OBJECTIVE: Develop and demonstrate a technology to rapidly purify bacteriophages (phages) of Gram-negative bacteria to a level suitable for human therapeutic use, free of endotoxin and of other bacterial remnants including other pyrogens and pathogen-associated molecular patterns (PAMPS), for application in treating recalcitrant multidrug resistant infections of the Warfighter. DESCRIPTION: Multidrug resistant (MDR) bacterial infections present a worldwide public health crisis, and MDR wound infections are an enduring challenge for the Military Health System. Treatment options are diminishing with the proliferation of MDR and lack of new antibiotic development. Multi-domain operations (MDO) envision medical evacuation delayed days to weeks, leaving wounded Warfighters and front-line medical providers in prolonged care (PC) scenarios of uncertain duration. Front-line providers will need to care for high volumes of wounded for days while retaining mobility, severely limiting interventions to prevent sepsis caused by polytraumatic wounds. Increasing MDR bacteria combined with PC will yield infection rates of 50-60% of the wounded, with a dramatic increase of sepsis-related deaths that could parallel those of previous conflicts such as Vietnam, Korea, or the World War II Pacific Theatre (in which medical support was delayed for weeks). To address these challenges, innovative and scalable solutions are urgently needed to amplify the capability of front-line providers to care for large numbers of wounded. The bacterial ESKAPEE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli) frequently colonize healthy military personnel and are common agents of persistent infections of traumatic and burn wounds that are biofilm associated and MDR. The diminution in effective antibiotic options and urgent need for wound infection and sepsis mitigation necessitates the development of novel effective antimicrobials with alternative mechanisms of antibacterial activity that can be used in combination with existing Standard of Care (SOC) antibiotics. Phage therapeutics are thus being developed for therapeutic and prophylactic purposes. Lytic or virulent phages are viruses of bacteria that specifically infect and kill their bacterial host species, including antibiotic resistant and MDR variants. Since they possess specificity to their bacterial targets, phages eliminate the infectious species without perturbing normal human microflora. Phages have demonstrated therapeutic efficacy against ESKAPEE infections in the laboratory and in domestic and farm animals, with promising results in expanded access human treatment and even in recent clinical trials when used in combination with antibiotics. Phage therapy has the potential to become an important adjunctive therapy against MDR bacterial infections in civilian and military patients, but robust randomized clinical trials must first be conducted to investigate the benefits of phage therapy with SOC compared with SOC alone. Standardized reliable and repeatable manufacturing of phage biologics is needed to obtain safe, uniform phage products the US Food & Drug Administration (FDA) can approve to go into clinical trials. As phage biologic products enter large-scale phase 3 clinical trials, and obtain licensure to enter production as approved therapeutics, the scalability of these manufacturing processes will be essential. Because phage manufacturing is universally done by propagating the virus on a susceptible host bacterium, a critical challenge is the removal of bacterial remnants after phage propagation, including the removal of lipopolysaccharide (LPS, also termed endotoxin), a component of Gram-negative bacteria. Many phages use parts of LPS as a receptor and thus bind LPS with moderate to high affinity. This makes endotoxin removal for the level of purity needed for parenteral routes of delivery a challenge. A high purity phage product is critical for intravenous administration, often used in expanded access phage therapy regimens as a precursor of broader therapeutic application. The purpose of this STTR is to provide a technology or process to enable the rapid purification of bacteriophages, including the removal of bacterial remnants, from a variety of Gram-negative bacterial targets. The envisioned system will purify a wide variety of phages that propagate on a variety of bacterial strains of the Gram-negative ESKAPEE pathogens (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli). This capability will be scalable to directly enable the manufacture of therapeutic phages and phage cocktails, to provide purified preparations of diverse phages with very low residual endotoxin at levels deemed acceptable by FDA for parenteral delivery. Future users of the system will use the technology developed for the purification of a wide variety of phages against any or all of these Gram-negative bacterial pathogens. The technology may be, but is not limited to, micro-filtration systems, microfluidics, centrifugation, nano-materials, gel or polymer matrix or any combination of relevant and novel technologies. The device can be a closed or open modular system. The following features will be critical to consider when proposing a technology: 1) System enables users to purify a variety of phages against a range of Gram-negative pathogens (K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter spp., and E. coli) over a 24 hour period, allowing multiple phages to be processed within a work day for small scale manufacturing and a significant improvement in efficiency to current large scale phage purification. 