WO2021258008A1

WO2021258008A1 - Compositions and methods for treating and preventing viral infection

Info

Publication number: WO2021258008A1
Application number: PCT/US2021/038117
Authority: WO
Inventors: Josh SMITH; Kenneth Hunter; Kristina KRUSE; Beth MCDOUGALL; Sally DUPREE
Original assignee: Immunacor Llc
Priority date: 2020-06-19
Filing date: 2021-06-18
Publication date: 2021-12-23

Abstract

Compositions and methods for treating and preventing viral infections using therapeutic viruses are described. These therapeutic viruses serve to activate and/or train cells of the innate immune system. Suitable therapeutic viruses include highly attenuated Poxviridae and are shown to elicit transcription of antiviral compounds in immune cells and to significantly reduce viral load in an animal model of SARS-CoV-2 infection.

Description

COMPOSITIONS AND METHODS FOR TREATING AND PREVENTING VIRAL

INFECTION

[0001] This application claims the benefit of United States Provisional Patent Application No. 63/041,247 filed on June 19, 2020. These and all other referenced extrinsic materials are incorporated herein by reference in their entirety. Where a definition or use of a term in a reference that is incorporated by reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein is deemed to be controlling.

Field of the Invention

[0002] The field of the invention is prophylaxis and treatment of viral infections.

Background

[0003] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Viruses are typically viewed as agents of disease, however some viruses have been explored as potentially useful therapeutic entities. For example, viruses that induce lysis and death in cancer cells (i.e. oncolytic viruses) have been investigated as a possible therapeutic agent for individuals with cancer. Unfortunately, such approaches have not been found to be sufficiently effective in vivo to find wide use. For example, International Patent Application Publication No. WO 2015/017915 (to Coffey et al.) proposes the use of genetically modified viruses of various species, including reovirus, Newcastle disease vims, vesicular stomatitis virus, adenovirus, vaccinia vims, herpes simplex vims, and ORF vims for the treatment of a wide variety of cancers in combination with treatment with a taxane. The application, however, is entirely prophetic and provides no evidence of actual efficacy. All publications identified herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. United States Patent No. 8,147,822 (to Bell et al.) describes the use of viruses that are not normally human pathogens to reduce the viability of tumor cells, specifically rhabdovirus, picomavirus, and vesicular stomatitis vims (VSV). Unfortunately, animal model studies indicated limited effectiveness when VSV was infused directly, with improved results seen when VSV infected cells were injected. In addition, pre-treatment with interferon appeared to be necessary in order to provide sufficient differentiation between neoplastic and normal cells. Similarly, United States Patent Application Publication No. 2003/0077819 (to Groene et al.) discusses methods in which Newcastle disease vims was administered in the form of vims-infected leukocytes or co-administered with leukocytes. Unfortunately, this application is also entirely prophetic, so there is no evidence of efficacy. It should be appreciated that a requirement for administration in the form of infected cells (or co-administration with infectable cells) greatly limits the practical application of such formulations.

[0005] As noted above, however, viral infection is typically seen as having neutral (e.g. harmless, asymptomatic infection) or harmful (e.g. disease causing) effects. Vimses typically have relatively small genomes, which provide gene products necessary for replication of the viral genome (e.g. HIV reverse transcriptase) as well viral proteins that participate in packaging and release of viral particles. Some vimses also express gene products that suppress the body’s normal defense mechanisms against viral infection, for example by dismpting apoptotic signaling within the infected cell or dismpting elements of the body’s immune response. Significant pathogens that do this include coronavimses (e.g., SARS-CoV-1, MERS, SARS- CoV-2), HCV, HIV, and influenza. Such dismptions of the body’s defenses can act to increase the severity and/or duration of viral infection, with a concomitant negative impact on the course of disease.

[0006] Coronavimses are among vimses that have been found to cross species barriers and induce moderate to severe disease in the recently transitioned species. A notable example of this is the global SARS-CoV-2 pandemic that began in 2019. While effective anti-viral treatments directed to the coronavims are in development, immunization has proven to be remarkably effective in preventing infection and reducing transmission. Unfortunately, politicization of the vaccination effort and hygiene-based efforts to reduce transmission has resulted in significant numbers of unvaccinated individuals that remain potential reservoirs for SARS-CoV-2. Symptomatic or asymptomatic infection of such unvaccinated individuals can provide both spread and a source of mutations of the SARS-CoV-2 vims, facilitating the development of strains that are resistant to conventional treatment and current vaccines.

[0007] Thus, there is still a need for safe and effective therapies that are useful in treating viral infections.

Summary of The Invention

[0008] The inventive subject matter provides compositions and methods that provide therapeutic viruses to a person in need of treatment for or prevention of infection with a pathogenic virus.

[0009] One embodiment of the inventive concept is a method of preventing and/or treating an infection by a pathogenic virus (such as a coronavirus, a hepatitis C vims, an HIV vims, an influenza vims, or SARS-CoV-2) by identifying an individual in need of treatment for infection by the pathogenic vims and administering a therapeutic vims to the individual, where the therapeutic vims is selected to stimulate innate immunity in the individual in need of treatment. The pathogenic vims can cause a dysregulation of innate immunity; the therapeutic vims is selected to overcome this dysregulation by stimulating innate immunity. The therapeutic vims is administered by application to a body surface (e.g., a mucus membrane) of the individual, application to the oral cavity of the individual, application to the gastrointestinal tract of the individual, injection into the individual, by inhalation by the individual (e.g., as a spray, mist, or suspended powder), and/or infusion into the individual. The therapeutic vims can be a poxviridae, a parapox vims, an avipox vims, or an influenza vims, and is preferably an attenuated vims. In some embodiments two or more therapeutic viruses are used in combination., and can be administered separately (e.g., on different schedules) or co administered. Such an additional therapeutic vims can be a poxviridae, a parapox vims, an avipox vims, or an influenza vims.

