WO2022229350A2

WO2022229350A2 - Single-stranded oligonucleotides for use in the medical treatment and/or prophylaxis of virus infections

Info

Publication number: WO2022229350A2
Application number: PCT/EP2022/061404
Authority: WO
Inventors: Anna-Lena Spetz
Original assignee: Tirmed Pharma Ab
Priority date: 2021-04-30
Filing date: 2022-04-28
Publication date: 2022-11-03
Also published as: WO2022229350A3

Abstract

The present disclosure relates to non-CpG single-stranded oligonucleotides (ssONs) with broad-spectrum antiviral activity for use in the treatment and/or prophylaxis of viral infections, such as Herpes simplex virus, Ebola virus, Lassa virus, Lyssa virus, rabies virus, measles virus, vesicular stomatitis virus (VSV), murine leukemia virus (MLV) and HIV-1, as well as multi-resistant HIV-1 infections, in a subject. Said ssONs have a length of at least 25 nucleotides and are stabilized by phosphorothioate internucleotide linkages and/or 2'-O-Methyl modifications.

Description

SINGLE-STRANDED OLIGONUCLEOTIDES FOR USE IN THE MEDICAL TREATMENT AND/OR PROPHYLAXIS OF VIRUS INFECTIONS

Field of the invention

The present disclosure relates to non-coding single-stranded oligonucleotides (ssONs) for use in the treatment and/or prophylaxis of viral infections caused by viruses such as Herpes simplex virus (HSV), Ebola virus, Lassa virus, Lyssa virus, rabies virus, measles virus, and Human immunodeficiency virus type 1 (HIV-1), including multi-resistant HIV-1. The ssONs have a length of at least 25 nucleotides and are stabilized by phosphorothioate internucleotide linkages and/or 2’-0-Methyl modifications. Background

Human immunodeficiency virus type 1 (HIV-1)

The Human immunodeficiency virus type 1 (HIV-1) has so far infected ~38 million people worldwide and in 2019, 690,000 people died from HIV and 1.7 million people became infected (https://www.unaids.org/en/resources/fact- sheet). Combination antiretroviral therapy (cART) has led to a pronounced decline in mortality and quality of life of people living with HIV-1 (Maiese EM et ai. Curr Med Res Opin. 2016;32(12):2039-46„ May MT, et al. AIDS. 2014;28(8) :1193-202.) However, treatment failure is not uncommon (Haggblom A, et al. PLoS One. 2015;10(12):e0145536.) and is highly related to adherence and acquired drug resistance (Biswas A, etal. Elife. 2019;8.,Chen et al. Biomed Res Int. 2020:5894124., Clutter DS, et al. Infect Genet Evol. 2016;46:292-307.). In addition, pretreatment drug resistance (PDR) is increasing world-wide (Andersson E, et al. AIDS. 2021 ;35(2):227-34., Gupta RK, et al. Lancet Infect Dis. 2018;18(3):346-55.). It is conceivable that HIV-1 subtype may influence the treatment outcome (Haggblom A, etal. Lancet HIV. 2016;3(4):e166-74).

Thus, despite improved HIV care in general, including the use of the efficient new class of integrase strand transfer inhibitors (INSTI), problems remain with drug resistance, side effects and increasing issues with pharmacokinetic interactions in elderly HIV-1 patients. Altogether, this shows the need of new categories of antiretroviral drugs.

Herpes genitalis

Herpes genitalis remains one of the most common sexually transmitted infections (STI) Mathew Jr J, Sapra A. etal. https://www.ncbi.nlm.nih.gov/books/NBK554427/). While the majority of cases are due to herpes simplex virus type 2 (HSV-2), rare but increasing cases have been found due to herpes simplex virus type 1 (HSV-1). Most commonly, viral replication occurs in epithelial tissue and establishes dormancy in sensory neurons, reactivating periodically as localized recurrent lesions. HSV-2 continues to be a common infection, affecting approximately 22% of adults ages 12 and older, representing 45 million adults in the United States alone (Mathew Jr J, Sapra etal. https://www.ncbi.nlm.nih.gov/books/NBK554427/).

While HSV-1 often affects the perioral region and can also cause genital lesions, however, HSV-2 is more commonly the consideration when patients present with genital lesions. Despite this, most outbreaks of the infection will present with nonspecific symptoms such as genital itching, irritation, and excoriations, which may delay diagnosis and treatment.

Anti-herpes viral agents include those that act as nucleoside analog- polymerase inhibitors and pyrophosphate analog-polymerase inhibitors. The mainstay of therapy remains acyclovir, which has some antiviral activity against all herpesviruses and has been FDA approved for the treatment and suppression of both HSV and VZV (Poole CL etal. Clin Ther. 2018 Aug;40(8):1282-1298). Other treatments include penciclovir (which is more often used as a topical therapy for HSV labialis) and ganciclovir (which has suppression activity against CMV). These medications are preferentially taken up by those cells already infected with the virus and stop viral replication (Poole CL, etal. Clin Ther. 2018 Aug;40(8):1282-1298). Standard therapy for HSV-2 infections include acyclovir, famciclovir and valacyclovir. Medications on the horizon include brincidofovir and maribavir (both against CMV) and valomaciclovir (activity against HSV, VZV, and EBV). However, patients seek alternative treatments that have a smaller side effect profile. Reported side effects include kidney toxicity and neutropenia when given at high doses. Given its chronicity of use, resistance has been reported in immunocompromised patients and those who are immunocompetent taking acyclovir as suppressive therapy for genital herpes (Poole CL,ef al. Clin Ther. 2018 Aug;40(8):1282-1298). There is no cure for HSV-2, early identification of symptoms, and prompt institution of pharmacotherapy can lead to early suppression of viral replication. The Herpes viruses as a family are responsible for significant neurological morbidity, and unfortunately, HSV- 2 persists in the seropositive individual for a lifetime.

Genital HSV-2 infection is known to be associated with an increased risk of HIV infection and there is a huge medical need to develop new treatments for HSV-2.

Ebola virus

Ebola virus (belonging to Filoviridae family) outbreaks typically occur due to transmissions from the animal kingdom to human beings, followed by human-to-human transmission and there is a medical need to develop treatment to contain new emerging strains of this virus family with high mortality rates (Jacob, S.T. etal. Nat Rev Dis Primers 6, 13 (2020)). https://doi.org/10.1038/s41572-020-0147-3.) To date, 12 distinct filoviruses have been described. The seven filoviruses that have been found in humans belong either to the genus Ebolavirus (Bundibugyo virus (BDBV), Ebola virus (EBOV), Reston virus (RESTV), Sudan virus (SUDV) and Ta^'i Forest virus (TAFV)) or to the genus Marburgvirus (Marburg virus (MARV) and Ravn virus (RAW) (Kuhn, J. H. etal. Nat. Rev. Microbiol. 17, 261-263 (2019) outlines the current official WHO International Classification of Diseases version 11 (ICD-11) subdivisions of filovirus disease (FVD), including Ebola virus disease (EVD). The WHO International Classification of Diseases Revision 11 (ICD- 11) of 2018 recognizes two major subcategories of filovirus disease (FVD): Ebola disease caused by BDBV, EBOV, SUDV or TAFV, and Marburg disease caused by MARV or RAW. Ebola virus disease is defined as a disease only caused by EBOV. Ebola virus disease has evolved as a global public health menace. Initially, the patients present with nonspecific influenza like symptoms and eventually terminate into shock and multiorgan failure. The first FDA approved treatment was Inmazeb (atoltivimab, maftivimab, and odesivimab), a mixture of three monoclonal antibodies for use against Zaire ebolavirus (Ebola virus) infection in adult and pediatric patients. The second drug, Ebanga, is a single monoclonal antibody and was approved in December 2020. Monoclonal antibodies (often abbreviated as mAbs) are proteins produced in a laboratory or other manufacturing facility that act like natural antibodies to stop a germ such as a virus from replicating after it has infected a person. These particular mAbs bind to a portion of the Ebola virus’ surface called the glycoprotein, which prevents the virus from entering a person’s cells https://www.cdc.gov/vhf/ebola/treatment/index.html.

