Abstract Hypochlorous acid (HOCl) is produced naturally by neutrophils and other cells to kill conventional microbes in vivo. Synthetic preparations containing HOCl can also be effective as microbial disinfectants. Here we have tested whether HOCl can also inactivate prions and other self-propagating protein amyloid seeds. Prions are deadly pathogens that are notoriously difficult to inactivate, and standard microbial disinfection protocols are often inadequate. Recommended treatments for prion decontamination include strongly basic (pH ≥~12) sodium hypochlorite bleach, ≥1 N sodium hydroxide, and/or prolonged autoclaving. These treatments are damaging and/or unsuitable for many clinical, agricultural and environmental applications. We have tested the anti-prion activity of a weakly acidic aqueous formulation of HOCl (BrioHOCl) that poses no apparent hazard to either users or many surfaces. For example, BrioHOCl can be applied directly to skin and mucous membranes and has been aerosolized to treat entire rooms without apparent deleterious effects. Here, we demonstrate that immersion in BrioHOCl can inactivate not only a range of target microbes, including spores of Bacillus subtilis, but also prions in tissue suspensions and on stainless steel. Real-time quaking-induced conversion (RT-QuIC) assays showed that BrioHOCl treatments eliminated all detectable prion seeding activity of human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, cervine chronic wasting disease, sheep scrapie and hamster scrapie; these findings indicated reductions of ≥103- to 106-fold. Transgenic mouse bioassays showed that all detectable hamster-adapted scrapie infectivity in brain homogenates or on steel wires was eliminated, representing reductions of ≥~105.75-fold and >104-fold, respectively. Inactivation of RT-QuIC seeding activity correlated with free chlorine concentration and higher order aggregation or destruction of proteins generally, including prion protein. BrioHOCl treatments had similar effects on amyloids composed of human α-synuclein and a fragment of human tau. These results indicate that HOCl can block the self-propagating activity of prions and other amyloids.

Author Summary Many serious diseases have been linked to pathogenic states of various proteins. These naturally occurring proteins can be corrupted to form aggregates such as prions and amyloids that propagate in and between tissues by acting as seeds that convert the normal form of the protein into more of the pathological form. For example, corrupted prion protein can cause fatal transmissible neurodegenerative diseases such as Creutzfeldt-Jakob disease in humans, chronic wasting disease in cervids and bovine spongiform encephalopathy. Other amyloid-forming protein aggregates are pathogenic in Parkinson’s, Alzheimer’s, and other diseases. The fact that prions and amyloids are composed predominantly of tough, tightly packed proteins makes them unusually resistant to conventional microbial disinfection procedures. Infectious prions can persist indefinitely in, or on, a variety of materials such as tissues, fluids, tools, instruments, and environmental surfaces, making it important to identify decontaminants that are effective without being dangerous or damaging. Here we show that hypochlorous acid, a disinfectant that is produced naturally by certain cells within the body, has strong anti-prion and anti-amyloid activity. We find that a non-irritating and broadly applicable hypochlorous acid preparation can disinfect prions in tissue homogenates and on stainless steel wires serving as surrogates for surgical instruments.

Citation: Hughson AG, Race B, Kraus A, Sangaré LR, Robins L, Groveman BR, et al. (2016) Inactivation of Prions and Amyloid Seeds with Hypochlorous Acid. PLoS Pathog 12(9): e1005914. https://doi.org/10.1371/journal.ppat.1005914 Editor: David Westaway, University of Alberta, CANADA Received: July 18, 2016; Accepted: September 4, 2016; Published: September 29, 2016 This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (BC), a gift to LR's department from BrioTech Inc and self-funding by BrioTech Inc (DT). The NIAID administration had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. However, the authors from BrioTech, Inc played roles in all of these aspects of the study. Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: DT is founder and CEO of BrioTech Inc, which sells BrioHOClTM. A gift from BrioTech was used to support the work at UW Bothell lead by LR. JFW is Chief Scientific Officer, corporate executive and shareholder of Briotech Inc.

