Virus-containing particles

Abstract

Particles are described that include a virus encapsulated in an amorphous silica shell, where the amorphous silica shell is deposited directly around the surface of the virus. Also described is a method of making the virus encapsulated in an amorphous silica shell, comprising concentrating or purifying the virus, suspending the virus in a buffer, hydrolyzing a silica precursor, directly contacting the hydrolyzed silica precursor with the surface of the virus in the buffer, and encapsulating the virus in the amorphous silica shell.

Description

[Technical field]

[0001] A particle containing a virus. [Background technology]

[0002] Virus-based therapeutics are increasingly being used in medicine. The first type of virus used in medicine is the bacteriophage. These can be used in "phage therapy" to treat bacterial infections. A number of well-studied phages have shown activity against the most problematic bacteria, including those with multidrug or antibiotic resistance. One of the biggest differences between bacteriophages and chemical antibiotics is that bacteriophages have a narrow host range. Each phage species is infectious for only a handful of bacterial strains. This feature can be extremely beneficial, as it can result in successful treatment of bacterial infections while leaving "good bacteria", e.g., the bacteria that make up the gut microbiome, unharmed. This prevents many of the unpleasant side effects associated with chemical antibiotics.

[0003] Bacteriophages have various characteristics and morphologies, but the majority of phages (e.g., T4 or phage K) exhibit a tailed morphology consisting of an icosahedral head, tail, and base that contains the genetic material. In addition, long tail fibers extend from the base that act as a detection device and can seek out the host organism. A second known morphology is the spherical phage, e.g., MS2. This phage is very similar in structure to many mammalian viruses and has a spherical capsid that stores the genetic material.

[0004] Other viruses include mammalian viruses that can be used for vaccination to establish protective immunity. Live attenuated or whole virus vaccines contain versions of live viruses that have been weakened or inactivated so that they do not cause severe disease. Other vaccines utilize mammalian viral vectors that can deliver genetic instructions to the body's cells.

[0005] However, a drawback of using viruses as therapeutics is their thermolability, since both the viral capsid and the genetic material tend to degrade when exposed to heat. As a result, vaccines often need to be refrigerated for storage and transportation. Certain viruses, including bacteriophage MS2, can only survive for short periods even at refrigerated temperatures, and as a result, cryogenic freezing in liquid nitrogen is required for long-term storage. This significantly increases the cost of storage and transportation of these phages and prevents their use in situations where such facilities are not available. Summary of the Invention

[0006] According to a first aspect of the present invention there is provided a particle comprising a virus encapsulated in an amorphous silica shell, the amorphous silica shell being deposited directly around the surface of the virus.

[0007] According to a second aspect of the invention, the method comprises concentrating or purifying a virus, suspending the virus in a buffer, Hydrolyzing a silica precursor, A method is provided which includes contacting a hydrolyzed silica precursor directly with the surface of the virus in a buffer to encapsulate the virus in an amorphous silica shell.

[0008] The method of the second embodiment may be used to make the particles of the first embodiment.

[0009] According to a third aspect of the invention there is provided the use of the method of the second aspect for the storage or preservation of a virus.

[0010] According to a fourth aspect of the invention there is provided a particle formed by the method of the second aspect.

[0011] According to a fifth aspect of the invention there is provided a dry powder comprising one or more particles of the first and / or fourth aspect.

[0012] According to a sixth aspect of the invention there is provided a pharmaceutical composition comprising one or more particles according to the first and / or fourth aspect. In some embodiments the pharmaceutical composition comprises at least two particles according to the first and / or fourth aspect, wherein the at least two particles comprise different viruses, for example two different bacteriophages.

[0013] According to a seventh aspect of the present invention there is provided a particle of the first and / or fourth aspect, a dry powder of the fifth aspect or a pharmaceutical composition of the sixth aspect for use in therapy.

[0014] According to an eighth aspect of the present invention there is provided a particle of the first and / or fourth aspect, a dry powder of the fifth aspect or a pharmaceutical composition of the sixth aspect for use as an antibiotic medicament, wherein the virus is a bacteriophage.

[0015] According to an aspect of the invention there is provided a non-therapeutic use of the particles of the first and / or fourth aspect, the dry powder of the fifth aspect or the pharmaceutical composition of the sixth aspect for use as an antimicrobial.

[0016] Also disclosed herein is a network of particles according to the first and / or fourth aspect. In some embodiments, the network of particles is in the form of a chain.

[0017] Also disclosed herein is a virus stabilized by an amorphous silica shell. The virus and the amorphous silica shell may be as defined herein.

[0018] Also disclosed herein are particles comprising a bacteriophage encapsulated in an amorphous silica shell, where the amorphous silica shell is deposited directly around the surface of the bacteriophage.

[0019] Also disclosed herein are particles comprising a virus encapsulated in an amorphous silica shell, wherein the particles do not comprise a polycationic polymer (eg, lysine).

[0020] Advantages of one or more of the above aspects The present invention provides particles comprising viruses that are substantially stabilized in an amorphous silica shell and have improved thermal stability compared to native, unencapsulated viruses. Surprisingly, after silica encapsulation and release, the viruses remain viable and capable of replicating.

[0021] The present invention also provides a method for making such particles. The method used to make the particles is a modified sol-gel method. In this method, hydrolyzed silica precursors (i.e., silica monomers) first directly interact with and deposit around the surface of the virus. Then, a silica shell grows or polymerizes around the surface of the virus. This ultimately results in the formation of a strong amorphous silica shell around the virus, protecting the virus from the denaturing conditions of the surroundings and preventing the degradation of the virus structure and the decomposition of genetic material.

[0022] Therefore, the method and resulting particles differ from typical methods used for silica encapsulation, which require an intermediate positively charged template to surround the virus particle prior to silica deposition. The method and resulting particles also differ from methods in which the silica structure is preformed and then loaded with a biological payload. Furthermore, in order for silica encapsulation of the virus to be achieved and differing from previous silica encapsulation methods, it was found that a concentration or purification step is key to successful silica encapsulation and particle growth. Other parameters were also found that are particularly favorable or optimal for silica encapsulation of the virus, so that the virus can be subsequently released with high recovery.

[0023] This is the first time that such a silica encapsulation / ensilication process has been demonstrated for such large complex biological structures containing genetic material. Previous studies and investigations have only demonstrated silica encapsulation of smaller, simpler protein-based biological targets lacking genetic material. Importantly and unexpectedly, the virus was able to withstand the encapsulation and concentration process and was able to replicate after release. Hence, this provides evidence that the genetic material of the virus can remain intact and survive the silica encapsulation process. Even more surprisingly, the silica encapsulated virus disclosed herein was able to generate plaques after release when stored for long periods at ambient temperature or even when exposed to high temperatures. Thus, the particles described herein provide a method to store and stabilize viruses, eliminating the need for refrigeration or cold chain for storage or transportation.

[0024] Surprisingly, the particles of the present invention, i.e., particles formed by the present method, have substantially different properties than previous silica-embedded / silica-encapsulated particles containing proteins or polypeptides. It has been found that the particles of the present invention have a larger median particle size and in fact the silica shell is also porous (i.e., and therefore in fact substantially more porous than silica-encapsulated proteins or polypeptides). For example, the particles disclosed herein may have a median particle size of at least 250 nm as determined by field emission scanning electron microscopy (SE-FEM), and more specifically, the median diameter is 250 nm to 1000 nm, or 250 to 600 nm, or 300 nm to 500 nm, or about 400 nm.

[0025] One or more embodiments of the invention may have one or more of the following features or additional advantages. In the particles disclosed herein, the virus encapsulated in the silica shell can be protected from ambient conditions, such as temperature or pH. In particular, the virus encapsulated in the silica shell is thermally stable, improving the thermal stability compared to native non-encapsulated viruses. In some cases, the virus has been shown to be stable after heating at 90° C. for 30 minutes and / or after long-term storage at ambient temperature (e.g., 21° C.). As a result, the particles disclosed herein can be used to store and / or stabilize viruses, eliminating the need for cold chain during storage or transportation. The particles disclosed herein may include a virus that is a bacteriophage. This is useful because bacteriophages can be used therapeutically, for example to treat bacterial infections.

[0026] The particles disclosed herein may be free of polycationic polymers, more specifically polylysine. This is advantageous since the addition of polycationic polymers increases the complexity of the particle and / or formulation, increasing the chance of side effects in the treatment while making regulatory approval more difficult to obtain. This also provides a particle that is less expensive and avoids any potential undesirable interactions between the polymer and silica that may impede the particle formulation and / or payload release. · The particles and methods disclosed herein are applicable to different types of viruses. Indeed, as demonstrated in the examples, the viruses may be DNA or RNA viruses and may have various shapes or morphologies. In some examples disclosed herein, the viruses are highly complex head-and-tail viruses (e.g., bacteriophage K, e.g., of the myoviridae family), whereas in other examples disclosed herein, the viruses are small spherical viruses (e.g., MS2, e.g., of the fiersviridae family). This shows that the methods disclosed herein are broadly applicable to different types of viruses. The methods disclosed herein are also broadly applicable to mammalian viruses, since they are structurally very similar to MS2. The particles disclosed herein can also include viruses of different molecular weights, including viruses with smaller molecular weights (e.g., MS2, with a molecular weight of about 50 kDa) and viruses with larger molecular weights (e.g., phage K), with molecular weights of more than 250 kDa or more than 500 kDa (e.g., phage K).

[0027] In the methods disclosed herein, the virus in the buffer is preferably at a concentration of 1×10 7 More than 1×10 PFU / ml, more preferably 8 More than 1×10 PFU / ml, or more preferably 9 Concentrations of greater than PFU / ml, or more preferably 1×10 9 ~1×10 10 The concentration of virus in the buffer is in PFU / mL. It has been found that a higher virus concentration in the buffer contributes to a higher recovery of the virus when released. · The methods disclosed herein may include a buffer comprising one or more salts, preferably one or more dicationic or monocationic metal salts. The one or more salts may stabilize the virus, increase virus recovery, increase the titer of the growth solution, and / or promote particle growth during silica encapsulation. In particular, the methods disclosed herein may include a buffer comprising one or more of a magnesium salt, a calcium salt, or a sodium salt. Dicationic salts including magnesium or calcium are believed to contribute to the stability of the lysate, and the absence of these salts reduces the titer of the growth phage solution. The presence of certain salts (e.g., sodium ions), where the salt ions can act as bridges between silica particles, is also believed to i) further contribute to the stability of the liquid lysate and ii) promote particle growth. In some examples disclosed herein, the buffer is an SM buffer or a modified SM buffer (e.g., an SM buffer in which 50 mM Trizma is replaced with glycine). Other suitable buffers may include PBS buffer, imidazole buffer (e.g., 50 mM imidazole buffer), bis-tris buffer, or sucrose buffer. Such buffers have been found to be more effective at silica encapsulation of viruses compared to Tris-only buffers.

[0028] In the methods disclosed herein, the ratio of hydrolyzed silica to virus in the buffer may be about 1:1 to 1:250, or 1:75 to 1:150, such as about 1:100. For higher ratios of virus to silica in the buffer, the silica embedding process is believed to be less uniform. In the methods disclosed herein, the pH of the contacting step is preferably slightly alkaline, e.g., a pH above 7, more preferably between 7.25 and 8.5, or between 7.25 and 8, e.g. about 7.5. The pH has been found to affect the rate of particle growth and can improve phage stability. Silica deposition and polymerization still works below pH 7 (e.g., at a pH of 6-7), but this process is much slower. In contrast, at higher pH (e.g., above pH 9), the integrity of the virus is affected. In some embodiments of the methods disclosed herein, the concentration or purification step is a polyethylene glycol (PEG) based concentration, for example using PEG 6000. This concentration method is very simple and fast, using readily available equipment and reagents that require little to no human time. This method allows for easy buffer exchange between the growth solution (TSB) and the working buffer (e.g., SM). In alternative embodiments, the concentration or purification step is by chloroform purification, filtration methods, size exclusion chromatography, ion exchange chromatography, or gradient centrifugation. These are other methods that can be used to generate a more pure product.

[0029] In the methods disclosed herein, the concentration or purification step is such that the virus in the buffer has a polydispersity index (PDI) of less than 0.2, more preferably less than 1.5. A lower PDI is believed to improve the silica encapsulation process because the silica is directed to the target as opposed to any residue or contaminants that may be present. In the method disclosed herein, the silica precursor is preferably a tetra-alkyl orthosilicate, more preferably tetra-ethyl orthosilicate, since TEOS leads to the less toxic and harmful by-product of ethanol, as opposed to TMOS, whose by-product is methanol. In the methods disclosed herein, the contacting step is preferably carried out for at least 5 minutes, or 10 minutes or more. The contacting step may be carried out for less than 30 minutes, or less than 20 minutes. As a result, the formation of particles is very simple and fast.

