WO2023083950A1 - Parapoxvirus for preparing for and treatment of respiratory virus infections in combination with immunomodulators - Google Patents

Parapoxvirus for preparing for and treatment of respiratory virus infections in combination with immunomodulators Download PDF

Info

Publication number
WO2023083950A1
WO2023083950A1 PCT/EP2022/081443 EP2022081443W WO2023083950A1 WO 2023083950 A1 WO2023083950 A1 WO 2023083950A1 EP 2022081443 W EP2022081443 W EP 2022081443W WO 2023083950 A1 WO2023083950 A1 WO 2023083950A1
Authority
WO
WIPO (PCT)
Prior art keywords
parapoxvirus
virus
immunomodulator
orf
respiratory
Prior art date
Application number
PCT/EP2022/081443
Other languages
French (fr)
Inventor
Peter Lischka
Helmut Buschmann
Sonakshi BHATTACHARJEE
Original Assignee
Aicuris Gmbh & Co. Kg
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Aicuris Gmbh & Co. Kg filed Critical Aicuris Gmbh & Co. Kg
Publication of WO2023083950A1 publication Critical patent/WO2023083950A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the invention relates to the treatment of respiratory virus infections and to preparing subjects for such an infection by administering a parapoxvirus in combination with immunomodulators. This is to assist the immune system in combatting the respiratory virus and to thereby prevent or to ameliorate symptoms of a respiratory virus disease.
  • Respiratory viruses are the most frequent causative agents of disease in humans, with significant impact on morbidity and mortality worldwide.
  • Common respiratory agents from several virus families are well adapted to efficient person-to-person transmission and circulate in a global scale.
  • Approximately one-fifth of all childhood deaths worldwide are related to acute respiratory infections (ARIs), particularly in impoverished populations of tropical regions, where ARI case-to-fatality ratios can be remarkably higher than in temperate regions of the world.
  • ARIs acute respiratory infections
  • respiratory viruses cause a great burden of diseases, only a few preventive or therapeutic interventions are currently available (Boncristiani et al., Encyclopedia of Microbiology, 2009: 500-518).
  • the respiratory viruses known to date do not account for all relevant respiratory virus diseases. Furthermore, new respiratory virus diseases emerge due to zoonosis. Recently, an outbreak of a coronavirus designated SARS-CoV-2 began in Wuhan, China, and has since spread all over the world. The illness caused by SARS-CoV-2, COVID-19 (coronavirus disease 2019) was declared to be a pandemic by the WHO on March 11, 2020 and it is still far from being controlled despite the development of a number of effective vaccines.
  • Parapoxvirus ovis causes acute dermal infections in goat and sheep, and while it leads to no serious disease in humans, it induces a complex innate and adaptive immune response in humans and treats infections with some specific viruses such as HBV (WO 2019/048640 Al).
  • HBV WO 2019/048640 Al
  • the invention relates, in one aspect, to a parapoxvirus agent for use, in combination with an immunomodulator, in (i) preparing a subject for a respiratory virus infection and/or (ii) treating a respiratory virus infection in a subject, wherein the respiratory virus is not a coronavirus.
  • the invention also relates to an immunomodulator for use, in combination with a parapoxvirus agent, in (i) preparing a subject for a respiratory virus infection and/or (ii) treating a respiratory virus infection in a subject, wherein the respiratory virus is not a coronavirus.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a) a parapoxvirus agent and b) sucrose and/or NaCl (preferably “and”) for use, in combination with an immunomodulator, in medicine (i.e. prophylaxis and/or therapy).
  • an immunomodulator for use, in combination with a pharmaceutical composition comprising a) a parapoxvirus agent and b) sucrose and/or NaCl (preferably “and”), in medicine (i.e. prophylaxis and/or therapy).
  • FIG. 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 +/- 5 pg/ml ConA. Where no ConA was added, PBS was added instead.
  • 5 pl/ml ConA was tested as a co-stimulus. Cultures were incubated for 3 days and supernatants were harvested. The supernatant was transferred to Vero-eGFP cells which were 2-4 hours later infected with SARS-CoV-2 to determine the antiviral activity of the respective supernatant 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).
  • CC mean positive control
  • VC mean negative control given as the mean virus control
  • the antiviral activity of the samples was calculated relative to the positive control CC mean and given in %.
  • FIG. 2 iPPVO D1701 and iPPVO NZ2 stimulate antiviral activity against SARS- CoV-2.
  • Whole-blood cultures were stimulated with iPPVO.
  • iPPVO vehicle served as a negative control.
  • iPPVO D1701 served as positive control.
  • Cultures were incubated for 3 days and supernatants were harvested. The supernatant was transferred to Vero-eGFP cells which were 2-4 hours later infected with SARS-CoV-2 to determine the antiviral activity residing in the respective supernatant samples and incubated for 5 days.
  • eGFP fluorescence as a measure for living cells which have been protected against SARS-CoV-2 due to the antiviral activity of cytokines in the respective supernatant was measured using the ImageJ software.
  • the antiviral activity of the positive control was set to 100 % (see upper dotted line).
  • the antiviral activity of the samples was calculated relative to the positive control and given in %.
  • the lower dotted line marks the value of the negative 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 lines represent means.
  • Figure 4 Survival of mice treated with iPPVO D1701 and infected with SARS- CoV-2. Animals reaching humane endpoints were euthanized. Overlapping lines are partially offset for better readability. Statistical evaluation was performed by Log-rank (Mantel-Cox) test, * indicates significant difference at p ⁇ 0.05.
  • FIG. 5 Body weight of mice treated with iPPVO D1701 and infected with SARS- CoV-2. Animals reaching humane endpoints were euthanized and are marked by a cross (f). Data are presented as means ⁇ standard errors. Statistical evaluation was performed by Multiple t-test (Holm-Sidak corrected), ** indicates significant difference at p ⁇ 0.01.
  • Figure 6 Clinical score of mice treated with iPPVO D1701 and infected with SARS-CoV-2. Animals reaching humane endpoints were euthanized and are marked by a cross (f). Data are presented as means ⁇ standard errors. Statistical evaluation was performed by Multiple t-test (Holm-Sidak corrected), * and ** indicate significant difference 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 geometric mean of each group. Each point represents one mouse. Reduction in viral load of iPPVO treated mice (left) is shown in fold reduction compared to placebo control (right). Statistical evaluation of the data was performed by Mann-Whitney U test in comparison to placebo control (ns: non-significant, **: p ⁇ 0.01).
  • FIG. 8 Body weight, clinical score and viral load in RSV infected mice. Aged and juvenile mice were infected with either 1X10 6 FFU or 5xl0 6 FFU of RSV on day 0. All animals were monitored daily for body weight (A) and clinical score (B). Animals reaching humane endpoints (> 20 points) were euthanized and are marked by a cross (f). Data are presented as means ⁇ standard errors. (C) Data points shown represent viral copy number of each animal with geometric mean of each group. Each point represents one mouse, whereby circles (•) indicate a survival of 6 days post infection and triangles indicates euthanized mouse according humane endpoints (A), hpi: hours post infection; dpi: days post infection
  • FIG. 9 Survival of RSV infected mice treated with iPPVO. Aged and juvenile mice received iPPVO by intravenous injection one day prior to RSV inoculation. Mice were with 5xl0 6 FFU of RSV on day 0. The percentages of surviving animals according to humane endpoints are shown. Overlapping lines are partially offset for better readability. Statistical evaluation of the data was performed by Mantel-Cox test in comparison to corresponding mock control (**: p ⁇ 0.01).
  • FIG 10 Body weight of RSV infected mice treated with iPPVO. Aged and juvenile mice received iPPVO by intravenous injection one day prior to RSV inoculation. Mice were infected with 5xl0 6 FFU of RSV on day 0. All animals were monitored daily for body weight. Animals reaching humane endpoints (> 20 points) were euthanized and are marked by a cross (f ). Data are presented as means ⁇ standard errors. Statistical evaluation of the data was performed by Mann-Whitney U-test in comparison to corresponding mock control (*: p ⁇ 0.05; **: p ⁇ 0.01).
  • Figure 11 Clinical score of RSV infected mice treated with iPPVO. Aged and juvenile mice received iPPVO by intravenous injection one day prior to RSV inoculation. Mice were infected with 5xl0 6 FFU of RSV on day 0. All animals were monitored daily for clinical score. Animals reaching humane endpoints (> 20 points) were euthanized and are marked by a cross (f). Data are presented as means ⁇ standard errors. Statistical evaluation of the data was performed by Mann-Whitney U-test in comparison to corresponding mock control (*: p ⁇ 0.05; **: p ⁇ 0.01).
  • Figure 12 Quantification of RSV-RNA load in mice lungs treated with iPPVO. Aged and juvenile mice received iPPVO by intravenous injection one day prior to RSV inoculation. Mice were infected with 5xl0 6 FFU of RSV on day 0. Data points shown represent viral copy number of each animal with geometric mean of each group. Each point represents one mouse, whereby circles (•) indicate a survival of 6 days post infection and triangles indicates euthanized mouse according humane endpoints (A). Reduction in viral load is shown in fold reduction compared to corresponding mock control. Statistical evaluation of the data was performed by Mann -Whitney U-test in comparison to corresponding mock control (ns: non-significant).
  • FIG. 13 PPVO acts synergistically with ribavirin in the treatment of cells infected with RSV.
  • CM conditioned medium
  • the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
  • the invention relates to a parapoxvirus agent for use, in combination with an immunomodulator, in (i) preparing a subject for a respiratory virus infection and/or (ii) treating a respiratory virus infection in a subject, wherein the respiratory virus is not a coronavirus and optionally is not an influenza virus.
  • the invention also relates to an immunomodulator for use, in combination with a parapoxvirus agent, in (i) preparing a subject for a respiratory virus infection and/or (ii) treating a respiratory virus infection in a subject, wherein the respiratory virus is not a coronavirus and optionally is not an influenza virus.
  • Non-limiting examples of respiratory viruses are respiratory syncytial virus (RSV), metapneumovirus (MPV), influenza virus (IV), parainfluenza virus (PIV), nipah virus (NiV), respiratory adenovirus (ADV), rhinovirus (RV), bocaparvovirus (BoV, previously termed bocavirus), hanta virus (HV, also termed Orthohantavirus), rift valley virus (RVFV), ebola virus (EBOV), marburg virus (MARV), lassa virus (LASV), lymphocytic choriomeningitis virus (LCMV), and variola virus (VARV, also known as smallpox virus).
  • the parapoxvirus is, in a preferred embodiment, Parapoxvirus ovis (PPVO).
  • PPVO Parapoxvirus ovis
  • PPVO Parapoxvirus ovis
  • ICTV International Committee on Taxonomy of Viruses
  • Exemplary strains are NZ2, NZ7, NZ10, D1701, OV/20, OV/7, OV/C2, OV/mi-90, OV-Torino, SAOO, Bo29, orfl l, Greek orf strain 155, and Greek orf strain 176.
  • the preferred strains is NZ2.
  • parapoxvirus agent is, in a preferred embodiment,
  • 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 stimulates 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 recognition receptor (PRR), e.g. TLR9.
  • PRR human pattern recognition receptor
  • the parapoxvirus fragment is a parapoxvirus protein.
  • Exemplary fragments are (exemplary coding sequence (nucleotide positions of SEQ ID NO: 1 representing strain NZ2) in parenthesis preceding the fragment): (3 to 539) ORF LI, (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 Ir, (8042 to 6684) ORF 2r, (9989 to 8070) ORF 3r, (11195 to 10062) ORF 4r, (11493 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
  • ORF 119 (121288 to 122256) ORF 119, (122350 to 123924) ORF 120, (123962 to 125566) ORF 121,
  • ORF 130 (131790 to 133283) ORF 130, (133246 to 133920) ORF 131, (133972 to 134370) ORF 132,
  • 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: 1 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, formaldehyde, glutaraldehyde, 2,2'-dithiodipyridine, or beta-propiolactone).
  • 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 immunomodulator. In one embodiment, it does not comprise genetic information encoding for a heterologous antigen, but it optionally comprises genetic information encoding for immunomodulator.
  • the parapoxvirus agent is a wildtype parapoxvirus.
  • it is a PPVO virion, more preferably an inactivated PPVO virion (iPPVO).
  • the parapoxvirus agent in particular iPPVO, is comprised in a pharmaceutical composition, preferably comprising sucrose and/or NaCl.
  • the immunomodulator is a drug capable of regulating or normalizing 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).
  • cytokine receptors e.g. antigen binding proteins or small compounds
  • antigen binding proteins or small compounds binding preferably neutralizing
  • soluble cytokine receptors e.g. for trapping cytokines. It does not include an antigen. It does also not include the parapoxvirus agent of the invention. In one embodiment, for reasons unrelated to the suitability for the use according to the invention, it does not include Concanavalin A.
  • the immunomodulator is capable of inducing an immune response, or capable of enhancing an immune response induced by the parapoxvirus agent (immunoinducer), e.g. it promotes an antiviral cell state in a subject.
  • the immunomodulator is capable of weakening or suppressing an immune response (immunosuppressor), e.g. it is an anti-inflammatory.
  • the immunomodulator is preferably an immunoinducer.
  • the immunomodulator is preferably an immunosuppressor.
  • the immunosuppressor is capable of weakening or suppressing a cytokine storm.
  • the immunomodulator may also have pleiotropic effects, i.e. it may be an immunosuppressor and an immunoinducer.
  • immunomodulators are thalidomide and analogs therof, ribavirin, corticosteroids and derivatives thereof (immunosuppressors such as dexamethasone, triamcinolone acetonide, triamcinolone hexacetonide, betamethasone sodium phosphate, betamethasone acetate and methylprednisolone acetate), bacillus of Calmette- Guerin (BCG, immunoinducer), an inducer of interferon activity (immunoinducers such as interferon, interferon alpha (IFN-alpha) B/D, a double-stranded (ds) RNA interferon (IFN) inducer, Ampligen (poly Lpoly C124), and the like), TLR agonists (immunoinducers such as triacylated lipoproteins, lipoteichoic acid, peptidoglycans, zymosan
  • immunosuppressors such as cyclosporine, tacrolimus and sirolimus), brilacidin, and immunomodulating antigen binding proteins (e.g. immunosuppressors such as baricitinib, tocilizumab, bamlanivimab, risankizumab, and lenzilumab).
  • a preferred immunomodulator is thalidomide or an analog therof.
  • Thalidomide and its analogues have pleiotropic immunomodulatory effects and can be used both for preparing and for treating.
  • the effects include anti-inflammatory effects (e.g. by TNF-a inhibition) and augmentation of T-cell (in particular CD8 T-cell) and NK cell function.
  • Thalidomide is a chiral drug and its R and S enantiomers show some differerences in their pharmacological effects. Thalidomide is present as a racemised mixture in vivo. However, included herein are also non-racemising, chirally stable R and S enantiomer derivatives such as a-fluoro-4-aminothalidomide.
  • Analogues of thalidomide are divided into class I analogs and class II analogs. Both classes are much more potent TNF-a inhibitors than thalidomide and they exhibit different pharmacological spectra.
  • Class I analogs also termed IMiDs (immunomodulatory imide drugs) do not inhibit phosphodiesterase (PDE) 4 but have broad inhibitory effects on the release of LPS-induced TNF-a, IL-10, IL-6 and IL-12 while increasing IL- 10 production.
  • PDE phosphodiesterase
  • these compounds also potently stimulate T cell proliferation as well as IL-2 and IFN-y production.
  • IMiDs generally have an amino group addition at the C4 location of the phthaloyl ring of thalidomide.
  • Class II analogs also termed SelCiDs (selective cytokine inhibitory drugs) are potent PDE4 inhibitors that suppress TNF-a production, have a modest inhibitory effect on LPS-induced IL- 10 and IL- 12, modestly increase anti-inflammatory IL- 10 generation, but do not affect IL-6 and T cell activation.
  • SelCiDs selective cytokine inhibitory drugs
  • the analogue is an IMiD.
  • IMiDs are lenalidomide (CC-5013), pomalidomide (actimid, CC-4047), ENMD-0995, CPS11, CPS45 and CPS49.
  • the analogue is a SelCiD.
  • exemplary SelCiDs are CC-3052, CC-1069 (SelCID-3), apremilast, PDA, PDP-Me, PDP, PEMN, 4APDPMe, 4NO2PDPMe, TFPDPMe, and PEMN.
  • the immunomodulator is comprised in a pharmaceutical composition.
  • the use of the parapoxvirus agent in combination with the immunomodulator is for “preparing and/or treating” a subject.
  • a subject is prepared with both parapoxvirus agent and immunomodulator in combination, or is treated with both parapoxvirus agent and immunomodulator in combination.
  • the subject is prepared with the parapoxvirus agent and treated with the immunomodulator or is prepared with the immunomodulator and treated with the parapoxvirus agent in combination.
  • the parapoxvirus agent is for preparing the subject and the immunomodulator is for preparing and/or treating the subject.
  • the subject is prepared with the parapoxvirus agent and treated with the immunomodulator and optionally the parapoxvirus agent.
  • a subject not yet infected with the respiratory virus is prepared for the infection with the parapoxvirus agent, in particular a subject having risk factors for contracting a respiratory virus infection and/or having risk factors for having a severe course of a respiratory virus infection as defined further below, and after the subject is infected with the respiratory virus, the subject is treated with the immunomodulator.
  • the subject may further be treated again with the parapoxvirus agent (boosting the effect of the preparing).
  • This most preferred embodiment can also be described as the immunomodulator, optionally in combination with the parapoxvirus agent, for use in treating a respiratory virus infection in a subject that has been prepared for the infection using the parapoxvirus agent.
  • parapoxvirus agent and immunomodulator are comprised in the same pharmaceutical composition, in fact it is preferred that the parapoxvirus agent and the immunomodulator are administered separately (i.e. not within the one composition, simultaneously or non-simultaneously). However, in some embodiments, e.g. wherein the parapoxvirus agent is used for boosting as described above, it is optionally comprised in the same composition as the immunomodulator. Preferably, parapoxvirus agent and immunomodulator, when administered separately, are administered non-simultaneously (i.e. not at the same time). Therein, it is generally envisaged that the parapoxvirus agent is administered prior to the immunomodulator. However, it is possible and may even be preferable that the immunomodulator is administered prior to the parapoxvirus agent. In both cases, the time between these admistrations may for example be 1-30 days, 1-20 days, 3-14 days or preferably 5-10 days.
