Methods and compositions related to high persistence phages

Abstract

Embodiments are directed to methods for measuring phage persistence in a timely and cost-effective manner by determination of surface charge and hydrophobicity. Other embodiments are directed to methods of enhancing persistence of phage preparations.

Description

METHODS AND COMPOSITIONS RELATED TO HIGH PERSISTENCE PHAGES

[0001] This International Patent Application claims priority to US Provisional Application 63 / 521,087 filed June 14, 2023 and US Provisional Application 63 / 568,395 filed March 21, 2024, each of which is incorporated herein by reference.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] None.BACKGROUND

[0003] Bacterial disease is progressively confounding medicine as increasingly more multi-drug resistant (MDR) bacteria evolve. The CDC reports that MDR bacteria have caused about 23,000 deaths (2.0 million cases) per year in 2013 and 35,000 deaths (2.8 million cases) per year in 2019 (URL cdc.gov / drugresistance / biggest-threats.html). Clostridium difficile infections are not included in these numbers. As more MDR bacteria evolve, the current therapeutic response is development of new antibiotics. However, without exception, MDR bacteria eventually evolve to bypass all antibiotics (Avershina et al., Front. Microbiol. 2021, 12, 707330; Bassetti and Garau, J. Antimicrob. Chemother. 2021 76(Suppl 4), iv23- iv37).

[0004] A promising alternative strategy for treating bacterial infections is anti-bacterial therapy using bacteria-infecting viruses or bacteriophages (phages) (URL cidrap.umn.edu / newsperspective / 2021 / 10 / scientists-biotechs-look-unlock-potential-phage- therapy; Serwer, AIMS Microbiol. 2017, 3, 706-12). The phages used are primarily doublestranded DNA, lytic (in contrast to lysogenic) phages. A mixture of several phages (called a phage cocktail) is given to a patient either IV or IP. The phages infect disease-causing bacteria and propagate, thereby killing most of the disease-causing bacteria. Importantly, phage therapy is refractory to the bacterial resistance problem because bacterial resistance toany one phage (which also always occurs) is managed by inexpensive and rapid isolation of new phages. The problem of MDR bacteria can be solved if phage therapy is improved.

[0005] Phage therapy started in the 1920s and continued through the early 1940s (reviewed in URL cidrap.umn.edu / newsperspective / 2021 / 10 / scientists-biotechs-look-unlock- potential-phage-therapy; Serwer, AIMS Microbiol. 2017, 3, 706-12). Phage therapy was successful with typhoid fever, caused by Salmonella typhimurium . However, success was not achieved with skin and tissue infections by Streptococcal and Staphylococcal bacterial strains. Low phage lifetime in blood (also called low phage persistence) appeared to be the reason (reviewed in Serwer et al., Antibiotics (Basel) 2021, 10:723). A recent revival of phage therapy for Acinetobacter baumannii infections generated a spectacular success followed by failure. The failure was associated with low phage persistence (reviewed in URL cidrap.umn.edu / newsperspective / 2021 / 10 / scientists-biotechs-look-unlock-potential-phage- therapy and Serwer et al., Antibiotics (Basel) 2021, 10:723). Success with murine Shigella dysenteriae infection was associated with high phage persistence (reviewed in Serwer et al., Antibiotics (Basel) 2021, 10:723). These results suggest that rapid, inexpensive screening for high persistence phages is needed for improving current phage therapy.

[0006] Current methods use phage lifetime in the blood of mice, which is expensive, especially in the cost of personnel, and takes about a week. Especially in cases of emergency, more rapid (about one day), up-scalable procedures are needed. Additionally, more rapid methods for determining or screening for phage persistence are needed.SUMMARY

[0007] A current limitation in phage therapies is low persistence of the phages in a treated organism. Persistence is the time phage is detectable in an organism, with blood persistence being the time a phage is detectable in the blood at levels comparable to what was originally inoculated. Typically, low persistence phages never achieve such blood levels when inoculated IP in mice. High persistence phages attain these blood levels for periods, thus far, as high as about 6 hours in mice. The current disclosure provides a solution to the problemsassociated with determining the probability that any given phage will have high-persistence phages or predicting persistence in a timely, cost effective and up-scalable manner.

[0008] Embodiments are directed to methods that measure physical properties that are, in both theory and practice, correlated with persistence. These physical properties of a target phage include average electrical surface charge density (G) and surface hydrophobicity (H). The methods can include native (intact phage) agarose gel electrophoresis (AGE). The rapid assessment of G and H can be used to provide more reliable phage therapy compositions with high probability of a sufficient persistence, for example for the treatment of bacterial infections, e.g., MDR; or cancer, e.g., when the phage is used as an anti-cancer drug delivery vehicle.

[0009] Certain embodiments are directed to a screening method for high persistence phage comprising: one or both of (i) determining a target phage surface charge (G) and / or (ii) determining hydrophobicity by measuring change of electrophoretic mobility in the presence of ionic detergent. In certain aspects a negative surface charge and / or a lower hydrophobicity identifies high persistence phage. In certain aspects the target phage surface charge is determined using gel electrophoresis. The gel electrophoresis can be a one- or two- dimensional native gel electrophoresis, for example native polysaccharide (e.g., agarose) gel electrophoresis.

[0010] Certain embodiments are directed to a phage composition comprising a phage selected for high persistence that confers high persistence to a second target phage. In certain aspects the conferring phage is a phage G.

[0011] Other embodiments are directed to a high persistence phage composition comprising a persistence factor.

[0012] Certain embodiments are directed to methods of treating an infectious disease or a metastatic cancer by administering a high persistence phage(s). In certain aspects an anticancer phage is loaded with an anti-cancer agent. Certain methods include delivering a drug to a subject comprising administering a drug loaded high persistence phage or drug loaded phage to a subject.

[0013] Still other embodiments are directed to methods of producing a high persistence phage comprising contacting a target phage with a persistence factor or a second phage selected for high persistence wherein high persistence is conferred or transferred to the target phage. Certain methods are directed to methods of increasing the persistence of a phage comprising incubating a first phage with a nongenomic persistence element of a second phage, wherein the nongenomic persistence element of the second phage is transferred to the first phage increasing the persistence of the first phage.

[0014] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0001] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0015] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0016] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0017] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0018] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and / or method that “comprises” a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps), but may include other elements (or components or features or steps) not expressly listed or inherent to the chemical composition and / or method.

[0019] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0020] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a chemical composition and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel character! stic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0021] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modificationswithin the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.DESCRIPTION OF THE DRAWINGS

[0022] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0023] FIG. 1 A-1B. Electron Microscopy of PrimeStore-Reagent Inactivated E. coli.

[0024] FIG. 2A-2B. Electron Microscopy of PrimeStore-Reagent Inactivated E. coli.

[0025] FIG. 3A-3B. Electron Microscopy of PrimeStore-Reagent Inactivated E. coli.

[0026] FIG. 4A-4B. Electron Microscopy of PrimeStore-Reagent Inactivated

[0027] B. thuringiensis.

[0028] FIG. 5. Multigel AGE analysis of phages T4 and clear-plaque phage G, with a phage T3 standard. AGE was performed for 16.0 hr at 1.0 V / cm, 25 °C, as described in (Serwer, Anal. Biochem. 1981, 112, 351-356) with Seakem Gold agarose (Lonza, Rockland, Maine, USA) running gels of the following six ^ values: 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6. The electrophoresis buffer was 0.09 M Tris-acetate, pH 8.4, 0.001 M MgCh. All three phages were analyzed in the same 18-track multigel. The preparation of phages has been previously described: T3 (Serwer et al., J. Mol. Biol. 1983, 170, 447-469), T4 (Serwer et al., Antibiotics (Basel) 2021, 10, 723), G (Roberts et al., Int. J. Mol. Sci. 2023, 24, 8941). Clear plaque phage G has a genomic sequence that differs from both the reference GenBank sequence (JN638751) and its parent at three single nucleotide locations. These changes, at co-ordinates 330356 (T>C), 343537 (G > A), and 454601 (G > A), would produce non-synonymous changes to the (functionally uncharacterized) gene products G_424 (p.V1452A), G_428 (p.G35S) and G_625 (p.G64S), respectively.

