Description The invention relates to a recombinant Mycobacterium cell for use in the prevention of an infectious disease. Particularly, the invention relates to the prevention or amelioration of a respiratory disorder caused by and/or associated with a virus infection, more particularly a Coronavirus infection. SARS-CoV-2 spreads rapidly throughout the world. SARS-CoV-2 is being transmitted via droplets and fomites during close unprotected contact between an infector and infectee. Health-care workers face an elevated risk of exposure to - and infection in general and of - SARS-CoV-2. (Huang, C. et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet (2020) doi: 10.1016/S0140-6736(20)30183-5.).

A large epidemic does seriously challenge the available hospital capacity, and would be augmented by absenteeism of healthcare professionals. Because hospital personnel is limited, it is imperative to ensure the safety, health and fitness of existing hospital personnel in order to safeguard continuous patient care. Strategies to prevent absenteeism of healthcare professionals are, therefore, desperately needed.

Bacille Calmette-Guerin (BCG) is a vaccine against tuberculosis, with protective non-specific effects against other respiratory tract infections in in vitro and in vivo studies, and reported significant reductions in morbidity and mortality. (Benn, C. S., Netea, M. G., Selin, L. K. & Aaby, P. A Small Jab - A Big Effect: Nonspecific Immunomodulation By Vaccines. Trends in Immunology (2013) doi: 10.1016/j. it.2013.04.004.)

A recombinant BCG strain expressing a phagolysosomal escape domain is described in WO 99/101496, the content of which is herein incorporated by reference. The phagolysosomal escape domain enables the strain to escape from the phagosome of infected host cells by perforating the membrane of the phagosome. In order to provide an acidic phagosomal pH for optimal phagolysosomal escape activity, a urease-deficient recombinant strain was developed. This strain is disclosed in WO 2004/094469, the content of which is herein incorporated.

WO 2012/085101, the content of which is herein incorporated, discloses that a recombinant BCG strain expressing membrane-perforating listeriolysin (Hly) of Listeria monocytogenes and devoid of urease C induces superior protection against aerogenic challenge with Mycobacterium tuberculosis ( Mtb ) as compared to parental BCG in a preclinical model. Further, it is shown that both the recombinant and the parenteral strain induce marked Th1 immune responses, whilst only the recombinant BCG strain elicits are profound Th17 response in addition. Tarancon et al (PLoS Pathog 16(4): e1008404) describe that the live attenuated tuberculosis vaccine MTBVAC induces trained immunity and confers protection against experimental lethal pneumonia elicited by administration of Streptococcus pneumoniae to mice. Miller et al (doi 10.1101/2020.03.24.20042937) describe a statistical correlation between universal BCG vaccination policy and reduced morbidity and mortality for COVID-19.

The present invention is based on a clinical human phase III study protocol, which provides evidence for the efficacy and safety of a recombinant urease- deficient and listeriolysin-expressing recombinant BCG strain in enhancing the immunity of a subject, particularly of a human subject, against a Coronavirus infection. This enhancement of the immunity leads to a prevention or an amelioration of the disease underlying the virus infection, e.g. COVID-19, including, but not limited to a shortening of the sickness time, a reduction of the occurrence and/or severity of symptoms and/or a reduction of mortality. A first aspect of the present invention is a pharmaceutical composition comprising a recombinant Mycobacterium cell, which comprises a recombinant nucleic acid molecule encoding a fusion polypeptide comprising:

(a) a domain capable of eliciting an immune response, and (b) a phagolysosomal escape domain, for use in the prevention and/or amelioration of a respiratory disorder caused by and/or associated with a virus infection, particularly with a Coronavirus infection, and more particularly with a SARS-CoV-2 infection. A further aspect of the present invention is a method for the prevention or amelioration of a respiratory disorder caused by and/or associated with a virus infection, particularly with a Coronavirus infection, and more particularly with a SARS-CoV-2 infection, in a subject in need thereof, comprising administering to said subject a pharmaceutical composition comprising a recombinant Mycobacterium cell, which comprises a recombinant nucleic acid molecule encoding a fusion polypeptide comprising:

(a) a domain capable of eliciting an immune response, and

(b) a phagolysosomal escape domain.

According to the present invention, a protocol is provided describing a phase III, double-blind, placebo-controlled, multi-center, clinical trial to assess the efficacy and safety of VPM1002 in reducing healthcare professionals absenteeism in the SARS-CoV-2 pandemic by modulating the immune system. The protocol is designed to assess the reduction of absenteeism among healthcare professionals with direct patient contacts during the epidemic phase of COVID19. Further, the protocol is designed to assess the reduction of hospital admission, intensive care unit (ICU) admission or death in healthcare professionals with direct patient contacts during the epidemic phase of COVID19.

Accordingly, the present invention further relates to the use of a pharmaceutical composition as described above for providing an immune- stimulatory effect when administered to a human subject, particularly to a human subject from a high-risk group such as hospital personnel, wherein said immune-stimulatory effect leads to a reduced susceptibility against a virus infection, particularly to a reduced susceptibility against a Coronavirus virus infection, and more particularly to a reduced susceptibility against a SARS-CoV-2 infection.