Introduction Prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal and untreatable neurodegenerative diseases. In humans, prion diseases include sporadic, variant and genetic forms of Creutzfeldt-Jakob disease (sCJD, vCJD and gCJD) as well as a number of other disorders [1–3]. Prion diseases of other species include classical bovine spongiform encephalopathy (C-BSE) [4], scrapie in sheep, goats [5] and rodents, and chronic wasting disease (CWD) of cervids [6, 7]. All mammalian prion diseases share an underlying molecular pathology that involves the conversion of the hosts’ normal form of prion protein, PrPC, to a misfolded, aggregated, infectious and pathological form, PrPSc [8, 9]. Compared to other types of pathogens, prions are unusual in that they lack a pathogen-specific nucleic acid genome, and tend to be particularly resistant to enzymatic, chemical, physical (eg. heat) or radiological inactivation [8, 10]. As a result, prions can resist complete inactivation under conditions that are typically used in medicine, the food industry, and agriculture to inactivate other types of pathogens. Current prion decontamination recommendations include incineration or harsh chemical treatments such as 1–2 N sodium hydroxide, 20–40% household bleach (20,000 ppm sodium hypochlorite) alone or, preferably, in combination with prolonged autoclaving to treat relevant materials or surfaces [10, 11]. Other effective treatments include enzymatic treatments with SDS [12], vaporized hydrogen peroxide [13] or 4% SDS in 1% acetic acid at 65–134°C [14, 15]. Environ LpH™ also inactivates prions [16, 17] but the active formulation of this acidic phenolic disinfectant has been removed from the market. Most, if not all, of the above treatments are potentially hazardous to the user and/or incompatible with various purposes. Thus, more safely and broadly applicable anti-prion reagents are needed. In humans, iatrogenic transmission of prion disease has occurred through the use of contaminated instruments, transplanted tissues or tissue extracts [2, 18]. The risk alone of iatrogenic transmission has been highly problematic when surgical procedures have been performed on patients who were later discovered to have sCJD [18]. Because routine disinfection procedures are not likely to be fully adequate for prions, the reuse of potentially contaminated tools or instruments on subsequent patients presents transmission risks. Prion decontamination is also a significant concern in autopsies and mortuary functions. In livestock, prion diseases can be spread via contaminated feeds and/or the environment [19, 20]. In cervids, the rampant spread of CWD threatens both captive and free-ranging populations in North America, Asia, and now Europe. With BSE, at least, there is also apparent zoonotic risk associated with contaminated beef and its handling in slaughter houses. Thus, prion disinfectants are needed that can be used routinely on potentially contaminated tools, instruments, and environmental surfaces to reduce the risks of prion transmission. Here we have tested a synthetic preparation of hypochlorous acid (HOCl), a reactive oxygen species that is produced naturally in vivo to inactivate pathogens. Synthetic formulations containing HOCl have been shown to kill bacteria, viruses, fungi and protozoans [21–23]. HOCl is the conjugate acid of hypochlorite, the sodium salt of which is the main component of household hypochlorite bleach. In concentrations recommended for prion inactivation, hypochlorite bleach is corrosive and highly basic, i.e. pH ≥~12, whereas HOCl solutions are weakly acidic, i.e. pH 3.7–6.3, and apparently safe for contact with skin and mucous membranes. For example, at least some HOCl formulations are used in cosmetics and topical skin treatments for humans and domestic animals (e.g. www.briotechinternational.com) and/or have strong antimicrobial activity at non-cytotoxic concentrations (e.g. [24]). Furthermore, electrolytically generated HOCl is acknowledged to be both powerful and benign enough to meet USDA standards for sanitation and safe food contact without need for rinsing (FSIS Directive 7120.1, Rev. 36, 6/29/16. US Dept. of Agriculture, pp 31–32). Many studies have described anti-microbial activities of HOCl, but only one has raised the possibility of anti-prion activity. In that study, a cycle of sonications and/or washes with electrolyzed basic (pH 11.9) and acidic (pH 2.7) water, with the latter presumed to contain HCl and HOCl, inactivated prions by ≥1 log 10 on steel wires [25, 26]. However, the role of HOCl in the anti-prion activity of this cyclic treatment remains unclear because, firstly, Cl 2 is also a prominent oxidizing species present in aqueous free-chlorine solutions at pH 2.7 [27]; and secondly, the pH 11.9 step may have been important given that basic solutions can have anti-prion activity [13, 16, 28, 29]. For the present study, we evaluated the anti-prion effects of a single unsonicated treatment with a mildly acidic, electrochemically-activated HOCl formulation (BrioHOCl) using both mouse bioassays [30, 31] and real time quaking-induced