A Recombinant Vesicular Stomatitis Virus Ebola Vaccine.

BACKGROUND: The worst Ebola virus disease (EVD) outbreak in history has resulted in more than 28,000 cases and 11,000 deaths. We present the final results of two phase 1 trials of an attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV)-based vaccine candidate designed to prevent EVD. METHODS: We conducted two phase 1, placebo-controlled, double-blind, dose-escalation trials of an rVSV-based vaccine candidate expressing the glycoprotein of a Zaire strain of Ebola virus (ZEBOV). A total of 39 adults at each site (78 participants in all) were consecutively enrolled into groups of 13. At each site, volunteers received one of three doses of the rVSV-ZEBOV vaccine (3 million plaque-forming units [PFU], 20 million PFU, or 100 million PFU) or placebo. Volunteers at one of the sites received a second dose at day 28. Safety and immunogenicity were assessed. RESULTS: The most common adverse events were injection-site pain, fatigue, myalgia, and headache. Transient rVSV viremia was noted in all the vaccine recipients after dose 1. The rates of adverse events and viremia were lower after the second dose than after the first dose. By day 28, all the vaccine recipients had seroconversion as assessed by an enzyme-linked immunosorbent assay (ELISA) against the glycoprotein of the ZEBOV-Kikwit strain. At day 28, geometric mean titers of antibodies against ZEBOV glycoprotein were higher in the groups that received 20 million PFU or 100 million PFU than in the group that received 3 million PFU, as assessed by ELISA and by pseudovirion neutralization assay. A second dose at 28 days after dose 1 significantly increased antibody titers at day 56, but the effect was diminished at 6 months. CONCLUSIONS: This Ebola vaccine candidate elicited anti-Ebola antibody responses. After vaccination, rVSV viremia occurred frequently but was transient. These results support further evaluation of the vaccine dose of 20 million PFU for preexposure prophylaxis and suggest that a second dose may boost antibody responses. (Funded by the National Institutes of Health and others; rVSV∆G-ZEBOV-GP ClinicalTrials.gov numbers, NCT02269423 and NCT02280408 .).

Z-Phe-Ala-diazomethylketone (PADK) disrupts and remodels early oligomer states of the Alzheimer disease Aß42 protein.

The oligomerization of the amyloid-ß protein (Aß) is an important event in Alzheimer disease (AD) pathology. Developing small molecules that disrupt formation of early oligomeric states of Aß and thereby reduce the effective amount of toxic oligomers is a promising therapeutic strategy for AD. Here, mass spectrometry and ion mobility spectrometry were used to investigate the effects of a small molecule, Z-Phe-Ala-diazomethylketone (PADK), on the Aß42 form of the protein. The mass spectrum of a mixture of PADK and Aß42 clearly shows that PADK binds directly to Aß42 monomers and small oligomers. Ion mobility results indicate that PADK not only inhibits the formation of Aß42 dodecamers, but also removes preformed Aß42 dodecamers from the solution. Electron microscopy images show that PADK inhibits Aß42 fibril formation in the solution. These results are consistent with a previous study that found that PADK has protective effects in an AD transgenic mouse model. The study of PADK and Aß42 provides an example of small molecule therapeutic development for AD and other amyloid diseases.

Submicromolar Aß42 reduces hippocampal glutamate receptors and presynaptic markers in an aggregation-dependent manner.

Synaptic pathology in Alzheimer's disease brains is thought to involve soluble Aß42 peptide. Here, sterile incubation in PBS caused small Aß42 oligomer formation as well as heterogeneous, 6E10-immunopositive aggregates of 80-100kDa. The high molecular weight aggregates (H-agg) formed in a time-dependent manner over an extended 30-day period. Interestingly, an inverse relationship between dimeric and H-agg formation was more evident when incubations were performed at 37°C as compared to 23°C, thus providing an experimental strategy with which to address synaptic compromise produced by the different Aß aggregates. H-agg species formed faster and to higher levels at 37°C compared to 23°C, and the two aggregate preparations were evaluated in hippocampal slice cultures, a sensitive system for monitoring synaptic integrity. Applied daily at 80-600nM for 7days, the Aß42 preparations caused dose-dependent and aggregation-dependent declines in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-d-aspartate (NMDA) receptor subunits as well as in presynaptic components. Unlike the synaptic effects, Aß42 induced only trace cellular degeneration that was CA1 specific. The 37°C preparation was less effective at decreasing synaptic markers, corresponding with its reduced levels of Aß42 monomers and dimers. Aß42 dimers decayed significantly faster at 37°C than 23°C, and more rapidly than monomers at either temperature. These findings indicate that Aß42 can self-aggregate into potent synaptotoxic oligomers as well as into larger aggregates that may serve to neutralize the toxic formations. These results will add to the growing debate concerning whether high molecular weight Aß complexes that form amyloid plaques are protective through the sequestration of oligomeric species.

