Alessandra Cenci

Inhibition of replication of HIV in primary monocyte/macrophages by different antiviral drugs and comparative efficacy in lymphocytes

Several anti‐HIV drugs acting on different steps of virus replication were tested in our experimental model of primary monocyte/macrophages; the results were compared with the activity found in lymphocytes. Nucleoside analogues (AZT, ddl, ddC, d4T, PMEA, 3TC etc.) show greater activity in macrophages (M/M) than in lymphocytes. In particular, the EC50 of AZT, ddC, and ddI in M/M is 2‐ to 100‐fold lower than that found in lymphocytes. This greater efficacy of nucleoside analogues in M/M depends on the enhancement of their chain‐terminating activity by the low levels of endogenous deoxynucleoside‐triphosphates (dNTP) usually found in resting cells such as M/M. Non‐nucleoside reverse transcriptase inhibitors (NNRTI) do not act as chain terminators (thus their antiviral effect is not related to the intracellular concentrations of dNTP); as a consequence the activity of TSAO, HEPT, TIBO, and other NNRTI tested in M/M is similar to that found in lymphocytes. Regarding inhibitors of binding and fusion of HIV, we found that their anti‐HIV activity is markedly decreased (or even nullified) when M/M are treated with cytokine activators of M/M function and enhancers of HIV replication. More relevant from a clinical standpoint, protease inhibitors are able to inhibit HIV replication in chronically infected macrophages cells carrying the proviral genome already integrated in the host genome). All other inhibitors of late stage of virus life cycle tested (antisense‐rev, anti‐tat, interferon‐α and ‐γ, phosphorothioate analogues, GLQ‐223, etc.) were totally inactive in chronically infected macrophages. The different effects of various classes of HIV inhibitors in lymphocytes and macrophages suggests that AIDS therapy should consider all aspects of the pathogenesis of HIV infection and must be restricted to drugs, or combinations of drugs, active against both lymphocytes and M/M in all body compartments where the virus hides and replicates. J. Leukoc. Biol. 62: 138–143; 1997.

Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes

Infection of macrophages (M/M) by human immunodeficiency virus (HIV) is a main pathogenetic event leading to neuronal dysfunction and death in patients with AIDS dementia complex. Alteration of viability of neurons and astrocytes occurs in vivo even without their infection, thus it is conceivable that HIV‐infected M/M may affect viability of such cells even without direct infection. To assess this hypothesis, we studied the effects of HIV‐infected M/M on an astrocytic cell‐line lacking CD4‐receptor expression. Exposure to supernatants of HIV‐infected M/M triggers complete disruption and apoptotic death of astrocytic cells. This effect is not related to HIV transmission from infected M/M, because HIV‐DNA and p24 production in astrocytic cells remained negative. Apoptotic death of astrocytes is mainly mediated by Fas ligand released in supernatants of HIV‐infected M/M (as demonstrated by complete reversal of such phenomenon by adding neutralizing antibodies against CD95 receptor). Treatment of astrocytic cells with recombinant (biologically active) Tat induces <10% apoptosis, and gp120 was totally ineffective. Treatment of HIV‐infected M/M with AZT completely reverses the proapoptotic effect of their supernatants on astrocytes, thus demonstrating that productive virus replication within M/M is required for the induction of astrocytic cell death.

Red blood cells mediated delivery of 9-(2-phosphonylmethoxyethyl)adenine to primary macrophages: efficiency, metabolism and activity against human immunodeficiency virus or herpes simplex virus

