EA044566B1 - METHODS FOR TREATING INFECTIONS USING BACTERIA - Google Patents

Description

This application claims priority under 35 US Code §119 to US Provisional Patent Application No. 62/737,762, filed September 27, 2018, the contents of which are incorporated herein by reference in their entirety.

State of the art

For viral infections such as hepatitis B (HBV) and human immunodeficiency virus (HIV), existing therapies can control viral replication, improve clinical status in the majority of treated patients, and lead to reduced mortality and morbidity. However, antiviral therapy is hampered by the resumption of viremia after cessation of treatment and the emergence of mutations leading to drug resistance. Most patients require lifelong treatment, and cure from chronic HBV or HIV infection is rarely achieved.

Cytokines and chemokines are known to play an important role in host defense against a wide range of viral infections, including hepatitis and HIV. Interferon-alpha (IFN-α) is approved for the treatment of hepatitis B infection, and several additional cytokines, including interferon-gamma (IFN-γ), interleukin12 (IL-12), interleukin-23 (IL-23), GM-CSF ( granulocyte-macrophage colony-stimulating factor) and tumor necrosis factor-alpha (TNF-α) are involved in host defense against or treatment of viral infections such as hepatitis and HIV.

Prophylactic vaccination against pathogens requires the provision of an antigenic determinant from the pathogen and an adjuvant that provides immune danger signals or their downstream effectors necessary to activate an immune response against the pathogen antigen. Therapeutic vaccines designed to treat a pre-existing infection depend on host recognition of antigenic determinants of the pathogen in an existing infection and also require adjuvants or their downstream effectors to mediate danger signal-mediated immune activation. In some cases, it is possible to enhance therapeutic vaccines by providing antigens derived from exogenous pathogens. Immune cells of both the innate and adaptive immune systems use pattern recognition receptors (PRRs) to perceive danger in the form of pathogen-associated molecular patterns (PAMPs). The best known family of PRRs consists of toll-like receptors (TLRs), found on virtually all immune cells. These receptors (TLR 1, 2, 3, 4, 5, 6, 7, 8, and 9) respond to products found in many different types of pathogens, including bacteria and viruses. Activation of TLR receptors leads to both direct and indirect activation of immune cell function and immune responses. Direct activation occurs by stimulating cell maturation, proliferation, and differentiation, whereas indirect activation occurs by inducing the secretion of cytokines and chemokines. A tenth TLR (TLR10) may act as a negative regulatory effector of immune function.

Because of the role of TLRs in host-mediated immune responses against pathogens, significant efforts have been made to develop TLR agonist adjuvants and therapeutics for the treatment of infections. A wide range of monospecific, purified, or synthetic TLR agonists have been prepared and tested in preclinical and clinical settings. TLR agonists are used as adjuvants in prophylactic vaccines. However, although activity against pathogens has been observed in therapeutic vaccine settings, these efforts have encountered significant challenges. Problems encountered included both lack of efficacy and excessive toxicity, indicating the need for further improvements in the prevention and, in particular, treatment of existing infections with TLR agonists.

Brief description of the invention

Due to the need to activate both the innate and adaptive immune responses for optimal immune defense, and due to the fact that pathogens contain multiple TLR agonists and other danger signaling components, it is proposed that for optimal anti-infective, including Antiviral therapy requires a therapeutic approach using multiple TLR agonists. Gram-negative bacteria are known to contain several TLR agonists and induce significant TLR agonist-associated immune responses, including cytokine secretion. However, wild-type Gram-negative bacteria that contain high levels of the TLR-4 agonist lipopolysaccharide (LPS) or endotoxin are highly toxic when administered intravenously, mainly due to the induction of excessive cytokine secretion by immune cells. Gram-negative bacteria also contain TLR 1/2, 5, and 9 agonists, which may contribute to both immune system stimulation and systemic toxicity.

The present invention is based on the surprising and unexpected discovery that treating Gram-negative bacteria to reduce their LPS-associated endotoxin activity, for example with polymyxin and glutaraldehyde, which can kill the bacteria, keep the bacteria intact and significantly reduce LPS levels, can at the same time increase the ability of bacteria to induce the secretion of cytokines by human immune cells. This is surprising, at least because it was expected that a significant reduction in LPS should also lead to a significant reduction in the amount of each of several cytokines released by immune cells exposed to treated bacteria compared with untreated wild-type bacteria.

Unexpectedly, as shown in table. 1, treated bacteria induced higher levels of 8 of 9 cytokines secreted by human immune cells compared to untreated wild-type bacteria when tested at the same concentrations of (untreated and treated) bacteria, despite the fact that treated bacteria only had 4 .94% of the LPS level present in untreated bacteria. Moreover, despite the significant reduction in LPS, treated bacteria induced higher levels of cytokines than monospecific TLR agonists (Table 2). The reduction in LPS-associated endotoxin activity resulting from treatment, which can be approximately 75-99% when measured using the Limulus amebocyte lysate (LAL) assay in vitro compared to untreated wild-type bacteria, may result in reduced toxicity. Without being limited to any particular theory, it is believed that the intact structure of the treated bacterial cells may help prevent or minimize the release of free LPS into certain compartments of the host subject. The results suggest that treated bacteria may elicit a better immune response than untreated bacteria or monospecific agonists, with reduced systemic toxicity compared to untreated bacteria.

Animal studies have also confirmed that such treated bacteria can inhibit existing viral infections of both HBV and HIV. In addition, compared with the standard of care (eg, entecavir for HBV), the treated bacteria exhibited a sustained and significant inhibitory effect long after treatment stopped, even though the initial inhibitory effect may take longer to manifest. It is also interesting that even though treatment with NSAIDs (eg, indomethacin) alone did not show observable antiviral effects, combination with NSAIDs may synergistically increase the effectiveness of the treated bacteria.

Accordingly, in one embodiment of the present invention, there is provided a method of treating an infection in a patient in need thereof, comprising administering to said patient an effective amount of a composition comprising a plurality of intact and substantially nonviable gram-negative bacterial cells that have been treated in a manner that results in decreased activity lipopolysaccharide (LPS)-associated endotoxin by at least 75%.

In yet another embodiment, the present invention provides a method of preventing infection in a subject in need thereof, comprising administering to said subject an effective amount of a composition comprising a plurality of intact and substantially non-viable gram-negative bacterial cells that have been treated in a manner that results in decreased activity of lipopolysaccharide (LPS). LPS)-associated endotoxin at least 90%, and pathogen-derived or pathogen-associated antigen.

In one embodiment, the present invention provides a method for preventing or treating an infection in a patient in need thereof. The method involves administering to said patient an effective amount of a composition comprising a plurality of intact and substantially non-viable gram-negative bacterial cells that have been treated in such a manner as to result in a reduction in lipopolysaccharide (LPS)-associated endotoxin (active LPS) activity by approximately 75-99% when measured by the Limulus amebocyte lysate (LAL) assay compared with untreated wild-type Gram-negative bacterial cells.

In some embodiments, the composition contains from about 0.01 to 100 ng of active LPS per kg of patient body weight. In some embodiments, the composition contains from about 0.02 to 20 ng of active LPS per kg of patient body weight. In some embodiments, the composition contains from about 0.1 to 10 ng of active LPS per kg of patient body weight.

In some embodiments, the composition contains from about 2 to 200 ng of active LPS per 1x10 ⁸ cells. In some embodiments, the composition contains from about 10 to 120 ng of active LPS per 1x10 ⁸ cells. In some embodiments, the composition contains from about 20 to 100 ng of active LPS per 1x10 ⁸ cells.

In some embodiments, intact and substantially nonviable gram-negative bacterial cells have been treated in a manner that results in a reduction in the level of LPS-associated endotoxin by approximately 85-98%. In some embodiments, intact and substantially nonviable gram-negative bacterial cells have been treated in a manner that results in a reduction in the level of LPS-associated endotoxin by approximately 90-98%.

In some embodiments, the infection is a viral infection, for example, caused by a virus selected from table. A. In some embodiments, the viral infection is caused by hepatitis B virus (HBV) or human immunodeficiency virus (HIV).

In one embodiment, a pharmaceutical dosage form is also provided, with

- 2 044566 containing a plurality of intact and substantially non-viable gram-negative bacterial cells that have been treated in a manner that results in a reduction in lipopolysaccharide (LPS)-associated endotoxin activity by approximately 75-99% as measured by the Limulus amebocyte lysate (LAL) assay ) compared to untreated wild-type Gram-negative bacterial cells, where the total LPS-associated endotoxin activity is equivalent to about 0.7 ng to 7000 ng active LPS, preferably equivalent to about 7 ng to 1400 ng active LPS.

Other embodiments also describe therapeutic compositions, vaccines, and dosage forms containing them.

Brief description of graphic materials

In fig. 1A-1D show the effects of treatment with entecavir, treated bacteria (mock bacteria), or a combination thereof in inhibiting HBV DNA production in vivo.

In fig. Figure 2 shows that mock bacteria, but not entecavir, reduced HBeAg levels in vivo.

In fig. 3A-D show the results of a longer-term in vivo HBV DNA production inhibition study.

In fig. 4A-D show the results of inhibition of HBsAg expression in vivo.

In fig. 5A-D show the results of inhibition of HBeAg expression in vivo.

