[Show abstract][Hide abstract] ABSTRACT: Prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep are fatal neurodegenerative diseases for which there is no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrPC) into a protease resistant infectious form (PrPsc or PrPres). Both in vitro (cell culture and cell free conversion assays) and in vivo (animal) studies have demonstrated the strong dependence of this conversion process on protein sequence homology between the initial prion inoculum and the host's own cellular prion protein. The presence of non-homologous (heterologous) proteins is often inhibitory to this conversion process. We hypothesize that the presence of heterologous prion proteins from one species might therefore constitute an effective treatment for prion disease in another species. To test this hypothesis, we infected mice intracerebrally with murine adapted RML-Chandler scrapie and treated them with heterologous prion protein (purified bacterially expressed recombinant hamster prion protein) or vehicle alone. Treated animals demonstrated reduced disease associated pathology, decreased accumulation of protease-resistant disease-associated prion protein, with delayed onset of clinical symptoms and motor deficits. This was concomitant with significantly increased survival times relative to mock-treated animals. These results provide proof of principle that recombinant hamster prion proteins can effectively and safely inhibit prion disease in mice, and suggest that hamster or other non-human prion proteins may be a viable treatment for prion diseases in humans.
PLoS ONE 07/2015; 10(7):e0131993. DOI:10.1371/journal.pone.0131993 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human immunodeficiency virus type 2 (HIV-2) is often distinguished clinically by lower viral loads, reduced transmissibility, and longer asymptomatic periods than for human immunodeficiency virus type 1 (HIV-1). Differences in the mutation frequencies of HIV-1 and HIV-2 have been hypothesized to contribute to the attenuated progression of HIV-2 observed clinically.
To address this hypothesis, we performed Illumina sequencing of multiple amplicons prepared from cells infected with HIV-1 or HIV-2, resulting in ~4.7 million read pairs and the identification of ~200,000 mutations after data processing. We observed that: (1) HIV-2 displayed significantly lower total mutation, substitution, and transition mutation frequencies than that of HIV-1, along with a mutation spectrum markedly less biased toward G-to-A transitions, (2) G-to-A hypermutation consistent with the activity of APOBEC3 proteins was observed for both HIV-1 and HIV-2 despite the presence of Vif, (3) G-to-A hypermutation was significantly higher for HIV-1 than for HIV-2, and (4) HIV-1 and HIV-2 total mutation frequencies were not significantly different in the absence of G-to-A hypermutants.
Taken together, these data demonstrate that HIV-2 exhibits a distinct mutational spectrum and a lower mutation frequency relative to HIV-1. However, the observed differences were primarily due to reduced levels of G-to-A hypermutation for HIV-2. These findings suggest that HIV-2 may be less susceptible than HIV-1 to APOBEC3-mediated hypermutation, but that the fidelities of other mutational sources (such as reverse transcriptase) are relatively similar for HIV-1 and HIV-2. Overall, these data imply that differences in replication fidelity are likely not a major contributing factor to the unique clinical features of HIV-2 infection.
[Show abstract][Hide abstract] ABSTRACT: Measures to prevent sexual mucosal transmission are critically needed, particularly to prevent transmission to young women at high risk in the microepidemics in South Africa that disproportionally contribute to the continued pandemic. To that end, microbicides containing anti-retroviral (ARV) agents have been shown to prevent transmission, but with efficacy limited both by adherence and pre-existing innate immune and inflammatory conditions in the female reproductive tract (FRT). Glycerol monolaurate (GML) has been proposed as a microbicide component to enhance efficacy by blocking these transmission-facilitating innate immune response to vaginal exposure. We show here in an especially rigorous test of protection in the SIV-rhesus macaque model of HIV-1 transmission to women, that GML used daily and before vaginal challenge protects against repeat high doses of SIV by criteria that include virological and immunological assays to detect occult infection. We also provide evidence for indirect mechanisms of action in GML-mediated protection. Developing a sustained formulation for GML delivery could contribute an independent, complementary protective component to an ARV-containing microbicide.