  • an immunomodulator as described herein includes the use of one or more immunomodulators, administered in combination (in the same composition, separately and then simultaneously or non-simultaneously).
  • the treating may further comprise administering one or more drugs and/or one or more therapies suitable for treating one or more symptoms of a respiratory virus.
  • the drug for example, may be a pain reliever, a sedative, an anti-fever drug, an anti-inflammatory drug such as IL- 10, or cough medication.
  • the one or more further therapies 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. delivering oxygen to the lungs without relying on breathing of the subject).
  • Passive therapies are non-pressurized oxygen therapy and pressurized oxygen therapy.
  • Active therapies are mechanical ventilation and extra-corporeal membrane oxygenation (ECMO, also a specific form of ECOS).
  • ECOS include ECMO, continuous renal replacement therapy (CRRT), hemofiltration (HF) and hemoperfusion (HP) e.g. to remove inflammatory cytokines, and left ventricular assistance.
  • CRRT continuous renal replacement therapy
  • HF hemofiltration
  • HP hemoperfusion
  • the parapoxvirus agent can be administered by any route, including but not limited to the intravenous, intramuscular, oral, parenteral, topical, intradermal, or subcutaneous route.
  • it can be administered directly to the respiratory tract, preferably to the lower respiratory tract, more preferably to the lung (pulmonary delivery).
  • it can be administered via inhalation (e.g. as an aerosol) or intranasally (e.g. as an aerosol or as drops).
  • inhalation e.g. as an aerosol
  • intranasally e.g. as an aerosol or as drops
  • it is administered intravenously.
  • the immunomodulator can be administered by any route, including those recited above.
  • the dose of the parapoxvirus agent is a pharmaceutically effective dose as can be determined by the skilled person.
  • the agent in case of the agent being a virion (live or inactivated), it can be in the range of from 10 6 to 10 10 particles.
  • the dose may be administered once or more than once for preparing or for treating, for example over a period of ⁇ 12 weeks, ⁇ 6 weeks, ⁇ 4 weeks, ⁇ 2 weeks, or ⁇ 1 week.
  • the respiratory virus is in general a human respiratory virus, i.e. a respiratory virus capable of infecting humans.
  • the respiratory virus may be any virus that can infect humans by respiratory transmission, in particular by droplet transmission and/or by airborne transmission. In one embodiment, it may be any virus that is capable of infecting the respiratory tract. However, the respiratory virus may also be capable of infecting other organs than the respiratory tract.
  • the respiratory virus may be a DNA or an RNA virus, although it is preferably an RNA virus.
  • viruses may be of the families pneumoviridae, e.g. a respiratory syncytial virus (RSV) or a metapneumovirus (MPV), orthomyxoviridae (e.g.
  • influenza virus IV
  • paramyxoviridae e.g. a parainfluenza virus, PIV, or a nipah virus, NiV
  • adenoviridae e.g. a respiratory adenovirus, ADV
  • picomaviridae e.g. a rhinovirus, RV
  • parvoviridae e.g. a bocaparvovirus, BoV, previously termed bocavirus
  • hantaviridae e.g. a hanta virus, HV, also termed Orthohantavirus
  • phenuiviridae e.g. a rift valley virus, RVFV
  • filoviridae e.g.
  • an ebola virus, EBOV, or a marburg virus MARV arenaviridae (e.g. a lassa virus, LASV, or a lymphocytic choriomeningitis virus, LCMV), or poxviridae (e.g. a variola virus, VARV, also known as smallpox virus).
  • MARV ebola virus
  • LASV lassa virus
  • LCMV lymphocytic choriomeningitis virus
  • poxviridae e.g. a variola virus, VARV, also known as smallpox virus.
  • species of respiratory viruses are RSV, IV, PIV, NiV, MPV, ADV, RV, BoV, HV, RVFV, species of the genus EBOV (including Bundibugyo, Reston, Sudan, Tai Forest, Zaire, and Bombali), MARV, LASV, LCMV and VARV.
  • the respiratory virus is selected from the group consisting of RSV, IV, PIV, MPV, ADV, RV and BoV.
  • RSV it may be subtype RSVA or RSVB.
  • IV it may be subtype IAV, IBV, ICV or IDV.
  • Exemplary IV serotypes are H5N1, H9N2, H7N7, H7N2, H7N3, H5N1, H5N6, H3N2v, H7N9, H6N1 and H10N8.
  • PIV it may be subtype HPIV-1, HPIV-2, HPIV-3 or HPIV-4.
  • MPV it may be subtype HPMV-A (Al or A2) or HPMV-B.
  • ADV it may be subtype A, B, C, D, E, F or G.
  • RV it may be subtype A, B or C.
  • BoV it may be subtype HBoV-1, HBoV-2, HBoV-3 or HBoV-4.
  • VARV it may be subtype major or minor.
  • the respiratory virus preferably is not a coronavirus (CoV).
  • the respiratory virus is also not TV, and/or also not varizella zoster virus (VZV), also for reasons unrelated to suitability of the use according to the invention.
  • the respiratory virus may also be IV if the parapoxvirus strain is NZ2.
  • a respiratory virus infection is defined by the entry of a respiratory virus into at least one cell of a subject and its replication in the at least one cell.
  • the infection is via airborne transmission. More preferably, it 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 of the larynx below the vocal folds, trachea, bronchi, bronchioles and the lungs including the respiratory bronchioles, alveolar ducts, alveolar sacs and alveoli).
  • the infection 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 respiratory virus-antigen-specific immune factor, preferably selected from the group consisting of B cells, follicular helper T cells (TFH cells), activated CD4 + T cells and CD8 + T cells (particularly also CD38 HLA-DR ), IgM antibodies, and IgG antibodies.
  • a respiratory virus-specific cytokine profile preferably in the blood or at the site of infection as specified above (most preferably in the lung).
  • cytokine profiles can be determined by the skilled person without undue burden, e.g. by taking a tissue sample, e.g. from the lung, infecting it with the respiratory virus and determining the expression of cytokines.
  • respiratory virus-specific cytokine profiles are (a) upregulation of one or more of, preferably all of IFN-I (e.g.
  • IFN- IFN-
  • IFN-II fFN-y
  • IFN-III IFN-kl, IFN-X2, and IFN-13
  • IL- Ibeta IL-6, IL-12, IL-8, MCP-1, MIP-lalpha, RANIES, CXCL1, CXCL2, CXCL5, and CXCL9, and preferably no upregulation of CXCL10; and (b) upregulation of one or more of, preferably all of IL-6, MCP-1, CXCL1, CXCL5, and/or CXLC10, and preferably no upregulation of any IFN and/or of any one or all of IL-lbeta, IL-12, IL-8, MIP-lalpha, RANTES, CXCL2, and CXCL9.
  • the preferred subject is human. While to the inventors’ best knowledge respiratory viruses infect all humans equally, there are risk factors for contracting a respiratory virus infection:
  • the area is preferably an ‘Affected Area’ as determined by a government authority or the WHO. “Recent” can mean within the incubation period of the respiratory virus, preferably within the past 27, 24, or preferably 19 days, more preferably 14 days or most preferably 10 days.
  • Contact can mean exposed to respiratory droplets of the person (e.g. within range of respiratory droplets in air or in contact with surfaces touched by person or by respiratory droplets).
  • a respiratory virus infection may or may not cause symptoms of a respiratory virus disease in a subject.
  • the terms “respiratory virus infection” and “respiratory virus disease” are distinguished herein by the presence of at least one respiratory virus disease symptom. As long as the infection is not accompanied by at least one symptom of a respiratory virus disease, it (or the subject) is asymptomatic (includes presymptomatic).
  • the term “respiratory virus disease” as used herein requires the presence of a respiratory virus infection and at least one symptom of a respiratory virus disease (also referred to herein as “symptomatic infection”).
  • Respiratory virus disease symptoms include nausea, vomiting and/or diarrhoea; headache; fever (>37.8°C); and organ (e.g. heart, lung, liver and/or kidney) failure. Further symptoms are selected from the group consisting of cough (with or without sputum; usually except VARV and RVFV); itchy or sore throat (usually except HV and RVFV); body (e.g.
  • “Usually except” herein means that the symptom occurs in the respriratory virurses of the invention not listed as exceptions above, and that the symptom may occur in some subjects infected with a virus listed as an exception, but that it is not indicative of or typical for the infection with that virus.
  • the respiratory virus disease is characterized by the presence of two or more, three or more, or four or more symptoms, preferably including one, two or all of fever (>37.8°C), headache, and optionally cough and/or breathing difficulty.
  • the respiratory virus disease is preferably a respiratory disease.
  • the respiratory virus 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 a respiratory virus disease.
  • Mild symptoms are selected from the group consisting of cough (with or without sputum), mild fever (>37.8°C to ⁇ 40°C), runny and/or blocked nose; fatigue; itchy or sore throat; headache; body (e.g. muscle and/or joint) pain; nausea, vomiting and/or diarrhoea; lymphophenia; conjunctivitis; wheezing; and croup.
  • Severe symptoms are selected from the group consisting of breathing difficulty, in particular acute respiratory distress syndrome; pneumonia; organ (e.g. heart, lung, liver and/or kidney) failure; cyanosis 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 a respiratory virus disease, (ii) need for hospitalization, in particular need for admission to intensive care unit (ICU) and/or need for oxygen therapy (as defined above), in particular for a respirator (also referred to as ventilator), and/or (iii) death of the subject.
  • a need for hospitalization is characterized by one or more of: clinical or radiological evidence of pneumonia, acute respiratory distress syndrome, and/or the presence of two or more symptoms including at least high 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 (age younger than 2 months: 60 or more breaths per minute; age 2 to 11 months: 50 or more breaths per minute, age 1 to 5 years: 40 or more breaths per minute, age > 5 years: 30 or more breaths per minute), respiratory distress and/or inadequate oxygenation (e.g. SpCh of 93% or less for adults, and 90% or less for children).
  • Severe complications are complications that are lethal when untreated and include, for example, septic shock, acute respiratory distress syndrome and organ failure.
  • a severe course can be characterized immunologically e.g. by the presence or development of a cytokine storm, in particular in the lung.
  • a respiratory virus 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.
  • the cytokine storm is preferably in the respiratory tract as defined for the infection above (including preferred parts thereof).
  • age i.e. old age (e.g. at least 40, 50, 60, 70, or 80 years with risk increasing with age) or young age (e.g. up to 5 years, 2 years or 6 months with risk increasing with lower age),
  • comorbidities preferably selected from the group consisting of cardiovascular disease, diabetes, obesity, respiratory disease (in particular chronic), hypertension, an immunocompromised state (e.g. AIDS), cancer, liver disease, kidney disease and lung disease.
  • an immunocompromised state e.g. AIDS
  • cancer e.g. cancer, liver disease, kidney disease and lung disease.
  • a particular comorbidity as risk factor is a respiratory disease (other than the respiratory virus disease), lung disease and/or an immunocompromised state.
  • the preparing of a subject is meant to make a subject readier (in the sense of more fortified or more protected immunologically) for a respiratory virus infection, which is afforded inter alia by the immunomodulation, in particular by the parapoxvirus agent, resulting in the medical use of the invention. It may also be described as promoting an antiviral cell state in a subject (as defined further below). It can also be termed “preparatory treatment”, “prophylactic treatment” or, with respect to the disease resulting from the invention, “preventative treatment” or “preventive treatment”. It is not meant to necessarily prevent a respiratory virus infection. As such, the subject is not yet infected by the respiratory virus.
  • the preparing can have one or more immunological effects or (preferably and) it can have one or more clinical effects, both once a subject is infected.
  • immunological effects are preferably selected from the group consisting of
  • the part of the subject is preferably the respiratory tract (upper or preferably lower respiratory tract, more preferably the lung).
  • (I-ii) is preferred over (I-i), and (I-iii) over (I-ii). Further (I-ii) may involve (I-i), and (I-iii) may involve (I-i) and/or (I-ii). (I-iv) is generally desired.
  • (C-ii) is preferred over (C-i), (C-iii) over (C-ii), (C-iv) 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 reduction of the degree of prospective symptoms refers to a prophylactic treatment of symptoms.
  • the parapoxvirus agent can be for use in one or more of (I-i) to (I-iv) and/or one or more of (C-i) to (C-vi) in a subject not yet infected by a respiratory virus.
  • 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).
  • the subject to be prepared is characterized by one or more risk factors for contracting a respiratory virus infection and/or by one or more risk factors for having a severe course of a respiratory virus disease.
  • the treating of a respiratory virus infection does not necessarily mean to treat or prevent a respiratory virus disease, although it is preferred that it does.
  • the treating can be of an asymptomatic infection (i.e. of an infected subject not having symptoms) or of the respiratory virus disease (i.e. of an infected subject having one or more symptoms).
  • it is a therapeutic treatment.
  • the treating can have one or more immunological effects or (preferably and) it can have one or more clinical effects.
  • immunological effects are preferably as defined for the preparing 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 (C-vi) as defined for the preparing above.
  • the clinical effects for a symptomatic infection are preferably selected from the group consisting of (C-i), (C-vi), both as defined for the preparing above, (C-vii) ameliorating one or more (or ideally all) symptoms of a respiratory virus disease, (C-viii) preventing, one or more (or ideally all) further symptoms of a respiratory virus disease, and
  • the parapoxvirus agent/immunomodulator according to the invention can be for use in one or more of (I-i) to (I-iv) and/or one or more of (C-i) to (C-ix) in a subject infected by the respiratory virus.
  • the parapoxvirus agent/immunomodulator according to the invention 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-ix).
  • the parapoxvirus agent/immunomodulator according to the invention is for use in preventing or treating a respiratory virus disease.
  • the subject to be treated may be asymptomatic.
  • the subject to be treated may have a respiratory virus disease.
  • the subject having a respiratory virus disease may not have any severe symptoms, e.g. it has only one or more mild symptoms.
  • it may have a severe course of a respiratory virus disease (excluding (iii) death; preferably (i) one or more severe symptoms).
  • the subject to be treated e.g. any of the afore-mentioned subjects except those already having a severe course of a respiratory virus disease
  • the subject is characterized by at least one of:
  • the subject is characterized by (ii) and/or (iii), (ii) and HScore of 169 or less, or (iii) and age 18 to 65 years or up to 5 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 respiratory virus diseases (Pehta et al., The Lancet Vol 395 March 28, 2020).
  • sHLH secondary haemophagocytic lymphohistoicytosis
  • An HScore of greater than 169 is 93% sensitive and 86% specific for sHLH.
  • the HScore is calculated using the following criteria: temperature ( ⁇ 38.4°C: 0 points, 38.4-39.4°C: 33 points, >39.4°C: 49 points), organomegaly (none: 0 points, hepatomegaly or splenomegaly: 23 points, hepatomegaly and splenomegaly: 38 points), number of cytopenias* (one lineage; 0 points, two lineages: 24 points, three linages: 34 points), triglycerides ( ⁇ 1.5 mmol/1: 0 points, 1.5-4.0 mmol/1: 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), serum aspartate aminotransferase ( ⁇ 30 IU/L: 0 points, >30 Il
  • fHIV positive or receiving long-term immunosuppressive therapy e.g. one or more of glucocorticoids, cyclosporine and/or azathioprine.
  • the subject to be treated is one that was prepared for a respiratory virus infection according to the invention.
  • the invention relates to a parapoxvirus agent for use as a medicament promoting an antiviral cell state in a subject (i) having a respiratory virus infection or (ii) characterized by one or more risk factors for contracting a respiratory virus infection and/or by one or more risk factors for having a severe course of a respiratory virus disease, in combination with an immunomodulator for use in preparing for or preferably treating the respiratory virus infection.
  • the respiratory virus preferably is not a coronavirus and optionally is not an influenza virus.
  • the invention also relates to the use of an immunomodulator for use in preparing for or preferably treating a respiratory virus infection, in combination with a parapoxvirus agent for use as a medicament promoting an antiviral cell state in a subject (i) having a respiratory virus infection or (ii) characterized by one or more risk factors for contracting a respiratory virus infection and/or by one or more risk factors for having a severe course of a respiratory virus disease.
  • the respiratory virus preferably is not a coronavirus and optionally is not an influenza virus.
  • the one or more risk factors for contracting a respiratory virus infection are selected from factors (i), (ii) and (iii) as defined above, e.g. (iii).
  • the one or more risk factors for having a severe course of a respiratory virus disease comprise at least (i) and (iv) as defined above, wherein the comorbidity is preferably a respiratory disease (in particular chronic), lung disease and/or an immunocompromised state.
  • the age of the subject is preferably at least 60, at least 70, or more preferably at least 80 years; or alternatively up to 5 years, preferably up to 2 years or more preferably up to 6 months.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a) a parapoxvirus agent and b) sucrose and/or NaCl for use, in combination with an immunomodulator, in medicine (i.e. prophylaxis and/or therapy).
  • the invention also relates to an immunomodulator for use, in combination with a pharmaceutical composition comprising a) a parapoxvirus agent and b) sucrose and/or NaCl, in medicine (i.e. prophylaxis and/or therapy).
  • the pharmaceutical composition may be formulated for routes of parapoxvirus administration as above (i.e. it may be for use by any route of administration). In a preferred embodiment, it is not formulated for oral administration (i.e. it is not for use by oral administration).
  • the use in medicine i.e. prophylaxis and/or therapy
  • the use in medicine preferably is (i) the preparing of a subject for a respiratory virus infection and/or (ii) the treating of a respiratory virus infection in a subject as described herein, although for any respiratory virus or the specific ones described herein (including CoV and IV).
  • the respiratory virus is not IV, not VZV and/or not CoV.
  • it is not CoV and optionally not IV.
  • the coronavirus is, in a preferred embodiment, an alphacoronavirus or a beta-coronavirus.
  • alpha-coronaviruses examples include HCoV-229E and CoV- NL63