[0029] FIG. 6. Transfer of high persistence from phage G to phage 0105phi7-2. To increase phage G persistence, the clear plaque mutant of phage G was passed through mouse blood three times, as previously described (Roberts et al., Int. J. Mol. Sci. 2023, 24, 8941).After each passage, a sample of blood was taken at 45 minutes after IP inoculation. Phages present were titered and then re-propagated in-gel (plate stock) as previously described (Roberts et al., Int. J. Mol. Set. 2023, 24, 8941). After the final passage and plate stock production, the phages were partially purified by differential pelleting (Roberts et al., Int. J. Mol. Sci. 2023, 24, 8941) (phage GH), which produced phages with an increase in persistence. Then, the phages were purified by rate zonal centrifugation through a sucrose gradient (Roberts et al., Int. J. Mol. Sci. 2023, 24, 8941), which caused loss of the persistence increase. The persistence increase was restored by adding an extract of uninfected cells to the high persistence selected phage G, but not to its unselected parent. In the current experiment, when GH was used, it was accompanied by 31.7 pl of uninfected cell extract (below). Phage 0105phi7-2 was obtained as described in (Roberts et al., Int. J. Mol. Sci. 2023, 24, 8941). Phages and phage mixtures indicated in the text were made and injected intraperitoneally into a 11-12 week-old female C57 / BL6 mouse, as previously described (Serwer et al., J. Mol. Biol. 1983, 170, 447-469.; Roberts et al., Int. J. Mol. Sci. 2023, 24, 8941) [35, 43], The total PFU inoculated were: 0105phi7-2, l.OxlO10; G, 1.0xl010in 180 pl final volume. At the times indicated on the horizontal axis, valueswere determined by procedures previously described (Roberts et al., Int. J. Mol. Sci. 2023, 24, 8941). The extract of uninfected cells was made by harvesting an uninfected bacterial lawn from 4 x 10 cm Petri plates and 2 x 15 cm in diameter Petri plates. The top agarose layer was harvested and pelleted (Roberts et al., Int. J. Mol. Sci. 2023, 24, 8941), and then resuspended in 1.0 ml of Milli-Q water (MilliporeSigma, Darmstadt, Germany). The cells were lysed by adding 1.7 mg lysozyme, followed by three cycles of freeze-thawing. This extract was clarified by pelleting at 10,000 rpm at 4 °C for 10 min (Beckman JLA 16.250 rotor, 15,000g).DESCRIPTION

[0030] The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any one embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope ofthe disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be an example of that embodiment, and not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.

[0031] Current limitations of phage therapy are (1) time required to isolate the needed phages, (2) difficulties in propagating phages to sufficient levels, and (3) time required and difficulty of purifying the phages. Phage therapy needs additional advances to bring phage therapy to FDA standards. One such advance is a rapid, inexpensive screening for high phage persistence. Currently phage persistence is measured by infectivity titer of the blood of a mouse after phage inoculation. This procedure takes about a week and is expensive.

[0032] The approach described herein use two physical phage properties that can be determined rapidly and inexpensively as indicators of persistence: average electrical surface charge density (G) and surface hydrophobicity (H). Native (intact phage) agarose gel electrophoresis (AGE) can be used for these determinations. These methods provide for increased reliability of phage therapy for MDR (and other) bacteria.

[0033] Another issue with phage therapy is the safety issues associated with the presence of pathogenic bacteria. The inventors develop a non-filtration, non-centrifugation, simple solution to this problem. A common and relatively inexpensive reagent is used, PrimeStore (Longhorn Vaccines and Diagnostics, San Antonio, TX), to inactivate both Gram-negative bacteria (e.g., Escherichia coll) and Gram-positive bacteria (e.g., Bacillus thuringiensis). No bacterial survivors were observed among 1010bacteria per ml. This procedure causes no detected inactivation of podophage T3, myophage T4 and siphophage 0105phi7-2. A 2x margin of safety for PrimeStore concentration exists for both bacterial inactivation and phage survival. Thus, more general applicability is expected. Comparable tests can be performed for each pathogenic bacterial strain / phage used. Electron microscopy reveals that the inactivated bacteria do not disintegrate but undergo changes in their cytoplasm (called ghosting), loss of some cytoplasm in the case of E. coli and phase separation in the case of B. thuringiensis.Thus, the inactivated, ghosted bacterial components can be removed by pelleting via low- speed centrifugation.

[0034] Other embodiments are directed to phage preparations that confer high persistence on other phages. Thus, even though phage therapy itself involves work with pathogenic hosts, for the purpose of enhancing the effectiveness of other phages, one would not have to use pathogenic hosts and could produce large amount of product efficiently. Persistence transference eliminates the requirement to either find or produce high persistence in a phage used for biomedicine.I. Assessing Phage Persistence

[0035] One issue with implementation of phage therapy is the development of rapid high- efficiency screens for phages with high persistence in organisms, organs, or body fluids such as blood. Phage therapy has been successful and has also failed. One reason for this unpredictability is low phage persistence. Some have assumed that phage persistence is always low. The inventors have shown dramatic variation in the persistence of closely related phages. One primary problem is how to screen for high-persistence phages rapidly and inexpensively. The historical ignoring of the “persistence problem” is reasonably assumed to be the cause of a lack of progress in phage therapy since the 1940s. Persistence is currently assayed by the time- and resource-consuming process of determining the infectivity titer vs. time of a phage in mouse blood.

[0036] Certain embodiments are directed to a procedure to determine a persistence indicator via a rapid multi-sample, inexpensive, chemical, and / or physical determination of phage characteristics. In certain aspects, persistence determination is complete in 10, 12, 14, 16, 18, 20, 22, 24 hours or less. The persistence assessment uses at least two phage characteristics for characterizing persistence: (i) average electrical surface charge density (G, e / nm2which is proportional to electrophoretic mobility in solution. To standardize results (producing a relative G), this number is normalized by dividing by the electrophoretic mobility for T3 phage, both in units of cm2 / (V.sec) and (ii) relative hydrophobicity (H), the latter determined by the change in a when the phage is bound by the hydrophobic end of adetergent that has a charged group projecting into solution. One reason for using relative H include the fact that measurements are more reliable if internally standardized. In certain aspects average electrical surface charge density and hydrophobicity can be determined by native gel electrophoresis. In certain embodiments a high persistence phage has a relative G of at least 0.5 , 0.75 or more.

[0037] Electrophoretic mobility in the absence of a gel (i.e., in solution) can be determined by extrapolation of in-gel electrophoretic mobility to a gel concentration of zero. This is best done in a gel, such as Seakem Gold agarose (Lonza, Rockland, Maine, USA) with (1) high strength to make possible the use of concentrations as low as 0.05% and (2) low electro-osmosis to minimize disturbance of the measurements by in-gel buffer movement. This can be performed in one-dimension by embedding several gels, each of different gel concentration, in a supportive higher concentration gel frame. It can also be done in two- dimensions when the concentration of the gel for the first dimension is lower than the concentration of the gel for the second dimension (2d-AGE when the gels are made of agarose).