PLoS ONE 06/2015; 10(6):e0129465. DOI:10.1371/journal.pone.0129465 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here we show how one can decompose the contribution of different levels of taxonomic classification in terms of their impact on differences in the microbiota when comparing two groups. First we demonstrate a difficulty in trying to estimate taxonomic effects at multiple levels simultaneously and demonstrate an approach to determining which taxa have differences in means that are identified. We then develop a model based on an approach that is popular in the RNA-Seq analysis literature and apply it to the problem of determining which taxa differ between two patient groups. This model provides a more powerful method than simpler alternatives. A Bayesian computational strategy is used to obtain exact inference. Simulation studies indicate that the procedure works as intended, and an application to the study of COPD demonstrates the method's practical utility. Software is provided for implementing the method.
Journal of computational biology: a journal of computational molecular cell biology 05/2015; 22(10). DOI:10.1089/cmb.2015.0021 · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antiretroviral therapy (ART) suppresses HIV replication in most individuals but cannot eradicate latently infected cells established before ART was initiated. Thus, infection rebounds when treatment is interrupted by reactivation of virus production from this reservoir. Currently, one or a few latently infected resting memory CD4 T cells are thought be the principal source of recrudescent infection, but this estimate is based on peripheral blood rather than lymphoid tissues (LTs), the principal sites of virus production and persistence before initiating ART. We, therefore, examined lymph node (LN) and gut-associated lymphoid tissue (GALT) biopsies from fully suppressed subjects, interrupted therapy, monitored plasma viral load (pVL), and repeated biopsies on 12 individuals as soon as pVL became detectable. Isolated HIV RNA-positive (vRNA+) cells were detected by in situ hybridization in LTs obtained before interruption in several patients. After interruption, multiple foci of vRNA+ cells were detected in 6 of 12 individuals as soon as pVL was measureable and in some subjects, in more than one anatomic site. Minimal estimates of the number of rebounding/founder (R/F) variants were determined by single-gene amplification and sequencing of viral RNA or DNA from peripheral blood mononuclear cells and plasma obtained at or just before viral recrudescence. Sequence analysis revealed a large number of R/F viruses representing recrudescent viremia from multiple sources. Together, these findings are consistent with the origins of recrudescent infection by reactivation from many latently infected cells at multiple sites. The inferred large pool of cells and sites to rekindle recrudescent infection highlights the challenges in eradicating HIV.
Proceedings of the National Academy of Sciences 02/2015; 112(10). DOI:10.1073/pnas.1414926112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human immunodeficiency virus (HIV) replication causes lymphoid tissue (LT) fibrosis, which causes CD4+ T-cell depletion. It is unknown whether people who spontaneously control HIV replication have LT fibrosis. We measured LT
fibrosis and CD4+ T cells in 25 HIV controllers, 10 noncontrollers, 45 HIV-positive individuals receiving therapy, and 10 HIV-negative individuals.
Controllers had significant LT fibrosis and CD4+ T-cell depletion, similar to noncontrollers, but the so-called Berlin patient (in whom HIV infection was cured) had near
normal LT. Thus, LT fibrosis occurs in all HIV-infected subjects, and current therapy does not reverse it. Reversal of fibrosis
during a curative intervention suggests that ongoing low-level virus production may maintain LT fibrosis.
The Journal of Infectious Diseases 10/2014; 211(7). DOI:10.1093/infdis/jiu586 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Cystic fibrosis (CF) patients exhibit a progressive decline in lung function accelerated by intermittent pulmonary exacerbations. There are urgent needs for clinically relevant biomarkers to aid in the diagnosis and management of a CF pulmonary exacerbation, in addition to providing insight into its pathophysiology. Club cell secretory protein (CCSP) is produced by bronchial epithelial cells, known to have anti-inflammatory properties and may play a role in CF pulmonary exacerbations. Our objective was to measure sputum CCSP concentration during hospitalizations for CF pulmonary exacerbation and during quarterly outpatient clinic visits for 2 years. We explored the correlations between CCSP concentration, lung function and markers of inflammation and infection.
