Major and Minor Group Rhinoviruses Elicit Differential Signaling and Cytokine Responses as a Function of Receptor-Mediated Signal Transduction

Major- and minor-group human rhinoviruses (HRV) enter their host by binding to the cell surface molecules ICAM-1 and LDL-R, respectively, which are present on both macrophages and epithelial cells. Although epithelial cells are the primary site of productive HRV infection, previous studies have implicated macrophages in establishing the cytokine dysregulation that occurs during rhinovirus-induced asthma exacerbations. Analysis of the transcriptome of primary human macrophages exposed to major- and minor-group HRV demonstrated differential gene expression. Alterations in gene expression were traced to differential mitochondrial activity and signaling pathway activation between two rhinovirus serotypes, HRV16 (major-group) and HRV1A (minor-group), upon initial HRV binding. Variances in phosphorylation of kinases (p38, JNK, ERK5) and transcription factors (ATF-2, CREB, CEBP-alpha) were observed between the major- and minor-group HRV treatments. Differential activation of signaling pathways led to changes in the production of the asthma-relevant cytokines CCL20, CCL2, and IL-10. This is the first report of genetically similar viruses eliciting dissimilar cytokine release, transcription factor phosphorylation, and MAPK activation from macrophages, suggesting that receptor use is a mechanism for establishing the inflammatory microenvironment in the human airway upon exposure to rhinovirus

Viral Linkage in HIV-1 Seroconverters and Their Partners in an HIV-1 Prevention Clinical Trial

Characterization of viruses in HIV-1 transmission pairs will help identify biological determinants of infectiousness and evaluate candidate interventions to reduce transmission. Although HIV-1 sequencing is frequently used to substantiate linkage between newly HIV-1 infected individuals and their sexual partners in epidemiologic and forensic studies, viral sequencing is seldom applied in HIV-1 prevention trials. The Partners in Prevention HSV/HIV Transmission Study (ClinicalTrials.gov #NCT00194519) was a prospective randomized placebo-controlled trial that enrolled serodiscordant heterosexual couples to determine the efficacy of genital herpes suppression in reducing HIV-1 transmission; as part of the study analysis, HIV-1 sequences were examined for genetic linkage between seroconverters and their enrolled partners.We obtained partial consensus HIV-1 env and gag sequences from blood plasma for 151 transmission pairs and performed deep sequencing of env in some cases. We analyzed sequences with phylogenetic techniques and developed a Bayesian algorithm to evaluate the probability of linkage. For linkage, we required monophyletic clustering between enrolled partners' sequences and a Bayesian posterior probability of ≥ 50%. Adjudicators classified each seroconversion, finding 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) indeterminate transmissions, with linkage determined by consensus env sequencing in 91 (84%). Male seroconverters had a higher frequency of unlinked transmissions than female seroconverters. The likelihood of transmission from the enrolled partner was related to time on study, with increasing numbers of unlinked transmissions occurring after longer observation periods. Finally, baseline viral load was found to be significantly higher among linked transmitters.In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner, illustrating the relevance of these methods in the design of future HIV-1 prevention trials in serodiscordant couples. A hierarchy of sequencing techniques, analysis methods, and expert adjudication contributed to the linkage determination process

HIV-2 Integrase Variation in Integrase Inhibitor-Naïve Adults in Senegal, West Africa

Antiretroviral therapy for HIV-2 infection is hampered by intrinsic resistance to many of the drugs used to treat HIV-1. Limited studies suggest that the integrase inhibitors (INIs) raltegravir and elvitegravir have potent activity against HIV-2 in culture and in infected patients. There is a paucity of data on genotypic variation in HIV-2 integrase that might confer intrinsic or transmitted INI resistance.We PCR amplified and analyzed 122 HIV-2 integrase consensus sequences from 39 HIV-2-infected, INI-naive adults in Senegal, West Africa. We assessed genetic variation and canonical mutations known to confer INI-resistance in HIV-1.No amino acid-altering mutations were detected at sites known to be pivotal for INI resistance in HIV-1 (integrase positions 143, 148 and 155). Polymorphisms at several other HIV-1 INI resistance-associated sites were detected at positions 72, 95, 125, 154, 165, 201, 203, and 263 of the HIV-2 integrase protein.Emerging genotypic and phenotypic data suggest that HIV-2 is susceptible to the new class of HIV integrase inhibitors. We hypothesize that intrinsic HIV-2 integrase variation at "secondary" HIV-1 INI-resistance sites may affect the genetic barrier to HIV-2 INI resistance. Further studies will be needed to assess INI efficacy as part of combination antiretroviral therapy in HIV-2-infected patients

HIV-1 outcompetes HIV-2 in dually infected Senegalese subjects with low CD4 counts