In fig. Figure 6 shows that the combination of indomethacin and mock bacteria (with or without entecavir) inhibited HBeAg expression in the liver of mice 27 weeks after cessation of treatment.

In fig. 7A-F show immunohistochemical analysis of HBcAg expression in the liver of mice 27 weeks after cessation of treatment.

In fig. 8A-C show inhibition of HIV blood levels by standard of care treatment or treated bacteria in humanized mice infected with HIV.

Detailed Description of the Invention

The following description presents illustrative embodiments of the present technology. However, it should be understood that such description is not intended to be limiting as to the scope of the present invention, but is instead provided as a description of illustrative embodiments.

As used herein, the following words, phrases and symbols generally have the meanings set forth below unless the context in which they are used indicates otherwise.

Compositions and methods for stimulating an immune response

Experimental examples of the present invention demonstrate that intact and nonviable gram-negative bacterial cells treated to significantly reduce LPS-associated endotoxin activity unexpectedly had an increased ability to induce cytokine secretion by immune cells. Thus, such treated bacterial cells are suitable for providing a safe and effective means for stimulating the immune response of a subject. The immune response may be directed against bacterial, fungal, parasitic or viral infections.

Thus, in accordance with one embodiment of the present invention, there is provided a method of stimulating an immune response in a subject in need thereof. In yet another embodiment, a method is provided for preventing or treating an infection in a patient in need thereof. In yet another embodiment, a method is provided for treating immunodeficiency in a patient in need thereof. In yet another embodiment, a method is provided for vaccinating a subject at risk of infection.

In some embodiments, the method comprises administering to the subject/patient an effective amount of a composition comprising a plurality of treated bacterial cells as disclosed herein. In some embodiments, the treated bacterial cells are intact and substantially nonviable gram-negative bacterial cells that have been treated to reduce lipopolysaccharide (LPS)-associated endotoxin activity and/or pyrogenicity.

Suitable bacterial organisms that can be used in the methods of the present invention are gram-negative and are derived from organisms that have LPS-associated endotoxin activity, such as wild-type organisms. The term gram-negative bacteria refers to bacteria that do not retain the original staining with a basic dye (such as crystal violet), which is part of the procedure known as Gram staining. In a typical Gram stain, cells are first fixed on a glass slide under heat and stained with a basic dye (such as crystal violet) that is absorbed by both Gram-negative and Gram-positive bacteria. The slides are then treated with a mordant (such as iodine for Gram stains) that binds to the base dye (such as crystal violet) and traps it in the cell. The cells are then washed with acetone or alcohol and then additionally stained with a second dye.

- 3 044566 of a different color (for example, safranin). Gram-positive organisms retain the original purple coloration, while Gram-negative organisms become discolored when washed with an organic solvent and therefore show additional coloration. Examples of Gram-negative bacteria include, but are not limited to, Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacteria spp., dtrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp. and Vibrio spp.

Gram-negative organisms include Enterobacteriaceae, a large family that, along with many harmless symbionts, includes many well-known pathogens such as Salmonella, E. coli, Yersinia pestis, Klebsiella and Shigella, Proteus, Enterobacter, Serratia and Citrobacter. Members of the family Enterobacteriaceae were named enterobacteriaceae because some of its members live in the intestines of animals.

In one embodiment, E. coli is selected as the organism. One particular strain under consideration is E. coli strain 2617-143-312, (Migula) Castellani and Chalmers (ATCC® 13070™). Additional is. coli strains that may be used include MG1655 (ATCC ® 47076) and KY8284 (ATCC® 21272).

The gram-negative organisms used in the methods of the present invention need not be recombinant organisms that contain or express DNA foreign to the wild-type form of the organism. However, in some embodiments, organisms may be modified to express certain non-native molecules, including, for example, pathogen antigens or immunostimulatory proteins.

The term lipopolysaccharide (LPS) refers to large molecules consisting of a lipid and a polysaccharide (glycophospholipid) joined by a covalent bond. LPS contains three parts: 1) O-antigen; 2) a central oligosaccharide and 3) lipid A. O-antigen is a repeating glycan polymer attached to a central oligosaccharide and contains the outermost domain of the LPS molecule. The central oligosaccharide attaches directly to lipid A and typically contains sugars such as heptose and 3-deoxy-O-mannooctulosonic acid (also known as KDO, ketodeoxyoctulosonate). Lipid A is a phosphorylated glucosamine disaccharide bound to several fatty acids. These fatty acids anchor LPS in the outer bacterial membrane, and the remaining LPS protrudes from the cell surface.

Endotoxin activity is inherent to the lipid A domain region of LPS and is thus also referred to as LPS-associated endotoxin activity. When bacterial cells are lysed by the immune system, membrane fragments containing LPS and lipid A are released into the circulation, causing fever (pyrogenicity) and potentially fatal shock (called endotoxic or septic shock). LPS toxicity is mediated by lipid A through interaction with cells of the mammalian immune system, a process leading to the secretion of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNFa) and interleukin-1-beta (IL-1e), which can have lethal consequences for the host.

LPS-associated endotoxin activity can be measured by methods well known in the art, including, for example, the Limulus amebocyte lysate (LAL) assay, which uses horseshoe crab blood and can detect very low levels of LPS. The presence of endotoxin activity will cause Limulus blood lysate to clot due to amplification through an enzymatic cascade. Gel clot, turbidimetric, and chromogenic forms of the LAL assay are commercially available.

Enzyme-linked immunosorbent assay (ELISA)-based endotoxin activity assays are also known, such as EndoLISA® from Hyglos, Munich, Germany. This assay uses an LPS-specific phage protein attached to a solid phase to capture LPS and, after a washing step, detects the presence of LPS by adding recombinant factor C which, when activated by LPS, cleaves the compound which then emits fluorescence. Factor C, present in Limulus amebocyte lysate, usually exists as a zymogen and is a primer for the coagulation cascade that occurs in the LAL test.

Pyrogenicity refers to the ability of an agent to cause fever in a subject. Pyrogenicity can be measured by the increase in rectal temperature in rabbits in response to intravenous administration of TLR agonists, organisms, or derivatives thereof.

Various methods are available to reduce endotoxin activity and/or pyrogenicity of gram-negative organisms. These methods involve treating the organisms with an agent that binds to LPS or disrupts its formation.

In one embodiment, reduction of endotoxin activity or pyrogenicity is achieved by treating the bacterial organisms with an endotoxin-inactivating antibiotic. A suitable such antibiotic is a polymyxin, including polymyxin B or polymyxin E. One skilled in the art will be able to determine the amount of antibiotic and the processing conditions. In one of

- 4 044566 embodiments The polymyxin, which is polymyxin B or polymyxin E, can be used at a concentration ranging from about 3 micrograms to 5000 micrograms per milliliter. In yet another embodiment, the concentration of polymyxin may be from about 200 micrograms to 5000 micrograms per milliliter. In one embodiment, the antibiotic is applied to the bacteria for 10 minutes to 4 hours, or about 30 minutes to about 3 hours.

In one embodiment, bacteria are grown in the presence of magnesium (Mg) in the form of MgCl ₂ . In one embodiment, the bacteria are treated with polymyxin in the presence of MgCl ₂ and at a temperature suitable to maintain the integrity of the bacteria. In one embodiment, the MgCl ₂ concentration in the growth medium is from about 0.5 mM to about 5.0 mM, or about 2 mM, and the MgCl ₂ concentration in the treatment medium is from about 5.0 mM to about 30 mM, or about 20 mM. In one embodiment, the temperature of the treatment environment is from about 2°C to about 10°C or about 4°C. Bacterial integrity is determined by recovery efficiency in a well-defined pellet after centrifugation at 3000xg for 10 minutes and by electron or optical microscopy with Gram staining. In a preferred embodiment, the recovery of bacteria after processing and washing is greater than about 80%, and the bacteria appear intact by optical or electron microscopy.

In yet another embodiment, reduction of endotoxin activity is achieved by treating the bacterial organisms with an antibiotic known to interfere with the biosynthesis of the KDO2lipid IVA complex. For example, Goldman et al., J

Bacteriol. 170(5):2185-91, 1988 describe antibacterial agents, including antibacterial agent III, which specifically inhibit the activity of CTP:CMP-3-deoxy-O-mannooctulosonate cytidylyltransferase and which are useful for blocking the incorporation of 3-deoxy-Dmannooctulosonate (KDO) into LPS gram-negative organisms. With the cessation of LPS synthesis, bacterial growth ceased. Attachment of KDO to LPS precursors, lipid IVA, is the main pathway for the formation of the lipid A-KDO complex in both S. typhimurium and E. coli. In one embodiment, the antibiotic is an antibacterial agent III and the gram-negative bacteria are treated with a suitable amount, such as, for example, 5 micrograms per milliliter to 500 micrograms per milliliter, for a suitable time, for example, 2 to 8 hours.

Likewise, the compound alpha-C-(1,5-anhydro-7-amino-2,7-dideoxy-Omannoheptopyranosyl)carboxylate is known to inhibit 3-deoxy-D-manno-octulosonate cytidyltransferase (CMP-KDO synthetase), a cytoplasmic enzyme which activates 3-deoxy-Omannooctulosonate (KDO) for incorporation into LPS (Nature. 1987 10-16;329(6135):162-4). Therefore, treating organisms with this compound may also reduce LPS-associated endotoxin activity.