  • beta-coronaviruses examples include SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-HKUl, and HCoV-OC43.
  • the coronavirus is a MERS or a SARS coronavirus, wherein preferably the SARS coronavirus is a virus of the species "severe acute respiratory syndrome-related coronavirus” as classified by the International Committee on Taxonomy of Viruses (ICTV). In a preferred embodiment, it is SARS-CoV-2.
  • the invention further relates to the following methods:
  • a method of administering a parapoxvirus agent and an immunomodulator to a subject (i) having a respiratory virus infection or (ii) characterized by one or more risk factors for contracting a respiratory virus infection and/or by one or more risk factors for having a severe course of a respiratory virus disease, wherein the respiratory virus is not a coronavirus and optionally is not an influenza virus.
  • a method of promoting an antiviral cell state in a subject comprising administering a parapoxvirus agent and an immunomodulator to the subject, wherein the respiratory virus is not a coronavirus and optionally is not an influenza virus.
  • a method of (i) preparing a subject for a respiratory virus infection and/or (ii) treating a respiratory virus infection in a subject comprising administering a parapoxvirus agent and an immunomodulator to the subject, wherein the respiratory virus is not a coronavirus and optionally is not an influenza virus.
  • a method of treating a respiratory virus disease in a subject comprising administering a parapoxvirus agent and an immunomodulator to the subject, wherein the respiratory virus is not a coronavirus and optionally is not an influenza virus.
  • a method administering (i) a pharmaceutical composition comprising (a) a parapoxvirus agent and (b) sucrose and/or NaCl, and (ii) an immunomodulator to a subject.
  • the method is preferably for prophylaxis and/or therapy.
  • parapoxvirus is not canine distemper virus (CDV)
  • the coronavirus is not porcine epidemic diarrhea virus (PEDV), and/or
  • the subject is not a pig (pig including sow and piglet).
  • identity refers to 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 of the 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.
  • MATRIX BLOSUM62, Gap Open: 10.0, Gap Extend: 0.5
  • MAFFT Matrix: Blosum62, Gap Open 1.53, Gap Extend 0.123
  • 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.
  • the "best sequence alignment" is defined as the alignment that produces 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.
  • variant refers generally to a modified version of the polynucleotide, e.g. a mutation, so one or more nucleotides of the polynucleotide may be mutated.
  • the variant is functional, meaning e.g. with regard to a virus that the virus is capable of infecting a host cell.
  • the functionality is generally that described for the polynucleotide the variant is from.
  • a “mutation” can be a nucleotide substitution, deletion and/or insertion (“and” may apply if there is more than one mutation). Preferably, it is a substitution.
  • respiratory transmission refers to the generation of aerosols (either droplet or small-particle aerosols) from the respiratory tract (e.g., nasal passages, trachea, or lungs) that then enter the airspace and cause infection by droplet or airborne spread. It involves inhalation of infectious aerosols suspended in the air and can involve aerosol particles of various sizes that either land on mucosal surfaces, such as the nose and mouth, or are inspired deeper than nose and mouth into the respiratory tract. It can also be described as an aerosol transmission from the respiratory tract of an infectious individual to mucosal surfaces or the resporiatory tract of an uninfected individual. Respiratory transmission includes both airborne transmission and droplet transmission.
  • airborne transmission refers to a virus transmission resulting from the inhalation of small respirable particles that remain infective over time and distance and can be dispersed over distances by air currents.
  • RSV, IV, PIV, NiV, MPV, ADV, RV, BoV, HV, RVFV, LCMV and VARV are known for airborne transmission.
  • other viruses such as EBOV, MARV, LASV can mutate towards the capability of airborne transmission, and airborne transmission has been shown for EBOV (to which MARV is very similar) by Osterholm et al. (mBio, Vol. 6, No.2, 1 May 2015).
  • droplet transmission refers to a direct contact transmission in which respiratory droplets carrying infectious pathogens transmit infection when they travel directly from the respiratory tract of an infectious individual to susceptible mucosal surfaces of the recipient. These droplets can be considered “propelled" from the infectious person and are not dispersed by air currents. All respiratory viruses referred to herein are capable of droplet transmission.
  • immunogen refers to an antigen that is capable of inducing an immune response.
  • Pattern recognition receptor refers to a germline-encoded host sensor which detects a pathogen-associated molecular pattern (P MP), which is specific for a pathogen, and/or a damage-associated molecular pattern (DAMP), which is specific for components of a host cell that are released during cell damage or death.
  • P MP pathogen-associated molecular pattern
  • DAMP damage-associated molecular pattern
  • a PRR can be a 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 adequate immune response to the antigen/immunogen of interest.
  • adjuvants examples include gel-like precipitates of aluminum hydroxide (alum); AIPO4; alhydrogel; bacterial products from the outer membrane of Gramnegative bacteria, in particular monophosphoryl lipid A (MPLA), lipopolysaccharides (LPS), muramyl dipeptides and derivatives thereof; Freund’s incomplete adjuvant; liposomes, 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 derivatives, in particular PamsCys; lipoarabinomannan;
  • MPLA monophosphoryl lipid A
  • LPS lip
  • 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 ingredient.
  • 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 IFN-a and/or p. Preferably it inhibits (i.e. prevents or at least reduces) virus replication in the cell and/or induces apoptosis (to prevent further virus replication 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 medicament. Thereby, it stops or at least slows down virus propagation.
  • Promotering includes an induction (e.g. for cells not yet in an antiviral state) and a reinforcement (i.e.
  • potentiation e.g. for cells already in an antiviral state
  • the former is preferred for a subject not (yet) having a respiratory virus infection, e.g. characterized by one or more risk factors as specified above, and the latter is preferred for a subject having a respiratory virus infection.
  • vector includes any vectors known to the skilled person including plasmid vectors, cosmid 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 encephalitis virus (VEE), Sindbis virus (SIN), semliki forest virus (SFV), and VEE-SIN chimeras), herpes virus vectors, measles virus vectors, pox virus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara (MV A), NYVAC (derived from the Copenhagen strain of vaccinia), and avipox vectors: canarypox (ALVAC) and fowlpox (FPV) vectors), and vesicular stomatitis virus vectors, or virus like particles.
  • Ad adenovirus
  • AAV adeno-
  • virus-like particle 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 penetration by the virus, which ensures efficient cell entry. Methods for producing particular VLPs are known in the art.
  • mitogen refers to an agent, e.g. a peptide or protein that induces a cell to begin cell division.
  • 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.
  • nucleoside analogue refers to a nucleoside containing a nucleic acid analogue and a sugar.
  • nucleotide analogue refers to a nucleotide containing a nucleic acid analogue, a sugar and a phosphate group with 1 to 3 phosphates.
  • 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.
  • antigen binding protein refers to antibodies and antibody-like proteins, including fragments and derivatives of antibodies.
  • Antigen binding proteins may comprise at least one variable domain, for example antibodies, domain antibodies (dAb), multiples of domain antibodies e.g.
  • a “domain antibody” or “dAb” is an immunoglobulin single variable domain which is capable of binding to an antigen.
  • An immunoglobulin single variable domain may be a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent, nurse shark and Camelid VHH dAbs.
  • Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Camelid-derived derived constructs like nanobodies are also included. Antibodies are a preferred embodiment.
  • composition relates to a composition comprising an active agent according to the invention and one or more pharmaceutically acceptable diluents, carriers, and/or preservatives.
  • Pharmaceutically acceptable diluents, carriers, and/or preservatives are described below and may in addition thereto, if the active agent is the parapoxvirus agent, include an (e.g. aqueous) medium suitable for structurally maintaining a parapoxvirus, in particular an inactivated parapoxvirus, or lyophilisate of this medium.
  • the kit may further comprise a leaflet with instructions for use of the parapoxvirus agent and the immunomodulator, preferably according to a medical use described herein.
  • the composition can be formulated for any route of administration as defined above.
  • Particular preferred pharmaceutical forms for the administration of the agents according to the invention are forms suitable for injectable use and include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • a solution or dispersion will include a solvent or dispersion medium, containing, for example, water-buffered aqueous solutions, e.g. biocompatible buffers, ethanol, 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-bacterial or anti-fungal agents, e.g. parabene, chlorobutanol, phenol, sorbic acid or thiomersal. Further, isotonic agents, such as sugars or salts, in particular sodium chloride may be incorporated in infusion or injection solutions.
  • Preferred diluents of the invention are water, physiological acceptable buffers, physiological acceptable buffer salt solutions or salt 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 magnesium stearate, talc, calcium stearate, zinc stearate, stearic acid, hydrogenated vegetable oil, leucine, glycerids and sodium stearyl fumarates, c) disintegrants such as starches, croscaramellose, sodium methyl cellulose, agar, bentonite, alginic acid, carboxymethyl cellulose, polyvinyl pyrrolidone and the like.
  • binders such as lactose, mannitol, crystalline sorb
  • 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. 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 have been developed and studied. Examples include liposomes, polymeric micelles, microspheres, nanoparticles, nanofibers, protein-drug conjugates, erythrocytes, virosomes and dendrimers. Different methods of attaching the drug to the carrier can be used, including adsorption, integration into the bulk structure, encapsulation, and covalent bonding.
  • PPVO virions stimulate the innate as well as the adaptive arm of the immune system.
  • APCs antigen-presenting cells
  • Test substances iPPVO of strain D1701 available from Pfizer
  • VeroE6-EGFP propagated in growth medium prepared by supplementing DMEM with 10% v/v heat-inactivated FCS and 5 mL sodium bicarbonate 7.5%, and cultured in T150 bottle and split 1/4 twice a week. Pen-strep added directly to the T150 bottle at a 1/100 dilution.
  • Coronavirus assay medium prepared by supplementing DMEM with 2% v/v heat- inactivated FCS and 5 mL sodium bicarbonate 7.5%. Preparation of samples
  • the plate was incubated at 37 °C, 5 % CO2 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.
  • Dilution of the samples 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 replication 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.
  • virus control VC
  • CC cell control
  • Preparation of the cell suspension A confluent culture (monolayer) of VeroE6-EGFP in T150 bottle was washed with DPBS and 10 mL trypsine 0.25%trypsine/EDTA was added. The bottle was incubated for 1 minute at room temperature, 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 of the cell suspension were seeded to each well of the Greiner plate.
  • FALCON CAT NO 352350 Cell Strainer
  • 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 detectable. 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 expected. Also 5 pg/ml ConA alone (in PBS) did 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 D1701 alone also had a clear positive effect on cell viability, and eGFP expression was further increased when iPPVO DI 701 was used in combination with 5 pg/ml ConA (which by itself showed no effect on eGFP expression at this concentration). Accordingly, iPPVO and ConA act synergistically.
  • Example 1 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 coronavirus.
  • Test substances iPPVO of strain NZ2 (chemically inactivated by binary ethyleneimine, BEI), internal designation AIC649, IxlO 9 lyophilized virions reconstituted in 1 ml water iPPVO of strain DI 701, see Example 1 iPPVO vehicle, negative control, see Example 1
  • Example 1 iPPVO and iPPVO vehicle were used alone. iPPVO D1701 shown to increase cell viability in Example 1 was used as a positive control in Example 2. Results
  • iPPVO DI 701 which in Example 1 was shown to be effective, was used as a positive control and as a 100% benchmark. Both PPVO strains DI 701 and NZ2 are active against coronavirus as shown by the comparison to the negative control. Accordingly, the promotion of an antiviral cell state which is effective against coronavirus is a hallmark of parapoxvirus in general and not only of the strain tested in Example 1.
  • the hamster infection model of SARS-CoV-2 described by Boudewijns et al. (STAT2 signaling as double-edged sword restricting viral dissemination 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 10 9 VP ofiPPVO one day prior to infection with SARS-CoV-2.
  • a second group of six hamsters is treated with iPPVO vehicle (placebo control) instead.
  • hamsters are anesthetized with ketamine/xylazine/atropine and inoculated intranasally with SARS-CoV-2 (1.89 x 10 6 TCIDso in 50 pL).
  • SARS-CoV-2 (1.89 x 10 6 TCIDso in 50 pL).
  • hamsters are euthanized by i.p. injection of 500 pL Dolethal (200 mg/mL sodium pentobarbital, Vetoquinol SA). Lungs are collected and infectious virus is quantified by endpoint virus titration. Efficacy is determined based on viral load in homogenized lung tissues on day 4 post-infection.
  • SARS-Cov-2 strain BetaCov/Belgium/GHB-03021/2020 (EPI ISL 109 407976
  • 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 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).
  • 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 debris.
  • Precellys bead disruption
  • 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 (TCID50) per mg tissue.
  • 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 10 5 compared to iPPVO-treated hamsters, in which the TCID50/mg lung tissue was measured to be 2.1 x 10 5 . See Figure 3.
  • mice are intravenously inoculated with 100 pl of iPPVO (1 vial dissolved in 500 pl supplied buffer, which equals a dose of IxlO 9 virus particles (VP)/animal) prior to coronavirus infection.
  • a second group of 10 mice serves as an infection control group (intravenous inoculation with 100 pl iPPVO buffer).
  • mice of both groups are infected intranasally under isoflurane anesthesia (inhalation of 3% isoflurane) with 900 focus forming units (FFU) of SARS-CoV-2 (German isolate) in 50 pl total volume. Mice are scored daily. Euthanasia is performed on day 10 after infection.
  • Test substances iPPVO strain of DI 701 see Example 1 - iPPVO vehicle (PPVO buffer only, i.e. without virus)
  • the clinical score was determined by adding the score of each clinical parameter according to Table 1 below. Animals were euthanised upon reaching a clinical score of >20 or scoring 20 for one clinical parameter (humane endpoints).
  • Viral RNA was isolated from 140 pl of homogenates using QIAamp Viral RNA Mini Kit (Qiagen). RT-qPCR reactions were performed using TaqMan® Fast Virus 1-Step Master Mix (Thermo Fisher) and 5 pl of isolated RNA as a template. Synthetic SARS-CoV2-RNA was used as a quantitative standard to obtain viral copy numbers.
  • 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.
  • mice of the iPPVO treatment group 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.
  • lOOpl iPPVO strain D1701, available from Pfizer, 230 IFN units reconstituted in 2 ml water
  • a control group 1 (low dose) is infected with a lower dose of RSV (lxlOE6) to show the impact of the high dose.
  • a control group 2 is infected with 5xlOE6 and sacrificed two days after infection to detect the presence of the applied virus.
  • a control group 3 contains young animals (“juvenile”, i.e. 8 weeks old; 5xlOE6) to compare the impact of age. Animals are scored daily. Blood sampling and euthanasia are performed on day 6 after infection (except control group 2). Earlier euthanasia is performed for mice reaching humane endpoints as determined by the clinical score. Response to treatment is assessed by a clinical score determination (clinical symptoms of infection) and body weight loss.
  • the clinical score was determined by adding the score of each clinical parameter according to Table 1 above. Animals were euthanised upon reaching a clinical score of >20 or scoring 20 for one clinical parameter (humane endpoints).
  • Viral RNA was isolated from 140 pl of homogenates using QIAamp Viral RNA Mini Kit (Qiagen). RT-qPCR reactions were performed using using Quantitect Probe RT-PCR Kit (Qiagen), SYBR Green detection and 5 pl isolated RNA as template. 10-fold dilutions of synthetic RSV-RNA of T7-transcripts were used as standards for the quantification of viral copy numbers.
  • the body weight of RSV infected mice not treated with iPPVO decreased over time for all groups, i.e. for aged mice (17 weeks old) infected with IxlO 6 FFU or with 5xl0 6 FFU and for juvenile mice (8 weeks old) infected with 5xl0 6 FFU. Both a high infection titre and age exacerbated the decrease (see Fig. 8 A). Correspondingly, the climical score increased overtime, with high infection titre and age leading to a higher score (see Fig. 8B). The viral load in aged animals increased from 6 hours post infection (hpi) to 2 days post infection (dpi). Until day 6, the viral load decreased in these animals, but it was still higher than at 6 hpi (see Fig. 8C).
  • the survival rate of iPPVO treated and RSV infected mice was significantly higher in comparison to untreated mice after 6 days for both aged and juvenile mice: 60% of the untreated juvenile mice and none of the aged mice survived RSV infection, whereas 100% of the iPPVO treated juvenile and aged mice survived RSV infection (see Fig. 9).
  • body weight, clinical score and viral load were improved in juvenile and aged mice by iPPVO treatment (see Figs. 10, 11 and 12).
  • ribavirin which is known to act immunomodulatory, e.g. by inhibiting production of TNF and IL-1, Ning etal., J Immunol. 1998;160:3487-3493
  • CM conditioned media
  • Fig. 13 B and C The results are shown in Fig. 13 B and C and in Table 2 below.
  • the virus yield was reduced as shown by the reduction of the number of RSV-infected (GFP-expressing) cells in a ribavirin dose-dependent fashion.
  • treatment in combination with iPPVO led to a stronger ribavirin dose-dependent reduction in the number of RSV-infected cells.
  • An increase in the CM concentration strengthened this effect (Fig. 13 B).
  • iPPVO and ribavirin have a synergistic antiviral activity. Accordingly, EC50 values were greatly reduced by the combination treatment compared to ribavirin alone (Table 2).