conversion (RT-QuIC) assays [32–36]. Animal bioassays are the gold standard tests for prion infectivity but are also costly, animal-intensive, and time-consuming—typically requiring months-years. RT-QuIC assays exploit the inherent self-propagating activity of prions by measuring a sample’s ability to seed the in vitro conversion of recombinant PrPC (rPrPC) into amyloid fibrils that enhance the fluorescence of thioflavin T (ThT) [32, 37]. Detection of RT-QuIC seeding activity correlates strongly with the presence of prion infections in mammalian hosts [32–49]. These assays are not only at least as sensitive as bioassays, but are also much more rapid, high throughput and cost-effective. Thus, our strategy was to first test effects of HOCl and other conventional anti-prion reagents using RT-QuIC, and then confirm any observed effects on infectivity using bioassays. Because of concerns about iatrogenic transmission of prion diseases via contaminated surgical instruments [18], and the tenacious binding and infectivity of prions bound to stainless steel [50, 51], we have not only tested HOCl inactivation of prions in brain homogenates (BH), but also prions on stainless steel wire as a surrogate for surgical instruments. The latter strategy has been employed previously for the evaluation of other disinfectants [12–15, 25, 26, 52]. To investigate whether HOCl might also inactivate other types of self-propagating amyloid seeds, we have also tested effects of BrioHOCl on amyloid seeds composed of human α-synuclein (α-syn) and tau. Aggregated forms of α-syn and tau are prominent pathological features of various proteinopathies including Parkinson’s and Alzheimer’s diseases respectively.

Discussion Numerous clinical and agricultural scenarios involving potential prion contamination would benefit from the availability of less harsh and more practical methods for inactivating prions. Here, we have demonstrated that weakly acidic BrioHOCl has strong anti-prion activity. In the case of hamster ScBH, we have shown a reduction in infectivity titer of ≥~105.75-fold by bioassay in mice (Table 1). This reduction is commensurate with the decrease in prion seeding activity measured by RT-QuIC. For sCJD, vCJD, C-BSE, CWD and sheep scrapie we have shown that BrioHOCl eliminates all detectable RT-QuIC seeding activity (Fig 2). Although we have not also done animal bioassays of BrioHOCl-treated brain homogenates containing these other strains, many prior studies have indicated that prion infection and RT-QuIC positivity in ex vivo samples are strongly correlated, and that RT-QuIC is at least as sensitive analytically as animal bioassays [32, 36, 37, 42, 43]. These results imply so far that if a disinfectant eliminates RT-QuIC seeding activity, it will likely also eliminate prion infectivity. However, we should note that the inverse is not necessarily true; that is, elimination of prion infectivity might not always be accompanied by loss of RT-QuIC seeding activity. For example, Environ LpH™ is much more effective at decreasing bioassayed scrapie infectivity ([16];Tables 1 and 2) than RT-QuIC seeding activity. This is not surprising given that synthetic recombinant PrP amyloids can have RT-QuIC seeding activity but no apparent infectivity in animals. Thus, infectious PrPSc comprises only a subset of PrP particles with RT-QuIC seeding activity, so treatments may neutralize the infectivity of PrPSc without proportionally affecting all possible types of PrP seeding activity. Accordingly, in future potential applications of RT-QuIC assays in screening for other anti-prion treatments, it would be advisable to use infectious tissue-derived prions/PrPSc as test specimens rather than non-infectious synthetic amyloid seeds. Although hits from such screens against PrPSc are likely to be effective against infectivity, it is possible that treatments that can neutralize infectivity without eliminating RT-QuIC seeding activity, like Environ LpH™, would be missed. From a mechanistic perspective, we have shown a positive dose-response relationship between the free Cl concentrations of BrioHOCl preparations and prion seed inactivation (Fig 6, Table 3), protein aggregation, and loss of detectable SDS-soluble protein monomers (Fig 8). The presence of Raman spectroscopy signals for HOCl but not hypochlorite ion, aqueous chlorine, or any other species that may arise during electrolysis of NaCl solutions [56] is consistent with the observed effects being attributable to HOCl. However, it remains possible that other as yet unidentified constituents or characteristics resulting from the manufacturing process contribute to the unusual stability and inactivating activity of BrioHOCl. Despite the high probability that our observations result primarily from the activity of HOCl, we cannot yet pinpoint a particular anti-prion mechanism of BrioHOCl. HOCl can covalently modify a number of different amino acid side chain moieties on proteins, including thiols, amines, aromatic amino acids, and backbone peptide bonds. HOCl reacts most rapidly with sulfur-containing amino acids. Oxidation of methionine