The reduced susceptibility against a virus infection results in the prevention and/or amelioration of a respiratory disorder caused by and/or associated with said virus infection. The virus may be any virus afflicting the mammalian, e.g. human respiratory system including the upper and lower airways. In certain embodiments, the virus is a Coronavirus. In further embodiments, the virus may an influenza virus or a Rhinovirus. The respiratory disorder relates to disorder afflicting the upper airways such as the oral cavity, the nasal cavity, the paranasal sinus, the pharynx and/or the throat, and/or the lower airways such as the bronchi and/or the lung.

According to certain embodiments of the invention, the reduced susceptibility to a SARS-CoV-2 infection leads to a prevention and/or amelioration of a Covid-19 disorder characterized by at least one of:

- a reduced infection rate, e.g. an infection rate reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50% versus a control group;

- a reduced sickness or absence of work time due to a viral infection, e.g. a sickness or absence of work time reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50% versus a control group;

- a reduced rate of COVID-19 symptoms such as increased temperature, e.g. fever of at least 38°C, and/or respiratory symptoms such as coughing and breathlessness, e.g. a rate of COVID-19 symptoms reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50% versus a control group; - a reduced rate of hospital admission, e.g. a rate of hospital admission reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50% versus a control group;

- a reduced rate of ICU admission, e.g. a rate of ICU admission reduced by at least 10%, at least 20% or at least 30% versus a control group

- a reduced mortality rate, e.g. a mortality rate reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50% versus a control group. The term “control group” refers to a group, which has not been treated with the composition of the invention, e.g. which has been treated with placebo, and which is not statistically different from the treatment group, i.e. the group, which has been treated with the composition of the invention. The active agent of the pharmaceutical composition is a live recombinant Mycobacterium cell, which comprises a recombinant nucleic acid molecule encoding a fusion polypeptide comprising (a) a domain capable of eliciting an immune response and (b) a phagolysosomal escape domain. The domain capable of eliciting an immune response is preferably an immunogenic peptide or polypeptide from a pathogen or an immunogenic fragment thereof.

The Mycobacterium cell is preferably an M. bovis cell, an M. tuberculosis cell, particularly an attenuated M. tuberculosis cell or other Mycobacteria, e.g. M. microti, M. smegmatis, M. canettii, M. marinum or M. fortuitum. More preferably, the cell is an attenuated recombinant M. bovis (BCG) cell, particularly an M. bovis BCG cell, more particularly a recombinant M. bovis BCG cell from strain Danish subtype Prague (Brosch et al., Proc. Natl. Acad. Sci. USA, 104 (2007), 5396-5601). In an especially preferred embodiment, the Mycobacterium cell is recombinant urease-deficient. In an especially preferred embodiment the ureC sequence of the Mycobacterium cell is inactivated (AUrec), e.g. by constructing a suicide vector containing a ureC gene disrupted by a selection marker gene, e.g. the hygromycin gene, transforming the target cell with the vector and screening for selection marker-positive cells having a urease negative phenotype. In an even more preferred embodiment, the selection marker gene, i.e. the hygromycin gene, is subsequently inactivated. In this embodiment, the cell is a selection marker-free recombinant Mycobacterium cell. Most preferably, the cell is selection marker-free recombinant BCG strain Danish subtype Prague characterized as recombinant BCG AUrec::Hly+.

The domain capable of eliciting an immune response is preferably selected from immunogenic peptides or polypeptides from M. bovis, M. tuberculosis or M. leprae or from immunogenic fragments thereof having a length of at least 6, preferably at least 8 amino acids, more preferably at least 9 amino acids and e.g. up to 20 amino acids. Specific examples for suitable antigens are Ag85B (p30) from M. tuberculosis, Ag85B (a-antigen) from M. bovis BCG, Ag85A from M. tuberculosis and ESAT-6 from M. tuberculosis and fragments thereof. In other embodiments, the domain capable of eliciting an immune response is selected from non-Mycobacterium polypeptides.

More preferably, the immunogenic domain is derived from the antigen Ag85B. Most preferably, the immunogenic domain comprises the sequence from aa.41 to aa.51 in SEQ ID No.2.

The recombinant nucleic acid molecule further comprises a phagolysosomal escape domain, i.e. a polypeptide domain which provides for an escape of the fusion polypeptide from the phagolysosome into the cytosol of mammalian cells. Preferably, the phagolysosomal escape domain is a Listeria phagolysosomal escape domain, which is described in US 5,733,151, herein incorporated by reference. More preferably, the phagolysosomal escape domain is derived from the listeriolysin gene (Hly) of L monocytogenes. Most preferably, the phagolysosomal domain is encoded by a nucleic acid molecule selected from: (a) a nucleotide sequence comprising nucleotides 211 - 1722 as shown in SEQ ID No.1, (b) a nucleotide sequence which encodes the same amino acid sequence as the sequence from (a), and (c) a nucleotide sequence hybridizing under stringent conditions with the sequence from (a) or (b). Apart from the nucleotide sequence depicted in SEQ ID No.1 the present invention also comprises nucleic acid sequences hybridizing therewith. In the present invention the term "hybridization" is used as defined in Sambrook et al. (Molecular Cloning. A laboratory manual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104). In accordance with the present invention the term "hybridization" is used if a positive hybridization signal can still be observed after washing for one hour with 1 X SSC and 0.1 % SDS at 55°C, preferably at 62° C and more preferably at 68°C, particularly for 1 hour in 0.2 X SSC and 0.1 % SDS at 55°C, preferably at 62°C and more preferably at 68°C. A sequence hybridizing with a nucleotide sequence as per SEQ ID