[0010] Another embodiment of the inventive concept is a composition for treatment of infection with a pathogenic vims, which includes an injectable formulation or an aerosol suspension of a therapeutic vims (such a coronavims, a hepatitis C vims, an HIV vims, an influenza vims, or SARS-CoV-2) in an amount that is effective to treat an individual in need of treatment for infection with the pathogenic vims. The first therapeutic virus is selected to stimulate innate immunity, and can be a poxviridae, a parapox vims, an avipox vims, or an influenza vims. The therapeutic vims is preferably an attenuated vims. The composition can be formulated for prophylactic treatment and/or treatment of an active infection. The composition can include two or more therapeutic viruses, which can be provided in combination or as a kit that includes separate vims formulations.

[0011] Another embodiment of the inventive concept is the use of a therapeutic vims treatment to treat infection by a pathogenic vims (such as coronavims, a hepatitis C vims, an HIV vims, an influenza vims, or SARS-CoV-2), where the therapeutic vims is selected to stimulate innate immunity. The therapeutic vims is administered application to a body surface (such as a mucus membrane), application to the oral cavity, application to the gastrointestinal tract, injection, inhalation (such as of a spray, mist, or powder) and/or infusion. The therapeutic vims can be a poxviridae, a parapox vims, an avipox vims, or an influenza vims, and is preferably attenuated. Treatment can be prophylactic treatment or treatment is treatment applied to active infection. In some embodiments incorporates two or more therapeutic viruses, which can be provided separately or in combination.

[0012] Another embodiment of the inventive concept is a method of enhancing innate immunity by identifying an individual in need of enhancement of innate immunity and administering a therapeutic vims selected to stimulate innate immunity to the individual. The therapeutic vims is administered by inhalation (e.g., of a mist, vapor, or powder), application to a body surface (such as a mucus membrane), application to the oral cavity, application to the gastrointestinal tract, injection, or infusion. The therapeutic vims can be a poxviridae, a parapox vims, an avipox vims, and an influenza vims, and is preferably attenuated. In some embodiments the individual additionally has a symptomatic viral infection, an asymptomatic viral infection, a chronic viral infection, and/or a chronic inflammatory condition. In some embodiments two or more therapeutic vimses are utilized, which can be provided separately or in combination. In some embodiments stimulation of innate immunity enhances a specific immune response in the individual, such as improving specificity or antibody titer of the specific immune response. [ 0013] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

Brief Description of The Drawings

[0014] FIG. 1 depicts types of epigenetic reprogramming in innate immune cells; DNA=deoxyribonucleic acid; miRNA=microRNA; RNA=ribonucleic acid.

[0015] FIG. 2 schematically depicts an enhanced innate immune response resulting from epigenetic reprogramming; M □= macrophages; NK=natural killer cells.

[0016] FIGs. 3A and 3B depict typical results of qPCR studies of cytokine gene expression in THP-1 cells in culture following treatment with therapeutic viruses of the inventive concept. Expression was characterized using an Inflammatory Gene Microarray (FIG. 3A) or an Interferon Gene TaqMan Array (FIG. 3B).

[0017] FIGs. 4A and 4B show results from animal trials of therapeutic viruses of the inventive concept. FIG. 4A schematically depicts the experimental protocol. FIG. 4B shows typical results of the application of therapeutic viruses of the inventive concept to an animal model of SARS-CoV-2 infection.

Detailed Description

[0018] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0019] The inventive subject matter compositions and methods useful for treatment of infection by a viral pathogen through application of a therapeutic virus. Such treatment can be prophylactic or directed to active infection. The therapeutic virus is selected to stimulate, enhance, or otherwise normalize one or more components of the innate immune response, and can be effective against viral pathogens that cause dysregulation of one or more aspects of the innate immune response. In preferred embodiments the therapeutic virus has been attenuated, does not result in symptomatic infection, and/or is readily treated. In some embodiments the therapeutic vims has been killed or inactivated. In other embodiments the therapeutic virus is capable of reproduction.

[0020] One should appreciate that the disclosed techniques provide many advantageous technical effects including safe and effective treatment of disease resulting from infection with a pathogenic virus.

[0021] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0022] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. [0023] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0024] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0025] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a therapeutic embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0026] Following its first safe use in 1796, the smallpox vaccination was refined and used worldwide, eventually leading to the eradication of smallpox. From the first introduction of the smallpox virus up to the time of smallpox eradication, physicians reported positive side effects in regard to other infections following a smallpox vaccination. Patients reported improvement in or elimination of wide range of disorders (from skin rashes to syphilis) reported improvement following a smallpox vaccination. More recently, researchers have observed positive indirect effects in both measles and BCG vaccination programs, with significant decreases in overall mortality that cannot be accounted for by simple prevention of the targeted disease.

[0027] Without wishing to be bound by theory, Inventors believe that such non-target beneficial effects of smallpox, measles, and BCG vaccines occur via the adaptation of the innate immune system (e.g. trained immunity) and a resultant long-term shift in cytokine balance and cell metabolism. [0028] The immune system of mammals and birds consists of an innate, primitive immune system (antigen-nonspecific or paraspecific) and an acquired or adaptive immune system (antigen specific). While distinct, these immune systems interact with each other. The innate immune system provides an immediate but broad and nonspecific defense against foreign substances, such as pathogens, toxins, and abnormal host cells. It also initiates and directs the development of the acquired immune response. The acquired immune system acts more slowly as it must first recognize each new pathogen (in the form of different antigens) and then activate various immune pathways, such as the clonal expansion of T cells and B cells, which produce antibodies tailored to that pathogen. Once the acquired immune system has encountered a pathogen, retention of specific memory B cells and memory T cells can provide rapid and increased protection when the body is re-infected with the same pathogen. Traditional immunization directed to specific infecting agents is directed to the acquired immune system and classical immunological memory.

[0029] The innate immune system adapts its function following stimulation (such as by infection and/or vaccination) and can protect the body against reinfection. This is referred to as the induction of innate immune memory or trained immunity. Recent studies have demonstrated that the basis of trained immunity is epigenetic reprogramming, that is, persistent alterations in gene expression and cell physiology that do not involve genetic changes such as mutations and recombination.

[0030] Following an initial stimulus, the innate immune system activates multiple cellular and humoral elements. While the specific pathways activated depend on the specific pathogen, the innate immune system typically activates macrophages, natural killer (NK) cells, and other innate lymphoid cells, and similarly upregulates the production of multiple cytokines in a cytokine cascade. Key cytokines that are often produced include interferons (interferon alpha [IFN-a], interferon beta [IFN-b], gamma interferon [IFN-g], interferon lambda [IFN-λ]), interleukins (IL-lb, IL-2, IL-6, IL-10, IL-12, IL-18), and tumor necrosis factor alpha (TNF-a), which interact with one another as well as activate other cytokines (Bennett, Round, and Leung, 2015) (Tannous and Ghanem, 2018). [0031] Additionally, activation of naive innate immune cells initiates epigenetic reprogramming, such as histone modification, DNA methylation, modulation of microRNA, and long-noncoding ribonucleic acid (RNA) expression (Netea et ah, 2016), as shown in FIG. 1. As a consequence of epigenetic reprogramming, these immune cells are trained. Even after the initial stimulus is cleared and the initial pro -inflammatory response has shifted to an anti-inflammatory response, the more stable epigenetic modifications can remain. Thus, when there is a secondary stimulus, the trained immune cells will respond more strongly than the naive cells, resulting in an enhanced innate immune response, as shown in FIG. 2.

[0032] Paramunity inducers or immunomodulators can be understood as vaccines that are designed to target and stimulate the innate immune system through the mechanisms described above. The overall result is improvement in immunological dysfunction and short-term protection against a wide variety of infections, as well as the ability to mount an enhanced innate immune response in the long-term.

[0033] Investigators have theorized that for some viruses (e.g., poxviruses) there can be a competition between protein epitopes responsible for paramunization and those responsible for conventional, antigen- specific immunization. Thus, the magnitude of the paramunization effect of a composition can be inversely proportional to that of the immunization effect. This is consistent with the following observations:

• Attenuation of different poxviruses over several hundred passages in cell cultures decreased the immunizing properties of the poxviruses but increased their paraspecific stimulating properties.

• Inactivation of the poxviruses (e.g., via irradiation and/or chemical treatment) caused a loss of their immunizing properties and an increase in their paramunizing properties.

Accordingly, Inventors believe that attenuation and inactivation (for example, by chemical treatment) of a viral composition can improve its properties in regard to activation and training of the paramune response.

[0034] As noted above many viral pathogens, in addition to reproducing within infected cells, generate gene products that dampen or otherwise dysregulate components of the innate immune response. Such elements include expression of peptides with antimicrobial activity, expression of soluble mediators, proteases, and expression of cell receptors. Examples of antimicrobial peptides include defensins. Examples of soluble mediators include cytokines such as TNF-a, IL- 1, IFN-a, IEN-g, IL-12, IL-15, IL-10, and TGF-b. Examples of proteases include Complement, Collectins, C reactive protein, Coagulation system proteins. Examples of cell receptors include TLRs, NLRs, CLRs, and RLRs. Viral pathogens that dysregulate the human innate immune response include, but are not limited to, hepatitis C virus (HCV), human immunodeficiency virus (HIV), and coronavirus (e.g. MERS-CoV, SARS-CoV, SARS-CoV2). Dysregulation of the innate immune response can extend survival of infected cells (thereby increasing productivity and/or burst size), and thereby increase the duration and/or severity of infection.

[0035] One embodiment of the inventive concept is a method of treating an infection by pathogenic viruses, for example pathogenic viruses that cause dysregulation of innate immunity and/or training the innate immune system, by administering a therapeutic vims that can safely and effectively correct such dysregulation to an individual that is need of treatment. Such a therapeutic vims can be selected and/or generated to enhance, increase, or otherwise normalize one or more aspects of the innate immune response. This mitigation can reduce the severity and/or duration of active, symptomatic infection, prevent or reduce the development of symptoms in an asymptomatic but infected individual, and/or prevent establishment of an infection in a non-inf ected individual upon exposure to the pathogenic vims. In some embodiments a single species or strain of therapeutic vims is administered. In other embodiments two or more species or strains of therapeutic vims is administered.

[0036] Another embodiment of the inventive concept is a method of treating an infection by pathogenic viruses that are not known to cause dysregulation of innate immunity and/or training of the innate immune system by administering a therapeutic vims that can safely and effectively provide stimulation of innate immunity and/r training of the innate immune system to an individual that is need of treatment. The therapeutic vims can be selected and/or generated to generally enhance or improve one or more aspects of the innate immune response. This enhancement or improvement can reduce the severity and/or duration of active, symptomatic infection, prevent or reduce the development of symptoms in an asymptomatic but infected individual, and/or prevent establishment of an infection in a non-infected individual upon exposure to the pathogenic virus. In some embodiments a single species or strain of therapeutic virus is administered. In other embodiments two or more species or strains of therapeutic virus is administered.

[0037] Suitable therapeutic viruses useful for methods of the inventive concept include, but are not limited to, double stranded DNA viruses (such as members of the Poxviridae family). Suitable therapeutic viruses of the Poxviridae family can belong to either the Chordopoxvirinae subfamily or the Entomopoxvirinae subfamily. Suitable therapeutic viruses of the Chordopoxvirinae subfamily can belong to the Avipoxvirus, Capripoxvirus, Centapoxvirus, Cervidpoxvirus, Crocodylidpoxvirus, Leporipoxvirus, Macropopoxvirus, Molluscipoxvirus, Mustelpoxvirus, or Parapox genera. Suitable therapeutic viruses can include wild type, mutated, attenuated, and/or genetically engineered Parapox ovis (i.e., Orf virus) and/or wild type, mutated, attenuated, and/or genetically engineered Avipox species. Specific examples, which the invention is not limited, are provided below.

[0038] An example of an embodiment of the inventive concept is a highly attenuated and then inactivated member of the poxviridae family, Parapoxvirus ovis virus ( PVI, such as strain D1701-V [DVB 13-01]), or an Avipoxvirus (AVI, such as strain fowlpox virus (isolate HP- 438[Munich]), passage 438 clone FP9 [DVB23-01]), or a mixture of these. A therapeutic virus can be generated, selected for, or otherwise provided in any suitable manner. In some embodiments the manner can be selected to address a typical point of entry for the pathogenic virus. Suitable routes for administration of a therapeutic virus include local application (for example, as a patch, a suspension, an aerosol, a lotion, a cream, or a gel) to a body surface. Suitable body surfaces include skin, the surface of the eye, and mucus membranes (e.g. mucus membranes of the upper respiratory tract, lower respiratory tract, oral cavity, urogenital system, and/or digestive tract). Another suitable route for administration is by injection (e.g. intradermal, intramuscular, intraperitoneal, or direct injection into an infected lesion) and/or infusion. Another suitable route for administration includes ingestion, for example of a liquid suspension, pill, tablet, or capsule. For example, in treating a pathogenic virus associate with upper and/or lower respiratory tract infection the therapeutic virus can be administered as an aerosol or spray that is inhaled. Similarly, in treating a pathogenic virus associated with infection of the intestinal tract the therapeutic vims(es) can be administered in the form of a delayed release pill or capsule that releases its contents into the lower digestive tract.

[0039] In other embodiments a therapeutic virus can be administered systematically. For example, a therapeutic virus can be administered by ingestion (e.g., of a liquid suspension, powder, pill, and/or capsule), injection (e.g., subcutaneous, subdermal intramuscular, and/or intraperitoneal injection), and or infusion.

[0040] A therapeutic vims can be any virus that enhances, increases, or otherwise normalizes one or more components of the innate immune response. Alternatively, or in addition, a therapeutic vims can be any vims that reduces replication, release, and/or infection by a pathogenic vims. In preferred embodiments the therapeutic vims does not cause symptomatic human infection, results in a mild infection with minimal and non-life threatening symptoms or is readily treatable. Examples of suitable viruses include a parapox vims, an avipox vims, and/or an influenza vims. The therapeutic can be provided as a ‘live’ (i.e., capable of reproduction) vims or a ‘killed’ (i.e., inactivated or incapable of reproduction) vims. In preferred embodiments a therapeutic vims is an attenuated strain (e.g., a strain prepared by repeated passage in cell culture). In some embodiments two or more of such therapeutic vimses can be used in combination.

[0041] In embodiments utilizing live therapeutic vims, the vims can be an attenuated vims. As noted above, attenuation can be performed by repeated passage through one or more cell lines, with the vims accumulating mutations that reduce its pathogenicity as it is passaged. The number of passages can be large, for example more than 100, 150, 200, 250, 300, 350, or more passages. For example, highly attenuated parapox vims strains are available through various repositories. Such attenuated parapox vims strains have been shown to stimulate innate immunity.

[0042] An example of an embodiment of the inventive concept is a highly attenuated and then inactivated Parapoxvims ovis vims ( PVI, such as strain D1701-V [DVB 13-01]) or an attenuated strain of Parapox ovis that is derived by repeated passage of the D1701 or D1701-V strain, that is used either alone or in combination with an Avipoxvims (AVI, such as strain fowlpox vims (isolate HP-438 [Munich]), passage 438 clone FP9 [DVB23-01]). Suitable formulations can provide from 106 to 1010 plaque-forming units (PFU) each of PVI and AVI in a suitable vehicle (e.g. sterile 0.8% or isotonic saline).

[0043] Inventors characterized the effects of an attenuated D1701-V strain of Parapox ovis (PPV) and an attenuated Avipox (AVI) on elaboration and expression of proteins associated with the innate immune response by the human promonocytic leukemia cell line THP- 1. The objective was to verify reports in the scientific literature that the poxviruses would stimulate human THP-1 cells to upregulate genes and secrete mediators related to the innate antiviral immune response.

[0044] The THP-1 cell line was obtained from ATCC (Rockville, MD) and maintained at 2 x 10⁵ cells/mL in RPMI 1640 medium supplemented with 10% FCS and 2 mmol/L L-glutamine. The cells grown under these conditions are phenotypically monocytes. For some experiments, THP-1 cells (2 x 10⁵ cells/mL) were differentiated from monocytes into macrophages using 162 nM (100 ng/mL) phorbol 12-myristate 13-acetate (PMA, Sigma- Aldrich) for at least 2 days. Differentiation of PMA-treated cells was completed by removing the PMA-containing media and incubating the cells in fresh RPMI 1640 (10% FCS, 1% L-glutamine) for at least 48 hours. Resting the cells without PMA assures complete differentiation of THP-1 cells to macrophages, as indicated by cell adherence, decrease in NF-KB gene clusters, high phagocytic capacity, and expression of differentiation dependent cell surface markers can be measured (e.g., CD 14, CD36, TLR-2, and CDllb/CD18) (Chanput et al. ,2014) .

[0045] Studies were performed to quantitatively evaluate the production of the following mediators by poxvirus -treated THP-1 cells differentiated into macrophages as described in Section 6.1: TNF-a, IL-6, IL-lb, IFN-a, and IFN-b. These mediators were chosen to represent three prototype inflammatory cytokines (TNF-a, IL-6, IL-lb) and two Type I interferons (IFN-a and IFN-b). Triplicate cultures of 2.0 x 10⁶ THP-1 cells were grown in 24-well plates were incubated for various amounts of time (typically 4-16 hours). Each culture was treated as described in Table 1. It should be noted that lipopolysaccharide (LPS) is utilized herein as a prototypic proinflammatory mediator. Polyinosinic: poly cy tidy lie acid (poly I:C) is a synthetic double-stranded RNA that has been used experimentally to mimic viral infections and the induction of anti- viral Type I interferons. After 4 hours of culture, supernatants were collected and tested for various mediators using a commercial ELISA.

[0046] Results from characterization of cytokines produced by treated and control THP- 1 cells using a commercial ELISA are summarized in Table 2.

a N = 3 b SD = Standard Deviation c Fold Increase = Treatment Mean/Medium Mean

Table 2 Infection with therapeutic poxviruses (PPV and/or AVI) has the greatest effect on the production of IFN-b, with some effects on all of the secreted mediators. The effect on IFN-oc was more modest than the effect on IFN-b. The prototypic inflammatory mediators LPS and b-glucan showed typical stimulation of TNF-oc, IL-6, and IL-lb, and lesser effects on the interferons. Poly-I:C, a prototypic stimulator of antiviral immunity had its greatest effect on the interferons, especially IFN-b. Of the two poxviruses, PPV was the most stimulatory. It was also notable in this experiment that the poxviruses did not induce high levels of the proinflammatoiy cytokines. This suggests that use of a therapeutic poxvirus preparation therapeutically would be unlikely to engender a "cytokine storm”.

[0047] Gene expression in THP-1 cells treated with therapeutic viruses as noted above was also characterized. To examine immune system stimulation engendered by treatment with PPV (i.e. Paravi-P) or the combination of PPV and AVI (i.e., Paravi), THP-1 cells were treated with the poxvirus preparations, total RNA was extracted from the treated cells, and the level of specific mRNAs measured utilizing RT-qPCR. In such gene expression experiments, positive controls included THP-1 cells treated with LPS or poly-I:C as described above.

[0048] Two primer arrays were used to probe immune system activation. One array was ThermoFisher’s Human Interferon Pathway TaqMan™ Array Plate 96 Plus (#4414285) which has 92 wells with the primers and probes for human interferon pathway genes and 4 reference genes. The other array was Real Time Primers’ Human Cytokine Primer Library I (Item# HCA-I) which contains 88 primer sets directed against cytokine genes and 8 housekeeping gene primer sets.

[0049] In these studies, human THP-1 monocytes, or THP-1 monocytes differentiated into macrophages were left untreated or treated with Paravi, Paravi-P, or positive controls (LPS or Poly I:C) as described in Table 1. After 3 hours, RNA was collected from the cultured cells and analyzed by RT-qPCR using either an Interferon Pathway TaqMan Array or the Human Cytokine Array. [0050] The RQ is the fold change compared to the calibrator (untreated sample THP-1 cells). The calibrator is set to an RQ value of 1. All treated samples are compared to the calibrator. When RQ values are positive (RQ > 1) it suggests up-regulation, while an RQ < 1 can suggest down-regulation. An RQ of 10 means that this gene is 10 times more expressed in the treated sample than in the control sample. An RQ of 0.1 means that the gene is 10 times less expressed. An RQ is considered significant when there is a minimum of two-fold change, i.e. RQ of more than 2 or less than 0.5. Data presented here is preliminary data testing the in vitro system, extraction method, and RT-qPCR systems.

[0051] Treated THP-1 cells were collected by pipetting off the media, adding TRIzol Reagent directly the cells in the culture plates, scraping and pipetting the cells and TRIzol into an Eppendorf tube. Samples were stored in the -20°C freezer prior to extraction. RNA extractions were run in a two-step process, the first step using the Zymo Direct-zol RNA MiniPrep Kit and the second step using the Zymo RNA Clean and Concentrator-25 Kit. A DNase treatment was included in both steps using NEB’s DNase I (cat #M0303S) for efficient removal of DNA. RNA was quantified using the QuantiFluor RNA System. Yields varied depending upon the number of cells and treatments they had received. RNA integrity (RIN) was examined by submitting samples to the Nevada Genomics Center for Agilent 6000 Nano Chip analysis with the Agilent Bioanalyzer. After establishing the two- step extraction protocol, the RINs were > 8.00.

[0052] The following primer arrays were used to probe immune system activation:

• ThermoFisher’s Human Interferon Pathway TaqMan™ Array Plate 96 Plus (#4414285) which has 92 wells with the primers and probes for human interferon pathway genes and 4 reference genes.

• Real Time Primers Human Cytokine Primer Library I (Item# HCA-I) which contains 88 primer sets directed against cytokine genes and 8 housekeeping gene primer sets. • A collection of twelve ThermoFisher TaqMan assays, shown in the table below, to focus in on nine interferon genes and including three endogenous controls GAPDH, HPRT1, and GUSB.

Table 3 provides details of nine Interferon TaqMan assays and three Endogenous control assays used.

[0053] Reverse Transcription (RT) to convert mRNA to cDNA was accomplished using ThermoFisher’s Superscript™ IVVILO™ (SSIV VILO) Master Mix (cat #11756050) which uses random primers and oligo (dT) 20. The cDNA was not quantified but moved directly to qPCR. Depending upon the assay being employed, qPCR was set-up with either ThermoFisher’s TaqMan Gene Expression Master Mix (cat #4369510) for the TaqMan arrays, or ThermoFisher’s PowerUp™ SYBR Green Master Mix (cat #A25780) for the Human Cytokine Primer Library. Ah qPCR was run on the QuantStudio3 platform. When TaqMan assays were run the data was analyzed using ThermoFisher’s Expression Suite software setting for the Singleplex Analysis calculations using the standard deviation 1 to determine the RQ Min and Max values, Benjamini-Hochberg false discovery rate to adjust p-values and setting the maximum Ct at 40 to include those with Ct over 40 in the calculations. [0054] In a typical study RT was started using 260ng of RNA per reaction and utilized the collection of twelve ThermoFisher TaqMan assays, shown in the Table 3 above, using RNA extracted from duplicate samples of PMA differentiated THP-1 cells treated with the immune system inducers listed below:

• Medium only

• LPS (lOng/mL)

• Poly I:C (10pg/mL)

• Glucan (5 particles/cell)

• Paravi (PPV and AVI, 10 virions/cell)

• Paravi (PPV and AVI, 5 virions/cell)

• Paravi-P (PPV, 10 virions/cell)

• Paravi-P (PPV, 5 virions/cell)

[0055] Table 4 shows the results for the three control immune system inducers, LPS and Poly I:C.

[0056] Typical data (generated using 260 ng/well of starting material) for stimulation using Paravi or Paravi-P induction is shown in Table 5. In studies loading 26ng/well the Paravi showed induction of IFNL1 and IL6, and loading 130ng/well the Paravi showed induction of IFNL2-3. Overall, it is apparent there is a trend for induction of IFNL1, IFNL2- 3, and IL6. Interestingly, a half dose Paravi (Paravi-0.5) does not generate the same induction trend. Paravi-0.5 shows induction of IFNG for both duplicates; however only one of the duplicate samples also shows induction of IFNL2 and IFNL4. Inventors believe that this is indicative of a dose-related response.

[0057] This shows that PPV (Paravi-P) and a combination of PPV and AVI (Paravi) can stimulate an antiviral interferon response in human monocytes. The data indicates that both Paravi and Paravi-P can induce IFNL1 and IFNL2-3.

[0058] In other studies, human THP-1 monocytes growing in culture were left untreated or treated with PPV (5 virions/cell), a combination of PPV and AVI (Paravi, 5 virions/cell), or the positive controls lipopolysaccharide (LPS, 10 ng/mL) and polyinosinic:polycytidylic acid (Poly I:C, 10 pg/mL). After 3 hours, RNA was collected from the cultured cells and analyzed by RT-qPCR using an Inflammatory Gene Microarray. The data shows that several genes related to the cytokine pathway were upregulated (RQ > 2) by both PPV and Paravi (FIG. 3A). In a similar study, human THP-1 monocytes growing in culture were treated as described and, after 3 hours, RNA was collected from the cultured cells and analyzed by RT- qPCR using an Interferon Gene TaqMan Array. The data shows that several genes related to the interferon signaling pathway were upregulated (RQ > 2) by both PPV and Paravi (FIG. 3B).

[0059] Direct antiviral effects of therapeutic viruses of the inventive concept were demonstrated using an animal model of SARS-CoV-2 infection. A typical treatment schedule is shown inf FIG. 4A. Syrian Hamster (Golden)were treated by intranasal route with 10,000 TCIDso of SARS-CoV-2 Hong Kong strain, then treated with either PPV or a combination of PPV and AVI administered intranasally (i.n.)or by intraperitoneal injection (i.p.)on days 2 and 5 post-infection with SARS-CoV-2. These untreated animals shed SARS-CoV-2 starting at day 2, and can infect contact animals. This is a model of mild disease in humans. Animals were sacrificed and tissues collected for characterization on day 8 post-infection with SARS-CoV- 2.

[0060] FIG. 4B shows the results of quantitative studies of SARS-CoV-2 virus recovery from lung tissue of untreated control animals and animals treated with PPV, AVI, and a combination of PPV and AVI. As shown, animals treated with a combination of PPV and AVI by intraperitoneal injection showed a large decrease (i.e., greater than 10-fold) in SARS- CoV-2 in their lung tissue. This is indicative of a strong synergistic effect between PPV and AVI under these conditions. In view of the demonstration of induction of anti-viral mediators in cell-based studies by both PPV and AVI, Inventors believe that similar effects can be provided using PPV, AVI, and/or other therapeutic poxviruses individually or in combination on optimization of dosage and administrative route. Similarly, Inventors believe that other routes of administration as detailed above can be effective upon further optimization of the therapeutic virus or combination of therapeutic viruses.

[0061] It should be appreciated that the amount of therapeutic virus utilized in treatment is dependent upon a variety of factors, including the therapeutic virus selected, the pathogenic virus being treated, the form in which the therapeutic virus is applied, and the nature of the intervention. Generally, it is considered that the therapeutic virus can be applied in concentrations ranging from 10 to 10¹⁰ viral particles per cubic centimeter of media (e.g. liquid volume of a droplet in an aerosol or suspension, volume of a liquid, gel or solid). For example, application as a spray or mist that is inhaled can require higher concentrations of therapeutic vims particles than application by infusion due to the much smaller volume delivered. Similarly, the amount of killed therapeutic vims used in treatment can be greater than that of live therapeutic vims, as the live therapeutic vims can have the capacity to reproduce within the individual being treated. Similarly, an effective amount of a therapeutic vims provided at an early and/or asymptomatic stage of infection with a pathogenic vims can be less than the amount that is effective at a later and more severe stage of infection.

[0062] As noted above treatment of the pathogenic vims is inclusive of prophylaxis, in which treatment with the therapeutic vims stimulates an innate immune response that prevents infection of the individual by a pathogen. The route for administration of the therapeutic vims can be selected based on the route for entry of the pathogenic vims. For example, inhalation of a spray or mist that incorporates a therapeutic vims can be effective in reducing the rate of infection by a pathogenic vims that infects or enters through the respiratory tract. Such prophylaxis can be applied prior to exposure to the pathogenic vims. For example, treatment with a therapeutic vims can provide effective prophylaxis when applied a month, a week, a day, an hour, less than five minutes, or immediately prior to exposure to exposure to the pathogenic vims.

Alternatively, treatment with a therapeutic vims can provide effective prophylaxis when applied following exposure or suspected exposure to a pathogenic vims. For example, effective prophylaxis can be provided by treatment with a therapeutic vims immediately following, within about 5 minutes, or within about 1 to 2 hours following exposure or suspected exposure to a pathogenic vims. It should be appreciated that this is different and distinct from conventional immunization to elicit a specific antibody response to the pathogenic vims, and that the therapeutic vims and the pathogenic vims can be different vims species or different vims strains.

[0063] Similarly, the innate immune system of an individual can be ‘trained’ by application of an attenuated and inactivated vims (or a combination of such vimses) to an individual. For example, such training can occur by epigenetic modification of cells of the innate immune system following exposure to one or more attenuated and inactivated vimses (e.g. a parapox vims and/or an avipox vims). Such training can provide a more rapid, vigorous, and/or effective innate immune response upon exposure of a treated individual to a pathogen (relative to an untreated individual). [0064] The Inventors believe that compositions that include one or more highly attenuated and/or inactivated viruses can also be useful in the treatment of any disease state or condition that can benefit from enhancement or training of the innate or specific immune responses. Such conditions can include chronic inflammatory conditions (e.g. inflammatory changes resulting from chronic viral infection), as well as bacterial infection, fungal infection, parasitic infection and/or their sequelae,

[0065] In some embodiments of the inventive concept the therapeutic vims is applied for treatment active infection. Such active infection can be asymptomatic or symptomatic. Such symptomatic infections can be mild or severe. The route of administration and/or dosage of the therapeutic vims can be selected based on the tissues involved and/or the severity of the disease. For example, a severe pulmonary infection can be treated by a combination of inhalation of an aerosol of the therapeutic vims in combination with an infusion of the same or a different therapeutic vims. Alternatively, an asymptomatic or mildly symptomatic individual infected with the same pathogenic vims can be effectively treated by application of inhalation of a spray or mist containing then therapeutic vims.

[0066] A treatment schedule for application of a therapeutic vims can range from single application or limited multiple applications to continuous (i.e. a continuous infusion, application of a mist through a ventilator) or periodic (application once a month, once a week, 2-3 times a week, daily, every 12 hours, every 8 hours, every 4 hours, or more frequently). Similarly, application of the therapeutic vims can be time delimited (e.g. for pathogenic vimses that result in infection that is eventually cleared, such as MERS-CoV, SARS-CoV, and SARS-CoV-2), and halted once the individual has sufficiently reduced symptoms or a sufficiently reduced pathogenic vims count. Alternatively, treatment with the therapeutic vims can be extended (e.g., for 6 months, a year, 2-5 years, 10 years, or over the life of the patient) where infection with the pathogenic vims is chronic (e.g. HIV, HBV, HCV, HSV).

[0067] In some embodiments two or more therapeutic vimses can be used in combination. Without wishing to be bound by theory the inventors believe that such a combination can provide a synergistic effect, in which the combined effect of the therapeutic vimses exceeds the sum of the effects of application of the individual therapeutic viruses when applied in similar amounts.

[0068] As noted above, viruses utilized in compositions of the inventive concept can be attenuated and/or inactivated. In some embodiments an attenuated virus is a result of an extensive attenuation process (e.g. over 300 passages) to substantially reduce virulence. In addition to the attenuation, an inactivation step can be applied to eliminate infectivity while maintaining a high degree of effect. An example of such an inactivation step is the use of 0.05% b-propiolactone (BPL). BPL acts on viral nucleic acids, alkylating the carboxyl- and hydroxyl- groups, thus making the virus replication incompetent (Chowdhury et ak, 2015). Characteristics of exemplary parapox virus (PVI) and avipox virus (AVI) characteristics so treated are summarized in Table 6.

[0069] In other embodiments one or more therapeutic viruses can be used in combination with conventional antiviral therapy. For example, a therapeutic virus can be used in combination with a known antiviral compound such as ribavirin. In some embodiments of the inventive concept a therapeutic antibody is provided in combination with an antibiotic and/or an anti-inflammatory drug. Such combinations can be provided by co-administration of different formulations, which can be provided on the same or different schedules. Alternatively, such co-administration can be provided as a single formulation that combines the effective components. Without wishing to be bound by theory the inventors believe that such a combination can provide a synergistic effect, in which the combined effect of the therapeutic vims and non-vims components of such therapy exceeds the sum of the effects of application of the individual viral and non-viral components when applied in similar amounts or in sub combinations.

[0070] In some embodiments of the inventive concept, enhancement or training of the innate immune response through application of one or more therapeutic vims(es) can be utilized to provide an improved or enhanced specific immune response in an individual so treated (relative to an immune response generated by the same specific antigenic stimulus provided in the absence of enhancement of the innate immune response). Such a specific immune response can be elicited by natural or environmental exposure to the antigen, or can be a result of inoculation (e.g. prophylactic immunization, controlled exposure, etc.). Suitable antigens that can generate a specific immune response include pathogens (e.g. a pathogenic virus, bacteria, fungus, or parasite), allergens (e.g. pollen, dust components, food allergens, venoms, etc.), and native antigens (molecules naturally present in the individual).

[0071] In some embodiments the enhancement to the immune response provides improved specificity of the immune response, narrowing the range of haptenic sites to which specific antibodies develop. Similarly, titer of specific antibodies can be increased. Inventors believe that such improved specificity can be realized through treatment with two or more attenuated and/or inactivated therapeutic viruses in combination, which can provide a synergistic effect (i.e. greater than additive effect) relative to the contributions of the individual therapeutic viruses.

[0072] As noted above, some embodiments of the inventive concept incorporate the use of two or more therapeutic antibodies. In some embodiments two or more therapeutic viruses that are to be used in combination can be provided as a single formulation. In other embodiments, subsets of the two or more therapeutic antibodies can be provided as separate formulations. This advantageously provides for different storage media and or conditions for therapeutic viruses with different stabilities under preferred storage conditions. For example, one or more therapeutic viruses can be provided in a fluid suspension, whereas a second therapeutic virus or set of therapeutic viruses can be provided as a lyophilized powder (e.g., to be reconstituted prior to use). Such separate formulations can be provided as a kit that includes two or more separate and distinct therapeutic virus formulations. Such formulations can be used in concert (for example, by blending prior to use), or can be applied separately. It should be appreciated that such separate application provides for different dosing schedules for components of such a kit or therapy.

[0073] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refer to at least one of something selected from the group consisting of A, B, C .... and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

Claims

CLAIMS What is claimed is:

1. A method of treating an infection by a pathogenic vims, comprising: identifying an individual in need of treatment for infection by the pathogenic virus; and administering a first therapeutic virus to the individual, wherein the therapeutic vims is selected to stimulate innate immunity.

2. The method of claim 1, wherein the pathogenic vims causes a dysregulation of innate immunity, wherein the therapeutic vims is selected to overcome the dysregulation by stimulating innate immunity.

3. The method of claim 1, wherein administration comprises at least one of application to a body surface, application to the oral cavity, application to the gastrointestinal tract, injection, and infusion.

4. The method of claim 1, wherein the body surface is a mucus membrane.

5. The method of one of claims 1 to 4, wherein the first therapeutic vims is applied as a spray or mist that is inhaled.

6. The method of one of claims 1 to 5, wherein the first therapeutic vims is selected from the group consisting of a poxviridae, a parapox vims, an avipox vims, and an influenza vims.

7. The method of one of claims 1 to 6, wherein the first therapeutic vims is an attenuated vims.

8. The method of one of claims 1 to 7, wherein the pathogenic vims is selected from the group consisting of a coronavims, a hepatitis C vims, an HIV vims, and an influenza vims.

9. The method of claim 8, wherein the pathogenic vims is SARS-CoV-2.

10. The method of one of claims 1 to 9, wherein treatment is prophylactic treatment.

11. The method of one of claims 1 to 9, wherein treatment is treatment applied to active infection.

12. The method of one of claims 1 to 9, wherein the individual in need of treatment is asymptomatic.

13. The method of one of claims 1 to 12, comprising application of a second therapeutic virus.

14. The method of claim 12, wherein the second therapeutic virus is co-administered with the first therapeutic vims.

15. The method of claim 13 or 14, wherein the second therapeutic virus is selected from the group consisting of a poxviridae, a parapox vims, an avipox vims, and an influenza vims.

16. A composition for treatment of infection with a pathogenic vims, comprising an injectable or an aerosol suspension of a first therapeutic vims in an amount effective for treatment of an individual in need of treatment for infection with the pathogenic vims, wherein the first therapeutic vims is selected to stimulate innate immunity.

17. The composition of claim 16, wherein the therapeutic vims is selected from the group consisting of a poxviridae, a parapox vims, an avipox vims, and an influenza vims.

18. The composition of claim 16 or 17, wherein the therapeutic vims is an attenuated vims.

19. The composition of one of claims 16 to 18, wherein the pathogenic vims is selected from the group consisting of a coronavims, a hepatitis C vims, an HIV vims, and an influenza vims.

20. The composition of claim 19, wherein the pathogenic vims is SARS-CoV-2.

21. The composition of one of claims 16 to 20, wherein treatment is prophylactic treatment.

22. The composition of one of claims 16 to 20, wherein treatment is treatment applied to active infection.

23. The composition of one of claims 16 to 22, comprising a second therapeutic vims.

24. The composition of claim 23, wherein the second therapeutic vims is provided in combination with the first therapeutic vims.

25. The composition of claim 23 or 24, wherein the second therapeutic virus is selected from the group consisting of a second poxviridae, a second parapox virus, a second avipox virus, and a second influenza virus.

26. Use of a first therapeutic virus in treatment of an infection by a pathogenic virus, wherein the first therapeutic virus is selected to stimulate innate immunity.

27. The use of claim 26, wherein the first therapeutic virus is administered by at least one of application to a body surface, application to the oral cavity, application to the gastrointestinal tract, injection, and infusion.

28. The use of claim 27, wherein the body surface is a mucus membrane.

29. The use of one of claims 26 to 28, wherein the first therapeutic virus is provided as a spray or mist that is suitable for being inhaled.

30. The use of one of claims 26 to 29, wherein the first therapeutic virus is selected from the group consisting of a poxviridae, a parapox virus, an avipox virus, and an influenza virus.

31. The use of one of claims 26 to 30, wherein the first therapeutic virus is an attenuated virus.

32. The use of one of claims 26 to 31, wherein the pathogenic virus is selected from the group consisting of a coronavirus, a hepatitis C virus, an HIV virus, and an influenza virus.

33. The use of claim 32, wherein the pathogenic virus is SARS-CoV-2.

34. The use of one of claims 26 to 33, wherein treatment is prophylactic treatment.

35. The use of one of claims 26 to 34, wherein treatment is treatment applied to active infection.

36. The use of one of claims 26 to 35, comprising use of a second therapeutic virus.

37. The use of claim 36, wherein the second therapeutic virus is provided in combination with the first therapeutic virus.

38. The use of claim 36 or 37, wherein the second therapeutic virus is selected from the group consisting of a second poxviridae a second parapox virus, a second avipox virus, and a second influenza virus.

39. A method of enhancing innate immunity, comprising: identifying an individual in need of enhancement of innate immunity; and administering a first therapeutic virus to the individual, wherein the therapeutic virus is selected to stimulate innate immunity.

40. The method of claim 39, wherein administration comprises at least one of application to a body surface, application to the oral cavity, application to the gastrointestinal tract, injection, and infusion.

41. The method of claim 40, wherein the body surface is a mucus membrane.

42. The method of one of claims 39 to 41, wherein the therapeutic virus is applied as a spray or mist that is inhaled.

43. The method of one of claims 39 to 42, wherein the first therapeutic virus is selected from the group consisting of a poxviridae, a parapox virus, an avipox virus, and an influenza virus.

44. The method of one of claims 39 to 43, wherein the first therapeutic virus is an attenuated virus.

45. The method of one of claims 39 to 44, wherein the individual has a condition selected from the group consisting of a symptomatic viral infection, an asymptomatic viral infection, a chronic viral infection, and a chronic inflammatory condition.

46. The method of one of claims 39 to 44, comprising application of a second therapeutic virus.

47. The method of claim 46, wherein the second therapeutic virus is co-administered with the first therapeutic virus.

48. The method of claim 46 or 47, wherein the second therapeutic virus is selected from the group consisting of a second poxviridae, a second parapox virus, a second avipox virus, and a second influenza virus.

49. The method of one of claims 39 to 48, wherein stimulation of innate immunity enhances a specific immune response in the individual.

50. The method of claim 48, wherein targeting, specificity, or antibody titer of the specific immune response is improved.