However, there is a need to develop new treatments as the surface glycoproteins recognized by mAbs often are subjected to viral mutations as a way to escape from the suppression.

Measles virus

Measles is a highly contagious, serious disease caused by a virus in the paramyxovirus family and it is normally passed through direct contact and through the air. Before the introduction of measles vaccine in 1963 and widespread vaccination, major epidemics occurred approximately every 2-3 years and measles caused an estimated 2.6 million deaths each year.

More than 140,000 people died from measles in 2018 - mostly children under the age of 5 years, despite the availability of a safe and effective vaccine.

The virus infects the respiratory tract, then spreads throughout the body. Measles is a human disease and is not known to occur in animals.

(https://www.who.int/news-room/fact-sheets/detail/measles). There is no specific antiviral therapy for measles. Medical care is supportive and to help relieve symptoms and address complications such as bacterial infections. Rabies virus

People are usually infected following a deep bite or scratch from an animal with rabies, and transmission to humans by rabid dogs accounts for up to 99% of cases. However, bat rabies is also an emerging public health threat.

Once clinical symptoms appear, rabies is virtually 100% fatal. In up to 99% of cases, domestic dogs are responsible for rabies virus transmission to humans. Yet, rabies can affect both domestic and wild animals. It is spread to people and animals through bites or scratches, usually via saliva. Every year, more than 29 million people worldwide receive a post-bite vaccination. This is estimated to prevent hundreds of thousands of rabies deaths annually (https://www.who.int/news-room/fact-sheets/detail/rabies).

Post-exposure prophylaxis (PEP) is the immediate treatment of a bite victim after rabies exposure. This prevents virus entry into the central nervous system, which results in imminent death. PEP consists of:

• Extensive washing and local treatment of the bite wound or scratch as soon as possible after a suspected exposure;

• a course of potent and effective rabies vaccine that meets WHO standards; and · the administration of rabies immunoglobulin (RIG), if indicated.

Starting the treatment soon after an exposure to rabies virus can prevent the onset of symptoms and death. However, no other anti-viral therapy is available for the treatment of rabies virus infections. Oligonucleotides

Oligonucleotides are short DNA or RNA molecules, oligomers, which have a wide range of applications.

Anti-sense oligonucleotides target messenger RNA (mRNA) and are thereby altering mRNA expression through a variety of mechanisms, including ribonuclease H mediated decay of the pre-mRNA, direct steric blockage, and exon content modulation through splicing site binding on pre-mRNA. Hence, anti-sense oligonucleotides are directed against a specific sequence thereby ultimately causing a regulatory effect. Another group of oligonucleotides are microRNA (miRNA) which are small single-stranded non-coding RNA molecules (containing about 20-22 nucleotides). MiRNA exert their function via base-pairing with complementary sequences with mRNA molecules. As a result, these mRNA molecules are silenced, by one or more of the following processes: (1 ) Cleavage of the mRNA strand into two pieces, (2) Destabilization of the mRNA through shortening of its poly(A) tail, and (3) Less efficient translation of the mRNA into proteins by ribosomes.

There are also known non-coding immunoregulatory oligonucleotides, that act by activation or inhibiting pattern recognition receptors such as the Toll-like receptors (TLR). CpG oligonucleotides (or CpG-ssON) are short single-stranded synthetic DNA or RNA molecules that contain a cytosine triphosphate nucleotide (“C”) followed by a guanine triphosphate nucleotide (“G”). It is known in the art that unmethylated CpG-containing nucleic acids stimulate the immune system and can be used to treat infectious diseases, allergy, asthma and other disorders (Scheiermann, J., and Klinman, D.M. 2014. Vaccine 32:6377-6389; Aryan, Z. and Rezaei, N. 2015. Curr Opin Allergy Clin Immunol. 15:568-74).

CpG sequences in ssDNA ligands have been shown to be indispensable for activation of TLR9, which plays a fundamental role in pathogen recognition and activation of innate immunity. The stimulatory effect of the ligand is lost when the CpG repeats are removed. Consequently, the TLR-mediated immunostimulatory effect has not been shown for single- stranded oligonucleotides lacking CpG motifs (“non-CpG ssON”).

It has been shown (Skold etal. 2012. Blood 120:768-777) that single- stranded DNA oligonucleotides (ssONs) inhibit TLR3-mediated responses in human monocyte-derived dendritic cells and in vivo in cynomolgus macaques. TLR3 is a key receptor for recognition of double-stranded RNA and initiation of immune responses against viral infections. It was shown by Skold etal. that human monocyte-derived dendritic cells up-regulate maturation markers and secrete pro-inflammatory cytokines on treatment with the synthetic TLR3 ligand polyinosine-polycytidylic acid (Poly(l:C)). Poly(l:C) is a synthetic agonist to for example TLR3 and is often used as an adjuvant in vaccines (see e.g. Duthie, M.S. et al. 2011. Immunol. Rev. 239(1 ):178-196). It was shown by Skold et al. that TLR3-mediated events were inhibited in cultures with CpG-ssON. Poly(l:C) activation of non-hematopoietic cells was also inhibited by CpG-ssON. The uptake of Poly(l:C) into cells was reduced in the presence of CpG-ssON, preventing TLR3 engagement from occurring. In cynomolgus macaques, the levels of pro-inflammatory cytokines in nasal secretions were reduced when ssONs were administered via the intranasal route. The ssON sequences used by Skold et al. were non-CpG-ssON SEQ ID NO: 1 and a CpG-ssON.

The present inventors have also shown (WO 2016/202779) that non- CpG-ssONs, such as the one shown as SEQ ID NO: 1 , are useful in the treatment or prophylaxis of disorders of the skin and/or subcutaneous tissue, including pruritus.

It has been shown (Baharom, F. et al. 2015. J. Immunol. 194:4422- 4430) that lipopolysaccharide or poly(l:C) stimulation of monocyte-derived dendritic cells results in type I IFN induction and reduced susceptibility to influenza A virus infection. In the paper by Skold, the present inventors showed that ssONs can inhibit poly l:C activation. A reduced TLR3-mediated response can according to the present state of the art be a risk factor leading to increased risk of virus infection as many viruses are susceptible to the anti viral effect of type 1 IFN. Flence, treatments with ssONs that inhibit TLR3 may lead to increased viral replication.

Synthetic oligodeoxynucleotides that can down-regulate cellular elements of the immune system have been developed and studied in preclinical models. These agents vary in sequence, mechanism of action, and cellular target(s) but there is often a lack of understanding on their mechanism of action (Bayik, D. et al. 2016. Pharmacol Res. 105:216-225).

For example, it has been shown (Gursel, I. et al. 2003. J Immunol.

171 (3):1393-1400) that repetitive elements, such as TTAGGG, present at high frequency in mammalian telomeres, down-regulate CpG-induced immune activation.

