Pascal Meylan

Individual Contributions of Mutant Protease and Reverse Transcriptase to Viral Infectivity, Replication, and Protein Maturation of Antiretroviral Drug-Resistant Human Immunodeficiency Virus Type 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease (PR) and reverse transcriptase (RT) inhibitors may display impaired infectivity and replication capacity. The individual contributions of mutated HIV-1 PR and RT to infectivity, replication, RT activity, and protein maturation (herein referred to as “fitness”) in recombinant viruses were investigated by separately cloning PR, RT, and PR-RT cassettes from drug-resistant mutant viral isolates into the wild-type NL4-3 background. Both mutant PR and RT contributed to measurable deficits in fitness of viral constructs. In peripheral blood mononuclear cells, replication rates (means ± standard deviations) of RT recombinants were 72.5% ± 27.3% and replication rates of PR recombinants were 60.5% ± 33.6% of the rates of NL4-3. PR mutant deficits were enhanced in CEM T cells, with relative replication rates of PR recombinants decreasing to 15.8% ± 23.5% of NL4-3 replication rates. Cloning of the cognate RT improved fitness of some PR mutant clones. For a multidrug-resistant virus transmitted through sexual contact, RT constructs displayed a marked infectivity and replication deficit and diminished packaging of Pol proteins (RT content in virions diminished by 56.3% ± 10.7%, and integrase content diminished by 23.3% ± 18.4%), a novel mechanism for a decreased-fitness phenotype. Despite the identified impairment of recombinant clones, fitness of two of the three drug-resistant isolates was comparable to that of wild-type, susceptible viruses, suggestive of extensive compensation by genomic regions away from PR and RT. Only limited reversion of mutated positions to wild-type amino acids was observed for the native isolates over 100 viral replication cycles in the absence of drug selective pressure. These data underscore the complex relationship between PR and RT adaptive changes and viral evolution in antiretroviral drug-resistant HIV-1.

Resistance to Nucleoside Analog Reverse Transcriptase Inhibitors Mediated by Human Immunodeficiency Virus Type 1 p6 Protein

ABSTRACT Resistance of human immunodeficiency virus type 1 (HIV-1) to antiretroviral agents results from target gene mutation within thepol gene, which encodes the viral protease, reverse transcriptase (RT), and integrase. We speculated that mutations in genes other that the drug target could lead to drug resistance. For this purpose, the p1-p6 gag -p6 pol region of HIV-1, placed immediately upstream ofpol, was analyzed. This region has the potential to alter Pol through frameshift regulation (p1), through improved packaging of viral enzymes (p6Gag), or by changes in activation of the viral protease (p6Pol). Duplication of the proline-rich p6Gag PTAP motif, necessary for late viral cycle activities, was identified in plasma virus from 47 of 222 (21.2%) patients treated with nucleoside analog RT inhibitor (NRTI) antiretroviral therapy but was identified very rarely from drug-naı̈ve individuals. Molecular clones carrying a 3-amino-acid duplication, APPAPP (transframe duplication SPTSPT in p6Pol), displayed a delay in protein maturation; however, they packaged a 34% excess of RT and exhibited a marked competitive growth advantage in the presence of NRTIs. This phenotype is reminiscent of the inoculum effect described in bacteriology, where a larger input, or a greater infectivity of an organism with a wild-type antimicrobial target, leads to escape from drug pressure and a higher MIC in vitro. Though the mechanism by which the PTAP region participates in viral maturation is not known, duplication of this proline-rich motif could improve assembly and packaging at membrane locations, resulting in the observed phenotype of increased infectivity and drug resistance.

Mycobacterium tuberculosis subverts innate immunity to evade specific effectors

The macrophage is the niche of the intracellular pathogen Mycobacterium tuberculosis. Induction of macrophage apoptosis by CD4+ or CD8+ T cells is accompanied by reduced bacterial counts, potentially defining a host defense mechanism. We have already established that M. tuberculosis-infected primary human macrophages have a reduced susceptibility to Fas ligand (FasL)-induced apoptosis. To study the mechanisms by which M. tuberculosis prevents apoptotic signaling, we have generated a cell culture system based on PMA- and IFN-γ-differentiated THP-1 cells recapitulating the properties of primary macrophages. In these cells, nucleotide-binding oligomerization domain 2 or TLR2 agonists and mycobacterial infection protected macrophages from apoptosis and resulted in NF-κB nuclear translocation associated with up-regulation of the antiapoptotic cellular FLIP. Transduction of a receptor-interacting protein-2 dominant-negative construct showed that nucleotide-binding oligomerization domain 2 is not involved in protection in the mycobacterial infection system. In contrast, both a dominant-negative construct of the MyD88 adaptor and an NF-κB inhibitor abrogated the protection against FasL-mediated apoptosis, showing the implication of TLR2-mediated activation of NF-κB in apoptosis protection in infected macrophages. The apoptosis resistance of infected macrophages might be considered as an immune escape mechanism, whereby M. tuberculosis subverts innate immunity signaling to protect its host cell against FasL+-specific cytotoxic lymphocytes.

