the switch, nucleotide reverse transcriptase inhibitor NRTI MRM prevalence was 29% (M184V:13.8%; any thymidine analogue mutation: 14.1%; K65R: 0.7%; K70E 0.4%); only three (2.1%) individuals showed INI major resistance mutations (Y143C/H/R [n = 1]; Y143C [n = 1]; N155H [n = 1]), and 82.0% of individuals received fully active B/F/TAF. Ninety-six wk after switch, the probability of VR was 5%, with only 12 events of VR at a median (IQR) viremia level of 284 (187-980) copies/mL, mainly transient. No significant associations between virological outcomes and genotypic susceptibility to B/F/TAF were observed. People who experienced previous INI failures showed a significantly higher adjusted hazard ratio (AHR [95% CI]) to experience VR under B/F/TAF (3.9 [1.1-13.4], P = 0.031). This AHR increased in people who experienced INI failures and received partially active B/F/TAF (5.5 [1.4-21.1], P = 0.013). CONCLUSION: Within 96 wk, a switch to B/F/TAF in individuals who were virologically suppressed ensured a very high rate of virological control in a clinical setting. Previous resistance alone did not affect B/F/TAF response. However, people who had previous INI failures were more prone to losing virological control under B/F/TAF.

Rapid and qualitative identification of SARS-CoV-2 mutations associated with variants of concern using a multiplex RT-PCR assay coupled with melting analysis.

OBJECTIVES: Considering the spread of new genetic variants and their impact on public health, it is important to have assays that are able to rapidly detect SARS-CoV-2 variants. METHODS: We retrospectively examined 118 positive nasopharyngeal swabs, first characterized by the Sanger sequencing, using the Simplexa® SARS-CoV-2 Variants Direct assay, with the aim of evaluating the performance of the assay to detect N501Y, G496S, Q498R, Y505H, E484K, E484Q, E484A, and L452R mutations. RESULTS: A total of 111/118 nasopharyngeal swabs were in complete agreement with the Sanger sequencing, whereas the remaining seven samples were not amplified due to the low viral load. The evaluation of the ability of the assay to detect the E484Q mutation was performed using a viral isolate of the SARS-CoV-2 Kappa variant, showing concordance in 15/15 samples. Simplexa® SARS-CoV-2 Variant Direct assay was able to detect mutation pattern of Alpha, Beta, Gamma, Delta, and Omicron variants with 100% specificity and 94% sensitivity, whereas 100% sensitivity and specificity for the Kappa variant was observed. CONCLUSION: The assay can be useful to obtain faster results, contributing to a prompt surveillance of SARS-CoV-2 variants; however, it requires to be confirmed by the Sanger method, especially in the case of pattern of mutations that are different from those expected and also requires updates as new variants emerge.

Analysis of HIV quasispecies and virological outcome of an HIV D+/R+ kidney-liver transplantation.

INTRODUCTION: Transplantation among HIV positive patients may be a valuable therapeutic intervention. This study involves an HIV D+/R+ kidney-liver transplantation, where PBMC-associated HIV quasispecies were analyzed in donor and transplant recipients (TR) prior to transplantation and thereafter, together with standard viral monitoring. METHODS: The donor was a 54 year of age HIV infected woman: kidney and liver recipients were two HIV infected men, aged 49 and 61. HIV quasispecies in PBMC was analyzed by ultra-deep sequencing of V3 env region. During TR follow-up, plasma HIV-1 RNA, HIV-1 DNA in PBMC, analysis of proviral integration sites and drug-resistance genotyping were performed. Other virological and immunological monitoring included CMV and EBV DNA quantification in blood and CD4 T cell counts. RESULTS: Donor and TR were all ART-HIV suppressed at transplantation. Thereafter, TR maintained a nearly suppressed HIV-1 viremia, but HIV-1 RNA blips and the increase of proviral integration sites in PBMC attested some residual HIV replication. A transient peak in HIV-1 DNA occurred in the liver recipient. No major changes of drug-resistance genotype were detected after transplantation. CMV and EBV transient reactivations were observed only in the kidney recipient, but did not require specific treatment. CD4 counts remained stable. No intermixed quasispecies between donor and TR was observed at transplantation or thereafter. Despite signs of viral evolution in TR, HIV genetic heterogeneity did not increase over the course of the months of follow up. CONCLUSIONS: No evidence of HIV superinfection was observed in the donor nor in the recipients. The immunosuppressive treatment administrated to TR did not result in clinical relevant viral reactivations.