[0038] To separately measure both the effective radius (RE) and the G of roughly spherical particles fractionated by AGE, 2d-AGE is performed with a first dimension gel dilute enough so that electrophoretic mobility is determined primarily by G. The dilute, first dimensional gel is stabilized by embedding it in a more concentrated gel used for the second dimension. To perform the second dimensional electrophoresis, the field / gel angle is rotated by 90° and electrophoresis is repeated with a second dimensional gel that is much more concentrated than the first dimensional gel.

[0039] The key to the 2d-AGE-based analysis of RE is that the percentage change in p is independent of G, when one compares p in the second dimension with p in the first, as first empirically confirmed in Serwer et al. (1986). Geometrically, this relationship implies that all particles of any given RE are on one line (called a size line) that extends from the effective origin of electrophoresis (O) through the center of the band formed by a particle. As the angle (0) between this line and the direction of the first electrophoresis decreases, RE increases. Thevalue of <5 is proportional to the distance migrated in the first dimension. With the use of standards of size known by small-angle x-ray scattering (Serwer et al., 1986; Serwer et al., 1989), differences in RE as small as 0.5% have been resolved by using radius of the effective pore (PE) values close to the RE’S of the particles analyzed (Casjens et al., 1992).

[0040] Native gels can be made of any polymer or combination of polymers that produces a gel in which the pore size is relatively consistent (or within a consistent range) for a given polymer concentration. Phage can be analyzed on agarose gels or the equivalent. In certain aspects the agarose gels are 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, to 1 percent agarose gels. As used herein “native” means nondenaturing or nondenatured and refers to conditions that do not disrupt intermolecular interactions within phages that allow them to maintain a structure that is found in nature. A “native gel” or “nondenaturing gel” is a gel that does not include denaturing agents such as denaturing detergents (for example, anionic detergents such as SDS or LDS) or chaotropes (urea, formamide, guanidine, potassium iodide, etc) sufficient to disrupt phage structure. Phages are remarkably resistant to detergents, which is why detergents can be used to determine hydrophobicity. Most all phages will remain native when other protein complexes denature and / or disassociate. Native (intact phage particle) agarose gel electrophoresis (AGE) can be performed in submerged, horizontal agarose slab gels. In certain aspects the electrophoresis buffer has a pH of 6.5 to 8.5 (for example a 0.09M Tris-Acetate, pH 8.4, 0.001M MgCh buffer). The buffer should be circulated to prevent the formation of a pH gradient. Temperature is typically maintained at 20 to 30 °C, e.g., 25 °C, and can be controlled ± 0.3 °C by circulation through a temperature- controlled water bath. Gels can be stained post-electrophoresis with a dye such as a fluorescent dye. The dye can be diluted into electrophoresis buffer in which the gel was submerged. Staining can be continued for 1 to 3 h or more, after which a digital photograph is taken. Packaged DNA can be expelled to increase staining to an extent greater than for unpackaged DNA. For this purpose, typically a gel is incubated at room temperature overnight in 0.002 M EDTA, pH 7.4 (Serwer and Wright, Biophysica 2022, 2:366-80). Incertain aspects gels can be subsequently protein-stained with Coomassie blue (Fang et al., J.Mol. Biol. 2008, 384: 1384-99).

[0041] To standardize the distance migrated by target phage (for example 0105phi7-2) the distance migrated by a target phage is divided by the distance migrated in the same gel by phage T3 ETS). TO remove effects of particle size and leave only effects of average electrical charge density (G), ET3 is extrapolated to an agarose gel percentage (A) of 0, which is called ETS(0), which is a measure of relative G. A value of ETS(0) can be converted to electrophoretic mobility, extrapolated to A of 0, by multiplying by the extrapolated electrophoretic mobility of phage T3, which is for example 1.5 x 10'4cm2 / (V.min) in 0.09 M Tris-Acetate, pH 8.4, 0.001M MgCh buffer.

[0042] It is contemplated that phage persistence increases as the magnitude of a negative G increases. This effect is based on electrostatic phage repulsion of internal epithelia (e.g., blood vessel linings, etc.), most of which are negatively charged. Hydrophobicity is determined because biological surfaces become stickier as H increases, as illustrated by the stickiness of hemoglobin in sickle cell anemia.

[0043] In initial studies at least 50 lytic, short-tail dsDNA phages (podophages) and 50 lytic, long, contractile tail dsDNA phages (myophages) will be tested. Podophages and myophages are both usually lytic. Persistence will be plotted versus standardized versions of both G and H to determine correlations.

[0044] Initial studies simultaneously measure persistence for several non-cross-plating phages in one IP-inoculated mouse. The ratio (R(|)) of the blood titer to amount of phage inoculated vs. time is measured. Then R([> value (R(|)) is averaged for 120 minutes after the time of peak of blood phage titer. The measured R([> is divided by the measured R([> of an internal standard, R(|)(s), z.e., R(|) / R(|)(s). The internal standard can be a high -persistence phage, e.g., T4 (Gonzalez et al., 2020, J. Mol. Biol. 432, 4139-53).

[0045] Use of R(|) / R(|)(s) removes effects of host variability. 5-6 different phages can be inoculated in one mouse. Therefore, we need roughly 10-15 mice for each 50 phages. For each phage, relative G is determined by AGE in a gel so dilute that electrophoretic mobility(p = velocity / electrical potential gradient) is not altered by gel sieving. In this case, p depends only on G. Podophages are small enough so that using a 0.3% high-purity agarose gel reduces gel sieving to insignificance. Values of p are (1) divided by p of phage T3 to minimize effects of variability of temperature (controlled ± 0.3 °C) and (2) obtained from 30-sample horizontal slab gels. Thus, only 4-5 runs will be needed to cover all the phages once.

[0046] The larger myophages will require a two-dimensional native gel electrophoresis to obtain a: ultra-low concentration first-dimension agarose gel; higher concentration second- dimension gel that embeds the first-dimension gel, four analyses per gel. Electron microscopy is used to determine if the length or structure of the tail influences persistence.

[0047] For each phage, the relative value of H is measured by determination of standardized G in the presence of the ionic detergent, e.g., sodium dodecyl sulfate (SDS), at 30 °C. SDS or the like does not inactivate phages. The hydrophobic end of SDS binds to hydrophobic regions on phage surfaces, and the negatively charged end projects into solution and increases the magnitude of negative G. Thus, the change of G in presence of SDS (Ac) is used as proportional to H. This H value has been shown to correlate well with other measures.

[0048] In certain studies native (intact phage) agarose gel electrophoresis (AGE) was used to analyze (1) the characteristics of the mutant phage and (2) the composition of postcentrifugation sucrose gradient fractions from loading experiments. The agarose gel had a concentration of 0.6% LE agarose. After polymerization, the gel was submerged beneath the electrophoresis buffer: 0.9 M Tris-acetate, pH 8.4, and 0.001 M MgC12 (Serwer et al., 2022). The samples loaded typically contained the sample, buffer, and a final concentration of 12.5% Nycodenz (Accurate Chemical & Scientific Corp., Westbury, N.Y.) The Nycodenz was used to increase density and, thus, ensure that the sample would load as intended.

[0049] After loading the samples into the wells, electrophoresis was performed at 1.0V / cm, 25 ± 0.3°C (Serwer et al., 2022). To prevent the formation of any pH gradient, buffer was circulated from one buffer tank to the other. The temperature of the buffer was controlledby running it through a glass coil submerged in a water bath. The gel then runs for 18 hours overnight.