No.1 under such washing conditions is a phagolysosomal escape domain encoding nucleotide sequence preferred by the subject invention.

A nucleotide sequence encoding a phagolysosomal escape domain as described above may be directly obtained from a Listeria organism or from any recombinant source e.g. a recombinant E.coli cell containing the corresponding Listeria nucleic acid molecule or a variant thereof as described above. Preferably, the recombinant nucleic acid molecule encoding for a fusion polypeptide contains a signal peptide encoding sequence. More preferably, the signal sequence is a signal sequence active in Mycobacteria, preferably in M.bovis, e.g. a native M.bovis signal sequence. A preferred example of a suitable signal sequence is the nucleotide sequence coding for the Ag85B signal peptide, which is depicted in SEQ ID No.1 from nucleotide 1 to 120.

Further, it is preferred that a peptide linker be provided between the immunogenic domain and the phagolysosomal escape domain. Preferably, said peptide linker has a length of from 5 to 50 amino acids. More preferably, a sequence encoding a linker as shown in SEQ ID No.1 from nucleotide 154 to 210 or a sequence corresponding thereto as regards the degeneration of the genetic code. The nucleic acid may be located on a recombinant vector. Preferably, the recombinant vector is a prokaryotic vector, i.e. a vector containing elements for replication or/and genomic integration in prokaryotic cells. Preferably, the recombinant vector carries the nucleic acid molecule of the present invention operatively linked with an expression control sequence. The expression control sequence is preferably an expression control sequence active in Mycobacteria, particularly in M.bovis. The vector can be an extrachromosomal vector or a vector suitable for integration into the chromosome. Examples of such vectors are known to the person skilled in the art and, for instance, given in Sambrook et at. supra.

The composition may administered to any subject, particularly a human subject, who is in risk of becoming infected with a pathogen, wherein the pathogen is particularly a virus, more particularly a Coronavirus, and even more particularly SARS-CoV-2. In certain embodiments, the composition is administered to a human subject, particularly to a human subject, who is a member of a high-risk group of being infected. In certain embodiments, the composition is administered to a human subject, who is a health-care professional, particularly a health-care professional working in a hospital, i.e. hospital personnel, and more particularly a health-care professional having direct contact with virus-infected patients, e.g. patients infected with SARS- CoV-2. The term “health-care professional” includes doctors, nurses, and other staff working in a hospital, as well RTV personnel, but also health care personnel working in private practices, nursing homes, retirement homes etc.

In certain embodiments, the composition is administered by injection, e.g. by subcutaneous, intramuscular or intradermal injection, particularly by intradermal injection. The composition is administered in an effective dose. For a human subject, the dose for an administration may be about 10 ⁴ to 10 ⁷ colony forming units (CFU), particularly about 10 ⁵ to 10 ⁶ CFU, and more particularly of about 2 x 10 ⁵ to 8 x 10 ⁵ CFU. In certain embodiments, the composition administered as a single dose. In further embodiments, the composition may be administered several times, e.g. at least 2 or 3 times, wherein the intervals between each administration may be at least one year, particularly between 3-5 years. Further, it is preferred to administer the composition without adjuvant.

The pharmaceutical composition comprises the recombinant Mycobacterial cell in living form. It may be a solid composition, e.g., a lyophilized or cryoconserved composition, which may be reconstituted with a suitable liquid carrier before use. In certain embodiments, the composition comprises a cryoprotectant, e.g. a carbohydrate such as glucose and/or dextran, and/or a polyalcohol. Alternatively, the preparation may be a liquid composition, e.g., a suspension.

Further, the invention is described in more detail by the following Figures and Examples. The immunotherapeutic agent “ VPM1002 " used in these examples is recombinant M. bovis (BCG) Danish subtype Prague with an inactivated ureC sequence (AUrec) and without functional selection marker gene, which expresses an Ag85B/Flly fusion protein as shown in SEQ ID No.2 (Hly+).

Example 1

A phase III, randomized, double-blind, placebo-controlled, multi-center, human clinical trial to assess the efficacy and safety of VPM1002 in reducing healthcare professionals’ absenteeism in the SARS-CoV-2 pandemic by modulating the immune system.

The trial design is schematically depicted in Figure 1 and described in detail as follows in Table 1. PROTOCOL SYNOPSIS Table 1