leads to the formation of sulfoxide, while disulfide bonds and oxy-acids are the products of cysteine oxidation [57]. Chlorination of lysines and tyrosines leads to formation of chloramines, and 3-Cl-tyrosine and 3,5-Cl-tyrosine residues, respectively. Tyrosines can also undergo dimerization via the formation of phenoxyl radicals, leading to protein crosslinking [58, 59]. Although less reactive than many amino acid side chains, backbone amide bonds can be chlorinated by excess HOCl leading to polypeptide fragmentation [60]. Any of these modifications, including those leading to further PrPSc aggregation by crosslinking, may modify or occlude seeding surfaces on PrPSc even without unfolding the protein, preventing PrPSc from converting PrP monomers into more PrPSc in vivo, or into recombinant PrP amyloid in vitro. A loss of specific infectivity with further aggregation of PrPSc would be consistent with previous observations that the most infectious prion particles are small and non-fibrillar [61]. We have shown activity of a HOCl formulation against prions in wet tissue homogenates and dried onto stainless steel wires. The ability to inactivate prions on stainless steel implements and instruments without damaging them would reduce risks of iatrogenic transmission in clinical settings, autopsy rooms and slaughter houses. Further work will be required to ascertain whether BrioHOCl can not only inactivate prions bound to stainless steel, but also to other types of materials such as those covering gastroscopes, broncoscopes and rhinoscopes. Although our results indicate that BrioHOCl’s active Cl content can decline over time, its stability on storage in sealed high density polyethylene vessels (half-life estimated to be 440 days), even under less-than-ideal warehouse storage conditions, is compatible with various practical applications. Certainly, further work is required to optimize storage vessel selection and fluid handling. Nevertheless, the speed and efficacy of prion inactivation even with test samples from barreled production lots >9 months old (Figs 1, 2, 4 and 5; Tables 1 and 2), together with the persistence of high level and rapid efficacy (4–7 LRV) against some of the most resistant microbes known (Bacillus and Aspergillus spores) (S1 Table), support the practical utility of BrioHOCl. Given the prolonged persistence of prions in the environment (e.g. [62–64]), it is important to have practical means of neutralizing prion infectivity in natural settings as well as procedure rooms, operating rooms, animal handling facilities and food processing plants. The generality of the effects of this HOCl formulation on proteins, as evidenced by the mobility shifts of many brain homogenate proteins (Fig 8), is consistent with its effects on PrPSc (Fig 8) and amyloid seeds of α-synuclein (Fig 9) and the tau fragment (Fig 10). This raises the intriguing possibility that HOCl could have even broader effects on pathological protein aggregates that are capable of seeding their own accumulation. Recent studies have indicated experimental transmissibilities of several protein misfolding processes such as those of Alzheimer disease, Parkinson disease, multiple systems atrophy [65, 66], and tauopathies (reviewed in [9, 67]). Although to our knowledge there is no clear evidence of transmissions of these diseases between humans, these studies have raised concerns that self-propagating protein amyloids, e.g. those composed of Aβ [68], α-synuclein, and tau, might pose risks of iatrogenic transmission via contaminated medical instruments or transplanted tissues. If the seeding activity associated with these various diseases can be inactivated by appropriate HOCl exposure as suggested by our study, then such potential transmission risks might be mitigated. Finally, as noted above, HOCl is produced naturally in vivo by a variety of “professional” phagocytes such as neutrophils, microglia and macrophages as part of innate immune mechanisms to inactivate microbial pathogens and trigger a variety of beneficial pathophysiological responses to injury. The fact that we have now shown that HOCl also has anti-prion activity in vitro raises the possibility that the same might be true in vivo. Many proteins can form misfolded oligomers and amyloid fibrils that can seed their own growth and accumulate in tissues to cause pathological changes. Protein quality control systems such as the unfolded protein response, chaperones, ubiquitination, proteasomes and autophagy can usually prevent the accumulation of misfolded proteins within cells [69]. However, it is less clear how organisms ordinarily cope with amyloid-like aggregates that escape these systems and accumulate inside or outside cells. Perhaps such aggregates can be recognized, and exposed to HOCl or other more stable products of the reactive oxygen burst such as N-chlorotaurine [70]. Such HOCl exposure might inactivate the self-propagating activity of protein aggregates and/or aid in their clearance. Further studies will be required to evaluate whether such a mechanism is a significant component of proteostasis in vivo.