Abstract

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

Description

PARAPOXVIRUS FOR PREPARING FOR AND TREATMENT OF RESPIRATORY
VIRUS INFECTIONS IN COMBINATION WITH IMMUNOMODULATORS
FIELD OF THE INVENTION
The invention relates to the treatment of respiratory virus infections and to preparing subjects for such an infection by administering a parapoxvirus in combination with immunomodulators. This is to assist the immune system in combatting the respiratory virus and to thereby prevent or to ameliorate symptoms of a respiratory virus disease.
BACKGROUND OF THE INVENTION
Respiratory viruses are the most frequent causative agents of disease in humans, with significant impact on morbidity and mortality worldwide. Common respiratory agents from several virus families are well adapted to efficient person-to-person transmission and circulate in a global scale. Approximately one-fifth of all childhood deaths worldwide are related to acute respiratory infections (ARIs), particularly in impoverished populations of tropical regions, where ARI case-to-fatality ratios can be remarkably higher than in temperate regions of the world. Although respiratory viruses cause a great burden of diseases, only a few preventive or therapeutic interventions are currently available (Boncristiani et al., Encyclopedia of Microbiology, 2009: 500-518).
The respiratory viruses known to date do not account for all relevant respiratory virus diseases. Furthermore, new respiratory virus diseases emerge due to zoonosis. Recently, an outbreak of a coronavirus designated SARS-CoV-2 began in Wuhan, China, and has since spread all over the world. The illness caused by SARS-CoV-2, COVID-19 (coronavirus disease 2019) was declared to be a pandemic by the WHO on March 11, 2020 and it is still far from being controlled despite the development of a number of effective vaccines.
Recurrent respiratory virus epidemics with the potential for pandemics show that these viruses continue to threaten the world as they emerge spontaneously, spread easily and can have severe consequences on human life, including not only health issues but also dramatic social and economic impacts. Therefore, there is a great medical need for prevention and treatment of the infection itself or of the disease symptoms caused by an infection. Furthermore, viruses are known to mutate and recombine often, so they present an ongoing challenge for disease control.
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 innate and adaptive immune response in humans and treats infections with some specific viruses such as HBV (WO 2019/048640 Al). Unexpectedly, the inventors found parapoxvirus to be useful for preparing subjects for and treating respiratory virus infections, in particular in combination with immunomodulators.
SUMMARY OF THE INVENTION
The invention relates, in one aspect, to a parapoxvirus agent for use, in combination with an immunomodulator, in (i) preparing a subject for a respiratory virus infection and/or (ii) treating a respiratory virus infection in a subject, wherein the respiratory virus is not a coronavirus. The invention also relates to an immunomodulator for use, in combination with a parapoxvirus agent, in (i) preparing a subject for a respiratory virus infection and/or (ii) treating a respiratory virus infection in a subject, wherein the respiratory virus is not a coronavirus.
In a further aspect, the invention relates to a pharmaceutical composition comprising a) a parapoxvirus agent and b) sucrose and/or NaCl (preferably “and”) for use, in combination with an immunomodulator, in medicine (i.e. prophylaxis and/or therapy). Regarding this aspect, the invention also relates to an immunomodulator for use, in combination with a pharmaceutical composition comprising a) a parapoxvirus agent and b) sucrose and/or NaCl (preferably “and”), in medicine (i.e. prophylaxis and/or therapy).
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 +/- 5 pg/ml ConA. Where no ConA was added, PBS was added instead. iPPVO vehicle +/- ConA or PBS +/- ConA, respectively, served as controls. 5 pl/ml ConA was tested as a co-stimulus. Cultures were incubated for 3 days and supernatants were harvested. The supernatant was transferred to Vero-eGFP cells which were 2-4 hours later infected with SARS-CoV-2 to determine the antiviral activity of the respective supernatant 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 negative control given as the mean virus control (VC mean). The antiviral activity of the samples was calculated relative to the positive control CC mean and given in %.
Figure 2: iPPVO D1701 and iPPVO NZ2 stimulate antiviral activity against SARS- CoV-2. Whole-blood cultures were stimulated with iPPVO. iPPVO vehicle served as a negative control. iPPVO D1701 served as positive control. Cultures were incubated for 3 days and supernatants were harvested. The supernatant was transferred to Vero-eGFP cells which were 2-4 hours later infected with SARS-CoV-2 to determine the antiviral activity residing in the respective supernatant samples and incubated for 5 days. eGFP fluorescence as a measure for living cells which have been protected against SARS-CoV-2 due to the antiviral activity of cytokines in the respective supernatant was measured using the ImageJ software. The antiviral activity of the positive control was set to 100 % (see upper dotted line). The antiviral activity of the samples was calculated relative to the positive control and given in %. The lower dotted line marks the value of the negative 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 lines represent means.
Figure 4: Survival of mice treated with iPPVO D1701 and infected with SARS- CoV-2. Animals reaching humane endpoints were euthanized. Overlapping lines are partially offset for better readability. Statistical evaluation was performed by Log-rank (Mantel-Cox) test, * indicates significant difference at p < 0.05.
Figure 5: Body weight of mice treated with iPPVO D1701 and infected with SARS- CoV-2. Animals reaching humane endpoints were euthanized and are marked by a cross (f). Data are presented as means ± standard errors. Statistical evaluation was performed by Multiple t-test (Holm-Sidak corrected), ** indicates significant difference at p < 0.01.
Figure 6: Clinical score of mice treated with iPPVO D1701 and infected with SARS-CoV-2. Animals reaching humane endpoints were euthanized and are marked by a cross (f). Data are presented as means ± standard errors. Statistical evaluation was performed by Multiple t-test (Holm-Sidak corrected), * and ** indicate significant difference 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 geometric mean of each group. Each point represents one mouse. Reduction in viral load of iPPVO treated mice (left) is shown in fold reduction compared to placebo control (right). Statistical evaluation of the data was performed by Mann-Whitney U test in comparison to placebo control (ns: non-significant, **: p < 0.01).
Figure 8: Body weight, clinical score and viral load in RSV infected mice. Aged and juvenile mice were infected with either 1X106 FFU or 5xl06 FFU of RSV on day 0. All animals were monitored daily for body weight (A) and clinical score (B). Animals reaching humane endpoints (> 20 points) were euthanized and are marked by a cross (f). Data are presented as means ± standard errors. (C) Data points shown represent viral copy number of each animal with geometric mean of each group. Each point represents one mouse, whereby circles (•) indicate a survival of 6 days post infection and triangles indicates euthanized mouse according humane endpoints (A), hpi: hours post infection; dpi: days post infection
Figure 9: Survival of RSV infected mice treated with iPPVO. Aged and juvenile mice received iPPVO by intravenous injection one day prior to RSV inoculation. Mice were with 5xl06 FFU of RSV on day 0. The percentages of surviving animals according to humane endpoints are shown. Overlapping lines are partially offset for better readability. Statistical evaluation of the data was performed by Mantel-Cox test in comparison to corresponding mock control (**: p < 0.01).
Figure 10: Body weight of RSV infected mice treated with iPPVO. Aged and juvenile mice received iPPVO by intravenous injection one day prior to RSV inoculation. Mice were infected with 5xl06 FFU of RSV on day 0. All animals were monitored daily for body weight. Animals reaching humane endpoints (> 20 points) were euthanized and are marked by a cross (f ). Data are presented as means ± standard errors. Statistical evaluation of the data was performed by Mann-Whitney U-test in comparison to corresponding mock control (*: p < 0.05; **: p < 0.01).
Figure 11: Clinical score of RSV infected mice treated with iPPVO. Aged and juvenile mice received iPPVO by intravenous injection one day prior to RSV inoculation. Mice were infected with 5xl06 FFU of RSV on day 0. All animals were monitored daily for clinical score. Animals reaching humane endpoints (> 20 points) were euthanized and are marked by a cross (f). Data are presented as means ± standard errors. Statistical evaluation of the data was performed by Mann-Whitney U-test in comparison to corresponding mock control (*: p < 0.05; **: p < 0.01).
Figure 12: Quantification of RSV-RNA load in mice lungs treated with iPPVO. Aged and juvenile mice received iPPVO by intravenous injection one day prior to RSV inoculation. Mice were infected with 5xl06 FFU of RSV on day 0. Data points shown represent viral copy number of each animal with geometric mean of each group. Each point represents one mouse, whereby circles (•) indicate a survival of 6 days post infection and triangles indicates euthanized mouse according humane endpoints (A). Reduction in viral load is shown in fold reduction compared to corresponding mock control. Statistical evaluation of the data was performed by Mann -Whitney U-test in comparison to corresponding mock control (ns: non-significant).
Figure 13: PPVO acts synergistically with ribavirin in the treatment of cells infected with RSV. A) Experimental approach as described in Example 6. B) Bar graph showing the reduction in RSV virus yield depending on ribavirin (RBV) dose at various dilutions of conditioned medium (CM) derived from donor PBMCs treated with PPVO. C) Logarithmic line graph showing the reduction in virus yield depending on ribavirin (RBV) dose (log concentration) for cells pretreated for 24h with various dilutions of conditioned medium (CM) derived from donor PBMCs treated with PPVO.
DETAILED DESCRIPTION OF THE INVENTION
Before the 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 invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have 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 multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety.
In the following, the elements of the invention will be described. These elements are listed with specific 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 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 elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
Throughout this specification 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 “consist of’. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents, unless the content clearly dictates otherwise. Generally, the conjunction “and” in “and/or” is preferred (with the exception of the preparing and/or treating referred to herein).
The invention relates to a parapoxvirus agent for use, in combination with an immunomodulator, in (i) preparing a subject for a respiratory virus infection and/or (ii) treating a respiratory virus infection in a subject, wherein the respiratory virus is not a coronavirus and optionally is not an influenza virus. The invention also relates to an immunomodulator for use, in combination with a parapoxvirus agent, in (i) preparing a subject for a respiratory virus infection and/or (ii) treating a respiratory virus infection in a subject, wherein the respiratory virus is not a coronavirus and optionally is not an influenza virus. Non-limiting examples of respiratory viruses are respiratory syncytial virus (RSV), metapneumovirus (MPV), influenza virus (IV), parainfluenza virus (PIV), nipah virus (NiV), respiratory adenovirus (ADV), rhinovirus (RV), bocaparvovirus (BoV, previously termed bocavirus), hanta virus (HV, also termed Orthohantavirus), rift valley virus (RVFV), ebola virus (EBOV), marburg virus (MARV), lassa virus (LASV), lymphocytic choriomeningitis virus (LCMV), and variola virus (VARV, also known as smallpox virus).
Parapoxvirus agent
The parapoxvirus is, in a preferred embodiment, Parapoxvirus ovis (PPVO). This includes any PPVO strain, preferably, any strain of the species PPVO as classified by the International Committee on Taxonomy of Viruses (ICTV). Exemplary strains are NZ2, NZ7, NZ10, D1701, OV/20, OV/7, OV/C2, OV/mi-90, OV-Torino, SAOO, Bo29, orfl l, Greek orf strain 155, and Greek orf strain 176. The preferred strains is 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 stimulates 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 recognition receptor (PRR), e.g. TLR9. In one embodiment, the parapoxvirus fragment is a parapoxvirus protein.
Exemplary fragments are (exemplary coding sequence (nucleotide positions of SEQ ID NO: 1 representing strain NZ2) in parenthesis preceding the fragment): (3 to 539) ORF LI, (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 Ir, (8042 to 6684) ORF 2r, (9989 to 8070) ORF 3r, (11195 to 10062) ORF 4r, (11493 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 13r, (20314 to 19736) ORF 14r, (20401 to 22101) ORF 15, (22125 to 22940) ORF 6, (23003 to 23866) ORF 17, (26908 to 23873) ORF 18r, (26926 to 27213) ORF 19, (27626 to 27216) ORF 20r, (29754 to 27616) ORF 21r, (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, (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 51102) 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, (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, (79816 to 79367) ORF 68r, (80529 to 79858) ORF 69r, (80774 to 80529 ORF 70r, (82815 to 80788) ORF 71r, (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 81r, (94060 to 93788) ORF 82r, (94238 to 94080) ORF 83r, (94508 to 94242) ORF 84r, (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, (100001 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 117114) ORF l l lr, (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) ORF 118,
(121288 to 122256) ORF 119, (122350 to 123924) ORF 120, (123962 to 125566) ORF 121,
(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) ORF 126,
(127851 to 128408) ORF 127, (128520 to 130076) ORF 128, (130105 to 131700) ORF 129,
(131790 to 133283) ORF 130, (133246 to 133920) ORF 131, (133972 to 134370) ORF 132,
(134418 to 134693) ORF 133a, (134402 to 134992) ORF Rl, (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: 1 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, formaldehyde, glutaraldehyde, 2,2'-dithiodipyridine, or beta-propiolactone). In 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 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).
In one preferred embodiment, the parapoxvirus agent, in particular iPPVO, is comprised in a pharmaceutical composition, preferably comprising sucrose and/or NaCl.
Immunomodulator
The immunomodulator is a drug capable of regulating or normalizing 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. antigen binding proteins or small compounds), antigen binding proteins or small compounds binding (preferably neutralizing) cytokines, and soluble cytokine receptors (e.g. for trapping cytokines). It does not include an antigen. It does also not include the parapoxvirus agent of the invention. In one embodiment, for reasons unrelated to the suitability for the use according to the invention, it does not include Concanavalin A.
In one embodiment, the immunomodulator is capable of inducing an immune response, or capable of enhancing an immune response induced by the parapoxvirus agent (immunoinducer), e.g. it promotes an antiviral cell state in a subject. In another embodiment, the immunomodulator is capable of weakening or suppressing an immune response (immunosuppressor), e.g. it is an anti-inflammatory. If used for preparing a subject and optionally for treating an infection (e.g. an asymptomatic infection), the immunomodulator is preferably an immunoinducer. If used only for treating a subject (i.e. the infection and preferably the disease), the immunomodulator is preferably an immunosuppressor. In one embodiment, the immunosuppressor is capable of weakening or suppressing a cytokine storm. The immunomodulator may also have pleiotropic effects, i.e. it may be an immunosuppressor and an immunoinducer.
Exemplary immunomodulators, next to those recited above, are thalidomide and analogs therof, ribavirin, corticosteroids and derivatives thereof (immunosuppressors such as dexamethasone, triamcinolone acetonide, triamcinolone hexacetonide, betamethasone sodium phosphate, betamethasone acetate and methylprednisolone acetate), bacillus of Calmette- Guerin (BCG, immunoinducer), an inducer of interferon activity (immunoinducers such as interferon, interferon alpha (IFN-alpha) B/D, a double-stranded (ds) RNA interferon (IFN) inducer, Ampligen (poly Lpoly C124), and the like), TLR agonists (immunoinducers such as triacylated lipoproteins, lipoteichoic acid, peptidoglycans, zymosan, Pam3CSK4, diacylated lipopeptides, HSPs, HMGB1, uric acid, fibronectin, ECM proteins, dsRNA, Poly I:C, LPS, lipoteichoic acid, P-defensin 2, fibronectin EDA, HMGB1, snapin, tenascin C, flagellin, ssRNA, CpG-A, Poly G10, Poly G3, unmethylated CpG DNA, PamCysPamSK4, Toxoplasma gondii profilin, and VSV), fungus-derived immunomodulators (e.g. immunosuppressors such as cyclosporine, tacrolimus and sirolimus), brilacidin, and immunomodulating antigen binding proteins (e.g. immunosuppressors such as baricitinib, tocilizumab, bamlanivimab, risankizumab, and lenzilumab). A preferred immunomodulator is thalidomide or an analog therof.
