Role of Direct Sexual Contact in Human Transmission of Monkeypox Virus, Italy.

The 2022 global mpox outbreak was driven by human-to-human transmission, but modes of transmission by sexual relationship versus sexual contact remain unclear. We evaluated sexual transmission of mpox by using monkeypox virus (MPXV) G2R-mRNA as a marker of ongoing viral replication through in vitro experiments. We analyzed clinical samples of 15 MPXV-positive patients in Italy from different biological regions by using the setup method. The presence of MPXV DNA, MPXV G2R-mRNA, or both in all analyzed lesion swab samples, independent of viral load, confirmed a higher infectivity risk from skin lesions. Positivity for MPXV G2R-mRNA in nasopharyngeal swabs was associated with high MPXV load, whereas positive results for MPXV G2R-mRNA were obtained only in the 2 semen samples with the lowest MPXV loads. Our results suggest that close or skin-to-skin contact during sexual intercourse is the main route of sexual transmission and that semen is a minor driver of infection, regardless of MPXV load.

Rapid Determination of SARS-CoV-2 Integrity and Infectivity by Using Propidium Monoazide Coupled with Digital Droplet PCR.

SARS-CoV-2 is a highly infectious virus responsible for the COVID-19 pandemic. Therefore, it is important to assess the risk of SARS-CoV-2 infection, especially in persistently positive patients. Rapid discrimination between infectious and non-infectious viruses aids in determining whether prevention, control, and treatment measures are necessary. For this purpose, a method was developed and utilized involving a pre-treatment with 50 µM of propidium monoazide (PMAxx, a DNA intercalant) combined with a digital droplet PCR (ddPCR). The ddPCR method was performed on 40 nasopharyngeal swabs (NPSs) both before and after treatment with PMAxx, revealing a reduction in the viral load at a mean of 0.9 Log copies/mL (SD ± 0.6 Log copies/mL). Furthermore, six samples were stratified based on the Ct values of SARS-CoV-2 RNA (Ct < 20, 20 < Ct < 30, Ct > 30) and analyzed to compare the results obtained via a ddPCR with viral isolation and a negative-chain PCR. Of the five samples found positive via a ddPCR after the PMAxx treatment, two of the samples showed the highest post-treatment SARS-CoV-2 loads. The virus was isolated in vitro from both samples and the negative strand chains were detected. In three NPS samples, SARS CoV-2 was present post-treatment at a low level; it was not isolated in vitro, and, when detected, the strand was negative. Our results indicate that the established method is useful for determining whether the SARS-CoV-2 within positive NPS samples is intact and capable of causing infection.

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.

Results of an interventional HIV testing programme in the context of a mpox (formerly monkeypox) vaccination campaign in Latium Region, Italy, August to October 2022.

HIV testing was offered to 2,185 people receiving mpox (formerly monkeypox) vaccination, who reported not being HIV positive. Among them 390 were current PrEP users, and 131 had taken PrEP in the past. Of 958 individuals consenting testing, six were newly diagnosed with HIV. Two patients had symptomatic primary HIV infection. None of the six patients had ever taken PrEP. Mpox vaccination represents an important opportunity for HIV testing and counselling about risk reduction and PrEP.

Multi-omics approach to COVID-19: a domain-based literature review.

BACKGROUND: Omics data, driven by rapid advances in laboratory techniques, have been generated very quickly during the COVID-19 pandemic. Our aim is to use omics data to highlight the involvement of specific pathways, as well as that of cell types and organs, in the pathophysiology of COVID-19, and to highlight their links with clinical phenotypes of SARS-CoV-2 infection. METHODS: The analysis was based on the domain model, where for domain it is intended a conceptual repository, useful to summarize multiple biological pathways involved at different levels. The relevant domains considered in the analysis were: virus, pathways and phenotypes. An interdisciplinary expert working group was defined for each domain, to carry out an independent literature scoping review. RESULTS: The analysis revealed that dysregulated pathways of innate immune responses, (i.e., complement activation, inflammatory responses, neutrophil activation and degranulation, platelet degranulation) can affect COVID-19 progression and outcomes. These results are consistent with several clinical studies. CONCLUSIONS: Multi-omics approach may help to further investigate unknown aspects of the disease. However, the disease mechanisms are too complex to be explained by a single molecular signature and it is necessary to consider an integrated approach to identify hallmarks of severity.

Role of HIV-1 matrix protein p17 variants in lymphoma pathogenesis.