Materials and Methods Anti-Prion reagents Pure Bright Germicidal Ultra Bleach® was used as the source for Na hypochlorite (6%). Environ LpH™ was obtained originally from Steris Inc. and had been stored for ≥6 years prior to use. It should be noted that this specific Environ LpH™ formulation differs from product sold with the same name in Europe [17]. BrioHOCl was produced from a saturated NaCl solution and filtered water in an electrochemical cell by a proprietary process of Briotech Inc., Woodinville, WA. We confirmed that HOCl is a primary active component of BrioHOCl, and that other potential electrochemical reaction products such as OCl- (hypochlorite) or molecular chlorine (Cl 2 ) were undetectable, by Raman spectroscopy (S1 Fig). Disinfectant treatments of BH suspensions 10% (w/v) brain homogenates (BH), defined as 10−1 tissue dilutions, were prepared as described previously [32] from brain tissue obtained from a scrapie-infected hamster, a BSE-infected cow (a gift from Dr. Kentaro Masujin, National Institute for Animal Health, Tsukuba, Japan), a scrapie-infected sheep (a gift from Dr. David Schneider, Animal Disease Research Unit, USDA-ARS, Pullman, WA), a human vCJD decedent (Drs. Kaetan Ladhani and Jillian Cooper at the CJD Resource Centre, NIBSC, Herts, UK), and a CWD-infected mule deer (Drs. Michael Miller, Colorado Department of Wildlife; Elizabeth Williams and Jean Jewell, University of Wyoming; and Terry Kreeger, Wyoming Game and Fish Department). In each case, 10% BH (w/v) was incubated at room temperature for the designated time in saline (0.9% NaCl), BrioHOCl, 1M NaOH, 40% household bleach (2.4% Na hypochlorite) or 2% Environ LpH™ at a ratio of 100:1, or 20:1 (v/v, disinfectant:BH) as specified. Following incubation, serial 10-fold dilutions were prepared in Sample Diluent (PBS, 0.1% SDS and Gibco N2 media supplement) and seeding activity was quantified by end-point dilution RT-QuIC analysis followed by Spearman-Kärber analysis to estimate the seeding dose giving positive reactions in 50% of the technical replicates [32]. In other experiments, a milder deactivation condition was used in which 10% (w/v) BH was incubated for only 5 min in saline or the designated disinfectants and then immediately diluted into Sample Diluent to greatly reduce continued effects of the disinfectants. Disinfection treatments of scrapie-coated wires Batches of sterile stainless steel suture wire (Havel, size 000), cut into 3–4mm lengths, were soaked in BH from normal (uninfected) or scrapie-infected animals at tissue dilutions of 10−3–10−10 for 1 h at room temperature, washed 3 times with a brief vortex in 1ml PBS, and left to dry in a sterile petri dish. Additional batches of wires coated with 10−3 scrapie-infected BH (ScBH) were further treated by submerging in disinfectants (BrioHOCl, 1M NaOH, 40% household bleach (2.4% hypochlorite), 2% Environ LpH™ or saline (mock disinfectant)). RT-QuIC analyses All RT-QuIC seeding assays were conducted using conditions similar to those described previously (eg. [32]) with variations described below. Hamster (90–231) recombinant prion protein (rPrPC) (Accession number K02234) or chimeric hamster-sheep rPrPC (Ha-S; Syrian hamster residues 23 to 137 followed by sheep residues 141 to 234 of the R 154 Q 171 polymorph [accession nos. K02234 and AY907689] [71]) were used as substrates in RT-QuIC experiments as indicated. Purification of hamster (90–231) and Ha-S rPrPC was conducted as previously described [72]. To measure prion seeding activity in brain tissue dilutions, 2 μl of each dilution was added to 98 μl RT-QuIC reaction solution to give final concentrations of 0.1 mg/ml rPrPC, 10 mM phosphate buffer (pH 7.4), 10 μM thioflavin T (ThT), 300 mM NaCl, 1 mM EDTA and 0.002% SDS. This final concentration of SDS in the reaction volume resulted from dilution of seed sample containing 0.1% SDS. Four technical replicate reaction wells at each dilution were set up in a 96-well plate. For analysis of wires, single wires were transferred into wells containing 100 μl of the RT-QuIC reaction solution with the 0.002% SDS final concentration added directly. The plates were then shaken in a temperature-controlled fluorescence plate reader (BMG FLUOstar) at 42°C unless indicated otherwise with cycles of 1 min double orbital shaking at 700 rpm and 1 min of rest [32]. ThT fluorescence was measured at 45-min intervals. To measure α-syn seeding activity recombinant α-syn purchased from rPeptide (Catalog # S 1001 1) was used as a substrate. α-Syn fibrils were generated in 20 mM Tris-HCl, 100 