Thalidomide and its analogues have pleiotropic immunomodulatory effects and can be used both for preparing and for treating. The effects include anti-inflammatory effects (e.g. by TNF-a inhibition) and augmentation of T-cell (in particular CD8 T-cell) and NK cell function. Thalidomide is a chiral drug and its R and S enantiomers show some differerences in their pharmacological effects. Thalidomide is present as a racemised mixture in vivo. However, included herein are also non-racemising, chirally stable R and S enantiomer derivatives such as a-fluoro-4-aminothalidomide. Analogues of thalidomide are divided into class I analogs and class II analogs. Both classes are much more potent TNF-a inhibitors than thalidomide and they exhibit different pharmacological spectra. Class I analogs, also termed IMiDs (immunomodulatory imide drugs) do not inhibit phosphodiesterase (PDE) 4 but have broad inhibitory effects on the release of LPS-induced TNF-a, IL-10, IL-6 and IL-12 while increasing IL- 10 production. In addition to their strong anti-inflammatory effects, like thalidomide, these compounds also potently stimulate T cell proliferation as well as IL-2 and IFN-y production. IMiDs generally have an amino group addition at the C4 location of the phthaloyl ring of thalidomide. Class II analogs, also termed SelCiDs (selective cytokine inhibitory drugs) are potent PDE4 inhibitors that suppress TNF-a production, have a modest inhibitory effect on LPS-induced IL- 10 and IL- 12, modestly increase anti-inflammatory IL- 10 generation, but do not affect IL-6 and T cell activation. Thus, one of the major distinctive differences between IMiDs and SelCiDs is that members of the latter class of drugs exhibit little or no T cell activation.
In some embodiments, which are useful for both preparing and for treating (in particular for preparing), the analogue is an IMiD. Exemplary IMiDs are lenalidomide (CC-5013), pomalidomide (actimid, CC-4047), ENMD-0995, CPS11, CPS45 and CPS49.
In other embodiments, which are also useful for both preparing and for treating (in particular for treating), the analogue is a SelCiD. Exemplary SelCiDs are CC-3052, CC-1069 (SelCID-3), apremilast, PDA, PDP-Me, PDP, PEMN, 4APDPMe, 4NO2PDPMe, TFPDPMe, and PEMN.
Preferably, the immunomodulator is comprised in a pharmaceutical composition.
Modes of use
According to the invention, the use of the parapoxvirus agent in combination with the immunomodulator (or vice versa) is for “preparing and/or treating” a subject. This includes that a subject is prepared with both parapoxvirus agent and immunomodulator in combination, or is treated with both parapoxvirus agent and immunomodulator in combination. In other embodiments, the subject is prepared with the parapoxvirus agent and treated with the immunomodulator or is prepared with the immunomodulator and treated with the parapoxvirus agent in combination. In preferred embodiments, the parapoxvirus agent is for preparing the subject and the immunomodulator is for preparing and/or treating the subject. In the most preferred embodiment, the subject is prepared with the parapoxvirus agent and treated with the immunomodulator and optionally the parapoxvirus agent. Accordingly, a subject not yet infected with the respiratory virus is prepared for the infection with the parapoxvirus agent, in particular a subject having risk factors for contracting a respiratory virus infection and/or having risk factors for having a severe course of a respiratory virus infection as defined further below, and after the subject is infected with the respiratory virus, the subject is treated with the immunomodulator. The subject may further be treated again with the parapoxvirus agent (boosting the effect of the preparing). This most preferred embodiment can also be described as the immunomodulator, optionally in combination with the parapoxvirus agent, for use in treating a respiratory virus infection in a subject that has been prepared for the infection using the parapoxvirus agent.
The use in “combination” as referred to herein does not mean that both parapoxvirus agent and immunomodulator are comprised in the same pharmaceutical composition, in fact it is preferred that the parapoxvirus agent and the immunomodulator are administered separately (i.e. not within the one composition, simultaneously or non-simultaneously). However, in some embodiments, e.g. wherein the parapoxvirus agent is used for boosting as described above, it is optionally comprised in the same composition as the immunomodulator. Preferably, parapoxvirus agent and immunomodulator, when administered separately, are administered non-simultaneously (i.e. not at the same time). Therein, it is generally envisaged that the parapoxvirus agent is administered prior to the immunomodulator. However, it is possible and may even be preferable that the immunomodulator is administered prior to the parapoxvirus agent. In both cases, the time between these admistrations may for example be 1-30 days, 1-20 days, 3-14 days or preferably 5-10 days.
The use of an immunomodulator as described herein includes the use of one or more immunomodulators, administered in combination (in the same composition, separately and then simultaneously or non-simultaneously).
The treating may further comprise administering one or more drugs and/or one or more therapies suitable for treating one or more symptoms of a respiratory virus. The drug, for example, may be a pain reliever, a sedative, an anti-fever drug, an anti-inflammatory drug such as IL- 10, or cough medication. The one or more further therapies 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. delivering oxygen to the lungs without relying on breathing of the subject). Passive therapies are non-pressurized oxygen therapy and pressurized oxygen therapy. Active therapies are mechanical ventilation and extra-corporeal membrane oxygenation (ECMO, also a specific form of ECOS). Examples for ECOS include ECMO, continuous renal replacement therapy (CRRT), hemofiltration (HF) and hemoperfusion (HP) e.g. to remove inflammatory cytokines, and left ventricular assistance.
The parapoxvirus agent can be administered by any route, including but not limited to the intravenous, intramuscular, oral, parenteral, topical, intradermal, or subcutaneous route. In a specific 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 via inhalation (e.g. as an aerosol) or intranasally (e.g. as an aerosol or as drops). Commonly, it is administered intravenously.
Similarly, the immunomodulator can be administered by any route, including those recited above.
The dose of the parapoxvirus agent (in fact of all agents described herein, including the immunomodulator) 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 preparing or for treating, for example over a period of < 12 weeks, < 6 weeks, < 4 weeks, < 2 weeks, or < 1 week.
Respiratory virus
The respiratory virus is in general a human respiratory virus, i.e. a respiratory virus capable of infecting humans. The respiratory virus may be any virus that can infect humans by respiratory transmission, in particular by droplet transmission and/or by airborne transmission. In one embodiment, it may be any virus that is capable of infecting the respiratory tract. However, the respiratory virus may also be capable of infecting other organs than the respiratory tract. The respiratory virus may be a DNA or an RNA virus, although it is preferably an RNA virus. For example, viruses may be of the families pneumoviridae, e.g. a respiratory syncytial virus (RSV) or a metapneumovirus (MPV), orthomyxoviridae (e.g. an influenza virus, IV), paramyxoviridae (e.g. a parainfluenza virus, PIV, or a nipah virus, NiV), adenoviridae (e.g. a respiratory adenovirus, ADV), picomaviridae (e.g. a rhinovirus, RV), parvoviridae (e.g. a bocaparvovirus, BoV, previously termed bocavirus), hantaviridae (e.g. a hanta virus, HV, also termed Orthohantavirus), phenuiviridae (e.g. a rift valley virus, RVFV), filoviridae (e.g. an ebola virus, EBOV, or a marburg virus MARV), arenaviridae (e.g. a lassa virus, LASV, or a lymphocytic choriomeningitis virus, LCMV), or poxviridae (e.g. a variola virus, VARV, also known as smallpox virus). Accordingly, non-limiting preferred examples of species of respiratory viruses are RSV, IV, PIV, NiV, MPV, ADV, RV, BoV, HV, RVFV, species of the genus EBOV (including Bundibugyo, Reston, Sudan, Tai Forest, Zaire, and Bombali), MARV, LASV, LCMV and VARV. These virus species are referred to herein as representrative examples for their families, and where referred to herein it is likewise meant that the family represented is also included in the respective embodiment. In a preferred embodiment, the respiratory virus is selected from the group consisting of RSV, IV, PIV, MPV, ADV, RV and BoV.
All species subtypes and serotypes are included. For example: For RSV, it may be subtype RSVA or RSVB. For IV, it may be subtype IAV, IBV, ICV or IDV. Exemplary IV serotypes are H5N1, H9N2, H7N7, H7N2, H7N3, H5N1, H5N6, H3N2v, H7N9, H6N1 and H10N8. For PIV, it may be subtype HPIV-1, HPIV-2, HPIV-3 or HPIV-4. For MPV, it may be subtype HPMV-A (Al or A2) or HPMV-B. For ADV, it may be subtype A, B, C, D, E, F or G. For RV, it may be subtype A, B or C. For BoV, it may be subtype HBoV-1, HBoV-2, HBoV-3 or HBoV-4. For VARV, it may be subtype major or minor.
The skilled person appreciates that new viruses emerge by antigenic drift and/or antigenic shift, leading to new strains as well as zoonotic strains, respectively. Such new viruses are within the scope of the above species/subtypes if classified accordingly by the International Committee on Taxonomy of Viruses (ICTV). Risk of zoonotic spillover can be predicted using online tools such as “Spillover Viral Risk Ranking” (https://spillover.global/).
For reasons unrelated to the suitability for the use according to the invention, the respiratory virus preferably is not a coronavirus (CoV). In one embodiment, the respiratory virus is also not TV, and/or also not varizella zoster virus (VZV), also for reasons unrelated to suitability of the use according to the invention. In a specific embodiment, the respiratory virus may also be IV if the parapoxvirus strain is NZ2.
Respiratory virus infection/disease
A respiratory virus infection is defined by the entry of a respiratory virus into at least one cell of a subject and its replication in the at least one cell. Preferably, the infection is via airborne transmission. More preferably, it 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 of the 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 respiratory virus-antigen-specific immune factor, preferably selected from the group consisting of B cells, follicular helper T cells (TFH 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 respiratory virus-specific cytokine profile, preferably in 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 taking a tissue sample, e.g. from the lung, infecting it with the respiratory virus and determining the expression of cytokines. Examples of respiratory virus-specific cytokine profiles are (a) upregulation of one or more of, preferably all of IFN-I (e.g. IFN- ), IFN-II (fFN-y), IFN-III (IFN-kl, IFN-X2, and IFN-13), IL- Ibeta, IL-6, IL-12, IL-8, MCP-1, MIP-lalpha, RANIES, CXCL1, CXCL2, CXCL5, and CXCL9, and preferably no upregulation of CXCL10; and (b) upregulation of one or more of, preferably all of IL-6, MCP-1, CXCL1, CXCL5, and/or CXLC10, and preferably no upregulation of any IFN and/or of any one or all of IL-lbeta, IL-12, IL-8, MIP-lalpha, RANTES, CXCL2, and CXCL9.
The preferred subject is human. While to the inventors’ best knowledge respiratory viruses infect all humans equally, there are risk factors for contracting a respiratory virus infection:
(i) Recent (within incubation period, i.e. within the past 27, 24, 19 or 14 days, preferably 10 days) presence in or travel from an area with ongoing spread of a respiratory virus infection. The area is preferably an ‘Affected Area’ as determined by a government authority or the WHO. “Recent” can mean within the incubation period of the respiratory virus, 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 a respiratory virus, in particular a person having one or more symptoms of a respiratory virus disease. “Contact” can mean exposed to respiratory droplets of the person (e.g. within range of respiratory droplets in air or in contact with surfaces touched by person or by respiratory droplets).
(iii) Being in an increased-risk-profession, which usually necessitates 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, a first responder like a paramedic, or a non-medical professional working in a medical institution, being a care worker (e.g. for children or the elderly), a retail worker (for instance in a supermarket or pharmacy), a teacher, a law enforcement officer, a correctional officer or a firefighter.
A respiratory virus infection may or may not cause symptoms of a respiratory virus disease in a subject. The terms “respiratory virus infection” and “respiratory virus disease” are distinguished herein by the presence of at least one respiratory virus disease symptom. As long as the infection is not accompanied by at least one symptom of a respiratory virus disease, it (or the subject) is asymptomatic (includes presymptomatic). The term “respiratory virus disease” as used herein requires the presence of a respiratory virus infection and at least one symptom of a respiratory virus disease (also referred to herein as “symptomatic infection”).
Respiratory virus disease symptoms include nausea, vomiting and/or diarrhoea; headache; fever (>37.8°C); and organ (e.g. heart, lung, liver and/or kidney) failure. Further symptoms are selected from the group consisting of cough (with or without sputum; usually except VARV and RVFV); itchy or sore throat (usually except HV and RVFV); body (e.g. muscle and/or joint) pain (usually except RSV and PIV); fatigue (usually except PIV, MPV, RV and LCMV); breathing difficulty (usually except VARV, RVFV, MARV and LCMV); pneumonia (usually except RVFV, EBOV, MARV and LCMV); conjunctivitis (usually except IV, RV, HV and NiV); rash, pustular rash or scabs (usually except RSV, IV, RV, HV and NiV); lymphopenia (usually except HV, VARV, NiV, RVFV, MARV and LCMV); runny and/or blocked nose (usually except HV, VARV, NiV, RVFV, MARV, LASV and LCMV); wheezing (usually except CoV, IV, VARV, RVFV, EBOV, MARV, LASV and LCMV); croup (usually except BoV, HV, VARV, NiV, RVFV, EBOV, MARV, LASV and LCMV; in particular in children up to 15 years old); and cyanosis (usually except PIV, MPV, ADV, RV, VARV, NiV, RVFV, EBOV, MARV, and LCMV). “Usually except” herein means that the symptom occurs in the respriratory virurses of the invention not listed as exceptions above, and that the symptom may occur in some subjects infected with a virus listed as an exception, but that it is not indicative of or typical for the infection with that virus.
Preferably, the respiratory virus disease is characterized by the presence of two or more, three or more, or four or more symptoms, preferably including one, two or all of fever (>37.8°C), headache, and optionally cough and/or breathing difficulty.
The respiratory virus disease is preferably a respiratory disease. The respiratory virus 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 a respiratory virus disease. Mild symptoms are selected from the group consisting of cough (with or without sputum), mild fever (>37.8°C to <40°C), runny and/or blocked nose; fatigue; itchy or sore throat; headache; body (e.g. muscle and/or joint) pain; nausea, vomiting and/or diarrhoea; lymphophenia; conjunctivitis; wheezing; and croup. Severe symptoms are selected from the group consisting of breathing difficulty, in particular acute respiratory distress syndrome; pneumonia; organ (e.g. heart, lung, liver and/or kidney) failure; cyanosis 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 a respiratory virus disease, (ii) need for hospitalization, in particular need for admission to intensive care unit (ICU) and/or need for oxygen therapy (as defined above), in particular for a respirator (also referred to as ventilator), and/or (iii) death of the subject. A need for hospitalization is characterized by one or more of: clinical or radiological evidence of pneumonia, acute respiratory distress syndrome, and/or the presence of two or more symptoms including at least high 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 (age younger than 2 months: 60 or more breaths per minute; age 2 to 11 months: 50 or more breaths per minute, age 1 to 5 years: 40 or more breaths per minute, age > 5 years: 30 or more breaths per minute), respiratory distress and/or inadequate oxygenation (e.g. SpCh of 93% or less for adults, and 90% or less for children). Severe complications are complications that are lethal when untreated 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 respiratory virus 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. Furthermore, the cytokine storm is preferably in the respiratory tract as defined for the infection above (including preferred parts thereof).
While a respiratory virus disease can take a severe course in any subject regardless of age or pre-existing condition, there are nevertheless risk factors for having a severe course of a respiratory virus disease, including:
(i) age, i.e. old age (e.g. at least 40, 50, 60, 70, or 80 years with risk increasing with age) or young age (e.g. up to 5 years, 2 years or 6 months with risk increasing with lower age),