[0030] In the methods disclosed herein, the methods do not include a templating step, for example using a polycationic polymer, such as polylysine. This is in contrast to known silica encapsulation methods, where polylysine is first used to form an intermediate layer around the biomolecule or biological entity prior to silica deposition. The resulting method of the present invention does not include a templating step and is therefore simpler and less complicated. The particles herein may be in the form of a dry powder comprising said particles. The dry powder may further improve particle long-term storage. Dry powders may also be easily formed since the particles of the present invention are precipitated in a solution followed by vacuum filtration and drying.

[0031] The particles herein may be formulated into a pharmaceutical composition, which may include one or more pharmaceutical carriers or adjuvants to aid in delivery of the particles. The pharmaceutical composition disclosed herein may comprise one or more particles disclosed herein. Advantageously, the pharmaceutical composition may comprise two particles disclosed herein, at least two particles comprising different viruses. This allows a cocktail of different viruses to be stabilized or administered simultaneously (i.e. for antibacterial action against different types of bacteria). In the case of bacteriophages, this is particularly beneficial for targeting a broader range or type of bacteria. [Brief description of the drawings]

[0032] [Figure 1]FIG. 1 illustrates an exemplary method according to the present invention for forming exemplary particles of the present invention. A solution of pre-hydrolyzed TEOS is added to a bacteriophage K suspension and mixed gently for 10 minutes. During this time, silica surrounds the phage molecules, forming silica particles, which begin to settle out of solution. After 10 minutes, the solution is vacuum filtered and air-dried. [Diagram 2] Top: Schematic diagram of a head and tail phage (e.g. T4 or phage K with features 1- protein capsid (i.e. capsid head), 2- genetic material, 3- collar, 4- whiskers, 5- tail outer, 6- base, and 7= legs) and bottom: spherical phage (MS2) with 1b- protein capsid, 2b genetic material, and 8- mature protein. [Diagram 3] DLS results of size analysis of 0.45 μm filtered concentrated bacteriophage K samples used in silica encapsulation / embedding. Left) Correlation diagram of sample analysis; Right) Size analysis of the sample. The bottom trace shows the estimated size as measured by scattered light intensity, while the top trace shows the size as measured by particle volume. [Figure 4] FIG. 1 shows a transmission electron microscope (TEM) image of grown and concentrated bacteriophage K. [Diagram 5] FIG. 1 shows a field emission scanning electron microscope (FE-SEM) image of fully silica encapsulated / embedded and dried bacteriophage K at 30,000× magnification. [Figure 6] Graph showing the effect of pH on the rate of particle size growth during silica encapsulation / embedding of bacteriophage K, with analysis obtained using dynamic light scattering (DLS) measurements. Each point is an average of an 11 second measurement window. The silica encapsulation / embedding measured used concentrated phage K lysate suspended in SM buffer and a silica to phage solution ratio of 1:100. The rate of silica polymerization and deposition onto the phage increases with the concentration of [OH-] until the particles become repelled from each other, which is seen at about pH 9. [Figure 7]FIG. 1 shows FE-SEM images showing silica encapsulated / embedded MS2 particles at 15,000× magnification. [Figure 8] Left) Plaque assay of native bacteriophage K at various dilutions (top row shows full confluence with phage lysis, middle row shows semi-confluence with lysis, bottom row shows ideal counting conditions with individual plaques visible); right) Plaque assay of silica encapsulated / embedded sample after release (i.e., sample no. 2 in Table 1). [Figure 9] Left) Plaque assay results obtained from silica encapsulated / embedded, heated and released bacteriophage K (i.e., sample no. 8 in Table 1) at dilutions ranging from undiluted to x10-2; Right: Plaque assay results of heated native (i.e., not silica embedded) phage k lysate. [Figure 10] Figure 1 shows images of agar plates used in plaque assays of silica-embedded, released and grown MS2 using various concentrations of HF for their release: a) shows results from samples taken after 5 hours of growth; b) shows results from samples taken after 24 hours of growth. [Figure 11] Figure 1 shows images of plaque assay agar plates of a) silica-embedded, heated to 90°C for 30 min and released bacteriophage K, b) native phage K lysate heated to 90°C for 30 min, c) silica-embedded, released bacteriophage K with clearly visible plaques, and d) native, unheated phage K lysate. [Figure 12] Top) Graph showing a plot of dV (log d) versus pore size for the BJH isotherm of bacteriophage K embedded in silica using a PHOS:lysate ratio of 1:10 (D is pore size and V is pore volume); bottom) Graph showing a plot of dV (log d) versus pore size for the BJH isotherm of phage K embedded in silica using a PHOS:lysate ratio of 1:40 (d is pore size and V is pore volume). [Figure 13]Figure 1 shows the main measurement time points of phage virion growth over time for the silica encapsulation / embedding process. Tail width is indicated by the lower line with measurements on the left main axis, and head diameter (top to bottom) is indicated by the upper line with measurements on the right axis. Error bars are 2x standard deviation of measurements. [Figure 14] FIG. 1 shows a transmission electron microscope (TEM) image of bacteriophage K captured 1 minute after silica encapsulation / embedding. Values ​​are in nm. H=head, T=tail, and S=silica nodule. [Figure 15] Figure 1 shows a TEM image of bacteriophage K after 2 minutes of silica encapsulation / embedding. Values ​​are in nm. H=head, T=tail, and S=silica nodule. [Figure 16] FIG. 1 shows a TEM image of bacteriophage K captured 5 min after silica encapsulation / embedding. Values ​​are in nm. H=head, T=tail, and S=silica nodule. [Figure 17] Figure 1 shows a TEM image of bacteriophage K after 7 minutes of silica encapsulation / embedding. Values ​​are in nm. H=head, T=tail, and S=silica nodule. [Figure 18] ITEM image of phage K 7 min after silica embedding. The outlines show two different individual phage virions incorporated into the same silica cluster network. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] As defined herein, the term "silica encapsulation" may be used interchangeably with "silica encapsulation."

[0034] As defined herein, "bacteriophage K" is a double-stranded DNA-based bacteriophage of the Myoviridae family. Bacteriophage K exhibits a classical tailed phage ultrastructure in morphology similar to bacteriophage T4, approximately 300 nm tail-to-head, with an icosahedral head, a long contractile tail, and a hexagonal / star-shaped base. Phage K is a polyvalent virus capable of infecting a range of host strains and is infectious for Staphylococcus aureus. Bacteriophage K is also known or referred to as Staphylococcus aureus phage K, Staphylococcus virus K, or phage K.

[0035] As defined herein, "bacteriophage MS2" is an icosahedral / isometric single-stranded RNA-based bacteriophage of the family Fyasviridae. Bacteriophage MS2 is also known or referred to as MS2.

[0036] As defined herein, "native virus" refers to a virus that is not silica encapsulated / embedded.

[0037] As used herein, the terms "treatment" and "treating" refer to an approach for obtaining beneficial or desired results in a subject, including prophylactic benefits and, in some cases, therapeutic benefits.

[0038] A "prophylactic effect" refers to delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0039] "Therapeutic benefit" refers to the eradication or amelioration of the underlying disorder under treatment. Therapeutic benefit may also be achieved by the eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that improvement is observed in the subject, although the patient may still be afflicted by the underlying disorder.

[0040] The term "comprising" (and related terms, such as "comprise" or "comprises" or "having" or "including") includes those embodiments, such as any composition of matter, composition, method, or process that "consists of" or "consists essentially of" the recited features. The terms "comprises" or "comprising" may be used interchangeably with "includes."

[0041] When ranges are used herein, it is intended to include all combinations and subcombinations of the ranges, as well as specific embodiments thereof. When referring to a number or numerical range, the term "about" means that the number or numerical range referred to is an approximation within experimental variation (or within statistical experimental error), and thus the number or numerical range may vary. Typical experimental variations may result, for example, from changes and adjustments required during scale-up from laboratory settings and manufacturing sites to large-scale settings.

[0042] It is intended that features of any dependent claim and / or embodiment described in this specification may be readily combined with features of any independent claim or other dependent claim described herein, unless the context clearly dictates otherwise.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Abbreviations used herein have their conventional meaning within the chemical and biological arts unless otherwise indicated.

[0044] particle Disclosed herein are particles that include a virus encapsulated in an amorphous silica shell. The particles can be formed using the methods disclosed herein.

[0045] The amorphous silica shell can be directly deposited around the surface of the virus. As defined herein, the term "directly deposited" refers to silica deposited on the surface of the virus, e.g., on the protein capsid of the virus, as opposed to being deposited on an intermediate polymer layer between the virus and the silica shell. With respect to the particles described herein, the amorphous silica shell electrostatically interacts with at least one positively charged amino acid on the surface of the virus.

[0046] The particles described herein may comprise a single virus or multiple viruses. In some embodiments, the particles comprise a single virus. In some embodiments, the particles comprise two or more, or three or more, or four or more, or five or more viruses.

[0047] In some cases, the particles of the present invention may have a larger median particle size than previously reported silica-encapsulated biomolecules. In some embodiments, the particles have a median particle size of at least 250 nm as determined by field emission scanning electron microscopy (SE-FEM), or at least 300 nm as determined by field emission scanning electron microscopy (SE-FEM), or at least 350 nm, or 400 nm or more. In some embodiments, the particles have a median particle size of 250 nm to 1000 nm as determined by field emission scanning electron microscopy (SE-FEM), or 250 nm to 750 nm, or 275 nm to 600 nm, or 300 nm to 500 nm, or in some cases, about 400 nm as determined by field emission scanning electron microscopy (SE-FEM). In some embodiments, the particles have a median particle size of less than 1000 nm as determined by field emission scanning electron microscopy (SE-FEM), or a median particle size of less than 900 nm, or less than 800 nm, or less than 700 nm, or less than 600 nm, or less than 500 nm as determined by field emission scanning electron microscopy (SE-FEM).

[0048] In some examples, the particles of the present invention may have increased porosity compared to previously reported silica-encapsulated biomolecules. In some embodiments, the amorphous silica shell is porous (i.e., contains one or more pores). In some embodiments, the silica shell is water permeable. In some embodiments, the silica shell is permeable to small molecules, i.e., molecules having a molecular weight of less than 500 Da, or less than 300 Da, or less than 100 Da, or molecules having a molecular weight of less than 50 Da. In some embodiments, the silica shell has a median pore size of less than 20 nm, or less than 15 nm, or less than 10 nm, as determined using the Barrett-Joyner-Halenda isotherm. In some embodiments, the particles disclosed herein have a median pore size of less than 25 nm, or less than 15 nm, or less than 10 nm, as determined using the Barrett-Joyner-Halenda isotherm. 2 / g or more than 30m 2In some embodiments, the particles disclosed herein may have a surface area (i.e., as determined by BET) of greater than 25 m / g. 2 / g~40m 2 / g, or 30m 2 / g~40m 2 / g of surface area.

[0049] virus The particles disclosed herein include viruses. As defined herein, the term "virus" may be used interchangeably with virion. A virus is a biological structure that includes at least genetic material (i.e., nucleic acid) and a capsid (i.e., a protein coat) that surrounds the genetic material. A virus may further include a lipid outer envelope. In some embodiments, a virus (e.g., a Myoviridae virus) may further include a collar, a tail, a base, or a combination thereof. In some embodiments, a virus may further include fibers (e.g., whiskers or legs). Because a virus further includes genetic material, the term "virus" is distinct and distinct from "virus-like particle." For the avoidance of doubt, the term "virus" does not include virus-like particles.

[0050] The virus may be any suitable virus. The virus may be a filamentous virus, an isometric (i.e., icosahedral) virus, a rod-shaped virus, a bottle-shaped virus, a lemon-shaped virus, a pleomorphic virus, or a head-and-tail virus. In some examples, the virus is a head-and-tail virus or an isometric virus.

[0051] In some embodiments, the virus is a non-enveloped virus. In some embodiments, the virus is a non-enveloped isometric virus (e.g., MS2).

[0052] In other embodiments, the virus is an enveloped virus, hi some embodiments, the virus is a non-enveloped head-and-tail virus (e.g., phage K).

[0053] In some embodiments, the virus is a bacteriophage (i.e., a virus that can infect and replicate within bacteria and archaea).

[0054] In some embodiments, the virus (i.e., bacteriophage) is a virus of the order belfryvirales, caudovirales, halopanivirales, haloruvirales, kalamavirales, ligamenvirales, mindivirales, norzivirales, petitvirales, primaviriales, timlovirales, tubulavirales, vinavirales, or durnavirales. In some examples, the bacteriophage is a norzivirales (e.g., MS2) or caudovirales (e.g., phage K) bacteriophage.