Although in decline after successful anti-HIV therapy, B-cell lymphomas are still elevated in HIV-1-seropositive (HIV+) persons, and the mechanisms are obscure. The HIV-1 matrix protein p17 persists in germinal centers long after HIV-1 drug suppression, and some p17 variants (vp17s) activate Akt signaling and promote growth of transformed B cells. Here we show that vp17s derived from four of five non-Hodgkin lymphoma (NHL) tissues from HIV+ subjects display potent B-cell growth-promoting activity. They are characterized by amino acid insertions at position 117-118 (Ala-Ala) or 125-126 (Gly-Asn or Gly-Gln-Ala-Asn-Gln-Asn) among some other mutations throughout the sequence. Identical dominant vp17s are found in both tumor and plasma. Three of seven plasma samples from an independent set of NHL cases manifested multiple Ala insertions at position 117-118, and one with the Ala-Ala profile also promoted B-cell growth and activated Akt signaling. Ultradeep pyrosequencing showed that vp17s with C-terminal insertions are more frequently detected in plasma of HIV+ subjects with than without NHL. Insertion of Ala-Ala at position 117-118 into reference p17 (refp17) was sufficient to confer B-cell growth-promoting activity. In contrast, refp17 bearing the Gly-Asn insertion at position 125-126 did not, suggesting that mutations not restricted to the C terminus can also account for this activity. Biophysical analysis revealed that the Ala-Ala insertion mutant is destabilized compared with refp17, whereas the Gly-Asn form is stabilized. This finding provides an avenue for further exploration of structure function relationships and new treatment strategies in combating HIV-1-related NHL.

Iron overload down-regulates the expression of the HIV-1 Rev cofactor eIF5A in infected T lymphocytes.

BACKGROUND: Changes in iron metabolism frequently accompany HIV-1 infection. However, while many clinical and in vitro studies report iron overload exacerbates the development of infection, many others have found no correlation. Therefore, the multi-faceted role of iron in HIV-1 infection remains enigmatic. METHODS: RT-qPCR targeting the LTR region, gag, Tat and Rev were performed to measure the levels of viral RNAs in response to iron overload. Spike-in SILAC proteomics comparing i) iron-treated, ii) HIV-1-infected and iii) HIV-1-infected/iron treated T lymphocytes was performed to define modifications in the host cell proteome. Data from quantitative proteomics were integrated with the HIV-1 Human Interaction Database for assessing any viral cofactors modulated by iron overload in infected T lymphocytes. RESULTS: Here, we demonstrate that the iron overload down-regulates HIV-1 gene expression by decreasing the levels of viral RNAs. In addition, we found that iron overload modulates the expression of many viral cofactors. Among them, the downregulation of the REV cofactor eIF5A may correlate with the iron-induced inhibition of HIV-1 gene expression. Therefore, we demonstrated that eiF5A downregulation by shRNA resulted in a significant decrease of Nef levels, thus hampering HIV-1 replication. CONCLUSIONS: Our study indicates that HIV-1 cofactors influenced by iron metabolism represent potential targets for antiretroviral therapy and suggests eIF5A as a selective target for drug development.

Slow or fast viral load decay as a predictor of residual viremia level in HIV-infected patients undergoing successful first-line cART.

Persistent residual viremia (RV) has been demonstrated in 70-90% of patients under successful cART. We analyzed the RV trend during the first year following cART-induced virological suppression (VS; HIVRNA <50 copies/ml) to identify predictors of achievement and maintenance of ultra-deep RV suppression (URVS; HIV-RNA <5 copies/ml) in 60 naïve patients. These patients were aligned at the time of reaching VS and were longitudinally tested with an ultrasensitive HIV-RNA assay. The influence of demographics, primary/chronic infection, pre-therapy HIV-RNA and CD4, cART regimen and time to reach VS on RV trends was evaluated. During the first year following VS, median RV levels steadily decreased. RV dropped below 5 copies/ml at least once in each patient, but URVS was maintained in 45% of patients. RV rebounded to levels fluctuating around 5-10 copies/ml while in the remaining 55% of patients. Predictors of early achievement and maintenance of stable URVS were fast (<12 weeks) VS achievement after the start of therapy, better pre-treatment viro-immunological conditions (lower viremia and higher CD4 before cART), and treatment initiation during primary infection. These findings emphasize the importance of an early onset of potent antiretroviral regimens. RV trends should be further studied in detail in the following years of cART.

In-depth analysis of compartmentalization of HIV-1 matrix protein p17 in PBMC and plasma.