Flanecak et al. (US 2011/0124715) discloses chemically modified oligonucleotides that are useful for inhibiting virus activity. The modified oligonucleotides have no more than 27 nucleic acid base units and comprise at least one GGGG sequence or at least two GGG sequences.

It is well known in the art that virus infections can be treated with oligonucleotides, polynucleotides or aptamers that specifically target virus- encoded molecules. For instance, Arnon etal. (US 2013/0079390) discloses a nucleic acid aptamer molecule capable of binding to hemagglutinin (a surface antigen on influenza virus).

Vaillant etal. (US 2012/0184601) discloses random sequence oligonucleotides that have antiviral activity. It was disclosed that HSV-1 , HSV- 2, CMV, Coxsackie virus B2, DHBV, Hantavirus, parainfluenza virus, HIV-1 , and vaccinia virus can be inhibited by ssONs in vitro. It was suggested that the random ssONs had a broad-spectrum antiviral activity with viruses where assembly and/or packaging and/or encapsidation of the viral genome was a required step in the replication. The inventors also stated that they could inhibit RSV by using ssONs, while they failed to inhibit viruses such as Influenza A (H3N2), Corona virus (HCoV-OC43), BVDV, rhinovirus (HGP) and adenovirus (Human Ad5).

Jarver etal. (WO 2019/048555), Poux etal. Frontiers Immunology 2019 Sep 12;10:2161. doi: 10.3389/fimmu.2019.02161 and Palsson etal. Frontiers Immunology 2020 Dec 8;11 :580547. doi:

10.3389/fimmu.2020.580547 further discloses ssON^'s with capacity to inhibit Influenza A H1 N1 and RSV infections.

Consequently, the results are ambiguous with regard to the structures and mechanisms of single-stranded oligonucleotides that are useful in the treatment of particular virus infections. In particular, there is an unmet need for methods for the treatment and prophylaxis of multi-resistant HIV-1 , Ebola, HSV-2, rabies virus, measles virus. Summary of the invention

The present invention is based on the inventors unexpected discovery that a specific group of non-CpG ssONs, which do not specifically target virus-encoded molecules, are capable of preventing viruses such as Human immunodeficiency virus type 1 (HIV-1), Herpes simplex virus 2 (HSV-2),

Ebola virus, Lassa virus, Lyssa virus, rabies virus, measles virus, vesicular stomatitis virus (VSV) and murine leukemia virus (MLV) from binding to the cell surface, thereby inhibiting viral infections using a different mechanism and with superior efficiencies compared to other agents previously described in the art, known to the applicant.

It is therefore an object of the present disclosure to provide single- stranded oligonucleotides (ssONs) for use in the treatment and/or prophylaxis of infections caused by viruses such as HIV-1 , HSV-2, Ebola virus, Lassa virus, Lyssa virus, rabies virus and measles virus, as well as VSV and MLV. These and other objects, which are evident to the skilled person from the present disclosure, are met by the different aspects of the invention as claimed in the appended claims and as generally disclosed herein.

Thus, in a first aspect of the disclosure, there is provided a single- stranded oligonucleotide (ssON) with broad-spectrum antiviral activity for use in the treatment and/or prophylaxis of an infection caused by a virus selected from the group consisting of Human immunodeficiency virus type 1 (HIV-1), Herpes simplex virus, Ebola virus, rabies virus, Lassa virus, Lyssa virus, measles virus, vesicular stomatitis virus (VSV) and murine leukemia virus (MLV) in a subject in need thereof, wherein: (a) the length of said ssON is between 25-40 nucleotides;

(b) either (i) at least 90% of the internucleotide linkages in said ssON are phosphorothioate internucleotide linkages; or (ii) said ssON comprises at least four phosphorothioate internucleotide linkages and at least four 2’-0-methyl modifications; and (c) said ssON does not contain any CpG motifs.

Typically, the ssON (i) is non-complementary to any human mRNA and/or viral gene; (ii) is not self-complementary; and/or (iii) does not comprise at least one GGGG sequence and/or does not comprise at least one GGG sequence ( e.g . TTAGGG).

As mentioned above, the length of the ssON is at least from 25 and at least to 40, such as 25-35, 25-30, 30-40, 35-40, or 30-35 nucleotides. Thus, a preferred length is 35 nucleotides.

In one embodiment of the invention, said ssON comprises at least six phosphorothioate internucleotide linkages and/or at least six 2’-0-methyl modifications. In another embodiment of the invention, all internucleotide linkages in said ssON are phosphorothioate internucleotide linkages.

The terms “phosphorothioate internucleotide linkages” and “PS linkages” refer to internucleotide linkages in which one of the non-bridging oxygens in the DNA phosphate (PO) backbone is replaced by sulfur. Preferably 95%, or more preferably 100%, of the internucleotide linkages in the ssON to be used according to the invention are phosphorothioate (PS) internucleotide linkages. Consequently, the invention includes the use of ssONs wherein some internucleotide linkages (such as one, two, three or more internucleotide linkages) are PO linkages without sulfur, while the remaining linkages are PS linkages. In cases where the ssON comprises phosphate groups in the 5’- terminal and/or 3-terminal, such phosphate groups may be modified (PS) or unmodified (PO) groups.

The term “2’-0-Methyl modifications” refers to nucleotide modifications wherein a methyl group is added to the 2’-hydroxyl group of the ribose moiety of a nucleoside.

The ssON to be used according to the invention may comprise additional chemical modifications. Chemically modified oligonucleotides are known in the art and disclosed in e.g. Jarver, P. et al. 2014. Nucleic acid therapeutics 24:37-47; and Deleavey, G.F. & Damha, M.J. 2012. Chemistry & Biology 19:937-954. Possible chemical modifications include e.g. LNA (Locked Nucleic Acid), wherein the ribose moiety is modified with an extra bridge connecting the 2’ oxygen and 4’ carbon. Further, the ssON could comprise a mix of ribose and deoxyribose as the five-carbon sugar. In addition, one or more nucleobases in the ssON could be modified. Oligonucleotide base modifications include methylation of cytosine to form 5- methylcytosine, and methylation of adenosine to form N6-methyladenosine.

The term “CpG motifs” will be understood to refer to immunostimulatory CpG oligonucleotides, i.e. short single-stranded synthetic nucleic acid molecules that contain a cytosine triphosphate deoxynucleotide (“C”) followed by a guanine triphosphate deoxynucleotide (“G”). The “p” refers to the phosphodiester or phosphorothioate link between consecutive nucleotides. Unmethylated CpG motifs are considered pathogen-associated molecular patterns (PAMPs) due to their abundance in microbial genomes but their rarity in vertebrate genomes. The CpG PAMP is recognized by the pattern recognition receptor (PRR) Toll-Like Receptor 9 (TLR9), which is constitutively expressed primarily in B cells and plasmacytoid dendritic cells (pDCs) in humans and other higher primates. Consequently, the invention does not include the use of ssONs comprising CpG motifs capable of stimulating a TLR9 response.

Suitably, the concentration of said ssON required to cause a 50% reduction in infectivity (ICso) of the virus is at most 1 x 10⁵M, such as at most 7.5 x 10⁶M, such as at most 5 x 10⁶M, such as at most 2.5 x 10⁶M, such as at most 2 x 10⁶M, such as at most 1.5 x 10⁶M, such as at most 1 x 10⁶M, such as at most 0.5 x 10⁶M.

