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While the HIV-1-glycan shield is known to shelter Env from the humoral immune response, its quantitative impact on antibody elicitation has been unclear. Here, we use targeted deglycosylation to measure the impact of the glycan shield on elicitation of antibodies against the CD4 supersite. We engineered diverse Env trimers with select glycans removed proximal to the CD4 supersite, characterized their structures and glycosylation, and immunized guinea pigs and rhesus macaques. Immunizations yielded little neutralization against wild-type viruses but potent CD4-supersite neutralization (titers 1: >1,000,000 against four-glycan-deleted autologous viruses with over 90% breadth against four-glycan-deleted heterologous strains exhibiting tier 2 neutralization character). To a first approximation, the immunogenicity of the glycan-shielded protein surface was negligible, with Env-elicited neutralization (ID) proportional to the exponential of the protein-surface area accessible to antibody. Based on these high titers and exponential relationship, we propose site-selective deglycosylated trimers as priming immunogens to increase the frequency of site-targeting antibodies.
Published by Elsevier Inc.
As observed during the 2013-2016 Ebola virus disease epidemic, containment of filovirus outbreaks is challenging and made more difficult by the lack of approved vaccine or therapeutic options. Marburg and Ravn viruses are highly virulent and cause severe and frequently lethal disease in humans. Monoclonal antibodies (mAbs) are a platform technology in wide use for autoimmune and oncology indications. Previously, we described human mAbs that can protect mice from lethal challenge with Marburg virus. We demonstrate that one of these mAbs, MR191-N, can confer a survival benefit of up to 100% to Marburg or Ravn virus-infected rhesus macaques when treatment is initiated up to 5 days post-inoculation. These findings extend the small but growing body of evidence that mAbs can impart therapeutic benefit during advanced stages of disease with highly virulent viruses and could be useful in epidemic settings.
Copyright © 2017, American Association for the Advancement of Science.
The HIV-1 envelope (Env) trimer is a target for vaccine design as well as a conformational machine that facilitates virus entry by transitioning between prefusion-closed, CD4-bound, and coreceptor-bound conformations by transitioning into a postfusion state. Vaccine designers have sought to restrict the conformation of the HIV-1 Env trimer to its prefusion-closed state as this state is recognized by most broadly neutralizing, but not nonneutralizing, antibodies. We previously identified a disulfide bond, I201C-A433C (DS), which stabilizes Env in the vaccine-desired prefusion-closed state. When placed into the context of BG505 SOSIP.664, a soluble Env trimer mimic developed by Sanders, Moore, and colleagues, the engineered DS-SOSIP trimer showed reduced conformational triggering by CD4. Here, we further stabilize DS-SOSIP through a combination of structure-based design and 96-well-based expression and antigenic assessment. From 103 designs, we identified one, named DS-SOSIP.4mut, with four additional mutations at the interface of potentially mobile domains of the prefusion-closed structure. We also determined the crystal structures of DS-SOSIP.4mut at 4.1-Å resolution and of an additional DS-SOSIP.6mut variant at 4.3-Å resolution, and these confirmed the formation of engineered disulfide bonds. Notably, DS-SOSIP.4mut elicited a higher ratio of tier 2 autologous titers versus tier 1 V3-sensitive titers than BG505 SOSIP.664. DS-SOSIP.4mut also showed reduced recognition of CD4 and increased thermostability. The improved antigenicity, thermostability, and immunogenicity of DS-SOSIP.4mut suggest utility as an immunogen or a serologic probe; moreover, the specific four alterations identified here, M154, M300, M302, and L320 (4mut), can also be transferred to other HIV-1 Env trimers of interest to improve their properties. One approach to elicit broadly neutralizing antibodies against HIV-1 is to stabilize the structurally flexible HIV-1 envelope (Env) trimer in a conformation that displays predominantly broadly neutralizing epitopes and few to no nonneutralizing epitopes. The prefusion-closed conformation of HIV-1 Env has been identified as one such preferred conformation, and a current leading vaccine candidate is the BG505 DS-SOSIP variant, comprising two disulfides and an Ile-to-Pro mutation of Env from strain BG505. Here, we introduced additional mutations to further stabilize BG505 DS-SOSIP in the vaccine-preferred prefusion-closed conformation. In guinea pigs, our best mutant, DS-SOSIP.4mut, elicited a significantly higher ratio of autologous versus V3-directed neutralizing antibody responses than the SOSIP-stabilized form. We also observed an improvement in thermostability and a reduction in CD4 affinity. With improved antigenicity, stability, and immunogenicity, DS-SOSIP.4mut-stabilized trimers may have utility as HIV-1 immunogens or in other antigen-specific contexts, such as with B-cell probes.