[0055] In some embodiments, the virus (i.e., bacteriophage) is selected from the group consisting of viridae, turriviridae, ackermannviridae, autographviridae, chaseviridae, demerecviridae, drexlerviridae, guenliviridae, herelleviridae, myoviridae, schi ... Family: Siphoviridae, Podoviridae, Rountreeviridae, Salasmaraviridae, Schitoviridae, Zobellviridae, Sphaerolipoviridae, Simuloviridae, Matshushitaviridae, Pleolipoviridae lipoviridae, tectiviridae, lipothrixviridae, rudiviridae, cystoviridae, atkinsviridae, duinviridae, phiasviridae, solspiviridae, microviridae, tristomaviridae, Family Blumeviridae, Steitzviridae, Inoviridae, Paulinoviridae, Plectroviridae, Corticoviridae, Ampullaviridae, Autolykivirida, Bicaudaviridae, Clavaviridae,The virus is selected from the family Finnlakeviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Halsviridae, Plasmaviridae, Portogloboviridae, Thaspiviridae, and Spiraviridae. In some examples, the virus is a Fyasviridae (e.g., MS2) or Myoviridae (e.g., phage K) virus.

[0056] In some embodiments, the virus (i.e., bacteriophage) is a virus of a species selected from bacteriophage MS2, bacteriophage Qβ (i.e., Qbeta), bacteriophage T4, bacteriophage K, bacteriophage f2, bacteriophage R17, and bacteriophage GA. In other embodiments, the bacteriophage species may be selected from bacteriophage mu, P1 phage, bacteriophage P2, enterobacteria lambda, bacteriophage T5, bacteriophage HK96, bacteriophage N15, bacteriophage T7, bacteriophage T3, bacteriophage Φ29, or bacteriophage P22. In some examples, the virus is bacteriophage MS2 or phage K.

[0057] In some embodiments, the virus is a mammalian virus. In some embodiments, the virus may be a virus of a family selected from Flaviviridae, Coronaviridae (i.e., coronaviruses), Adenoviridae, Herpesviridae, Poxviridae, Parvoviridae, Reoviridae (i.e., including rotaviruses), Retroviridae, Togaviridae, Orthomyxoviridae (i.e., including influenza viruses), and Hepadnaviridae. In some embodiments, the mammalian virus is an isometric mammalian virus.

[0058] In some embodiments, the mammalian virus is a vaccine or part of a vaccine (i.e., or the mammalian virus is suitable for use as a vaccine to establish immunity in humans / mammals). In some embodiments, the mammalian virus is an inactivated / killed virus. In other embodiments, the mammalian virus is a live virus, preferably a live attenuated virus. In some embodiments, the mammalian virus is a viral vector vaccine.

[0059] The viruses described herein contain genetic material. In some embodiments, the viruses are RNA viruses (i.e., contain RNA genetic material). The RNA viruses may contain single-stranded or double-stranded RNA. In some examples, the RNA viruses contain single-stranded RNA, and the single-stranded RNA may be positive-sense RNA or negative-sense RNA. In some examples, the RNA viruses contain positive-sense single-stranded RNA (e.g., phage MS2). The RNA viruses may contain linear or circular RNA, e.g., linear dsRNA, linear ssRNA, circular dsRNA, or circular ssRNA. In some embodiments, the viruses are DNA viruses (i.e., contain DNA genetic material). The DNA viruses may contain single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). The DNA viruses may contain linear or circular DNA, e.g., linear dsDNA, linear ssDNA, circular dsDNA, or circular ssDNA. In some examples, the DNA viruses contain double-stranded linear DNA (e.g., bacteriophage K).

[0060] The virus may have any suitable molecular weight. In some embodiments, the virus has a molecular weight of more than 55 kDa, or more than 100 kDa, or more than 250 kDa, or more than 500 kDa, or more than 1 MDa, or more than 25 MDa, or more than 50 MDa, or more than 100 MDa. In some embodiments, the virus has a molecular weight of less than 250 MDa, or less than 200 MDa, or less than 100 MDa, or less than 50 MDa, or less than 25 MDa, or less than 1 MDa, or less than 500 kDa, or less than 250 kDa, or less than 100 kDa. In some embodiments, the virus has a molecular weight of 55 kDa to 250 MDa. In some examples, the virus has a molecular weight of 1 MDa to 250 MDa or 10 MDa to 250 MDa. In some examples, the virus has a molecular weight of about 33 MDa (i.e., phage K) or 56 kDa (i.e., MS2).

[0061] In preferred embodiments, the virus is viable and / or exhibits no or minimal loss of biological activity (i.e., after release from the silica shell). In preferred embodiments, the virus is capable of infecting a host cell (i.e., after release from the silica shell). In preferred embodiments, the virus is capable of replicating in a host cell (i.e., after release from the silica shell). For embodiments in which the virus is a bacteriophage, the host cell is a bacterium or archaea. In some embodiments, the bacterium is Escherichia coli (e.g., E. coli), Staphylococcus (e.g., Staphylococcus aureus), Streptococcus, Pseudomonas, Shigella, or Vibrio.

[0062] For embodiments in which the virus is a mammalian virus, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the mammalian cell is a bovine cell, a sheep cell, a pig cell, a goat cell, a dog cell, a cat cell, a donkey cell, a deer cell, a horse cell, or a rodent cell (e.g., a mouse cell or a guinea pig cell), a fox cell, a wolf cell, a bat cell, a coyote cell, a raccoon cell, a skunk cell, a ferret cell, a monkey cell, a primate cell, a hare cell, a rabbit cell, a bear cell, or a mongoose cell.

[0063] Silica Shell In some embodiments, the silica shell is deposited directly around the entire surface of the virus. In alternative embodiments, the silica shell is deposited directly around a majority of the surface of the virus, i.e., about 50% of the surface of the virus, or about 60%, or about 70%, or about 80%, or about 85%, or about 90%, or about 90%, or about 99%, or about 99% of the surface of the virus. This can be determined by TEM-EDX (transmission electron microscopy energy dispersive X-ray spectroscopy) or FE-SEM-EDX (field emission scanning electron microscopy energy dispersive X-ray spectroscopy).

[0064] The silica shell is an amorphous silica shell. The amorphous silica shell refers to a silica shell that is non-crystalline. The silica shell is preferably a polymeric matrix of silica, i.e., covalently bonded silica. In the present invention, the silica shell is in direct contact with the surface of the virus, for example, the protein capsid of the virus, more specifically, at least one positive amino acid residue that constitutes the protein capsid of the virus. The direct contact is preferably an electrostatic interaction. In a preferred embodiment, the silica shell is not covalently bonded to the surface of the virus.

[0065] In some embodiments, the silica shell is non-uniform or substantially non-uniform (i.e., the silica shell has a non-uniform thickness around the surface of the virus). For example, for a head-and-tail virus, the silica shell may be thicker around the head compared to the tail. In embodiments where the virus includes a base, the silica shell may also be thicker around the base compared to other portions of the virus surface (e.g., the tail).

[0066] The silica shell may be of any suitable morphology. In the examples disclosed herein, the silica shell has a spheroidal morphology. In some embodiments, the silica shell has a spherical morphology. In some embodiments, the silica shell has an ellipsoidal morphology.

[0067] Particle composition As defined above, the particles include a virus and an amorphous silica shell.

[0068] In some embodiments, the particles include one or more salts. In preferred embodiments, the one or more salts may include dicationic or monocationic metal ions. In some embodiments, the dicationic metal ions are or include magnesium ions or calcium ions or combinations thereof. In some embodiments, the monocationic metal ions are or include sodium ions. In some embodiments, the one or more salts include one or more of magnesium ions, calcium ions, and sodium ions. In some embodiments, the particles include a buffer, such as a component of SM buffer or any buffer described elsewhere herein. In some examples, the one or more salts include magnesium sulfate and / or sodium chloride. The presence of certain salts is believed to contribute to the stability of the liquid lysate and may also promote particle growth. In particle growth, salt ions may act as bridges between silica particles.

[0069] In some embodiments, the particles may further comprise one or more small molecules. In some embodiments, the one or more small molecules may be an amino acid. In some embodiments, the amino acid is glycine. In some embodiments, the electrostatic interaction may be facilitated by the presence of a small molecule, such as an amino acid, or more specifically, glycine. In some embodiments, the particles may be free of Trizma (i.e., tromethamine).

[0070] In some embodiments, the particles may further comprise a stabilizing component (i.e., to stabilize or further stabilize the virus). In some embodiments, the stabilizing component may comprise gelatin.

[0071] In a preferred embodiment, the particles do not include polycationic polymeric material.In a preferred embodiment, the particles do not include polylysine.

[0072] In some embodiments, the particles may include one or more excipients. The one or more excipients may be selected from an emulsifier, an osmolyte, a co-solvent, an antimicrobial, or a combination thereof. An example of an emulsifier may be Tween 20 or Tween 80. An example of an osmolyte includes sucrose. An example of a co-solvent may be sorbitol and / or glycerol. An example of an antimicrobial includes phenol and / or benzethonium chloride. In some embodiments, the particles may include one or more adjuvants. In some embodiments, the adjuvant may be selected from alum, monophosphoryl lipid (e.g., MPLA), a squalene-based adjuvant (e.g., AS03 or MF59), AS04 (e.g., a combination of MPLA and alum), or a CpG DNA adjuvant (e.g., CpG 1018).

[0073] characteristics In the particles disclosed herein, the virus may be protected from ambient conditions. In some cases, the virus may be protected from temperature. In some cases, the virus may be protected from acidic or basic pH.

[0074] In the particles disclosed herein, the virus may be thermally stable, or at least the virus has improved thermal stability compared to the native virus. In some cases, the particle is stable after heating at 90°C for at least 5 minutes, or at least 10 minutes, or at least 20 minutes, or at least 30 minutes. In some cases, the particle is stable after storage at 21°C for at least 24 hours, or at least 48 hours, or at least 1 week, or at least 1 month, or at least 3 months, or at least 1 year. In some cases, the particle is stable after storage at 4°C for at least 1 week, or at least 1 month, or at least 3 months, or at least 1 year. The stability measurement can be determined by plaque assay. That is, after release, the virus is substantially intact and substantially functional to replicate and / or form plaques.

[0075] In the particles disclosed herein, the virus can be stable to acid or base. The particles disclosed herein can be exposed to low pH, for example, ≦pH 4.0, ≦pH 3.5, or ≦pH 3.0. When subsequently released from the particle, the virus is substantially intact and substantially functional. In contrast, a virus that is not silica-embedded is likely to denature and lose its function under these conditions.

[0076] method Disclosed herein is a method for making a virus encapsulated in an amorphous silica shell, comprising: Concentrating or purifying the virus, suspending the virus in a buffer, Hydrolyzing a silica precursor, The method involves contacting a hydrolyzed silica precursor directly around the surface of the virus in a buffer to encapsulate the virus in an amorphous silica shell.

[0077] The method can be used to form the particles described herein. The virus can be any suitable virus described herein. The amorphous silica shell can be as described elsewhere herein.

[0078] In some embodiments, the method further comprises growing the virus prior to concentrating or purifying the virus. In some embodiments, the growth is solid phase or liquid phase growth. The growth comprises culturing the virus in the presence of bacteria in a growth buffer. In some examples, the growth buffer is or comprises Tryptic Soy Broth (TSB) (i.e., including modified TSB buffer), Luria Broth (LB), Super Optimal broth with Catabolite repression (SOC), Super Optimal Broth (SOB), Terrific Broth (TB), YT Broth (2xYT Broth).

[0079] The method includes a concentration or purification step. The concentration or purification step may be by any suitable concentration or purification method. In some embodiments, the concentration or purification is selected from PEG-based concentration, gradient centrifugation, size exclusion chromatography, ion exchange chromatography, chloroform purification, or filtration methods. In a preferred embodiment, the concentration or purification is from viruses in a lysate (i.e., viruses in a growth buffer). In a preferred embodiment, the concentration or purification is a PEG-based concentration, where the PEG is polyethylene glycol. In some examples, the PEG has an average molecular weight of 3000-12000 g / mol, or 3500-10000 g / mol, or 4000-80000 g / mol, or 5000-7000 g / mol, or about 6000 g / mol. In some examples, the PEG-based concentration includes PEG 6000, i.e., PEG with an average molecular weight of 6000 g / mol. In some examples, the PEG-based enrichment comprises adding 1-15% w / w, or 5-12.5% ​​w / w, or 7-10% w / w PEG to the virus (i.e., a viral lysate). In some examples, the PEG-based enrichment comprises adding at least 5% w / w, or at least 7% w / w, or at least 10% w / w PEG to the virus (e.g., a viral lysate). In some embodiments, the enrichment or purification comprises chloroform purification.