In yet another embodiment, reduction of endotoxin activity is achieved by treating the organisms with an LPS inhibitor. For example, the bacterial cyclic lipopeptide surfactin has been shown to bind to lipid A, inhibiting its activity (J Antibiot 2006 59(1):35-43).

In addition to LPS-associated endotoxin, various other components of gram-negative organisms may induce or contribute to pyrogenicity and septic shock, including outer membrane proteins, fimbriae, pili, lipopeptides, and lipoproteins (reviewed by Jones, M., Int. J. Pharm. Compd., 5(4):259-263, 2001). Pyrogenicity can be measured in rabbits by a method well known in the art, involving assessment of rectal temperature after intravenous administration of suspected pyrogens.

It was found that treatment of a Gram-negative organism with a combination of polymyxin B and glutaraldehyde resulted in a 30-fold reduction in pyrogenicity as measured in rabbits. In one embodiment, 1000 micrograms per milliliter (μg/ml) of polymyxin B and 1% glutaraldehyde was used to achieve a 30-fold reduction in pyrogenicity as measured in rabbits. Pyrogenicity is reduced by a combination of the reaction of polymyxin B with LPS and the reactivity of glutaraldehyde with LPS and/or other bacterial components. In these circumstances, glutaraldehyde plays a dual role by also neutralizing bacteria.

Thus, in one embodiment, a method is proposed for reducing the endotoxin activity and pyrogenicity of a gram-negative bacterial microorganism, and neutralizing it by treating these bacteria with a combination of 1000 μg/ml polymyxin B and 1% glutaraldehyde. In yet another embodiment, gram-negative bacteria are treated with a combination of polymyxin B in a dose range of about 3 μg/ml to about 1000 μg/ml and glutaraldehyde in a dose range of about 0.1% to about 1.0%. In a further embodiment, the dosage range of polymyxin B is from about 100 μg/ml to about 1000 μg/ml, and glutaraldehyde is used in the dosage range from about 0.5% to about 1.0%. In addition, gram-negative bacteria can be treated, for example, with a dose range of polymyxin B from about 1000 μg/ml to about 3000 μg/ml, and glutaraldehyde is used in a dose range of about 0.5% to about 1.0% . In yet another aspect, gram-negative bacteria can be treated, for example, with a dosage range of polymyxin B from about

3000 μg/ml to about 5000 μg/ml, and glutaraldehyde is used in a dosage range of about 0.5% to about 2.0%.

In some embodiments, intact and substantially nonviable gram-negative bacterial cells have at least about a 70% reduction in LPS-associated endotoxin activity (eg, as measured by a LAL assay) compared to untreated wild-type bacteria. In some embodiments, the reduction is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 .5%, 99.9%, 99.95% or 99.98%. In some embodiments, the reduction is no more than about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%. In some embodiments, the reduction is from about 70% to about 99.99%, from about 80% to about 99.99%, from about 90% to about 99.5% or 99%, from about 91% to about 99% , from about 92% to about 98%, from about 93% to about 97%, from about 94% to about 96%, from about 94.5% to about 95.5%, from about 94% to about 97%, from about 95% to about 98%, from about 96% to about 99%, from about 97% to about 99.5%, or from about 98% to about 99.9%, without limitation.

In some embodiments, certain residual levels of active LPS are preferred. For example, in some embodiments, the composition of the present invention contains from about 1 to 200 ng of active LPS per 1x108 cells. In some embodiments, it contains from about 2 to 200 ng, from about 5 to 150 ng, from about 5 to 120 ng, from about 10 to 120 ng, from about 20 to 100 ng, from about 20 to 50 ng, from about 10 up to 50 ng of active LPS per 1x108 cells.

In some embodiments, intact and substantially nonviable gram-negative bacterial cells have at least about a 70% reduction in pyrogenicity (eg, as measured by an in vivo rabbit assay) compared to untreated wild-type bacteria. In some embodiments, the reduction is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 .5%, 99.9%, 99.95% or 99.98%. In some embodiments, the reduction is no more than about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%. In some embodiments, the reduction is from about 70% to about 99.99%, from about 80% to about 99.99%, from about 90% to about 99.5% or 99%, from about 91% to about 99% , from about 92% to about 98%, from about 93% to about 97%, from about 94% to about 96%, from about 94.5% to about 95.5%, from about 94% to about 97%, from about 95% to about 98%, from about 96% to about 99%, from about 97% to about 99.5%, or from about 98% to about 99.9%, without limitation.

As stated above, in addition to LPS-associated endotoxin, various other components of gram-negative organisms can also induce or contribute to pyrogenicity, such as outer membrane proteins, fimbriae, pili, lipopeptides and lipoproteins. In some embodiments, intact and substantially nonviable gram-negative bacterial cells are treated such that reduction in pyrogenicity is achieved by both reducing LPS-associated endotoxin activity and reducing non-LPS-associated pyrogenicity, such as by inactivating, removing, or blocking outer membrane proteins, fimbriae, pili, lipopeptides or lipoproteins. In some embodiments, the reduction in non-LPS pyrogenicity is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95% or 99.98%. In some embodiments, the reduction is no more than about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, 99.5%, 99.9%, 99.95%, 99.98% or 99.99%.

Bacteria to be administered in accordance with the methods of the present invention are rendered nonviable or substantially nonviable either before administration or become so after administration. The term nonviable means that the organisms are neutralized by treatment with an exogenous agent and/or they contain a mutation that results in the inability of the organisms to survive in a mammalian host. Essentially, non-viable bacteria are strains whose viability has been reduced by at least 80%, 85%, 90%, 95%, 99% or more.

Bacteria can be rendered nonviable by treatment with a compound such as polymyxin. Polymyxin binds to LPS and disrupts membrane integrity as bacteria divide, with reduced viability as a result of cell membrane permeability. If viability decreases using this method, measures must be taken to prevent cell lysis and maintain the cells intact.

Another approach is to grow bacterial strains with conditional mutations in the LPS biosynthetic pathway that are repressed during growth and then undergo nonpermissive conditions that activate the mutation and impair LPS biosynthesis. In each case, the procedure used is one that renders the bacteria nonviable by determining in each situation the optimal treatment time or dose of compound such that viability is substantially lost while maintaining significant integrity of the bacterial cell. In cases where nonviability is less than 100%, bacteria containing a mutation that prevents further proliferation of viable bacteria in the mammalian host can be used (for example, the diaminopimelic acid auxotroph described by Bukhari and Taylor, J. Bacteriol. 105(3):844-854 , 1971 and Curtiss et al., Immunol. Invest. 18(l-4):583-596, 1989).

Diseases and conditions

Intact and substantially non-viable gram-negative bacterial cells as disclosed herein are useful for enhancing the immune system of a subject and are thus useful for preventing or treating diseases and conditions through an improved immune response. The intact and essentially non-viable gram-negative bacterial cells disclosed herein may also be used as vaccines or immunological adjuvants for subjects at risk of developing such diseases or conditions.

Treatment or implementation of treatment is an approach to obtaining favorable or desired results, including clinical results. Beneficial or desired clinical results may include one or more of the following: a) inhibiting a disease or condition (eg, reducing one or more symptoms resulting from a disease or condition and/or reducing the severity of a disease or condition); b) slowing or stopping the development of one or more clinical symptoms associated with a disease or condition (eg, stabilizing a disease or condition, preventing or delaying the worsening or progression of a disease or condition, and/or preventing or delaying the spread (eg, metastasis) of a disease or condition ); and/or c) alleviation of the disease, that is, regression of clinical symptoms (for example, improving the course of the disease, providing partial or complete remission of the disease or condition, enhancing the effect of another drug, slowing the progression of the disease, improving quality of life and/or prolonging survival).

Prophylaxis or the implementation of prophylaxis means any treatment of a disease or condition that does not produce clinical symptoms of the disease or condition. In some embodiments, the bacterial cells may be administered to a subject (including a human) who is at risk of developing or has a family history of a disease or condition.

Subject refers to an animal, such as a mammal (including a human), that has been or will be the subject of treatment, observation, or experiment. The methods described in this application may be applicable in human therapy and/or veterinary medicine. In some embodiments, the subject is a mammal, such as a human, dog, cat, cow, sheep, etc. In one embodiment, the subject is a human.

In some embodiments, the diseases or conditions being treated are infectious diseases. In some embodiments, the infection is caused by bacteria, fungi, parasites, or viruses. In particular, the bacterial cells disclosed herein may be uniquely suited for the treatment of viral infections, such as those caused by the viruses listed in Table 1. And, optionally with the help of an additional anti-infective agent.

