Mapping the complete glycoproteome of virion-derived HIV-1 gp120 provides insights into broadly neutralizing antibody binding

The surface envelope glycoprotein (SU) of Human immunodeficiency virus type 1 (HIV-1), gp120SU plays an essential role in virus binding to target CD4+ T-cells and is a major vaccine target. Gp120 has remarkably high levels of N-linked glycosylation and there is considerable evidence that this “glycan shield” can help protect the virus from antibody-mediated neutralization. In recent years, however, it has become clear that gp120 glycosylation can also be included in the targets of recognition by some of the most potent broadly neutralizing antibodies. Knowing the site-specific glycosylation of gp120 can facilitate the rational design of glycopeptide antigens for HIV vaccine development. While most prior studies have focused on glycan analysis of recombinant forms of gp120, here we report the first systematic glycosylation site analysis of gp120 derived from virions produced by infected T lymphoid cells and show that a single site is exclusively substituted with complex glycans. These results should help guide the design of vaccine immunogens

Interaction and flocculation of spherical colloids wetted by a surface-induced corona of paranematic order

Particles dispersed in a liquid crystal above the nematic-isotropic phase transition are wetted by a surface-induced corona of paranematic order. Such coronas give rise to pronounced two-particle interactions. In this article, we report details on the analytical and numerical study of these interactions published recently [Phys. Rev. Lett. 86, 3915 (2001)]. We especially demonstrate how for large particle separations the asymptotic form of a Yukawa potential arises. We show that the Yukawa potential is a surprisingly good description for the two-particle interactions down to distances of the order of the nematic coherence length. Based on this fact, we extend earlier studies on a temperature induced flocculation transition in electrostatically stabilized colloidal dispersions [Phys. Rev. E 61, 2831 (2000)]. We employ the Yukawa potential to establish a flocculation diagram for a much larger range of the electrostatic parameters, namely the surface charge density and the Debye screening length. As a new feature, a kinetically stabilized dispersion close to the nematic-isotropic phase transition is found.Comment: Revtex v4.0, 16 pages, 12 Postscript figures. Accepted for publication in Phys. Rev.

Reconstruction of a first-order phase transition from computer simulations of individual phases and subphases

We present a new method for investigating first-order phase transitions using Monte Carlo simulations. It relies on the multiple-histogram method and uses solely histograms of individual phases. In addition, we extend the method to include histograms of subphases. The free energy difference between phases, necessary for attributing the correct statistical weights to the histograms, is determined by a detour in control parameter space via auxiliary systems with short relaxation times. We apply this method to a recently introduced model for structure formation in polypeptides for which other methods fail.Comment: 13 pages in preprint mode, REVTeX, 2 Figures available from the authors ([email protected], [email protected]

Sequence randomness and polymer collapse transitions

Contrary to expectations based on Harris' criterion, chain disorder with frustration can modify the universality class of scaling at the theta transition of heteropolymers. This is shown for a model with random two-body potentials in 2D on the basis of exact enumeration and accurate Monte Carlo results. When frustration grows beyond a certain finite threshold, the temperature below which disorder becomes relevant coincides with the theta one and scaling exponents definitely start deviating from those valid for homopolymers.Comment: 4 pages, 4 eps figure

Nucleic Acid, Antibody, and Virus Culture Methods to Detect Xenotropic MLV-Related Virus in Human Blood Samples

The MLV-related retrovirus, XMRV, was recently identified and reported to be associated with both prostate cancer and chronic fatigue syndrome. At the National Cancer Institute-Frederick, MD (NCI-Frederick), we developed highly sensitive methods to detect XMRV nucleic acids, antibodies, and replication competent virus. Analysis of XMRV-spiked samples and/or specimens from two pigtail macaques experimentally inoculated with 22Rv1 cell-derived XMRV confirmed the ability of the assays used to detect XMRV RNA and DNA, and culture isolatable virus when present, along with XMRV reactive antibody responses. Using these assays, we did not detect evidence of XMRV in blood samples (N = 134) or prostate specimens (N = 19) from two independent cohorts of patients with prostate cancer. Previous studies detected XMRV in prostate tissues. In the present study, we primarily investigated the levels of XMRV in blood plasma samples collected from patients with prostate cancer. These results demonstrate that while XMRV-related assays developed at the NCI-Frederick can readily measure XMRV nucleic acids, antibodies, and replication competent virus, no evidence of XMRV was found in the blood of patients with prostate cancer