[0080] In some embodiments, the enrichment or purification comprises gradient centrifugation. In some embodiments, the gradient centrifugation utilizes a gradient medium selected from sucrose, cesium chloride, iodixanol, sorbitol, Histodenz, or dextran. In some embodiments, the enrichment or purification comprises size exclusion or ion exchange chromatography. In some embodiments, the enrichment or purification comprises a filtration method.

[0081] The concentrated or purified virus may be added to any suitable buffer to form concentrated virus in the buffer. In a preferred embodiment, the concentrated or purified virus is suspended in the buffer. In some embodiments, the buffer comprises one or more salts, preferably the one or more salts comprise dicationic or monocationic metal ions. In some embodiments, the one or more salts may comprise dicationic metal ions. In some embodiments, the one or more salts may comprise one or more magnesium or calcium. In some embodiments, the one or more salts may comprise monocationic metal ions, and the monocationic metal ions may comprise sodium. In some examples, the one or more salts may comprise magnesium sulfate and / or sodium chloride. In some embodiments, the buffer comprises one or more dicationic metal salts at a concentration of 0.1 mM to 50 mM, or about 1 mM to 25 mM, or about 5 mM to 11 mM, or about 8 mM. In some embodiments, the buffer comprises one or more dicationic metal salts at a concentration of greater than 0.5 mM, or greater than 1 mM, or greater than 2 mM, or greater than 3 mM, or greater than 4 mM, or greater than 5 mM, or greater than 6 mM, or greater than 7 mM, or greater than about 8 mM. In some embodiments, the dicationic metal salt preferably comprises magnesium, more preferably magnesium sulfate.

[0082] In some embodiments, the buffer comprises a sodium salt at a concentration of 0.1 mM to 500 mM, or about 50 mM to 200 mM, or about 100 mM. In some embodiments, the buffer comprises one or more dicationic metal salts at a concentration of greater than 0.1 mM, or greater than 1 mM, or greater than 5 mM, or greater than 10 mM, or greater than 25 mM, or greater than 50 mM, or greater than 75 mM, or about 100 mM. In a preferred embodiment, the sodium salt is preferably sodium chloride.

[0083] In some embodiments, the buffer may include one or more small molecules. In some embodiments, the buffer includes one or more amino acids. In some embodiments, the buffer includes glycine. In some embodiments, the buffer includes an amino acid, such as glycine, at a concentration of at least 1 mM, or at least 5 mM, or at least 10 mM, or at least 25 mM, or 50 mM or more. In some embodiments, the buffer includes glycine at a concentration of 1 mM to 100 mM, or about 10 mM to 80 mM, or about 25 mM to 75 mM, or about 40 mM to 60 mM, or about 50 mM.

[0084] In some embodiments, the buffer may include one or more stabilizers. In some embodiments, the buffer may include gelatin. In some embodiments, the buffer may include at least 0.01 g / mL gelatin, or at least 0.5 g / mL, or about 0.1 g / mL gelatin.

[0085] In some embodiments, the buffer comprises or is a SM buffer, a modified SM buffer (e.g., a glycine modified SM buffer), a PBS buffer, an imidazole buffer, a bis-tris buffer, or a sucrose buffer. In some cases, the buffer is not a Tris buffer (i.e., a buffer containing only Trizma and water). In some cases, the buffer does not contain Trizma.

[0086] In certain embodiments, after purification and concentration, the virus in the buffer has a polydispersity index (PDI) determined using DLS analysis of less than 0.2, more preferably less than 0.15, or less than 0.14, or less than 0.13, or less than 0.12. In some examples, the virus in the buffer has a PDI of 0.1 to 0.15.

[0087] In preferred embodiments, enriching involves concentrating the concentration of the virus (i.e., the virus in the buffer is increased in concentration or PFU / mL). In some embodiments, concentrating involves centrifugation of the virus. Centrifugation can be performed at any suitable speed, for example, between 5,000×G and 200,000×G. In some embodiments, for example, for phage K, centrifugation is performed at a speed of less than 25000×g, or preferably less than 16000×g.

[0088] In a preferred embodiment, after concentration and purification, the virus in the buffer (i.e., concentrated virus in buffer) is about 1×10 7 More than 1×10 PFU / ml, more preferably 8 More than 1×10 PFU / ml, more preferably 9 Concentrations of more than 1×10 PFU / ml are preferred 8 PFU / ml ~ 1×10 10 PFU / ml or 1 x 10 9 PFU / ml ~ 1×10 10 It is present in concentrations ranging from PFU / ml.

[0089] The method of the present invention also includes a hydrolysis step that includes hydrolysis of a silica precursor. The resulting product is a hydrolyzed silica precursor, which may include silica coordinated by four oxygen atoms. In a preferred embodiment, the hydrolyzed silica precursor is, for example, a silicic acid monomer of formula, for example, Si(OH)4. The prehydrolysis step preferably occurs in the absence of viruses. In some embodiments, the prehydrolysis step is carried out until the solution is single-phase. In some embodiments, the prehydrolysis step occurs for about 20-120 minutes, or for about 40-80 minutes, or for about 20-120 minutes, or for about 40-80 minutes. In some embodiments, the prehydrolysis step occurs for at least 20 minutes, or for at least 40 minutes, or for more than 60 minutes, or for at least 20 minutes, or for at least 40 minutes, or for more than 60 minutes. In some embodiments, the prehydrolysis step occurs for less than 120 minutes, or for less than 90 minutes, or for less than 60 minutes, or for less than 120 minutes, or for less than 90 minutes, or for less than 60 minutes.

[0090] [ka]

[0091] The silica precursor may be any suitable silica precursor, i.e., any molecule that hydrolyzes to form silica, more preferably a silica monomer as described above. In some embodiments, the silica is covalently bonded to one or more alkoxide groups (i.e., the silica precursor is an alkoxysilane). In some embodiments, the silica precursor is a tetra-alkyl orthosilicate. In some embodiments, the alkyl of the tetra-alkyl orthosilicate is a C1-C6 alkyl. Examples of silica starting materials include tetra-methoxy-orthosilicate (TMOS), tetra-ethoxy-orthosilicate (TEOS), tetra-propoxy-orthosilicate (TPOS), tetra-butoxy-orthosilicate (TBOS), and tetra(ethoxymethoxy)silane. In some embodiments, the tetra-alkyl orthosilicate includes or is tetra-ethyl orthosilicate.

[0092] Hydrolysis of the silica starting material is typically carried out at an acidic pH. The acidic pH may be below 4.5, 3.5, 3.0, 2.5, or 2.0. In some cases, the step of hydrolyzing the silica precursor is carried out at a pH of 3.0 or less. A preferred acidifying agent is HCl, for example 32% HCl. The acidifying agent catalyzes the hydrolysis of TEOS. In some cases, a 1:1 ratio of tetraethylorthosilicate solution to HCl is used.

[0093] The method further comprises contacting the hydrolyzed silica precursor directly with the surface of the virus in the buffer to encapsulate the virus in an amorphous silica shell. In some embodiments, the contacting comprises adding the hydrolyzed silica to the virus in the buffer, preferably by stirring the mixture. A schematic of this process is shown in FIG. 1. In this step, the hydrolyzed silica precursor is preferably added to the virus in the buffer. In some embodiments, the hydrolyzed silica precursor (i.e., silicic acid monomers) is first contacted with the surface of the virus (e.g., by electrostatic interactions between the silica and the protein capsid), and then the silica shell grows and polymerizes around the surface of the virus. This may otherwise be referred to as silica embedding around the virus, and the resulting particles may be referred to as silica-embedded viruses.

[0094] The encapsulation process, which forms a silica shell around the surface of the virus, differs in several aspects from typical sol-gel methods. Silica embedding starts with silicic acid monomers that polymerize around the surface of the virus itself when added to the virus in a buffer. In particular, hydrolyzed silica precursors are typically attracted to positive residues on the surface of the virus and electrostatically bind to the virus surface. Subsequent silica monomers then link silica chains, resulting in the growth of a silica shell around the virus surface. This method can be contrasted with previous studies involving silica coating of viruses using typical sol-gel methods. In the previous studies, instead, a polymerized silica network is formed prior to contact with the biomolecule. Unlike previous silica encapsulation methods, silica is instead deposited around the surface of the virus, as opposed to being deposited around a template or intermediate structure formed around the virus (e.g., using a polycationic polymer). The methods of the present invention may not include a templating step, such as where an intermediate layer is formed around the virus using a polycationic polymer, such that the silica does not directly contact the surface of the virus. In some embodiments, the method may not include a polycationic polymer. In some embodiments, the method may not include polylysine.

[0095] The contacting can be carried out at a pH > 6. In a preferred embodiment, the contacting is carried out at a pH above 7 or above 7.25, preferably the pH is between 7.25 and 8.5, for example about 7.5.

[0096] The contacting can be performed for any suitable time. In some embodiments, the contacting is performed for at least 5 minutes, preferably at least 10 minutes. In some embodiments, the contacting is performed for less than 60 minutes, or less than 45 minutes, or less than 20 minutes, or less than 10 minutes, or less than 5 minutes, or less than 2 minutes, or 1 minute, or less than 30 seconds. In some embodiments, the contacting is performed for 10 to 20 minutes.

[0097] The contacting step can be carried out using any suitable ratio of hydrolyzed silica precursor:virus in buffer. In some embodiments, the ratio of hydrolyzed silica precursor:virus in buffer is 1:1 to 1:250, more preferably 1:10 to 1:250. In a preferred embodiment, the ratio of hydrolyzed silica to virus in buffer is about 1:75 to 1:150, for example, about 1:100. In some embodiments, the ratio of hydrolyzed silica to virus in buffer is 1:≧10, or 1:≧25, or 1:≧50, or 1:≧75, or 1:≧100.

[0098] The contacting step can be carried out at any suitable temperature, hi some embodiments, the contacting may be carried out at a temperature of from 4 to 40°C, or from 4 to 25°C, or from 15 to 25°C, or from 15 to 20°C.

[0099] In some embodiments, the resulting particles are allowed to settle. In some embodiments, the method further comprises a vacuum filtration step, i.e., the particles are vacuum filtered. In some embodiments, the particles may be further subjected to drying, e.g., air drying.

[0100] dry powder The present invention also provides a dry powder comprising a particle of the present invention, more preferably a plurality of particles of the present invention. The dry powder can be obtained by the method described in the second embodiment, followed by vacuum filtration and drying. The dry powder can comprise at least one first particle comprising a first virus, and at least one second particle comprising a virus different from the virus in the first particle (i.e., a second virus).

[0101] Pharmaceutical Compositions The present invention also provides a pharmaceutical composition comprising one or more particles of the present invention, more preferably a plurality of particles of the present invention. In some embodiments, the particles of the present invention may be in the form of a dry powder as described above. The pharmaceutical composition may comprise at least one first particle comprising a first virus, and at least one second particle comprising a virus (i.e., a second virus) different from the virus in the first particle. The pharmaceutical composition may otherwise be referred to as a composition.

[0102] The pharmaceutical composition may further comprise one or more pharmaceutical excipients or adjuvants.

[0103] In some embodiments, the one or more excipients may be selected from an emulsifier, an osmolyte, a co-solvent, an antimicrobial, or a combination thereof. An example of an emulsifier may be Tween 20 or Tween 80. An example of an osmolyte includes sucrose. An example of a co-solvent may be sorbitol and / or glycerol. An example of an antimicrobial includes phenol and / or benzethonium chloride. In some embodiments, the particles may include one or more adjuvants. In some embodiments, the adjuvant may be selected from alum, monophosphoryl lipid (e.g., MPLA), a squalene-based adjuvant (e.g., AS03 or MF59), AS04 (e.g., a combination of MPLA and alum), or a CpG DNA adjuvant (e.g., CpG 1018).

[0104] treatment The particles described herein may be for use in therapy. The particles described herein may be used in methods of treatment or prevention of disease. In some embodiments where the virus is a bacteriophage, the virus may be for use as an antibacterial pharmaceutical (i.e., phage therapy). In some embodiments where the virus is a mammalian virus (e.g., a live attenuated virus or an inactivated virus), the virus may be for use as a vaccine (i.e., to provide protective immunity against infection), e.g., the infection is a bacterial infection or a viral infection. In some embodiments, the particles for use are administered by any suitable means, e.g., intramuscularly, subcutaneously, topically, intranasally, or orally.

[0105] non-therapeutic use Also disclosed herein is a non-therapeutic use of the particles, powders, or pharmaceutical compositions described herein, where the virus is a bacteriophage, for use as an antimicrobial. In some examples, the particles described herein can be used as antimicrobials, where the particles are applied to a surface (i.e., the bacteriophage is stored on the surface). In some embodiments, the surface can include, but is not limited to, a structure, a packaging material, a tool or part of a device (e.g., a medical instrument), or a biological material, such as a plant surface. This can make the surface harmful to bacterial growth.