mM NaCl, pH 7.4 through constant shaking at 1000 rpm while incubating at 37°C for 5 d in a Eppendorf Thermomixer R. 20 μL of these fibrils, or fibrils treated with HOCl as described, were used to seed a reaction mix containing final concentrations of 104 mM Tris, 20 mM NaCl, 10 μM ThT, and 30 μM α-syn, at pH 7.5. Seeded reactions were incubated at 37°C with the shake-rest cycles and reading parameters the same as for RT-QuIC. For tau-based RT-QuIC reactions, the cysteine-free K19 tau fragment was expressed and purified as previously described [55, 73] with modifications. Synthetic tau seed was generated and seeding assays with fluorescence detection were performed in HEPES-buffered saline solutions containing low molecular weight heparin by following the protocol described [55] with modifications such as periodic shaking in a 96-well plate rather than sonication in tubes. All RT-QuIC experiments were set up such that the plate readers would give a ThT fluorescence negative control baseline of around 50,000 rfu (relative fluorescence units). These readers have a fluorescence saturation signal of 260,000 rfu. Following collection the experimental data was normalized such that the baseline signal of the lowest negative control was set at 0% and the saturation signal of 260,000 rfu was set at 100%. The individual traces graphed are the averages of the 4 wells for each dilution tested. For Spearman-Kärber analyses of end-point dilution RT-QuIC experiments [32, 74], individual reaction wells were judged to be positive at 50 h when the signal exceeded 50% of the saturation signal. The seeding dose (± S.E.) giving ThT positivity in 50% of technical replicate wells (SD 50 ) was calculated as described using the S.E. “smoothing” procedure to account for small group size [68]. Mice Homozygous, tg7 mice on a C57BL/10 background were bred at RML and used for all bioassay experiments. Creation of the original tg7 mice has been described previously [31]. The tg7 mice used in these bioassays over-express hamster PrP (approximately 5-fold) under the control of the endogenous mouse PrP promoter and do not express any mouse PrP. Mouse bioassay of tissue suspensions Following the 100:1 Disinfectant treatments of BH suspensions described above a 10−3 dilution of ScBH was further diluted in serial 10-fold increments into PBS for inoculation into mice. The following dilutions of treated ScBH were tested: Saline treatment group, 10−4 through 10−10; BrioHOCl, 10−3 through 10−8; NaOH, 10−5 through 10−8; bleach, 10−5 through 10−8; Environ LpH™, 10−5 through 10−9. The dilutions selected for bioassay were based on expected levels of infectivity or, in some situations, the disinfectants were further diluted prior to inoculation to prevent acute toxicity (i.e., for NaOH, bleach and Environ LpH™). Each dilution was inoculated intracerebrally into groups of 4 tg7 mice. For the inoculation, mice were anesthetized with isoflurane and inoculated in the left brain hemisphere with 30 μl of dilutions of disinfectant- or saline-treated ScBH. Following inoculation mice were monitored for onset of scrapie. Mice were euthanized when they displayed advanced stages of scrapie including poor grooming, kyphosis, ataxia, wasting, delayed response to stimuli, and somnolence. Following euthanasia brains were removed and flash frozen for biochemical analysis. Infectivity titers were calculated for each experimental group using the Spearman-Kärber formula [32]. Mouse bioassay of steel-bound scrapie infectivity For wire implantation, experimental groups were 3–8 mice (Table 2). Tg7 mice were anesthetized with isoflurane gas and the dorsal surface of the mouse skull was shaved, ophthalmic ointment was applied to protect each eye, and the dorsal surface of the skull was scrubbed with chlorhexidine surgical scrub. Each mouse was then positioned in a stereotactic device and isoflurane anesthesia was provided via nose cone. Using aseptic technique a midline incision was made on the skin of the skull to expose the bregma landmark. The drill was positioned at a location 1 mm anterior to bregma and 1.7 mm to the left, lateral side of midline (above the striatum). A small hole was drilled at this location and a 3–4 mm pre-treated stainless steel wire was inserted. Bone wax was used to seal the defect in the skull once the wire was is in place. The incision was closed with 5–0 PDS suture in a cruciate pattern. Mice were placed in heated cages following surgery until fully recovered. Each mouse received 0.2 mg/kg buprenorphine (Buprenex) subcutaneously immediately