Nucleocapsid Protein Zinc-Finger Mutants of Simian Immunodeficiency Virus Strain Mne Produce Virions That Are Replication Defectivein Vitroandin Vivo

AbstractAll retroviruses (except the spumaretroviruses) contain a nucleocapsid (NC) protein that encodes one or two copies of the Zn2+-finger sequence -Cys-X2-Cys-X4-His-X4-Cys-. This region has been shown to be essential for recognition and packaging of the genomic RNA during virion particle assembly. Additionally, this region has been shown to be involved in early infection events in a wide spectrum of retroviruses, including mammalian type C [e.g., murine leukemia virus (MuLV)], human immunodeficiency virus type 1 (HIV-1),Rous sarcomavirus, and other retroviruses. Mutations in the two Zn2+-fingers of the NC protein of simian immunodeficiency virus strain Mne [SIV(Mne)] have been generated. The resulting virions contained the normal complement of processed viral proteins with densities indistinguishable from wild-type SIV(Mne). All of the mutants had electron micrograph morphologies similar to those of immature particles observed in wild-type preparations. RNA packaging was less affected by mutations in the NC protein of SIV(Mne) than has been observed for similar mutants in the MuLV and HIV-1 systems. Nevertheless,in vitroreplication of SIV(Mne) NC mutants was impaired to levels comparable to those observed for MuLV and HIV-1 NC mutants; replication defective NC mutants are typically 105- to 106-fold less infectious than similar levels of wild-type virus. One mutant, ΔCys33–Cys36, was also found to be noninfectiousin vivowhen mutant virus was administered intravenously to a pig-tailed macaque. NC mutations can therefore be used to generate replication defective virions for candidate vaccines in the SIV macaque model for primate lentiviral diseases

Ability of herpes simplex virus vectors to boost immune responses to DNA vectors and to protect against challenge by simian immunodeficiency virus

AbstractThe immunogenicity and protective capacity of replication-defective herpes simplex virus (HSV) vector-based vaccines were examined in rhesus macaques. Three macaques were inoculated with recombinant HSV vectors expressing Gag, Env, and a Tat-Rev-Nef fusion protein of simian immunodeficiency virus (SIV). Three other macaques were primed with recombinant DNA vectors expressing Gag, Env, and a Pol-Tat-Nef-Vif fusion protein prior to boosting with the HSV vectors. Robust anti-Gag and anti-Env cellular responses were detected in all six macaques. Following intravenous challenge with wild-type, cloned SIV239, peak and 12-week plasma viremia levels were significantly lower in vaccinated compared to control macaques. Plasma SIV RNA in vaccinated macaques was inversely correlated with anti-Rev ELISPOT responses on the day of challenge (P value<0.05), anti-Tat ELISPOT responses at 2 weeks post challenge (P value <0.05) and peak neutralizing antibody titers pre-challenge (P value 0.06). These findings support continued study of recombinant herpesviruses as a vaccine approach for AIDS

Outcomes of highly active antiretroviral therapy in the context of universal access to healthcare: the U.S. Military HIV Natural History Study