[0106] Method of release Also disclosed herein are methods for releasing viruses from particles of the invention.

[0107] The virus may be released prior to its use for administration to a subject, for example prior to injection, in some embodiments, after release, the virus may be mixed with an additional adjuvant or pharmaceutical carrier prior to administration to the subject.

[0108] In some embodiments, the method includes contacting the silica shell with a solution (i.e., release solution) containing fluoride ions at an acidic pH (i.e., to generate HF). The solution may include sodium fluoride acidified, for example, using HCl. The method may further include stirring the mixture. In some embodiments, the contacting may be for about 10 minutes to about 2 hours, or 30 minutes to about 90 minutes. The solution may have a pH of less than 6, or less than 5, or less than 4, or less than 3, and in some cases about 2.9. The HF concentration of the solution is preferably 3 mg / mL or less, or 2.5 mg / mL or less, or 2 mg / mL or less, or 1.5 mg / mL or less, or 1 mg / mL or less, or 0.5 mg / mL or less. In some embodiments, the total concentration of HF contacting the particles is less than 0.5 mg / mL. In some cases, the solution includes SM buffer or water (i.e., deionized water).

[0109] In other embodiments, the virus may be released in a manner that does not involve contacting the particles with fluoride ions at an acidic pH (e.g., for isometric viruses such as MS2). In some embodiments, the virus is released by incubating the particles (e.g., in growth buffer at 37°C). EXAMPLES

[0110] Materials and Methods material Growth media Tryptic Soy Agar (TSA) and Tryptic Soy Broth (TSB), TEOS, HCl, NaF, Trizma base, and chloroform were purchased from Sigma Aldrich.

[0111] Solid phage growth Phage K Bacteriophage K and the S. aureus (MSSA H560) host strain were kindly provided by Dr. S. Milo of the Jenkins Group, University of Bath. A single colony from the provided MSSA streak plate was placed into 10 mL of tryptic soy broth (TSB) solution (Sigma Aldrich) and incubated overnight at 37°C with shaking. Approximately 15 mL of tryptic soy agar (TSA) gel was poured into sterilin 90 mm circular Petri dishes to form the base for bacterial growth. A top soft agar layer was created by adding 100 μL of the overnight bacterial solution and 100 μL of the provided phage lysate to 3 mL of TSB solution containing 0.7% TSA. This 3 mL soft agar was poured into the TSA gel dishes, allowed to set, and then incubated stationary at 37°C for 18 hours. After incubation, 3 mL of SM buffer (pH 7.5) was added to each dish and the dishes were incubated under the previous conditions for an additional 4 hours. After incubation, approximately 3 mL of free SM buffer per plate was pipetted into a centrifuge tube and spun at 10,000 RPM for 10 minutes at 4° C. The top layer was collected, filtered through a 0.22 μm millex filter, and stored at 4° C.

[0112] MS2 Protocol provided by ATCC with phage and host strain E. coli 15597. Bacterial cultures were thawed at room temperature and streak plated onto clean TSA petri dishes. The plates were incubated overnight at 37°C. Once fully grown, one colony was selected from the plate and placed into 10 mL of TSB to form an overnight solution of bacteria and incubated at 37°C for 18 hours.

[0113] One mL of this overnight culture was pipetted onto the lyophilized phage sample and carefully inverted to rehydrate the phage. Approximately 15 mL of tryptic soy agar (TSA) gel was poured into Sterilin 90 mm round petri dishes to form the base for bacterial growth. 100 μL of the overnight solution and 100 μL of the provided phage lysate were added to 3 mL of TSB solution containing 0.7% TSA to create a top soft agar layer. 3 mL of this soft agar was poured into the TSA gel dishes, allowed to set, and then incubated stationary at 37°C for 18 hours. After incubation, 3 mL of SM buffer (pH 7.5) was added to each dish and incubated on the dishes for an additional 4 hours. After incubation, approximately 3 mL of free SM buffer per plate was pipetted into a centrifuge tube and spun at 10,000 RPM for 10 minutes at 4°C. The top layer was collected, filtered through a 0.22 μm millex filter, collected and stored at 4°C.

[0114] Small-scale enrichment of bacteriophage K 10 mL of impure phage lysate was centrifuged (Beckman Coulter Avanti J-26S with JA-25-50 rotor) at 15,300×g, 3° C. for 20 min with gentle acceleration and no mechanical deceleration. The supernatant solution was then added to a container in an ice bath on a magnetic stirrer. NaCl (Sigma-Aldrich) was slowly added under gentle stirring until a final concentration of 2.3% w / w was reached. Polyethylene glycol M / W 6000 (PEG-6000) was then added under gentle stirring until a final concentration of % w / w was reached. The beaker was covered with aluminum foil while maintaining gentle stirring and left for 1 h, after which the ice bath was removed from stirring and placed in the refrigerator at 4° C. overnight. The chilled phage lysate was then centrifuged as before at 15,300×g for 20 min at 3° C. with slow acceleration and no mechanical deceleration (Beckman Coulter Avanti J-26S with JA-25-50 rotor). The supernatant was discarded and the sediment was resuspended in a small volume of SM buffer (pH 7.5). The suspension was then transferred to a clean centrifuge bottle, the previous bottle was rinsed, and the wash was added to the suspension in the “clean” bottle. The suspension was then centrifuged further at 13,000×g for 4 min at 23° C. with slow acceleration and no mechanical deceleration to facilitate precipitation of the PEG-6000 (Beckman Coulter Avanti J-26S with JA-25-50 rotor). The supernatant was collected and stored at 4° C. for later use.

[0115] Liquid-phase growth of bacteriophage K 30 mL of TSB was inoculated with a bacterial freezer stock and incubated at 37° C. with shaking at 180 RPM for approximately 18 hours to create an overnight bacterial solution. 10 mL of this overnight culture was added to each of 3×600 mL medic flasks containing 400 mL fresh TSBss. The medic flasks were incubated at 37° C. with shaking at 170 RPM for exactly 18 hours. The contents of the flasks were then centrifuged (Beckman Coulter Allegra 64R with TA-10-250 rotor) at 15,300×g for 20 minutes at 3° C. and the supernatant was retained.

[0116] Large-scale enrichment of phages The supernatant collected from the liquid phase growth was transferred to a 2L plastic beaker in an ice bath on a magnetic stirrer. With constant gentle stirring (approximately 150 RPM), NaCl was gradually added to a final concentration of 2.3% w / w. Once dissolved, PEG-6000 was added to a final concentration of 7% w / w for phage k or 10% w / w for the optimized MS2 method, also under gentle stirring. The beaker was then covered with foil and allowed to stir for 1 hour before being transferred to a 4°C cold room overnight. Any sediment was discarded and the solution was then centrifuged (Beckman Coulter Allegra 64R with a TA-10-250 rotor) at 15,300×g, 3° C., for 20 minutes (K) or 40 minutes (MS2), with slow acceleration (K), no mechanical deceleration (K), or fast acceleration (MS2), and high mechanical deceleration (MS2). The supernatant was discarded and the pellet resuspended in SM buffer to a total resuspension volume of 150 mL. For phage k, the suspension was then centrifuged at 13,000×g at 23° C. for 4 min with slow acceleration and no mechanical deceleration (Beckman Coulter Avanti J-26S with JA-25-50 rotor), after which the supernatant was transferred to a long-term storage vessel. For MS2, the phage are centrifuged for 40 min at higher acceleration and deceleration as detailed above.

[0117] Chloroform purification of MS2 Add an equal volume of CHCl3 to 40 mL of concentrated MS2 lysate. Vortex mix the mixture and then centrifuge at 3,000 x G for 15 min. The mixture should then separate into three distinct layers. Use a pipette to collect the top aqueous layer and transfer it to a long-term storage container at 4-8 °C.

[0118] Silica embedding of phages Preparation of pre-hydrolyzed silica: 40 mL of a 1:1 mixture of tetraethyl orthosilicate (TEOS) and HO along with 40 μL of 32% HCl was stirred at 700 RPM for approximately 40 minutes until the solution was single phase, then the stirring speed was reduced to 125 RPM for 20 minutes. This solution is the pre-hydrolyzed silica solution.

[0119] In a separate beaker, add 90 mL of SM buffer for phage K or SM (glycine) buffer for MS2, and concentrated high titer (>1.00 × 10 9 10 mL of phage lysate (PFU / mL) was mixed and gently stirred at 125 RPM to ensure they were thoroughly mixed. 1 mL of PHOS was added to the phage suspension and the mixture was stirred at 125 RPM for 10 minutes. After this time, the mixture was filtered under vacuum through a 0.7 μm microglass fiber filter (Fisher Scientific). The filter and material were allowed to dry in a fume hood until a dry powder material was obtained. The powder was then removed from the filter and placed in an Eppendorf vial for long-term storage.

[0120] Plaque assay A 10 mL bacterial overnight solution was made. 100 μL / dish was added to 3 mL / dish of warmed TSB containing 0.7% (w / v) TSA and allowed to stand for 10 min. 3 mL of the inoculated 0.7% TSA solution was added to each TS agar plate and allowed to stand under sterile conditions. 100 μL of phage lysate was added to 900 μL SM buffer to obtain a 10× dilution, and this process was repeated serially to generate a dilution range of 10-1 to 10-9. 10 μL of each dilution was pipetted three times onto agar dishes containing three different dilutions per dish. After the spots were dried under sterile conditions, the dishes were incubated (37°C, 18 h). Once incubated, visible plaques were counted to obtain plaque forming units per milliliter (PFU / mL) titers.

[0121] Release of silica-embedded phages by HF 10 mg of the silica-embedded powder was placed in a 1 mg / mL solution of NaF in SM buffer, pH 2.90. This was rotated end-over-end for 1 hour, after which 2 mL of this solution was transferred to a 40 mL culture of 1-hour-old bacteria. This was then incubated at 37°C, 170 RPM for 24 hours. It was then filtered through a 0.45 μm filter and stored at 4°C for assay.

[0122] In optimization experiments of NaF / HF release, 96-well plate concentrated phage lysates suspended in SM buffer were mixed with increasing concentrations of NaF / HF buffer. This mixture was then added to 18-h-old bacterial cultures in TSB and optical density (measured at 595 nm) was used to determine the presence of bacterial growth and therefore phage survival.

[0123] Release of MS2 silica-embedded phages without HF A small chunk (40 mg) of silica-embedded MS2 powder was added to 1 mL of TSBss and incubated for 1 h, then added to 400 mL of 1-h-old E. coli and incubated for an additional 24 h. These samples were then subjected to plaque assays.

[0124] Results and Discussion Phage concentration / purification Phage K Initial attempts to silica encapsulate native phage K lysate were unsuccessful. Using 10:1 lysate to prehydrolyzed silica in 90 mL SM buffer, the solution was mixed for 10 minutes with no turbidity or particle formation observed. Further mixing still resulted in no change and no visible formation of silica particles. Ultimately, it was found that only properly concentrated and purified phage lysate was suitable for silica embedding. Silica embedding of concentrated phage lysate resulted in almost instantaneous turbidity of the solution and formation of silica nanoparticles. The results of these tests indicated that only properly concentrated phage lysate was suitable for this silica embedding process.

[0125] Phage lysates were concentrated using the concentration method described above using PEG 6000, although chloroform purification could also be used to purify the lysate. Importantly, the PEG 6000 concentration method also had the advantage of lowering the volume of the lysate by 10-fold and increasing the average PFU / mL of the concentrated lysate by 10×. The PEG concentration method removed most of the unwanted bacterial debris, and subsequent centrifugation of the mixture and resuspension in SM buffer effectively removed the TS broth.

[0126] Concentrating bacteriophage K utilizing PEG 6000 allows for a lysate of increased purity to be obtained for further testing and experimentation. After concentration, DLS analysis showed that the concentrated solution was much less polydisperse compared to the non-concentrated solution, with a calculated PDI of 0.118 compared to the non-concentrated solution, which had a PDI of 0.384. After concentration, the correlation plot obtained from DLS analysis showed a rapid initial drop in signal correlation at approximately 10 μS, followed by a steep slope without a peak shoulder (see Figure 3). These results indicate a relatively small particle size with a highly monodisperse distribution and no impurities.

[0127] As the enrichment process was successfully confirmed, the enriched lysate was subjected to the same plaque assay process as the impure lysate, and a plaque count of 5.4 × 10 was obtained, approximately 1 log higher than the impure lysate. 12 A titer of PFU / mL ±11% was obtained.

