Community Engagement newsletter, Faculty of Veterinary Science, Winter, 2012

Mangaung community receives support from Onderstepoort / Willem Engelbrecht, Helzna Krikke, Ingrid Metz, Janine Lombard, Marelize Greyling and Willem Janson -- Fighting animal abuse one school at a time / Ruche Harmse, Taryn Light, Ansu Visser, Este van Coetzen, Megan Naude, Nadia Strydom and Tess Tjasink -- Veterinary education and “Vets for the kids” / Patrick Ntsibande, Nyeleti Manganyi, Noluthando Ndashe, Thapelo Makae, Khulekani Lukhele and Sabelo Magagula -- soVETo k9 Mobile clinic outreach / Daleen Bester, Gideon Stemmet, Ian Gibson, Kobus Rabe, Louw Grobler and Rhynard de RidderNews articles with colour photos about the various community engagement projects of the Faculty of Veterinary Science, University of Pretoria.ab201

Validation of CyTOF Against Flow Cytometry for Immunological Studies and Monitoring of Human Cancer Clinical Trials

Flow cytometry is a widely applied approach for exploratory immune profiling and biomarker discovery in cancer and other diseases. However, flow cytometry is limited by the number of parameters that can be simultaneously analyzed, severely restricting its utility. Recently, the advent of mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system, allowing simultaneous interrogation of a large number of parameters. This is important for deep interrogation of immune responses and particularly when sample sizes are limited (such as in tumors). Our goal was to compare the accuracy and reproducibility of CyTOF against flow cytometry as a reliable analytic tool for human PBMC and tumor tissues for cancer clinical trials. We developed a 40+ parameter CyTOF panel and demonstrate that compared to flow cytometry, CyTOF yields analogous quantification of cell lineages in conjunction with markers of cell differentiation, function, activation, and exhaustion for use with fresh and viably frozen PBMC or tumor tissues. Further, we provide a protocol that enables reliable quantification by CyTOF down to low numbers of input human cells, an approach that is particularly important when cell numbers are limiting. Thus, we validate CyTOF as an accurate approach to perform high dimensional analysis in human tumor tissue and to utilize low cell numbers for subsequent immunologic studies and cancer clinical trials

Characterization of Structural and Binding Properties of 4E-BP2

Eukaryotic initiation factor-4E (eIF4E) controls the rate of cap-dependent translation initiation and is in turn exquisitely regulated by 4E-BPs. 4E-BP2 binds eIF4E with the highest affinity and is implicated in cancer, and metabolic and neurological disorders. Herein we use NMR, ITC and fluorescence to characterize 4E-BP2 structural and binding properties. Isolated 4E-BP2 is intrinsically disordered, but possesses some transient secondary structural propensities. eIF4E, however, is folded but has a disordered N-terminus. The eIF4E:4E-BP2 interaction is tight (Kd = 10-9 nM) and involves 4E-BP2 C-terminal and canonical binding regions, and the disordered eIF4E N-terminus. 4E-BP2 remains largely disordered upon binding to eIF4E. Noteworthy, high affinity interactions are not necessarily mediated by static structures, and 4E-BP2 binding is not the simple “disorder-to-order” transition observed in many interactions involving disordered proteins. This study offers molecular insights into 4E-BP2 functionality, and lays a foundation for development of novel therapies for cancer and neurological disorders.MAS

Interactions between HIV accessory proteins and host factors BST2 and CD4: at the interface of virus dissemination and persistence

