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

Interferon-α Improves Phosphoantigen-Induced Vγ9Vδ2 T-Cells Interferon-γ Production during Chronic HCV Infection

In chronic HCV infection, treatment failure and defective host immune response highly demand improved therapy strategies. Vγ9Vδ2 T-cells may inhibit HCV replication in vitro through IFN-γ release after Phosphoantigen (PhAg) stimulation. The aim of our work was to analyze Vγ9Vδ2 T-cell functionality during chronic HCV infection, studying the role of IFN-α on their function capability. IFN-γ production by Vγ9Vδ2 T-cells was analyzed in vitro in 24 HCV-infected patients and 35 healthy donors (HD) after PhAg stimulation with or without IFN-α. The effect of in vivo PhAg/IFN-α administration on plasma IFN-γ levels was analyzed in M. fascicularis monkeys. A quantitative analysis of IFN-γ mRNA level and stability in Vγ9Vδ2 T-cells was also evaluated. During chronic HCV infection, Vγ9Vδ2 T-cells showed an effector/activated phenotype and were significantly impaired in IFN-γ production. Interestingly, IFN-α was able to improve their IFN-γ response to PhAg both in vitro in HD and HCV-infected patients, and in vivo in Macaca fascicularis primates. Finally, IFN-α increased IFN-γ-mRNA transcription and stability in PhAg-activated Vγ9Vδ2 T-cells. Altogether our results show a functional impairment of Vγ9Vδ2 T-cells during chronic HCV infection that can be partially restored by using IFN-α. A study aimed to evaluate the antiviral impact of PhAg/IFN-α combination may provide new insight in designing possible combined strategies to improve HCV infection treatment outcome

Viral parameters influencing clinical long-term non progression in HIV-1 infected subjects

A very small portion of the HIV-1 infected population is constituted by individuals, called long- term non progressors (LTNP), in whom after even a decade or more, no visible deterioration of the immune system is recognized and the infection remains therefore asymptomatic. The reasons for such "natural" prolonged well-being can be attributed to both the virus and the infected host and their understanding is likely to give important information for the development of an effective HIV- vaccine and for immune reconstitution therapy. The aim of the present thesis was to investigate and to evaluate parameters suspected to influence long-term non progression, and in specific the HIV-replication dynamic, the genetic "defects" in viral regulatory genes and the genetic susceptibility of the host. A multiple competitor PCR (mcPCR) assay was established, evaluated and finally used for quantifying HIV-1 DNA in blood cell samples from LTNP and from patients with advanced disease. A significantly higher FHV-1 DNA copy number was obtained in the latter subjects. Quantification of viral RNA and DNA loads was done in a longitudinal study, starting in 1993, in 20 HIV-infected subjects with 7 years of clinical non-progression. At study end in 1998, the 20 patients were classified as slow progressors (n=12) or LINP (n=8). Slow progressors displayed higher plasma HIV-1 RNA levels than LTNP already at inclusion, while HIV-1 DNA levels were comparable in the two groups. Slow progressors thereafter showed a continuously increasing RIV-1 RNA level, which was more pronounced than in LTNP. The majority of LTNP had a stable or a declining HIV- 1 DNA level. In a particular subject with almost undetectable plasma HIV-1 RNA and a remarkable progressive reduction of the HIV-1 DNA between 1994 and 1998, the evolution of HIV-1 env quasispecies heterogeneity in PBMC was analyzed. A very low heterogeneity was present in all the samples. The analysis of the quantification data, the phylogenetic distances and the amino acid conformation in the V3 indicated that the quasispecies present until 1996 had been substituted by a different one in 1998. Truncation or subtle mutations in HIV-1 long terminal repeat (LTR) and in the nef gene have been described in LTNP and suggested to be associated with an attenuated viral pathogenesis. We analyzed nef and LTR alleles in a large cohort of 21 LTNP and in a group of progressors. An untruncated intact nef open reading frame was observed as a major sequence in all LTNP. In the LTR, no specific mutations were discovered in LTNP, and none of the observed mutations was associated with in vivo HIV-1 RNA or DNA levels. To investigate if particular mutations in the nef are linked to LTNP, the amino acid sequences obtained from our cohort were compared with the previously reported sequences of 16 American LTNP and of 36 progressors. None of the substitutions in known functional sites of the Nef protein was linked to LTNP, but a valine/isoleucine at the variable position 1 1 was strongly associated with both European and American LTNP. A lower interpatient viral diversity was found in LTNP, but phylogenetic analysis did not showed any specific clustering of the LTNP sequences. The correlation between the presence of a deletion (A) in the human CCR5 gene, disease progression and HIV-1 specific immune responses was analyzed in 79 HIV-1 infected patients. The prevalence of the CCR5 allele was lower among patients with rapid progression as compared to patients with slow progression or to 25 HIV-1 uninfected blood donors. The antibody titers against gp41 and V3MN peptides in patients with a CCR5/CCR5 genotype were not significantly different from those in pair-matched CCR5/CCR5 controls. In conclusion, (1) LTNP express in general low and stable amounts of HIV-1 in plasma and stable, or even declining, cellular HIV-1 DNA levels; (2) the viral population in LTNP may exhibit a low heterogeneity; (3) defects in the nef and LTR are rare in LTNP and are not associated to a particular pattern of viral loads; (4) the heterozygous CCR5 mutation prolongs the AIDS-free interval, but is not associated to HIV-1 env antibody pattern nor is it a specific marker for LTNP