[0128] During optimization of the enrichment process for phage K, it was found that high acceleration and deceleration within the centrifugation process can result in mechanical and shear stress on the phages, and at higher centrifugation speeds, the enriched lysates had lower titer values ​​than the optimized method shown above. TEM analysis of these enriched solutions showed that some of the phage virions had missing or fractured heads, causing premature release of genetic material. Decreasing the centrifugation speed and removing the auxiliary deceleration along the optimized enrichment process shown above was found to alleviate the mechanical and shear stress on the phages. TEM analysis of these samples showed that they contained very few destroyed or spent phages and a large amount of intact virions (see Figure 4).

[0129] MS2 Although the concentration and purification methods developed for bacteriophage K could easily be applied to MS2, the above methods resulted in a volume of lysate with a lower titer compared to the phage K results. To further increase the titer values ​​of the phage lysate, a larger amount of PEG 6000 was added to the concentration step of MS2 compared to phage K (i.e., 10% w / w instead of 7% w / w). This may be because MS2 is smaller and therefore higher levels of PEG are more effective at salting or crystallizing the virions out of solution. Additionally, a more severe centrifugation step was used for MS2 after PEG concentration compared to phage K. Performing a longer and more severe centrifugation step is believed to be less damaging due to the simpler structure of MS2. After this optimization, the plaque assay of the lysate resulting from these two modifications yielded a 3.50×10 10 PFU / ml. TEM analysis of concentrated MS2 showed abundant globular mass with no signs of spent or disrupted phage capsids.

[0130] Further purification of MS2 using chloroform The concentrated MS2 lysates were further purified by mixing the concentrated MS2 lysates with an equal volume of chloroform and then vortexing to ensure that the two solutions were thoroughly mixed. This was followed by a low speed centrifugation step. Plaque assays and DLS analysis of these purified solutions were performed. The titers of these purified solutions were 3.7×10 7 PFU / ml. Thus, with the excessive purification steps, DLS analysis showed very minimal increase in purity, indicating that a second or further purification step is not necessary.

[0131] Silica encapsulation / embedding of phage K Using a 1:100 ratio of prehydrolyzed silica to buffer, concentrated phage samples were silica-embedded for 10 min, then vacuum filtered through glass microfiber paper and air-dried for >48 h. A schematic of silica encapsulation is shown in Figure 1. 9Batch silica embedding of 10 mL of concentrated phage lysate at PFU / mL typically yielded 159.3 mg ± 55.6 mg (2 × SD) of silica-embedded powder. BCA protein assays showed that 1 mL of phage lysate of the same titer yielded 1.33 mg / mL protein concentration.

[0132] In addition to the required concentration step, the method itself was also further modified to improve the encapsulation process compared to silica encapsulation previously used for small polypeptides. A higher pH of 7.5 was successfully used as a replacement of the Tris buffer with a sterile SM buffer for phage K, or a modified SM buffer containing glycine for phage MS2. NaCl and MgSO4 salts form the main components of the SM buffer.

[0133] The collected images of silica-embedded phage K showed a highly networked structure consisting of individual spheres (see FIG. 5). The spheres themselves had a larger median diameter than previous silica-encapsulated biomolecules. The average particle size was found to have a median of approximately 400 nm as determined by FE-SEM imaging. Furthermore, the visible surface of the spheres appeared to be much smoother than expected. The silica shell was also found to be porous (see further discussion below).

[0134] Increasing pH was found to be a contributing factor to the rate of silica embedding. Investigation of the effect of pH on the rate of silica embedding showed that increasing pH resulted in an increase in the reaction rate until pH 9.0 was reached, where the net charge of the solution prevented the formation of silica monomers required for the reaction (see Figure 6). The increased solubility of silica at higher pH is also believed to contribute to the particles growing to a larger size. In addition, it was found that the buffer composition used for silica embedding is important, and the choice of buffer affects the recovery of phage. Double cationic salts (e.g., Mg 2+ or Ca 2+It is believed that the addition of salts (including SM buffer) is important for the growth of bacteriophages, and the absence of these salts reduced the titer of the grown phage solution. Furthermore, it is believed that the presence of sodium salts may contribute to the stability of the liquid lysate. In particular, sodium salts can prevent aggregation and the relief of hydrostatic pressure that can be fatal to virus or phage capsids. The presence of sodium ions was found to promote particle growth and make the final silica nanoparticles larger in size after 10 minutes compared to those created in the absence of sodium ions. Overall, it was found that the addition of salts at a mildly basic pH (e.g., SM buffer) creates an environment more favorable for particle size growth than rapid aggregation and precipitation.

[0135] The concentration of phage in the lysate also appears to be important. >1×10 7 Phage lysate used for silica embedding in PFU / mL was found to contribute to more successful silica embedding and release, resulting in the highest residual release titers.

[0136] It was also found that a particular ratio of phage lysate to pre-hydrolyzed silica was optimal: for higher ratios of lysate to silica, the silica embedding process appears to be less uniform, resulting in most phage virions remaining more or less intact, while other virions are completely silica-embedded.

[0137] Silica embedding of MS2 Silica embedding of MS2 was modified from that described for phage K in that a modified SM buffer was used replacing 50 mM Trizma base with 50 mM glycine. This was found to have improved the electrostatic charge between the silica and the phage and facilitated the silica embedding process. Silica embedding with this buffer produced turbidity after a few minutes of stirring and numerous silica nanoparticles were produced after a few minutes of stirring. Silica embedding of MS2 using SM(G) buffer produced an average of 146.25 mg (± 3.46%) of powder. FE-SEM imaging of the powder is shown in Figure 7. The appearance of the particles is similar to that seen for phage K. Individual spherical particles form chains that unite into a distinctive silica network. Individual spheres are 300-500 nm in diameter and the morphology of the spheres is less uniform than phage K, with some particles having an ellipsoidal shape. The surface of the particles is also different compared to phage K particles. MS2 shows a significantly more porous appearance compared to phage K.

[0138] BCA assay of MS2 powder revealed that the powder contained 17.5±1.9% protein and 82.5±1.9% silica.

[0139] Release of bacteriophage K from silica shells. HF emission A low concentration hydrogen fluoride buffer can be used to dissolve the silica shell surrounding the phage without causing significant damage to the product.

[0140] Bacteriophage K was found to be more sensitive to mild acidity than previously tested proteins, and therefore the concentration of NaF / HF buffer used for release was optimized.

[0141] Since the concentration of HF used in the protein test is 4 mg / mL, the 7.91x dilution gives a tolerance of the phage to 0.51 mg / mL HF. Increasing the acid concentration above this value resulted in the disruption of the phage ultrastructure.

[0142] However, the silica that coats the phage is preferentially digested by the acid. This means that the concentration of the release solution must contain enough acid to completely dissolve the silica shell with an excessive concentration of less than 0.51 mg / mL. Table 1 shows the experimental results of sample release of silica-embedded phage K with various concentrations of HF and time scales of bacterial incubation. 9 The use of 2 mg / mL HF at a release of approximately 10 mg PFU / mL silica-embedded phage was the highest concentration of HF tolerated by the silica-embedded phage. The use of higher concentrations of HF or lower titer powders resulted in the death of the silica-embedded phage.

[0143] [Table 1]

[0144] The above results suggest that the released samples can be used to form plaque forming units even after silica embedding and subsequent release. The most successful released samples show approximately 6 log loss of plaque forming units. This reduction seems large, but importantly, it poses little overall problem for the success of the bacteriophage. 8This is because only a small number of phages are needed to grow a phage population back to high titer values ​​or higher. The number of viable phage in the silica-embedded and released bacteriophage K powder is therefore more than sufficient for this regrowth to occur. One of these was sample 9, which was stored at ambient conditions for its 18 month (day) lifespan, with temperatures ranging from approximately 10 to 35°C. Images of this plaque assay are shown in Figure 8 below. The presence of a dark spot on a light lawn corresponds to a single "plaque forming unit" or phage virion. Counting these individual spots and factoring in sample volume and dilution can give a value for infectious phage particles in a given volume, quoted as PFU / mL. The PFU / mL of native non-silica-embedded phage lysate can be compared to the PFU / mL obtained from silica-embedded and released phage to indicate phage viability during the process. Growth of new bacteriophage within bacterial host cultures from released silica-embedded samples indicates that the virulence of silica-embedded phages can be stored.

[0145] For the silica-embedded MS2 phage, lower levels of HF were also used to release the particles. HF concentrations of 1.5 and 3.0 mg / mL were used to release approximately 10 mg of silica-embedded MS2 for 1 hour. 4 mL of each release was then added to 40 mL of 1-hour-old E. coli and incubated. Sampling was performed at 5 and 24 hours of incubation time. Each sample was then plaque assayed as a standard. All samples harvested produced relatively high concentrations of plaques. Each spot showed either full or semi-confluent (see Figure 10). Although the exact value of the titer cannot be established from the images obtained, it is clear that MS2 is significantly easier to release than silica-embedded phage K and therefore can be replicated to much higher titers.

[0146] HF-free emission The release of silica-embedded MS2 shows one major difference when compared to other silica-embedded targets. When added to a mature bacterial host culture in liquid suspension, the phage is able to replicate without prior removal of the silica shell using hydrofluoric acid. A small chunk (40 mg) of silica-embedded MS2 powder was added to 1 mL of TSBss and incubated for 1 h, then added to 400 mL of 1-h-old E. coli and incubated for an additional 24 h. These samples were then plaque assayed. The results show high titer values ​​for silica-embedded MS2, which does not use hydrofluoric acid to destroy the silica shell. These values ​​are in the same order of magnitude as native MS2, which is not natively silica-embedded.

[0147] The silica-embedded powder used was over 8 weeks old and had been stored at room temperature in Eppendorf vials. Although there were no natural controls stored for that period at the same temperatures used during this experiment, it can be said with confidence that the natural MS2 lysate would lose all viability after this period at 2-8°C, let alone about 20°C. As a result, it is clear that the MS2 growth from the silica-embedded powder cannot be due to phage adsorbed to the silica surface, but must result from the release of intact silica-embedded bacteriophage into the TSB and bacterial cultures.

[0148] Stability Testing Using a suitable release method, the protection that the silica coat provides to the phage was established by looking at encapsulated phage K.

[0149] Thermal stability characterization of native phage K showed that the phage cannot survive at temperatures above 70°C for 30 minutes. As a result, by heating silica-embedded phage at 90°C for 30 minutes and then releasing the silica-embedded phage, any plaques generated from the release would be purely silica-embedded, rather than silica-embedded and adsorbed to the surface of the silica particles. It would also show that silica-embedding provides improved thermal stability, as opposed to native liquid lysate. A sample of silica-embedded phage K was heated in an oven at 90°C for 30 minutes, and once cooled, the silica-embedded sample was released according to the most successful set of parameters, after which a plaque assay was performed alongside the heated native sample, the unheated native sample, and the unheated silica-embedded and released sample. The results are shown in the scanned image of the agar plate shown in Figure 9.

[0150] For MS2, silica-embedded phage was also stored at room temperature for several weeks. These conditions would remove all viability in a native liquid lysate. The reason is that the guidance provided by the ATCC points out that phage MS2 is in liquid suspension and a rapid loss of titer is expected as early as 2 days when kept at 2-8 °C. As a result, cryogenic freezing is required for MS2 storage. These results indicate that silica embedding of MS2 can protect MS2 against thermal degradation.

[0151] porous SYBR safe dyes and BCA test The use of fluorescent DNA dyes was used to determine whether the silica shell of silica-embedded phage K was porous enough to allow small to medium sized molecules to pass through the channels and interact with the virions enveloped inside. The high level of fluorescence detected upon addition of the dye to the silica-embedded phage would indicate that the dye was able to penetrate the silica shell and successfully bind to either the DNA inside the phage capsid or the free DNA in solution. However, SYBR Safe dyes are not specific and may also result in false positives from contaminating DNA in the solution, e.g. bacterial DNA left over from growth. Testing of phage K powder with SYBR Safe dyes is summarized in Table 2 below.

[0152] Table 2: Table showing the results of fluorescence measurements of native and silica-embedded phage K samples with SYBR Safe DNA dye. Vortexed phage K is native concentrated lysate that was vigorously vortexed to encourage the release of phage DNA. [Table 2]

[0153] The results show that when mixed with SYBR Safe dye, silica-embedded phage exhibits comparable levels of fluorescence to native phage lysates, 21,921 AU versus 37,445 AU, respectively. Despite the relatively high uncertainty values ​​of the fluorescence readings, there is a significant difference between the results of silica-embedded phage mixed with SYBR Safe and those of silica-embedded phage without added dye, indicating that the silica powder itself contributes very little to the fluorescence signal. Therefore, we can confidently say that the silica shell is porous enough to allow SYBR Safe molecules to penetrate and interact with the DNA contained inside.

[0154] BCA assay Similar to the use of SYBR safe dye, the copper ions and the necessary potassium tartrate in the BCA mixture should be able to migrate through the pore channels of the silica coat and interact with the protein peptide backbone and amino acid residues to produce the light blue and light purple complexes characteristic of the reaction. Results from the BCA assay of silica-embedded phage K powder are shown in Table 3. This information can be further strengthened by performing similar tests using BCA reagents instead of SYBR safe dye.