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Table A

List of viruses

Virus Genus, family Adeno-associated virus Dependovirus, Parvoviridae Aichi virus Kobuviras, Picomaviridae Australian bat lyssavirus Lyssavirus, Rhabdoviridae Polyomavirus BK Polyomaviras, Polyomaviridae Banna virus Seadomavirus, Reoviridae Barma forest virus Alphaviras, Togaviridae Bunyamwera virus Orthobunyaviras, Bunyaviridae Bunyavirus La Crosse Orthobunyavirus, Bunyaviridae

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Mountain hare bunyavirus Orthobunyaviras, Bunyaviridae Marmoset herpesvirus Lymphocryptoviras, Herpesviridae Chandipur virus Vesiculovirus, Rhabdoviridae Chikungunya virus Alphavirus, Togaviridae Cosavirus A Cosavirus, Picomaviridae Cowpox virus Orthopoxvirus, Poxviridae Coxsackie virus Enterovirus, Picomaviridae Crimean-Congo hemorrhagic fever virus Nairovirus, Bunyaviridae Dengue virus Flavivirus, Flaviviridae Dhori virus Thogotovirus, Orthomyxoviridae Dugbe virus Nairovirus, Bunyaviridae Duvenhage virus Lyssavirus, Rhabdoviridae Eastern equine encephalitis virus Alphavirus, Togaviridae Ebola virus Ebolavirus, Filoviridae Echovirus Enterovirus, Picomaviridae Encephalomyocarditis virus Cardiovims, Picomaviridae Epstein-Barr virus Lymphocryptovirus, Herpesviridae European bat lyssavirus Lyssavirus, Rhabdovirus GB virus C/hepatitis G virus Pegivirus, Flaviviridae Hantaan virus Hantavirus, Bunyaviridae Hendra virus Henipavims, paramyxoviridae Hepatitis A virus Hepatovims, picomaviridae Hepatitis B virus Orthohepadnavirus, Hepadnaviridae Hepatitis C virus Hepacivims, Flaviviridae Hepatitis E virus Hepevirus, not assigned Hepatitis delta virus Deltavims, not assigned Horsepox virus Orthopoxvirus, Poxviridae Human adenovirus Mastadenovirus, Adenoviridae Human astrovirus Mamastrovirus, Astroviridae Human coronavirus Alphacoronavirus, Coronaviridae Human cytomegalovirus Cytomegalovirus, Herpesviridae Human enterovirus 68, 70 Enterovirus, Picomaviridae Human herpesvirus 1 Simplexvirus, Herpesviridae Human herpesvirus 2 Simplexvirus, Herpesviridae Human herpesvirus 6 Roseolovirus, Herpesviridae Human herpesvirus 7 Roseolovirus, Herpesviridae Human herpesvirus 8 Rhadinovims, Herpesviridae AIDS virus Lentivirus, Retroviridae Human papillomavirus 1 Mupapillomavirus, Papillomaviridae Human papillomavirus 2 Alphapapillomavirus, Papillomaviridae

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Human papillomavirus 16.18 Alphapapillomavirus, Papillomaviridae Human parainfluenza Re spiro virus, Paramyxoviridae Human parvovirus B19 Erythrovirus, Parvoviridae Human respiratory syncytial virus Orthopneumovirus, Pneumoviridae Human rhinovirus Enterovirus, Picomaviridae Human SARS coronavirus Betacoronavirus, Coronaviridae Human spumaretrovirus Spumavirus, Retroviridae Human T-lymphotropic virus Deltaretrovirus, Retroviridae Human torovirus Torovirus, Coronaviridae Influenza A virus Influenzavirus A, Orthomyxoviridae Influenza B virus Influenzavirus B, Orthomyxoviridae Influenza C virus Influenzavirus C, Orthomyxoviridae Isfahan virus Vesiculovirus, Rhabdoviridae Polyomavirus JC Polyomavirus, Polyomaviridae Japanese encephalitis virus Flavivirus, Flaviviridae Arenavirus Junin Arenavirus, Arenaviridae Polyomavirus KI Polyomavirus, Polyomaviridae Kunjin Virus Flavivirus, Flaviviridae Lagos bat virus Lyssavirus, Rhabdoviridae Lake Victoria Marburgvirus Marburgvirus, Filoviridae Langat virus Flavivirus, Flaviviridae Laos virus Arenavirus, Arenaviridae Lordsdale virus Norovirus, Caliciviridae Scottish sheep encephalomyelitis virus Flavivirus, Flaviviridae Lymphocytic choriomeningitis virus Arenavirus, Arenaviridae Machupo virus Arenavirus, Arenaviridae Mayaro virus Alphavirus, Togaviridae Coronavirus MERS Betacoronavirus, Coronaviridae measles virus Morbilivirus, Paramyxoviridae Mengo encephalomyocarditis virus Cardiovirus, Picomaviridae Merkel cell polyomavirus Polyomavirus, Polyomaviridae Mokola virus Lyssavirus, Rhabdoviridae Molluscum contagiosum virus Molluscipoxvirus, Poxviridae Monkeypox virus Orthopoxvirus, Poxviridae Mumps virus Rubulavirus, Paramyxoviridae Murray Valley Encephalitis Virus Flavivirus, Flaviviridae New York virus Hantavirus, Bunyavirus Nipah virus Henipavirus, Paramyxoviridae Norwalk virus Norovirus, Caliciviridae O'nyong-nyong virus Alphavirus, Togaviridae

- 10 044566

Orff virus Parapoxviras, Poxviridae Oropush virus Orthobunyaviras, Bunyaviridae Pichinde virus Arenavirus, Arenaviridae Poliovirus Enterovirus, Picomaviridae Phlebovirus Punta Toro Phlebovirus, Bunyaviridae Puumala virus Hantavirus, Bunyaviras Rabies virus Lyssaviras, Rhabdoviridae Rift Valley fever virus Phlebovirus, Bunyaviridae Rosavirus A Rosaviras, Picomaviridae Ross River virus Alphaviras, Togaviridae Rotavirus A Rotavirus, Reoviridae Rotavirus B Rotavirus, Reoviridae Rotavirus C Rotavirus, Reoviridae Rubella virus Rubiviras, Togaviridae Sagiyama virus Alphaviras, Togaviridae Salivirus A Salivirus, Picomaviridae Sicilian mosquito fever virus Phlebovirus, Bunyaviridae Sapporo virus Sapoviras, Caliciviridae Semliki Forest Virus Alphaviras, Togaviridae Virus Seoul Hantavirus, Bunyaviras Simian foam virus Spumaviras, Retroviridae Monkey virus 5 Rubulavirus, Paramyxoviridae Sindbis virus Alphaviras, Togaviridae Southampton virus Norovirus, Caliciviridae St. Louis encephalitis virus Flaviviras, Flaviviridae Powassan tick-borne virus Flaviviras, Flaviviridae Hepatitis TTV virus Alphatorqueviras, Anelloviridae Tuscany virus Phlebovirus, Bunyaviridae Uukuniemi virus Phlebovirus, Bunyaviridae Vaccinia virus Orthopoxvirus, Poxviridae Varicella zoster virus Varicelloviras, Herpesviridae Variola virus Orthopoxvirus, Poxviridae Venezuelan equine encephalitis virus Alphaviras, Togaviridae Vesicular stomatitis virus Vesiculovirus, Rhabdoviridae Western equine encephalitis virus Alphaviras, Togaviridae Polyomavirus WU Polyomaviras, Polyomaviridae West Nile virus Flaviviras, Flaviviridae Yaba monkey tumor virus Orthopoxvirus, Poxviridae Yaba virus-like disease Orthopoxvirus, Poxviridae Yellow fever virus Flaviviras, Flaviviridae Zika virus Flaviviras, Flaviviridae

In some embodiments, the disease being treated is an HBV infection. In some embodiments, the disease being treated is an HIV infection.

Dosing and frequency

In some embodiments, the level of LPS-associated endotoxin activity of intact and substantially nonviable gram-negative bacterial cells administered to a subject may be determined. In one embodiment, the input composition contains

- 11 044566 approximately 0.01 to 200 ng of active LPS per kg of subject's body weight.

The term active LPS refers to LPS in a composition that is capable of exhibiting LPS-associated endotoxin activity, for example, as measured by the LAL assay, where 5-9 endotoxin units (EU) are considered equivalent to 1 ng of active LPS based on a standard LPS preparation. The amount of active LPS in a composition can be described as the mass of uninhibited LPS capable of exhibiting the same level of LPS-associated endotoxin activity as the composition.

In some embodiments, the administered composition (eg, a composition having LPS-associated endotoxin activity of intact and substantially nonviable gram-negative bacterial cells) contains from about 0.01 to 150 ng of active LPS per kg of subject body weight. In some embodiments, the composition administered contains from about 0.02 to 150 ng, from about 0.02 to 100 ng, from about 0.05 to 100 ng, from about 0.05 to 100 ng, from about 0.05 to 50 ng, about 0.05 to 20 ng, about 0.1 to 20 ng, about 0.1 to 10 ng, about 0.1 to 5 ng, about 0.2 to 100 ng, about 0 .2 to 50 ng, about 0.2 to 20 ng, about 0.2 to 10 ng, about 0.2 to 5 ng, about 0.3 to 90 ng, about 0.4 to 80 ng , about 0.5 to 70 ng, about 0.6 to 60 ng, about 0.7 to 50 ng, about 0.8 to 40 ng, about 0.9 to 30 ng, about 1 to 20 ng, about 2 to 15 ng, or about 3 to 12 ng of active LPS per kg body weight of the subject.