<p>Abstract</p> <p>Background</p> <p>To examine the outcomes of highly-active antiretroviral therapy (HAART) for individuals with free access to healthcare, we evaluated 2327 patients in a cohort study composed of military personnel and beneficiaries with HIV infection who initiated HAART from 1996 to the end of 2007.</p> <p>Methods</p> <p>Outcomes analyzed were virologic suppression (VS) and failure (VF), CD4 count changes, AIDS and death. VF was defined as never suppressing or having at least one rebound event. Multivariate (MV) analyses stratified by the HAART initiation year (before or after 2000) were performed to identify risk factors associated with these outcomes.</p> <p>Results</p> <p>Among patients who started HAART after 2000, 81% had VS at 1 year (N = 1,759), 85% at 5 years (N = 1,061), and 82% at 8 years (N = 735). Five years post-HAART, the median CD4 increase was 247 cells/ml and 34% experienced VF. AIDS and mortality rates at 5 years were 2% and 0.3%, respectively. In a MV model adjusted for known risk factors associated with treatment response, being on active duty (versus retired) at HAART initiation was associated with a decreased risk of AIDS (HR = 0.6, 95% CI 0.4-1.0) and mortality (0.6, 0.3-0.9), an increased probability of CD4 increase ≥ 50% (1.2, 1.0-1.4), but was not significant for VF.</p> <p>Conclusions</p> <p>In this observational cohort, VS rates approach those described in clinical trials. Initiating HAART on active duty was associated with even better outcomes. These findings support the notion that free access to healthcare likely improves the response to HAART thereby reducing HIV-related morbidity and mortality.</p

PKQuest: capillary permeability limitation and plasma protein binding – application to human inulin, dicloxacillin and ceftriaxone pharmacokinetics

BACKGROUND: It is generally assumed that the tissue exchange of antibiotics is flow limited (complete equilibration between the capillary and the tissue water). This assumption may not be valid if there is a large amount of plasma protein binding because the effective capillary permeability depends on the product of the intrinsic capillary permeability (PS) and the fraction of solute that is free in the blood (fw(B)). PKQuest, a new generic physiologically based pharmacokinetic software routine (PBPK), provides a novel approach to modeling capillary permeability in which the only adjustable parameter is the PS of muscle. METHODS: All the results were obtained by applying PKQuest to previously published human pharmacokinetic data. RESULTS: The PKQuest analysis suggests that the highly protein bound antibiotics dicloxacillin and ceftriaxone have a significant capillary permeability limitation. The human muscle capillary PS of inulin, dicloxacillin and ceftriaxone was 0.6, 13 and 6 ml/min/100 gm, respectively. The ceftriaxone protein binding is non-linear, saturating at high plasma concentrations. The experimental ceftriaxone data over a wide range of intravenous inputs (0.15 to 3 gms) was well described by PKQuest. PKQuest is the first PBPK that includes both permeability limitation and non-linear binding. CONCLUSIONS: Because of their high degree of plasma protein binding, dicloxacillin and ceftriaxone appear to have a diffusion limited exchange rate between the blood and tissue and are not flow limited as had been previously assumed. PKQuest and all the examples are freely available at

Loss of CD103+ DCs and Mucosal IL-17+ and IL-22+ Lymphocytes is Associated with Mucosal Damage in SIV Infection

HIV/SIV disease progression is associated with multifocal damage to the GI tract epithelial barrier that correlates with microbial translocation and persistent pathological immune activation but the underlying mechanisms remain unclear. Investigating alterations in mucosal immunity during SIV infection, we found that damage to the colonic epithelial barrier was associated with loss of multiple lineages of IL-17-producing lymphocytes, cells that microarray analysis showed express genes important for enterocyte homeostasis, including IL-22. IL-22-producing lymphocytes were also lost after SIV infection. Potentially explaining coordinate loss of these distinct populations, we also observed loss of CD103+ DCs after SIV infection which associated with loss of IL-17 and IL-22-producing lymphocytes. CD103+ DCs expressed genes associated with promotion of IL-17/IL-22+ cells, and co-culture of CD103+ DCs and naïve T-cells led to increased IL17A and RORc expression in differentiating T-cells. These results reveal complex interactions between mucosal immune cell subsets providing potential mechanistic insights into mechanisms of mucosal immune dysregulation during HIV/SIV infection, and offer hints for development of novel therapeutic strategies to address this aspect of AIDS virus pathogenesis