[0155] Table 3: Table showing the results of absorbance measurements of the BCA analysis of native and silica-embedded phage K material. [Table 3]

[0156] The results obtained from the BCA analysis of the silica-embedded phage powders show that the absorbance values ​​of the silica-embedded samples are much lower than those of the native or released phage (1.70 or 1.23 mg / mL), but still significantly higher than the buffer value (0.11 mg / mL). The value of the powder without added BCA reagent shows the absorbance value due to the powder itself. This value is much lower than the silica-embedded samples, again indicating that the silica shell is porous enough to allow the necessary reagents to traverse the pore structure and generate the chelated copper complexes required for the analysis. This data reinforces the data collected from the SYBR safe dye analysis and proves that the silica shell is porous enough to allow the passage of small / medium sized molecules to the virions.

[0157] Nitrogen BET Three silica-embedded phage samples were also tested, each embedded in silica using different pre-hydrolyzed silica:lysate ratios, 1:1, 1:10, and 1:40. Using the BET isotherm the surface area of ​​the material can be calculated and these values ​​can be compared between samples to show how porous they are. Further analysis using the Barrett-Joyner-Halenda (BJH) isotherm allows for a map of the pore size distribution which gives a value for the number of pores of a particular size. The BET values ​​of the samples are shown in Table 4.

[0158] Table 4: Surface area of ​​native and silica-embedded phage K material. [Table 4]

[0159] The surface area of ​​the samples shows several important results: the surface area of ​​all phage samples is larger than that of previously encapsulated materials, such as lysozyme.

[0160] The BJH isotherms for the 1:10 and 1:40 samples are shown in Figure 12. These graphs show that for the silica-embedded phage samples, the overwhelming majority of the pores in the samples are below 10 nm in diameter. In the range of 0-10 nm, the pores are distributed in very tight clusters, producing a sharp peak that indicates a very small range of diameters for these pore clusters.

[0161] Between 10 and 20 nm there is only one peak that is much broader than the others, indicating a more even distribution of pore sizes. Above 20 nm there is almost no pore volume in the phage sample.

[0162] Visualization of silica encapsulation by TEM The large size of phage K allows the use of electron microscopy to view silica growth over the length of a silica encapsulation or embedding experiment. The silica embedding method used for the visualization experiments differs slightly from the silica embedding method discussed above, because the process described above generates particulates very rapidly and is not amenable to sampling to show differences in particle growth.

[0163] The differences used were aimed at slowing down the rate of the silica embedding process. In particular, the pH of the reaction was lowered from 7.5 to 6.00. In addition to lowering the pH, the reaction was performed in an ice bath to keep the temperature at a level that further slowed down the rate of the reaction, also providing an environment that promotes phage survival. We chose time points used to sample the modified silica embedding process that do not coincide with the normal process times to facilitate visualization.

[0164] Samples were taken from the modified silica embedding batch process at various time points, blotted and stained onto carbon grids to stop the reaction and provide a freeze frame of the sample at that particular silica embedding time point.

[0165] The images obtained from viewing each sample at time points throughout the silica embedding process clearly showed the initial formation and growth of the silica coat on the phage virions, as well as the incorporation of the virions into the silica network that is unique to the sol-gel process. Furthermore, these images have measurable size differences around the main structural components of the phage molecule, providing visible evidence of the initial growth sites of silica on molecules and regions that it prefers. This provides strong indications for our earlier hypothesis that silica is attracted to positively charged regions of the target's surface during silica embedding, and that after that initial step, it grows and spreads along the surface of the phage.

[0166] Each image collected from the TEM analysis of the silica-embedded samples was analyzed using ImageJ software. The cardinal points of the phage virion were measured to allow for comparison of the physical size of the virions throughout the silica embedding process. These cardinal points were head diameter bottom to top, head diameter left to right, average tail width over the tail length from at least five measurements, and tail length from bottom of head to top of base if the virion exhibited a straight tail.

[0167] Main characteristic measurements Measurements of tail width and head diameter were collected together and averaged per time point. The number of measurements was based on the number of clearly visible virions at each time point. Measurements of tail width consisted of 15 at 1 and 2 min, 25 at native and 5 min, and 10 at the 7 min time point. Measurements of head diameter consisted of 18 at 1 and 2 min, 30 at 5 and native, and 12 at 7 min. The error of the size measurements obtained was ±2 standard deviations at each time point and is quoted as a percentage of the mean value. This data is shown in Table 2 and graphically represented in Figure 13. The standard deviation of the tail width increases significantly with increasing silica growth. This is expected because silica growth over the tail length forms specific localized nodule growth and regular deposition of silica over the entire length of the tail, resulting in different measurement widths depending on where the measurements are taken.

[0168] Images captured at each time point show clear differences in the morphology and appearance of the silica surrounding the virions at the first stage of silica embedding. Figures 14-17 below show TEM images of bacteriophage K through various stages of silica embedding. The main characteristic measurements are shown in red and annotated with the associated size (nm). Silica nodule growth is highlighted in yellow in each image. Even in samples taken at 1 minute of silica embedding, the differences from the native sample are evident. Figure 14 shows some virions after 1 minute of silica embedding. The phage ultrastructure is still largely visible, but the silica in the solution has begun to blur the image details. The beginnings of some silica growth can be discerned, but were not yet prominent enough to affect the physical measurements. The beginnings of nodule growth (especially around the base of the virion) are more evident in these images.

[0169] Figure 15 shows TEM images collected 2 minutes after silica embedding. The initial silica growths faintly visible along the length of the phage tail and across the head in Figure 10 are much larger and easily discernible at this point. In addition to the increase in nodule growth size, the overall size of the silica coat at 2 minutes is noticeably larger within the measurements of phage structure (Table 2). At this point, there is an average difference of 18.94% in the width of the tail coat and 6.94% in the diameter of the head coat between virions measured at 1 and 2 minutes.

[0170] These measurements also increase in images taken 5 minutes after silica embedding, but at a lower rate, with the virion average size seeing an increase of 13.56% and 2.90% in tail coat width and head coat diameter, respectively. However, both of these increases are smaller than the measurement error (see Figure 16). Virions observable in images captured after 7 minutes show an increase in growth between the 1-2 minute and 2-5 minute images, with an increase of 22.07% and 4.25% in tail coat width and head coat diameter, respectively (see Figure 17). Due to imaging issues, the increasingly thick silica gel formation in the solution measurements after 7 minutes was not recorded.

[0171] Silica growth preference The base of the phage molecule appears to be preferentially favored by the silica monomers compared to the rest of the phage. The base assembly shows large clumps of silica deposited after only 2 minutes. These clumps of silica formed on the phage base appear to be the beginning of a silica gel network that is unique to the sol-gel process used in silica embedding. These networks entrap the phage virions at the head and base and then cover the entire virion. Comparing the data shown in Table 5.1 together with the captured images, it is easy to see that the silica coat shows growth over the experiment, not only in a uniform layer around the phage virions but also in irregular nodular growth especially around the base and head groups of the phage. The collected images show that silica embedding is a very gentle process with uniform deposition of silica throughout the phage body as well as large non-uniform accumulation of silica from several sites on the phage body and head surface. This non-uniform growth spreads from the initial nucleation site on the phage body and spins to form the silica network that is unique to the modified sol-gel process. Each silica sphere may contain one or several silica-embedded phages. This is reinforced by what is shown in Figure 18, which shows an ITEM image of phage K 7 minutes after silica embedding. The contours show that two different individual phage virions are incorporated into the same silica cluster network.

[0172] The end result of the silica embedding process under standard silica embedding described above after 10 min at the standard speed of silica embedding is a collection of smooth network-like spheroids or spheres of about 400-500 nm in diameter, the electron micrograph of which is shown in Figure 5. This figure was collected after application of the standard phage-silica embedding as opposed to the modified phage-silica embedding used during the visualization process.

[0173] Results of porosity tests of silica-embedded phage powders indicate that silica-embedded bacteriophages are more porous than some previous biopharmaceutical targets, such as lysozyme.

[0174] conclusion It was found that silica-embedded bacteriophage were able to produce plaques even when stored at ambient temperature for much longer periods than native phage lysates. This indicates that silica-embedded bacteriophage are suitable for long-term ambient temperature storage, where native liquid phage lysates would otherwise degrade. This is reinforced by the fact that silica-embedded phage powders are extremely resilient to high temperatures and do not lose any additional infectivity compared to non-heated silica-embedded powders. TEM images collected from visualization of silica embedding of phage K indicate that the process is extremely gentle, allowing structures as complex as tailed phage, including genetic material, to survive intact. The deposited silica appears to have no or minimal impact on the structural integrity of the virus.

[0175] To the best of our knowledge, this is the first time that a similar silica encapsulation process has been applied to a biomolecule as large and complex as an intact virion, without the presence of intermediate polycationic structures. Moreover, the resulting particles were found to have different properties than previous silica-embedded proteins or polypeptides. The particles had a much larger median diameter, while also being more porous.

[0176] The results obtained show that silica embedding is suitable for application to a wide range of viruses, including both i) bacteriophage K, a DNA bacteriophage virus with a highly complex structure, and ii) MS2, an RNA bacteriophage virus with a less complex structure. MS2 has a large structure similar to many mammalian viruses, and therefore this study also serves as a proof of concept that these methods can be used for mammalian viruses, especially other simple, spherical viruses such as influenza and rotaviruses.

[0177] In particular, the inventors have found that concentration of the viral lysate is necessary prior to silica embedding. Although concentration methods using PEG 6000 or chloroform are described above, the inventors believe that different purification or concentration methods may be used prior to silica embedding. The inventors have also found that certain parameters of the concentration and / or silica embedding method may result in the highest viable release titers.

[0178] Phage K may be considered infectious and preserved in infectivity even when subjected to silica embedding and release. Fresh phage stocks can be grown indefinitely from the released phage solution. The virus can replicate and replenish its virion levels, so silica embedding does no permanent damage to the infectivity of the virus when applied to a pathogenic target. Furthermore, the bacteriophage was silica-embedded and stored at room temperature for extended periods of time while retaining its infectivity. Over the course of the test, phage K showed about a 4 log reduction in concentration. Given how unstable the phage is at room temperature, both silica-embedded phages showed much greater resilience to extreme temperatures. No further reduction in titer was observed after heating at 90°C for 30 minutes. This indicates improved survival of the phage at room temperature compared to the native lysate, and indicates that the silica-embedded samples can survive for extended periods of use without refrigeration or cold chain.

[0179] Supplementary Information Details of materials and characterization methods Tryptic soy agar (TSA) from Sigma-Aldrich contains 15 g / L agar, 15 g / L casein peptone (pancreatic), 5 g / L sodium chloride, 5 g / L soy peptone (papainic). Mix with ddH2O and autoclave at 121°C for 15 minutes.

[0180] Tryptic soy broth (TSB) from Sigma-Aldrich contains casein peptone (pancreas) 17 g / L, dipotassium phosphate 2.5 g / L, glucose 2.5 g / L, sodium chloride 5 g / L, soy peptone (papain digest) 3 g / L. Prepare with ddH2O and autoclave at 121°C for 15 min.

[0181] Tryptic soy broth with salts (TSBss) from Sigma-Aldrich contains casein peptone (pancreas) 17g / L, dipotassium phosphate 2.5g / L, glucose 2.5g / L, sodium chloride 5g / L, soy peptone (papain digest) g / L. Also contains NaCl 5.7g / L, MgSO4.7H2O 2.09g / L. Made with ddH2O and autoclaved at 121°C for 15 min.

[0182] PEG-6000 was purchased from VWR. NaCl, MgSO4.7H2O, gelatin, and glycine were purchased from Fisher Scientific. Bacteriophage K and host Staphylococcus aureus H560 were provided by Dr. S. Milo and Professor A. Jenkins, University of Bath.

[0183] Saline magnesium buffer (SM buffer) contains NaCl 5.7g / L, MgSO4.7H2O 2.09g / L, Trizma base 7.88g / L, and gelatin 0.1g / L. pH 7.5 with HCl, mixed with ddH2O, and autoclaved at 121℃ for 15 minutes.

[0184] Normal saline magnesium buffer with glycine (SM(G) buffer) contains NaCl 5.7g / L, MgSO4.7H2O 2.09g / L, glycine 3.75g / L, and gelatin 0.1g / L. pH 7.5 with HCl, prepared with ddH2O, and autoclaved at 121℃ for 15 minutes.

[0185] Tris buffer contained 7.88 g / L Trizma base, prepared with ddH2, and autoclaved at 121°C for 15 min.