The amount of active LPS administered to a subject may vary depending on the type of subject and disease. For some animals, such as mice, rats and dogs, higher amounts (eg, 250-500 times higher compared to humans and rabbits) of active LPS can be used effectively. Accordingly, in some embodiments, the composition administered contains from about 10 to 100,000 ng of active LPS per kg of body weight of the subject. In some embodiments, the composition administered contains about 20 to 50,000 ng, about 30 to 40,000 ng, about 40 to 30,000 ng, about 50 to 20,000 ng, about 0.7 to 10,000 ng, about 80 to 8,000 ng , about 90 to 7000 ng, about 100 to 6000 ng, about 200 to 5000 ng, or about 500 to 5000 ng of active LPS per kg body weight of the subject.

The number of bacterial cells administered to a subject can be determined based on the required amount of active LPS and the level of endotoxin activity of the bacterial cells. The amount may also depend on various factors, including age, body weight, general health, sex, diet, time of administration, route of administration and rate of elimination, drug combination, and the severity of the particular disease in the subject receiving therapy. For example, the dosage may be expressed as the number of bacterial cells described herein per kilogram of subject body weight (mg/kg). Dosages of approximately 10,000 to 100,000,000 cells/kg may be suitable. In some embodiments, from about 100,000 to 10,000,000 cells/kg may be suitable. In other embodiments, a dosage of 1,000,000 to 5,000,000 cells/kg may be suitable. Standardization can also be made based on body surface area expressed in square meters ( ^m2 ). In some embodiments, dosages of about 10,000 to 100,000,000 cells/m ² may be suitable. In some embodiments, from about 100,000 to 10,000,000 cells/m ² may be suitable. In other embodiments, a dosage of 1,000,000 to 5,000,000 cells/m ² may be suitable. Rating according to the body weight of the subject is particularly appropriate when adjusting dosages between subjects of significantly different size, for example, when using a drug in both children and adults, or when converting the effective dosage in non-human subjects, such as a dog , in a dosage suitable for humans.

Bacterial cells are usually administered after a disease or condition has been diagnosed. In some embodiments, administration begins within 24 hours of infection, or within 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days of infection. In some embodiments, administration begins at least 24 hours after infection, or at least 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after infection. In some embodiments, administration begins at any time before or after infection and may be administered as needed.

Administration may also begin before actual infection or before infection is diagnosed, as a prophylactic vaccine or prophylaxis.

Combination therapies

In one embodiment, the bacterial cells disclosed herein may be used in combination with one or more additional therapeutic agents that are used and/or developed to treat infections.

In some embodiments, one or more additional therapeutic agents may be cyclooxygenase (COX) enzyme inhibitors, such as NSAIDs, including 6MNA (6methoxy-2 naphthylacetic acid), aspirin, carprofen, diclofenac, fenoprofen, flufenamate, flu

- 12 044566 biprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, naproxen, niflumic acid, piroxicam, sulindac sulfide, suprofen, tenidap, tolmetin, tomoxiprole, zomepirac, celecoxib, etodolac, meloxicam, nimesulide, diisopropyl fluorophosphate, L745, 337, NS398, rofecoxib, SC58125, S-aminosalicylic acid, ampirone, diflunisal, nabumetone, paracetamol, resveratrol, salicin, salicylic aldehyde, sodium salicylate, sulfasalazine, sulindac, tamoxifen, ticlopidine and valeryl salicylate.

In some embodiments, one or more additional therapeutic agents may be agonists of stimulatory immune checkpoints, such as CD27, CD28, CD40, CD122, CD137, OX40, GITR, and ICOS, or antagonists of inhibitory immune checkpoints, such as A2AR, B7-H3 , B7-H4, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, TIM-3 and VISTA.

Non-limiting examples of one or more additional therapeutic agents also include abacavir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, balavir, cidofovir, combivir, dolutegravir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, ecoliver, famciclovir, fomivirsen, fosamprenavir, foscarnet, phosphonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitor, type III interferon, type II interferon, type I interferon, interferon, lamivudine, lopinavir , loviride, maraviroc, moroxidine, metisazone, nelfinavir, nevirapine, nexavir, nitazoxanide, nucleoside analogues, norvir, oseltamivir (Tamiflu®), peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitor, raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, sofosbuvir, stavudine, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, Truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir and zidovudine.

In one embodiment, the additional therapeutic agent is interferon alpha.

In some embodiments, the gram-negative organisms include a polynucleotide encoding a nonbacterial protein, such as, for example, virus-specific antigens or immune stimulating proteins. In some embodiments, the gram-negative organisms include a polynucleotide encoding a bacterial antigen, which may be derived from the same or another bacterial organism, including a gram-positive organism or another pathogen. As used herein, an antigen is any molecule that can be recognized by an immune response - an antibody or an immune cell.

Pharmaceutical compositions, dosage forms and methods of administration

Disclosed herein are various compositions suitable for treating diseases or conditions. In one embodiment, a composition or dosage form is provided comprising a plurality of intact and substantially non-viable gram-negative bacterial cells that have been treated in a manner that results in a reduction in lipopolysaccharide (LPS)-associated endotoxin activity by approximately 90-99% when measured with using the Limulus amebocyte lysate (LAL) assay compared to untreated wild-type Gram-negative bacterial cells, where the total LPS-associated endotoxin activity is equivalent to approximately 0.7 ng to 7000 ng active LPS, approximately 7 ng to 7000 ng active LPS, approximately 7 ng to 1400 ng active LPS or approximately 70 ng to 1400 ng active LPS.

The compositions can be used as adjuvants or biological response modifiers. As used herein, the terms adjuvant and biological response modifier refer to any substance that enhances the immune response to an antigen. Thus, an adjuvant or biological response modifier is used to stimulate a more active immune system response to a foreign antigen or pathogen or disease-related organism. However, in some embodiments, recombinant forms of gram-negative bacteria that express, for example, viral proteins or human immune system activation proteins, such as cytokines or chemokines, are provided for use in the disclosed methods. In an alternative embodiment, purified immune activation proteins, such as cytokines or chemokines, are mixed with the gram-negative organisms before administration or are administered before or after the gram-negative organisms.

In some embodiments, the intact and substantially nonviable gram-negative bacterial cells in the dosage form have at least about a 70% reduction in LPS-associated endotoxin activity (eg, as measured by the LAL assay) compared to untreated wild-type bacteria. In some embodiments, the reduction is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 .5%, 99.9%, 99.95% or 99.98%. In some embodiments, the reduction is no more than about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%. In some embodiments, the reduction is from about 70% to about 99.99%, from about 80% to about 99.99%, from about 90% to about 99.5% or 99%, from about 91% to about 99% , from about 92% to about 98%, from about 93% to about 97%, from about 94% to about 96%, from about 94.5% to about 95.5%, from about 94% to about 97%, from about 95% to about 98%, from about 96% to about 99%, from about 97% to about 99.5%, or from about 98% to about 99.9%, without limitation.

In some embodiments, the intact and substantially nonviable gram-negative bacterial cells in the dosage form have at least about a 70% reduction in pyrogenicity (eg, as measured by an in vivo rabbit assay) compared to untreated wild-type bacteria. In some embodiments, the reduction is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 .5%, 99.9%, 99.95% or 99.98%. In some embodiments, the reduction is no more than about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%. In some embodiments, the reduction is from about 70% to about 99.99%, from about 80% to about 99.99%, from about 90% to about 99.5% or 99%, from about 91% to about 99% , from about 92% to about 98%, from about 93% to about 97%, from about 94% to about 96%, from about 94.5% to about 95.5%, from about 94% to about 97%, from about 95% to about 98%, from about 96% to about 99%, from about 97% to about 99.5%, or from about 98% to about 99.9%, without limitation.

In some embodiments, the reduction in non-LPS pyrogenicity is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95% or 99.98%. In some embodiments, the reduction is no more than about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, 99.5%, 99.9%, 99.95%, 99.98% or 99.99%.

In some embodiments, the viability of the substantially nonviable bacteria is reduced by at least 80%, 85%, 90%, 95%, 99%, or more.

In some embodiments, the dosage form includes from about 10,000 to about 1x10 ¹⁰ such bacterial cells. In some embodiments, the dosage form includes from about 100,000 to about 1x10 ⁸ such bacterial cells. In some embodiments, the dosage form includes from about 1,000,000 to about 1x10 ⁷ such bacterial cells. In some embodiments, the dosage form includes from about 1x10 ⁶ to about 1x10 ⁸ such bacterial cells.

In some embodiments, the composition further includes a second therapeutic/antiviral agent. In some embodiments, the second therapeutic agent is an NSAID (non-steroidal anti-inflammatory drug). In some embodiments, the second therapeutic agent is a cyclooxygenase inhibitor, preferably selected from the group consisting of 6MNA, aspirin, carprofen, diclofenac, fenoprofen, flufenamate, flubiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, naproxen, niflumic acid , piroxicam, sulindac sulfide, suprofen, tenidap, tolmetin, tomoxiprol, zomepirac, celecoxib, etodolac, meloxicam, nimesulide, diisopropyl fluorophosphate, L745, 337, NS398, rofecoxib, SC58125, S-aminosalicylic acid, ampirone, diflunisal, on bumeton, paracetamol, resveratrol , salicin, salicylic aldehyde, sodium salicylate, sulfasalazine, sulindac, tamoxifen, ticlopidine, valeryl salicylate and combinations thereof.