HIV-1 p17 plays an important role in the virus life-cycle and disease pathogenesis. Recent studies indicated a high heterogeneity of p17. A high number of insertions in the p17 carboxy-terminal region have been more frequently detected in patients with non-Hodgkin lymphoma (NHL), suggesting a role of altered p17 in lymphomagenesis. Based on p17 heterogeneity, possible PBMC/plasma compartmentalization of p17 variants was explored by ultra-deep pyrosequencing in five NHL patients. The high variability of p17 with insertions at the carboxy-terminal region was confirmed in plasma and observed for the first time in proviral genomes. Quasispecies compartmentalization was evident in 4/5 patients. Further studies are needed to define the possible role of p17 quasispecies compartmentalization in lymphomagenesis.

Longitudinal analysis of HIV-1 coreceptor tropism by single and triplicate HIV-1 RNA and DNA sequencing in patients undergoing successful first-line antiretroviral therapy.

OBJECTIVES: Maraviroc has been shown to be effective in patients harbouring CCR5-tropic HIV-1. While this CCR5 antagonist has initially been used in salvage therapy, its excellent safety profile makes it ideal for antiretroviral treatment simplification strategies in patients with suppressed plasma viraemia. The aim of this study was to compare HIV-1 tropism as detected in baseline plasma RNA and peripheral blood mononuclear cell (PBMC) DNA prior to first-line therapy and to analyse tropism evolution while on successful treatment. METHODS: HIV-1 tropism was determined using triplicate genotypic testing combined with geno2pheno[coreceptor] analysis at a 10% false positive rate in 42 patients. Paired pre-treatment plasma RNA and PBMC DNA and two subsequent PBMC DNA samples (the first obtained after reaching undetectable plasma HIV-1 RNA and the second after at least 2 years of suppression of plasma viraemia) were evaluated. RESULTS: Coreceptor tropism was completely concordant in paired pre-treatment RNA and DNA, with 26.2% of HIV-1 sequences predicted to be non-CCR5-tropic. During follow-up, coreceptor tropism switches were detected in 4 (9.5%) patients without any preferential direction. Although false positive rate discrepancies within triplicates were common, the rate of discordance of coreceptor tropism assignment among triplicate results in this mostly CCR5-tropic dataset was only 2.1%, questioning the added value of triplicate testing compared with single testing. CONCLUSIONS: HIV-1 coreceptor tropism changes during virologically successful first-line treatment are infrequent. HIV-1 DNA analysis may thus support the choice of a CCR5 antagonist in treatment switch strategies; however, maraviroc treatment outcome data are required to confirm this option.

Evolution of HIV-1 tropism at quasispecies level after 5 years of combination antiretroviral therapy in patients always suppressed or experiencing episodes of virological failure.

OBJECTIVES: Tropism evolution of HIV-1 quasispecies was analysed by ultra-deep pyrosequencing (UDPS) in patients on first-line combination antiretroviral therapy (cART) always suppressed or experiencing virological failure episodes. METHODS: Among ICONA patients, two groups of 20 patients on cART for ≥5 years, matched for baseline viraemia and therapy duration, were analysed [Group I, patients always suppressed; and Group II, patients experiencing episode(s) of virological failure]. Viral tropism was assessed by V3 UDPS on plasma RNA before therapy (T0) and on peripheral blood mononuclear cell proviral DNA before-after therapy (T0-T1), using geno2pheno false positive rate (FPR) (threshold for X4: 5.75). For each sample, quasispecies tropism was assigned according to X4 variant frequency: R5, <0.3% X4; minority X4, 0.3%-19.9% X4; and X4, ≥20% X4. An R5-X4 switch was defined as a change from R5/minority X4 in plasma/proviral genomes at T0 to X4 in provirus at T1. RESULTS: At baseline, mean FPR and %X4 of viral RNA were positively correlated with those of proviral DNA. After therapy, proviral DNA load significantly decreased in Group I; mean FPR of proviral quasispecies significantly decreased and %X4 increased in Group II. An R5-X4 switch was observed in five patients (two in Group I and three in Group II), all harbouring minority X4 variants at T0. CONCLUSIONS: UDPS analysis reveals that the tropism switch is not an 'on-off' phenomenon, but may result from a profound re-shaping of viral quasispecies, even under suppressive cART. However, episodes of virological failure seem to prevent reduction of proviral DNA and to accelerate viral evolution, as suggested by decreased FPR and increased %X4 at T1 in Group II patients.