Influence of host cell type and V3 loop of the surface glycoprotein on susceptibility of human immunodeficiency virus type 1 to polyanion compounds.

Dextran sulfate is a potent inhibitor of human immunodeficiency virus (HIV) binding and replication in lymphocytic cell lines. In this study, we demonstrate that the effect of dextran sulfate and heparin depends on the host cell type and on the V3 loop, the principal neutralizing determinant of HIV gp120. In particular, when dextran sulfate was tested on primary human macrophages infected with macrophage-tropic viruses, enhancement of infection was observed in 6 of 11 independent macrophage preparations and with 5 of 13 primary HIV isolates. Our in vitro observations might explain why enhanced HIV replication was observed in HIV-infected patients treated with dextran sulfate. Images

Seroepidemiology of Herpes Simplex virus type 1 and 2 in Western and Southern Switzerland in adults aged 25–74 in 1992–93 : a population-based study

BackgroundGenital herpes is one of the most prevalent sexually-transmitted diseases, and accounts for a substantial morbidity. Genital herpes puts newborns at risk for very severe disease and also increases the risk of horizontal HIV transmission. It thus stands as an important public health problem. The recent availability of type-specific gG-based assays detecting IgG against HSV-1 and HSV-2 allows to establish the prevalence of each subtype. Worldwide, few data have been published regarding the seroprevalence in general populations of HSV-2, the major causative agent for genital herpes, while no data exist regarding the Swiss population.MethodsTo evaluate the prevalence of IgG antibodies against HSV-1 and HSV-2 in Switzerland, we used a population-based serum repository from a health examination survey conducted in the Western and Southern area of Switzerland in 1992–93. A total of 3,120 sera were analysed by type-specific gG-based ELISA and seroprevalence was correlated with available volunteers characteristics by logistic regression.ResultsOverall, seroprevalence rates were 80.0 ± 0.9% (SE, 95% CI: 78.1–81.8) for HSV-1 and 19.3 ± 0.9% (SE, 95% CI: 17.6–21.1) for HSV-2 in adults 35–64 year old. HSV-1 and HSV-2 seroprevalence increased with age, with a peak HSV-2 seroprevalence in elderly gentlemen, possibly a seroarcheological evidence of sexually transmitted disease epidemics during World War II. Risk factors for HSV-2 infection included female sex, marital status other than married, and size of town of residence larger than 1500 inhabitants. Unexpectedly and conversely to HSV-1, HSV-2 seroprevalence increased with educational level. HSV-2 infection was less prevalent among HSV-1 infected individuals when compared to HSV-1 uninfected individuals. This effect was most apparent among women at high risk for HSV-2 infection.ConclusionsOur data demonstrate that by the early nineties, HSV-2 had spread quite largely in the Swiss population. However, the epidemiology of HSV-2 in Switzerland presents paradoxical characteristics, e.g. positive correlation with education level, that have not been observed elsewhere.

Valganciclovir in Adult Solid Organ Transplant Recipients Pharmacokinetic and Pharmacodynamic Characteristics and Clinical Interpretation of Plasma Concentration Measurements

Valganciclovir and ganciclovir are widely used for the prevention of cytomegalovirus (CMV) infection in solid organ transplant recipients, with a major impact on patients’ morbidity and mortality. Oral valganciclovir, the ester prodrug of ganciclovir, has been developed to enhance the oral bioavailability of ganciclovir. It crosses the gastrointestinal barrier through peptide transporters and is then hydrolysed into ganciclovir. This review aims to describe the current knowledge of the pharmacokinetic and pharmacodynamic characteristics of this agent, and to address the issue of therapeutic drug monitoring.Based on currently available literature, ganciclovir pharmacokinetics in adult solid organ transplant recipients receiving oral valganciclovir are characterized by bioavailability of 66±10% (mean ± SD), a maximum plasma concentration of 3.1 ± 0.8 mg/L after a dose of 450 mg and of 6.6 ± 1.9 mg/L after a dose of 900 mg, a time to reach the maximum plasma concentration of 3.0±1.0 hours, area under the plasma concentration-time curve values of 29.1±5.3mg · h/L and 51.9±18.3mg · h/L (after 450 mg and 900 mg, respectively), apparent clearance of 12.4 ± 3.8 L/h, an elimination half-life of 5.3 ± 1.5 hours and an apparent terminal volume of distribution of 101 ±36 L. The apparent clearance is highly correlated with renal function, hence the dosage needs to be adjusted in proportion to the glomerular filtration rate. Unexplained interpatient variability is limited (18% in apparent clearance and 28% in the apparent central volume of distribution). There is no indication of erratic or limited absorption in given subgroups of patients; however, this may be of concern in patients with severe malabsorption.The in vitro pharmacodynamics of ganciclovir reveal a mean concentration producing 50% inhibition (IC50) among CMV clinical strains of 0.7 mg/L (range 0.2–1.9 mg/L). Systemic exposure of ganciclovir appears to be moderately correlated with clinical antiviral activity and haematotoxicity during CMV prophylaxis in high-risk transplant recipients. Low ganciclovir plasma concentrations have been associated with treatment failure and high concentrations with haematotoxicity and neurotoxicity, but no formal therapeutic or toxic ranges have been validated.The pharmacokinetic parameters of ganciclovir after valganciclovir administration (bioavailability, apparent clearance and volume of distribution) are fairly predictable in adult transplant patients, with little interpatient variability beyond the effect of renal function and bodyweight. Thus ganciclovir exposure can probably be controlled with sufficient accuracy by thorough valganciclovir dosage adjustment according to patient characteristics. In addition, the therapeutic margin of ganciclovir is loosely defined. The usefulness of systematic therapeutic drug monitoring in adult transplant patients therefore appears questionable; however, studies are still needed to extend knowledge to particular subgroups of patients or dosage regimens.