Virological response and resistance profile in highly treatment-experienced HIV-1-infected patients switching to dolutegravir plus boosted darunavir in clinical practice.

OBJECTIVES: We evaluated the virological response and resistance profile in combined antiretroviral therapy (cART)-experienced HIV-1-infected patients starting a dual therapy with dolutegravir (DTG) and boosted darunavir (bDRV) for the first time. METHODS: Survival analyses were used to evaluate virological success (VS) and virological rebound (VR) in viraemic and virologically suppressed patients, respectively. Major resistance mutations (MRMs) and genotypic susceptibility score (GSS) were evaluated at baseline and after switch. RESULTS: Overall, 130 patients [62 (47.7%) viraemic; 68 (52.3%) virologically suppressed] were retrospectively analysed. At the moment of switch, 81.5% accumulated one or more MRM [protease inhibitor (PI), 35.7%; nucleoside(t)ide reverse transcriptase inhibitor (NRTI), 77.5%; non-NRTI, 69.0%; integrase inhibitor (INI), 10.1%), but 77.7% harboured strains fully susceptible to DTG + bDRV. In viraemic patients, the overall probability of VS by 12 months of treatment was 91.7%. In virologically suppressed patients, the overall probability of VR was 10.5% by 24 months after therapy start. Patients with previous time under virological suppression ≤ 6 months showed a higher VR probability compared with others (37.5% vs. 6.7%, P < 0.002). Among 13 non-responding patients for whom a genotypic resistance test result at failure was available, only two (15.4%) accumulated further resistance in integrase (Y143C/H/R; S147G and N155H) and protease (V32I, L33F, I54L). CONCLUSIONS: In highly treatment-experienced patients, the use of dual therapy based on DTG + bDRV appears to be a very good regimen for switch therapy, with a high rate of virological control in both viraemic and virologically suppressed patients. Among non-responding patients, the selection of further resistance is a rare event.

Molecular Transmission Dynamics of Primary HIV Infections in Lazio Region, Years 2013-2020.

Molecular investigation of primary HIV infections (PHI) is crucial to describe current dynamics of HIV transmission. Aim of the study was to investigate HIV transmission clusters (TC) in PHI referred during the years 2013-2020 to the National Institute for Infectious Diseases in Rome (INMI), that is the Lazio regional AIDS reference centre, and factors possibly associated with inclusion in TC. These were identified by phylogenetic analysis, based on population sequencing of pol; a more in depth analysis was performed on TC of B subtype, using ultra-deep sequencing (UDS) of env. Of 270 patients diagnosed with PHI during the study period, 229 were enrolled (median follow-up 168 (IQR 96-232) weeks). Median age: 39 (IQR 32-48) years; 94.8% males, 86.5% Italians, 83.4% MSM, 56.8% carrying HIV-1 subtype B. Of them, 92.6% started early treatment within a median of 4 (IQR 2-7) days after diagnosis; median time to sustained suppression was 20 (IQR 8-32) weeks. Twenty TC (median size 3, range 2-9 individuals), including 68 patients, were identified. A diagnosis prior to 2015 was the unique factor associated with inclusion in a TC. Added value of UDS was the identification of shared quasispecies components in transmission pairs within TC.

Virological and Serological Characterisation of SARS-CoV-2 Infections Diagnosed After mRNA BNT162b2 Vaccination Between December 2020 and March 2021.