Suitably, said ssON prevents binding of the virus to the cell surface and/or subsequent fusion of the virus to the cell membrane.

In some embodiments of the invention, the infection is caused by a virus selected from the viral family groups HIV-1 , Ebola virus, Herpes simplex virus (HSV), rabies virus, measles virus, Lassa virus, Lyssa virus, vesicular stomatitis virus (VSV), and murine leukemia virus (MLV), such as HIV-1 ,

HSV, Ebola virus and rabies virus.

In another embodiment of the invention, said infection is caused by an HIV-1 virus, such as a multi-resistant HIV-1.

In another embodiment, said infection is caused by a Herpes simplex virus, such as HSV-2.

In another embodiment, said infection is caused by Ebola virus. Preferably, the ssON to be used according to the invention has a “sequence independent” mode of action, does not have antisense activity and/or is not complementary to a gene. More specifically, not more than 15, and preferably not more than 10, consecutive nucleotides in said ssON are complementary with any human mRNA sequence. Consequently, the ssON is essentially “non-complementary” with any human mRNA sequence. The term “non-complementary” will be understood to refer to nucleic acid sequences that are not capable of precise pairing (of purine or pyrimidine bases between the two strands of nucleic acids sequences) under moderate or stringent hybridization conditions (i.e. 5-10°C below T_m). In particular, the ssON is non- complementary to nucleotide sequences coding for receptor proteins, e.g. Toll-like receptors, such as TLR3 or TLR9, or any other protein which recognize DAMPs (Damage-associated molecular pattern) or PAMPs. It will thus be understood that the ssONs to be used according to the invention are not “antisense” molecules that are complementary to a messenger RNA (mRNA) strand transcribed within a cell or encoded by viral nucleic acids.

Further, the ssON to be used according to the invention does typically not target any specific viral gene or polypeptide. As mentioned in the background section, it is known in the art that virus infections can be treated with oligonucleotides, polynucleotides or aptamers that specifically target virus-encoded molecules. For instance, Arnon etal. (US 2013/0079390) discloses a nucleic acid aptamer molecule capable of binding to hemagglutinin (a surface antigen on influenza virus).

It will thus be understood that according to the invention, the ssON is useful based on its tertiary structure, i.e. the spatial organization, and not solely dependent on its primary structure, i.e. the sequence of nucleotides. Flence, non-coding single-stranded oligonucleotides can bind to proteins expressed on the cell surface containing RNA binding domains, of which some recognize primarily the shape of the RNA. The class of ssONs disclosed in the present invention has the capacity to bind to such proteins and thereby also block binding of viruses, which are strictly relying on binding to cell surfaces in order to enter cells and start replication. It is known in the art that different types of viruses use different cell receptors to bind to cell surfaces. In contrast, the class of ssONs disclosed herein has surprisingly been found to block the binding to the cell surface independently of the virus- specific cellular receptors.

A person having ordinary skill in the art will be able to identify oligonucleotide sequences which are “non-complementary” as defined according to the present invention. For instance, the skilled person could use well-known tools such as the BLAST algorithm as implemented online by the US National Center for Biotechnology Information. See e.g. Madden, T. 2013. The BLAST Sequence Analysis Tool. The NCBI Handbook [Internet], 2^nd edition. (www.ncbi.nlm.nih.gov/books/NBK153387).

Preferably, the ssON is not self-complementary. The term “not self complementary” will be understood to mean that the ssON does not have any self-complementary sequences that would allow two ssONs to dimerize, or that would allow parts of the oligonucleotide to fold and pair with itself to form stem loops. It is well-known that stem loop (also referred to as “hair-pin” loop) base pairing can occur in single-stranded DNA or RNA. It occurs when two regions of the same strand, usually complementary when read in opposite directions, base-pair to form a double helix that ends in an unpaired loop.

A person having ordinary skill in the art will be able to identify self complementary sequences by comparing parts of the ssON sequence and detecting whether Watson-Crick base pairing (CG and AT/AU) could occur. Alternatively, a software tool such as Oligo Calc: Oligonucleotide Properties Calculator (www.basic.northwestern.edu/biotools/oligocalc.html) could be used to detect self-complementary sequences. Models for self-dimerization and hairpin formation in oligonucleotides are known in the art and are described in e.g. Hilbers, C.W. 1987. Anal Chem 327:70; Serra, M.J. 1993. Nucleic Acids Res 21 :3845-3849; and Vallone, P.M. 1999. Biopolymers. 50:425-442. As a general rule, at least 5 base pairs would be required for self-dimerization, and at least 4 base pairs would be required for hair-pin formation. Consequently, preferably the ssON as defined above does not comprise more than 3 consecutive nucleotides that could form base pairs with another sequence of 3 consecutive nucleotides at the same ssON molecule. Preferably, the ssON is not composed of single repeats or dinucleotide repeats, as this reduces efficacy to binding to the cells (disclosed by the inventors in Jarver etal. Sci Reports 2018 page 15841 DOI 10.1038/s41598- 018-33960-4) and instead leads to binding to virus as disclosed by Vaillant et al. (US 2012/0184601 ), which is not what the ssON of the present invention does to exert its function and inhibit viral infections with superior efficiencies compared to previously described agents.

Typically, the ssONs to be used according to the invention do not comprise TTAGGG sequences shown by Gursel etal. {supra) to down- regulate CpG-induced immune activation. Typically, the ssONs do not comprise GGG or GGGG sequences disclosed by Hanecak etal. (US 2011/0124715).

Preferably, the ssON is a single-stranded oligodeoxynucleotide (ssODN). However, the invention also provides the use of ssONs that are stabilized single-stranded RNA (ribonucleic acid) molecules. As will be understood by the skilled person, when the ssON is an oligodeoxynucleotide, the monosaccharides in the ssON are 2’-deoxyribose. However, in the present context the term “ssODN” also includes oligonucleotides comprising one or more modified monosaccharides such as 2’-0-methylribose.

The inventors have described the use of their invention in the context of 27 ssONs. Thus, in preferred aspects of the invention, the ssON comprises or consists of polynucleotide having a sequence of any one of SEQ ID NOs: 1-4, 7, 8, 13-27 in the Sequence Listing. More preferably, the ssON comprises or consists of the sequence shown as SEQ ID NO: 1.

In a further preferred aspect of the invention, at least 30% of the nucleobases in the ssON are chosen from A (Adenine) and T (Thymine) and U (Uracil). Preferably, at least 35%, 40%, 45%, 50%, 55%, or 60% of the nucleobases in the ssON are chosen from A, T and U. When the ssON is an oligodeoxynucleotide (ssODN), containing deoxyribose as its pentose component, the nucleobases are normally chosen from A and T. When the ssON is a ribonucleotide containing ribose, the nucleobases are normally chosen from A and U. However, the ssONs according to the invention could include synthetic variants which may differ from naturally occurring oligonucleotides. For instance, the ssON could comprise a deoxyuridine moiety (i.e. uracil bound to deoxyribose). The ssON could also comprise nucleobase analogues, which are well known in the art and include e.g. xanthine, hypoxanthine, 7-methylguanine, 5-methylcytosine, and 5- hydroxymethylcytosine.