HIV-1 RNA monitoring with a dual-target diagnostic assay: A case report.

In a restricted subset of people living with HIV-1 (PLWH) on antiretroviral therapy (ART) with persistent suppressed viral load (i.e., pol-based HIV-RNA repeatedly undetected), a dual-target (pol and LTR) diagnostic assay for HIV-RNA monitoring can measure quantifiable levels of viral loads (VL) above 30 copies/mL exclusively through the amplification of the LTR region, while the pol target results undetected. We report a patient who shows high levels of HIV-RNA detected exclusively through amplification of the LTR region while undetected by the pol region, during a long monitoring period, from 2018 to date. In this follow-up, the ART was modified without reaching LTR-based undetected HIV-RNA values. Immunological and virological parameters remained optimal with a progressive and steady gain of the CD4/CD8 ratio. The clinical history of this patient, shows that LTR-based viremia above 50 copies/mL can be found occasionally or persistently in the plasma of PLWH under suppressive ART, even at high levels. Based on previous studies, VL detected and quantified exclusively through the amplification of the LTR region corresponds to partial or incomplete HIV-RNA transcripts, which cannot trigger new infections. Interestingly, changes in ART do not eliminate repeated findings of these unusual viral elements.

Human and Viral microRNA Expression in Acute and Chronic HIV Infections.

Human and viral microRNAs (miRNAs) are involved in the regulation of gene transcription, and the establishment of their profiles in acute (AHI) and chronic (CHI) HIV infections may shed light on the pathogenetic events related to different phases of HIV disease. Next-generation sequencing (NGS) of miRNA libraries was performed, and the reads were used to analyze miRNA differential expression in the plasma with AHI and CHI. Functional analysis was then undertaken to investigate the biological processes characterizing the two phases of HIV infection. Except for hsa-miR-122-5p, which was found in 3.39% AHI vs. 0.18% CHI, the most represented human miRNAs were similarly represented in AHI and CHI. However, when considering the overall detected miRNAs in AHI and CHI, 15 displayed differential expression (FDR p < 0.05). Functional analysis identified 163 target mRNAs involved in promoting angiogenesis activation in AHI versus CHI through the action of hsa-miR10b-5p, hsa-miR1290, hsa-miR1-3p, and hsa-miR296-5p. The viral miRNAs detected, all belonging to herpesviruses, accounted for only 0.014% of total reads. The present data suggest that AHI patients exhibit strong innate immune activation through the upregulation of hsa-miR-122-5p and early activation of angiogenesis. More specific investigations are needed to study the role of viral miRNAs in HIV pathogenesis.

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.

Development and validation of a nanoplate-based digital PCR assay for absolute MPXV quantification.

Quantification of mpox virus (MPXV) across different human body anatomical sites can provide insights about the most likely transmission routes, so methods able to release absolute and exact quantitative values of MPXV DNA are crucial. Here, we optimized a new QIAcuity digital PCR (dPCR) protocol for the detection and quantification of MPXV DNA in clinical samples and assessed the performance of the assay by comparing the results obtained in 144 biological samples with those resulting from the use of an in-house real-time PCR (qPCR). Overall, the concordance between the two assays was 95%, with samples identified concordantly as MPXV DNA positive and having a mean number of copies per µl of 1708 (95% CI: 107-2830 copies/µl). The remaining samples gave discordant results, with 5 out of 7 detected with the QIAcuity dPCR assay but not with the in-house qPCR. MPXV DNA levels measured by QIAcuity dPCR were strongly correlated with the Ct values detected by in-house qPCR and with those detected by another dPCR assay previously developed in our laboratories. The QIAcuity dPCR assay may be a robust and easy-to-perform method for MPXV DNA quantification in several biological samples.

Kinetics of TTV Loads in Peripheral Blood Mononuclear Cells of Early Treated Acute HIV Infections.

Torquetenovirus (TTV) is the most abundant component of the human blood virome and its replication is controlled by a functioning immune system. In this study, TTV replication was evaluated in 21 people with acute HIV infection (AHI) and immune reconstitution following antiretroviral therapy (ART). PBMC-associated TTV and HIV-1 DNA, as well as plasma HIV-1 RNA, were measured by real-time PCR. CD4 and CD8 differentiation, activation, exhaustion, and senescence phenotypes were analyzed by flow cytometry. Thirteen healthy donors (HD) and twenty-eight chronically infected HIV individuals (CHI), late presenters at diagnosis, were included as control groups. TTV replication in AHI seems to be controlled by the immune system being higher than in HD and lower than in CHI. During ART, a transient increase in TTV DNA levels was associated with a significant perturbation of activation and senescence markers on CD8 T cells. TTV loads were positively correlated with the expansion of CD8 effector memory and CD57+ cells. Our results shed light on the kinetics of TTV replication in the context of HIV acute infection and confirm that the virus replication is strongly regulated by the modulation of the immune system.

