Safety and Immunogenicity of Novel Adenovirus Type 26- and Modified Vaccinia Ankara-Vectored Ebola Vaccines: A Randomized Clinical Trial.

IMPORTANCE: Developing effective vaccines against Ebola virus is a global priority. OBJECTIVE: To evaluate an adenovirus type 26 vector vaccine encoding Ebola glycoprotein (Ad26.ZEBOV) and a modified vaccinia Ankara vector vaccine, encoding glycoproteins from Ebola virus, Sudan virus, Marburg virus, and Tai Forest virus nucleoprotein (MVA-BN-Filo). DESIGN, SETTING, AND PARTICIPANTS: Single-center, randomized, placebo-controlled, observer-blind, phase 1 trial performed in Oxford, United Kingdom, enrolling healthy 18- to 50-year-olds from December 2014; 8-month follow-up was completed October 2015. INTERVENTIONS: Participants were randomized into 4 groups, within which they were simultaneously randomized 5:1 to receive study vaccines or placebo. Those receiving active vaccines were primed with Ad26.ZEBOV (5 × 10(10) viral particles) or MVA-BN-Filo (1 × 10(8) median tissue culture infective dose) and boosted with the alternative vaccine 28 or 56 days later. A fifth, open-label group received Ad26.ZEBOV boosted by MVA-BN-Filo 14 days later. MAIN OUTCOMES AND MEASURES: The primary outcomes were safety and tolerability. All adverse events were recorded until 21 days after each immunization; serious adverse events were recorded throughout the trial. Secondary outcomes were humoral and cellular immune responses to immunization, as assessed by enzyme-linked immunosorbent assay and enzyme-linked immunospot performed at baseline and from 7 days after each immunization until 8 months after priming immunizations. RESULTS: Among 87 study participants (median age, 38.5 years; 66.7% female), 72 were randomized into 4 groups of 18, and 15 were included in the open-label group. Four participants did not receive a booster dose; 67 of 75 study vaccine recipients were followed up at 8 months. No vaccine-related serious adverse events occurred. No participant became febrile after MVA-BN-Filo, compared with 3 of 60 participants (5%; 95% CI, 1%-14%) receiving Ad26.ZEBOV in the randomized groups. In the open-label group, 4 of 15 Ad26.ZEBOV recipients (27%; 95% CI, 8%-55%) experienced fever. In the randomized groups, 28 of 29 Ad26.ZEBOV recipients (97%; 95% CI, 82%- 99.9%) and 7 of 30 MVA-BN-Filo recipients (23%; 95% CI, 10%-42%) had detectable Ebola glycoprotein-specific IgG 28 days after primary immunization. All vaccine recipients had specific IgG detectable 21 days postboost and at 8-month follow-up. Within randomized groups, at 7 days postboost, at least 86% of vaccine recipients showed Ebola-specific T-cell responses. CONCLUSIONS AND RELEVANCE: In this phase 1 study of healthy volunteers, immunization with Ad26.ZEBOV or MVA-BN-Filo did not result in any vaccine-related serious adverse events. An immune response was observed after primary immunization with Ad26.ZEBOV; boosting by MVA-BN-Filo resulted in sustained elevation of specific immunity. These vaccines are being further assessed in phase 2 and 3 studies. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT02313077.

Bicaudal D2, dynein, and kinesin-1 associate with nuclear pore complexes and regulate centrosome and nuclear positioning during mitotic entry.

BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D, an evolutionarily conserved adaptor between microtubule motors and their cargo that was previously shown to link vesicles and mRNP complexes to the dynein motor. Here, we identified a G2-specific role for BICD2 in the relative positioning of the nucleus and centrosomes in dividing cells. By combining mass spectrometry, biochemical and cell biological approaches, we show that the nuclear pore complex (NPC) component RanBP2 directly binds to BICD2 and recruits it to NPCs specifically in G2 phase of the cell cycle. BICD2, in turn, recruits dynein-dynactin to NPCs and as such is needed to keep centrosomes closely tethered to the nucleus prior to mitotic entry. When dynein function is suppressed by RNA interference-mediated depletion or antibody microinjection, centrosomes and nuclei are actively pushed apart in late G2 and we show that this is due to the action of kinesin-1. Surprisingly, depletion of BICD2 inhibits both dynein and kinesin-1-dependent movements of the nucleus and cytoplasmic NPCs, demonstrating that BICD2 is needed not only for the dynein function at the nuclear pores but also for the antagonistic activity of kinesin-1. Our study demonstrates that the nucleus is subject to opposing activities of dynein and kinesin-1 motors and that BICD2 contributes to nuclear and centrosomal positioning prior to mitotic entry through regulation of both dynein and kinesin-1.

Rab6 regulates transport and targeting of exocytotic carriers.