In this prospective, longitudinal cohort study, expectorated sputum, blood and lung function data were collected from 45 CF patients during 68 hospitalizations for pulmonary exacerbation and 193 clinic visits. Sputum CCSP concentration was measured and sputum and blood were assayed with a panel of inflammatory cytokines. We used a repeated measures model to compare log transformed sputum CCSP concentrations across multiple time points and to correlate those concentrations with related clinical variables.
Our population had a mean age of 29 (16-58 years), and a median FEV(1) %predicted of 60% (18-105%). Sputum CCSP concentration was significantly lower in the initial, interim and final exacerbation samples (p=0.0021, p=0.0005 and p=0.0274, respectively) compared to outpatient visits. Sputum CCSP concentration was negatively associated with sputum neutrophil elastase concentration (p=0.0373). Patients with Pseudomonas aeruginosa mucoid had a significantly lower sputum CCSP concentration (p=0.0129).
Sputum CCSP concentration is associated with CF pulmonary exacerbation.
[Show abstract][Hide abstract] ABSTRACT: Even with prolonged antiretroviral therapy (ART), many human immunodeficiency virus-infected individuals have <500 CD4+ T cells/µL, and CD4+ T cells in lymphoid tissues remain severely depleted, due in part to fibrosis of the paracortical T-cell zone (TZ) that impairs
homeostatic mechanisms required for T-cell survival. We therefore used antifibrotic therapy in simian immunodeficiency virus-infected
rhesus macaques to determine whether decreased TZ fibrosis would improve reconstitution of peripheral and lymphoid CD4+ T cells. Treatment with the antifibrotic drug pirfenidone preserved TZ architecture and was associated with significantly
larger populations of CD4+ T cells in peripheral blood and lymphoid tissues. Combining pirfenidone with an ART regimen was associated with greater preservation
of CD4+ T cells than ART alone and was also associated with higher pirfenidone concentrations. These data support a potential role
for antifibrotic drug treatment as adjunctive therapy with ART to improve immune reconstitution.
The Journal of Infectious Diseases 09/2014; 211(5). DOI:10.1093/infdis/jiu519 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Principles to guide design of an effective vaccine against HIV are greatly needed, particularly to protect women in the pandemic's epicenter in Africa. We have been seeking these principles by identifying correlates of the robust protection associated with SIVmac239Δnef vaccination in the SIV-rhesus macaque animal model of HIV-1 transmission to women. We identified one correlate of SIVmac239Δnef protection against vaginal challenge as a resident mucosal system for SIV-gp41 trimer Ab production and neonatal FcR-mediated concentration of these Abs on the path of virus entry to inhibit establishment of infected founder populations at the portal of entry. In this study, we identify blocking CD4(+) T cell recruitment to thereby inhibit local expansion of infected founder populations as a second correlate of protection. Virus-specific immune complex interactions with the inhibitory FcγRIIb receptor in the epithelium lining the cervix initiate expression of genes that block recruitment of target cells to fuel local expansion. Immune complex-FcγRIIb receptor interactions at mucosal frontlines to dampen the innate immune response to vaginal challenge could be a potentially general mechanism for the mucosal immune system to sense and modulate the response to a previously encountered pathogen. Designing vaccines to provide protection without eliciting these transmission-promoting innate responses could contribute to developing an effective HIV-1 vaccine.
The Journal of Immunology 08/2014; 193(6). DOI:10.4049/jimmunol.1400822 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We sought design principles for a vaccine to prevent HIV transmission to women by identifying correlates of protection conferred by a highly effective live attenuated SIV vaccine in the rhesus macaque animal model. We show that SIVmac239Δnef vaccination recruits plasma cells and induces ectopic lymphoid follicle formation beneath the mucosal epithelium in the rhesus macaque female reproductive tract. The plasma cells and ectopic follicles produce IgG Abs reactive with viral envelope glycoprotein gp41 trimers, and these Abs are concentrated on the path of virus entry by the neonatal FcR in cervical reserve epithelium and in vaginal epithelium. This local Ab production and delivery system correlated spatially and temporally with the maturation of local protection against high-dose pathogenic SIV vaginal challenge. Thus, designing vaccines to elicit production and concentration of Abs at mucosal frontlines could aid in the development of an effective vaccine to protect women against HIV-1.
