Within-Host Evolution Of Hiv-1: Novel Pathways Of Virus Escape From Cellular And Humoral Immunity

Longitudinal HIV-1 single genome sequencing (SGS), which permits unambiguous genetic characterization of circulating viral strains without introduction of PCR error, can be used to identify sites in the viral genome that are under selective pressure. Following transmission, the earliest sites under positive selection often fall in cytotoxic T lymphocyte (CTL) epitopes. During escape from CTL immune pressure, viral sequences typically exhibit nonsynonymous mutations within the span of the cognate T cell epitope. I applied SGS to study sequence evolution in the HIV-1 5’ leader sequence, which is thought to be translationally silent. I observed mutational patterns consistent with CTL escape and demonstrated that the HIV-1 5’ leader expresses T cell antigens from non-canonical one-off AUG codons (e.g. CUG). While these non-canonical start codons can be mutated during CTL escape, a reverse transcriptase overextension error periodically restores a one-off AUG within the 5’ leader. As infection ensues, sites under selection within the gene encoding the viral envelope glycoprotein (Env) often fall within autologous neutralizing antibody epitopes. In a subset of individuals, the strain-specific neutralizing antibody response develops into a broadly cross-reactive neutralizing antibody (bnAb) response. To understand what factors influence bnAb ontogeny, I used SGS to study Env evolution both during natural infection and immunotherapy. I found viral diversification in bnAb contact residues and divergence of the virus population into multiple persistent lineages to precede bnAb development. Taken together, these data demonstrate that longitudinal HIV-1 SGS can be used to discover novel aspects of virus biology and host-pathogen interactions

Computer program for Bessel and Hankel functions

A set of FORTRAN subroutines for calculating Bessel and Hankel functions is presented. The routines calculate Bessel and Hankel functions of the first and second kinds, as well as their derivatives, for wide ranges of integer order and real or complex argument in single or double precision. Depending on the order and argument, one of three evaluation methods is used: the power series definition, an Airy function expansion, or an asymptotic expansion. Routines to calculate Airy functions and their derivatives are also included

Modeling and Simulation of Coating Growth on Nanofibers

This work presents modeling and simulation results of a procedure to coat nanofibers and core-clad nanostructures with thin film materials using plasma enhanced physical vapor deposition. In the experimental effort that motivates the modeling, electrospun polymer nanofibers are coated with metallic materials under different operating conditions to observe changes in the coating morphology. The modeling effort focuses on linking simple models at the reactor, nanofiber, and atomic levels to form a comprehensive model. Numerical simulations that link the concentration field with the evolution of the coating free surface predict that as the Damkohler number is increased the coating morphology changes from a wavy to a nodular to a dendritic needle-type form as observed experimentally

Modeling, Simulation, and Experiments of Coating Growth on Nanofibers

This work is a comparison of modeling and simulation results with experiments for an integrated experimental/modeling investigation of a procedure to coat nanofibers and core-clad nanostructures with thin film materials using plasma enhanced physical vapor deposition. In the experimental effort, electrospun polymer nanofibers are coated with metallic materials under different operating conditions to observe changes in the coating morphology. The modeling effort focuses on linking simple models at the reactor level, nanofiber level and atomic level to form a comprehensive model. The comprehensive model leads to the definition of an evolution equation for the coating free surface around an isolated nanofiber. This evolution equation was previously derived and solved under conditions of a nearly circular coating, with a concentration field that was only radially dependent and that was independent of the location of the coating free surface. These assumptions permitted the development of analytical expressions for the concentration field. The present work does not impose the above-mentioned conditions and considers numerical simulations of the concentration field that couple with level set simulations of the evolution equation for the coating free surface. Further, the cases of coating an isolated fiber as well as a multiple fiber mat are considered. Simulation results are compared with experimental results as the reactor pressure and power, as well as the nanofiber mat porosity, are varied. (C) 2008 American Institute of Physics

Effects of Calcium β-HMB Supplementation During Training on Markers of Catabolism, Body Composition, Strength and Sprint Performance

Calcium β-hydroxy β-methylbutyrate (HMB) supplementation has been reported to reduce catabolism and promote gains in strength and fat free mass in untrained individuals initiating training. However, the effects of HMB supplementation on strength and body composition alterations during training in athletes is less clear. This study examined the effects of 28-d of calcium HMB supplementation during intense training on markers of catabolism, body composition, strength, and sprint performance. In a double-blind and randomized manner, 28 NCAA division I-A football players were matched-paired and assigned to supplement their diet for 28-d during winter resistance/agility training (~8 hr/wk) with a carbohydrate placebo supplement (P) or the P supplement with 3 g/day of HMB as a calcium salt (HMB). Prior to and following supplementation: dietary records and fasting blood samples were obtained; body composition was determined via DEXA; subjects performed maximal effort bench press, barbell back squat, and power clean isotonic repetition tests; and, subjects performed a repeated cycle ergometer sprint test (12 x 6-s sprints with 30-s rest recovery) to simulate a 12-play drive in football. Results revealed no significant differences between the placebo and HMB supplemented groups in markers of catabolism, muscle/liver enzyme efflux, hematological parameters, body composition, combined lifting volume, or repetitive sprint performance. Results indicate that HMB supplementation (3 g/day) during off-season college football resistance/agility training does not reduce catabolism or provide ergogenic benefit

Multiscale Modeling, Simulations, and Experiments of Coating Growth on Nanofibers. Part Ii. Deposition