Phylogenetic and Phylodynamic Analysis of Delta Strains Circulating in Italy

The hepatitis delta virus (HDV) exhibits high genetic and evolutionary variability and is classified into eight genotypes (HDV-1 to -8). HDV-1 is the most widespread genotype worldwide and includes several subtypes. It predominates mainly in Europe, the Middle East, North America, and Northern Africa, and is associated with both severe and mild forms of liver disease. In this study, we performed phylogenetic and phylodynamic analyses of HDV strains circulating in Regione Lazio, Italy, to understand when these strains were introduced into the Lazio region and to define their genetic variability in Italy. Fifty HDV RNA positive patient samples were amplified using a nested RT-PCR approach targeting the HDV R0 region and sequenced. A phylogenetic tree of patient-derived sequences and reference sequences representing HDV-1 to -8 was constructed using the GTRGAMMA model in RAxML v8. The results indicated that HDV-1 was the predominant genotype with HDV-1d being the most frequently inferred subtype. HDV-1 sequences clustering with subtypes 1b and 1e were also identified. A phylodynamic analysis of HDV-1 sequences employing a Bayesian birth-death model inferred a clock rate of 3.04 × 10−4 substitutions per site per million years, with a 95% Highest Posterior Density (HPD) interval of 3.45 × 10−5 to 5.72 × 10−4. A Bayesian birth-death analysis with tree calibration based on a sample dating approach indicated multiple original sources of infection (from the late 1950s to late 1980s). Overall, these results suggest that HDV sequences from the native Italian and non-Italian patients analyzed in this study represent multiple lineages introduced across a wide period. A common ancestral origin should be excluded

Simple and Reliable Method to Quantify the Hepatitis B Viral Load and Replicative Capacity in Liver Tissue and Blood Leukocytes

BACKGROUND: A functional cure of chronic hepatitis B (CHB) is feasible, but a clear view of the intrahepatic viral dynamics in each patient is needed. Intrahepatic covalently closed circular DNA (cccDNA) is the stable form of the viral genome in infected cells, and represents the ideal marker of parenchymal colonization. Its relationships with easily accessible peripheral parameters need to be elucidated in order to avoid invasive procedures in patients. OBJECTIVES: The goal of this study was to design, set up, and validate a reliable and straightforward method for the quantification of the cccDNA and total DNA of the hepatitis B virus (HBV) in a variety of clinical samples. PATIENTS AND METHODS: Clinical samples from a cohort of CHB patients, including liver biopsies in some, were collected for the analysis of intracellular HBV molecular markers using novel molecular assays. RESULTS: A plasmid construct, including sequences from the HBV genome and from the human gene hTERT, was generated as an isomolar multi-standard for HBV quantitation and normalization to the cellular contents. The specificity of the real-time assay for the cccDNA was assessed using Dane particles isolated on a density gradient. A comparison of liver tissue from 6 untreated and 6 treated patients showed that the treatment deeply reduced the replicative capacity (total DNA/cccDNA), but had limited impact on the parenchymal colonization. The peripheral blood mononuclear cells (PBMCs) and granulocytes from the treated and untreated patients were also analyzed. CONCLUSIONS: A straightforward method for the quantification of intracellular HBV molecular parameters in clinical samples was developed and validated. The widespread use of such versatile assays could better define the prognosis of CHB, and allow a more rational approach to time-limited tailored treatment strategies

Simple and reliable method to quantify the hepatitis B viral load and replicative capacity in liver tissue and blood leukocytes

BACKGROUND: A functional cure of chronic hepatitis B (CHB) is feasible, but a clear view of the intrahepatic viral dynamics in each patient is needed. Intrahepatic covalently closed circular DNA (cccDNA) is the stable form of the viral genome in infected cells, and represents the ideal marker of parenchymal colonization. Its relationships with easily accessible peripheral parameters need to be elucidated in order to avoid invasive procedures in patients. OBJECTIVES: The goal of this study was to design, set up, and validate a reliable and straightforward method for the quantification of the cccDNA and total DNA of the hepatitis B virus (HBV) in a variety of clinical samples. PATIENTS AND METHODS: Clinical samples from a cohort of CHB patients, including liver biopsies in some, were collected for the analysis of intracellular HBV molecular markers using novel molecular assays. RESULTS: A plasmid construct, including sequences from the HBV genome and from the human gene hTERT, was generated as an isomolar multi-standard for HBV quantitation and normalization to the cellular contents. The specificity of the real-time assay for the cccDNA was assessed using Dane particles isolated on a density gradient. A comparison of liver tissue from 6 untreated and 6 treated patients showed that the treatment deeply reduced the replicative capacity (total DNA/cccDNA), but had limited impact on the parenchymal colonization. The peripheral blood mononuclear cells (PBMCs) and granulocytes from the treated and untreated patients were also analyzed. CONCLUSIONS: A straightforward method for the quantification of intracellular HBV molecular parameters in clinical samples was developed and validated. The widespread use of such versatile assays could better define the prognosis of CHB, and allow a more rational approach to time-limited tailored treatment strategies