Macrophage Migration Inhibitory Factor Reduces the Growth of Virulent Mycobacterium tuberculosis in Human Macrophages

ABSTRACT Macrophage migration inhibitory factor (MIF) is a key mediator of the innate immune system and plays a crucial role in the host response to bacterial infections. Its role in immunity to intracellular pathogens has not been well studied. Here, we show that MIF released by infected human macrophages inhibits the growth of virulent Mycobacterium tuberculosis.

Infection of the central nervous system caused by varicella zoster virus reactivation: a retrospective case series study

BACKGROUNDnRecent data suggest that varicella zoster virus (VZV)-associated complications of the central nervous system (CNS) are more common and diverse than previously thought. The main purpose of this article is to describe the clinical characteristics and the outcome of patients suffering from meningitis and encephalitis caused by VZV reactivation.nnnMETHODSnA retrospective case study of adult patients (≥16 years old) diagnosed with a VZV reactivation in the CNS was performed. The cases were identified by a qualitative PCR DNA assay of the cerebrospinal fluid (CSF) at the Regional Hospital of Lugano between January 1, 2003 and July 31, 2010.nnnRESULTSnEleven out of 519 CSF samples (2.1%), submitted from patients with a clinical diagnosis of viral meningitis or encephalitis, were positive for VZV. A vesiculo-pustular skin eruption was observed in only five patients (45%). In six cases (55%), a systemic inflammatory syndrome was absent. The clinical outcome was favorable in eight patients (73%). Only one out of 11 patients (9%) died. The four patients with encephalitis had a less favorable prognosis: one patient recovered without residual neurological sequelae; two had a chronic neuropsychological handicap, speech difficulties, facial nerve palsy, and focal seizures; one patient died. We estimated an annual incidence rate of VZV infection of the CNS of 1.02/100,000 inhabitants for southern Switzerland.nnnCONCLUSIONSnScreening of CSF for VZV by PCR is recommended for all patients with encephalitis and for those with viral meningitis of unclear origin in order to better target antiviral treatment.

Virological and immunological characteristics of HIV treatment failure.

BackgroundResistance to antiretroviral treatment is prevalent. There is limited knowledge of the determinants of disease evolution in subjects infected with multidrug-resistant HIV (MDR-HIV). MethodsInfectivity, replication, chemokine receptor usage, and env, gag, protease and reverse transcriptase sequence analysis was performed for MDR-HIV isolates from 14 HIV-infected individuals and compared to wild-type HIV isolates from individuals naive to antiretroviral treatment. Expression of CD45RO/RA, Ki67 and interferon-γ and CD4 proliferative response to various antigens was determined for individuals infected with MDR-HIV and compared to that in individuals with optimal suppression of viral replication. ResultsInfectivity and replication are diminished for various MDR-HIV isolates, usually in the context of an increase in CD4 and CD4+CD45RA+ T-cell counts. However, a number of MDR-HIV isolates are associated with high in vivo viraemia and pronounced immunosuppression, and display in vitro levels of infectivity and replication comparable to those of wild-type strains. No specific genetic sequence or chemokine receptor usage predicted the fitness of an MDR isolate. ConclusionsDespite the biological diversity of resistant viruses and the range of host responses observed, our descriptive analysis indicates that viral factors play a role in determining the degree of immune damage observed in the context of MDR-HIV infection.

Cytomegalovirus infection and new-onset post-transplant diabetes mellitus.

Abstract:u2002 We analyzed data from all consecutive kidney transplant patients at our institution between April 2003 and October 2006. We found 15 cases of late‐onset cytomegalovirus (CMV) infection, two of which developed concurrent post‐transplant diabetes mellitus (PTDM). In these two cases, PTDM was transient and normal glucose tolerance was achieved after an eight‐wk therapeutic course of oral valganciclovir. These findings suggest that CMV infection after organ transplantation may be associated with concurrent PTDM. The distinct causative relationship is yet to be determined.