This work is Part II of an integrated experimental/modeling investigation of a procedure to coat nanofibers and core-clad nanostructures with thin-film materials using plasma-enhanced physical vapor deposition. In the experimental effort, electrospun polymer nanofibers are coated with aluminum materials under different operating conditions to observe changes in the coating morphology. This procedure begins with the sputtering of the coating material from a target. Part I [J. Appl. Phys. 98, 044303 (2005)] focused on the sputtering aspect and transport of the sputtered material through the reactor. That reactor level model determines the concentration field of the coating material. This field serves as input into the present species transport and deposition model for the region surrounding an individual nanofiber. The interrelationships among processing factors for the transport and deposition are investigated here from a detailed modeling approach that includes the salient physical and chemical phenomena. Solution strategies that couple continuum and atomistic models are used. At the continuum scale, transport dynamics near the nanofiber are described. At the atomic level, molecular dynamics (MD) simulations are used to study the deposition and sputtering mechanisms at the coating surface. Ion kinetic energies and fluxes are passed from the continuum sheath model to the MD simulations. These simulations calculate sputtering and sticking probabilities that in turn are used to calculate parameters for the continuum transport model. The continuum transport model leads to the definition of an evolution equation for the coating-free surface. This equation is solved using boundary perturbation and level set methods to determine the coating morphology as a function of operating conditions. (c) 2005 American Institute of Physics

Multiscale Modeling, Simulations, and Experiments of Coating Growth on Nanofibers. Part I. Sputtering

This paper is Part I of an integrated experimental/modeling investigation of a procedure to coat nanofibers and core-clad nanostructures with thin-film materials using plasma-enhanced physical vapor deposition. In the experimental effort, electrospun polymer nanofibers are coated with aluminum under varying operating conditions to observe changes in the coating morphology. This procedure begins with the sputtering of the coating material from a target. This paper focuses on the sputtering process and transport of the sputtered material through the reactor. The interrelationships among the processing factors for the sputtering and transport are investigated from a detailed modeling approach that describes the salient physical and chemical phenomena. Solution strategies that couple continuum and atomistic models are used. At the continuum scale, the sheath region and the reactor dynamics near the target surface are described. At the atomic level, molecular-dynamics (MD) simulations are used to study the sputtering and deposition mechanisms. Ion kinetic energies and fluxes are passed from the continuum sheath model to the MD simulations. These simulations calculate sputtering and sticking probabilities that in turn are used to calculate parameters for the continuum reactor model. The reactor model determines the concentration field of the coating material. (c) 2005 American Institute of Physics

Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA

HIV-1-infected individuals harbor a latent reservoir of infected CD4⁺ T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones

Staged induction of HIV-1 glycan–dependent broadly neutralizing antibodies

A preventive HIV-1 vaccine should induce HIV-1–specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies have not been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third variable loop (V3) of the HIV-1 envelope protein require limited SHM, the V3-glycan epitope is an attractive vaccine target. By studying the cooperation among multiple V3-glycan B cell lineages and their coevolution with autologous virus throughout 5 years of infection, we identify key events in the ontogeny of a V3-glycan bnAb. Two autologous neutralizing antibody lineages selected for virus escape mutations and consequently allowed initiation and affinity maturation of a V3-glycan bnAb lineage. The nucleotide substitution required to initiate the bnAb lineage occurred at a low-probability site for activation-induced cytidine deaminase activity. Cooperation of B cell lineages and an improbable mutation critical for bnAb activity defined the necessary events leading to breadth in this V3-glycan bnAb lineage. These findings may, in part, explain why initiation of V3-glycan bnAbs is rare, and suggest an immunization strategy for inducing similar V3-glycan bnAbs

Effective Treatment of SIVcpz-Induced Immunodeficiency in a Captive Western Chimpanzee

Abstract Background Simian immunodeficiency virus of chimpanzees (SIVcpz), the progenitor of human immunodeficiency virus type 1 (HIV-1), is associated with increased mortality and AIDS-like immunopathology in wild-living chimpanzees (Pan troglodytes). Surprisingly, however, similar findings have not been reported for chimpanzees experimentally infected with SIVcpz in captivity, raising questions about the intrinsic pathogenicity of this lentivirus. Findings Here, we report progressive immunodeficiency and clinical disease in a captive western chimpanzee (P. t. verus) infected twenty years ago by intrarectal inoculation with an SIVcpz strain (ANT) from a wild-caught eastern chimpanzee (P. t. schweinfurthii). With sustained plasma viral loads of 105 to 106 RNA copies/ml for the past 15 years, this chimpanzee developed CD4+ T cell depletion (220 cells/μl), thrombocytopenia (90,000 platelets/μl), and persistent soft tissue infections refractory to antibacterial therapy. Combination antiretroviral therapy consisting of emtricitabine (FTC), tenofovir disoproxil fumarate (TDF), and dolutegravir (DTG) decreased plasma viremia to undetectable levels (<200 copies/ml), improved CD4+ T cell counts (509 cell/μl), and resulted in the rapid resolution of all soft tissue infections. However, initial lack of adherence and/or differences in pharmacokinetics led to low plasma drug concentrations, which resulted in transient rebound viremia and the emergence of FTC resistance mutations (M184V/I) identical to those observed in HIV-1 infected humans. Conclusions These data demonstrate that SIVcpz can cause immunodeficiency and other hallmarks of AIDS in captive chimpanzees, including P. t. verus apes that are not naturally infected with this virus. Moreover, SIVcpz-associated immunodeficiency can be effectively treated with antiretroviral therapy, although sufficiently high plasma concentrations must be maintained to prevent the emergence of drug resistance. These findings extend a growing body of evidence documenting the immunopathogenicity of SIVcpz and suggest that experimentally infected chimpanzees may benefit from clinical monitoring and therapeutic intervention