Role of zika virus envelope protein domain iii as a target of human neutralizing antibodies

Zika virus (ZIKV) is a flavivirus that is structurally highly similar to the related viruses, dengue virus (DENV), West Nile virus, and yellow fever virus. ZIKV causes an acute infection that often results in mild symptoms but that can cause severe disease in rare instances. Following infection, individuals mount an adaptive immune response, composed of antibodies (Abs) that target the envelope (E) glycoprotein of ZIKV, which covers the surface of the virus. Groups have studied monoclonal antibodies and polyclonal immune sera isolated from individuals who recovered from natural ZIKV infections. Some of these antibodies bind to domain III of E (EDIII), but the functional importance of these antibodies is unknown. In this study, we aimed to determine if EDIII is a major target of the potent serum neutralizing antibodies present in people after ZIKV infection. By generating a chimeric virus containing ZIKV EDIII in a DENV4 virus backbone, our data show a minor role of EDIIItargeting antibodies in human polyclonal neutralization. These results reveal that while monoclonal antibody (MAb) studies are informative in identifying individual antibody epitopes, they can overestimate the importance of epitopes contained within EDIII as targets of serum neutralizing antibodies. Additionally, these results argue that the major target of human ZIKV neutralizing antibodies resides elsewhere in E; however, further studies are needed to assess the epitope specificity of the neutralizing response at the population level. Identification of the major epitopes on the envelope of ZIKV recognized by serum neutralizing antibodies is critical for understanding protective immunity following natural infection and for guiding the design and evaluation of vaccines. IMPORTANCE Zika virus is a flavivirus that was recently introduced to Latin America, where it caused a massive epidemic. Individuals infected with ZIKV generate an immune response composed of antibodies which bind to the envelope (E) protein. These anti-E antibodies are critical in protecting individuals from subsequent infection. Multiple groups have found that many ZIKV antibodies bind to domain III of E (EDIII), suggesting that this region is an important target of neutralizing antibodies. Here, we generated a chimeric virus containing ZIKV EDIII in a dengue virus backbone to measure ZIKV EDIIIspecific antibody responses. We found that while polyclonal ZIKV immune serum contains antibodies targeting EDIII, they constitute only a small fraction of the total population of antibodies that neutralize ZIKV. Further studies are needed to define the main targets on the viral envelope recognized by human neutralizing antibodies, which is critical for guiding the development of ZIKV vaccines

Optimization of Surface Display of DENV2 e Protein on a Nanoparticle to Induce Virus Specific Neutralizing Antibody Responses

The dengue virus (DENV) causes over 350 million infections, resulting in ∼25,000 deaths per year globally. An effective dengue vaccine requires generation of strong and balanced neutralizing antibodies against all four antigenically distinct serotypes of DENV. The leading live-attenuated tetravalent dengue virus vaccine platform has shown partial efficacy, with an unbalanced response across the four serotypes in clinical trials. DENV subunit vaccine platforms are being developed because they provide a strong safety profile and are expected to avoid the unbalanced immunization issues associated with live multivalent vaccines. Subunit vaccines often lack immunogenicity, requiring either a particulate or adjuvanted formulation. Particulate formulations adsorbing monomeric DENV-E antigen to the particle surface incite a strong immune response, but have no control of antigen presentation. Highly neutralizing epitopes are displayed by DENV-E quaternary structures. To control the display of DENV-E and produce quaternary structures, particulate formulations that covalently attach DENV-E to the particle surface are needed. Here we develop a surface attached DENV2-E particulate formulation, as well as analysis tools, using PEG hydrogel nanoparticles created with particle replication in nonwetting templates (PRINT) technology. We found that adding Tween-20 to the conjugation buffer controls DENV-E adsorption to the particle surface during conjugation, improving both protein stability and epitope display. Immunizations with the anionic but not the cationic DENV2-E conjugated particles were able to produce DENV-specific and virus neutralizing antibody in mice. This work optimized the display of DENV-E conjugated to the surface of a nanoparticle through EDC/NHS chemistry, establishing a platform that can be expanded upon in future work to fully control the display of DENV-E