In a particularly preferred embodiment, the ssON is one wherein:

(a) the length of the ssON is at least 25 nucleotides {e.g. at least 28 nucleotides);

(b) either (i) at least 90% of the internucleotide linkages in the ssON are phosphorothioate internucleotide linkages; or (ii) the ssON comprises at least four phosphorothioate internucleotide linkages or at least four 2’-0- methyl modifications; and

(c) the ssON does not contain any CpG motifs. Preferably, (i) the ssON does not comprise at least one GGGG sequence and/or does not comprise at least one GGG sequence {e.g. TTAGGG); and/or (ii) the ssON comprises at least three different nucleotides selected from the group consisting of A, C, G and T, or at least four different nucleotides selected from the group consisting of A, C, G and T.

In a particularly preferred embodiment, the ssON {e.g. ss ODN) is one wherein:

(b) either (i) at least 90% of the internucleotide linkages in the ssON are phosphorothioate internucleotide linkages; or (ii) the ssON comprises at least four phosphorothioate internucleotide linkages and/or at least four 2’-0- methyl modifications;

(c) the ssON does not contain any CpG motifs; and

(d) (i) the ssON is non-complementary to any human mRNA and/or viral gene and/or (ii) the ssON is not self-complementary. Preferably, (i) the ssON does not comprise at least one GGGG sequence and/or does not comprise at least one GGG sequence {e.g. TTAGGG); and/or (ii) the ssON comprises at least three different nucleotides selected from the group consisting of A, C, G and T, or at least four different nucleotides selected from the group consisting of A, C, G and T.

In a particularly preferred embodiment, the ssON ( e.g . ss ODN) is one wherein:

(b) either (i) at least 90% of the internucleotide linkages in the ssON are phosphorothioate internucleotide linkages; or (ii) the ssON comprises at least four phosphorothioate internucleotide linkages and/or at least four 2’-0- methyl modifications; and

(c) the ssON does not contain any CpG motifs; and

(d) the ssON does not target {e.g. bind specifically to) any specific viral {e.g. HIV-1 , HSV-2 or Ebola) gene or polypeptide. Preferably, (i) the ssON does not comprise at least one GGGG sequence and/or does not comprise at least one GGG sequence {e.g. TTAGGG); and/or (ii) the ssON comprises at least three different nucleotides selected from the group consisting of A, C, G and T, or at least four different nucleotides selected from the group consisting of A, C, G and T.

In a particularly preferred embodiment, the ssON {e.g. ss-ODN) is one wherein:

(c) the ssON does not contain any CpG motifs;

(d) the ssON does not target {e.g. bind specifically to) any specific viral gene or polypeptide; and

(e) (i) the ssON is non-complementary to any human mRNA and/or viral gene and/or (ii) the ssON is not self-complementary. Preferably, (i) the ssON does not comprise at least one GGGG sequence and/or does not comprise at least one GGG sequence {e.g. TTAGGG); and/or (ii) the ssON comprises at least three different nucleotides selected from the group consisting of A, C, G and T, or at least four different nucleotides selected from the group consisting of A, C, G and T.

As described in the Examples, the inventors have described the invention in the context of 27 ssONs. Thus, in an especially preferred embodiment, the ssON comprises or consists of a polynucleotide having a sequence of any one of SEQ ID NOs: 1 -27 listed in Table 1 , or the complementary sequence thereof. Particularly preferred ssONs amongst these sequences are SEQ ID NOs: 1 -4, 7, 8, 13-27, such as SEQ ID NO: 1.

In a further embodiment, the ssON comprises or consists of a polynucleotide having a sequence sharing at least 76% sequence identity with a sequence of any one of SEQ ID NOs: 1 -27 listed in Table 1 , or a complementary sequence thereof, such as at least 76%, 77%, 78%, 79%, 80% sequence identity, and more preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% sequence identity, and even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence of any one of SEQ ID NOs: 1 -27 listed in Table 1 , or a complementary sequence thereof. Again, particularly preferred ssONs amongst these sequences are SEQ ID NOs: 1-4, 7, 8, 13-27, such as SEQ ID NO: 1.

By the term "sequence identity", we include the meaning of a quantitative measure of the degree of homology between two nucleic acid sequences or between two nucleic acid sequences of equal length. If the two sequences to be compared are not of equal length, they must be aligned to give the best possible fit, allowing the insertion of gaps or, alternatively, truncation at the ends of the nucleotide sequences. The sequence identity can be calculated, wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences, preferably sequence identity is calculated over the full-length reference as provided herein. A gap is typically counted as non-identity of the specific residue(s).

With respect to all embodiments of the invention relating to nucleotide sequences, the percentage of sequence identity between one or more sequences may also be based on alignments using the NCBI BLAST software https://blast.ncbi. nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_S PEC=blast2seq&LINK_LOC=align2seq) or the clustalW software (http:/www.ebi. ac.uk/clustalW/index.html), for example with default settings. Default settings for nucleotide sequence alignments in clustalW are: Alignment=3Dfull, Gap Open 10.00, Gap Ext. 0.20, Gap separation Dist. 4, DNA weight matrix: identity (IUB).

In a further embodiment, the ssON comprises or consists of a polynucleotide having a sequence of any one of SEQ ID NOs: 1 -27 listed in Table 1 , or a complementary sequence thereof, wherein up to ten nucleotides are replaced by another nucleotide ( e.g . up to 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides are replaced by another nucleotide). For example, the ssON may comprise or consist of a polynucleotide having a sequence of any one of SEQ ID NOs: 1 -27 listed in Table 1 , or a complementary sequence thereof, wherein between 2-10, such as 3-9 or 4-8 nucleotides are replaced by another nucleotide. Again, particularly preferred ssONs amongst these sequences are SEQ ID NOs: 1-4, 7, 8, 13-27.

For the avoidance of doubt, when ssONs are defined by reference to any of the sequences in Table 1 below, it will be appreciated that the ssONs do not need to contain each and every modification depicted in Table 1 in respect of any of those sequences. Preferably, when the ssONs are defined by reference to any of the sequences in Table 1 below, they do contain each and every modification depicted in Table 1 in respect of whichever sequence, however, it is not essential. The ssON may simply be defined by reference to the nucleotide sequence of a particular sequence depicted in Table 1 , without any chemical modifications or with different chemical modifications to those depicted in Table 1.

The first aspect of the invention includes the use of a single-stranded oligonucleotide (ssON) with broad-spectrum antiviral activity in the manufacture of a medicament for the treatment and/or prophylaxis of an infection caused by a virus selected from the group consisting of Fluman immunodeficiency virus type 1 (HIV-1 ), Ebola virus, rabies virus, Lassa virus, Lyssa virus, HSV-2 virus and measles virus as well as VSV and MLV in a subject.

The first aspect of the invention also includes a method for the treatment and/or prophylaxis of an infection caused by a virus selected from the group consisting of Human immunodeficiency virus type 1 (HIV-1), Ebola virus, rabies virus, Lassa virus, Lyssa virus, HSV-2 virus and measles virus as well as VSV and MLV in a subject, comprising administering an ssON to the subject.

In both instances, preferences for the ssON include those described above. Particularly preferred ssONs are SEQ ID NOs: 1-4, 7, 8, 13-27.

For the avoidance of doubt, it will be appreciated that, in any aspect of the invention described herein, the ssON does not include any of the ssONs mentioned in Table 21 of W02006/042418 (see pages 127-129c, for example REP2006, REP2018, REP2029, REP2028, REP2033, REP2055, REP2056 and REP2057) incorporated herein by reference, and does not include any of the ssONs mentioned in Table 1 and/or Table 7 of US2011/0124715 (see pages 6, 7, 15 and 16) incorporated herein by reference.