In some embodiments, the second therapeutic agent is a stimulatory immune checkpoint agonist, preferably selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, and iCoS. In some embodiments, the second therapeutic agent is an inhibitory immune checkpoint antagonist, preferably selected from the group consisting of A2AR, B7-H3, B7-H4, CTLA-4, IDO, KIR, LAG3, PD-1, PDL1, TIM- 3 and VISTA.

In some embodiments, the second therapeutic agent is selected from the group consisting of abacavir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, balavir, cidofovir, combivir, dolutegravir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz , emtricitabine, enfuvirtide, entecavir, ecoliver, famciclovir, fomivirsen, fosamprenavir, foscarnet, phosphonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitor, type III interferon, type II interferon, type I interferon, interferon , lamivudine, lopinavir, loviride, maraviroc, moroxidine, metisazone, nelfinavir, nevirapine, nexavir, nitazoxanide, nucleoside analogues, norvir, oseltamivir (Tamiflu®), peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitor, raltegravir a, ribavirin , rimantadine, ritonavir, pyramidin, saquinavir, sofosbuvir, stavudine, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, vira- 14 044566 midine, zalcitabine, zanamivir, zidovudine and their combinations.

In some embodiments, the second therapeutic agent is interferon alpha.

The compositions described herein can be prepared in a variety of ways for use in the methods described herein. In one embodiment, the composition contains the organisms described throughout this application and a pharmaceutically acceptable carrier.

The term pharmaceutically acceptable carriers refers to any diluents, excipients or carriers that can be used in the compositions. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, whey proteins such as human serum albumin, buffering agents such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as magnesium sulfate, protamine sulfate, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block copolymers , polyethylene glycol, cryopreservatives such as trehalose and mannitol, and lanolin. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, the standard reference in the art.

The compositions are formulated for pharmaceutical administration to a mammal, preferably a human. Such pharmaceutical compositions according to the present invention can be administered by various routes, including parenterally. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

Sterile injectable forms of the compositions may be an aqueous or oily suspension. These suspensions can be prepared in accordance with methods known in the art, using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol. Acceptable carriers and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are traditionally used as a solvent or suspending medium. For this purpose, any neutral, fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are suitable for the preparation of injectable preparations, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated variants. These oily solutions or suspensions may also contain a long-chain alcohol diluent or dispersing agent, such as carboxymethylcellulose or similar dispersing agents commonly used in the preparation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants such as Tweens, Spans and other emulsifiers or bioavailability enhancers commonly used to prepare pharmaceutically acceptable solid, liquid or other dosage forms may also be used for formulation purposes. The compositions may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.

Pharmaceutical compositions can be administered in either single or multiple doses. The pharmaceutical composition can be administered by various routes, including, for example, rectal, buccal, intranasal and transdermal routes. In certain embodiments, the pharmaceutical composition may be administered by intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, or subcutaneous.

One route of administration is parenteral, for example by injection. Forms in which the pharmaceutical compositions described herein may be formulated for injection include, for example, aqueous or oleaginous suspensions or emulsions with sesame oil, corn oil, cottonseed oil or peanut oil, as well as elixirs, mannitol, dextrose or sterile aqueous solution and similar pharmaceutical carriers.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, trehalose, starches, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup and methylcellulose. The formulations may further include lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweeteners; and flavoring additives.

- 15 044566

Examples

The following examples are included to demonstrate specific embodiments of the present invention. It will be clear to those skilled in the art that the methods disclosed in the examples below are methods that work well in the practice of the present invention, and thus may be considered to constitute specific embodiments thereof. However, those skilled in the art in light of the present disclosure should appreciate that many changes may be made to the specific embodiments disclosed and still achieve the same or a similar result without departing from the spirit and scope of the present invention.

Example 1: Preclinical characterization of the efficacy of a systemically administered multiple toll-like receptor (TLR) agonist.

This example tested the hypothesis that significant reduction without complete elimination of LPS activity, coupled with neutralization and stabilization of non-pathogenic Gram-negative bacteria, could provide a multi-TLR product that could safely and effectively induce antitumor immune responses when administered intravenously.

Non-pathogenic Gram-negative E. coli were treated with polymyxin B and glutaraldehyde under neutralizing and cell-stabilizing conditions, resulting in a greater than 90% reduction in LPS endotoxin activity and pyrogenicity (treated bacteria, also called mock bacteria or simply Mock bacteria). Endotoxin activity and pyrogenicity were quantified using Limulus amebocyte lysate and in vivo rabbit assays. Bacterial integrity was assessed using electron and light microscopy. Antitumor activity was determined using standard syngeneic and xenograft tumor models.

Treated bacteria showed a 3-fold reduction in short-term toxicity in vivo compared to untreated bacteria. The induction of antitumor cytokine secretion by mouse and human peripheral blood mononuclear cells (PBMCs) was not impaired compared with untreated bacteria, which was unexpected. Treatment with treated bacteria (i.v.) provided significant monotherapeutic antitumor activity against orthotopic colorectal carcinoma in mice and metastatic pancreatic carcinoma in mice.

Synergistic combination activity, including eradication of established tumors with a therapeutic index of up to 10, was observed in combination with IL-2 or low-dose cyclophosphamide (LDC) in mouse models of colorectal carcinoma, with subcutaneous (SC) LDC in a non-Hodgkin lymphoma (NHL) model. in mice and with LDC plus SC rituximab in a human NHL model. Synergistic antitumor activity was also observed in combination with a low-dose nonsteroidal anti-inflammatory drug (NSAID) in a mouse model of metastatic pancreatic carcinoma. Additionally, tumor clearance was observed in combination with NSAIDs and was enhanced by the addition of anti-PD1 therapy SC in a mouse model of hepatocellular carcinoma. Optimal (80–100%) tumor clearance was shown to be mediated by natural killer (NK), CD4+ and CD8+ T cells. Immunological memory (80–100% and partial), defined by rejection of subsequent tumor challenge, has been demonstrated in both immunocompetent and innate-only conditions, respectively.

Example 2: Induction of cytokine secretion by treated bacteria.

In this example, the ability of treated bacteria to induce cytokine secretion from human peripheral blood mononuclear cells was studied.

Non-pathogenic Gram-negative E. coli were treated with polymyxin B and glutaraldehyde under neutralizing and cell-stabilizing conditions, resulting in a reduction in LPS endotoxin activity and pyrogenicity by more than 90% (treated bacteria, mock bacteria, or simply Mock bacteria), see

Example 1 and US Patent No. 9,265,804 B2. Endotoxin activity and pyrogenicity were quantified using Limulus amebocyte lysate and in vivo rabbit assays. Bacterial integrity was assessed by light microscopy after Gram staining.

1x10 to 1x108 untreated or treated bacteria in 10-fold increments were incubated with 2.5x105 human peripheral blood mononuclear cells (PBMCs) in RPMI culture medium containing 2.5 mM glutamine, 10% human serum, and 1% penicillin and streptomycin at 37°C with 5% CO ₂ for 48 hours. Supernatants were collected by centrifuging the plates at 1000 rpm for 10 minutes and stored at -80°C. Luminex analysis of cytokine levels was performed using Millipore Human Cytokine/Chemokine Magnetic Bead HCYTOMAG-60K and HSTCMAG-28SK panels. Cytokine levels were interpolated from the calibration curve using 5-point nonlinear regression analysis, where approximation = (A+((BA)/(1+(((BE)/(EA))*((x/C) ^A D))))) . Interpolated data were normalized to vehicle control or unstimulated control and analyzed. PBMCs from fresh normal peripheral blood leukapheresis products (ALLCells, Alameda, CA) were isolated using a Ficoll-Paque PLUS gradient (Ficoll-Paque PLUS, catalog no. 17-144-02, density 1.077 +/- 0.001 g/ml from GE Healthcare BioSciences, Pittsburgh, PA). The carrier for bacteria was sodium phosphate buffer (PBS) without calcium and with 2 mM MgCl ₂ .

The results shown are the peak levels determined for each cytokine using the same dose of untreated or treated bacteria. The treated bacteria contained 4.94% LPS of the level of untreated bacteria, per bacterium, as measured by the in vitro Limulus amebocyte lysate (LAL) assay.

Table 1

Cytokine levels from PBMCs induced by treated bacteria

Secretion of human PBMCs in vitro Raw bacteria Treated bacteria Cytokine 48-hour peak, pg/ml (average of three parallel assays), at the same bacterial dose for each cytokine GM-CSF 1094 1197 IFNy 175866 47488* IL-Ιβ 11976 17651 IL-6 78422 98534 IL-8 126942 166769 IL-10 6970 7670 IL-12p70 176 528 IL-23 0 119 TN Fa 49782 77919

The results presented are the peak levels determined for each cytokine that were observed at the same dose of untreated and treated bacteria, with the exception of IFNy, which peaked at a lower dose for untreated bacteria and was compared with the same dose of treated bacteria.