(ii) male gender,
(iii) smoking, and
(iv) having one or more comorbidities, preferably selected from the group consisting of cardiovascular disease, diabetes, obesity, respiratory disease (in particular chronic), hypertension, an immunocompromised state (e.g. AIDS), cancer, liver disease, kidney disease and lung disease. At particular risk are subjects having risk factors (i) and (iv) in combination; a particular comorbidity as risk factor is a respiratory disease (other than the respiratory virus disease), lung disease and/or an immunocompromised state. Preparing a subject
The preparing of a subject is meant to make a subject readier (in the sense of more fortified or more protected immunologically) for a respiratory virus infection, which is afforded inter alia by the immunomodulation, in particular by the parapoxvirus agent, resulting in the medical use of the invention. It may also be described as promoting an antiviral cell state in a subject (as defined further below). It can also be termed “preparatory treatment”, “prophylactic treatment” or, with respect to the disease resulting from the invention, “preventative treatment” or “preventive treatment”. It is not meant to necessarily prevent a respiratory virus infection. As such, the subject is not yet infected by the respiratory virus.
Thus, the preparing can have one or more immunological effects or (preferably and) it can have one or more clinical effects, both once a subject is infected. These immunological effects are preferably selected from the group consisting of
(I-i) increasing the number of one or more of respiratory virus-antigen-specific immune factors (as defined above) in the subject or in a part of the subject,
(I-ii) inhibiting (i.e. reducing or stopping) respiratory virus replication in the subject or in a part of the subject,
(I-iii) reducing the respiratory virus load in the subject or in a part of the subject, and
(I-iv) preventing or ameliorating a respiratory virus-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-ii) is preferred over (I-i), and (I-iii) over (I-ii). Further (I-ii) may involve (I-i), and (I-iii) may involve (I-i) and/or (I-ii). (I-iv) is generally desired.
The clinical effects are preferably selected from the group consisting of
(C-i) reducing contagiousness,
(C-ii) delaying the onset of the respiratory virus disease,
(C-iii) reducing the degree of one or more prospective symptoms of the respiratory virus disease, preferably of one or more prospective severe symptoms of the respiratory virus disease, (C-iv) preventing one or more symptoms of the respiratory virus disease, preferably one or more severe symptoms of the respiratory virus disease,
(C-v) preventing a severe course of the respiratory virus disease, and
(C-vi) preventing the respiratory virus disease.
Therein, (C-ii) is preferred over (C-i), (C-iii) over (C-ii), (C-iv) 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 reduction 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 (I-i) to (I-iv) and/or one or more of (C-i) to (C-vi) in a subject not yet infected by a respiratory virus. 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 prepared is characterized by one or more risk factors for contracting a respiratory virus infection and/or by one or more risk factors for having a severe course of a respiratory virus disease.
Treating a subject
The treating of a respiratory virus infection (i.e. of a subject that is infected) does not necessarily mean to treat or prevent a respiratory virus 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 the respiratory virus disease (i.e. of an infected subject having one or more symptoms). As opposed to the preparatory treatment described above, it is a therapeutic treatment.
The treating can have one or more immunological effects or (preferably and) it can have one or more clinical effects. These immunological effects are preferably as defined for the preparing 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 (C-vi) as defined for the preparing above. The clinical effects for a symptomatic infection (i.e. on respiratory virus disease) are preferably selected from the group consisting of (C-i), (C-vi), both as defined for the preparing above, (C-vii) ameliorating one or more (or ideally all) symptoms of a respiratory virus disease, (C-viii) preventing, one or more (or ideally all) further symptoms of a respiratory virus disease, and
(C-ix) ameliorating, preferably preventing, a severe course of a respiratory virus disease. Therein, “further symptoms” are those not yet characterizing the symptomatic infection.
Accordingly, the parapoxvirus agent/immunomodulator according to the invention can be for use in one or more of (I-i) to (I-iv) and/or one or more of (C-i) to (C-ix) in a subject infected by the respiratory virus. In other words, the parapoxvirus agent/immunomodulator according to the invention 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-ix). In a preferred embodiment, the parapoxvirus agent/immunomodulator according to the invention is for use in preventing or treating a respiratory virus disease.
As indicated above, the subject to be treated may be asymptomatic. Alternatively, the subject to be treated may have a respiratory virus disease. The subject having a respiratory virus disease may not have any severe symptoms, e.g. it has only one or more mild symptoms. In another embodiment, it may have a severe course of a respiratory virus disease (excluding (iii) death; preferably (i) one or more severe symptoms). In a preferred embodiment, the subject to be treated (e.g. any of the afore-mentioned subjects except those already having a severe course of a respiratory virus disease) is characterized by one or more risk factors for having a severe course of a respiratory virus 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 all,
(ii) age up to 75, 70 or preferably 65 years, more preferably 18 to 65 years, or alternatively up to 5 years or preferably up to 2 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 age 18 to 65 years or up to 5 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 respiratory virus diseases (Pehta et al., The Lancet Vol 395 March 28, 2020). An HScore of greater than 169 is 93% sensitive and 86% specific for sHLH. The HScore is calculated using the following criteria: temperature (<38.4°C: 0 points, 38.4-39.4°C: 33 points, >39.4°C: 49 points), organomegaly (none: 0 points, hepatomegaly or splenomegaly: 23 points, hepatomegaly and splenomegaly: 38 points), number of cytopenias* (one lineage; 0 points, two lineages: 24 points, three linages: 34 points), triglycerides (<1.5 mmol/1: 0 points, 1.5-4.0 mmol/1: 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), serum aspartate aminotransferase (<30 IU/L: 0 points, >30 IlJ/L: 19 points), haemophagocytosis on bone marrow aspirate (no: 0 points, yes: 35 points), and known immunosuppression f (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 110000 platelets per mm3 or less, or all of these criteria combined. fHIV positive or receiving long-term immunosuppressive therapy (e.g. one or more of glucocorticoids, cyclosporine and/or azathioprine).
In a preferred embodiment, the subject to be treated is one that was prepared for a respiratory virus infection according to the invention.
Further medical use aspects
In a further medical use aspect, the invention relates to a parapoxvirus agent for use as a medicament promoting an antiviral cell state in a subject (i) having a respiratory virus infection or (ii) characterized by one or more risk factors for contracting a respiratory virus infection and/or by one or more risk factors for having a severe course of a respiratory virus disease, in combination with an immunomodulator for use in preparing for or preferably treating the respiratory virus infection. The respiratory virus preferably is not a coronavirus and optionally is not an influenza virus.
The invention also relates to the use of an immunomodulator for use in preparing for or preferably treating a respiratory virus infection, in combination with a parapoxvirus agent for use as a medicament promoting an antiviral cell state in a subject (i) having a respiratory virus infection or (ii) characterized by one or more risk factors for contracting a respiratory virus infection and/or by one or more risk factors for having a severe course of a respiratory virus disease. The respiratory virus preferably is not a coronavirus and optionally is not an influenza virus.
The one or more risk factors for contracting a respiratory virus infection are selected from factors (i), (ii) and (iii) as defined above, e.g. (iii).
In another embodiment, the one or more risk factors for having a severe course of a respiratory virus disease comprise at least (i) and (iv) as defined above, wherein the comorbidity is preferably a respiratory disease (in particular chronic), lung disease and/or an immunocompromised state. In addition, the age of the subject is preferably at least 60, at least 70, or more preferably at least 80 years; or alternatively up to 5 years, preferably up to 2 years or more preferably up to 6 months.
In yet a further medical use aspect, the invention relates to a pharmaceutical composition comprising a) a parapoxvirus agent and b) sucrose and/or NaCl for use, in combination with an immunomodulator, in medicine (i.e. prophylaxis and/or therapy). The invention also relates to an immunomodulator for use, in combination with a pharmaceutical composition comprising a) a parapoxvirus agent and b) sucrose and/or NaCl, in medicine (i.e. prophylaxis and/or therapy). The pharmaceutical composition may be formulated for routes of parapoxvirus administration as above (i.e. it may be for use by any route of administration). In a preferred embodiment, it is not formulated for oral administration (i.e. it is not for use by oral administration).
The use in medicine (i.e. prophylaxis and/or therapy) according to these further medical use aspects preferably is (i) the preparing of a subject for a respiratory virus infection and/or (ii) the treating of a respiratory virus infection in a subject as described herein, although for any respiratory virus or the specific ones described herein (including CoV and IV). In one embodiment, the respiratory virus is not IV, not VZV and/or not CoV. Preferably, it is not CoV and optionally not IV. If included, the coronavirus is, in a preferred embodiment, an alphacoronavirus or a beta-coronavirus. 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 syndrome-related coronavirus” as classified by the International Committee on Taxonomy of Viruses (ICTV). In a preferred embodiment, it is SARS-CoV-2.
Methods of the invention
The invention further relates to the following methods:
A method of administering a parapoxvirus agent and an immunomodulator to a subject (i) having a respiratory virus infection or (ii) characterized by one or more risk factors for contracting a respiratory virus infection and/or by one or more risk factors for having a severe course of a respiratory virus disease, wherein the respiratory virus is not a coronavirus and optionally is not an influenza virus.
A method of promoting an antiviral cell state in a subject (i) having a respiratory virus infection or (ii) characterized by one or more risk factors for contracting a respiratory virus infection and/or by one or more risk factors for having a severe course of a respiratory virus disease, the method comprising administering a parapoxvirus agent and an immunomodulator to the subject, wherein the respiratory virus is not a coronavirus and optionally is not an influenza virus.
A method of (i) preparing a subject for a respiratory virus infection and/or (ii) treating a respiratory virus infection in a subject, the method comprising administering a parapoxvirus agent and an immunomodulator to the subject, wherein the respiratory virus is not a coronavirus and optionally is not an influenza virus. A method of treating a respiratory virus disease in a subject, the method comprising administering a parapoxvirus agent and an immunomodulator to the subject, wherein the respiratory virus is not a coronavirus and optionally is not an influenza virus.
A method administering (i) a pharmaceutical composition comprising (a) a parapoxvirus agent and (b) sucrose and/or NaCl, and (ii) an immunomodulator to a subject. The method is preferably for prophylaxis and/or therapy.
Definitions and embodiments described herein regarding the medical uses according to the invention also apply to these methods.
Disclaimers
In a select embodiment, one or more of the following three disclaimers apply to the invention:
1) the parapoxvirus is not canine distemper virus (CDV),
2) the coronavirus is not porcine epidemic diarrhea virus (PEDV), and/or
3) the subject is not a pig (pig including sow and piglet).
Definitions and further embodiments of the invention
The specification uses a variety of terms and phrases, which have certain meanings as defined below. Preferred meanings are to be construed as preferred embodiments of the aspects of the 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 polynucleotide sequences refers to 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 of the 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" is defined as the alignment that produces 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 generally to a modified version of the polynucleotide, e.g. a mutation, so one or more nucleotides of the polynucleotide may be mutated. Generally, the variant is functional, meaning e.g. with regard to a virus that the virus is capable of infecting a host cell. The functionality is generally that described for the polynucleotide the variant is from. A “mutation” can be a nucleotide substitution, deletion and/or insertion (“and” may apply if there is more than one mutation). Preferably, it is a substitution.
The term “respiratory transmission”, as used herein, refers to the generation of aerosols (either droplet or small-particle aerosols) from the respiratory tract (e.g., nasal passages, trachea, or lungs) that then enter the airspace and cause infection by droplet or airborne spread. It involves inhalation of infectious aerosols suspended in the air and can involve aerosol particles of various sizes that either land on mucosal surfaces, such as the nose and mouth, or are inspired deeper than nose and mouth into the respiratory tract. It can also be described as an aerosol transmission from the respiratory tract of an infectious individual to mucosal surfaces or the resporiatory tract of an uninfected individual. Respiratory transmission includes both airborne transmission and droplet transmission.
The term “airborne transmission” as used herein refers to a virus transmission resulting from the inhalation of small respirable particles that remain infective over time and distance and can be dispersed over distances by air currents. For example, RSV, IV, PIV, NiV, MPV, ADV, RV, BoV, HV, RVFV, LCMV and VARV are known for airborne transmission. However, other viruses such as EBOV, MARV, LASV can mutate towards the capability of airborne transmission, and airborne transmission has been shown for EBOV (to which MARV is very similar) by Osterholm et al. (mBio, Vol. 6, No.2, 1 May 2015).
The term “droplet transmission” as used herein refers to a direct contact transmission in which respiratory droplets carrying infectious pathogens transmit infection when they travel directly from the respiratory tract of an infectious individual to susceptible mucosal surfaces of the recipient. These droplets can be considered "propelled" from the infectious person and are not dispersed by air currents. All respiratory viruses referred to herein are capable of droplet transmission.
The term “immunogen” refers to an antigen that is capable of inducing an immune response.
“Pattern recognition receptor” or “PRR”, as used herein, refers to a germline-encoded host sensor which detects a pathogen-associated molecular pattern (P MP), which is specific for a pathogen, and/or a damage-associated molecular pattern (DAMP), which is specific for components of a host cell that are released during cell damage or death. A PRR can be a 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 adequate immune response to the antigen/immunogen of interest. Examples of adjuvants that may be used in the context of the composition according to the invention are gel-like precipitates of aluminum hydroxide (alum); AIPO4; alhydrogel; bacterial products from the outer membrane of Gramnegative bacteria, in particular monophosphoryl lipid A (MPLA), lipopolysaccharides (LPS), muramyl dipeptides and derivatives thereof; Freund’s incomplete adjuvant; liposomes, 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 derivatives, in particular PamsCys; lipoarabinomannan; peptidoglycan; zymosan; heat shock proteins (HSP), in particular HSP 70; dsRNA and synthetic derivatives thereof, in particular Poly EC; polycationic peptides, in particular poly-L-arginine; taxol; fibronectin; flagellin; imidazoquinoline; cytokines with adjuvant activity, in particular GM- CSF, interleukin-(IL-)2, IL-6, IL-7, IL-18, type I and II interferons, in particular IFN-y, TNF- a; 25-dihydroxyvitamin D3 (calcitriol); and synthetic oligopeptides, in particular MHC-II- presented 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 ingredient.