[0050] Post electrophoresis, the gel was photographed using a short-wave ultraviolet transilluminator (Ultra-Violet Products Model TM-36) before staining to identify any intrinsic sample fluorescence. After the first photograph, the nucleic acid in the gel was stained with a 1 / 250 dilution of GelStar (green peak fluorescence emission; Lonza) for 3 hours at room temperature. After staining, the gel was photographed again and the GelStar was observed while illuminated with ultraviolet radiation. To expel DNA packaged in the phage, the gel was submerged in 0.002 M EDTA, pH 7.4. The gel was then left overnight (~24 hours) and then photographed again the next day. Expelling DNA increases the intensity of the GelStar fluorescence.

[0051] Sometimes these gels were subsequently protein-stained with Fast Coomassie Blue. This procedure begins by submerging the gel in -200 mL of Fast Coomassie blue stain (Fischer). After staining for -1 hour the stain was removed with a syringe. The agarose gel was then covered with 2x the amount of Fast Coomassie de-staining solution. This solution was replaced frequently until the protein bands were visible and the gel was translucent again.II. Persistence Factor

[0052] Certain embodiments are directed to conditioning a low persistence target phage(s) to increase persistence of the target phage(s). A transferrable factor has been identified in an adapted Phage G preparation. Phage G is a bacteriophage with a remarkably large capsid (-180 nm in diameter at the 5-fold vertex) and packaged genome length (626 kbp) compared to the median genome size of 52 kbp derived from available phage sequences. Phage G was originally isolated and characterized using a host bacterium classified as Bacillus megaterium, now known to be a Lysinibacillus . Phage G has the largest capsid and genome of any known propagated phage. The major capsid protein is gp27. Trimers of the decoration protein, gp26, sit on the 3 -fold axes and are thought to enhance the interactions of the hexameric capsomeres of gp27, for other phages encoding decoration proteins. Phage G’sdecoration protein is longer than what has been reported in other phages, and it is suspect the extra interaction surface area helps stabilize the capsid. The phage G host can be a Lysinibacillus .

[0053] Phage G can be selected, by serial mouse passage, followed by propagation, for an increase in the time that it resides in the blood of a living mouse, i.e., high persistence. Multiple-mouse-passaged phage G levels in blood, at any given time, are higher, by a factor of 100-1000, than the levels of the original phage G, unselected. However, the high persistence is lost after purification of phage G by rate zonal centrifugation in a sucrose gradient, as though the high persistence is caused by a phage bound factor that can be dissociated. A host-encoded factor is involved in generating the high persistence as found by adding a lysate of host cells to a phage preparation before measuring persistence. For example, a host cell can be Lysinibacillus, a bacterium. Importantly, when a low persistence phage is co-mouse inoculated with a high-persistence, partially purified phage G, the persistence of phage G decreases and the persistence of the originally low persistence phage increases. The low persistence phage is unrelated to phage G. This does not work if phage G has not been selected for high persistence. An arbitrary low persistence phage can be converted to a high -persistence phage by association with the persistence factor.

[0054] This persistence conversion has potential for use in two areas: (1) phage therapy of infectious disease, where a major limitation is low persistence of the phages used and (2) use of phages as drug delivery vehicles, where high persistence is also critical.

[0055] Phage therapy and the use of phages as DDVs rely on the ability of the phage to resist removal from the blood (persistence). Previous studies on phage therapy have not considered persistence to be a priority. However, persistence could play an important role in a successful or failed therapeutic attempt (Serwer et al., 2021; Pires et al., 2020). Past research contains correlations between high phage persistence and successful phage therapy (Serwer et al., 2021). High persistence is also needed when choosing a DDV. The use of a T4 phage as a DDV requires evasion by T4 of innate immune responses long enough to travel to tumor environments and release drug.

[0056] Fortunately, phage T4 has persistence high enough to remain alive for 4-6 hours in the blood (Serwer et al., 2021). Phage T4 also has another critical property. T4 has a gate that can be opened to load drug and closed to transport and deliver drug (Serwer & Wright, 2022). Gating opens the opportunity to increase the amount of tumor-delivered drug to the point that drug-resisting cancer cell mutations, as well as drug toxicity become less problematic (Pan et al., 2016; Gewirtz et al., 2010).

[0057] Loading Bacteriophage. Taking into consideration the characteristics of chemotherapeutic drugs and the gating and high persistence of phage T4, preliminary drug loading and delivery experiments were conducted. They incubated partially purified T4 phage with either ethidium or bleomycin at 58°C for 1.0 hour. The phage was then fractionated by rate zonal centrifugation in a sucrose gradient and assayed by native agarose gel electrophoresis (AGE) to test the stability of phage loading. The previously loaded ethidium was detected with ultraviolet light excited fluorescence (orange fluorescence). The AGE gel was then subjected to 2 hours of Gel Star staining (stains DNA). After staining, the presence of partially assembled T4 phages (green fluorescence) was visible while the ethidium-loaded phages remained orange because of their impermeability to GelStar. A 0.002 M concentration of EDTA was then added to the gel (EDTA causes the phage to break open). The partial replacement of the orange ethidium fluorescence with green GelStar fluorescence after incubation with EDTA for 3 hours was seen. Lastly, incubated with EDTA for 16 hours, shows a total replacement of orange ethidium fluorescence with green GelStar fluorescence because of the expulsion of DNA from the capsid of intact phage T4. Data from this experiment suggested that phage T4 has a gate that can open to load specific compounds and then be closed in a leakproof state (Serwer & Wright, 2022). The gating procedure of the T4 phage is a novel characteristic that could be used in the development of DDVs for improved tumor targeting of drug loaded in phage T4.

[0058] After previously loading ethidium, the T4 phage is incubated with bleomycin to test the ability of loading anti-tumor compounds. After loading T4 phage with bleomycin under the same procedures as ethidium the loaded phage was assayed by AGE for bleomycinfluorescence. Although bleomycin fluorescence is weaker and difficult to identify due to the background of light scattering in an agarose gel, fluorescence was still detected in the AGE before GelStar staining.

[0059] Bleomycin is primarily used as an antineoplastic agent and has received a wide variety of FDA approvals for therapeutic usage (Brandt & Gerriets, 2023). Bleomycin has been used in cancer treatment due to its ability to damage DNA through oxidation by binding metal ions (Petering et al., 1990). Comparative to other chemotherapy drugs, bleomycin does have some adverse effects including pulmonary toxicity (Patil et al., 2016). However, having a targeted, gated, non-leaking DDV could increase efficacy while reducing the toxicity of bleomycin.III. Examples

[0060] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.EXAMPLE 1

[0061] Phage Therapy. Phage Therapy is the therapeutic use of bacteria-infecting (harmless to humans) viruses (phages). Phages infect disease-causing bacteria, propagate, thereby killing the disease-causing bacterial host cells. Phage therapy is refractory to bacterial resistance. One problem to be solved is developing a method of rapid prediction of the probability of high resistance to innate immune system removal, i.e., high persistence. These methods need to translate ‘persistence’ safely to phages propagated on highly pathogenic, e.g., MDR bacterial strains.