The activity of the treated bacteria was compared with several toll-like receptor agonists (TLRa) and isolated LPS. The experiment was carried out as described in table. 1. Toll-like receptor agonists (TLRa) were obtained from InvivoGen (San Diego, CA) and experimentally titrated as follows; CpG ODN 2006 (No. tlrl-2006, from 0.005 to 5 micromol), poly(1:C) (No. tlrlpic, from 0.001 to 100 micrograms/ml), R848 (No. tlrl-r848, from 0.1 to 100 micrograms/ml) ml) and E. coli LPS (No. tlrl-pb5lps, from 10 to 1 x10 ⁶ picograms/ml). TLR agonist stock solutions were prepared according to the manufacturer's recommendations within their recommended solubility limits, and the results represent peak cytokine levels determined for each TLRa and bacteria treated. The results are presented in the table below. 2.

table 2

Cytokine induction compared to individual TLR agonists

CpG ODN (TLR9a) Poly(1:C) (TLR3a) R848 (TLR 7/8a) LPS (TLR4a) Treated bacteria Cytokine pg/ml (average peak value of the full titration curve) GM-CSF 0 0 87 175 1197 IFNy 7 103 31324 29416 91475 IL-Ιβ 1 22 9990 4631 17651 IL-6 241 129 40555 54174 98534 IL-8 2436 1452 116135 143459 166769 IL-10 374 8 940 3542 7670 IL-12p70 4 18 253 109 528 TN Fa 51 208 33393 24944 77919

Example 3: Testing treatment of viral infection with treated bacteria in animals.

In this example, treated bacteria that can be prepared as shown in Examples 1 and 2 were tested for their activity in treating viral infections in animals.

The animal models of hepatitis B (HBV) infection used in this example can be any of the following: hydrodynamic injection model, FVB/N model, adenoviral delivery model, adeno-associated virus model, hepatitis virus infection model

- 17 044566 marmots, chimpanzee model, tupaia model, HBV transgenic mouse model, HBVTrimera model and chimeric human liver model. Suitable animal models of HIV can also be used to test the activity of treated bacteria in preventing or treating HIV infection.

Preparations of treated bacteria with total LPS activity as measured by the in vitro Limulus amebocyte lysate (LAL) assay in various ranges are administered to animals infected with the virus or expressing viral genes. In some animals, additional immunostimulating or antiviral agents will be used as combination therapy. Untreated animals and animals treated with other immunostimulating or antiviral agents were used as controls. The treated bacteria are believed to exhibit effective antiviral activity in these animal models.

Example 4 Inhibition of HBV in mice.

Adeno-associated virus/hepatitis B virus (AAV/HBV) is a recombinant AAV that carries the replicable human HBV genome. Taking advantage of the high hepatotropicity of AAV genotype 8, the human HBV genome can be efficiently delivered into mouse liver cells. Infection of immunocompetent mice with AAV/HBV can lead to prolonged HBV viremia, including the release of human HBV virions, HBsAg, and HBeAg into the blood, mimicking chronic HBV infection in patients. The AAV/HBV model can be used to evaluate the in vivo activity of various types of anti-HBV agents. In addition, this model is suitable for the evaluation of immunomodulators (see, for example, Huang et al., Int. J. Onc. v39 pp1511-1519, 2011).

rAAV8-1.3HBV, genotype D, was purchased from Beijing FivePlus Institute of Molecular Medicine (batch number A2018092406). The virus stock solution with ^1χ10Λ12 viral genomes (vg)/ml was diluted to 5χ10Λ11 vg/ml with sterile sodium phosphate buffer (PBS) and 1x10Λ11 vg. AAV/HBV at 200 μl was injected into 5-week-old male C57BL/6 mice 31 days before dosing (day -31).

Blood samples (-100 μl) were collected in K2-EDTA-coated tubes through submandibular bleeding on days -17, -7 and -4. Samples were centrifuged at 7000 g (4°C) for 10 minutes to collect plasma. At least 40 μl of plasma samples were prepared to determine HBV DNA levels by qPCR and HBs antigen (HBsAg) and HBe antigen (HBeAg) levels by enzyme-linked immunosorbent assay (ELISA). The remaining plasma samples were stored at -80°C for postmortem analyses. Based on plasma HBV, HBsAg, HBeAg DNA levels and body weight on days -17, -7 and -4, mice with appropriate levels of viral infection were selected and randomly divided into 7 groups with 5 mice in each group on day -1. All mice were evenly distributed in each group to ensure that there were no significant differences between each group in terms of HBV DNA levels, HBsAg, HBeAg and body weight on day -4.

Mock bacteria were prepared as described in Example 1 and US Pat. No. 9,265,804 B2, with an LPS endotoxin activity range of approximately 2000 to 6000 endotoxin units (EU) or approximately 250 to 750 ng of LPS per 10Λ9 neutralized bacterial cells. Groups of animals received no treatment, received entecavir (ETV) at a dose of 0.005 mg/kg orally (PO) daily on days 034, Sham at a dose of 2χ10Λ8 neutralized bacteria per mouse injected into the tail vein IV on days 1, 2, 8, 9, 15, 16, 22, 23, 29 and 30, or ETV and Imitation. Plasma samples were obtained weekly and analyzed for HBV DNA, HBsAg, and HBeAg as described above. Levels of HBV DNA (copies), HBsAg, and HBeAg in mice from the treated groups were compared with those in the untreated group at each matched time point using unpaired, nonparametric Mann-Whitney statistical analysis.

Fig. 1 demonstrates that standard of care (ETV) therapy significantly inhibited HBV DNA production within 3 days of dosing, reducing plasma levels in 5/5 mice to the lower limit of quantitation (120 copies/μL plasma) by 28 day of daily dosing (day 59 postinfection) (Fig. 1B). Mock also significantly inhibited HBV DNA production, reducing plasma levels in 3/5 mice to the lower limit of quantitation on day 28 of twice-weekly dosing (day 59 postinfection) (Fig. 1C). Inhibition of HBV DNA by ETV + Mock was similar to ETV alone, demonstrating the lack of antagonistic interaction up to this point upon administration of the compound (Fig. 1D).

No inhibition of HBsAg or HBeAg production was observed with ETV administration. However, Mock was found to inhibit HBeAg production on day 28 of dosing (day 59 postinfection) (Figure 2).

In summary, this example demonstrates that, as with standard of care (ETV), mock bacteria can also significantly inhibit HBV replication, although the mock bacteria take longer to exhibit inhibitory activity, consistent with the immunological basis mechanism of action of Imitation. However, unlike ETV, which

- 18 044566 inhibited only HBV DNA production, mimic bacteria also have the advantage of inhibiting HBe antigen production.

Example 5: Long-term HBV inhibition.

This experiment was conducted to determine whether a nonsteroidal anti-inflammatory drug (NSAID) could enhance the antiviral activity of Mock bacteria if Mock antiviral activity is lost after treatment was stopped, and the potential toxic effects of ETV, Mock, and ETV + Mock treatment. The experiment was performed as described in Example 4, except that the AAV/HBV virus was injected on day -29 relative to the start of dosing. Plasma HBV DNA, HBsAg and HBeAg levels were determined on days -15, -8 and -1 relative to the start of dosing on day 0, and the ETV dose was increased to 0.1 mg/kg. In addition, all groups presented in FIG. 3, 4, 5 and 6, indomethacin (NSAID) (10 μg/ml in drinking water) was administered daily. Dosing was carried out over 5 weeks as in Example 4, with plasma levels of HBV DNA, HBsAg and HBeAg determined weekly and biweekly for 27 weeks after cessation of treatment. Inhibitory activity was determined by comparing the treated groups at each time point with the control group (indomethacin alone) at the same time point using unpaired, nonparametric Mann-Whitney statistical analysis.

At the end of the experiment, the liver of each mouse was divided into several sections, flash frozen in liquid nitrogen immediately after collection, and stored at -80°C. Liver sections were used to detect HBV DNA, and additional liver sections from each mouse were fixed in 10% neutral buffered formalin (NBF) for approximately 24 hours, then transferred for standard dehydration and paraffin embedding. Paraffin blocks were sectioned for hematoxylin-eosin (H&E) staining and histopathological analysis, as well as immunohistochemical (IHC) analysis for HBsAg and HBcAg.

Fig. 3A demonstrates that indomethacin alone did not inhibit HBV DNA production. At the same time, FIG. 3 (B, C, and D) demonstrates that ETV, Mock, or ETV + Mock inhibited HBV DNA production in the presence of indomethacin similar to what was observed in the absence of indomethacin (Fig. 1). No weight loss was observed with ETV treatment. Transient body weight loss of 6% per day was observed during 1 week of treatment with Sham (±ETV), 1.3% (without ETV) and 3.4% (+ETV) per day during 2 weeks Sham treatment, 0.8% (no ETV) and 2.0% (+ETV) for one day during the third week of treatment, and no weight loss was observed during the fourth and fifth weeks of Sham treatment (±ETV).

Statistically significant inhibition of HBV DNA was lost at the end of the experiment (day 253), 27 weeks after stopping ETV treatment (Figure 3B), but inhibition remained statistically significant at day 253 in the Sham (Figure 3C) and Sham + ETV groups. (Fig. 3D). This demonstrates that Shams have a longer lasting inhibitory effect compared to ETV.

Indomethacin + ETV did not inhibit HBsAg or HBeAg compared to indomethacin alone (Figures 4A,B and 5A,B). However, addition of Mock (i.e., indomethacin + Mock or indomethacin + Mock + ETV) inhibited the production of HBsAg and HBeAg compared to indomethacin alone (Figures 4 A, C, D and 5 A C, D). Since neither Mock alone nor indomethacin alone inhibited HBsAg production, these results demonstrate a synergistic interaction between Mock and indomethacin in inhibiting HBsAg.