Red blood cells (RBC) may act as selective carriers of drugs to macrophages, an important reservoir of viruses such as human immunodeficiency virus (HIV) and herpes simplex virus type 1 (HSV-1). We therefore assessed the incorporation of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a potent inhibitor of HIV and HSV-1) into RBC, its delivery to macrophages and its activity against HIV or HSV-1. Loading of PMEA in artificially aged opsonized RBC affords significant levels of intracellular PMEA. RBC metabolize PMEA to its active congener PMEA-diphosphate, although with low efficiency. Exposure of macrophages to RBC-encapsulated PMEA inhibits the replication of both HIV and HSV-1 (about 90% inhibition at the highest RBC:macrophages ratios) even if RBC were removed before virus challenge. By contrast, the antiviral activity of free PMEA removed before virus challenge was irrelevant at concentrations up to 150-fold higher than the 50% effective concentration (EC50). Finally, the antiviral effect of RBC-encapsulated PMEA correlates with PMEA levels in macrophages about 500-fold higher than those achieved by free PMEA (at concentrations 10-fold higher than the EC50). The efficacy of RBC-mediated delivery to macrophages of PMEA (and perhaps of compounds with shorter intracellular half-lives) warrants further studies in infectious diseases involving phagocytizing cells as main targets of the pathogen.

Correlates of infection and molecular characterization of blood-borne HIV, HCV, and HBV infections in HIV-1 infected inmates in Italy: An observational cross-sectional study

AbstractCoinfection of blood-borne hepatitis B and hepatitis C viruses (HBV and HCV, respectively) in human immunodeficiency virus type 1 (HIV-1)-positive individuals frequently occurs in inmate population and peculiar viral strains and patterns of virological markers may be observed.Plasma from 69 HIV-1-positive inmates was obtained from 7 clinical centers connected with correctional centers in different towns in Italy. HIV, HBV, and HCV markers were tested by commercial assays. Virus genotyping was carried out by sequencing the protease and reverse transcriptase-encoding region (PR-RT region) for HIV and a region encompassing the NS5B gene for HCV and subsequent phylogenetic analysis.Twelve over 14 HIV-subtyped inmates were infected with HIV-1 subtype B strains. The 2 non-B strains belonged to subtype G and CRF02_AG, in an Italian and a Gambian patient, respectively. Variants carrying the K103N and Y181C resistance mutations to non-nucleoside reverse transcriptase inhibitors (NNRTIs) were found in 2 out of 9 patients naive for combined antiretroviral therapy (cART) (22.2%). Most HIV-positive patients (92.8%) showed evidence of past or present HBV and/or HCV infection. Prevalence of HBV and HCV was 81.2% for both viruses, whereas prevalence of HBV/HCV coinfection was 69.6%. A significantly higher presence of HCV infection was found in Italians [odds ratio (OR) 11.0; interval 1.7–80.9] and in drug users (OR 27.8; interval 4.9–186.0). HCV subtypes were determined in 42 HCV or HBV/HCV-coinfected individuals. HCV subtypes 1a, 3a, 4d, and 1b were found in 42.9%, 40.5%, 14.3%, and 2.4% of inmates, respectively. Low titers of HBV DNA in HBV DNA positive subjects precluded HBV subtyping.The high prevalence of HBV and HCV coinfections in HIV-infected inmates, as well as the heterogeneity of HIV and HCV subtypes suggest the need to adopt systematic controls in prisons to monitor both the burden and the genetic forms of blood-borne viral infections, in order to apply targeted therapeutic interventions.

May Phylogenetic Analysis Support Epidemiological Investigation in Identifying the Source of HIV Infection