Kinetics of viral DNA in body fluids and antibody response in patients with acute Monkeypox virus infection.

We report the follow-up laboratory investigation of three MPXV cases infected in May-June 2022 from diagnosis to disease resolution, monitoring viral shedding in different body fluids and antibody kinetics. Out of 138 non-lesion samples, viral DNA was found in 92.3% saliva, 85.7% semen, 86.2% oropharyngeal swabs, 51.7% plasma, 46.1% stool, and 9.5% urine samples. Viral load quantified by digital PCR widely varied, but tend to be higher in oropharyngeal swabs, saliva, and stool. Replication competent virus was recovered from four out of seventeen samples, including 1 saliva, 1 oropharyngeal swabs, 1 semen, and 1 stool. The analysis of the antibody kinetics revealed that IgM, IgA, and IgG antibodies were detected within two weeks post-symptoms onset for all three patients, with IgG detected early on at day 4-8 and IgM and IgA showing lower titers along the time frame of the study. Antibody levels increased during the second week of illness with IgG reaching high titers.

Ultra-deep sequencing reveals hidden HIV-1 minority lineages and shifts of viral population between the main cellular reservoirs of the infection after therapy interruption.

Viral quasispecies population dynamics between monocytes and T-lymphocytes were analyzed in patients after highly active antiretroviral therapy (HAART) interruption, during a follow-up of 3-6 months. V3 env region underwent ultra-deep pyrosequencing. Co-receptor usage prediction was performed by Position Specific Score Matrix Analysis. Phylogenetic trees were constructed to evaluate the relationships between the variants. Gene flow was also investigated. Even though at the moment of therapy interruption monocyte-derived HIV-1 genomes presented higher genetic heterogeneity than that of T-lymphocytes, at subsequent times, this difference in genetic heterogeneity disappeared, due to different waves of expansion and reduction of quasispecies variability associated with monocytes and T-lymphocytes. Phylogenetic analysis and gene flow evaluation supported the hypothesis of extensive interchange of variants between cellular compartments of the infection. A spread of proviral X4 lineages hidden in monocytes to T cells was observed, but this was not associated with an overall shift towards CXCR4 using variants during the observation period.

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.

Virological and Immunological Outcomes of an Intensified Four-Drug versus a Standard Three-Drug Antiretroviral Regimen, Both Integrase Strand Transfer Inhibitor-Based, in Primary HIV Infection.

The optimal therapeutic approach for primary HIV infection (PHI) is still debated. We aimed to compare the viroimmunological response to a four- versus a three-drug regimen, both INSTI-based, in patients with PHI. This was a monocentric, prospective, observational study including all patients diagnosed with PHI from December 2014 to April 2018. Antiretroviral therapy (ART) was started, before genotype resistance test results, with tenofovir/emtricitabine and either raltegravir plus boosted darunavir or dolutegravir. Cumulative probability of virological suppression [VS] (HIV-1 RNA< 40 cp/mL), low-level HIV-1 DNA [LL-HIVDNA] (HIV-1 DNA < 200 copies/106PBMC), and CD4/CD8 ratio ≥1 were estimated using Kaplan−Meier curves. Factors associated with the achievement of VS, LL-HIVDNA, and CD4/CD8 ≥ 1 were assessed by a Cox regression model. We enrolled 144 patients (95.8% male, median age 34 years): 110 (76%) started a four-drug-based therapy, and 34 (24%) a three-drug regimen. Both treatment groups showed a comparable high probability of achieving VS and a similar probability of reaching LL-HIVDNA and a CD4/CD8 ratio ≥1 after 48 weeks from ART initiation. Higher baseline HIV-1 RNA and HIV-1 DNA levels lowered the chance of VS, whereas a better preserved immunocompetence increased that chance. Not statistically significant factors associated with LL-HIVDNA achievement were found, whereas a higher baseline CD4/CD8 ratio predicted the achievement of immune recovery. In PHI patients, the rapid initiation of either an intensified four-drug or a standard three-drug INSTI-based regimen showed comparable responses in terms of VS, viral reservoir size, and immunological recovery.