[0186] Bacteriophage MS2 lyophilized lysate - obtained from ATCC, product number 15597-B1, batch number 70008569

[0187] Other materials: UA Zero Stain -Agar Scientific 200 Mesh, Copper Supported Carbon / Formva Electron Microscopy Grids -Agar Scientific Sodium Chloride - Fisher Scientific UK Magnesium Sulfate Heptahydrate - Fisher Scientific UK Trizma Base - Sigma Aldrich Type B gelatin from bovine skin - Sigma Aldrich Hydrochloric acid (32%) - Sigma Aldrich Polyethylene glycol M / W 6000 (PEG-6000) - Sigma Aldrich Tetraethyl orthosilicate - Sigma Aldrich Sodium Fluoride - Sigma Aldrich Glycine - Fisher Scientific UK

[0188] Visualization of silica embedding by TEM Physiological magnesium saline (SM) buffer was adjusted to pH 6. A 1:100 ratio of prehydrolyzed silica to this SM buffer was used. 1000 μL of concentrated 0.22 μm filtered phage K lysate was transferred to a 1.5 mL Eppendorf vial. To this was added 100 μL of prehydrolyzed silica and a timer was started. The Eppendorf vial was gently inverted. After 45 seconds, a sample was taken and immediately blotted onto a freshly glow discharged carbon grid. One to two drops of 1% uranyl acetate stain were used. The entire sampling process took approximately 15 seconds, meaning that each sample was a 60 second silica embedding time. Multiple samples were taken from the same silica embedding run and analyzed using a Tecnai 12 TEM (Wolfson Bio-imaging Facility, University of Bristol) equipped with a BioTwin Spirit objective system at 120 kV accelerating voltage and an FEI Eagle 4k × 4k CCD camera. If it was necessary to obtain a more extensive image, several acquisitions were repeated.

[0189] TEM Using freshly glow-discharged carbon grids, a small volume of lysate (5–10 μL) was pipetted onto the grid. The sample was allowed to sit on the grid for 30 s, after which excess liquid was blotted using filter paper. The grid was washed twice with ddH2O and excess water blotted as before. Two drops of 1% uranyl acetate solution or UAZero Stain (Agar Scientific) were added to the grid and allowed to sit for 30 s, after which excess liquid was carefully blotted with filter paper. The grid was then allowed to air dry for approximately 3 h before use.

[0190] The TEM used was a JEOL / EO JEM-2100+ equipped with a LaB6 electron gun and an accelerating voltage of 120 kV (Materials Analysis Suite, University of Bath). The grids used were Agar Scientific 200 mesh formva / carbon copper backing grids. The TEM used was a Tecnai 12 TEM equipped with a BioTwin Spirit objective lens system at 120 kV accelerating voltage and an FEI Eagle 4k×4k CCD camera (Wolfson Bio-imaging Facility, University of Bristol). The grids used were Agar Scientific 200 mesh formva / carbon copper backing grids.

[0191] FE-SEM A fresh carbon / mica film was glued to the SEM sample holder. The dried silica embedding powder was ground to the appropriate size using a mortar and pestle and then spread onto the film. The samples and holder were stored under vacuum until use. Just prior to analysis, each sample was sputter coated with a 20 μm layer of chromium. The sputter coating used was a Quorum 150VS Plus and the SEM instrument used was a JEOL FESEM6301F equipped with a field emission electron gun and using an accelerating voltage of 5 kV (University of Bath, Materials Analysis Suite).

[0192] Dynamic Light Scattering All samples used were filtered through 0.22 μm Millex syringe filters into disposable polystyrene microcuvettes that were thoroughly rinsed with both ddH2O and sample. Samples were analyzed using a 173° measurement angle, each time at standard temperature and pressure, using various sampling times and replicates. The instrument used was a Malvern Panalytical Zetasizer Nano ZS equipped with a 633 nm He-Ne laser.

[0193] DLS analysis was used to determine the monodispersity of the lysates. The Malvern Panalyticals Zetasizer software used a metric called the "polydispersity index" (PDI) that indicates the purity of a sample. Values ​​closer to 0 are pure, whereas values ​​closer to 1 are impure and a mixture of many different particle types.

[0194] Bicinchoninic acid (BCA) assay A 96-well microtiter plate is used. 100 μL of 0.2 M NaOH was added to 100 μL of 2 mg / mL bovine serum albumin (BSA) and mixed well. This was then diluted to produce 0.8, 0.6, 0.4, 0.2, and 0 mg / mL BCA solutions in 0.1 M NaOH. 10 μL of these dilutions were then pipetted into the microtiter plate to produce a calibration series spanning 1.0 to 0 mg / mL BSA. Protein solutions to be analyzed were pipetted either undiluted or diluted with 0.1 M NaOH to yield concentrations in the range of the BSA calibration series depending on the estimated concentration of the protein solution.

[0195] The BCA solution was prepared by mixing BCA Reagent A and BCA Reagent B in a 50:1 ratio, then pipetting 200 μL of this mixture into each well of a 96-well plate. The plate was then covered and incubated at 37° C. for 30 min before being analyzed with a microplate reader (PHERAstar FS, BMG LABTECH).

[0196] DNA fluorescence The DNA dye used was Invitrogen - SYBR Safe DNA Gel Stain. 10,000x concentrate in DMSO. Batch no. 1988947 samples were pH adjusted using either HCl or NaOH to a pH of 7-8. 900μL of sample was pipetted into an Eppendorf vial. SYBR Safe dye was diluted twice using 10μL of dye in 990μL MilliQ water followed by 100μL of this dilution in 900μL MilliQ water to reach a final dilution of 1,000x. 100μL of this dilution was added to the Eppendorf vial containing the sample. After gently inverting the vial several times, 200μL was pipetted into a black plastic 96-well plate. These 96-well plates were incubated at 37°C for 5 min before being analyzed using a microplate reader (PHERAstar FS, BMG LABTECH) at 509nm.

[0197] Sodium dodecyl sulfate (SDS) pulsed gel electrophoresis (PAGE) Phage samples were run using precast gradient gels (Bio-Rad Mini-PROTEAN TGX Precast 8-16%) with Novex Sharp unstained protein standards (Life Technologies, Thermo Fisher Scientific) as reference ladders in the left and right most wells. In case of gel warping, these ladders can be compared and results extrapolated. 20 μL of phage lysate sample was added to 10 μL of SDS-PAGE sample buffer and then incubated at 95°C for 10 min. The precast gel was fixed into an assembly cassette (Mini Protean3, Biorad) and electrophoresis buffer was added, after which the reference ladder and phage sample were pipetted into the sample lane. The cassette was run for 45 min at ≤200V. Once complete, the gel was removed from the cassette and stained with ~20 mL of Pageblue at room temperature with rocking, followed by destaining with ddH2O at room temperature with rocking. Gels were imaged in an Epi Chemi II Darkroom (UVP) equipped with a Hamamatsu C4742-98 digital camera using ClearLive software.

[0198] Differential Scanning Calorimetry (DSC) A 700 μL sample of concentrated native phage (e.g., phage K) was pipetted into a stainless steel cell and placed in the instrument along with a control containing blank SM buffer. The sample was heated from 20°C to 150°C in 1°C increments and then cooled back to 20°C.

[0199] Nitrogen physisorption, BET and BJH All samples were carefully measured in sample tubes and thoroughly degassed overnight. The equipment was leak tested before each sample analysis. The type of gas used was N2 at 77 K. The tolerances and equilibration times are shown in Table 5. The equipment used was a Quantachrome Anton Paar - Autosorb- iQ-C.

[0200] Table 5: Tolerances and equilibration times used for nitrogen physisorption analysis of silica-embedded bacteriophage [Table 5]

[0201] High temperature stability test 1 mL of phage lysate and 50 mg of silica-embedded phage were placed in a small vial. These were placed in a 90° C. oven and heated for 30 minutes. The silica-embedded samples were released as described above, and both the native and silica-embedded released samples were subjected to plaque assays.

[0202] Long-term stability testing 10 mL of phage lysate and 50 mg of silica-embedded phage were placed in small vials. These vials were left in ambient laboratory atmosphere. On alternate days, 100 μL of native lysate was taken, diluted in SM buffer, and plaque assayed at room temperature to determine phage reduction over time. When no more PFUs were detected from the phage, silica-embedded samples were released and plaque assayed to determine their titer.

[0203] pH characterization 100 mL of pH-adjusted SM buffer was used for each pH point, and the pH was adjusted using either HCl diluted in milli-q water or NaOH dissolved in milli-q water. 1 mL of concentrated phage lysate was added to each pH-adjusted buffer, and the mixture was stirred at 125 RPM for 30 minutes. Immediately after stirring, the mixture was diluted in pH 7.5 SM buffer to the standard plaque assay range (up to ×10 -9 ), and standard plaque assays were performed.

Claims

1. A particle comprising a virus encapsulated in an amorphous silica shell, the amorphous silica shell being deposited directly around the surface of the virus.

2. 10. The particles of claim 1, wherein the particles have a median diameter of at least 250 nm as determined by field emission scanning electron microscopy, optionally the particles have a median diameter of 250 nm to 1000 nm, or preferably 300 nm to 500 nm.

3. The particle of claim 1 , wherein the virus is capable of replicating in a host cell.

4. The particle of claim 1 , which does not contain polylysine.

5. 10. The particle of claim 1, wherein the silica shell has a spheroidal morphology.

6. 10. The particle of claim 1, wherein the virus encapsulated in the silica shell is protected from ambient conditions, optionally the ambient conditions being temperature or pH.

7. 2. The particle of claim 1, wherein the virus encapsulated in the silica shell is thermally stable, preferably the virus is stable after heating at 90°C for 30 minutes.

8. The particle of claim 1 , wherein the virus is a DNA virus or an RNA virus.

9. The particle of claim 1 , wherein the virus is a bacteriophage.

10. 10. The particle of claim 9, wherein the virus is (i) a Myoviridae virus, such as bacteriophage K, or (ii) a Fyasviridae virus, such as phage MS2.

11. The particle of claim 1 , wherein the virus is a mammalian virus.

12. 1. A method for producing a virus encapsulated in an amorphous silica shell, comprising: Concentrating or purifying the virus, suspending the virus in a buffer, Hydrolyzing a silica precursor, The method comprises contacting a hydrolyzed silica precursor directly with the surface of the virus in a buffer to encapsulate the virus in an amorphous silica shell.

13. 1 x 10 virus in buffer 7 Concentrations of greater than 1 x 10 PFU / ml, more preferably greater than 1 x 10 8 PFU / ml ~ approx. 1×10 10 13. The method of making the particles of claim 12, wherein the particles are present at a concentration of

14. 13. The method for producing the virus of claim 12, wherein the buffer comprises one or more salts including one or more of a magnesium salt, a calcium salt, or a sodium salt, and optionally the buffer is SM buffer, modified SM buffer, PBS, imidazole buffer, sucrose buffer, or bis-tris buffer.

15. 13. A method of making the particles of claim 12, wherein the enrichment or purification is by PEG-based enrichment.

16. 13. A method of making the particles of claim 12, wherein the virus in the buffer has a polydispersity index (PDI) of less than 0.2, more preferably less than 0.

15.

17. 13. A method for making particles according to claim 12, wherein the silica precursor is a tetra-alkyl orthosilicate, preferably tetra-ethyl orthosilicate (TEOS).

18. 13. The method of making particles according to claim 12, wherein the ratio of hydrolyzed silica to virus in the buffer is about 1:75 to 1:150, for example about 1:

100.

19. 13. A method for making particles according to claim 12, wherein the contacting is carried out for at least 5 minutes, preferably at least 10 minutes, and / or the contacting is carried out at a pH above 7, preferably between pH 7.25 and 8.5, for example at a pH of about 7.

5.

20. 13. Use of the method according to claim 12, wherein the method is used for storing or preserving a virus.

21. 20. A particle formed by the method of any one of claims 12 to 19.

22. A dry powder comprising the particles of claim 1.

23. 23. A pharmaceutical composition comprising: (i) one or more particles according to any one of claims 1 to 11, and / or a dry powder according to claim 22; or (ii) at least two particles according to any one of claims 1 to 11, wherein the at least two particles comprise different viruses.

24. 23. A particle according to any one of claims 1 to 11, a dry powder according to claim 22 or a pharmaceutical composition comprising a particle according to any one of claims 1 to 11 or a dry powder according to claim 22 for use in therapy, preferably for use as an antibacterial medicament, wherein the virus is a bacteriophage.

25. 23. Non-therapeutic use of a particle according to any one of claims 1 to 11, a dry powder according to claim 22, or a pharmaceutical composition comprising a particle according to any one of claims 1 to 11 or a dry powder according to claim 22, for use as an antimicrobial, wherein the virus is a bacteriophage.

Images