Reactivation of latent viruses in individuals receiving rituximab for new onset type 1 diabetes.

BACKGROUND: Rituximab has been successfully used as an experimental therapy in different autoimmune diseases. Recently, a double-blind placebo-controlled phase-2 study in early onset type 1 diabetes showed that rituximab delayed progression of the disease. However, like with any immunosuppressive therapy, there is a concern of opportunistic viral reactivations with the use of rituximab, including herpes and polyomaviruses. OBJECTIVES: To study the incidence of new infections and reactivations with BK, JC, Epstein-Barr and cytomegalovirus (BKV, JCV, EBV and CMV) in T1D participants in the phase-2 rituximab study. STUDY DESIGN: Subjects received 4 weekly doses of rituximab (N = 57) or placebo (N = 30) during the first month of study. Blood samples obtained at weeks 0, 12, 26, 56 and 78 were assayed for CMV, EBV, BKV and JCV by real-time DNA PCR and serology. RESULTS: EBV reactivations were diagnosed by PCR in 25% of placebo, but none of rituximab recipients (p < 0.01). There were no episodes of CMV viremia in either treatment group. BKV viremias were significantly more common in the rituximab recipients (9%) compared with placebo controls (0, p < 0.01). No JCV reactivations were detected in this study, but among 6 rituximab and 2 placebo recipients who seroconverted for JCV during the study, only one rituximab recipient had detectable viremia. All infections were asymptomatic. CONCLUSIONS: Four doses of rituximab administered to individuals with early onset T1D decreased the incidence of asymptomatic EBV reactivations, as predicted by the rituximab-mediated elimination of memory B-cells, but increased the frequency of asymptomatic viremias caused by polyomaviruses.

Lytic and latent EBV gene expression in transplant recipients with and without post-transplant lymphoproliferative disorder.

BACKGROUND: Epstein-Barr virus (EBV) is associated with post-transplant lymphoproliferative disorder (PTLD), which has significant morbidity and mortality in transplant recipients. To devise prophylactic measures, we need predictors of PTLD and a better understanding of the physiopathogenesis of the disease. OBJECTIVES: To identify a molecular pattern of EBV gene products in blood that is specific to PTLD and can be used for the diagnosis of this disease. STUDY DESIGN: We evaluated the ratio between latent and replicating EBV nucleic acids in individuals with PTLD by comparison with transplant recipients without PTLD and immunocompetent hosts with EBV DNA-emia. Subjects were prospectively identified between July 2009 and October 2010 at the University of Colorado Hospital. EBV DNA, LMP-2A Latency III and BZLF1 Lytic genes mRNA were quantified using real-time PCR. RESULTS: We found that PTLD subjects (N = 7) had significantly higher EBV DNA-emia compared with non-transplant immunocompetent subjects (N = 69; p<0.0001), and transplant recipients without PTLD (N = 105; p<0.0001). The ratios between LMP-2A and BZLF1 mRNA in transplant recipients were significantly lower than in non-transplant subjects (p = 0.04). However, PTLD and non-PTLD transplant recipients displayed similar ratios. CONCLUSIONS: These results suggest that EBV replication makes a larger contribution to the circulating EBV DNA in transplant recipients compared with immunocompetent hosts. Transplant recipients seem to lose control over EBV replication, which may contribute to the development of PTLD.

Human metapneumovirus.