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 IFN-a and/or p. Preferably it inhibits (i.e. prevents or at least reduces) virus replication in the cell and/or induces apoptosis (to prevent further virus replication 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 medicament. Thereby, it stops or at least slows down virus propagation. “Promoting” includes an induction (e.g. for cells not yet in an antiviral state) and a reinforcement (i.e. potentiation) (e.g. for cells already in an antiviral state). Although not limited thereto, the former is preferred for a subject not (yet) having a respiratory virus infection, e.g. characterized by one or more risk factors as specified above, and the latter is preferred for a subject having a respiratory virus infection.
The term “vector” as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid 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 encephalitis virus (VEE), sindbis virus (SIN), semliki forest virus (SFV), and VEE-SIN chimeras), herpes virus vectors, measles virus vectors, pox virus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara (MV A), NYVAC (derived from the Copenhagen strain of vaccinia), and avipox vectors: canarypox (ALVAC) and fowlpox (FPV) vectors), and vesicular stomatitis virus vectors, or virus like particles. As used herein, the term "virus-like 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 penetration 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 “nucleotide analogue” refers to a nucleotide 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 “antigen binding protein” as used herein refers to antibodies and antibody-like proteins, including fragments and derivatives of antibodies. Antigen binding proteins may comprise at least one variable domain, for example antibodies, domain antibodies (dAb), multiples of domain antibodies e.g. dumbbells, dAb-dAb in-line fusions, Fab, Fab', FAb2, F(ab')2, Fv, ScFv, diabodies, mAbdAbs, DVD-lgs, affibodies, heteroconjugate antibodies, bispecifics and the like. A “domain antibody” or “dAb” is an immunoglobulin single variable domain which is capable of binding to an antigen. An immunoglobulin single variable domain may be a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent, nurse shark and Camelid VHH dAbs. Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Camelid-derived derived constructs like nanobodies are also included. Antibodies are a preferred embodiment.
The term “pharmaceutical composition” relates to a composition comprising an active agent according to the invention and one or more pharmaceutically acceptable diluents, carriers, and/or preservatives. Pharmaceutically acceptable diluents, carriers, and/or preservatives are described below and may in addition thereto, if the active agent is the parapoxvirus agent, include an (e.g. aqueous) medium suitable for structurally maintaining a parapoxvirus, in particular an inactivated parapoxvirus, or lyophilisate of this medium. The kit may further comprise a leaflet with instructions for use of the parapoxvirus agent and the immunomodulator, preferably according to a medical use described herein. The composition can be formulated for any route of administration as defined above. Particular preferred pharmaceutical forms for the administration of the agents according to the invention are forms suitable for injectable use and include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile 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, ethanol, 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-bacterial or anti-fungal agents, e.g. parabene, chlorobutanol, phenol, sorbic acid or thiomersal. Further, isotonic agents, such as sugars or salts, in particular sodium chloride may be incorporated in infusion or injection solutions. Preferred diluents of the invention are water, physiological acceptable buffers, physiological acceptable buffer salt solutions or salt 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 magnesium stearate, talc, calcium stearate, zinc stearate, stearic acid, hydrogenated vegetable oil, leucine, glycerids and sodium stearyl fumarates, c) disintegrants such as starches, croscaramellose, sodium methyl cellulose, agar, bentonite, alginic 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. 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 have been developed and studied. Examples include liposomes, polymeric micelles, microspheres, nanoparticles, nanofibers, protein-drug conjugates, erythrocytes, virosomes and dendrimers. Different methods of attaching the drug to the carrier can be used, including adsorption, integration 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 specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be covered by the 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 stimulate the innate as well as the adaptive arm of the immune system.
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 activates 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 supernatant containing soluble factors like cytokines are harvested. These supernatants are assayed for their activity on coronavirus.
Test substances iPPVO of strain D1701 (available from Pfizer); 230 IFN units reconstituted in 2 ml water iPPVO vehicle (PPVO buffer only, i.e. without virus), negative control
PBS, negative control
Concavalin (Con) A, co-stimulus, stock solution 1 mg/ml 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 in growth medium prepared by supplementing DMEM with 10% v/v heat-inactivated FCS and 5 mL sodium bicarbonate 7.5%, and cultured in T150 bottle and split 1/4 twice a week. Pen-strep added directly to the T150 bottle at a 1/100 dilution.
Coronavirus assay medium: prepared by supplementing DMEM with 2% v/v heat- inactivated FCS and 5 mL sodium bicarbonate 7.5%. Preparation of samples
Per well in a 96 well round-bottom plate: 120 pl cell medium for whole blood incubation + 20 pl ConA or PBS + 20 pl iPPVO, iPPVO vehicle or PBS + 40 pl whole blood.
The plate was incubated at 37 °C, 5 % CO2 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 replication 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.
Preparation of the cell suspension: A confluent culture (monolayer) of VeroE6-EGFP in T150 bottle was washed with DPBS and 10 mL trypsine 0.25%trypsine/EDTA was added. The bottle was incubated for 1 minute at room temperature, 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 of the cell suspension were seeded to 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 all 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 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 detectable. 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 expected. Also 5 pg/ml ConA alone (in PBS) did 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 D1701 alone (in PBS) also had a clear positive effect on cell viability, and eGFP expression was further increased when iPPVO DI 701 was used in combination with 5 pg/ml ConA (which by itself showed no effect on eGFP expression at this concentration). Accordingly, iPPVO and ConA act synergistically.
These results show the efficacy of parapoxvirus in promoting an antiviral cell state which is protective and inhibits coronavirus replication 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 synergistically, 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 coronavirus.
See Example 1.
Test substances iPPVO of strain NZ2 (chemically inactivated by binary ethyleneimine, BEI), internal designation AIC649, IxlO9 lyophilized virions reconstituted in 1 ml water iPPVO of strain DI 701, see Example 1 iPPVO vehicle, negative control, see Example 1
Further materials, preparation of samples, coronavirus assay
See Example 1. iPPVO and iPPVO vehicle were used alone. iPPVO D1701 shown to increase cell viability in Example 1 was used as a positive control in Example 2. Results
The results are shown in Fig. 2. iPPVO DI 701, which in Example 1 was shown to be effective, was used as a positive control and as a 100% benchmark. Both PPVO strains DI 701 and NZ2 are active against coronavirus as shown by the comparison to the negative control. Accordingly, the promotion of an antiviral cell state which is effective against coronavirus is a hallmark of parapoxvirus in general and not only of the strain tested in Example 1.
EXAMPLE 3
Aim
Investigating the effect of parapoxvirus on the infectious 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 dissemination 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 ofiPPVO 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 TCIDso in 50 pL). At day 4 post-infection (pi), hamsters are euthanized by i.p. injection of 500 pL Dolethal (200 mg/mL sodium pentobarbital, Vetoquinol SA). Lungs are collected and infectious virus is quantified by endpoint virus titration. Efficacy is determined based on viral load in homogenized lung tissues on day 4 post-infection.
Animals
Syrian Golden Hamster, females, 6-10 weeks old
SARS-Cov-2
SARS-Cov-2 strain BetaCov/Belgium/GHB-03021/2020 (EPI ISL 109 407976|2020- 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 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-point 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 debris. To quantify 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 (TCID50) 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 4
Aim
Investigating the effects of parapoxvirus on coronavirus infection in a mouse model.
10 mice are intravenously inoculated with 100 pl of iPPVO (1 vial dissolved in 500 pl supplied buffer, which equals a dose of IxlO9 virus particles (VP)/animal) prior to coronavirus infection. A second group of 10 mice serves as an infection control group (intravenous inoculation with 100 pl iPPVO buffer). One day after treatment, mice of both groups are infected intranasally under isoflurane anesthesia (inhalation of 3% isoflurane) with 900 focus forming units (FFU) of SARS-CoV-2 (German isolate) in 50 pl total volume. Mice are scored daily. Euthanasia is performed on day 10 after infection. Earlier euthanasia is performed for mice reaching humane endpoints as determined by the clinical score. Response to treatment is assessed by a clinical score determination (clinical symptoms of infection) and body weight loss. Also, SARS-CoV-2 load is determined in lungs and brains. Animals
Transgenic K18-hACE2 mouse
Test substances iPPVO strain of DI 701, see Example 1 - iPPVO vehicle (PPVO buffer only, i.e. without virus)
Determination of clinical score
The clinical score was determined by adding the score of each clinical parameter according to Table 1 below. Animals were euthanised upon reaching a clinical score of >20 or scoring 20 for one clinical parameter (humane endpoints).
Table 1: Clinical score.
Figure imgf000035_0001
Figure imgf000036_0001
Determination of organ viral load
Organs were homogenized in 2 ml PBS. Viral RNA was isolated from 140 pl of homogenates using QIAamp Viral RNA Mini Kit (Qiagen). RT-qPCR reactions were performed using TaqMan® Fast Virus 1-Step Master Mix (Thermo Fisher) and 5 pl of isolated RNA as a template. Synthetic SARS-CoV2-RNA was used as a quantitative standard to obtain viral copy numbers.
Results
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 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 euthanasia 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 of the iPPVO treatment group 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 beneficial effect of PPVO treatment on the 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.
EXAMPLE 5
Aim
Investigating the effects of parapoxvirus on RSV infection in a mouse model.
Groups of BALB/c mice (each n=5; “aged”, i.e. 17 weeks old) are used for the intravenous inoculation with lOOpl iPPVO (strain D1701, available from Pfizer, 230 IFN units reconstituted in 2 ml water) one day before RSV infection. Mock infection groups (also n=5) are inoculated with buffer only. One day after, all groups (except the infection control group) are lightly anesthetized by inhalation of 3% isoflurane and infected intranasally with 5xlOE6 pfu of RSV-A2 Long strain in 40 pl total volume. A control group 1 (low dose) is infected with a lower dose of RSV (lxlOE6) to show the impact of the high dose. A control group 2 is infected with 5xlOE6 and sacrificed two days after infection to detect the presence of the applied virus. A control group 3 contains young animals (“juvenile”, i.e. 8 weeks old; 5xlOE6) to compare the impact of age. Animals are scored daily. Blood sampling and euthanasia are performed on day 6 after infection (except control group 2). Earlier euthanasia is performed for mice reaching humane endpoints as determined by the clinical score. Response to treatment is assessed by a clinical score determination (clinical symptoms of infection) and body weight loss.
Determination of clinical score
The clinical score was determined by adding the score of each clinical parameter according to Table 1 above. Animals were euthanised upon reaching a clinical score of >20 or scoring 20 for one clinical parameter (humane endpoints).
Determination of viral load
Mice were euthanized according to humane endpoints or latest at day 6 post infection and lungs were homogenized in 2 ml PBS. Viral RNA was isolated from 140 pl of homogenates using QIAamp Viral RNA Mini Kit (Qiagen). RT-qPCR reactions were performed using using Quantitect Probe RT-PCR Kit (Qiagen), SYBR Green detection and 5 pl isolated RNA as template. 10-fold dilutions of synthetic RSV-RNA of T7-transcripts were used as standards for the quantification of viral copy numbers.
Results
The body weight of RSV infected mice not treated with iPPVO decreased over time for all groups, i.e. for aged mice (17 weeks old) infected with IxlO6 FFU or with 5xl06 FFU and for juvenile mice (8 weeks old) infected with 5xl06 FFU. Both a high infection titre and age exacerbated the decrease (see Fig. 8 A). Correspondingly, the climical score increased overtime, with high infection titre and age leading to a higher score (see Fig. 8B). The viral load in aged animals increased from 6 hours post infection (hpi) to 2 days post infection (dpi). Until day 6, the viral load decreased in these animals, but it was still higher than at 6 hpi (see Fig. 8C).
The survival rate of iPPVO treated and RSV infected mice was significantly higher in comparison to untreated mice after 6 days for both aged and juvenile mice: 60% of the untreated juvenile mice and none of the aged mice survived RSV infection, whereas 100% of the iPPVO treated juvenile and aged mice survived RSV infection (see Fig. 9). Correspondingly, body weight, clinical score and viral load were improved in juvenile and aged mice by iPPVO treatment (see Figs. 10, 11 and 12).
Overall, these data clearly demonstrate a beneficial effect of PPVO treatment on the clinical outcome of RSV infected mice, suggesting an improvement of the clinical situation of RSV infected patients, including a delay in the onset of symptoms and a milder course of the disease.
EXAMPLE 6
Aim
Showing the effects of parapoxvirus in combination with a further immunomodulator on RSV infection.
Approach
The antiviral activity of a further immunomodulatory compound (ribavirin, RBV, which is known to act immunomodulatory, e.g. by inhibiting production of TNF and IL-1, Ning etal., J Immunol. 1998;160:3487-3493) as a function of preincubation with conditioned media (CM) derived from iPPVO treated donor hPBMCs was determined by a GFP -based virus yield reduction assay in cell culture.
3 x 104 Hep-2 cells/well were cultured in 96-well plates in 200 pl of cell culture medium. 24 h after plating of cells medium was aspirated and cells were incubated for 24 h with different dilutions (1 : 10; 1 :50; 1 :250) of CM obtained from iPPVO treated PBMCs. Thereafter, medium was aspirated again and cells were infected with the GFP expressing virus strain RSV strain A2 (MOI 0.2) in 100 pl medium. Following a 4 h adsorption period, the virus inoculum was replaced with 200 pl fresh medium containing serial dilutions of ribarivin spanning five different concentrations ranging from 100 pM to 4 pM. All drug concentrations were tested at least in duplicate and the final concentration of DMSO in all assays was <1%. Thereafter, plates were incubated at 37°C for 4 days. In order to determine relative titers of infectious progeny virus per well, cell lysates were serially diluted in cell culture medium and used for infection of confluent Hep-2 cells seeded in 96 well plates (3xl04 cells/well). Cultures were incubated for 24 h, medium was removed (washed twice with PBS and replaced with PBS) and GFP positive cells per well were counted across a range of virus dilutions using automated fluorescence microscopy. Given that the number of infected cells within a given dilution reflects the yield of infectious progeny virus after drug action, drug effects were calculated as a percentage of reduction of GFP positive cells in the presence of each drug concentration compared to the number of infected (GFP positive) cells determined in the absence of drug(s). Furthermore, EC50 values (drug concentrations producing 50% reduction in virus yield) were determined. All assays were performed at least in duplicate and standard deviations were calculated. The experimental approach is shown in Fig. 13 A.
Statistical analysis
Statistical analysis and curve fitting was performed using Prism 9 (GraphPad, La Jolla, CA, USA).
Results
The results are shown in Fig. 13 B and C and in Table 2 below. The virus yield was reduced as shown by the reduction of the number of RSV-infected (GFP-expressing) cells in a ribavirin dose-dependent fashion. Unexpectedly, treatment in combination with iPPVO led to a stronger ribavirin dose-dependent reduction in the number of RSV-infected cells. An increase in the CM concentration strengthened this effect (Fig. 13 B). This demonstrates that iPPVO and ribavirin have a synergistic antiviral activity. Accordingly, EC50 values were greatly reduced by the combination treatment compared to ribavirin alone (Table 2).
Table 2: Anti-RSV potency of PPVO conditioned media (CM) + immunomodulator
Figure imgf000040_0001