Oligomeric state of the ZIKV E protein defines protective immune responses

The current leading Zika vaccine candidates in clinical testing are based on live or killed virus platforms, which have safety issues, especially in pregnant women. Zika subunit vaccines, however, have shown poor performance in preclinical studies, most likely because the antigens tested do not display critical quaternary structure epitopes present on Zika E protein homodimers that cover the surface of the virus. Here, we produce stable recombinant E protein homodimers that are recognized by strongly neutralizing Zika specific monoclonal antibodies. In mice, the dimeric antigen stimulate strongly neutralizing antibodies that target epitopes that are similar to epitopes recognized by human antibodies following natural Zika virus infection. The monomer antigen stimulates low levels of E-domain III targeting neutralizing antibodies. In a Zika challenge model, only E dimer antigen stimulates protective antibodies, not the monomer. These results highlight the importance of mimicking the highly structured flavivirus surface when designing subunit vaccines

Physiological temperatures reduce dimerization of dengue and Zika virus recombinant envelope proteins

The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary. The sRecE proteins from DENV and ZIKV crystallize as native-like dimers, but studies in solution suggest that these dimers are marginally stable. To better understand the challenges associated with creating stable sRecE dimers, we characterized the thermostability of sRecE proteins from ZIKV and three DENV serotypes, DENV2– 4. All four proteins irreversibly unfolded at moderate temperatures (46 –53 °C). At 23 °C and low micromolar concentrations, DENV2 and ZIKV were primarily dimeric, and DENV3– 4 were primarily monomeric, whereas at 37 °C, all four proteins were predominantly monomeric. We further show that the dissociation constant for DENV2 dimerization is very temperature-sensitive, ranging from <1 M at 25 °C to 50 M at 41 °C, due to a large exothermic enthalpy of binding of 79 kcal/mol. We also found that quaternary epitope antibody binding to DENV2– 4 and ZIKV sRecE is reduced at 37 °C. Our observation of reduced sRecE dimerization at physiological temperature highlights the need for stabilizing the dimer as part of its development as a subunit vaccine

Human antibody response to Zika targets type-specific quaternary structure epitopes

The recent Zika virus (ZIKV) epidemic in the Americas has revealed rare but serious manifestations of infection. ZIKV has emerged in regions endemic for dengue virus (DENV), a closely related mosquito-borne flavivirus. Cross-reactive antibodies confound studies of ZIKV epidemiology and pathogenesis. The immune responses to ZIKV may be different in people, depending on their DENV immune status. Here, we focus on the human B cell and antibody response to ZIKV as a primary flavivirus infection to define the properties of neutralizing and protective antibodies generated in the absence of preexisting immunity to DENV. The plasma antibody and memory B cell response is highly ZIKV type–specific, and ZIKV-neutralizing antibodies mainly target quaternary structure epitopes on the viral envelope. To map viral epitopes targeted by protective antibodies, we isolated 2 type-specific monoclonal antibodies (mAbs) from a ZIKV case. Both mAbs were strongly neutralizing in vitro and protective in vivo. The mAbs recognize distinct epitopes centered on domains I and II of the envelope protein. We also demonstrate that the epitopes of these mAbs define antigenic regions commonly targeted by plasma antibodies in individuals from endemic and nonendemic regions who have recovered from ZIKV infections

Primaerenergieeinsparung und Emissionsminderung durch Einsatz neuer regelbarer Brenner zur industriellen Verfeuerung von Prozessgasen mit schwankender Zusammensetzung Schlussbericht