Human metapneumovirus (hMPV) is a common pathogen that can cause both upper and lower respiratory tract infections, particularly in children, elderly adults, and immunocompromised hosts. Since its initial identification in 2001, hMPV has been isolated from individuals with acute respiratory tract infections (ARTIs) in virtually every continent. Serological studies indicate that it has caused human infection since 1958 or earlier. The epidemiology and clinical manifestations of hMPV are similar to those of the human respiratory syncytial virus (hRSV). HMPV has a seasonal variation: it circulates in late winter to early spring in temperate climates; late spring and summer in tropical regions. In young children, symptoms range from mild upper respiratory tract infections to severe lower respiratory tract infections (eg, bronchiolitis, croup, and pneumonia). In adults, hMPV reinfection typically presents with colds and flulike clinical manifestations. The disease is more severe (sometimes lethal) in immunocompromised hosts. Reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive test with which to detect hMPV infection. Direct detection of hMPV antigens with an immunofluorescent antibody test is available but is less sensitive than PCR. Antibody testing is used mainly for retrospective diagnosis (≥ fourfold increase in titer or seroconversion) and for epidemiological studies. The mainstay of treatment of hMPV infections is supportive. Ribavirin has similar activity in vitro and in animal models against hRSV and hMPV, but its efficacy in vivo is unproven. Monoclonal antibodies have activity in murine models but are not available in humans. Several vaccines are promising in animal models, but their safety and efficacy have not yet been evaluated in humans.

Two-way Bayesian hierarchical phylogenetic models: An application to the co-evolution of gp120 and gp41 during and after enfuvirtide treatment.

Enfuvirtide (ENF) is a fusion inhibitor that prevents the entry of HIV virions into target cells. Studying the characteristics of viral evolution during treatment and after a treatment interruption can lend insight into the mechanisms of viral evolution and fitness. Although interruption of anti-HIV therapy often results in rapid emergence of an archived "wild-type" virus population, previous work from our group indicates that when only ENF is interrupted, viral gp41 continues to evolve forward and resistance mutations are lost due to back-mutation and remodeling of the envelope protein. To examine the co-evolution of gp120 and gp41 during ENF interruption we extend the Bayesian Hierarchical Phylogenetic model (HPM). Current HPMs enforce conditional independence across all outcomes while biologically all gene regions within a patient should return the same tree unless recombination confers an evolutionary selective advantage. A two-way-interaction HPM is proposed that provides middle ground between these two extremes and allows us to test for differences in evolutionary pressures across gene regions in multiple patients simultaneously. When the model is applied to a well-characterized cohort of HIV-infected patients interrupting ENF we find that across patients, the virus continued to evolve forward in both gene regions. Overall, the hypothesis of independence over dependence between the gene regions is supported. Models that allow for the examination of co-evolution over time will be increasingly important as more therapeutic classes are developed, each of which may impact other through novel and complex mechanisms.

Enfuvirtide cerebrospinal fluid (CSF) pharmacokinetics and potential use in defining CSF HIV-1 origin.

BACKGROUND: Enfuvirtide is a potent inhibitor of systemic HIV-1 replication, but its penetration into the human central nervous system (CNS) has not been analysed. Here, we define cerebrospinal fluid (CSF) enfuvirtide pharmacokinetics and present a case illustrating the use of enfuvirtide as a probe to trace the origins of CSF HIV-1 quasispecies. METHODS: Enfuvirtide CSF pharmacokinetics were assessed in 18 CSF and plasma sample pairs from four HIV-1-infected individuals. Enfuvirtide levels were measured by liquid chromatography tandem mass spectrometry using known standards and controls that included spiked CSF samples from untreated, HIV-negative individuals. A segment of the gp41 coding region encompassing the heptad repeat HR-1 and HR-2 domains was amplified from selected CSF and plasma samples and independent clones sequenced to assess resistance-associated mutations. RESULTS: CSF and plasma samples obtained between 2 and 20 h after enfuvirtide injection showed plasma concentrations similar to previous reports (mean 3.687 SD +/- 1.828 mg/ml) with prolonged decay. By contrast, enfuvirtide in all CSF samples was below the assay detection limit of 0.025 mg/ml. In one individual, who developed a transient increase in CSF HIV-1 RNA, seven of seven CSF and plasma clones had identical enfuvirtide resistance-associated V38A mutations, suggesting that the CSF quasispecies derived from that of blood. CONCLUSIONS: Enfuvirtide penetration into CSF is negligible; thus, in clinical settings, where direct CNS drug exposure is crucial, this drug Is not likely to directly contribute to the local therapeutic effect. Enfuvirtide can be used as a tool to dissect the origin of the CNS virus.