The invention provides ssONs as disclosed above for use in the treatment and/or prophylaxis of medical conditions in mammals, in particular humans, wherein the route of administration is selected from parenteral, intramuscular, subcutaneous, epidermal, intradermal intraperitoneal, intravenous, mucosal delivery, oral, sublingual, dermal, transdermal, topical, buccal, inhalation, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop or mouthwash.

The ssON is soluble in water and has effects after topical delivery to mucosal surfaces.

The ssON can be systemically administered in an amount from 1 pg/kg to 10 mg/kg body weight; preferably from about 1 pg/kg to about 1 mg/kg; more preferably from about 1 pg/kg to about 100 pg/kg.

In a further aspect, the invention provides a pharmaceutical composition comprising an ssON as defined above, together with a pharmaceutically acceptable carrier for use in the treatment and/or prophylaxis of an infection caused by a virus selected from the group consisting of Human immunodeficiency virus type 1 (HIV-1), Ebola virus, rabies virus, Lassa virus, Lyssa virus, HSV-2 virus and measles virus as well as VSV and MLV. In a preferred embodiment, said infection is caused by HIV-1. As used herein a “pharmaceutical composition” is a pharmaceutically active compound that has been admixed with conventional pharmaceutical carriers and excipients (/. e., vehicles) and used in the form of aqueous solutions, tablets, capsules, gels and the like. Optionally, the pharmaceutical composition may contain other pharmaceutically acceptable components, such as buffers, surfactants, antioxidants, viscosity modifying agents, preservatives and the like.

The ssON for use according to the invention may be administered as a monotherapy, or together with additional therapeutic agent(s). In one embodiment said ssON and said additional therapeutic agent(s) are formulated as a pharmaceutical composition further comprising pharmaceutically acceptable carrier(s). Said additional therapeutic agent(s) may be a dipeptide.

Suitably, the subject in need of treatment and/or prophylaxis is a human subject.

Brief description of the figures

Figure 1. ssON inhibiting HIV-1 binding, fusion and infection of cells.

Figure 2. Drug sensibility assays of the combinations of ssON with WG-am or VQ-am.

Figure 3A and B. ssON inhibiting HIV-1 replication independent of the subtype and co-receptor usage.

Figure 4. ssON exhibiting antiviral activity against various pseudotyped particles.

Figure 5. ssON exhibiting antiviral activity against HSV-2 and measles virus but not against Zika, encephalomyocarditis virus (EMCV) and SARS- CoV-2.

Figure 6. ssON exhibiting antiviral activity against HSV-2 in vivo. Examples

Summary

The inventors have surprisingly discovered that non-CpG ssONs, as exemplified herein with SEQ ID NO: 1 , are useful for the inhibition of HIV-1 , including multi-resistant HIV-1 , HSV-2, Ebola virus, rabies virus, measles virus, vesicular stomatitis virus (VLV) and murine leukemia virus (MLV), with higher potency as compared with those disclosed by e.g. Replicor. Surprisingly, the antiviral effect is in the low nM concentrations against multiple viruses, which can be compared with the ssONs described to act directly on viruses by Replicor in US 9,616083 B2 and US 8,008270 B2, which are in the low mM concentration. The inventors also unexpectedly found that the ssONs described herein were more potent against the viruses described and exemplified herein, including HIV-1 , HSV-2, Ebola etc. compared to against other viruses such as, influenza virus.

As shown in Example 1 below, ssONs were found to inhibit HIV-1 infection of cells by preventing binding of the virus to the cell surface and subsequent fusion to the cell membrane. Cells were pre-treated with ssON and then exposed to either R5- and X4 tropic HIV-1 strains. There was a marked and highly significant reduction in viral infection compared with untreated cells or cells treated with ssONs composed of 15 nucleotides. Efficacy of the ssON used was 11-15nM. Further, as shown in Example 2, there was significant reduction of viral infection using ssON also in combination with another treatment modality, as exemplified using dipeptides. As shown in Example 3, ssON displayed a surprising inhibition of HIV-

1 infection regardless of HIV-1 subtype and despite the notion of multiresistant strains. This is surprising considering ssONs capacity to inhibit binding and fusion to the cell as the highly variable and diverse HIV-1 Env protein is binding to cells using CD4 as main host receptor or CCR5 or CXCR4 as co-receptors. Examples 4 and 5 further shows ssON capacity to act as a broad-spectrum antiviral. However, depending on the virus family, the IC50 measured in vitro varies and some viruses such as for example Zika virus was not inhibited at all. General Materials and Methods

Synthesis of oligonucleotides:Svnthetic. endotoxin-free, oligonucleotides were synthesized according to methods known in the art, as disclosed in e.g. Artificial DNA: Methods and Applications

(Khudyakov, Y.E. & Howard A. Fields, H.A., Eds.) CRC Press, 2002 (ISBN 9780849314261).

Compounds: Amide forms of valylglutamine (VQ) and tryptophylglycine (WG) were synthesized and purchased from Pepscan (Pepscan Presto, Lelystad, the Netherlands) with a purity of >95%. A 35 bases long fully phosphorothioate (PS)-modified oligonucleotide, designated ssON, with sequence: 5’-G AAGTTTT G AGGTTTT GAAGTT GTTGGTGGTGGTG-3’ (SEQ ID NO:1)and control oligonucleotide ctrON (5'GGTTTTGAAGTTGTT3’) (SEQ ID NO:6) was purchased from Integrated DNA Technologies (Coralville, Iowa, USA).

Clinical specimens: Stored plasma samples (n= 30) from patients who were given ART were randomly selected from the HIV-1 cohort at Karolinska University Hospital, Stockholm, Sweden. Subtyping using the pol-region revealed: HIV-1 A, n=11 ; HIV-1 B, n=4; HIV-1 C, n=5; HIV-1 D, n=3; HIV-1 F, n=3; HIV-1 G, n=4. Of these, A1 , 01_AE and 02_AG were grouped as A-like viruses (n= 5), while A6 (n= 6) was categorized as an independent group HIV-1 A6 sub subtype. The co-receptor usage of the chimeric viruses was predicted using Geno2Pheno (co-receptor) 2.5 (Vicenti I, etal. J Clin Virol. 2019;111 :12-8). Ten out of 30 samples had at least one drug resistance mutation (DRM) (L74I, L74M, and/or Q148R) causing resistance to integrase strand inhibitors (INSTI),

2 of 30 samples had DRM (V82A and I50V) to protease inhibitors (PI) and 14 of 30 samples had DRM to reverse transcriptase inhibitor (RTI). The DRM were identified using the MiDRMpol pipeline (Aralaguppe SG, et al. Viruses. 2019;11 (9)). Cells: All cell lines were cultured in Dulbecco's modified Eagle's medium

(DMEM, Gibco) containing 100 units/ml penicillin, 100pg/ml streptomycin, 2 mM L-glutamine. Vero E6 ( Cercopithecus aethiops derived epithelial kidney) medium was supplemented with 2.5% heat-inactivated fetal calf serum (FCS), 1 mM sodium pyruvate, and 1x non-essential amino acids. Caco-2 (human epithelial colorectal adenocarcinoma), TZM.bl cell line (NIH AIDS Research and Reference Reagent Program, USA) and HEK-293T cells (ATCC, USA) cells were supplemented with 10% FCS.

Peripheral blood mononuclear cells (PBMCs) were isolated on a Ficoll-Paque Plus density gradient (Merck S.L.). Prior to treatment with the antiviral compounds, PBMCs were stimulated with the mitogen phytohemagglutinin (PFIA) for 48 hours (2 pg/mL; Thermo Fisher Scientific, Waltham, MA, USA).