[0062] Pathogen free phages. Due to the absence of a membrane in lytic phages, bacterial membrane disruption could be a preferential and rapid means of killing contaminating host pathogens leaving phage intact and functional. One approach uses a common and relatively inexpensive reagent toxic to viral membranes (PrimeStoreTM-Longhom Vaccine and Diagnostics, Bethesda, MD).Table 1. Effect of PrimeStore-Reagent on Representative Gram- and Gram+ OrganismsTable 2. Effect of PrimeStore-Reagent on Various Bacteriophages

[0063] PrimeStore-Reagent treatment of bacterial and phage preparations constitutes ‘proof of principle’ of an efficient and inexpensive means for: ( / > effective (100 %) killing of bacteria (109 / ml) without inactivating phages. (2) potential safe handling of pathogen propagated phage.EXAMPLE 2BIOPHYSICAL BREAKTHROUGHS PROJECTED FOR THE PHAGE THERAPY OF BACTERIAL DISEASE

[0064] Past anti-bacterial use of bacteriophages (phage therapy) is already well reviewed as a potential therapeutic response to the emergence of multidrug-resistant, pathogenic bacteria. Phage therapy has been limited by the following. (1) The success rate is too low for routine use and FDA approval. (2) Current strategies of routine phage characterization do not sufficiently improve the success rate of phage therapy. (3) The stability of many phages at ambient temperature is not high enough to routinely store and transport phages at ambient temperature. New and previous data is presented that we interpret as introductory to biophysically and efficiently transforming phage therapy to needed effectiveness. Included are (1) procedure and preliminary data for the use of native gel electrophoresis (a low-cost procedure) for projecting the therapy effectiveness of a newly isolated phage, (2) data that suggest a way to achieve stabilizing of dried, ambient temperature phages via polymer embedding, and (3) data that suggest means to increase the blood persistence, and therefore the therapy effectiveness, of what would otherwise be a relatively low-persistence phage.

[0065] The problem of multi-drug resistant (MDR) bacterial pathogens is now well documented and reviewed. In summary, antibiotic therapy has been compromised, and sometimes thwarted, by MDR pathogens. The reason is that the pathogens are resistant to either most or all antibiotics (reviews: (Gajic et al., J. Hosp. Infect. 2023, 737, 1-7; Domingues et al., Int. J. Mol. Sci. 2023, 24, 11438; Cohen et al., Genome Med. 2019, 11, 45; Mbewana and Msagati, Can. J. Infect. Dis Med. Microbiol. 2023, Nov 30, 6659212; Kunz Coyne et al., Infect. Dis. Ther. 2022, 11, 661-682; Giovagnorio et al., Antibiotics 2023, 12, 1621; Shrivastava et al., J. Res. Med. Sci. 2018, 23, 21; 2019 AR Threats Report. Centers for Disease Control and Infection, US Department of Health and Human Services. URL cdc.gov / DrugResistance / Biggest-Threats.html. Accessed 26 January, 2023; Centers for Disease Control and Infection, US Department of Health and Human Services. Antibiotic resistance threats in the United States. URL cdc.gov / drugresistance / pdf / threats-report / 2019-ar-threats-report-508.pdf, Accessed 24 December, 2023; Antimicrobial resistance collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 2022, 399, 629-655). The consequence is known to be lethal for roughly 48,000 citizens of the USA in 2019, when Clostridioides difficile (not typically pathogenic, but made pathogenic by long-term antibiotic use) is included. Worldwide, the estimate is at least 4.95 million deaths in 2019, with the top six pathogens being Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. MDR bacterial infections in 2019 were the third leading cause of death worldwide, behind heart attacks and stroke. However, ambiguity often exists for both in-hospital and outpatient cause of death. By one informed calculation, the number of MDR-bacteria caused deaths is higher, possibly as much as almost 7x higher in the US (Burnham et al., Infect. Control Hosp. Epidemiol. 2019, 40, 112-113).

[0066] Therapeutic use of anti-bacterial viruses (bacteriophages or phages) is an obvious anti-bacterial measure (phage therapy) to counter infections by MDR pathogenic bacteria. However, as previously reviewed (Hatfull et al., Annu. Rev. Med. 2022, 73, 197-211; Azevedo et al., J. Burn Care Res. 2022, 43, 336-342; Onallah et al., Open Forum Infect. Dis. 2023, 10, ofad221; Diallo and Dublanchet, Antibiotics 2023, 12, 751; Dqbrowska, Med. Res. Rev. 2019, 39, 2000-2025; Dedrick et al., Clin. Infect. Dis. 2023, 76, 103-112; Adesanya et al., AIMS Microbiol. 2020, 6, 204-230; McCallin et al., Viruses 2019, 11, 343; Gorski et al., Antibiotics (Basel) 2020, 9, 827), phage therapy sometimes works and too often does not. Phage therapy is not yet an FDA-approved procedure in the US unless performed on a compassionate use basis, e.g., with a patient in otherwise hopeless condition (Dedrick et al., Clin. Infect. Dis. 2023, 76, 103-112; Adesanya et al., AIMS Microbiol. 2020, 6, 204-230; McCallin et al., Viruses 2019, 11, 343; Gorski et al., Antibiotics (Basel) 2020, 9, 827). Nonetheless, clinical trials are in progress, with the goal of answering the question of whether or not phage therapy works (Dedrick et al., Clin. Infect. Dis. 2023, 76, 103-112; McCallin et al., Viruses 2019, 11, 343; Gorski et al., Antibiotics (Basel) 2020, 9, 827; Ali et al., Microb. Pathog. 2023, 181, 106199; Jault et al., Lancet Infect. Dis. 2019, 19, 35-45). The subtext ofthis question is, however, in conflict with the history of phage therapy. Our point here is that more appropriate is the question of which phages should be used and how. This point is emphasized by the spectacular successes that have occurred in the context of too many failures.

[0067] Phages to Use and Not to Use for Phage Therapy

[0068] Known Past Basics. A clear plaque is criterion #1 for choosing phages for phage therapy. The reason is that clear plaque-forming phages are the most likely to be lytic, i.e., bacterial lysis-inducing without capacity for lysogeny, i.e., without capacity to become a replicating component of the bacterial genome (Hatfull et al., Annu. Rev. Med. 2022, 73, 197-211; Azevedo et al., J. Burn Care Res. 2022, 43, 336-342; Onallah et al., Open Forum Infect. Dis. 2023, 10, ofad221; Diallo and Dublanchet, Antibiotics 2023, 12, 751; Dqbrowska, Med. Res. Rev. 2019, 39, 2000-2025; Dedrick et al., Clin. Infect. Dis. 2023, 76, 103-112; Adesanya et al., AIMS Microbiol. 2020, 6, 204-230; McCallin et al., Viruses 2019, 11, 343; Gorski et al., Antibiotics (Basel) 2020, 9, 827; Hyman, Pharmaceuticals (Basel) 2019, 12, 35; Howard-Varona et al., ISME J. 2017, 11, 1511-1520). The preparation used for phage therapy is typically a mixture of several phages (phage cocktail); multi-phage cocktails appear to be the current standard.

[0069] Lysogenic phages are avoided because of the carrying, by some lysogenic phages, of genes encoding either bacterial toxins (Hyman, Pharmaceuticals (Basel) 2019, 12, 35) or antibiotic resistance (Hyman, Pharmaceuticals (Basel) 2019, 12, 35; Howard-Varona et al., ISME J. 2017, 11, 1511-1520). Verification of the absence of these latter genes can be achieved by whole genome DNA sequencing (Howard-Varona et al., ISME J. 2017, 11, 1511-1520)

[0025] , This is criterion #2 because of the potential of gene transfer to bacterial hosts. Historically, other criteria appear to have been used but were not well documented. For example, one gets the impression from the second paragraph of (Desranleau, Can. J. Public Health 1949, 40, 473-478) that, in the 1930s, the favored anti-typhoid fever phages made relatively small clear plaques. However, no definitive, rigorous statement was made. The early days of phage therapy appear to have been characterized by fly-by-the-seat-of-your-pants phage selection. One imagines that some of the same is occurring even today. Our data- generated opinion is that more precise criteria are needed to reach a next-generation phage therapy that is FDA approvable.