2) System removes bacterial remnants of the production host strain including endotoxin and other pyrogens to acceptable levels for clinical use. The FDA acceptable limit of endotoxin for intravenous delivery is 5 EU per kg per h, thus 300 EU or lower is sought per 109 - 1010 plaque forming units (pfu) phage per dose (https://www.americanpharmaceuticalreview.com/Featured-Articles/353977-Calculating-Endotoxin-Limits-for-Drug-Products/). 3) System will be scalable from laboratory-level phage production and small-scale manufacturing (yields in the 1010 to 1011 pfu/mL range) to large-scale cGMP manufacturing of drug products. PHASE I: This phase is focused on the design of a proof-of-concept prototype technology that enables rapid phage purification from different Gram-negative bacterial hosts. During this phase, the performer may focus on endotoxin removal from a variety of phages against at least two Gram-negative ESKAPEE bacterial hosts, including P. aeruginosa since it can be challenging for the production of phages with low endotoxin levels. System does not need to be integrated in this phase but should have scalable workflow. At the end of this phase, a working prototype technology will efficiently purify phages from at least two bacterial species. Comparison with published methods can be used to assess the efficiency or rapidity of phage purification. PHASE II: During this phase, the technology/device will be integrated. The workflow from Phase I will be refined to expand the proof-of-concept to a product enabling rapid purification of diverse phages against diverse strains of the listed target species. Critical features of the technology will be addressed including measures of purity including endotoxin removal, efficiency and scalability. Testing will be controlled, rigorous and reproducible. PHASE III DUAL USE APPLICATIONS: Efforts during this phase should address the US Army Medical Materiel Development Activity and the office of Warfighter Protection and Acute Care small business. This phase will focus on scaling production, marketing of the developed technology to distributors, and contracts. Accompanying application instructions, simplified procedures, and training materials are drafted in a multimedia format for use and integration of the product into market. The end-state for this product is a commercially viable technology for phage product manufacturing in the industry that will also be used for phage product development by the Department of Defense. Performer should pursue a commercial path to improve phage manufacturing processes within the bio-pharma arena, but also across medical and educational institutes involved in personalized medical treatments and in product development. The developed process will be integrated into tailored phage therapy and into larger scale industrial manufacturing of phage products for human and veterinary health applications. This product will be broadly applicable to the civilian health care system for wound care, especially against MDR infections. The Contractor may collaborate with the Walter Reed Army Institute of Research (WRAIR) team in optimizing and validating the system. Potential funding sources include Medical Technical Enterprise Consortium and Congressionally Directed Medical Research Programs' mechanisms including the Peer Reviewed Medical, Combat Readiness-Medical and Joint Warfighter Medical Research Programs. REFERENCES: 1. Vento TJ, Cole DW, Mende K, Calvano TP, Rini EA, Tully CC, Zera WC, Guymon CH, Yu X, Cheatle KA, Akers KS, Beckius ML, Landrum ML, Murray CK. Multidrug-resistant gram-negative bacteria colonization of healthy US military personnel in the US and Afghanistan. BMC Infect Dis. 2013;13:68. 2. Akers KS, Mende K, Cheatle KA, Zera WC, Yu X, Beckius ML, Aggarwal D, Li P, Sanchez CJ, Wenke JC, Weintrob AC, Tribble DR, Murray CK; Infectious Disease Clinical Research Program Trauma Infectious Disease Outcomes Study Group. Biofilms and persistent wound infections in United States military trauma patients: a case-control analysis. BMC Infect Dis. 2014 Apr 8;14:190. 3. Nikolich MP, Filippov AA. Bacteriophage therapy: Developments and directions. Antibiotics (Basel). 2020 Mar 24;9(3):135. 4. Luong T, Salabarria AC, Edwards RA, Roach DR. Standardized bacteriophage purification for personalized phage therapy. Nat Protoc. 2020 Sep;15(9):2867-2890. 5. Aslam S, Lampley E, Wooten D, Karris M, Benson C, Strathdee S, Schooley RT. Lessons learned from the first 10 consecutive cases of intravenous bacteriophage therapy to treat multidrug-resistant bacterial infections at a single center in the United States. Open Forum Infect Dis. 2020 Aug 27;7(9):ofaa389. KEYWORDS: Bacterial infections, multidrug resistance, bacteriophages, phages, antibacterials, phage purification, endotoxin removal, phage manufacturing