The Journal of Immunology 08/2014; 193(6). DOI:10.4049/jimmunol.1400820 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alternative polyadenylation (APA) is an evolutionarily conserved mechanism for regulating gene expression. Transcript 3' end shortening through changes in polyadenylation site usage occurs following T cell activation, but the consequences of APA on gene expression are poorly understood. We previously showed that GU-rich elements (GREs) found in the 3' untranslated regions of select transcripts mediate rapid mRNA decay by recruiting the protein CELF1/CUGBP1. Using a global RNA sequencing approach, we found that a network of CELF1 target transcripts involved in cell division underwent preferential 3' end shortening via APA following T cell activation, resulting in decreased inclusion of CELF1 binding sites and increased transcript expression. We present a model whereby CELF1 regulates APA site selection following T cell activation through reversible binding to nearby GRE sequences. These findings provide insight into the role of APA in controlling cellular proliferation during biological processes such as development, oncogenesis and T cell activation.
[Show abstract][Hide abstract] ABSTRACT: Antiretroviral therapy can reduce HIV-1 to undetectable levels in peripheral blood, but the effectiveness of treatment in suppressing replication in lymphoid tissue reservoirs has not been determined. Here we show in lymph node samples obtained before and during 6 mo of treatment that the tissue concentrations of five of the most frequently used antiretroviral drugs are much lower than in peripheral blood. These lower concentrations correlated with continued virus replication measured by the slower decay or increases in the follicular dendritic cell network pool of virions and with detection of viral RNA in productively infected cells. The evidence of persistent replication associated with apparently suboptimal drug concentrations argues for development and evaluation of novel therapeutic strategies that will fully suppress viral replication in lymphatic tissues. These strategies could avert the long-term clinical consequences of chronic immune activation driven directly or indirectly by low-level viral replication to thereby improve immune reconstitution.
Proceedings of the National Academy of Sciences 01/2014; 111(6). DOI:10.1073/pnas.1318249111 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of this discovery-level investigation was to use mass spectrometry to identify low mass compounds in bronchoalveolar lavage fluid from lung transplant recipients that associate with bronchiolitis obliterans syndrome.
Bronchoalveolar lavage fluid samples from lung transplant recipients were evaluated for small molecules using ESI-TOF mass spectrometry and correlated to the development of bronchiolitis obliterans syndrome. Peptides associated with samples from persons with bronchiolitis obliterans syndrome and controls were identified separately by MS/MS analysis.
The average bronchoalveolar lavage fluid MS spectrum profile of individuals that developed bronchiolitis obliterans syndrome differed greatly compared to controls. Controls demonstrated close inter-sample correlation (R = 0.97+/-0.02, average+/-SD) while bronchiolitis obliterans syndrome showed greater heterogeneity (R = 0.86+/-0.09, average+/-SD). We identified 89 features that were predictive of developing BOS grade 1 and 66 features predictive of developing BOS grade 2 or higher. Fractions from MS analysis were pooled and evaluated for peptide content. Nearly 10-fold more peptides were found in bronchiolitis obliterans syndrome relative to controls. C-terminal residues suggested trypsin-like specificity among controls compared to elastase-type enzymes among those with bronchiolitis obliterans syndrome.
Bronchoalveolar lavage fluid from individuals with bronchiolitis obliterans syndrome has an increase in low mass components detected by mass spectrometry. Many of these features were peptides that likely result from elevated neutrophil elastase activity.