Reovirus infection or ectopic expression of outer capsid protein micro1 induces apoptosis independently of the cellular proapoptotic proteins Bax and Bak.

Mammalian orthoreoviruses induce apoptosis in vivo and in vitro; however, the specific mechanism by which apoptosis is induced is not fully understood. Recent studies have indicated that the reovirus outer capsid protein µ1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed µ1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of µ1 activated both the extrinsic and intrinsic apoptotic pathways with activation of initiator caspases-8 and -9 and downstream effector caspase-3. Activation of both pathways was required for µ1-induced apoptosis, as specific inhibition of either caspase-8 or caspase-9 abolished downstream effector caspase-3 activation. Similar to reovirus infection, ectopic expression of µ1 caused release into the cytosol of cytochrome c and smac/DIABLO from the mitochondrial intermembrane space. Pancaspase inhibitors did not prevent cytochrome c release from cells expressing µ1, indicating that caspases were not required. Additionally, µ1- or reovirus-induced release of cytochrome c occurred efficiently in Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (MEFs). Finally, we found that reovirus-induced apoptosis occurred in Bax(-/-)Bak(-/-) MEFs, indicating that reovirus-induced apoptosis occurs independently of the proapoptotic Bcl-2 family members Bax and Bak.

Positive lysosomal modulation as a unique strategy to treat age-related protein accumulation diseases.

Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and α-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator Z-Phe-Ala-diazomethylketone (PADK). Such positive modulators of the lysosomal system have been studied in the well-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Active cathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulation reduced the pre-existing PHF deposits, restored tubulin structure and transport, and recovered synaptic components. Further proof-of-principle studies used Alzheimer disease mouse models. It was recently reported that systemic PADK administration caused dramatic increases in cathepsin B protein and activity levels, whereas neprilysin, insulin-degrading enzyme, α-secretase, and ß-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Aß) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Aß(1-38) peptide corresponded with decreased levels of Aß(1-42), supporting the lysosome's antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomal system, identifying lysosomal modulation as an avenue for disease-modifying therapies. From the in vitro and in vivo findings, unique lysosomal modulators represent a minimally invasive, pharmacologically controlled strategy against protein accumulation disorders to enhance protein clearance, promote synaptic integrity, and slow the progression of dementia.

Nonpeptidic lysosomal modulators derived from z-phe-ala-diazomethylketone for treating protein accumulation diseases.

Lysosomes are involved in protein turnover and removing misfolded species, and their enzymes have the potential to offset the defect in proteolytic clearance that contributes to the age-related dementia Alzheimer's disease (AD). The weak cathepsin B and L inhibitor Z-Phe-Ala-diazomethylketone (PADK) enhances lysosomal cathepsin levels at low concentrations, thereby eliciting protective clearance of PHF-τ and Aß42 in the hippocampus and other brain regions. Here, a class of positive modulators is established with compounds decoupled from the cathepsin inhibitory properties. We utilized PADK as a departure point to develop nonpeptidic structures with the hydroxyethyl isostere. The first-in-class modulators SD1002 and SD1003 exhibit improved levels of cathepsin up-regulation but almost complete removal of cathepsin inhibitory properties as compared to PADK. Isomers of the lead compound SD1002 were synthesized, and the modulatory activity was determined to be stereoselective. In addition, the lead compound was tested in transgenic mice with results indicating protection against AD-type protein accumulation pathology.

Protective effects of positive lysosomal modulation in Alzheimer's disease transgenic mouse models.

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid ß peptide (Aß) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aß(1-42). Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aß pathology. Systemic PADK injections in APP(SwInd) and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aß immunostaining as well as Aß(x-42) sandwich ELISA measures in APP(SwInd) mice of 10-11 months. More extensive Aß deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aß(1-38) occurs as Aß(1-42) levels decrease in the mouse models, indicating that PADK treatment leads to Aß truncation. Associated with Aß clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aß(1-42) accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aß pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders.