OA21061A - Parapoxvirus for conditioning for and treatment of coronavirus infections. - Google Patents

Abstract

The present invention relates to the treatment of coronavirus infections and to preparing subjects for such an infection by administering a parapoxvirus. This treatment is to assist the immune system in combatting the virus and to thereby prevent and to ameliorate symptoms of coronavirus disease.

Description

PARAPOXVIRUS FOR. CONDITIONING FOR AND TREATMENT OF CORONAVIRUS INFECTIONS

FIELD OF THE INVENTION

The présent invention relates to the treatment of coronavirus infections and to preparing subjects for such an infection by administering a parapoxvirus. This treatment is to assist the 10 immune system in combatting the virus and to thereby prevent and to ameliorate symptoms of coronavirus disease.

BACKGROUND OF THE INVENTION

Coronaviruses are enveloped, positive-sense singie-stranded RNA viruses, which cause diseases in mammals and birds. In humans, they cause respiratory infections, including the common cold, aithough some viruses such as SARS and MERS coronaviruses can be léthal.

Novel coronaviruses emerge periodically in different areas around the world. SARS-CoV (severe acute respiratory syndrome coronavirus) occurred in 2002 and caused 916 deaths during 20 the épidémie, having a mortality rate of about 12%. MERS-CoV (middle east respiratory syndrome coronavirus) emerged in 2012, with 543 dead reported and a mortality rate of about 39%.

Recently, an outbreak of a coronavirus designated SARS-CoV-2 began in Wuhan, China, and has sînee spread ail over the world. The illness caused by SARS-CoV-2, COVID25 19 (coronavirus disease 2019) was declared to be a pandémie by the WHO on March 11,2020.

COV1D-19 ranges in severity from asymptomatic or mild to severe, and a significant portion of patients with clinically évident infection develop severe disease. The case fatalîty rate varies by country and is thought to be between 2% and 3%, but the true mortality rate is uncertain, because the total number of cases îs unknown. While SARS-CoV-2 apparently has a lower 30 mortality rate than SARS-CoV and MERS-CoV, it appears to be more infectious and has been able spread around the world, with around 3.66 million confirmed cases and 257,000 confirmed deaths by May 6, 2020 and presumably many more unconfirmed ones. The knowledge of COVID-19 is incomplète and evolving.

The récurrent coronavirus épidémies show that these viruses continue to threaten the world as they emerge spontaneously, spread easily and can hâve severe conséquences on human life, including not only health issues but also dramatic social and économie impacts. Therefore, there is a great medical need for prévention and treatment. However, since coronaviruses are known to mutate and recombine often, they présent an ongoing challenge for disease control. Neither vaccines nor antiviral drugs for preventing or treating human coronavirus infections are currently available. A vast number of antivirals are being tested around the world for an effect on coronavirus, with promising candidates failing.

Unexpectedly, the inventors found parapoxvirus to be useful for conditioning subjects for and treating coronavirus infections. Parapoxvirus ovis (PPVO) causes acute dermal infections in goat and sheep, and while it leads to no serious disease in humans, it induces a complex înnate and adaptive immune response in humans and treats infections with some spécifie viruses such as HBV (WO 2019/048640 Al). However, the immune response induced by parapoxvirus is complex and not necessarily useful for treating viral infections in general. Therefore, parapoxvirus could not hâve been expected to hâve utility in conditioning subjects for and treating coronavirus infections.

SUMMARY OF THE INVENTION

In a first aspect, the présent invention relates to a parapoxvirus agent for use in (i) conditioning a subject for a coronavirus infection or (îî) treating a coronavirus infection in a subject. In a second aspect, the invention relates to a composition or a kit comprising a parapoxvirus agent and a coronavirus agent. In a third aspect, the présent invention relates to a pharmaceutical composition or a kit comprising a parapoxvirus agent for the medical use according to the invention, wherein the pharmaceutical composition or the kit comprises a parapoxvirus agent and preferably a further médicament.

LEGENDS TO THE FIGURES

Figure 1: Stimulation of inactivated parapoxvirus (iPPVO) induced antiviral activity against SARS-CoV-2 that was enhanced by combinatorial approaches. Wholeblood cultures were stimulated with iPPVO +1- 5 pg/ml ConA. Where no ConA was added,

PBS was added instead. iPPVO vehicle +/- ConA or PBS +/- ConA, respectively, served as

Controls. 5 μΐ/ml ConA was tested as a co-stimulus. Cultures were incubated for 3 days and supernatants were harvested. The supematant was transferred to Vero-eGFP cells which were

2-4 hours later infected with SARS-CoV-2 to détermine the antiviral activity of the respective supematant samples and incubated for 5 days. eGFP fluorescence as a measure for living cells was analyzed using the ImageJ software. The mean of the cell control to which no virus was added served as positive control (CC mean) and was set to 100 % (see upper dotted line). Cells infected with SARS-CoV2 but not treated gave the highest possible destruction of living cells and served as négative control given as the mean virus control (VC mean). The antiviral activity ofthe samples was calculated relative to the positive control CC mean and given in %.

Figure 2: iPPVO DI701 and iPPVO NZ2 stimulate antiviral activity against SARSCoV-2. Whole-blood cultures were stimulated with iPPVO. iPPVO vehicle served as a négative control. iPPVO DI701 served as positive control. Cultures were incubated for 3 days and supernatants were harvested. The supematant was transferred to Vero-eGFP cells which were 2-4 hours later infected with SARS-CoV-2 to détermine the antiviral activity residing in the respective supematant samples and incubated for 5 days. eGFP fluorescence as a measure for living cells which hâve been protected against SARS-CoV-2 due to the antiviral activity of cytokines in the respective supematant was measured using the ImageJ software. The antiviral activity of the positive control was set to 100 % (see upper dotted line). The antiviral activity ofthe samples was calculated relative to the positive control and given in %. The lower dotted line marks the value of the négative control.

Figure 3: Lung titres of infectious SARS-CoV-2 particles in hamsters treated with iPPVO NZ2. Each dot represents an animal of the respective group. Horizontal Unes represent means.

Figure 4: Survival of mice treated with iPPVO DI701 and infected with SARSCoV-2. Animais reaching humane endpoints were euthanized. Overlappîng lines are partially offset for better readability. Statistical évaluation was performed by Log-rank (Mantel-Cox) test, * indicates signifîcant différence at p < 0.05.

Figure S: Body weight of mice treated with iPPVO D1701 and infected with SARSCoV-2. Animais reaching humane endpoints were euthanized and are marked by a cross (f). Data are presented as means ± standard errors. Statistical évaluation was performed by Multiple t-test (Holm-Sidak corrected), ** indicates signifîcant différence at p < 0.01.

Figure 6: Clinical score of mice treated with iPPVO DI701 and infected with SARS-CoV-2. Animais reaching humane endpoints were euthanized and are marked by a cross (f). Data are presented as means ± standard errors. Statistical évaluation was performed by

Multiple t-test (Holm-Sidak corrected), * and ** indicate significant différence at p < 0.05 and p < 0.01, respectively.

Figure 7; Quantification of SARS-CoV-2 viral loads in mice lungs and brains. Data points represent the viral copy number of each animal with géométrie mean of each group. Each point represents one mouse. Réduction in viral load of iPPVO treated mice (left) is shown in fold réduction compared to placebo control (right). Statistical évaluation of the data was performed by Mann-Whitney U test in comparison to placebo control (ns: non-significant, **: p < 0.01 ).

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, ail technical and scientific terms used herein hâve the same meanings as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in “A multilîngual glossary of biotechnologîcal terms: (IUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Kôlbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Several documents are cited throughout the text of this spécification. Each of the documents cited herein (including ail patents, patent applications, scientific publications, manufacturer^ spécifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety.

In the following, the éléments of the present invention will be described. These éléments are listed with spécifie embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments, which combine the explicitly described embodiments with any number of the disclosed and/or preferred éléments. Furthermore, any permutations and combinations of ail described éléments in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Throughout this spécification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising , are to be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. In preferred embodiments, “comprise” can mean “consistof”. As used in this spécification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents, unless the content clearly dictâtes otherwise.

[n a first aspect, the present invention relates to a parapoxvirus agent for use in (i) conditionîng a subject for a coronavirus infection or (ii) treating a coronavirus infection în a subject.

Parapoxvirus agent

The parapoxvirus is, în a preferred embodiment, Parapoxvirus ovis (PPVO). This includes any PPVO strain, preferably, any straîn of the species PPVO as classified by the International Committee on Taxonomy of Viruses (ICTV). Exemplary strains are NZ2, NZ7, NZ10, DI 701, OV/20, OV/7, OV/C2, OV/mi-90, OV-Torino, SAOO, Bo29, orfl 1, Greek orf strain 155, and Greek orf strain 176. Preferred strains are NZ2 and DI 701, especially NZ2.

The parapoxvirus agent is, in a preferred embodiment, (a) the parapoxvirus itself or a fragment thereof, or (b) an agent that comprises genetic information encoding for (a).

In a more preferred embodiment, it is (i) a live parapoxvirus virion, (ii) an inactivated parapoxvirus virion, (iii) a fragment of (i) or (ii), (iv) a nucleic acid encoding for any of (i) to (iii), (v) a vector comprising the nucleic acid of (iv), or (vi) a cell comprising the nucleic acid of (iv) or the vector of (v).

The live parapoxvirus virion may or may not be attenuated. Attenuated parapoxvirus virions are known in the art, e.g. virions lacking a virulence gene such as vegf-e and/or gif. The fragment can be recognized by the immune system and preferably stimulâtes an immune response (i.e. it is an antigen and preferably an immunogen) and can be any fragment, although preferably it is a fragment that is bound by a human pattern récognition receptor (PR.R), e.g. TLR9. In one embodiment, the parapoxvirus fragment is a parapoxvirus protein.

Exemplary fragments are (exemplary coding sequence (nucléotide positions of SEQ ID

NO: 3 representing strain NZ2) in parenthesis preceding the fragment): (3 to 539) ORF L1, (781 to 449) ORF L2r, (1933 to 1664) ORF L3r, (3269 to 2790) ORF L4r, (2799 to 3851) ORF L5, (2962 to 3753) ORF L6, (3784 to 3122) ORF L7r, (4341 to 4129) ORF L8r, (4904 to 4428)

ORF lar, (6517 to 4970) ORF 1 r, (8042 to 6684) ORF 2r, (9989 to 8070) ORF 3r, (11195 to 10062) ORF 4r,(l 1493 to 11227) ORF 5r, (11802 to 12038) ORF 6, (12358 to 12080) ORF 7r, (13980 to 12364) ORF 8r, (14826 to 14053) ORF 9ar, (15080 to 15394) ORF 10, (16838 to 15423) ORF Hr, (19021 to 16847) ORF 12r, (19704 to 19156) ORF i3r, (20314 to 19736) ORF 14r, (20401 to 22101) ORF 15, (22125 to 22940) ORF 6, (23003 to 23866) ORF 17, 10 (26908 to 23873) ORF 18r, (26926 to 27213) ORF 19, (27626 to 27216) ORF 20r, (29754 to

27616) ORF 21 r, (32217 to 29800) ORF 22r, (33380 to 32418) ORF 23r, (33602 to 33393 ORF 24r, (34466 to 33612) ORF 25r, (34735 to 34502) ORF 26r, (35905 to 34739) ORF 27r, (37194 to 35905) ORF 28r, (37200 to 39248) ORF 29; 41037 to 39229) ORF 30r, (41374 to 42066) ORF 31, (42336 to 41731) ORF 32r, (42407 to 41997) ORF 33r, (42410 to 43765) ORF 34, 15 (43770 to 43958) ORF 35, (43980 to 44534) ORF 36, (45727 to 44537) ORF 37r, (45760 to

46557) ORF 38, (46567 to 47568) ORF 39, (47572 to 48303) ORF 40, (48352 to 48621) ORF 41, (49887 to 48634) ORF 42r, (49917 to 50793) ORF 43, (50719 to 51 102) ORF 44, (51059 to 51511) ORF 44a, (51584 to 52591) ORF 45, (52509 to 53066) ORF 46, (53523 to 53023) ORF 47r, (53607 to 57473) ORF 48, (58070 to 57528) ORF 49r, (57700 to 58662) ORF 50, 20 (59674 to 58673) ORF 51r, (62089 to 59678) ORF 52r, (62198 to 62881) ORF 53, (62909 to

63862) ORF 55, (63858 to 64271) ORF 56, (64309 to 66831) ORF 57, (67266 to 66799) ORF 58r, (67803 to 67273) ORF 58ar, (67915 to 68607) ORF 59, (68624 to 70984) ORF 60, (70994 to 72898) ORF 61, (72938 to 73507) ORF 62, (73540 to 74211) ORF 63, (76120 to 74207) ORF 64r, (76749 to 76186) ORF 65r, (77698 to 76799) ORF 66r, (79343 to 77709) ORF 67r, 25 (79816 to 79367) ORF 68r, (80529 to 79858) ORF 69r, (80774 to 80529 ORF 70r, (82815 to

80788) ORF 71 r, (83835 to 82834) ORF 72r, (83874 to 85583) ORF 73, (85535 to 84402) ORF 74r, (88096 to 85574) ORF 75r, (87759 to 88667) ORF 76, (88920 to 88642) ORF 77r, (91652 to 88938) ORF 78r, (91667 to 92674) ORF 79, (93466 to 92681) ORF 80r, (93761 to 93486) ORF 81 r, (94060 to 93788) ORF 82r, (94238 to 94080) ORF 83r, (94508 to 94242) ORF 84r, 30 (95571 to 94498) ORF 85r, (96187 to 95600) ORF 86r, (96202 to 97665) ORF 87, (97915 to

97643) ORF 88r, (98251 to 99537) ORF 89, (99537 to 99974) ORF 90, (l 00001 to 101140)

ORF 91, (101168 to 104650 ORF 92, (106354 to 104795) ORF 93r, (107947 to 106400) ORF

94r, (108256 to 107990) ORF 95r, (108719 to 108300) ORF 96r, (109679 to 108738) ORF 97r, (109861 to 109682) ORF 98r, (110830 to 10033) ORF 99r, (110208 to 110417) ORF 100, (110469 to 110651) ORF 100a, (110915 to 111397) ORF 101, (111419 to 111913 ORF 102, (111949 to 112485) ORF 103, (112593 to 113450) ORF 104, ¢113323 to 112967) ORF I05r, (113526 to 114152) ORF 106, (114199- to 115236) ORF 107, (115353 to 115787 ORF 108, (115859 o 116551) ORF 109, (116729 to 117523) ORF 110, (117572 to 1171 14) ORF 111 r, 5 (117423 to 118085) ORF 12, (118968 to 118375) ORF 114r, (118508 to 119119) ORF’ 115, (119588 to 120202) ORF 116, (120314 to 21231) ORF 117, (121380 to 123920) ORF118, (121288 to 122256) ORF 119, (122350 to 123924) ORF 120, (123962 to 125566) ORF121, (125193 to 124591)ORF 122r, (125689 to 123935 ORF 123r, (123839 to 123297) ORF 123ar, (125652 to 126170) ORF 124, (126121 to 125699) ORF 125r, (126279 to 127769) ORF126, (127851 to 128408) ORF 127, (128520 to 130076) ORF 128, (130105 to 131700) ORF129, (131790 to 133283) ORF 130, (133246 to 133920) ORF 131, (133972 to 134370) ORF132, (134418 to 134693) ORF 133a, (134402 to 134992) ORF Ri, (134853 to 134419) ORF R2r, (135628 to 135897) ORF R3, (136780 to 137112) ORF R4, and (137558 to 137022) ORF R5r. The group of fragments includes homologs of the exemplary fragments listed above, i.e.

homologous fragments of another parapoxvirus (e.g. species or strains as described above). It also includes functional variants of the exemplary fragments listed above (the function being as defined for the fragments generally above). Thus, the group of fragments includes variants with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or preferably at least 99% sequence identity to those fragments encoded by the exemplary coding sequences of SEQ ID NO: 3 listed above.

The inactivated parapoxvirus virion can be obtained by any means known in the art suitable for inactivating enveloped viruses, for example by physical inactivation (e.g. exposure to heat, specifically pasteurization, or UV light) or by Chemical inactivation (e.g. by exposure to low pH, to a detergent or to an inactivation agent such as ethyleneimine, binary ethyleneimine, formaldéhyde, glutaraldehyde, 2,2'-dithiodipyridine, or beta-propiolactone). In 25 a preferred embodiment, the inactivated parapoxvirus virion is an ethyleneimine- or binary ethyleneimine-inactivated virion, preferably a binary ethyleneimine-inactivated virion.

The parapoxvirus agent may comprise genetic information (DNA or RNA) encoding for a heterologous (i.e. not of parapoxvirus) antigen, and/or an immunomodulator, or it may alternatively not comprise genetic information encoding for a heterologous antigen and/or an 30 immunomodulator. In one embodiment, it does not comprise genetic information encoding for a heterologous antigen, but it optionally comprises genetic information encoding for immunomodulator.

In a preferred embodiment, the parapoxvirus agent is a wildtype parapoxvirus. Preferably it is a PPVO virion, more preferably an inactivated PPVO virion (iPPVO).

Modes of use

The parapoxvirus agent can be used alone or in combination with a further treatment regimen for (i) conditioning a subject for a coronavîrus infection or (ii) treating a coronavîrus infection in a subject.

In a preferred embodiment, both of the conditioning and the treating, the further treatment regimen can comprise administering a coronavîrus agent.

Specifically for the conditioning, the further treatment regimen can comprise administering a further immune stimulatory agent (e.g. an adjuvant, a ILR agonist, a PRR, e.g. a cytosolic PRR such as RIG-I-like receptors including RIG-I, MDA5 and LGP2, or a mitogen, particularly a T cell mitogen or activator such as phytohemagglutinin (PHA), Concanavalin (Con) A, wheat germ agglutinin (WGA), pokeweed mitogen (PWM), a lectin or an anti-CD3 antibody or antigen-binding fragment thereof), or a B cell mitogen or activator such as PWM, lipopolysaccharide (LPS), an anti-CD40 antibody or antigen-binding fragment thereof, or an anti-(B cell antigen receptor) antibody or antigen-binding fragment thereof. The immune stimulatory agent preferably promûtes an antiviral cell State. Specifically for the treating, the further treatment regimen can comprise administering one or more further médicaments and/or one or more further thérapies (non-medicament/drug treatments) suitable for the treatment of a subject infected by coronavîrus or having coronavîrus disease. The one or more further médicaments are preferably selected from the group consisting of a further immune stimulatory agent (as exemplified above), an antiviral, an antibiotic, an adjuvant, a glucocorticoid, an antihypertensive drug (e.g. an ACE inhibitor), a hypoglycaemic drug, an anti-shock drug, and a drug suitable for treating one or more symptoms of coronavîrus disease and/or to accompany the one or more further thérapies (e.g. a pain reliever, a sédative, an anti-fever drug, an antiinflammatory drug such as IL-10, cough médication or an immunosuppressant). The one or more further thérapies are preferably selected from the group consisting of oxygen therapy and extra-corporeal organ support (ECOS). Oxygen therapy can be passive (i.e. relying on breathing of the subject) or active (i.e. deiivering oxygen to the lungs without relying on breathing of the subject). Passive thérapies are non-pressurized oxygen therapy and pressurized oxygen therapy. Active thérapies are mechanical ventilation and extra-corporeal membrane oxygénation (ECMO, also a spécifie form of ECOS). Examples for ECOS include ECMO, continuons rénal replacement therapy (CRRT), hémofiltration (HF) and hemoperfusion (HP) e.g. to remove inflammatory cytokines, and left ventricular assistance.

Spécifie examples of further médicaments for conditioning and treating are: Ivermectin, TMPRSS2 inhibitors (e.g. ammonium chloride, serine protease inhibitor camostat mesyîate) optionally together with a CatB/L inhibitor (e.g. E-64d), Favipiravir, Pimodivir, Baloxavir optionally together with Marboxil, IL-6 blockers (e.g. sarilumab, tocilizumab), Sarilumab, 5 Zitivekumab, lopinavir optionally together with ritonavir, chloroquine, hydroxychloroquine, Remdesivir, α-Ketoamides (e.g. cyclopentylmethyl, cyclohexylmethyl or α-Ketoamide 13b), Thalidomide, Maraviroc, Tenofovir optionally together with Disoproxil, Dolutegravir, Nelfinavir, Raltegravir, Tipranavir and Type I interferons (IFN-1, specifically IFN-βΙ). Preferred examples are Remdesivir and Thalidomide.

] Q The parapoxvirus agent can be administered by any route, including but not limited to the întravenous, intramuscular, oral, parentéral, topical, intradermal, or subeutaneous route. ïn a spécifie embodiment, it can be administered directly to the respiratory tract, preferably to the lower respiratory tract, more preferably to the lung (pulmonary delivery). As such, it can be administered as an aérosol and/or via inhalation.

The dose of the parapoxvirus agent (in fact of ail agents described herein, including a further immune stimulatory agent, a further médicament and a coronavirus agent) is a pharmaceutically effective dose as can be determined by the skilled person. For instance, in case of the agent being a virion (live or inactivated), it can be in the range of from 10⁶ to 10¹⁰ particles. The dose may be administered once or more than once, for example over a period of 20 <12 weeks, < 6 weeks, < 4 weeks, < 2 weeks, or < 1 week.

Coronavirus

The coronavirus is, in a preferred embodiment, an alpha-coronavirus or a betacoronavirus. More preferably, it is a respiratory coronavirus, i.e. a coronavirus that causes a 25 respiratory disease. Examples of alpha-coronaviruses are HCoV-229E and CoV-NL63, and examples of beta-coronaviruses are SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-HKUl, and HCoV-OC43. It is preferred that the coronavirus is a MERS or a SARS coronavirus, wherein preferably the SARS coronavirus is a virus of the species “severe acute respiratory symirome-related coronavirus” as classified by the International Committee on Taxonomy of 30 Viruses (ICTV). In the most preferred embodiment, it is SARS-CoV-2.

The skilled person appréciâtes that coronaviruses mutate and that new strains of SARSCoV-2 are isolated daily, with 461 strains having been isolated by April 6, 2020. Thus, in terms of a sequence définition, a preferred virus of the invention has a genome comprising or consisting of a sequence according to SEQ ID NO: 1 (original strain of the 2019 outbreak “Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-I”, NCBI Reference Sequence NC_045512.2, version of March 30, 2020) or a variant thereof with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or preferably at least 99% sequence identity. Alternative!y or in addition, a preferred virus of the invention has a genome comprising or consisting of a 5 sequence according to SEQ ID NO: 2 (a SARS-CoV-2 consensus sequence of 153 genome assemblies) or a variant thereof with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or preferably at least 99% sequence identity. It is preferred that the variant is classified as a strain of SARS-CoV-2 by the International Committee on Taxonomy of Viruses (1CTV).

Coronavirus agent

A coronavirus agent generally is an agent that is suitable for treating a coronavirus infection. It can be (a) the coronavirus itself or a fragment thereof which is recognized by and preferably stimulâtes an immune response (e.g. an antigen and preferably immunogen), (b) an agent that comprises genetic information encoding for (a), or (c) an agent which interfères with 15 the infection of a cell by the coronavirus or with the réplication of the coronavirus within a cell (in a therapeutic setting; is capable upon administration of an effective amount thereof to a subject infected with coronavirus to interfère with the infection of a cell by the coronavirus or with the réplication of the coronavirus within a cell). The coronavirus agent is preferably selected from the group consisting of (i) an inactivated coronavirus, (ii) an antigenic, preferably immunogenic fragment of coronavirus (e.g. the spîke protein or a fragment thereof), (iii) a nucleic acid encoding for (i) or (ii), (iv) a vector comprising the nucleic acid of (iii), (v) a cell comprising the nucleic acid of (iii) or the vector of (iv), (vi) an antibody or an antigen-binding fragment thereof binding to coronavirus or a fragment thereof, preferably the envelope, (vii) a nucleotide/nucleoside analogue, preferably coronavirus-specific, (viii) a coronavirus-specific RNAi molécule, (ix) a small molécule, an antibody or an antigen-binding fragment thereof, binding to a site of proiein-protein interaction (PPI) between a coronavirus protein and a subject protein, and (x) blood plasma of a subject that has recovered from a coronavirus infection, preferably from coronavirus disease.

A coronavirus can be inactivated as described for parapoxvirus above.

The PPI of (ix) is preferably an interaction of a SARS-CoV-2 protein or homologue thereof of another coronavirus and a subject protein (Uniprot protein ID of each subject protein is <name recited below>_HUMAN) selected from - SARS-CoV2 E and AP3B1, BRD4, BRD2, CWC27, ZCH1S or CTL2;

- SARS-CoV2 M and MPPB, YIF1A, AT1B1, ACADM, ETFA, STOM, VKGC, VATA,

PSMD8, REEP5, MPPA, AN06, PREP, ZNT9, FAKD5, ZNT7, GCP3, COQ8B, SAAL1, REEP6, INT4, ODC, GCP2, SYTM, RTN4, FA8AI, AASS, AKP8L, AAR2 or BZW2

- SARS-CoV2 N and U3IP2, PABP1, CSK22, CSK2B, G3BPL PABP4, LARP1, FA98A, SNIP1, RENT1, MOV10, G3BP2, DDX21, RBM28 or RL36;

- SARS-CoV2 Spike and ACE2, G0GA7 or ZDHC5;

- SARS-CoV2 nspl and DPOLA, PRI1, PRI2, DPOA2, GT251 or PKP2;

- SARS-CoV2 nsplO and AP2A2, ALR, ERGI1, AP2M1 orGRPEl;

- SARS-CoV2 nspl 1 and TBCA;

- SARS-CoV2 nspl2 and SBNO1, BCRD, AKAP8, MYCB2, SLU7, RIPK1, UBP2L, 15 TYSDI, PDZ11, PRC2B, UBAP2, ZN318, CRTC3, UBP54, Z3H7A, LAR4B, RBM41, HTF4, PP1L3 or PKHA5;

- SARS-CoV2 nspl 3 and TBKB1, CTRO, HSBP1, PCNT, FRI OP, KAP2, KAPCA, KAP3, RADI, CENPF, TLE1, TLE3, TLE5, G0GA3, GOGA2, GOGB1, GRAP1, CE350, MYOME, CP135, CEP68, CNTRL, RB6I2, GCC2, CLIP4, NIN, CEI 12, MIPOI, UBP13, 20 GCC1, JKIP1, CK5P2, AKAP9, GORS1, FYCO1, CA050, CP250, TBK1, FIOOK1 or NINL;

- SARS-CoV2 nspl4 and AGAL, IMDH2 or SIR5;

- SARS-CoV2 nspl5 NTF2, ARF6 or RNF41 ;

- SARS-CoV2 nsp2 and S27A2, IF4E2, NCPR, GDSl, WASC4, FKB15 or GGYF2;

- SARS-CoV2 nsp4 and IDE, ΤΙΜΙΟ, ALG11, PO210, TIM29, DJC11, ΤΙ0Β orTIM9;

- SARS-CoV2 nsp5 and HDAC2;

- SARS-Co V2 nsp5_C 145A and GPX1 or TRM1 ;

- SARS-CoV2 nspôand ATP5L, VASI, SGMR1 or ATI 33;

- SARS-CoV2 nsp7 and ADAS, CYB5B, ACSL3,NB5R3, RALA, COMT, RAB5C, RAB7A, 30 RAB8A, RAB2A, RAB10, RAB14, RHOA, RAB1A, GBB1, GBG5, LMAN2, MOGS, TOIP1, MARC1, QSOX2, HS2ST, NDUF2, SCPDL, SCRB1, NAT14, DCAKD, F162A, TIM14, SELS, PGES2 or RAB1 8;

- SARS-CoV2 nsp8 and MPP10, SRP72, ATEl, NSD2, SRP19, SRP54, RT25, DDX10, LARP7, MEPCE, NGDN, EX0S8, SYNM, N0L1O, CCD86, SPCS, EXOS5, EX0S3, AATF, HECD1, RT02, RT05, EX0S2 or RT27;

- SARS-CoV2 nsp9 and T2FB, FBNl, FBN2, NU214, NUP62, DCAF7, IF4H, NUP54, 5 MIB1, NEK9, ZN503, NUP88, NUP58, MAT2B or FBLN5;

- SARS-CoV2 orflO and PPT1, CUL2, MA7D1, ΤΗΤΡΑ, ZY11B, TIM8B, RBX1,ELOC or ELOB;

- SARS-CoV2 orDa and HMOX1, TRI59, AR6P6, VPS39, CLCC 1, VPS11, SUN2 or ALG5;

- SARS-CoV2 orf3b and STML2;

1Û - SARS-CoV2 orf6 and NUP98, RAE1L or MTCH ! ;

- SARS-CoV2 orf7a and HEAT3 or MDN1 ;

- SARS-CoV2 orf8 and PL0D2, TOR1 A, STC2, TP A, ITB i, CISD3, CO6A1, PVR, DNMT1, LYOX, PCSK6, INHBE, NPC2, MFGM, 0S9, NPTX1, PLGT2, PLGT3, ERO1B, PLD3, FXRD2, CHSS2, PUSL1, EMC1, GGH, ERLEC, Il 7RA, NGLY1, H6ST2, SDF2, NEUR1, 15 GDF 15, TM2D3, ERP44, EDEM3, SIL1, 0FUT1, SMOC1, PKHF2, FXL12, UGGG2, CHPF2, ATSl, HY0U1, FKBP7, ADAM9 or FKB10;

- SARS-CoV2 orf9b and NHRF1, CHM2A, CSDE1, TOM70, MARK3, MARK2, DPH5, DCTP1, MARRI, PTBP2 or BAG5; and

- SARS-CoV2 orf9c and UBXN8, GPAA1, WFS1, MRP1, PAR2, SCAP, D19L1, SGMR2, 20 ZNT6, TAPTl, ERMP1, NLRX1, RETR3, PIGO, FACR2, ECSIT, ALG8, TM39B, GHITM, ACAD9, NFIP2, BCS1, CIA30, TMED5, NDUB9 or PIGS.

An antibody or an antigen-binding fragment thereof to a PPI site can be generated by routine methods. For instance, antibodies or antigen-binding fragments thereof to one of the interacting proteins can be generated and then screened for interfering with the interaction.

The small molécule of (ix) is preferably selected from the group consisting of

Loratadîne, Daunorubicin, Midostaurin, Ponatinib, Silmitasertib, Valproic Acid, Haloperidol, Metformin, Migalastat, S-verapamil, Indomethacin, Ruxolîtinîb, Mycophenolic acid, Entacapone, Ribavirin, E-52862, Merimepodib, RVX-208, XL413, AC-55541, Apîcidin, AZ3451, AZ8838, Bafilomycin Al, CCT 365623, GB110, H-89, JQ1, PB28, PD-144418, RS30 PPCC, TMCB, UCPH-101, ZINC 1775962367, ZINC4326719, ZINC4511851,

ZINC95559591, 4E2RCat, ABBV-744, Camostat, Captopril, CB5083, Chloramphenicol,

Chloroquine, PI4K-II10 inhibitor Compound 10 (Rutaganira, F. U. et al., J. Med. Chem. 59,

1830-1 839 (2016)), Cyclophilin inhibitor Compound 2 (Mackman, R. L. et al., J. Med. Chem.

61, 9473-9499 (2018)), CPI-0610, Dabrafenib, DBeQ, dBET6, IHVR-19029, Lînezolsd,

Lisinopril, Minoxidil, ML240, MZ1, Nafamostat, Pevonedistat, PS3061, Rapamycin (Sirolimus), Sanglifehrin A, Sapanisertib (ΓΝΚ128/Μ1Ν128), FK-506 (Tacrolimus), Ternatin 4 (DA3), Tigecycline, Tomivosertib (eFT-508), Verdinexor, WDB002, Azacitidine, Seiinexor, and Zotatifin (eFT226).

Coronavirus infection/dîsease

A coronavirus infection is defined by the entry of coronavirus into at least one cell of a subject and its réplication in the at least one cell. Preferably, the infection is an infection of the respiratory tract, including the upper respiratory tract (nose and nasal passages, paranasal sinuses, the pharynx, and the portion of the larynx above the vocal folds (cords)) and the lower respiratory tract (portion ofthe larynx below the vocal folds, trachea, bronchi, bronchioles and the lungs including the respiratory bronchioles, alveolar ducts, alveolar sacs and alveoli). More preferably, it is an infection of the lower respiratory tract, most preferably of the lungs (including one or more of respiratory bronchioles, alveolar ducts, alveolar sacs and alveoli). The infection can further be characterized immunologically by the presence of at least one coronavirus-antigen-specific immune factor,preferably selected from the group consisting of B cells, follicular helperT cells (Tri cells), activated CD4 T cells and CD8’ T cells (particularly also CD38⁺HLA-DR⁺), IgM antibodies, and IgG antibodies. Alternatively or in addition, it can be characterized immunologically by a coron avirus-speci fie cytokine profile, preferably în the blood or at the site of infection as specified above (most preferably in the lung). These profiles can be determined by the skilled person without undue burden, e.g. by takîng a tissue sample, e.g. from the lung, infecting it with the coronavirus and determining the expression of cytokines. Examples of coronavirus-specîfic cytokine profiles are (a) for SARS-CoV: upregulation of one or more of, preferably ail of IFN-I (e.g. IFN-β), IFN-II (IFN-γ), 1FN-III (IFN-λΙ, IFN-À2, and 1FN-À3), IL-lbeta, IL-6, IL-12, IL-8, MCP-1, MIP-la!pha, RANTES, CXCL1, CXCL2, CXCL5, and CXCL9, and preferably no upregulation of CXCL10; and (b) for SARS-CoV-2: upregulation of one or more of, preferably ail of IL-6, MCP-1, CXCL1, CXCL5, and/or CXLC10, and preferably no upregulation of any IFN and/or of any one or ail of IL-lbeta, 1L12, IL-8, MlP-lalpha, RANTES, CXCL2, and CXCL9.

The preferred subject is human. While to the inventors’ best knowledge coronavirus infects ail humans equally, there are risk factors for contracting a coronavirus infection:

(i) Recent (within incubation period, i.e. within the past 27, 24, or 19 days, preferably 14 (CDC,

NHC)orlO(WHO) days) presence in ortravel from an area with ongoing spread of coronavirus infection. The area is preferably an 'Affected Area’ as determined by the WHO. “Recent’ can mean within the incubation period ofthe coronavirus, preferably within the past 27, 24, or preferably 19 days, more preferably 14 days or most preferably 10 days, (ii) Contact with a person infected with coronavirus, in particular a person having one or more symptoms of coronavirus disease. ‘Contact can mean exposed to respîratory droplets of the 5 person (e.g. within range of respîratory droplets in air or in contact with surfaces touched by person or by respîratory droplets).

(iiî) Being in an increased-risk-profession, which usuaîly nécessitâtes being in contact with the general public (contact being defined as above) regularly (e.g. at least daily or at least weekly), e.g. being a medical professional, in particular in a hospital, being a care worker (e.g. for 10 children or the elderly) or retail worker (for instance in a supermarket or pharmacy).

A coronavirus infection may or may not cause symptoms of coronavirus disease in a subject. The tenns “coronavirus infection” and “coronavirus disease” are distinguished herein by the presence of at least one coronavirus disease symptom. As long as the infection is not accompanîed by at least one symptom of coronavirus disease, it (or the subject) is asymptomatic 15 (includes presymptomatic). The term “coronavirus disease” as used herein requîtes the presence of a coronavirus infection and at least one symptom of coronavirus disease (also referred to herein as “symptomatic infection”).

Coronavirus symptoms include dry cough, fever (>37.8°C), runny and/or blocked nose, fatigue, breathing difficulty, pneumonia, organ (e.g. heart, lung, liver and/or kîdney) failure, 20 îtchy throat, headache, joint pain, nausea, diarrhoea, shivering, lymphophenia, loss of smell and/or loss of taste. Preferably, the coronavirus disease is characterized by the presence of two or more, three or more, or four or more symptoms, preferably including one or two or more of dry cough, fever (>37.8°C), breathing difficulty, loss of smell and/or loss of taste.

The coronavirus disease is preferably a respîratory disease (e.g. SARS or MERS), more 25 preferably Covid-19. The coronavirus disease can take a mild (non-severe) or a severe course. A mild course is characterized by the presence of one or more only mild symptoms (i.e. no severe symptoms) of coronavirus disease. Mild symptoms are selected from the group consisting of dry cough, mild fever (>37.8°C to <40°C), runny and/or blocked nose, fatigue, îtchy throat, headache, joint pain, nausea, diarrhoea, shivering, lymphophenia, loss of smell and 30 loss of taste. Severe symptoms are selected from the group consisting of breathing difficulty in particular acute respîratory distress syndrome, pneumonia, organ (e.g. heart, lung, liver and/or kidney) failure and high fever (>40°C). A severe course is characterized clinically by one or more of (i) the presence of one or more severe symptoms of coronavirus disease, (ii) need for hospitalization, in particular need for admission to intensive care unit (1CU) and/or need for oxygen therapy (as defined above), în particular for a respirator (also referred to as ventilator), and/or (iii) death ofthe subject. A need for hospitalization is characterized by one or more of: clinicai or radiological évidence of pneumonia, acute respiratory distress syndrome, and/or the presence of two or more symptoms including at least fever and persistent cough (with or without sputum), in particular persistent cough (e.g. of at least 3 days). A need for ICU admission is characterized by severe pneumonia and/or one or more severe complications. Severe pneumonia is characterized by tachypnea (âge younger than 2 months: 60 or more breaths per minute; âge 2 to 11 months: 50 or more breaths per minute, âge 1 to 5 years: 40 or more breaths per minute, âge > 5 years: 30 or more breaths per minute), respiratory distress and/or inadéquate oxygénation (e.g. SpCh of 93% or less for adults, and 90% or less for children). Severe complications are complications that are léthal when untreaîed and include, for example, septic shock, acute respiratory distress syndrome and organ failure. Alternatively or in addition, a severe course can be characterized immunologically e.g. by the presence or development of a cytokine storm, in particular in the lung. A coronavirus cytokine storm is characterized by hyperinflammation, preferably by an increase (vs. infected but asymptomatic subject, preferably as after the incubation period, or average or mean of a plurality of such subjects) of one or more of IL-2, IL-7, TNF-alpha, MIP-lalpha, MCP-1, G-CSF and/or CXCL10, wherein the increase can be characterized as excessive and/or uncontrolled. In particular, the cytokine storm is a Covid-19 cytokine storm. Furthermore, the cytokine storm is preferably in the respiratory tract as defined for the infection above (including preferred parts thereof).

While the coronavirus disease can take a severe course in any subject regardless of âge or pre-existing condition, there are nevertheless risk factors for having a severe course of coronavirus disease, including:

(i) âge, e.g. at least 40, 50, 60, 70, or 80 years with risk increasing with âge, (ii) male gender, (iii) smoking, and (iii) having one or more comorbidities, preferably selected from the group consisting of cardiovascular disease, diabètes, respiratory disease (in particular chronic), hypertension, AIDS, cancer, liver disease, kidney disease and lung disease. A particular comorbidity as risk 30 factor is a respiratory disease.

Conditkming a subject

The conditioning of a subject is meant to préparé a subject for a coronavirus infection, which is afforded by the immunomodulation resulting in the medical use of the invention. It can also be termed “preparatory treatment”, “prophylactic treatment” or, with respect to the disease resulting from the invention, “preventatîve treatment” or “préventive treatment”. It is not meant to necessarily prevent a coronavirus infection. As such, the subject is not yet infected by coronavirus.

Thus, the conditioning can hâve one or more immunological effects or (preferably and) it can hâve one or more clinical effects, both once a subject is infected. These immunological effects are preferably selected from the group consisting of (I-î) increasing the number of one or more of coronavirus-antigen-specific immune factors (as defined above) in the subject or in a part ofthe subject, (I-ii) inhibiting (i.e. reducing or stopping) coronavirus réplication in the subject or in a part of the subject, (I-iii) reducing the coronavirus load in the subject or in a part of the subject, and (I-iv) preventing or ameliorating a coronavirus-specific cytokine profile or a cytokine storm (as defined above) in the subject or in a part of the subject.

Therein, the part of the subject is preferably the respiratory tract (upper or preferably lower respiratory tract, more preferably the lung). (I-iî) is preferred over (1-i), and (I-iii) over (I-ii). Further (I-ii) may involve (I-i), and (I-iii) may involve (i-i) and/or (I-iî). (I-iv) is generally desired.

The clinical effects are preferably selected from the group consisting of (C-i) reducing contagiousness, (C-îi) delaying the onset of coronavirus disease, (C-iii) reducing the degree of one or more prospective symptoms of coronavirus disease, preferably of one or more prospective severe symptoms of coronavirus disease, (C-iv) preventing one or more symptoms of coronavirus disease, preferably one or more severe symptoms of coronavirus disease, (C-v) preventing a severe course of coronavirus disease, and (C-vi) preventing coronavirus disease.

Therein, (C-ii) is preferred over (C-i), (C-iii) over (C-iî), (C-îv) over (C-iii), (C-v) over (C-iv), and (C-vi) over (C-v).

Prospective symptoms are symptoms that are not yet present but can or will arise. Thus, the réduction of the degree of prospective symptoms refers to a prophylactic treatment of symptoms.

Accordingly, the parapoxvirus agent can be for use in one or more of (1-i) to (I-iv) and/or one or more of (C-i) to (C-vi) in a subject not yet infected by coronavirus. In other words, the parapoxvirus agent is capable upon administration of an effective amount thereof to achieve one or more of (I-i) to (I-iv) and/or one or more of (C-i) to (C-vi).

In a preferred embodiment, the subject to be conditioned îs characterized by one or more risk factors for contracting a coronavirus infection and/or by one or more risk factors for having 5 a severe course of coronavirus disease.

In another embodiment, the subject to be conditioned is selected by a method according to the corresponding further aspect of the invention described below.

Treating a subject

The treating of a coronavirus infection (i.e. of a subject that is infected) is not meant necessarily to treat or prevent coronavirus disease, although it is preferred that it does. As such, the treating can be of an asymptomatic infection (i.e. of an infected subject not having symptoms) or of coronavirus disease (i.e. of an infected subject having one or more symptoms).

The treating can hâve one or more immunologica! effects or (preferably and) it can hâve 15 one or more clinical effects. These immunological effects are preferably as defined for the conditioning above, for either an asymptomatic infection or for the disease. The clinical effects for an asymptomatic infection are preferably selected from the group consisting of (C-i) to (Cvi) as defined for the conditioning above. The clinical effects for a symptomatic infection (i.e. on coronavirus disease) are preferably selected from the group consisting of (C-i), (C-vi), both as defined for the conditioning above, (C-viî) ameliorating one or more (or ideally ail) symptoms of coronavirus disease, (C-viii) preventing, one or more (or ideally ail) further symptoms of coronavirus disease, and (C-ix) ameliorating, preferably preventing, a severe course of coronavirus disease.

Therein, “further symptoms” are those not yet characterizing the symptomatic infection.

Accordingly, the parapoxvirus agent can be for use in one or more of (I-î) to (I-iv) and/or one or more of (C-i) to (C-ix) în a subject infected by coronavirus. In other words, the parapoxvirus is capable upon administration of an effective amount thereof to achieve one or more of (1-i) to (I-iv) and/or one or more of (C-i) to (C-ix). In a preferred embodiment, the parapoxvirus agent is for use in preventing or treating coronavirus disease.

As indicated above, the subject to be treated may be asymptomatic. Alternatively, the subject to be treated may hâve coronavirus disease. The subject having coronavirus disease may not hâve any severe symptoms, e.g. it has only one or more mild symptoms. In another embodiment, it may hâve a severe course of coronavirus disease (excluding (ni) death; preferably (i) one or more severe symptoms). In a preferred embodiment, the subject to be treated (e.g. any ofthe afore-mentioned subjects except those already having a severe course of coronavirus disease) is characterized by one or more risk factors for having a severe course of coronavirus disease.

In a further preferred embodiment, the subject is characterized by at least one of:

(i) no cytokine storm in the lung, and preferably no cytokine storm at ail, (îi) âge up to 75, 70 or preferably 65 years, more preferably 18 to 65 years, and/or (iii) HScore of 169 or less, preferably 150 or less, 130 or less, or 110 or less, Preferably, the subject is characterized by (ii) and/or (iii), (ii) and HScore of 169 or less, or (iii) and âge 18 to 65 years old.

The HScore generates a probability for the presence of secondary haemophagocytic lymphohistoicytosis (sHLH), a hyperinflammatory syndrome with a cytokine storm profile similar to that of coronavirus disease (Pehta et al., The Lancet Vol 395 March 28, 2020). An HScore of greater than 169 is 93% sensitive and 86% spécifie for sHLH. The HScore is calculated using the following criteria: température (<38.4°C: 0 points, 38.4-39.4°C: 33 points, ] 5 >39.4°C: 49 points), organomegaly (none: 0 points, hepatomegaly or splenomegaly: 23 points, hepatomegaly and splenomegaly: 38 points), number of cytopenias* (one lîneage; 0 points, two lineages: 24 points, three linages: 34 points), triglycérides (<1.5 mmol/1: 0 points, 1.5-4.0 mmol/l: 44 points, >4.0 mmol/1: 64 points), fibrinogen (>2.5 g/L: 0 points, <2.5 g/L: 30 points), ferritin (<2000 ng/ml: 0 points, 2000-60000 ng/ml: 35 points, >6000 ng/ml: 50 points), sérum aspartate amînotransferase (<30 1U/L: 0 points, >30 1U/L: 19 points), haemophagocytosis on bone marrow aspirate (no: 0 points, yes: 35 points), and known immunosuppression! (no: 0 points, yes: 18 points). *Defined as either haemoglobin concentration of 9.2 g/dL or less (<5.71 mmol/L), a white blood cell count of 5000 white blood cells per mm³ or less, or platelet count of ] 10000 platelets per mm³ or less, or ail of these criteria combined. fHIV positive or receiving 25 long-term immunosuppressive therapy (e.g. one or more of glucocorticoids, cyclosporine and/or azathioprine).

In another embodiment, the subject to be treated îs selected by a method according to the corresponding further aspect ofthe invention described below.

Further medical use

In a further medical use aspect, the invention relates to a parapoxvirus agent for use as a médicament promoting an antiviral (preferably anticoronaviral) cell state in a subject (i) having a coronavirus infection or (ii) characterized by one or more risk factors for contracting a coronavirus infection and/or by one or more risk factors for having a severe course of coronavirus disease.

In one embodiment, the one or more risk factors for contracting a coronavirus infection are selected from factors (i) and (ii) as defined above.

In another embodiment, the one or more risk factors for having a severe course of coronavirus disease comprise at least (ii) and (iii), or (ii) and (iv) as defined above, or comprise at least a comorbidity selected from respîratory disease (in particular chronic) and lung disease. In addition, the âge of the subject is preferably at least 60, at least 70, or more preferably at least 80 years.

The phrase “promoting an antiviral cell state” refers to the promotion of the transcription of cellular antiviral genes coding for host defence proteins, e.g. as promoted by lFN-α and/or β. Preferably it inhibits (i.e. prevents or at least reduces) virus réplication in the cell and/or induces apoptosis (to prevent further virus réplication in the cell), and more preferably it reduces the amount of infectious virions released by an infected cells compared to a cell in which the antiviral cell state was not promoted by the médicament. Thereby, it stops or at least slows down virus propagation. “Promoting” includes an induction (e.g. for cells not yet in an antî viral state) and a re inforce ment (i.e. potentialion) (e.g. for cells aiready in an antiviral state). Although not limited thereto, the former is preferred fora subject not (yet) having a coronavirus infection, e.g. characterized by one or more risk factors as specified above, and the latter is preferred for a subject having a coronavirus infection.

Methods of treatment

The invention further relates to the following methods

A method of administering a parapoxvirus agent to a subject (i) having a coronavirus infection or (ii) characterized by one or more risk factors for contracting a coronavirus infection and/or by one or more risk factors for having a severe course of coronavirus disease.

A method of promoting an antiviral (preferably anticoronaviral) cell state in a subject (i) having a coronavirus infection or (ii) characterized by one or more risk factors for contracting a coronavirus infection and/or by one or more risk factors for having a severe course of coronavirus disease, the method comprising administering a parapoxvirus agent to the subject.

A method of (i) conditioning a subject for a coronavirus infection or (ii) treating a coronavirus infection in a subject, the method comprising administering a parapoxvirus agent to the subject.

A method of treating coronoavirus disease in a subject, the method comprising administering a parapoxvirus agent to the subject.

Définitions and embodiments described below, in particular under the header 'Définitions and further embodiments of the invention' apply to the first aspect of the invention.

Jn a second aspect, the invention relates to a composition or a kit comprising a parapoxvirus agent and a coronavirus agent.

In a preferred embodiment, the composition or kit further comprises one or more pharmaceutically acceptable diluents, carriers, and/or preservatives; and/or it is a pharmaceutical composition or kit. Pharmaceutically acceptable diluents, carriers, and/or preservatives are described below and may in addition thereto include an (e.g. aqueous) medium suitable for structurally maintaining a parapoxvirus, in particular an inactivated parapoxvirus, or lyophilisate of this medium. Specifically, the composition or kit may be for any of the medical uses of the invention described herein.

The composition can be formulated for any route of administration as defined above. Also, it can be formulated for simultaneous or separate administration or for simultaneous or separate release of the parapoxvirus agent and the coronavirus agent.

In a preferred embodiment, the kit comprises a composition of the second aspect.

In another embodiment, the kit comprises a leaflet with instructions for use of the parapoxvirus agent and the coronavirus agent, or of the composition of the second aspect, preferably according to a medical use described herein.

Définitions given and embodiments described with respect to the first aspect apply also to the second aspect, in as far as they are applicable. Also, définitions and embodiments described below, in particular under the header 'Définitions and further embodiments of the invention' apply to the composition or kit of the second aspect.

In a third aspect, the invention relates to a pharmaceutical composition or kit for any of the medical uses ofthe invention described herein, wherein the pharmaceutical composition or kit comprises a parapoxvirus agent and preferably a further médicament. The pharmaceutical composition usually comprises one or more pharmaceutically acceptable diluents, carriers, and/or preservatives.

In a preferred embodiment, the kit comprises a composition ofthe third aspect.

In another embodiment, the kit comprises a leaflet with instructions for use of the parapoxvirus agent and the further médicament, or of the composition of the third aspect, preferably according to a medical use described herein.

Définitions given and embodiments described with respect to the first and second aspect (e.g. formulation) apply also to the third aspect, in as far as they are applicable. Also, définitions and embodiments described below, in particular under the header 'Définitions and further embodiments of the invention' apply to the composition or kit of the third aspect.

In a further aspect, the invention relates to a method of selecting a subject for any ofthe medical uses of the invention described herein, wherein the method comprises the steps of (i) determining the presence or absence of a cytokine storm in the subject, and (ii) selecting the subject if a cytokine storm îs absent.

In one embodiment, step (i) comprises determining the HScore of the subject, and step (ii) comprises selecting the subject if the HScore is 169 or less.

In another embodiment, step (i) comprises detectîng in a biological sample ofthe subject one or more signs of hyperînflammation, and step (ii) comprises selecting the subject if no sign of hyperînflammation is détectable. Hyperînflammation therein is an increased level, e.g. excessive and/or uncontrolled, (vs. infected but asymptomatic subject, preferably as after the incubation period, or average or mean of a plurality of such subjects) of one or more of IL-2, 1L-7, TNF-alpha, MIP-1 alpha, MCP-i, G-CSF and/or CXCL10; or of one or more of IL-6, ferritin, platelet count and/or érythrocyte sédimentation rate (ESR). The biological sample is preferably selected from the group consisting of blood or a blood-derived sample (e.g. plasma or sérum), sputum, saliva, and a sample derived from the lung (e.g. by bronchoscopy including bronchial lavage, bronchial alveolar lavage, bronchial brushing, and bronchial abrasion).

The method of the fourth aspect is preferably an ex vivo method.

Définitions given and embodiments described with respect to the first, second and third aspect apply also to the fourth aspect, in as far as they are applicable. Also, définitions and embodiments described below, in particular under the header 'Définitions and further embodiments of the invention' apply to the method of the fourth aspect.

Disclaimers

In a select embodiment, one or more of the following disclaimers apply to the invention:

the parapoxvirus is not canine distemper virus (CDV), the coronavîrus îs not porcine épidémie diarrhea virus (PEDV), and/or the subject is not a pig (pig including sow and piglet).

Définitions and further embodiments of the invention

The spécification uses a variety of terms and phrases, which hâve certain meanings as 5 defmed below. Preferred meanings are to be construed as preferred embodiments of the aspects ofthe invention described herein. As such, they and also further embodiments described in the following can be combined with any embodiment of the aspects of the invention and in particular any preferred embodiment of the aspects of the invention described above.

The term identity or identical in the context of polynucléotide sequences refers to 10 the number of residues in the two sequences that are identical when aligned for maximum correspondence. Specifically, the percent sequence identity of two sequences is the number of exact matches between two aligned sequences divided by the length ofthe shorter sequence and multiplied by 100. Alignment tools that can be used to align two sequences are well known to the person skilled in the art and can, for example, be obtained on the World Wide Web, e.g.

Needle (EMBOSS) (https://www.ebi.ac.uk/Tools/psa/emboss_needle/), MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/), MAFFT (http://www.ebi.ac.uk/Tools/msa/ mafft/) or WATER (http://www.ebi.ac.uk/Tools/psa/ emboss_water/). The alignments between two sequences may be carried out using default parameters settings, e.g. for Needle preferably MATRIX: BLOSUM62, Gap Open: 10.0, Gap Extend: 0.5, for MAFFT preferably: Matrix:

Blosum62, Gap Open 1.53, Gap Extend 0.123, for WATER polynucleotides preferably; MATRIX: DNAFULL, Gap Open: 10.0, Gap Extend 0.5 and for WATER polypeptides preferably MATRIX: BLOSUM62, Gap Open: 10.0, Gap Extend: 0.5. Those skilled in the art understand that it may be necessary to introduce gaps in either sequence to produce a satisfactory alignment. The best sequence alignment îs defined as the alignment that produces 25 the largest number of aligned identical residues while having a minimal number of gaps.

Preferably, it is a global alignment, which includes every residue in every sequence in the alignment.

The term “variant” refers generaily to a modified version of the polynucleotide, e.g. a mutation, so one or more nucléotides of the polynucleotide may be mutated. Generaily, the 30 variant is functional, meaning e.g. with regard to a virus that the virus îs capable of infecting a host cell. The functionality is generaily that described for the polynucleotide the variant is from. A “mutation” can be a nucléotide substitution, délétion and/or insertion (“and” may apply if there is more than one mutation). Preferably, it is a substitution.

The term “antigen” refers to any substance, e.g. protein or peptide, that is capable of being bound by an antibody, a B cell receptor and/or a T cell receptor. An antigen comprises at least one epitope preferably comprising at least 8 amino acids and more preferably comprising between 8 and 17 amino acids. The epitope can be a T cell and/or a B cell epitope. AT cell 5 epitope is an epitope that can be presented on the surface of an antigen-presenting cell, where ît is bound to an MHC molécule. In humans, professional antigen-presenting cells are specialized to present MHC class II peptides, whereas most nucleated somatic cells present MHC class I peptides. T cell epitopes presented by MHC class I molécules are typically peptides between 8 and 1 1 amino acids in length, whereas MHC class II molécules present 10 longer peptides, e.g. 13-17 amino acids in length. A B cell epitope is an epitope that is recognised as three-dimensional structures on the surface of native antigens by B cells. Epitopes can be predicted with in silico tools, e.g. the online B- or T-cell prédiction tools of the IEDB Analysis Resource.

The term “immunogen” refers to an antigen that is capable of inducing an immune 15 response.

“Pattern récognition receptor” or “PRR”, as used herein, refers to a germline-encoded host sensor which detects a pathogen-associated molecular pattern (PAMP), which is spécifie for a pathogen, and/or a damage-associated molecular pattern (DAMP), which is spécifie for components of a host cell that are released during cell damage or death. A PRR can be a 20 membrane-bound PRR (cell-surface PRR) or a cytosolic PRR.

An “adjuvant” is a substance that accelerates, prolongs and/or enhances the quality and/or strength of an immune response to an antigen/immunogen, in comparison to the administration of the antigen alone, thus, reducing the quantity of antigen/immunogen necessary, and/or the frequency of injection necessary in order to generate an adéquate immune 25 response to the antigen/immunogen of interest. Examples of adjuvants that may be used in the context of the composition according to the present invention are gel-like précipitâtes of aluminum hydroxide (alum); AIPO4; alhydrogel; bacterial products from the outer membrane of Gram-negative bacteria, in particular monophosphoryl lipid A (MPLA), lipopolysaccharides (LPS), muramyl dipeptides and dérivatives thereof; Freund’s incomplète adjuvant; liposomes, 30 in particular neutral liposomes, liposomes containing the composition and optionally cytokines; non-ionic block copolymers; ISCOMATRIX adjuvant (Drane et al., 2007); unmethylated DNA comprising CpG dinucleotides (CpG motif), in particular CpG ODN with a phosphorothioate (PTO) backbone (CpG PTO ODN) or phosphodiester (PO) backbone (CpG PO ODN); synthetic lipopeptide dérivatives, in particular PamsCys; lipoarabi nom annan; peptîdoglycan;

zymosan; heat shock proteins (HSP), in particular HSP 70; dsRNA and synthetic dérivatives thereof, in particular Poly J:C; polycationic peptides, in particular poly-L-arginine; taxol; fibronectin; flagellin; imidazoquinolîne; cytokines with adjuvant activity, in particular GMCSF, interleukin-(IL-)2, IL-6, IL-7, IL-18, type I and II interferons, in particular IFN-γ, TNFa; 25-dihydroxyvjtamin D3 (caicitriol); and synthetic oligopeptides, in particular MHC-IIpresented peptides. Non-ionic block polymers containing polyoxyethylene (POE) and polyoxypropylene (POP), such as POE-POP-POE block copolymers may be used as an adjuvant (Newman et al., 1998). This type of adjuvant is particularly useful for compositions comprising nucleic acids as active ingrédient.

The term “recombinant” refers in particular to a virus that is modified to comprise a heterologous polynucleotide sequence in its genome.

The term “immunomodulator” refers to a drug used to regulate or normalize the immune system by inducing, enhancing, suppressing and/or weakening an immune response in a subject (“and” meaning that some parts of the immune system are selectively induced or enhanced and others are selectively suppressed or weakened). Examples are cytokines, non-cytokine agonists or antagonists of cytokine receptors (e.g. antibodies or small compounds), antibodies or small compounds binding (preferably neutralizing) cytokines, and soluble cytokine receptors (e.g. for trapping cytokines). It does not include an antigen.

The term “vector” as used herein includes any vectors known to the skilied person including plasmîd vectors, cosmîd vectors, phage vectors such as lambda phage, viral vectors such as adenovirus (Ad) vectors), adeno-associated virus (AAV) vectors, alphavirus vectors (e.g., Venezuelan equine encephalîtis virus (VEE), sîndbis virus (SIN), semlikî forest virus (SFV), and VEE-SIN chimeras), herpes virus vectors, measles virus vectors, pox virus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC (derived from the Copenhagen strain of vaccinia), and avipox vectors: canarypox (ALVAC) and fbwlpox (FPV) vectors), and vesicular stomatitis virus vectors, or virus like particîes. As used herein, the term virus-lîke particle or VLP refers to a non-replicating, empty viral shell. VLPs are generally composed of one or more viral proteins, such as, but not limited to those proteins referred to as capsid, coat, shell, surface and/or envelope proteins. They contain functional viral proteins responsible for cell pénétration by the virus, which ensures efficient cell entry. Methods for producing particular VLPs are known in the art.

The term “mitogen” refers to an agent, e.g. a peptide or protein that induces a cell to begin cell division. The term “cell activator” refers to an agent, e.g. a peptide or protein that binds and cross-links cell receptors, e.g. T cell or B cell receptors.

The term “nucleoside analogue” refers to a nucleoside containing a nucleic acid analogue and a sugar. The term “nucléotide analogue” refers to a nucléotide containing a nucleic acid analogue, a sugar and a phosphate group with 1 to 3 phosphates. The term nucleic acid analogue” refers to a compound that is structurally similar to RNA and DNA in that it has one or more of phosphate backbone, pentose sugar and/or the nucleobase altered such that it has different base pairing and/or base stacking properties. Examples include peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA).

The term “RNAi molécule” refers to a molécule capable of neutralizing mRNA molécules în a target-sequence-specîfic manner. Examples include dsRNA, miRNA and siRNA.

Particular preferred pharmaceutical forms for the administration ofthe agents according to the invention are forms suitable for injectable use and include stérile aqueous solutions or dispersions and stérile powders for the extemporaneous préparation of stérile injectable solutions or dispersion. Typically, such a solution or dispersion will include a solvent or dispersion medium, containing, for example, water-buffered aqueous solutions, e.g. biocompatible buffers, éthanol, polyol, such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants or vegetable oils. Infusion or injection solutions can be accomplished by any number of art-recognized techniques including but not limited to addition of preservatives like anti-bacterîal or anti-fungal agents, e.g. parabene, chlorobutanol, phénol, sorbic acid or thiomersal. Further, isotonie agents, such as sugars or salts, in particular sodium chloride may be incorporated in infusion or injection solutions.

Preferred diluents of the present invention are water, physiological acceptable buffers, physiological acceptable buffer sait solutions or sait solutions. Excipients which can be used with the various pharmaceutical forms of the pharmaceutical according to the invention can be chosen from the following non-limiting list:

a) binders such as lactose, mannitol, crystalline sorbitol, dibasic phosphates, calcium phosphates, sugars, microcrystalline cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, polyvinyl pyrrolidone and the like,

b) lubricants such as magnésium stéarate, talc, calcium stéarate, zinc stéarate, stearic acid, hydrogenated vegetable oil, leucine, glycerids and sodium stearyl fumarates,

c) disintegrants such as starches, croscaramellose, sodium methyl cellulose, agar, bentonite, algînic acid, carboxymethyl cellulose, polyvinyl pyrrolidone and the like.

The term “pharmaceutically acceptable carrier” refers to any substrate which serves to improve the selectivity, effectiveness, and/or safety of drug administration. Such carriers can be used to control the release of a drug into systemic circulation. This can be accomplîshed either by slow release ofthe drug over a long period oftime (typically diffusion) or by triggered release at the drug’s target by some stimulus, such as changes in pH, application of heat, and activation by light. Carriers can also be used to improve the pharmacokinetic properties, specifically the bioavailability, of many drugs with poor water solubility and/or membrane permeability. A wide variety of drug carrier Systems hâve been developed and studied. Examples include liposomes, polymeric micelles, microspheres, nanopartîcles, nanofibers, protein-drug. conjugales, érythrocytes, virosomes and dendrimers. Different methods of attaching the drug to the carrier can be used, including adsorption, intégration into the bulk structure, encapsulation, and covalent bonding.

Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with spécifie preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such spécifie embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvions to those skilled in the relevant fields are întended to be covered by the présent invention.

***

The invention is described by way of the following examples which are to be construed as merely illustrative and not limitative of the scope of the invention.

EXAMPLE 1

Aim

Testing the antiviral activity of inactivated parapoxvirus orf (iPPVO) virions against coronavirus. PPVO virions stîmulate the innate as well as the adaptive arm of the immune system.

Approach

In whole blood, APCs (antigen-presenting cells) take up the iPPVO virion, which leads to their activation and the release of cytokines. This in turn activâtes T cells as well as NK cells which also start to proliferate and release cytokines. After 3 days of incubation the cells are spun down and the supematant containing soluble factors like cytokines are harvested. These supernatants are assayed for their activity on coronavirus.

Test substances iPPVO of strain DI701 (available from Pfizer); 230 IFN unitsreconstituted in 2 ml water iPPVO vehicle (PPVO buffer only, i.e. without virus), négative control

PBS, négative control

Concavalin (Con) A, co-stimulus, stock solution 1 mg/mt PBS = 10X (used in well at 5 pg/ml)

Further materials

Whole blood of a healthy donor: 9 ml whole blood freshly collected in a Sarstedt Monovette, Li-Heparin

Medium for whole blood incubation: RPMI (500 ml) supplemented with 1% GlutaMAX™ and 0.25 ml Heparin-Sodium (5000 U)

Cells of coronavirus assay: VeroE6-EGFP propagated în growth medium prepared by supplementing DMEM with 10% v/v heat-inactivated FCS and 5 mL sodium bicarbonate 7.5%, and cultured in Tl 50 bottle and split 1/4 twice a week. Pen-strep added directiy to the Tl50 bottle at a 1/100 dilution.

Coronavirus assay medium: prepared by supplementing DMEM with 2% v/v heatinactivated FCS and 5 mL sodium bicarbonate 7.5%.

Préparation of samples

Per well in a 96 well round-bottom plate: 120 μI cell medium for whole blood incubation + 20 pl ConA or PBS + 20 μΙ iPPVO, iPPVO vehicle or PBS + 40 μΐ whole blood.

The plate was incubated at 37 °C, 5 % CO? for 72 hrs and then centrifuged for 5 min at 230 g. The supernatant was aliquoted to new 96 well plates (plate layout maintained). Sample plates were frozen at -80 °C until further use.

Coronavirus assay

Dilution of the samples (test substance as prepared above): 100 pL assay medium were added to wells of a Greiner Bio One 655090 plate and 15 pL were removed again from wells (except from wells for Controls of this assay: virus control (VC) controlling for virus réplication and effect on cells, and cell control (CC) for controlling cell viability without virus). 15 pL from wells of sample plate was added to wells of which 15 pL medium was removed.

Préparation of the cell suspension: A confluent culture (monolayer) of VeroE6-EGFP in Tl 50 bottle was washed with DPBS and 10 mL trypsine 0.25%trypsine/EDTA was added.

The bottle was incubated for I minute at room température, and trypsine/EDTA was removed except for 2 mL. The bottle was then incubated for 15 minutes at 37°C. After incubation, the cells were resuspended in 10 mL assay medium, passed through a Cell Strainer (FALCON CAT NO 352350) and counted using coulter (3 samples of 10 pL in 10 mL were counted). 3000 cells were then resuspended in 50 pL assay medium and 50 pL ofthe cell suspension were seeded to 10 each well of the Greiner plate.

Assay: Plates with cell suspension as prepared above were incubated overnight (37°C / 5%CO2), after which SARS-CoV-2 (strain BetaCoV/Belgium/GHB-03021/2020, stock titer 4.8 x 10⁷ TCID50/ml) was added to ail wells (except the cell control wells) at a final dilution of 1/200,0000 in 200 pl/well (3000 cells/well, resulting in an MOI of 0.016 TCIDso/cell). The 15 plates were incubated at 37°C / 5% CO₂ for 5 days, after which GFP signal was measured using whole-well fluorescence, HCI (High Content Imaging) and the ImageJ software. Reduced EGFP expression is a marker for virus-induced cytopathic activity. See also Ivens et al. (Journal of Virological Methods 129, 2005, 56-63).

Results

The results are shown in Fig. 1. Cells not infected with coronavirus (CC mean) were viable and expressed eGFP. Cells infected with coronavirus (VC mean) died and no eGFP expression was détectable. Results for PBS and iPPVO vehicle alone were similar to VC mean, i.e. PBS and iPPVO vehicle alone had no effect on cell viability, i.e. against coronavirus, as 25 expected. Also 5 pg/ml ConA alone (in PBS) dîd not retain eGFP expression, and it also did not affect eGFP expression when given together with iPPVO vehicle as shown by the comparison to ConA alone in PBS. iPPVO DI701 alone (in PBS) also had aclear positive effect on cell viability, and eGFP expression was further increased when iPPVO DI701 was used in combination with 5 pg/ml ConA (which by itself showed no effect on eGFP expression atthis 30 concentration). Accordingly, ÎPPVO and ConA act synergistically.

These results show the efficacy of parapoxvirus in promoting an antiviral cell State which is protective and inhibits coronavirus réplication and propagation and thereby the infection of further cells. This effect can be improved by adding an immune stimulatory agent such as ConA, which acts synergistîcally, i.e. the efficacy of iPPVO is enhanced in a combinatorial approach.

EXAMPLE 2

Aim

Determining whether the proof-of-principle shown in Example 1 applies to parapoxvirus in general by testing the antiviral activity of a further parapoxvirus strain against coronavîrus.

Approach

See Example 1.

Test substances iPPVO of strain NZ2 (chemîcally inactivated by binary ethyleneimine, BEI), internai désignation AIC649, IxiO⁹ lyophilized virions reconstituted in 1 ml water iPPVO ofstraîn D1701, see Example l iPPVO vehicle, négative control, see Example 1

Further materials. préparation of samples. coronavîrus assay

See Example 1. iPPVO and iPPVO vehicle were used alone. ÎPPVO D1701 shown to increase cell vîability in Example 1 was used as a positive control în Example 2.

Results

The results are shown in Fig. 2. iPPVO DI701, which in Example 1 was shown to be effective, was used as a positive control and as a 100% benchmark. Both PPVO strains DI701 and NZ2 are active against coronavîrus as shown by the comparison to the négative control. Accordingly, the promotion of an antiviral cell State which is effective against coronavîrus is a hallmark of parapoxvirus in general and not only ofthe strain tested in Example 1.

EXAMPLE 3

Aim

Investigating the timing of parapoxvirus addition, specifically determining how the antiviral activity of parapoxvirus shown in Examples 1 and 2 is modulated by adding the supernatants at different time points to the cells ofthe coronavîrus assay.

Approach

See Examples 1 and 2, but with different time points for adding the supernatants of the sample plate to the cells of the coronavirus assay (before or after the infection of the VeroE6EGFP cells with SARS-CoV-2).

Test substances iPPVO of strain NZ2, see Example 2 iPPVO of strain DI 701, see Example 1 iPPVO vehicle, négative control, see Example 1

ConA, see Example 1

Further materials, préparation of samples. coronavirus assay

See Examples 1 and 2. Whole blood was incubated with iPPVO or iPPVO vehicle (alone or in combination with ConA). Supernatants were generated and added at different timepoints before or after the infection of the VeroE6-EGFP cells with the coronavirus (SARS-CoV-2).

EX AMPLE 4

Aim

Testing further combinatorial approaches, specifically confirming that the strength of the antiviral activity of parapoxvirus shown in Examples 1, 2, and 3 can be enhanced in combinatorial approaches using further combination partners, e.g. an immune stimulatory agent or an antiviral as described herein. This includes investîgatîng the timing of parapoxvirus and combination partner addition (parapoxvirus to blood samples and combination partner to blood sample or directly to cells of coronavirus assay), i.e. relative to each other, and also the timing of adding the coronavirus relative to parapoxvirus and combination partner. It also includes testing the influence of the concentrations of parapoxvirus and combination partner.

Approach

See Examples 1, 2, and 3, but with different time points for adding the supernatants (of blood treated with parapoxvirus and/or combination partner, at the same or different time points) of the sample plate to the cells of the coronavirus assay (before or after the infection of the VeroE6-EGFP cells with SARS-CoV-2), and with different time points for adding the combination partner (if not already added to the blood sample) to the cells of the coronavirus assay (before or after the supernatant addition and/or the SARS-CoV-2 addition).

Test substances iPPVO of strain NZ2, see Example 2 iPPVO of strain D1701, see Example 1 iPPVO vehicle, négative control, see Example 1

ConA, see Example 1 (combination partner control)

Thalidomide (combination partner)

Remdesîvir (combination partner)

Further materials. préparation of samples, coronavirus assay

See Examples 1 and 2. Whole blood was incubated with iPPVO or iPPVO vehicle (alone or in combination with one or more combination partners). Supernatants were added at different timepoînts before or after the infection ofthe VeroE6-EGFP cells with the coronavirus (SARSCoV-2). One or more combination partners not used for incubation ofthe blood samples were added at different time points to the VeroE6-EGFP cells (before both the supernatant and the coronavirus, after both or between the two). Different concentrations of the combination partners were used.

EXAMPLE 5

Aim

Investigating the effect of parapoxvirus on the infections viral load of coronavirus in the lung in a hamster model.

Approach

The hamster infection model of SARS-CoV-2 described by Boudewijns et al. (STAT2 signaling as double-edged sword restricting viral dissémination but driving severe pneumonia in SARS-CoV-2 infected hamsters. bioRxiv 2020, 2020.04.23.056838) is used. Six hamsters are treated via the intraperitoneal (i.p.) route with 1.6 x 10⁹ VP of iPPVO one day prior to infection with SARS-CoV-2. A second group of six hamsters is treated with iPPVO vehicle (placebo control) instead. For infection, hamsters are anesthetized with ketamine/xylazine/atropine and inoculated intranasally with SARS-CoV-2 (1.89 x 10⁶ TCID50 in 50 pL). Atday 4 post-infection (pi), hamsters are euthanized by i.p. injection of 500 pL Dolethal (200 mg/mL sodium pentobarbital, Vétoquinol SA). Lungs are collected and infectious virus is quaniified by endpoint virus titration. Efficacy is determined based on viral load in homogenized lung tissues on day 4 post-infection.

Animais

Syrian Golden Hamster, females, 6-10 weeks old

SARS-Cov-2

SARS-Cov-2 strain BetaCov/Belgîum/GHB-03021/2020 (EPI ISL 109 407976|202010 02-03) was recovered from a nasopharyngeal swab taken from a RT-qPCR confirmed asymptomatic patient who returned from Wuhan, China in the beginning of February 2020. A close relation with the prototypic Wuhan-Hu-1 2019-nCoV (GenBank accession 112 number MN908947.3) strain was confirmed by phylogenetic analysis. Infectious virus was isolated by serial passaging on HuH7 and Vero E6 cells (Boudewijns et al., supra)', passage 6 virus was 15 used for the study described here. The titre of the virus stock was determined by end-point dilution on Vero E6 cells by the Reed and Muench method (supra).

Test substances iPPVO strain of NZ2, see Example 2

- iPPVO vehicle (PPVO buffer only, i.e. without virus)

End-poînt virus titration

Lung tissues were homogenized using bead disruption (Precellys) in 350 pL minimal essential medium and centrifuged (10,000 rpm, 5 min, 4°C) to pellet the cell débris. To quantify 25 infectious SARS-CoV-2 particles, endpoint titrations were performed on confluent Vero E6 cells in 96 well plates. Viral titres were calculated using the Reed and Muench method (Reed and Muench, The American Journal of Hygiene, 1938. 27(3): p. 493-497) and the Lindenbach calculator and were expressed as 50% tissue culture infectious dose (TC1D50) per mg tissue.

Results

Prophylactic treatment with iPPVO reduced the number of infectious SARS-CoV-2 particles in the lung: The mean value of TCID50/mg lung tissue of placebo treated hamsters was measured to be 6.3 x 10⁵ compared to iPPVO-treated hamsters, in which the TCID50/mg lung tissue was measured to be 2.1 x 10⁵. See Figure 3.

EXAMPLE 6

Atm

Investigating the effects of parapoxvirus on coronavîrus infection in a mouse model.

Approach mice are intravenously inoculated with 100 μΐ of iPPVO (1 vial dissolved in 500 μΐ supplied buffer, which equals a dose of 1x10⁹ virus particles (VP)/animal) prior to coronavîrus infection. A second group of 10 mice serves as an infection control group (intravenous inoculation with 100 μΐ iPPVO buffer). One day after treatment, mice of both groups are înfected intranasally under isofiurane anesthésia (inhalation of 3% îsoflurane) with 900 focus forming units (FFU) of SARS-CoV-2 (German isolate) in 50 μΐ total volume. Mice are scored daily. Euthanasia is performed on day 10 after infection. Earlîer euthanasia is performed for mice reaching humane endpoînts as determined by the clinical score. Response to treatment is assessed by a clinical score détermination (clinical symptoms of infection) and body weight loss. Also, SARS-CoV-2 load is determined in lungs and brains.

Animais

Transgenic K18-hACE2 mouse

Test substances iPPVO strain of DI701, see Example 1 iPPVO vehicle (PPVO buffer only, î.e. without virus)

Détermination of clinical score

The clinical score was determined by adding the score of each clinical parameter according to Table 1 below. Animais were euthanised upon reaching a clinical score of >20 or scoring 20 for one clinical parameter (humane endpoînts).

Table 1: Clinical score.

Clinical parameter	Score criteria	Score	Welfare measures
Body weight réduction /	Up to max. 7 % réduction or increase / balanced skelétal-m use ular ratio.	0
body condition	8-10 % réduction / visible skeletal attachments in the area of the spine (sunken)	5	Offer moistened food

	11-19 % réduction / prominent skeletal structure spine and beginning at the pelvis (emacîated)	10	Offer moistened food, présentation to veterinarian and lead expérimenter, administration of metamizole in the drinkîng water
20 % réduction ! very prominent skeletal structure of spine and pelvic bones (emacîated)	20	Euthanasia
General condition (coat, eyes, respiration)	Open, clear eyes; coat: smooth, shiny and close fitting; clean and dry orifices; even, hardly visible respiration.	0
Coat somewhat dull, partly erect (piloerection).	2
Coat alîogether slightly shaggy and possibly erect; body openings unkempt; clear sécrétion (smear film) on one or both eyes.	5	Cleaning of body orifices, eye ointment
Very shaggy/straggly coat, body openings slightly stuck together, eye(s) no longer fully open.	10	2 x daily adspection, offer moistened food, cleaning of body orifices, eye ointment, présentation to veterinarian and lead expérimenter, administration of metamizole in the drinkîng water
Coat very shaggy and very puffy general impression with strongly stuck or reddened or swollen eyes or completely closed eyes, visibly increased breathing with possibly relieving posture	20	Euthanasia
Behaviour (spontaneous and provoked)	regular food ! water intake, active and attentive/curious (exploration behaviour), social contacts, nestbuilding instinct, personal hygiene	0
Slightly reduced activity or slight hyperkinetîcs	5	Offer moistened food
Clearly reduced activity, possibly séclusion from the group with defeuce against cage mates, provoked behaviour: Aggressiveness when touched /grabbing or reduced reaction to touch.	10	2 x daily adspection, offer moistened feed, présentation to veterinarian and lead expérimenter, administration of metamizole via drinkîng water
Strongly reduced activity, no exploration behaviour and/or nest-building instinct, strongly reduced social behaviour up to self-isolation, apathy; Provoked behaviour: Sounds of pain when grasping and/or no reaction to touch.	20	Euthanasia
Posture / motor function (includes rare S A RS-	Normal gait, including raising on hind legs; normal spreadîng of hînd limbs when lifted by tail	0
Gait abnonnalities with slowed movement on cage floor; hind legs not fully spread when lifted on tail	5	Offer moistened food

CoV-2 spécifie neurological déficits).	Reduced movement pattern with stiff gait, coordination problems; when lîfted by tail, hind legs not spread and paralysis of one limb	10	2 x daily adspection, offer moistened feed, présentation to veterinarian and lead expérimenter, administration of metamizole via drinking water
Curved posture (tucked up abdomen, painful hump); toe-tapping gait; Tremor / paralysis (dragging behind one limb); when lifted by the tail hind legs not spread and paralysis oftwo limbs	20	Euthanasîa

Détermination of organ viral load

Organs were homogenized in 2 ml PBS, Viral RNA was isolated from 140 μΐ of homogenates using QIAamp Viral RNA Mini Kit (Qiagen). RT-qPCR reactions were 5 performed using TaqMan® Fast Virus 1 -Step Master Mix (Thermo Fisher) and 5 μΐ of isolated RNA as a template, Synthetic SARS-CoV2-RNA was used as a quantitative standard to obtain viral copy numbers.

Results ] o The survival rate of iPPVO treated and SARS-CoV-2 infected mice was significantly higher in comparison to untreated mice after 10 days: 40% in the iPPVO treated group compared to 20% in the control group, Also, mortality was delayed. See Figure 4.

In line with this, the decrease in body weight and onset of clinical signs/symptoms were delayed in the iPPVO treatment group. At days 4 to 6 after SARS-CoV-2 infection, body weight 15 was higher and the clinical score was lower in the iPPVO treated mice compared to the control group. At day 7 this was reversed due to euthanasîa of mice in the control group that reached humane endpoints, i.e. of the mice worst affected by SARS-CoV-2 infection. These mice were not included in the data points after being euthanised, so means of the control group include only the healthiest mice from day 7 on. At days 7 and 8, mice ofthe iPPVO treatment group 20 reached human endpoints and had to be euthanised. Body weight and clinical score were similar in the surviving mice of both treatment and control groups (day 9 and 10). See Figures 5 and 6.

The SARS-CoV-2 viral load was reduced dramatically in both lungs ( 16x compared to the control group) and brains (664x compared to placebo) of infected mice. See Figure 7.

Overall, these data clearly demonstrate a bénéficiai effect of PPVO treatment on the 25 clinical outcome of SARS-CoV-2 infected mice, suggesting an improvement of the clinical situation of SARS-CoV-2 infected patients, including a delay in the onset of symptoms and a milder course of the disease.

Claims (15)

1. A Parapoxvirus ovis (PPVO) agent for use in (i) preparing a subject for a coronavirus infection or (ii) treating a coronavirus infection in a subject.

2. The PPVO agent for use according to claim 1, wherein the PPVO agent is:

(a) PPVO itself or a fragment thereof, or (b) an agent that comprises genetic information encoding for (a).

3. The PPVO agent for use according to claim 1 or 2, wherein the PPVO agent is an inactivated PPVO virion.

4. The PPVO agent for use according to any one of claims 1 to 3, wherein the coronavirus îs a SARS coronavirus.

5 (iii) a nucleic acid encoding for (i) or (ii), (iv) a vector comprising the nucleic acid of (iii), (v) a cell comprising the nucleic acid of (iii) or the vector of (iv), (vi) an antibody or an antigen-binding fragment thereof bindîng to coronavirus or a fragment thereof,

5 (iii) a nucleic acid encoding for (i) or (ii), (îv) a vector comprising the nucleic acid of (iii), (v) a cell comprising the nucleic acid of (iii) or the vector of (iv), (vi) an antibody or an antigen-binding fragment thereof binding to coronavirus or a fragment thereof,

5. The PPVO agent for use according to any one of claims 1 to 4, wherein the coronavirus is SARS-CoV-2 or a variant thereof with at least 80% sequence îdentity to SEQ ID NO: 1 or 2.

6. The PPVO agent for use according to any one of claims 1 to 5. wherein the subject to be prepared is characterized by one or more risk factors for contracting a coronavirus infection and/or by one or more risk factors for having a severe course of coronavirus disease.

7. The PPVO agent for use according to any one of claims 1 to 5, wherein the subject to be treated îs asymptomatic.

8. The PPVO agent for use according to any one of claims 1 to 5, wherein the subject to be treated has coronavirus disease.

9. The PPVO agent for use according claim 8, wherein the subject has either no severe symptoms, or has a severe course of coronavirus disease.

10 (vîi) a nucleotide/nucleoside analogue, (viiî) a coronavirus-specific RNAi molécule, (ix) a small molécule, an antibody or an antigen-binding fragment thereof, bindîng to a site of proteîn-protein interaction (PPI) between a coronavirus protein and a subject protein, and

10 (vii) a nucleotide/nucleoside analogue, (viii) a coronavirus-specific RNAi molécule, (ix) a small molécule, an antibody or an antigen-binding fragment thereof, binding to a site of protein-protein interaction (PPI) between a coronavirus protein and a subject protein, and

15 (x) blood plasma of a subject that has recovered from a coronavirus infection;

the further médicament is selected from the group consisting of an immune stimulatory agent, an antiviral, an antibiotic, an adjuvant, a glucocortîcoid, an antihypertensive drug, an hypoglycaemic drug, an anti-sbock drug, and a drug suitable for treating one or more symptoms of coronavirus disease; and/or

20 - the one or more further thérapies are selected from the group consisting of oxygen therapy and extra-corporeal organ support (ECOS).

10. The PPVO agent for use according to any one of claims 1 to 9, wherein the use for (i) comprises use of a coronavirus agent and/or an immune stîmulatory agent for preparing the subject, and the use for (ii) comprises use of a coronavirus agent, one or more further médicaments and/or one or more further non-drug treatments for treating the coronavirus infection.

11. The PPVO agent for use according claim 10, wherein the coronavirus agent îs selected from the group consisting of:

(î) an inactivated coronavirus, (iî) an antigenic fragment of coronavirus,

12. A composition or a kit comprising a PPVO agent and a coronavirus agent.

25

13. A pharmaceutical composition or kit for use in (i) preparing a subject for a coronavirus infection or (ii) treating a coronavirus infection in a subject, wherein the pharmaceutical composition or kit comprises a PPVO agent.

14. The composition or kit of claim 12 or the pharmaceutical composition of kit of claim 30 13, wherein the PPVO agent is (a) PPVO itself or a fragment thereof, or (b) an agent that comprises genetic information encoding for (a), or (c) an inactivated PPVO virion.

T* 15. The composition or kit of claim 12, wherein the coronavirus agent is selected from the group consisting of:

(i) an inactivated coronavirus, (ii) an antigenic fragment of coronavirus,

15 (x) blood plasma of a subject that has recovered from a coronavirus infection.