Tetherin/BST2 and CD4 are increasingly being recognized as chief modulators of human immunodeficiency virus (HIV) dissemination and persistence. BST2 is a type I interferon (IFN)-stimulated restriction factor that inhibits HIV-1 release by tethering nascent Env-containing virions at the surface of infected cells, and is counteracted by HIV-1 Vpu. Vpu also acts in concert with Nef to down-regulate expression of the virus primary receptor, CD4. The interactions between Vpu and the host factors BST2 and CD4 are still ill-defined, and a subject of intense investigation. In the first part of the study, we sought to elucidate determinants within Vpu that govern BST2 antagonism. We identify highly conserved residues adjacent to the functionally important Vpu transmembrane domain (TMD) that regulate the ability of Vpu to bind and co-localize with BST2. Moreover, these residues, particularly a glutamic acid residue positioned immediately following the TMD, are a determinant not only for efficient targeting of BST2, but also for binding and degradation of CD4. Mechanistically, our data suggest a role of these residues in maintenance of the Vpu TMD conformational configuration such that interactions with membrane-associated host targets are favoured. The second part of the research investigates the roles of BST2 and CD4 in clearance of infected cells via antibody-dependent cell-mediated cytotoxicity (ADCC), an important element of the anti-HIV response. We show that CD4 sensitizes infected cells to ADCC mediated by CD4-induced (CD4i), non-neutralizing antibodies (nNAbs) whose binding depends on CD4-Env interactions. Accumulation of CD4 on infected cells unmasked epitopes on Env that are recognized by these CD4i nNAbs. We further demonstrate that BST2-mediated virion tethering, which increases the amount of Env epitopes at the cell surface, enhances ADCC mediated by CD4-independent broadly neutralizing antibodies (bNAbs) and, to a lesser extent, by nNAbs. Importantly, HIV uses its Vpu and Nef proteins to target CD4 and BST2 and, in so doing, shields infected cells from lysis. In accordance with this, WT virus-infected cells, which are virtually not susceptible to nNAb-mediated ADCC, can be sensitized via blocking Vpu and Nef. Similarly, while bNAbs can mediate ADCC against WT virus-infected cells, inactivating Vpu and Nef significantly potentiates their killing. Moreover, increasing BST2-mediated virion crosslinking using IFNα treatment enhances ADCC susceptibility. Furthermore, the data reveal that reactivated HIV latent cells are susceptible, albeit poorly, to ADCC lysis. Likewise, their killing could be enhanced by both exogenous IFN administration and removal of the viral accessory proteins.Altogether, this work emphasizes a novel role of BST2 and CD4 towards elimination of infected cells, as well as a mechanism by which HIV Vpu and Nef function synergistically to protect infected cells from ADCC, thereby promoting viral persistence. As such, strategies aimed at enhancing ADCC activity via treatments with IFN and/or small molecules targeting these accessory proteins represent a promising avenue for protection from HIV acquisition, post-infection control of HIV, as well as enhanced clearance of latent viral reservoirs in "Shock and Kill" cure approaches. To this effect, the study identifies a vulnerable region within Vpu that may represent a feasible target for development of Vpu inhibitors.Les protéines Tetherin/BST2 et CD4 sont de plus en plus reconnues comme étant les principaux modulateurs de la diffusion et de la persistance du virus de l'immunodéficience humaine (VIH). BST2 est un facteur de restriction qui est induit par l'interféron (IFN) de type I; elle inhibe la relâche des nouveaux virions détenant des glycoprotéines Env à la surface des cellules infectées, et est contrecarrée par la protéine Vpu du VIH-1. Vpu agit également de concert avec Nef pour réguler négativement l'expression de CD4, le récepteur primaire du virus. Les interactions entre Vpu et les facteurs de l'hôte BST2 et CD4 sont encore mal définies et restent un sujet de recherche intense. Dans la première partie de cette étude, nous avons cherché à élucider les déterminants de Vpu qui gouvernent son activité anti-BST2. Nous identifions des acides aminés hautement conservés adjacents au domaine transmembranaire (TMD) de Vpu qui sont importants pour la liaison et la co-localisation de Vpu avec BST2. De plus, ces acides aminés, en particulier l'acide glutamique positionné immédiatement après le TMD, ont un rôle dans la dégradation de CD4. D'un point de vue fonctionnel, nos données suggèrent que ces acides aminés contribuent au maintien de la configuration conformationnelle du TMD de Vpu de sorte que les interactions de Vpu avec certaines molécules membranaires de l'hôte sont favorisées.La deuxième partie de ce travail étudie les rôles de BST2 et de CD4 dans l'élimination des cellules infectées via la cytotoxicité cellulaire dépendante des anticorps (ADCC), un élément important de la réponse anti-VIH. Nous montrons que CD4 sensibilise les cellules infectées à l'ADCC induite par les anticorps non-neutralisants (nNAbs) induits par CD4 (iCD4) dont la liaison dépend des interactions CD4-Env. L'accumulation de CD4 sur des cellules infectées démasque des epitopes de l'Env qui sont reconnus par ces nNAbs CD4i. Nous démontrons en outre que l'ancrage du virion par BST2, qui augmente la quantité d'épitopes Env à la surface cellulaire, améliore l'ADCC médiée par des anticorps neutralisants à large spectre indépendants de CD4 (bNAbs) et, dans une moindre mesure, par les nNAbs. Il est important de noter que le VIH utilise ses protéines Vpu et Nef pour cibler CD4 et BST2 et, ce faisant, protège les cellules infectées de la lyse. Conformément à cela, les cellules infectées par le virus WT, qui ne sont pratiquement pas sensibles à l'ADCC à médiation par nNAb, peuvent être sensibilisées via Vpu et Nef. De même, alors que les bNAbs peuvent servir de médiateur ADCC contre les cellules infectées par le virus WT, l'inactivation de Vpu et Nef potentialise de manière significative leur destruction. De plus, l'augmentation de virions retenus par BST2 suite à l'ajout d'IFNα accentue la susceptibilité à l'ADCC. De plus, les données révèlent que les cellules latentes infectées au VIH qui sont par suite réactivées sont susceptibles, bien que faiblement, à la lyse par ADCC. Leur destruction pourrait être accélérée par l'administration d'IFN exogène et par l'élimination des protéines accessoires virales.Dans l'ensemble, ce travail met l'accent sur un nouveau rôle de BST2 et de CD4 dans l'élimination des cellules infectées, ainsi que sur un mécanisme par lequel les protéines Vpu et Nef du VIH fonctionnent de manière synergique pour protéger les cellules infectées de l'ADCC, favorisant ainsi la persistance virale. Ainsi, les stratégies visant à améliorer l'activité de l'ADCC par l'intermédiaire de traitements avec de l'IFN et / ou de petites molécules ciblant ces protéines accessoires représentent une voie prometteuse pour une protection prophylactique contre le VIH, le contrôle post-infection du VIH ainsi qu'une élimination plus efficace des réservoirs viraux latents par l'approche "Shock and Kill". À cet effet, l'étude identifie une région vulnérable dans Vpu qui peut représenter une cible intéressante pour le développement d'inhibiteurs de Vpu