BACKGROUND: Vaccines for coronavirus disease 2019 (COVID-19) are proving to be very effective in preventing severe illness; however, although rare, post-vaccine infections have been reported. The present study focuses on virological and serological features of 94 infections that occurred in Lazio Region (Central Italy) between 27 December 2020, and 30 March 2021, after one or two doses of mRNA BNT162b2 vaccine. METHODS: We evaluated clinical features, virological (viral load; viral infectiousness; genomic characterisation), and serological (anti-nucleoprotein Ig; anti-Spike RBD IgG; neutralising antibodies, nAb) characteristics of 94 post-vaccine infections at the time of diagnosis. Nasopharyngeal swabs (NPSs) and serum samples were collected in the framework of the surveillance activities on SARS-CoV-2 variants established in Lazio Region (Central Italy) and analysed at the National Institute for Infectious Diseases "L. Spallanzani" in Rome. RESULTS: The majority (92.6%) of the post-vaccine infections showed pauci/asymptomatic or mild clinical course, with symptoms and hospitalisation rate significantly less frequent in patients infected after full vaccination course as compared to patients who received a single dose vaccine. Although differences were not statistically significant, viral loads and isolation rates were lower in NPSs from patients infected after receiving two vaccine doses as compared to patients with one dose. Most cases (84%) had nAb in serum at the time of infection diagnosis, which is a sub-group of vaccinees, were found similarly able to neutralise Alpha and Gamma variants. Asymptomatic individuals showed higher nAb titres as compared to symptomatic cases (median titre: 1:120 vs. 1:40, respectively). Finally, the proportion of post-vaccine infections attributed either to Alpha and Gamma variants was similar to the proportion observed in the contemporary unvaccinated population in the Lazio region, and mutational analysis did not reveal enrichment of a defined set of Spike protein substitutions depending on the vaccination status. CONCLUSION: Our study conducted using real-life data, emphasised the importance of monitoring vaccine breakthrough infections, through the characterisation of virological, immunological, and clinical features associated with these events, in order to tune prevention measures in the next phase of the COVID-19 pandemic.

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.

Identification of gp120 polymorphisms in HIV-1 B subtype potentially associated with resistance to fostemsavir.

OBJECTIVES: We evaluated natural resistance to the new antiretroviral fostemsavir and its potential association with other HIV-1 gp120 polymorphisms. METHODS: A total of 1997 HIV-1 B subtype gp120 sequences from the Los Alamos HIV Database were analysed for mutation prevalence at fostemsavir resistance-associated positions and potential association with other gp120 polymorphisms. The role of each fostemsavir resistance-related position and the correlated gp120 mutations, both in protein stability and in reducing the binding affinity between antibody and/or T cell lymphocyte epitopes and the MHC molecules, was estimated. RESULTS: The prevalence of fostemsavir resistance mutations was as follows: L116Q (0.05%), S375H/M/T (0.55%/1.35%/17.73%, the latter being far less relevant in determining resistance), M426L (7.56%), M434I (4.21%) and M475I (1.65%). Additionally, the M426R polymorphism had a prevalence of 16.32%. A significantly higher prevalence in X4 viruses versus R5 viruses was found only for S375M (0.69% versus 3.93%, P = 0.009) and S375T (16.60% versus 22.11%, P = 0.030). Some fostemsavirv resistance positions positively and significantly correlated with specific gp120 polymorphisms: S375T with I371V; S375M with L134W, I154V and I323T; M475I with K322A; and M426R with G167N, K192T and S195N. The topology of the dendrogram suggested the existence of three distinct clusters (bootstrap ≥0.98) involving these fostemsavir resistance mutations and gp120 polymorphisms. Interestingly, all clustered mutations are localized in class I/II-restricted T cell/antibody epitopes, suggesting a potential role in immune HIV escape. CONCLUSIONS: A low prevalence of known fostemsavir resistance mutations was found in the HIV-1 B subtype. The detection of novel HIV-1 gp120 polymorphisms potentially relevant for fostemsavir resistance deserves new in-depth in vitro investigations.