post-surgery. Following implantation tg7 mice were monitored for onset of scrapie. Mice that developed disease were euthanized. At the time of euthanasia, all the wires were confirmed to be in place and showed no signs of deterioration. Active chlorine and other BrioHOCl characteristics Hach reagent kits for Total (active, free) Chlorine (Hach Company, Loveland, CO) were used for determination of the active Cl content of the BrioHOCl formulation, after validation by comparison of manual iodometric and digital titration results on 33 samples (6 replicates each). Thereafter the digital Hach device was used (4 replicates per sample) to measure active Cl in all samples used for inactivation experiments with PrPSc, and for antimicrobial efficacy testing. Titratable free Cl concentrations were also measured in archived samples at Briotech (oldest 34 months), and, to establish the Cl trends, in a serially sampled lot of BrioHOCl. The latter were stored in sealed ~100 mL aliquots in HDPE bottles at 21°C, and prepared specifically for this purpose. All other HOCl samples used throughout this study were derived from routine production electrolysis runs at the manufacturing plant. Product from each lot was stored in different vessel types (100 mL up to 4 L bottles, and 220 L barrels, all HDPE) in uncontrolled temperature warehouse environments (3.5°C to 35°C). Small vessels were sealed with aluminum caps, and drums lids were tightly sealed to avoid exposure to air (known to be deleterious), but no optimization of storage conditions was attempted for materials used herein. The pH, oxidation-reduction potential (ORP, in mV) and conductivity were recorded for all samples using a Hach Multi Parameter meter (Model HQ40d). ORP targeted at production was +1140 mV, at pH 3.9. Starting active Cl concentrations were varied in production lots during electrolysis, depending on intended applications. Generally these values ranged between 175 and 350 ppm active Cl, with background NaCl concentrations of either 0.9 or 1.8%. Solutions with both NaCl backgrounds were tested in RT-QulC assays. UV/Vis spectrophotometry Test solutions were loaded into 1 mL quartz cuvettes, and spectra obtained using a BioMate 3S UV-Visible Spectrophotometer. The instrument was blanked using Nanopure water, and test solutions consisted of undiluted BrioHOCl at selected time points in the sequential sampling of product stored at room temperature. Absorbance was measured from 190–400 nm. Raman spectroscopy Spectra were obtained using a Renishaw InVia Raman microscope. Spectra were observed using an excitation wavelength of 785 nm with undiluted BrioHOCl in a 1 mL quartz cuvette. The acquisition time for each scan was 20 s, and 100 acquisitions were accumulated. A deionized water blank was scanned in the same manner, and subtracted from the test sample data using Igor software (WaveMetrics). Protein gel analysis PrPSc, purified as previously described [75], hamster ScBH at a dilution of 10−1, α-synuclein, or Lewy bodies isolated from the brains of a Lewy body dementia patient, as described [76], were pretreated with saline or BrioHOCl solutions as indicated in the figure legends. Following treatment, samples were diluted with equal volumes of 2X sample buffer (125 mM Tris-HCl pH 6.8, 5% glycerol, 6 mM EDTA, 10% SDS, 0.04% bromophenol blue, 48% urea, 8% 2-mercaptoethanol) and boiled for 5 min. Equal volumes of samples were run on 10% Bis-Tris NuPAGE gels (Invitrogen) and used for subsequent Deep Purple protein stain per manufacturer’s instructions (GE Healthcare) or the protein transferred to an Immobilon P membrane (Millipore) using the iBlot Gel Transfer System (Life Technologies). PrP was detected using rabbit PrP antisera R30 (1:10,000; residues 90–104) [77, 78] and alkaline-phosphatase conjugated secondary antibody (1:5000; Jackson ImmunoResearch). α-Synuclein was detected using mouse Anti-α-Synuclein Clone 42 antibody (1:1000; BD Transduction Laboratories) and alkaline-phosphatase conjugated secondary antibody (1:2000; Jackson ImmunoResearch). Tau was detected using a tau antibody (anti-tau ab64193, Abcam) as the primary antibody. Ethics statement All mice were housed at the Rocky Mountain Laboratory (RML) in an AAALAC-accredited facility in compliance with guidelines provided by the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research Council). Experimentation followed RML Animal Care and Use Committee approved protocol #2015–070 in compliance with guidelines provided by the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research Council).