For the improvement of the energy utilisation and the reduction of emissions from natural and process gas fired plants new adjustable burners were developed. These burners ensure a complete and low emission combustion for different gas types or varying gas properties and compositions. In a first step different conventional burner types were investigated concerning flame stability, burnout and emissions. Beside well-known results form previous research projects and from literature investigations with standard burners at test facilities, mathematical modelling and operational measurements at industrial facilities were carried out. Based on these results simple adjustments for industrial gas burners were developed. In cooperation with burner suppliers three different burner prototypes were designed and built. The experiments at a burner test facility showed that the adjustable burners can be used in a wider range than the conventional burners. The results of this research project show the possibility to employ adjustable burners for the use of different fuel gases. In dependence of operational conditions and gas composition the mixing principle and parameters are recommended to ensure stable combustion and lowest emissions at the same time. (orig.)Zur Verbesserung der Energieausnutzung und Senkung der Schadstoffemissionen von erd- und prozessgasbefeuerten Anlagen wurden neuartige regelbare Brenner entwickelt. Diese Brenner gewaehrleisten bei dem Einsatz unterschiedlicher Gasarten oder bei schwankenden Brenngaseigenschaften oder -zusammensetzungen eine vollstaendige und schadstoffarme Verbrennung. Zunaechst wurden verschiedene konventionelle Brennerkonstruktionen im Hinblick auf Flammenstabilitaet, Ausbrand und Emissionsverhalten untersucht und klassifiziert. Hierzu wurden neben bekannten Ergebnissen aus vorhergehenden Forschungsarbeiten und verfuegbaren Literaturangaben, Heissversuche mit konventionellen Brennern an Pruefstaenden, numerische Berechnungen und Betriebsmessungen an industriellen Thermoprozessanlagen durchgefuehrt und ausgewertet. Aufbauend auf diesen Ergebnisen wurden einfache Regelkonzepte fuer industrielle Gasbrenner entwickelt, Brennerprototypen in Zusammenarbeit mit mittelstaendischen Brennerherstellern ausgelegt und gefertigt und mit Hilfe numerischer Berechnungsverfahren bewertet. Die Ergebnisse der umfangreichen Untersuchungen an einer Brennerversuchsanlage zeigen, dass durch den Einsatz der Regelmechanismen der Einsatzbereich der Brenner - im Vergleich zur konventionellen statistischen Ausfuehrung - deutlich erweitert werden kann. Die Ergebnisse dieses Forschungsvorhabens bieten die Moeglichkeit bei Neu- und Umbaumassnahmen an Industrieoefen und industriellen Feuerungen, die auf den Einsatz wechselnder Brenngase angewiesen sind, regelbare Brenner einzusetzen. Abhaengig von der vorgesehenen Betriebsweise und den Gasarten koennen Empfehlungen fuer Mischprinzip und Einstellparameter gegeben werden, die eine stabile Flamme bei geringsten Emissionen erwarten lassen. (orig.)SIGLEAvailable from TIB Hannover: RA 3271(5.45.027)+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Comprehensive visualization of multimodal cardiac imaging data for assessment of coronary artery disease: first clinical results of the SMARTVis tool

In clinical practice, both coronary anatomy and myocardial perfusion information are needed to assess coronary artery disease (CAD). The extent and severity of coronary stenoses can be determined using computed tomography coronary angiography (CTCA); the presence and amount of ischemia can be identified using myocardial perfusion imaging, such as perfusion magnetic resonance imaging (PMR). To determine which specific stenosis is associated with which ischemic region, experts use assumptions on coronary perfusion territories. Due to the high variability between patient's coronary artery anatomies, as well as the uncertain relation between perfusion territories and supplying coronary arteries, patient-specific systems are needed. We present a patient-specific visualization system, called Synchronized Multimodal heART Visualization (SMARTVis), for relating coronary stenoses and perfusion deficits derived from CTCA and PMR, respectively. The system consists of the following comprehensive components: (1) two or three-dimensional fusion of anatomical and functional information, (2) automatic detection and ranking of coronary stenoses, (3) estimation of patient-specific coronary perfusion territories. The potential benefits of the SMARTVis tool in assessing CAD were investigated through a case-study evaluation (conventional vs. SMARTVis tool): two experts analyzed four cases of patients with suspected multivessel coronary artery disease. When using the SMARTVis tool, a more reliable estimation of the relation between perfusion deficits and stenoses led to a more accurate diagnosis, as well as a better interobserver diagnosis agreement. The SMARTVis comprehensive visualization system can be effectively used to assess disease status in multivessel CAD patients, offering valuable new options for the diagnosis and management of these patients