[0070] Next Generation Biophysical Screening of Phages: Average Electrical Surface Charge Density (a)- Our main point here is that the key objective is to develop additional phage screening criteria that bring phage therapy to the point that its success rate is at least as high as the success rate of the use of antibiotics with non-MDR bacterial pathogens. High speed is important because the observed (Oechslin, Viruses 2018, 10, 351; Egido et al., FEMS Microbiol Rev 2022, 46, fuab048) development of bacterial resistance to phages implies that the phages used for phage therapy cocktails will have to be periodically changed to keep the cocktail effective. In general, the relatively high speed and low cost of phage therapy -based response to increased bacterial resistance is anticipated to be a major advantage of phage therapy in relation to antibiotic therapy.

[0071] A data-suggested screening criterion is the rate of a patient’s innate immune clearance of the phage (reviews D^browska, Med. Res. Rev. 2019, 39, 2000-2025; Marchi et al., iScience 2023, 26, 106004; Van Belleghem et al., Viruses 2018, 11, 10; Roach et al., Cell Host Microbe 2017, 22, 38-47)). If one makes the pessimistic assumption of high clearance rate for all phages, one might conclude that phage therapy will never be routinely effective. In contrast, phages are found to sometimes enhance the innate immune clearing of bacteria, which would enhance phage therapy (Van Belleghem et al., Viruses 2018, 11, 10; Roach et al., Cell Host Microbe 2017, 22, 38-47). The complexity involved suggests an approach that does not depend on detailed knowledge of innate immunity pathways.

[0072] Specifically, a relatively optimistic assumption is that (1) the key parameter is, indeed, the rate at which inoculated phages are removed from circulation by innate immunity, and (2) this rate varies significantly among phages. If so, then lowering this rate is the key to improving the success rate of phage therapy.

[0073] To test the accuracy of this latter assumption, in a controlled way, recently, we(Serwer, P.; Wright, E.T.; De La Chapa, J.; Gonzales, C.B. Basics for improved use ofphages for therapy. Antibiotics (Basel) 2021, 10, 723) assembled (1) a collection of four phages and (2) bacterial hosts to selectively plate each of the phages. These phages and bacteria were used to perform the first test of the blood lifetime (to be called persistence) of four different phages in one mouse. The blood titers (plaque-forming units [PFU] per ml), normalized to the total PFU injected (approximately the same for all four phages), varied by about 10,000x at 2-4 hours after intraperitoneal injection. Two phages, including phage T3, had relatively high persistence and two, including the T3 relative, T7, had low persistence. Notably, the difference in persistence between T3 and T7, which are the same size and genetically related (Issinger and Falk, Arch Virol. 1976, 52, 217-231), indicates that size is not the only persistence-determining factor involved, although it might be one of several persistence-determining factors.

[0074] T3 and T7 differ in average electrical surface charge density (c), as determined by extrapolation of gel electrophoretic migration to a gel concentration of 0 for both T3 and T7 (Serwer et al., J. Mol. Biol. 1983, 170, 447-469). In solution (without a gel), electrophoretic mobility depends only on c for particles the size of the smallest and larger phages. The c of both T3 and T7 is negative with T3 being 1.3x more negative than T7. Correlation of more negative c with higher persistence has been observed for red blood cells; the sialyation of glycophorin, on the surface of red blood cells, generates a negative c relatively high in magnitude (Jan and Chien, J. Gen. Physiol. 1973, 61, 638-654). Although more data are needed for phages, our working assumption is that, relative to phage T7, the co-existence of the more negative T3 c and the higher T3 persistence are part of a c / persistence correlative pattern. That is to say, rapid determination of c is likely to be one of possibly several criteria useful to determine the probability that a newly isolated phage will have high persistence.

[0075] Biophysical Technique

[0076] Basics of Native Agarose Gel Electrophoresis (AGE) - For comparing c values, a relatively rapid, inexpensive, and accurate procedure is to perform native (intact phage) agarose gel electrophoresis (AGE) of the two phages to be compared, according to the following protocol. (1) Embed agarose gels of different agarose percentage, A, (running gels)in a single more concentrated, physically supportive agarose frame (frame gel). Use an ultrastrong, ultra-low electro-osmosis agarose for the running gels (Electro-osmosis is electrophoresis generated motion of water and will distort the results unless kept low.). (2) Determine distance migrated vs. A for both phages during electrophoresis. (3) Extrapolate distance migrated to an A of 0 and compare the result for the two phages.

[0077] For any newly characterized phage, we have simplified the data processing by dividing this extrapolated distance by the extrapolated distance for phage T3, obtained in the same multigel. This is a process of normalization. A normalized c will be called G\. We use ON, rather than the physical concept, G, because (1) s is an imprecise concept, complicated by lack of knowledge of the surface of slip, with some details implicit in the Debye-Hiickel model (Zhang et al., Molecules 2023, 28, 2042), and (2) no reason exists to perform an analysis beyond ON at this point. We use T3 as the normalization standard because of the high persistence and high stability of T3. The stability includes stability in the presence of chelating agents and several buffers, which has made T3 a standard used for AGE of chromatin, for example (Baker et al., Nucleic Acids Res. 2009, 37, 5019-5031; Fletcher et al., Biochemistry 1994, 33, 10859-10863).

[0078] That having been said, we know that persistence is dependent on more than ON. Specifically, phage G, a myophage, is a low persistence phage (Roberts et al., Int. J. Mol. Sci. 2023, 24, 8941). The value of ON, however, is 1.20 (plot indicated by G in FIG. 5; the T3 plot is indicated by T3 in FIG. 5), in contradiction to the correlation of ON > 1.0 with high persistence. Nonetheless, high persistence phage T4 (also a myophage), has a ON of 0.99 (T4 plot in FIG. 5), consistent with this correlation. Phage G is also the largest phage that has been laboratory-propagated; it has a genome about 3x the length of the T4 genome and a capsid about 60% larger. Correlation of large size with relatively high innate immune uptake by macrophages has been observed for latex spheres (Petithory et al., Nanomaterials (Basel) 2021, 11, 1963). Thus, relatively large size might explain the relatively low persistence of phage G. Of course, the above analysis needs data from more phages. Also, other phage biophysical characteristics are likely to be needed for a complete analysis. These mightinclude density of hydrophobic patches on surface of the capsid, which can also be electrophoretically determined (Bastin et al., Virology 2022, 568, 23-30).

[0079] Further Phage Characterization by AGE - An advantage of multigel-AGE is that one can use the sieving effects of the running gels to obtain information about the size and shape of particles being analyzed. Specifically, as the overall size of particles increases, the slope of the distance migrated vs. A plots increases at the lowest A values. A previous study of rod-shaped phages reveals that the effective radius (7?E), as determined by this slope, is closest to the radius of a sphere that has the surface area of the non-spherical particle (Griess et al., Biopolymers 1990, 29, 1277-1287). Thus, by use of AGE alone, one can estimate the 7?E of a phage for possible use in determining the probability of high persistence. AGE can be performed directly on phage plaques, without phage purification (Serwer et al., AppL Theor. Electrophor. 1995, 4, 211-217). Thus, both Ry. and ON can be determined without purifying phage particles, which might be especially important for high-persistence screening in an emergency, such as a bacteremia projected to be otherwise rapidly lethal.

[0080] Furthermore, even some information about phage asymmetry can be obtained from multigel-AGE. Specifically, the phage G plot in FIG. 5 has upward curvature at the highest A values, as emphasized in FIG. 5 by the dashed line that continues the straight line generated at the lowest A values. The opposite curvature is generated at the higher A values by more spherical particles, including podophages, such as T3 and T7. The upward curvature indicates either a long tail phage (myophage or siphophage) or a rod-shaped phage. Although not yet explored in detail, discrimination of these two can be made by determining the dependence on the magnitude of the electrical field used for AGE.