Supporting Information S1 Fig. Raman spectroscopy of BrioHOCl. https://doi.org/10.1371/journal.ppat.1005914.s001 (DOCX) S1 Table. Antimicrobial efficacy of BrioHOCl in ASTM E2315 Time vs. Kill suspension test protocol using lot samples of different ages. https://doi.org/10.1371/journal.ppat.1005914.s002 (DOCX) S2 Fig. RT-QuIC seeding activity tolerance for BrioHOCl. RT-QuIC analysis was performed with Hamster (90–231) recombinant prion protein substrate at 42°C using 2μl per well of normal brain homogenate (gray) or hamster scrapie brain homogenate at a tissue dilution of 5x10-8 as reaction seed in the presence of 0 (red) or 0.001, 0.01, 0.1, 1 & 10% BrioHOCl (blue) is indicated. In each case BrioHOCl concentrations were added directly to the reaction volume in the wells. Each trace represents the average ThT fluorescence of four technical replicate wells normalized between baseline and maximal signal and graphed here as a function of time. https://doi.org/10.1371/journal.ppat.1005914.s003 (DOCX) S3 Fig. Tolerance of α-synuclein RT-QuIC assay for BrioHOCl. Direct addition of BrioHOCl to α-synuclein RT-QuIC reactions seeded with a 10-2 dilution of an artificial α-syn seed. While direct addition of the equivalent to a 10-2 dilution (1.8% HOCl, blue line) partially interfered with the reaction (compared to the no HOCl control, orange line), 10-3 (0.18%), 10-4 (0.018%), and 10-5 (0.0018%) dilution equivalents of HOCl had no effect on the reaction kinetics when directly added to the reaction without preincubation with the α-syn seed. https://doi.org/10.1371/journal.ppat.1005914.s004 (DOCX)

Acknowledgments We thank Michael Mettrick and Drs. Karin Peterson and Christina Orrù for critical review of the manuscript. We also thank Lynne and Gregory Raymond for technical assistance. We thank Anita Mora, Ryan Kissinger and Austin Athman for graphics assistance; and Jeff Severson for animal husbandry. The Raman spectral analysis was conducted, with advice from Micah Glaz, at the Molecular Analysis Facility, a National Nanotechnology Coordinated Infrastructure site at the University of Washington which is supported in part by the National Science Foundation (grant ECC-1542101), the University of Washington, the Molecular Engineering & Sciences Institute, the Clean Energy Institute, and the National Institutes of Health.

Author Contributions Conceptualization: BC DT AGH BR AK LRS BRG ES LR DT JFW MM. Funding acquisition: BC DT LR. Investigation: AGH BR AK BRG ES KP VD MM LC LR DT JFW. Methodology: AGH DT BR AK BRG ES MM BC JFW LRS LR. Project administration: BC JFW LRS LR DT. Resources: DT GZ JFW LRS LR. Supervision: BC DT JFW LR. Visualization: AGH BR AK BRG ES LR. Writing – original draft: BC AGH BR AK BRG ES. Writing – review & editing: BC AGH BR AK AK BRG ES JFW LRS LR GZ.