Editor: The human immunodeficiency virus (HIV) is characterized by great genetic heterogeneity driven by several factors, such as the lack of proofreading ability of the reverse transcriptase (RT), the rapid turnover of HIV-1 in vivo, host selective immune pressures, and recombination events during replication. In particular, HIV-1 group M subtypes are responsible for most of the infections worldwide. At the end of the year 2008, in Italy, 60,346 persons were cumulatively diagnosed with AIDS. Overall, it is estimated that 140,000/150,000 HIV-positive people live within the country. At the beginning of the epidemic the most common route of HIV transmission was needle sharing among intravenous drug users (IDUs) whereas in more recent years heterosexual contacts have become the leading cause of infection (44.7%). Phylogenetic analysis is an important way to trace epidemiological relationship in cases with dubious or unknown links. Moreover, phylogeny is also an easy way to describe and to identify groups of viral isolates forming genetically related sister clades, which appear to be more closely related to each other. We describe here a case of HIV-1 infection in a 24-year-old Italian soldier in service in Kabul, Afghanistan, where his military command was placed. The epidemiological investigation was conducted in three different phases. In September 2009, during a laboratory investigation in the Military Barracks based on demand screening, he was found to be positive for anti-HIV antibodies (by an ELISA test), which was confirmed by Western blot. The viral load was 39,882 copies/ml and the CD4 cell count was 568 cells/ml. During the month of October he was hospitalized in Catanzaro Hospital for diagnostic controls. His viral load was of 18,000 copies/ml and his CD4 cell count was 701 cells/ml. The epidemiological investigation did not identify any known risk factors for HIV infection (i.e., drug use, tattoos, homosexual or heterosexual contact, biological fluid exposure). Thus, we tried to reconstruct the epidemiological network using the pol viral sequences (1271 nucleotides) detected by a sample collected in November 2009, before starting antiretroviral therapy. RNA extraction, polymerase chain reaction (PCR) amplification, and sequences were obtained as previously described. The pol gene sequence was aligned and compared with reference sequences of the HIV-1 pol genes downloaded from the HIV Los Alamos database (htpp://hiv-web.lanl .gov) as previously described; the HIV-1 pol sequences were obtained with the Blast similarity search (using the Italian soldier’s sequence as the query sequence). HIV-1 subtype B pol sequences obtained by Blast similarity search analysis are reported in Supplementary Table 1 (Supplementary data are available online at www.liebertonline.com/aid). The tree was generated using the F84 model of substitution using both neighbor-joining (NJ) and maximum-likelihood (ML) tree building methods. The evolutionary model was chosen as the best-fitting nucleotide substitution model according to the hierarchical likelihood ratio test (HLRT) implemented with Model Test v3.0 software. The statistical robustness and reliability of the branching order within each phylogenetic tree were confirmed by bootstrap analysis using 1000 replicates for the NJ tree and with the zero branch length test for the ML tree. All calculations were performed with PAUP*4.0 software. SimPlot software version 3.2 and the split-decomposition analysis were used to investigate the recombinant. Separate phylogenetic analyses of the subalignments generated by the different break-points were also used to better investigate the supposed recombinant sequences. Phylogenetic analysis classified our pol gene sequence as a ‘‘nonpure’’ B subtype and showed that the patient’s isolate was closely related to B subtype sequences from Spain, forming a significant monophyletic cluster (bootstrap value >70%) (Fig. 1). Because our sequence was in a marginal position within the cluster, SimPlot and split-decomposition analyses were performed and our sequence was found to be a possible BFB recombinant form; however, we classified it as ‘‘BUB’’ (U for unclassified) since the F trait and the break-point of recombination were under the threshold value (70%) useful for statistical significance (Supplementary Figs. 1 and 2). The young man was enrolled in the peace military mission of the ISAF (International Security Assistance Force). In this context, soldiers from different nations (Italy, the United States,

HIV fitness and resistance as covariates associated with the appearance of mutations under antiretroviral treatment

The ability of human immunodeficiency virus (HIV) strains to replicate in human target cells represents a major driving force of the progression of the disease. Despite antiretroviral treatment, HIV overcomes drug pressure by adding new (compensatory) mutations, appearing in a specific and sequential order, that modulate its replication capacity and favour viral escape. In the case of M184V (a mutation involving the catalytic site of HIV reverse transcriptase), no pathways of viral escape have been defined so far; it is thus conceivable that the mutated virus maintains a relatively low replicative capacity. At the time of interruption of specific viral pressure (lamivudine in the case of M184V), wild-type virus easily overgrows mutated strains. A deep molecular analysis (90 clones) conducted on proviral DNA of lymphocytes demonstrates that M184V strains are no longer detected in plasma and proviral DNA shortly after interruption of therapeutic regimens including lamivudine (even if a new therapeutic regimen has been started). This supports the concept that the low fitness of M184V strains is not easily compensated by additional mutations. Taken together, the results suggest that the assessment of viral fitness, either direct (through biological methods) or indirect (through the identification of specific mutations that affect the replicative capacity), may provide substantial advancements in the definition of the long-term efficacy of antiretroviral therapy.