[0081] The Potential Future of Dry Phage Therapy Cocktails: An Improved Source of Phages

[0082] Phage storage-inactivation is an obvious problem for liquid phage cocktails. This problem was recognized via (1) early phage characterizations (d’Herelle, F.H.D. The Bacteriophage and Its Behavior,' The Williams & Wilkins Company: Baltimore, MD, USA, 1926) and (2) review of early phage therapy failures (review (Dublanchet and Fruciano,Breve histoire de la phagotherapie [A short history of phage therapy], Med. Mai. Infect. 2008, 38, 415-420. French)). Typical long-term laboratory storage of phages manages this problem by freezing, typically at temperatures between -70 and -80 °C in the presence of a cryoprotectant. The PS-laboratory submerges a plaque in growth medium with 10% dextran 10 before freezing at -70 °C. Doing this in a biomedical context is expensive and potentially limiting to the use of phage therapy. A further complication is that phage G, for example, is not multi-year stable when stored this way. This may also be true for other phages.

[0083] A simpler, less expensive, less machine failure- and power failure-susceptible strategy would be storage of phages dry and at ambient temperature. The following observation indicates that one way to do this, for at least some phages, is to air-dry the phages in the presence of a phage-stabilizing agent. When a ranch-animal proximal soil is dry enough so that molds do not grow during storage at room temperature, this soil is a prolific source of phages (Serwer et al., Virology 2004, 329, 412-424; Serwer et al., Virol. J. 2007, 4, 21). The key point here is the phage survival of drying. In our case, the dry environmental phages were (1) exposed to environmental temperatures above 50 °C and (2) dried in air (no vacuum). The pre-drying presence of phages is expected because non-dried samples of ranch animal excretions are found by others to be a prolific source of phages (Montso et al., Front. Public Health 2019, 7, 355; Letarov et al., J. Applied Microbiol. 2009, 107, 1-13; Clokie et al., Bacteriophage 2011, 1, 31-45).

[0084] A new-phage yield of over 50 phages per week per person is a reasonable expectation for dry, ranch animal-proximal soil. Some of these soil-associated phages are so stabilized that they can be (have been) isolated 18-years after taking of the soil sample. The assumption is that soil-associated, phage-protective agents are involved. Candidates include the polymers that bacteria secrete to form biofilms (review (Sharma et al., Microorganisms 2023, 11, 1614)).

[0085] Also, in-liquid phage stabilizing compounds exist and work on physical principles that should be applicable to all phages and in near-dry conditions. These compounds do not penetrate phages and, thus, generate a phage-stabilizing osmotic pressure across the DNA-containing protein shell of the capsid. This osmotic effect was first demonstrated for phages P22 and T7 (Serwer et al., J. Virol. 1983, 75, 665-671). It was later demonstrated in more quantitative detail for phage lambda. Its use was a critical part of the determination of the energetics of partial phage DNA packaging (Evilevitch et al., Biophys. J. 2008, 97, 1110- 1120; Grayson et al., Virology 2006, 348, 430-436).

[0086] Studies in this general direction have been conducted for (1) stabilizing phages for aerosol delivery (Flint et al., Cells 2023, 12, 2057; Kohler et al., Nat. Commun. 2023, 77, 3629), (2) freeze drying and (3) in-air spray drying (Clark et al., Cryobiol. 1973, 10, 351-360; Jonczyk-Matysiak et al., Expert Rev. Anti. Infect. Ther. 2019, 17, 583-606; Malik et al., Adv. Colloid Interface Sci. 2017, 249, 100-133; Wdowiak et al., Pharmaceutics 2022, 77, 1936). Spray drying is closest to what happens in the environment. These studies appear to have been conducted without awareness of the points made in the previous paragraph, a condition that continues to the most recent review Wdowiak et al. Nonetheless, when one averages a very large amount of data presented in Wdowiak et al. the conclusion is drawn that, among the sugars tested as drying-stabilizers, trehalose and sucrose, both non-reducing disaccharides, are the most effective. Other sugars tested include two disaccharides, both reducing: maltose (a-linked) and lactose (P-linked). Given the above effects of nonpenetration of phages, the anomeric-to-anomeric carbon linkage of trehalose and sucrose may be the source of relatively high effectiveness, via the provision of non-penetration. In any case, a uniform phage stabilization strategy best assists phage therapy. In our opinion, use of basic biophysical principles, such as those in the previous paragraph, is the best way to arrive at this condition.

[0087] Enhancing Phage Persistence: Future Quantification of Persistence

[0088] One can increase the murine blood persistence of phages lambda and P22 by serial exposure to murine blood, each exposure followed by (mutagenic) phage propagation (Merril et al., Proc. Natl. Acad. Sci. U S A 1996, 93, 3188-3192). The higher persistence version of phage lambda is (1) a mutant, with an identified amino acid change (glutamic acid to lysine) in the major protein of the DNA-encapsulating outer shell of the lambda capsid, and (2) moreeffective in murine phage therapy (Merril et al.). This study represents the logical beginning of next-generation phage therapy.

[0089] Of course, one cannot be sure that the observed amino acid change is a direct cause of the high persistence. For example, mutations in other genes, not sequenced, may have been the cause via change in the conformation of the major capsid protein. Nonetheless, biomedically, this experiment reveals that directed evolution might be a way to increase phage persistence before the use of phages in phage therapy cocktails. However, biomedically using directed evolution introduces a time delay and a cost increase that makes phage therapy less of a prospect. A better approach would be introduction to existing phage therapy cocktails of an agent that increases the persistence of those phages already isolated but of low persistence.

[0090] We accidentally discovered a possible way to do this. We subjected low persistence phage G to three serial passages through murine blood, each passage interspersed with propagation of phage G, although propagation was in non-mutagenic conditions. High persistence was, indeed, achieved for partially purified (FIG. 6) phages. The persistence progressively increased with passage number. The intermediate-persistence steps suggested the existence of several mutations. However, not a single mutation had occurred in the genome of the final product, as determined by whole-genome, Illumina sequencing. The intermediate-persistence phage G was not sequenced. The initial phage G was a clear plaque mutant, which had been selected to improve the accuracy of phage titers (FIG. 5).

[0091] Furthermore, the high persistence, phenotype-only variant of phage G, after the final repropagation and partial purification (to be called phage GH), depended on a host factor for its high persistence. When further purified by rate zonal centrifugation, phage Gu lost its high persistence. However, the high persistence of phage GH was recovered by addition of an extract of uninfected host cells. Some details are in FIG. 6.

[0092] The key observation was that host extract-boosted, purified phage GH was found to transfer its high persistence to low-persistence phage 0105phi7-2. This is shown in FIG. 6 where normalized in-blood phage titer (Rp) is plotted vs. time;is the plaque-forming units(PFU) per ml of blood, divided by the total PFU inoculated intraperitoneally (IP). The persistence for unaltered phage G (G plot in FIG. 6) confirmed the low phage G persistence. The plot for host extract-boosted phage GH (GH plot in FIG. 6) showed a 10-100x increase in persistence depending on time. In addition, the low persistence of phage 0105phi7-2 (FIG. 6; 7-2 plot) was increased when phage GH was mixed with phage 0105phi7-2 and then IP inoculated (7-2 + GH plot FIG. 6). This increase was accompanied by a decrease in persistence of host extract-boosted phage GH (GH + 7-2 plot FIG. 6).