PLoS ONE 01/2014; 9(1):e84471. DOI:10.1371/journal.pone.0084471 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Even with prolonged antiretroviral therapy (ART), many human immunodeficiency virus-infected individuals have <500 >CD4+ T cells/μL, and CD4+ T cells in lymphoid tissues remain severely depleted, due in part to fibrosis of the paracortical T-cell zone (TZ) that impairs homeostatic mechanisms required for T-cell survival.We therefore used antifibrotic therapy in simian immunodeficiency virus-infected rhesus macaques to determine whether decreased TZ fibrosis would improve reconstitution of peripheral and lymphoid CD4+ T cells. Treatment with the antifibrotic drug pirfenidone preserved TZ architecture and was associated with significantly larger populations of CD4+ T cells in peripheral blood and lymphoid tissues. Combining pirfenidone with an ART regimen was associated with greater preservation of CD4+ T cells than ART alone and was also associated with higher pirfenidone concentrations. These data support a potential role for antifibrotic drug treatment as adjunctive therapy with ART to improve immune reconstitution.
[Show abstract][Hide abstract] ABSTRACT: Live-attenuated SIV vaccines (LAVs) have been the most effective to date in preventing or partially controlling infection by wild-type SIV in non-human primate models of HIV-1 transmission to women acting by mechanisms of protection that are not well understood. To gain insights into mechanisms of protection by LAVs that could aid development of effective vaccines to prevent HIV-1 transmission to women, we used in situ tetramer staining to determine whether increased densities or changes in the local distribution of SIV-specific CD8 T cells correlated with the maturation of SIVΔnef vaccine-induced protection prior to and after intra-vaginal challenge with wild-type SIVmac251. We evaluated the immunodominant Mamu-A1*001:01/Gag (CM9) and Mamu-A1*001:01/Tat (SL8) epitope response in genital and lymphoid tissues, and found that tetramer+ cells were present at all time points examined. In the cervical vaginal tissues, most tetramer+ cells were distributed diffusely throughout the lamina propria or co-localized with other CD8 T cells within lymphoid aggregates. The distribution and densities of the tetramer+ cells at the portal of entry did not correlate with the maturation of protection or change after challenge. Given these findings, we discuss the possibility that changes in other aspects of the immune system, including the quality of the resident population of virus-specific effector CD8 T cells could contribute to maturation of protection, as well as the potential for vaccine strategies that further increase the size and quality of this effector population to prevent HIV-1 transmission.
PLoS ONE 12/2013; 8(12):e81623. DOI:10.1371/journal.pone.0081623 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Management goals for HIV are shifting from chronic viral suppression to functional cure; i.e. control of HIV in the absence of combination antiretroviral therapy (cART). Assessment of such novel treatments will require interruption of standard cART, yet structured treatment interruptions (STIs) have generally been avoided given safety concerns. This study was designed to assess the safety, as determined by resumption of virologic control after treatment re-initiation, maintenance of CD4 T cell count, and lack of clinical adverse events, of a short-term STI in patients with preserved CD4 T cell count and virologic suppression on cART.
Methods: 14 HIV-infected individuals with CD4 count >350 cells/mm3 and virologic suppression on cART were enrolled in an STI treatment protocol designed to characterize HIV reservoirs. All subjects underwent interruption of cART at study initiation. Peripheral blood CD4 count and HIV viral load were assessed 3 times weekly while off of cART. Once HIV RNA was detected in peripheral blood, samples for the reservoir study were collected, genotype was obtained and cART was restarted. CD4 count and HIV viral load were followed weekly until viral load was undetectable then intermittently for up to 90 days.
Results: After cART discontinuation, median time to HIV viremia was 14 days (range 5-29). At cART re-initiation, median viral load was 871 copies/mL (60-23,402 copies/mL). Longer duration of HIV infection and lower reported CD4 nadir were associated with more rapid virologic rebound (p=0.019 and 0.016). Longer lifetime exposure to ART was associated with more prolonged time to viremia (p=0.029). Once cART was restarted, all subjects achieved peripheral blood viral suppression, with median time to suppression being 14 days (7-47). CD4 count at treatment resumption did not differ from study initiation (p=0.18), no new resistance-conferring mutations were detected in any subject and none had symptoms associated with reactivation of viral replication. No subjects developed virologic failure during post-STI follow up period.
Conclusion: Brief interruption of cART in patients with preserved CD4 count and virologic suppression is safe for the evaluation of novel therapies to test for cure of HIV.
IDWeek 2012 Meeting of the Infectious Diseases Society of America; 10/2012