Fig. 6 demonstrates that in the presence of indomethacin, Sham or Sham + ETV, but not ETV, inhibited HBeAg expression in the liver of mice 27 weeks after cessation of treatment. In fig. 7 shows IHC analysis of HBcAg expression in the liver of mice treated with Mock vehicle (ie, only the buffer used in the Mock composition) and compound. The combination of indomethacin and Sham again reduced HBcAg expression in 2 of 5 mice compared with indomethacin alone, and the combination of indomethacin, Sham, and ETV reduced HBcAg expression in 5 of 5 mice compared with indomethacin + ETV or indomethacin + Sham treatment.

This example demonstrates that NSAIDs such as indomethacin are unable to inhibit HBV but can significantly enhance the antiviral activity of the mimic bacteria in a synergistic manner. Compared with standard of care ETV, the mock bacteria demonstrated a longer-lasting and sustained treatment effect. The combination of Sham and ETV resulted in significant improvements compared with either treatment alone, particularly with regard to long-term follow-up after treatment cessation.

Example 6 Inhibition of HIV infection.

To study the ability of mock bacteria to inhibit human immunodeficiency virus (HIV) infection in vivo, the human immune system was recapitulated in NOD/Shi-scid/IL2Rγnull-specific female immunodeficient (NCG) mice (Charles River) using hematopoietic stem cells isolated from human umbilical cord blood. Mice were engrafted with cord blood-derived CD34+ hematopoietic stem and progenitor cells (French Blood Institute) after myeloablative chemotherapy (Manfroi et al., Can. Res. v77, pp1097-1107

2017). Engraftment consisted of intravenous injection of 105 CD34+ cells. Engraftment rates were monitored by flow cytometry testing for human CD45+ cells among all blood leukocytes.

After 14 weeks of engraftment, matched hu mice were inoculated with 80 ng of HIV-1 (NL4-3 HIV1/Clade B; X4-tropic virus) by i.p. injection. Plasma viremia was determined at week 5 using qRT-PCR. Other methods were as described in Wenzel et al., J. Virol. Doi:10.1128/JVI.00907-19 (accepted manuscript published online August 2, 2019). Only HIV mice with a viral load of more than 10 ⁴ copies/ml were used in the experiment. At week 5, mice were randomly assigned to groups based on their degree of humanization, HIV load, and percentage of CD4+ T cells in the blood.

The groups received Sham vehicle twice weekly for 5 weeks, highly active antiretroviral therapy (HAART or tri-therapy) consisting of 2.4 mg lamivudine, 2.35 mg tenofovir disoproxil and 19.2 mg raltegravir per day, added in feed pellets for 6 weeks, or neutralized mock bacteria (prepared as described in Example 4), 6x10 ⁷ IV (tail vein) twice a week for 5 weeks.

Blood (100 μl) was collected biweekly from the retro-orbital sinus into EDTA-coated tubes. Plasma was separated from cells by centrifugation and incubated for 30 min at 56°C to inactivate HIV before freezing at -80°C. Viral RNAs were isolated from 40 μl of frozen inactivated plasma using an automated nucleic acid purification device (Arrow, NorDiag) and a viral RNA extraction kit (DiaSorin, no. 12-08-02). Plasma HIV viral load was determined by qRT-PCR using the Generic HIV Charge Virale kit (Biocentric, no. TR001-440IC, lot 0089/08A). The detection limit was set at 1000 copies/ml. Values below this threshold were considered undetected. Inhibitory activity was determined by comparing the treated groups at each time point with the vehicle group at the same time point using an unpaired, nonparametric Mann-Whitney statistical analysis. Asterisks indicate statistically significant inhibition.

Fig. 8 demonstrates that HAART therapy inhibited blood levels of HIV two weeks after initiation of treatment and up to three weeks after cessation of treatment (Fig. 8B). Mock bacteria did not inhibit HIV levels during treatment, but inhibition was observed starting four weeks after treatment stopped and lasted for eleven weeks, with the exception of week 19 (Figure 8C). The delayed inhibitory response suggested that the mimic bacteria were capable of inducing an immune response against HIV infection. One mouse death each was recorded in the Sham vehicle-treated group and the HAART-treated group. There were no deaths in the Sham-treated group.

Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by one skilled in the art to which this invention relates.

The inventions clearly described herein may be suitably practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, the terms containing, including, etc. are to be understood in a broad sense and without limitation. Further, the terms and expressions used herein are used as descriptive and non-limiting terms, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, and it is understood that various modifications are possible within the scope claimed invention.

Thus, it should be understood that while the present invention has been specifically described by preferred embodiments and optional features, modifications, improvements, and variations of the inventions disclosed herein may be made by one skilled in the art, and such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods and examples presented in this application represent preferred embodiments, are illustrative and are not intended to limit the scope of the present invention.

The invention has been described herein broadly and in general terms. Each of the more specific generic features and subgeneric groups of features covered by the general disclosure also forms part of the present invention. This includes a general description of the invention with a condition or negative limitation excluding any item of kind, regardless of whether the excluded feature is specifically mentioned in this application.

In addition, if features or aspects of the present invention are described in terms of Markush groups, those skilled in the art will appreciate that the present invention is also thus described in terms of any individual member or subset of members of the Markush group.

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Claims (7)

All publications, patent applications, patents and other references mentioned in this application are expressly incorporated by reference in their entirety to the same extent as if each had been incorporated by reference individually. In the event of any conflict, this specification, including definitions, governs. It should be understood that although the present invention has been described in connection with the above embodiments, the foregoing description and examples are intended to be illustrative and not to limit the scope of the present invention. Other aspects, advantages and modifications within the scope of the present invention will be apparent to those skilled in the art to which the present invention relates. CLAIM 1. The use of a composition for the production of a medicament for the prevention or treatment of a viral infection in a patient in need thereof, wherein said composition contains a plurality of intact and substantially non-viable gram-negative bacterial cells that have been treated in such a way as to result in a decrease in the activity of lipopolysaccharide (LPS) -associated endotoxin by 75-99% when measured using the Limulus amebocyte lysate (LAL) assay compared to untreated wild-type Gram-negative bacterial cells, wherein said bacterial cells are Salmonella or Escherichia cells, and wherein said treatment is polymyxin treatment at temperatures from 2°C to 10°C and glutaraldehyde. 2. Use according to claim 1, characterized in that said composition contains from 0.01 to 100 ng, preferably from 0.02 to 20 ng, more preferably from 0.1 to 10 ng of active LPS per kg of body weight of the specified patient. 3. Use according to claim 1 or claim 2, characterized in that said composition contains from 2 to 200 ng, preferably from 10 to 120 ng, more preferably from 20 to 100 ng of active LPS per 1x10 ⁸ cells. 4. Use according to any one of claims 1 to 3, characterized in that said intact and essentially non-viable gram-negative bacterial cells have been treated in such a way as to result in a reduction in the level of LPS-associated endotoxin by 85-98%, preferably by 90% - 98%. 5. Use according to any one of claims 1 to 4, characterized in that said viral infection is caused by hepatitis B virus (HBV) or human immunodeficiency virus (HIV). 6. Use according to any one of claims 1 to 5, characterized in that said composition additionally contains a second therapeutic agent, wherein said second therapeutic agent: a) is a cyclooxygenase inhibitor, preferably selected from the group consisting of 6MNA (6-methoxy-2 naphthylacetic acid), aspirin, carprofen, diclofenac, fenoprofen, flufenamate, flubiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, naproxen, niflumic acid, piroxicam, sulindac sulfide, suprofen, tenidap, tolmetin, tomoxiprol, zomepirac, celecoxib, etodolac, meloxicam, nimesulide, diisopropyl fluorophosphate, L745,337, NS398, rofecoxib, SC58125, S-aminosalicylic acid, amp. she, diflunisal, nabumetone , paracetamol, resveratrol, salicin, salicylic aldehyde, sodium salicylate, sulfasalazine, sulindac, tamoxifen, ticlopidine, valeryl salicylate and combinations thereof; b) is a stimulating immune checkpoint agonist, preferably selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR and ICOS; c) is an inhibitory immune checkpoint antagonist, preferably selected from the group consisting of A2AR, B7-H3, B7-H4, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, TIM-3 and VISTA ; d) selected from the group consisting of abacavir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, balavir, cidofovir, combivir, dolutegravir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvir tida, entecavir, ecoliver, famciclovir, fomivirsen, fosamprenavir, foscarnet, phosphonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitor, type III interferon, type II interferon, type I interferon, interferon, lamivudine, lopinavir, lovir ida , maraviroc, moroxidine, metisazone, nelfinavir, nevirapine, nexavir, nitazoxanide, nucleoside analogues, norvir, oseltamivir (Tamiflu®), peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitor, raltegravir, ribavirin, rimantadine, ritonavir, pyramidin, saquinavir, sofosbuvir, stavudine, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, Truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, zidovudine and combinations thereof; or f) is interferon alpha or pegylated interferon. 7. Use according to any one of claims 1-6, characterized in that said gram-negative bacteria -