[0093] As a beginning in quantifying persistence, we define high persistence as maintaining aabove 0.03 for at least 4 hours. At 107phages IP inoculated for phage therapy and 107pathogenic bacteria per ml of blood, this should be sufficient to clear a phage-susceptible bacteremia, assuming a latent period of less than 45 minutes and a burst size of at least 10, which are probably pessimistic assumptions for most lytic phages during phage therapy. More precise calculations, persistence definitions and projections will be made as more data are obtained.

[0094] Implications for FDA Clearance: Safety and Effectiveness

[0095] The first point to note here is that phage therapy is biomedicine. Antibiotic therapy is what might be called chemo-medicine, even though many antibiotics were originally isolated from organisms. Thus, a complete re-think is needed for FDA clearance. To illustrate this point, think about the use of leeches to prevent and remove blood clots, which is also biomedicine and is FDA approved. No two leeches are exactly the same. Criteria to be used are (1) statistical in character and (2) include a range of possibilities, even after applying statistics. This problem exists and has, indeed, been solved even for some complex chemo-medicines. These include tetanus toxoid-bacterial polysaccharide conjugate vaccines such as Prevnar and Pneumovax. These vaccines are expected to have components that vary in size and even composition. Size variability has been found empirically by a procedure of two-dimensional AGE; this procedure was developed for the characterization of phages (Tietz et al., Electrophoresis 1991, 72, 46-54).

[0096] In the case of phage therapy, we start from a good position. In the short term (weeks), no phage has ever been found to harm either a human or an animal at the levels used for phage therapy. These levels are typically 107- 108PFU. This non-toxicity is not surprising in that titers per ml of this magnitude are sometimes recorded for endogenous human phages in healthy people (Lusiak-Szelachowska et al., Microorganisms 2020, 8, 2012). To test for possibly rare, short-term adverse reactions, a skin allergy-like test, based on a dry phage therapy cocktail (above), might be developed and FDA-required. The long-term effects of phage therapy are not empirically known, and some data suggest that the possibility of negative long-term effects is significant (Podlacha et al., Int. J. Mol. Sci. 2024, 25, 2107).

[0097] Of course, rapid phage-killing of bacteria potentially creates the problem of toxic levels of bacterial endotoxin (Foster et al., Int. J. Mol. Sci. 2023, 24, 16185). Post-phage therapy, the occurrence of fever spikes, possibly endotoxin-caused, has been observed. Indeed, this occurrence is presumably a criterion for therapy success. A second FDA requirement might be standards for the time and characteristics of fever monitoring after phage therapy begins.

[0098] In the area of therapy effectiveness, dry phage therapy cocktails can be as easily screened as liquid phage preparations. If a cocktail is stabilization agent-embedded on, for example, a non-biological membrane, a small piece of this membrane can be cut away and placed on a Petri dish-contained agar layer that has confluent propagating pathogenic bacteria. Post-incubation, a clear ring, of size to be determined, around the membrane would be a criterion for cocktail effectiveness. This screening (1) is the way that disk-diffusion tests are done for antibiotics and (2) would be performed after previous screening for high persistence.

[0099] For extensive wounds, a piece of the phage cocktail-membrane can be placed in one region of the wound to test for effectiveness. If effective, then more membrane pieces would be distributed. Several different cocktails could be simultaneously tested. Here again, the procedure used could be regulated by the FDA. Apparently, a therapeutic procedure of this type is not practical for antibiotics, presumably because of diffusion-induced lowering ofantibiotic concentration. However, the in-wound replication of phages should make this therapeutic procedure practical.

[0100] The problem of endotoxin removal from phage therapy cocktails is likely to be limiting for FDA approval. We recently found a reagent and method that can be used to remove over 99% of the in vitro endotoxin activity while also completely inactivating representative Gram -positive and Gram-negative bacteria (Chambers et al., Biophysica 2023, 3, 558-568). Procedures of this type should simplify FDA-clearance of phage therapy cocktails and also have application when removal of endotoxin is limiting for reducing inflammation and reducing pain, for example, after endodontic procedures.

[0101] Contextual Significance of Phage Therapy

[0102] Rather than developing new text to describe the origin of the problem of obtaining new antibiotics for MDR bacteria, we quote here the 2022 Annual Report of the drug company, Pfizer, a company that produces multivalent vaccines for both Streptococcus pneumoniae and Neisseria meningitidis (in this quote, AMR = antimicrobial resistance). M robust pipeline of new antimicrobials is essential to restoring the balance against increasing rates of AMR. However, significant economic hurdles have made research and development in this area a challenge. No novel class of antibiotics has been launched for almost 40 years, and even when newly approved treatments come to market, they may be used sparingly to support good antimicrobial stewardship practices — making it difficult to recover the high cost associated with development. New reimbursement models that more fully reflect the complete value of antimicrobials are critical ''

[0103] The point missed in the above quotation is, obviously, the potential of covering the limitations of antibiotics with the use of phage therapy. Nonetheless, the therapeutic value of phages is a concept already embraced by Pfizer, although in the context of gene therapy. Again, corporate development of anti-bacterial phage therapy appears to be limited by both phage isolation and phage characterization.

[0104] The following quotes make this point from the perspective of active phage therapy trials. “Currently, the therapeutic potential of phages is substantially limited by the greatvariation in phage susceptibility and a relatively small repertoire of therapeutically useful phages" (Hatfull, Pulm. Ther. 2023, 9, 91-107). “Although several scientific organizations / societies recognized that phage therapy could be of key value in modern wound care, specific aspects are critical for a burn surgeon and might represent pitfalls discouraging phage therapy adoption in burn wound management; in particular, the unavailability of consensual therapeutic guidelines / regulatory policies and the lack of laboratorial support that might be predictive of its efficacy' (J. Burn Care Res. 2022, 43, 336-342). “One major limitation that we faced was the lack of available human pharmaceutical-grade phage preparations, which still remains a bottleneck in our system and a limiting step in each phage request timeline ” (Open Forum Infect. Dis. 2023, 10, ofad221). “Phage treatment of Mycobacterium infections is challenging due to the limited repertoire of therapeutically useful phages, but favorable clinical outcomes in patients lacking any other treatment options support continued development of adjunctive phage therapy for some mycobacterial infections" (Clin. Infect. Dis. 2023, 76, 103-112).

[0105] Conclusion

[0106] The data indicate that solving the MDR bacteria problem is possible with phage therapy. Doing this will take key problem-identifying / targeting research. This research is likely to (1) be enhanced by biological factors not necessarily anticipated in advance, and (2) succeed via use of already established principles and techniques of biophysics and phage biology.

Claims

CLAIMS1. A screening method for high persistence phage comprising:(i) determining a target phage surface charge (o);(ii) determining hydrophobicity by measuring change of electrophoretic mobility in the presence of ionic detergent; wherein a negative surface charge and lower hydrophobicity identifies high persistence phage.

2. The method of claim 1, wherein the target phage surface charge is determined using gel electrophoresis.

3. The method of claim 2, wherein the gel electrophoresis is one- or two- dimensional native agarose gel electrophoresis.

4. A phage composition comprising a phage configured to confer high persistence on a second target phage.

5. The phage of claim 4, wherein the phage is a phage G.

6. A high persistence phage composition comprising a persistence factor.

7. A method of treating an infectious disease or a metastatic cancer by administering a high persistence phage(s).

8. A method of producing a high persistence phage comprising contacting a target phage with a persistence factor or a second phage selected for high persistence.

9. A method of delivering a drug to a subject comprising administering a drug loaded high persistence phage to a subject.

10. A method of increasing the persistence of a phage comprising incubating a first phage with a nongenomic persistence element of a second phage, wherein the nongenomic persistence element of the second phage is transferred to the first phage increasing the persistence of the first phage.

Images