Copyright © 2017 American Society for Microbiology.
Recent studies have suggested that antibody-mediated protection against the Ebolaviruses may be achievable, but little is known about whether or not antibodies can confer cross-reactive protection against viruses belonging to diverse Ebolavirus species, such as Ebola virus (EBOV), Sudan virus (SUDV), and Bundibugyo virus (BDBV). We isolated a large panel of human monoclonal antibodies (mAbs) against BDBV glycoprotein (GP) using peripheral blood B cells from survivors of the 2007 BDBV outbreak in Uganda. We determined that a large proportion of mAbs with potent neutralizing activity against BDBV bind to the glycan cap and recognize diverse epitopes within this major antigenic site. We identified several glycan cap-specific mAbs that neutralized multiple ebolaviruses, including SUDV, and a cross-reactive mAb that completely protected guinea pigs from the lethal challenge with heterologous EBOV. Our results provide a roadmap to develop a single antibody-based treatment effective against multiple Ebolavirus infections.
Copyright © 2016 Elsevier Inc. All rights reserved.
Mammalian gestation and pregnancy are fast evolving processes that involve the interaction of the fetal, maternal and paternal genomes. Version 1.0 of the GEneSTATION database (http://genestation.org) integrates diverse types of omics data across mammals to advance understanding of the genetic basis of gestation and pregnancy-associated phenotypes and to accelerate the translation of discoveries from model organisms to humans. GEneSTATION is built using tools from the Generic Model Organism Database project, including the biology-aware database CHADO, new tools for rapid data integration, and algorithms that streamline synthesis and user access. GEneSTATION contains curated life history information on pregnancy and reproduction from 23 high-quality mammalian genomes. For every human gene, GEneSTATION contains diverse evolutionary (e.g. gene age, population genetic and molecular evolutionary statistics), organismal (e.g. tissue-specific gene and protein expression, differential gene expression, disease phenotype), and molecular data types (e.g. Gene Ontology Annotation, protein interactions), as well as links to many general (e.g. Entrez, PubMed) and pregnancy disease-specific (e.g. PTBgene, dbPTB) databases. By facilitating the synthesis of diverse functional and evolutionary data in pregnancy-associated tissues and phenotypes and enabling their quick, intuitive, accurate and customized meta-analysis, GEneSTATION provides a novel platform for comprehensive investigation of the function and evolution of mammalian pregnancy.
© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
Once thought to be an artifact of microsomal systems, atypical kinetics with cytochrome P450 (CYP) enzymes have been extensively investigated in vitro and found to be substrate and species dependent. Building upon increasing reports of heterotropic CYP activation and inhibition in clinical settings, we screened a compound library of clinically approved drugs and various probe compounds to identify the frequency of heterotropism observed with different drug classes and the associated CYP enzymes thereof (1A2, 2C9, 2D6, and 3A4/5). Results of this screen revealed that the prescribed androgen receptor antagonist flutamide activated the intrinsic midazolam hydroxylase activity of CYP3A in human hepatic microsomes (66%), rat and human hepatocytes (36 and 160%, respectively), and in vivo in male Sprague-Dawley rats (>2-fold, combined area under the curve of primary rat in vivo midazolam metabolites). In addition, a screen of the pharmacologically active metabolite 2-hydroxy-flutamide revealed that this principle metabolite increased CYP3A metabolism of midazolam in human microsomes (30%) and hepatocytes (110%). Importantly, both flutamide and 2-hydroxy-flutamide demonstrated a pronounced increase in the CYP3A-mediated metabolism of commonly paired medications, nifedipine (antihypertensive) and amiodarone (antiarrhythmic), in multispecies hepatocytes (100% over baseline). These data serve to highlight the importance of an appropriate substrate and in vitro system selection in the pharmacokinetic modeling of atypical enzyme kinetics. In addition, the results of our investigation have illuminated a previously undiscovered class of heterotropic CYP3A activators and have demonstrated the importance of selecting commonly paired therapeutics in the in vitro and in vivo modeling of projected clinical outcomes.
Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.
Earlier we reported that low molecular weight (LMW) peptides accumulate in aging human lens tissue and that among the LMW peptides, the chaperone inhibitor peptide αA66-80, derived from α-crystallin protein, is one of the predominant peptides. We showed that in vitro αA66-80 induces protein aggregation. The current study was undertaken to determine whether LMW peptides are also present in guinea pig lens tissue subjected to hyperbaric oxygen (HBO) in vivo. The nuclear opacity induced by HBO in guinea pig lens is the closest animal model for studying age-related cataract formation in humans. A LMW peptide profile by mass spectrometry showed the presence of an increased amount of LMW peptides in HBO-treated guinea pig lenses compared to age-matched controls. Interestingly, the mass spectrometric data also showed that the chaperone inhibitor peptide αA66-80 accumulates in HBO-treated guinea pig lens. Following incubation of synthetic chaperone inhibitor peptide αA66-80 with α-crystallin from guinea pig lens extracts, we observed a decreased ability of α-crystallin to inhibit the amorphous aggregation of the target protein alcohol dehydrogenase and the formation of large light scattering aggregates, similar to those we have observed with human α-crystallin and αA66-80 peptide. Further, time-lapse recordings showed that a preformed complex of α-crystallin and αA66-80 attracted additional crystallin molecules to form even larger aggregates. These results demonstrate that LMW peptide-mediated cataract development in aged human lens and in HBO-induced lens opacity in the guinea pig may have common molecular pathways.
Copyright © 2015 Elsevier Ltd. All rights reserved.
The naked mole rat (Heterocephalus glaber) displays exceptional longevity, with a maximum lifespan exceeding 30 years. This is the longest reported lifespan for a rodent species and is especially striking considering the small body mass of the naked mole rat. In comparison, a similarly sized house mouse has a maximum lifespan of 4 years. In addition to their longevity, naked mole rats show an unusual resistance to cancer. Multi-year observations of large naked mole-rat colonies did not detect a single incidence of cancer. Here we identify a mechanism responsible for the naked mole rat's cancer resistance. We found that naked mole-rat fibroblasts secrete extremely high-molecular-mass hyaluronan (HA), which is over five times larger than human or mouse HA. This high-molecular-mass HA accumulates abundantly in naked mole-rat tissues owing to the decreased activity of HA-degrading enzymes and a unique sequence of hyaluronan synthase 2 (HAS2). Furthermore, the naked mole-rat cells are more sensitive to HA signalling, as they have a higher affinity to HA compared with mouse or human cells. Perturbation of the signalling pathways sufficient for malignant transformation of mouse fibroblasts fails to transform naked mole-rat cells. However, once high-molecular-mass HA is removed by either knocking down HAS2 or overexpressing the HA-degrading enzyme, HYAL2, naked mole-rat cells become susceptible to malignant transformation and readily form tumours in mice. We speculate that naked mole rats have evolved a higher concentration of HA in the skin to provide skin elasticity needed for life in underground tunnels. This trait may have then been co-opted to provide cancer resistance and longevity to this species.
Microneedles (MNs) provide a minimally invasive means to enhance skin permeability by creating micron-scale channels (micropores) that provide a drug delivery pathway. Adequate formation of the micropores is critical to the success of this unique drug delivery technique. The objective of the current work was to develop sensitive and reproducible impedance spectroscopy techniques to monitor micropore formation in animal models and human subjects. Hairless guinea pigs, a Yucatan miniature pig, and human volunteers were treated with 100 MN insertions per site following an overnight prehydration period. Repeated measurements were made pre- and post-MN treatment using dry and gel Ag/AgCl electrodes applied with light verses direct pressure to hold the electrode to the skin surface. Impedance measurements dropped significantly post-MN application at all sites (p < 0.05, irrespective of electrode type or gel application), confirming micropore formation. In the Yucatan pig and human subjects, gel electrodes with direct pressure yielded the lowest variability (demonstrated by lower %relative standard deviation), whereas dry electrodes with direct pressure were superior in the guinea pigs. These studies confirm that impedance measurements are suitable for use in both clinical and animal research environments to monitor the formation of new micropores that will allow for drug delivery through the impermeable skin layers.
Copyright © 2013 Wiley Periodicals, Inc.