HIV-1 infection of TZM-bl cells (Fig 1 ): Virus stocks of the R5-tropic and X4-tropic HIV-1 NL4-3 92TH014 derivative were generated by transient transfection of 293T cells as described (J. Munch, et al. Cell 2007, DOI 10.1016/j.cell.2007.10.014). Virus stocks were harvested 48 h post transfection and frozen at -80°C. The infection rate of the HIV-1 stocks were quantified by titrating viral stock on TZM-bl cells. For infection, 10,000 TZM-bl cells, a HeLa cell line derivative that expresses high amounts of human CD4, CCR5 and CXCR4 and contain a B-galactosidase gene under the control of the HIV-1 long terminal repeat (LTR) promotor were seeded in 96 well plates. Next day, oligonucleotides were titrated and added to cells for 2h, followed by infection of cells. Three days post infection, viral infectivity was determined using a galactosidase screen kit from Tropix as recommended by the manufacturer b- Galactosidase activities were quantified as relative light units (RLU) per second with an Orion Microplate luminometer (Berthold). Values in the absence of compounds was set to 100% infection. p24 ELISA to detect HIV-1 binding to cells: To determine binding of R5- tropic HIV-1 to TZM-bl cells, 150,000 TZM-bl cells were seeded into 24 well plates. One day later, oligonucleotides, CRO or AS1411 were titrated and added to cells for 2h, followed by adding virus for 1 h at 4°C. Next, unbound virus was removed by washing with PBS, detached and lysed with 1% Triton X-100 for 1 h at 37°C. Cell associated HIV-1 capsid antigen was detected using an in-house p24 ELISA (J. Munch, et al. Cell 2007, DOI 10.1016/j.cell.2007.10.014). Values in the absence of compounds was set to 100 % binding. HIV-1 fusion assay: To determine fusion of HIV-1 to cells, a BlaM-Vpr fusion assay was performed as described before (M. Cavrois, et al. Nat. Biotechnol. 2002, 20, 1151 ). Briefly, to produce virions that incorporate a b- lactamase Vpr (BlaM-Vpr) protein chimera, HEK293T cells were cotransfected with pNL4-3 92TH014 proviral DNA, pCMV-BlaM-Vpr, and pAdVAntage vectors as previously described. For the fusion assay, 80,000 TZM-bl were seeded in 96 well plates. Next day, oligonucleotides were titrated and added to cells for 2h, followed by adding infection normalized virions containing BlaM- Vpr at 37°C for 2.5 h. Cells were washed and loaded with CCF2 dye in C02- independent medium and incubated overnight at room temperature. Next day cells were fixed with 4% PFA and the change in emission fluorescence of CCF2 after cleavage by the BlaM-Vpr chimera was monitored with a BD LSRII. Values in the absence of compounds was set to 100% fusion.

HIV-1 infection of moDC and T cells: Isolation, differentiation and HIV-1 infection of immature monocyte derived dendritic cells moDC) were performed as described in (Mohanram V, et al. J Immunol. 2013;190(7):3346- 53). Briefly, human CD14+ monocytes were isolated from buffy coats using the RosetteSep Monocyte Enrichment Kit (StemCell) followed by Ficoll centrifugation (Lymphoprep; Stemcell). Monocytes were differentiated into moDC by culturing the cells in complete RPMI medium (RPMI 1 ,640, 1 mM sodium pyruvate, 10mM HEPES, 2mM L-glutamine, 1% Penicillin / Streptomycin, and 10% FBS) supplemented with GM-CSF (250 ng/ml_; PeproTech) and rlL-4 (6.5 ng/ml_; R&D Systems) for 6 days prior to HIV-1 infection.

CD4+ T cells were isolated from buffy coats using Rosette Sep CD4+ T cell Enrichment Kit (StemCell) followed by by Ficoll centrifugation (Lymphoprep; Stemcell). CD4+ T cells were activated with anti-human CD3 (10pg/ml; clone OKT 3; Ortho Biotech Inc., Raritan, NJ) and soluble anti human CD28 (2ug/ml; L293; BD Biosciences, San Diego, CA) for 48 hours in in complete RPMI medium prior to HIV-1 infection in the presence of rlL-2 (Mohanram V, et al. PLoS One. 2011 ;6(6):e21171 ). ssON (0.5mM) (SEQ ID NO:1 ) was added to MoDCs and T cells just prior to infection with HIV-1 BaL. For infection, 3000-6000 50% tissue culture infectious dose/million cells of HIV-1 BaL were used (Smed-Sorensen A, et al. Blood. 2004;104(9):2810-7). The percentage of infected moDCs and T cells were determined by intracellular p24 staining after five days of infection, as previously described (Mohanram V, et al. PLoS One. 2011 ;6(6):e21171 and Mohanram V, et al. J Immunol. 2013;190(7):3346-53). Cells were first stained for cell surface markers (CD1 a, CD3, CD4) and dead/live marker (LIVE/DEAD Fixable aqua, Thermofisher) then fixed and permeabilized (Fix/Perm BD) prior to incubation for 1-2 h at 4°C with a p24 mAb (clone KC57; Coulter, Hialeah, FL) or the corresponding isotype control Ab. Intracellular p24 expression was assessed by a Fortessa flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star). Gates were set on CD1a+ (moDC) or CD3+ T cells. The data is based on five donors and the significant differences were assessed by non-parametric Mann-Whitney test.

Recombinant virus production (Figs 2 and 3): Viral stocks of CCR5-tropic R5-HIV-1 NLAD8 and CXCR4-tropic X4-HIV-1 NL4.3 laboratory strains were obtained by transient transfection of pNL(AD8) and pNL(4.3) plasmids, respectively, (NIH AIDS Research and Reference Reagent Program) in HEK- 293T cells (ATCC, Manassas. VA, USA). Supernatants were collected at 48h and 72h. Viral stocks were clarified by centrifugation prior to evaluating the viral titer by HIV-1 p24gag ELISA kit (INNOTEST® Innogenetics, Belgium).

Briefly, viral RNA was extracted using the QIAmp viral RNA extraction kit (Qiagen, Hilden, Germany) from 140pL of plasma. The env region encoding for the gp120 fragments (HXB2: 6443-8439) were cloned into pMN plasmid following pMN plasmid nlgoMIV and Mlu1 (New England Biolabs, USA), following ligation using T4 DNA ligase (New England Biolabs), as previously described (Njenda DT, A et al. J Antimicrob Chemother. 2018;73(10):2721 -8). The chimeric viruses were produced by transient transfection of the plasmids into the HEK-293T cell line using FuGENE HD Transfection Reagent (Promega, USA) and harvested 48h and 72h later by collection of the cell-free supernatant via centrifugation; aliquots were stored at -80°C.