Constitutive exocytosis delivers newly synthesized proteins, lipids, and other molecules from the Golgi apparatus to the cell surface. This process is mediated by vesicles, which bud off the trans-Golgi network, move along cytoskeletal filaments, and fuse with the plasma membrane. Here, we show that the small GTPase Rab6 marks exocytotic vesicles and, together with the microtubule plus-end-directed motor kinesin-1, stimulates their processive microtubule-based transport to the cell periphery. Furthermore, Rab6 directs targeting of secretory vesicles to plasma-membrane sites enriched in the cortical protein ELKS, a known Rab6 binding partner. Our data demonstrate that although Rab6 is not essential for secretion, it controls the organization of exocytosis within the cellular space.

STIM1 is a MT-plus-end-tracking protein involved in remodeling of the ER.

Stromal interaction molecule 1 (STIM1) is a transmembrane protein that is essential for store-operated Ca(2+) entry, a process of extracellular Ca(2+) influx in response to the depletion of Ca(2+) stores in the endoplasmic reticulum (ER) (reviewed in [1-4]). STIM1 localizes predominantly to the ER; upon Ca(2+) release from the ER, STIM1 translocates to the ER-plasma membrane junctions and activates Ca(2+) channels (reviewed in [1-4]). Here, we show that STIM1 directly binds to the microtubule-plus-end-tracking protein EB1 and forms EB1-dependent comet-like accumulations at the sites where polymerizing microtubule ends come in contact with the ER network. Therefore, the previously observed tubulovesicular motility of GFP-STIM1 [5] is not a motor-based movement but a traveling wave of diffusion-dependent STIM1 concentration in the ER membrane. STIM1 overexpression strongly stimulates ER extension occurring through the microtubule "tip attachment complex" (TAC) mechanism [6, 7], a process whereby an ER tubule attaches to and elongates together with the EB1-positive end of a growing microtubule. Depletion of STIM1 and EB1 decreases TAC-dependent ER protrusion, indicating that microtubule growth-dependent concentration of STIM1 in the ER membrane plays a role in ER remodeling.

Nonhuman primate to human immunobridging to infer the protective effect of an Ebola virus vaccine candidate.

It has been proven challenging to conduct traditional efficacy trials for Ebola virus (EBOV) vaccines. In the absence of efficacy data, immunobridging is an approach to infer the likelihood of a vaccine protective effect, by translating vaccine immunogenicity in humans to a protective effect, using the relationship between vaccine immunogenicity and the desired outcome in a suitable animal model. We here propose to infer the protective effect of the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen with an 8-week interval in humans by immunobridging. Immunogenicity and protective efficacy data were obtained for Ad26.ZEBOV and MVA-BN-Filo vaccine regimens using a fully lethal EBOV Kikwit challenge model in cynomolgus monkeys (nonhuman primates [NHP]). The association between EBOV neutralizing antibodies, glycoprotein (GP)-binding antibodies, and GP-reactive T cells and survival in NHP was assessed by logistic regression analysis. Binding antibodies against the EBOV surface GP were identified as the immune parameter with the strongest correlation to survival post EBOV challenge, and used to infer the predicted protective effect of the vaccine in humans using published data from phase I studies. The human vaccine-elicited EBOV GP-binding antibody levels are in a range associated with significant protection against mortality in NHP. Based on this immunobridging analysis, the EBOV GP-specific-binding antibody levels elicited by the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen in humans will likely provide protection against EBOV disease.

BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures.

Cytoplasmic dynein is the major microtubule minus-end-directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein-dynactin interaction are poorly understood. In this study, we focus on dynein-dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N-dynein-dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end-directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors.

A role for the Rab6B Bicaudal-D1 interaction in retrograde transport in neuronal cells.

The Rab6 subfamily of small GTPases consists of three different isoforms: Rab6A, Rab6A' and Rab6B. Both Rab6A and Rab6A' are ubiquitously expressed whereas Rab6B is predominantly expressed in brain. Recent studies have shown that Rab6A' is the isoform regulating the retrograde transport from late endosomes via the Golgi to the ER and in the transition from anaphase to metaphase during mitosis. Since the role of Rab6B is still ill defined, we set out to characterize its intracellular environment and dynamic behavior. In a Y-2H search for novel Rab6 interacting proteins, we identified Bicaudal-D1, a large coiled-coil protein known to bind to the dynein/dynactin complex and previously shown to be a binding partner for Rab6A/Rab6A'. Co-immunoprecipitation studies and pull down assays confirmed that Bicaudal-D1 also interacts with Rab6B in its active form. Using confocal laser scanning microscopy it was established that Rab6B and Bicaudal-D1 co-localize at the Golgi and vesicles that align along microtubules. Furthermore, both proteins co-localized with dynein in neurites of SK-N-SH cells. Live cell imaging revealed bi-directional movement of EGFP-Rab6B structures in SK-N-SH neurites. We conclude from our data that the brain-specific Rab6B via Bicaudal-D1 is linked to the dynein/dynactin complex, suggesting a regulatory role for Rab6B in the retrograde transport of cargo in neuronal cells.