Characterisation of HIV-1 molecular transmission clusters among newly diagnosed individuals infected with non-B subtypes in Italy.

OBJECTIVE: We evaluated the characteristics of HIV-1 molecular transmission clusters (MTCs) in 1890 newly diagnosed individuals infected with non-B subtypes between 2005 and 2017 in Italy. METHODS: Phylogenetic analyses were performed on pol sequences to characterise subtypes/circulating recombinant forms and identify MTCs. MTCs were divided into small (SMTCs, 2-3 sequences), medium (MMTCs, 4-9 sequences) and large (LMTCs, ≥10 sequences). Factors associated with MTCs were evaluated using logistic regression analysis. RESULTS: 145 MTCs were identified and involved 666 individuals (35.2%); 319 of them (16.9%) were included in 13 LMTCs, 111 (5.9%) in 20 MMTCs and 236 (12.5%) in 112 SMTCs. Compared with individuals out of MTCs, individuals involved in MTCs were prevalently Italian (72.7% vs 30.9%, p<0.001), male (82.9% vs 62.3%, p<0.001) and men who have sex with men (MSM) (43.5% vs 14.5%, p<0.001). Individuals in MTCs were also younger (median (IQR) years: 41 (35-49) vs 43 (36-51), p<0.001) and had higher CD4 cell count in comparison with individuals out of MTCs (median (IQR): 109/L: 0.4 (0.265-0.587) vs 0.246 (0.082-0.417), p<0.001). The viral load remained stable between the two groups (median (IQR) log10 copies/mL: 4.8 (4.2-5.5) vs 5.0 (4.3-5.5), p=0.87). Logistic regression confirmed that certain factors such as being MSM, of Italian origin, younger age and higher CD4 cell count were significantly associated with MTCs. CONCLUSIONS: Our findings show that HIV-1 newly diagnosed individuals infected with non-B subtypes are involved in several MTCs in Italy. These MTCs include mainly Italians and MSM and highlight the complex phenomenon characterising the HIV-1 spread. This is important especially in view of monitoring the HIV epidemic and guiding the public health response.

Genetic divergence of HIV-1 B subtype in Italy over the years 2003-2016 and impact on CTL escape prevalence.

HIV-1 is characterized by high genetic variability, with implications for spread, and immune-escape selection. Here, the genetic modification of HIV-1 B subtype over time was evaluated on 3,328 pol and 1,152 V3 sequences belonging to B subtype and collected from individuals diagnosed in Italy between 2003 and 2016. Sequences were analyzed for genetic-distance from consensus-B (Tajima-Nei), non-synonymous and synonymous rates (dN and dS), CTL escapes, and intra-host evolution over four time-spans (2003-2006, 2007-2009, 2010-2012, 2013-2016). Genetic-distance increased over time for both pol and V3 sequences (P < 0.0001 and 0.0003). Similar results were obtained for dN and dS. Entropy-value significantly increased at 16 pol and two V3 amino acid positions. Seven of them were CTL escape positions (protease: 71; reverse-transcriptase: 35, 162, 177, 202, 207, 211). Sequences with ≥3 CTL escapes increased from 36.1% in 2003-2006 to 54.0% in 2013-2016 (P < 0.0001), and showed better intra-host adaptation than those containing ≤2 CTL escapes (intra-host evolution: 3.0 × 10-3 [2.9 × 10-3-3.1 × 10-3] vs. 4.3 × 10-3 [4.0 × 10-3-5.0 × 10-3], P[LRT] < 0.0001[21.09]). These data provide evidence of still ongoing modifications, involving CTL escape mutations, in circulating HIV-1 B subtype in Italy. These modifications might affect the process of HIV-1 adaptation to the host, as suggested by the slow intra-host evolution characterizing viruses with a high number of CTL escapes.