Claims

1. A parapoxvirus agent for use, in combination with an immunomodulator, in (i) preparing a subject for a respiratory virus infection and/or (ii) treating a respiratory virus infection in a subject, wherein the respiratory virus is not a coronavirus.
2. An immunomodulator for use, in combination with a parapoxvirus agent, in (i) preparing a subject for a respiratory virus infection and/or (ii) treating a respiratory virus infection in a subject, wherein the respiratory virus is not a coronavirus.
3. The parapoxvirus agent for use of claim 1 or the immunomodulator for use of claim 2, wherein the parapoxvirus agent is PPVO of the strain NZ2, preferably wherein the parapoxvirus agent is iPPVO of the strain NZ2.
4. The parapoxvirus agent for use of claim 1 or 3 or the immunomodulator for use of claims 2 or 3, wherein the respiratory virus is selected from the group consisting of a respiratory syncytial virus, an influenza virus, a parainfluenza virus, a metapneumovirus, a respiratory adenovirus, a rhinovirus, a bocaparvovirus, a hanta virus and a variola virus.
5. The parapoxvirus agent for use of any one of claims 1, 3 or 4 or the immunomodulator for use of any one of claims 2-4, wherein the respiratory virus is not an influenza virus.
6. The parapoxvirus agent or the immunomodulator for use of claim 3, wherein the respiratory virus is an influenza virus.
7. The parapoxvirus agent for use of any one of claims 1 or 3-6 or the immunomodulator for use of any one of claims 2-6, wherein the immunomodulator is an immunoinducer if the immunomodulator is used for preparing the subject, and wherein the immunomodulator is an immunosuppressor if the immunomodulator is used for treating the infection and preferably a respiratory virus disease.
8. The parapoxvirus agent for use of any one of claims 1 or 3-7 or the immunomodulator for use of any one of claims 2-7, wherein the immunomodulator is both an immunoinducer and an immunosuppressor.
9. The parapoxvirus agent or the immunomodulator for use of claim 8, wherein the immunomodulator is thalidomide or an analog thereof.
10. The parapoxvirus agent or the immunomodulator for use of claim 8, wherein the analog is an IMiD or a SelCiD.
11. The parapoxvirus agent for use of any one of claims 1 or 3-10 or the immunomodulator for use of any one of claims 2-10, wherein the parapoxvirus agent and the mmunomodulator are administered separately and preferably non-simultaneously.
12. The parapoxvirus agent or immunomodulator for use of claim 11, wherein the parapoxvirus agent is administered prior to the immunomodulator.
13. The parapoxvirus agent for use of any one of claims 1 or 3-12 or the immunomodulator for use of any one of claims 2-12, wherein the parapoxvirus agent is for use in preparing the subject for the respiratory virus infection and optionally in treating the respiratory virus infection, and wherein the immunomodulator is for use in treating the respiratory virus infection.
14. A pharmaceutical composition comprising a) a parapoxvirus agent and b) sucrose and/or NaCl for use, in combination with an immunomodulator, in medicine.
15. An immunomodulator for use, in combination with a pharmaceutical composition comprising a) a parapoxvirus agent and b) sucrose and/or NaCl, in medicine.
PCT/EP2022/081443 2021-11-10 2022-11-10 Parapoxvirus for preparing for and treatment of respiratory virus infections in combination with immunomodulators WO2023083950A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP21207405 2021-11-10
EP21207405.8 2021-11-10

Publications (1)

Publication Number Publication Date
WO2023083950A1 true WO2023083950A1 (en) 2023-05-19

Family

ID=78789623

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2022/081443 WO2023083950A1 (en) 2021-11-10 2022-11-10 Parapoxvirus for preparing for and treatment of respiratory virus infections in combination with immunomodulators

Country Status (1)

Country Link
WO (1) WO2023083950A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10122451A1 (en) * 2000-07-11 2002-04-04 Bayer Ag Use of strains of the Parapoxvirus ovis for the production of antiviral and anti-cancer drugs
US20030021769A1 (en) * 2000-07-11 2003-01-30 Olaf Weber Use of strains of parapoxvirus ovis for producing antiviral medicaments and medicaments against cancer
WO2006005529A1 (en) * 2004-07-13 2006-01-19 Aicuris Gmbh & Co. Kg Parapoxviruses in combination with other antiviral agents for the treatment of hiv/aids
WO2019048640A1 (en) 2017-09-07 2019-03-14 Aicuris Gmbh & Co. Kg Combination therapies of hepatitis b virus (hbv)-infected individuals using parapoxvirus ovis (ppvo) and at least one further antiviral drug

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10122451A1 (en) * 2000-07-11 2002-04-04 Bayer Ag Use of strains of the Parapoxvirus ovis for the production of antiviral and anti-cancer drugs
US20030021769A1 (en) * 2000-07-11 2003-01-30 Olaf Weber Use of strains of parapoxvirus ovis for producing antiviral medicaments and medicaments against cancer
WO2006005529A1 (en) * 2004-07-13 2006-01-19 Aicuris Gmbh & Co. Kg Parapoxviruses in combination with other antiviral agents for the treatment of hiv/aids
WO2019048640A1 (en) 2017-09-07 2019-03-14 Aicuris Gmbh & Co. Kg Combination therapies of hepatitis b virus (hbv)-infected individuals using parapoxvirus ovis (ppvo) and at least one further antiviral drug

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. MN908947.3
BONCRISTIANI ET AL., ENCYCLOPEDIA OF MICROBIOLOGY, vol. 2009, pages 500 - 518
BOUDEWIJNS ET AL.: "STAT2 signaling as double-edged sword restricting viral dissemination but driving severe pneumonia in SARS-Col 2 infected hamsters", BIORXIV, 2020
IVENS ET AL., JOURNAL OF VIROLOGICAL METHODS, vol. 129, 2005, pages 56 - 63
LIN FONG-YUAN ET AL: "Suppression of influenza virus infection by the orf virus isolated in Taiwan", JOURNAL OF VETERINARY MEDICAL SCIENCE, vol. 77, no. 9, 1 January 2015 (2015-01-01), JP, pages 1055 - 1062, XP055904069, ISSN: 0916-7250, DOI: 10.1292/jvms.14-0663 *
NING ET AL., JLMMUNOL, vol. 160, 1998, pages 3487 - 3493
OSTERHOLM ET AL., MBIO, vol. 6, no. 2, 1 May 2015 (2015-05-01)
PEHTA ET AL., THE LANCET, vol. 395, 28 March 2020 (2020-03-28)
REEDMUENCH, THE AMERICAN JOURNAL OF HYGIENE, vol. 27, no. 3, 1938, pages 493 - 497

Similar Documents

Publication Publication Date Title
Ross et al. Single dose combination nanovaccine provides protection against influenza A virus in young and aged mice
US20230346915A1 (en) Production Of Vaccines Comprising Inactivated SARS-COV-2 Viral Particles
CA3165957A1 (en) Methods, compositions, and vaccines for treating a virus infection
EP3906929A1 (en) Letermovir for use in the prevention and the treatment of coronavirus infections
US20220401553A1 (en) Parapoxvirus for conditioning for and treatment of coronavirus infections
WO2023083950A1 (en) Parapoxvirus for preparing for and treatment of respiratory virus infections in combination with immunomodulators
JP7412353B2 (en) Diltiazem for use in the treatment of microbial infections
Adam et al. A modified porous silicon microparticle potentiates protective systemic and mucosal immunity for SARS-CoV-2 subunit vaccine
WO2023083943A1 (en) Parapoxvirus for preparing for and treatment of respiratory virus infections in combination with antivirals
JP5897700B2 (en) Avian metapneumovirus in oncolysis
JP2013540825A (en) Viral vaccine and preparation method thereof
OA21061A (en) Parapoxvirus for conditioning for and treatment of coronavirus infections.
WO2023083951A1 (en) Parapoxvirus for preparing for and treatment of respiratory syncytial virus infections
WO2023201882A1 (en) Use of lentinan in treating sars-cov-2 infection
CN113583970B (en) A medicine for preventing or resisting coronavirus transmission and pathopoiesia, and its preparation method
Narasimhan et al. Single dose combination nanovaccine provides protection against influenza A virus in young and aged mice
US20220133877A1 (en) Multivalent Live-attenuated Influenza Vaccine for Prevention and Control of Equine Influenza Virus (EIV) in Horses
US20150174236A1 (en) Viral vaccine and process for preparing the same
WO2022175436A1 (en) Influenza virus defective interfering particles for use in the prophylactic or therapeutic treatment of coronaviridae infection
WO2023230479A1 (en) Combined vaccine containing infectious bronchitis virus attenuated massachusetts and recombinant lasota virus expressing arkansas spike
JP5257364B2 (en) Feline influenza vaccine and method of use

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22818616

Country of ref document: EP

Kind code of ref document: A1