Thesis (Master's)--University of Washington, 2012Dual infection with HIV-1 and HIV-2, which is not uncommon in West Africa, has important implications for transmission, progression, and antiretroviral therapy. Few studies have examined HIV viral dynamics in this setting. We compared HIV-1 and HIV-2 viral loads from 65 dually infected, antiretroviral therapy-naïve Senegalese subjects. Participants provided demographic information and blood, oral fluid, and cervicovaginal lavage (CVL) or semen samples for virologic and immunologic testing. Associations between HIV-1 and HIV-2 levels in plasma, PBMC, oral and genital samples were assessed using linear regression models with generalized estimating equations to account for subjects with multiple samples over time. In analyses adjusting for CD4 count, age, sex, and commercial sex work, HIV-1 RNA levels were significantly higher than HIV-2 levels in semen, CVL, and oral fluids. HIV-1 and HIV-2 PBMC viral DNA loads were similar in those with CD4 counts above 500 cells/μl. However, compared to those with high CD4 counts, subjects with CD4 counts below 500 cells/μl had higher HIV-1 and lower HIV-2 DNA levels. In plasma, subjects with CD4 counts above 500 cells/&mul had mean HIV-1 plasma RNA viral loads approximately one log10 copies/ml higher than HIV-2, with HIV-1 levels significantly higher and HIV 2 levels showing a trend toward lower mean viral loads among subjects with CD4 counts below 500 cells/μl. Our data are consistent with the hypothesis that with decreasing CD4 counts and HIV disease progression, HIV-1 outcompetes HIV-2 in dually-infected individuals. This finding may help explain the differences in epidemiology between HIV-1, HIV-2, and HIV-dual infection

Three Main Mutational Pathways in HIV-2 Lead to High- Level Raltegravir and Elvitegravir Resistance: Implications for Emerging HIV-2 Treatment Regimens

Human immunodeficiency virus type 2 (HIV-2) is intrinsically resistant to non-nucleoside reverse transcriptase inhibitors and exhibits reduced susceptibility to several of the protease inhibitors used for antiretroviral therapy of HIV-1. Thus, there is a pressing need to identify new classes of antiretroviral agents that are active against HIV-2. Although recent data suggest that the integrase strand transfer inhibitors raltegravir and elvitegravir may be beneficial, mutations that are known to confer resistance to these drugs in HIV-1 have been reported in HIV-2 sequences from patients receiving raltegravir-containing regimens. To examine the phenotypic effects of mutations that emerge during raltegravir treatment, we constructed a panel of HIV-2 integrase variants using site-directed mutagenesis and measured the susceptibilities of the mutant strains to raltegravir and elvitegravir in culture. The effects of single and multiple amino acid changes on HIV-2 replication capacity were also evaluated. Our results demonstrate that secondary replacements in the integrase protein play key roles in the development of integrase inhibitor resistance in HIV-2. Collectively, our data define three major mutational pathways to high-level raltegravir and elvitegravir resistance: i) E92Q+Y143C or T97A+Y143C, ii) G140S+Q148R, and iii) E92Q+N155H. These findings preclude the sequential use of raltegravir and elvitegravir (or vice versa) for HIV-2 treatmen

Dose-response plots from spreading-infection assays of GSK232 antiviral activity, which were performed in CEMss cells.

Data points indicate the amount of infectious virus produced in GSK232-treated cultures relative to the amount produced in cultures that received solvent only (no-drug controls). Each point is the mean of four cultures that were maintained in parallel. Error bars indicate ±1 SD and, when not visible, are smaller than the symbols. IC50 values were calculated for the HIV-1 isolates using a four-parameter regression model in GraphPad Prism 6.0 as described in the Materials and Methods; the regression curves are shown as dashed lines. R2 values are also shown as calculated in Prism.</p

Cytotoxicity of GSK232 in CEMss, MAGIC-5A, and 293T/17 cell cultures.

Cytotoxicity of GSK232 in MAGIC-5A, 293T/17, and CEMss cells. ATP levels in cell lysates were quantified using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega Corp, Madison, WI) as described in the Materials & methods. Luminescence was quantified using a Victor3 Multi-Label plate reader (PerkinElmer Inc., Akron, OH). Cell viability (% of no-drug control) was calculated as the magnitude of the luminescence signal in each culture well relative to the average signal from two cultures that received solvent only. Each datum point is the mean of two values from two assay wells. Error bars indicate ±1 SD and when not visible are smaller than the symbols. (PDF)</p

Examples of dose-response plots showing the activity of GSK232 against HIV-1 in the single-cycle assay.

Data points indicate the amount of infectious virus produced in GSK232-treated 293T/17 cells relative to the amount produced in cultures that received solvent only (no-drug controls). Each point is the mean of four cultures that were maintained in parallel. Error bars indicate ±1 SD and, when not visible, are smaller than the symbols. IC50 values were calculated using a four-parameter regression model in GraphPad Prism 6.0 as described in the Materials and Methods. R2 values for the regressions are also shown as calculated in Prism. (PDF)</p

Susceptibility of HIV-1, HIV-2, and SIV isolates to GSK2838232 in spreading infections of CEMss cells.

Susceptibility of HIV-1, HIV-2, and SIV isolates to GSK2838232 in spreading infections of CEMss cells.</p

Chemical structures of the HIV-1 maturation inhibitors discussed in this study.

The structure of GSK232 was adopted from Johnson et al. (70). Bevirimat was adopted from PubChem CID entry 457928. GSK795 was based on the structure reported by Nowicka-Sans et al. (49). GSK254 was adopted from Dicker at al. (45). All structures were drawn in Marvin JS version 21.14.0 (ChemAxon Int., International; available at https://chem.nlm.nih.gov/chemidplus).</p