MOESM2 of Conserved residues within the HIV-1 Vpu transmembrane-proximal hinge region modulate BST2 binding and antagonism

Additional file 2: Figure S2. BST2 binding capacity of Vpu TMD mutants. Co-IP following co-transfection of HEK293T cells with a proviral construct encoding WT Vpu, E28A/L33A or the indicated Vpu TMD mutants and a BST2 expressor. Below each blot is the extent of BST2 binding of each Vpu mutant, relative to WT Vpu (set at 100%). For each condition, BST2 binding efficiency was determined from the ratio obtained from densitometric analyses of the intensities of Vpu- and BST2-related band signals in the immunoprecipitated fractions. The asterisk denotes an Ab-related band

MOESM3 of Conserved residues within the HIV-1 Vpu transmembrane-proximal hinge region modulate BST2 binding and antagonism

Additional file 3: Figure S3. E28 is important for efficient degradation of BST2. Shown is a representative Western blot indicating the steady state levels of BST2 in mock-infected HeLa cells (lane 1) as well as in Hela cells following infections with VSV-G-pseudotyped HIV-1 viruses encoding WT Vpu and the indicated Vpu mutants (lanes 2–7). Below the blot is the extent of BST2 expression based on densitometric analyses of the intensities of BST2-related band signals obtained from each Vpu mutant, relative to dU (set at 100%)

Community Engagement newsletter, Faculty of Veterinary Summer, December, 2015

Education all round / Nadia Saunderson -- A day at CLAW / Jennifer Gerner -- Veterinary science students making a difference! There is no gift greater than the gift of knowledge / Elmien Durieux -- Reconnecting with our roots: future Vets and Vet Nurses visit Hluvukani Clinic / Nyeleti Manganyi -- You snooze, you lose... It’s called passion / Khulekani Lukhele -- A rush to save as many as possible! / Sabelo Magagula.News articles with colour photos about the various community engagement projects of the Faculty of Veterinary Science, University of Pretoria.ab201