Drug susceptibility assay (PSA): DSA was performed to determine the antiviral activity of ssON, WG-am, VQ-am alone and also their combinations against the reference viruses (R5-HIV-1ADS or X4-HIV-1 NL4.3) and the chimeric viruses derived from 30 ART treated patients (HIV-1 A, n=11 of which six were HIV-1 A6; HIV-1 B, n=4; HIV-1 C, n=5; HIV-1 D, n=3; HIV-1 F, n=3; HIV-1 G, n=4;). Drugs were serially diluted in culture medium, ranging from 1mM to 1mM for dipeptides and from 1mM to 1nm for ssON, and then, added in triplicate in 96- well plates that had been seeded 24h before the start with 10⁴ TZM-bl cells/well. The viruses were added to each well at 100 TCID50/well. In addition, DSA was also perform in PHA activated PBMCs for 48h, (2x10⁵ cells/well in 200mI_). PBMCs pre-activated with PHA for were seeded in round bottom 96 well plates and treated with the compound combinations and challenged with an HIV-1 infection. Three days later, supernatants were collected and 100mI_ were added to TZM.bl cells, which had been seeded in 96-well plates at a density of 10⁴ cells/100 mI_ per well the day before. HIV-1 replication was determined after quantification of luciferase activity (relative light units) using the Bright-Glo Luciferase Assay System (Promega, USA) 48h post-infection. Drug concentrations required for inhibiting virus replication by 50% (EC50) were calculated from a dose-response curve using non-linear regression analysis (GraphPad Prism, version 8.0.1 ; GraphPad Software, La Jolla, CA, USA). The synergistic profile of the compounds were determined by using CalcuSyn software (Biosoft, Cambridge, UK), based on the median effect principle (Chou TC, Talalay P. Adv Enzyme Regul. 1984;22:27-55). Chou- Talalay’s combination indices (Cls) were calculated using methods derived from the mass-action principle, which follows the following equation where fa is the fractional inhibition caused by the drug relative to the no drug control; 1 and 2 represent the individual action of the drugs; and C represents the combined action of the drug combination. Cl values < 0.9, 0.9 < Cl < 1.1 and Cl > 1.1 indicate synergy, additivity and antagonism, respectively.

The DSA experiments were performed with three technical replicates for each virus with the specified dynamic concentration range of the drug, and at least two independent analyses (biological replicates) were performed. The reproducibility of the DSA was assessed based on the 95% Cl obtained for the drug EC50 and the degree of correlation between technical replicates. The output for the drug EC50 results was used to compute the fold change value for each virus relative to NL4.3 before being exported to GraphPad Prism.

Statistical Analysis: Statistics were presented as means +/- standard deviation (SD) unless otherwise noted. Parametric and/or non-parametric statistical tests were performed as appropriate and are listed in the respective figure legends and tables. Statistical significance was accepted when P<0.05. Statistical analysis was performed using GraphPad Prism 6 (GraphPad, Inc) and CalcuSyn (Biosoft).

HSV-2 infection of Vero cells: HSV-2 (333) e(GFP) was made and kindly provided by Patricia G. Spear, Department of Microbiology-Immunology, Northwestern University, Chicago, USA (J. M. Taylor, et al. Cell Host Microbe 2007, 2, 19). Stocks were produced by inoculation of Vero E6 cells with a multiplicity of infection (MOI) of 0.01 . 2 days later, virus was harvested. To this end, supernatant was collected, centrifuged with 1500 rpm for 5 min to remove cell debris and stored at -80°C until further usage. For infection, 6,000 Vero E6 cells were seeded in 96 well plates. Next day, a 5-fold dilution series of oligonucleotides or acyclovir was produced and added to cells for 2h, followed by infection of cells with a MOI of 0.2. Highest concentration of oligonucleotides and acyclovir on cells was 10 mM and 200mM, respectively. Two days later infection induced cell death was assessed using an MTT assay. Values in the absence of compounds was set to 100 % induced cell death.

Measles virus infection of TZM-bl cells: Measles virus (MeV, Schwarz strain) was kindly provided by Karl-Klaus Conzelmann (Ludwig-Maximilians- University of Munich) and propagated on Vero E6 cells as previously described (L. Koepke, etal. Sci. Rep. 2020, 10, 12241 and E. Braun etal. Cell Rep. 2019, 27, 2092).

For infection, 20,000 TZM-bl cells were seeded in 96 well plates. Next day, a 5-fold dilution series of oligonucleotides or heparin was produced and added to cells for 2h, followed by infection of cells with a MOI of 0.1 . 72 h later, cells were detached, fixed with 4% PFA for 30 min and analyzed by flow cytometry for GFP⁺ cells. Values in the absence of compounds was set to 100% infection. ZIKA virus infection of Vero cells: For propagation, 70% confluent Vero E6 cells in 175 cm² cell culture flasks were inoculated with Zika virus isolate MR766 (G. W. A. Dick, et al. Trans. R. Soc. Trop. Med. Hyg. 1952, 46, 509) in 5 ml medium for 2 h, before 40 ml medium was added. After 3 to 5 days when cytopathic effect was visible, supernatant was harvested. For infection, 6,000 Vero E6 cells were seeded in 96 well plates. The next day, a 5-fold dilution series of oligonucleotides or PSVBS was added to cells for 2 h, followed by infection of cells with Zika virus at a MOI of 0.2. Two days after infection, Zika virus E protein expression (4G2; Absolute Antibody #Ab00230-2.0) was quantified via cell-based immunodetection assay as described. Values in the absence of compounds was set to 100% infection.

EMCV infection of FIEK293T cells: Encephalomvocarditis virus (EMCV, EMC strain) was purchased from ATCC (#VR-129B) and propagated as previously described (L. Koepke, et al. Sci. Rep. 2020, 10, 12241 and K. M. J. Sparrer, et al. Nat. Microbiol. 2017, 2, 1543). For infection, 30,000 FIEK293T cells were seeded in 96 well plates. Next day, oligonucleotides or quinacrine were titrated and added to cells for 2h, followed by infection of cells with a MOI of 0.1. Highest concentration of oligonucleotides and quinacrine on cells was 10 mM and 40 pg/ml, respectively. As soon a cytopathic effect was visible (24- 26h after infection), an MTT assay was performed to indicate infection induced cell death. Values for untreated controls were set to 100 % induced cell death.

Infection assays with pseudotvoed viral particles: Pseudotyped particle production for Influenza, Ebola, VSV, MLV, Rabies, Lyssa, Lassa and SARS- CoV-2 were as described in (Schandock F, et al. Adv Health Mater. 2017 Dec;6(23):1700748 and Weil T, et al. J Am Chem Soc. 2020 Oct 7;142(40):17024-17038). For infection, 10,000 HEK293T cells were seeded in 96 well plates. Next day, oligonucleotides were titrated and added to cells for 2h, followed by infection of cells with infection normalized pseudotyped particles. Highest concentration of oligonucleotides was 10 pM. 48 hours post transduction, transduction rates were assessed by measuring luciferase activity in cell lysates with a commercially available kit (Promega). Values in the absence of compounds was set to 100% transduction. Cell viability assay: Cytotoxicity of the compounds was assessed using the MTT assay. Supernatant was removed and replaced by 100 mI MTT solution (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) (0.5 mg/ml). After incubation at 37°C and 5% CO2 for 2.5 h, the supernatant was removed and 100 mI 1 :1 DMSO-Ethanol solution was added. The absorption was measured at 590 nm with baseline-corrected at 650 nm using VersaMax Microplate Reader.

HSV-2 challenge in mice: Female BALB/c mice (8 weeks old) were obtained from commercial supplier (Charles River, Sulzfeld, Germany). The mice were randomly assigned into groups of 6 animals and kept under standard conditions in isolated ventilated cages. Experimental animals were acclimated for a minimum of 6 days prior to infection. All animal experiments were performed in accordance with the EU Directive 2010/63/EU for animal experiments and were approved by local authorities. BALB/c mice were anaesthetized with isoflurane for intranasal inoculation using 10⁴ PFU FISV-2 (MS strain, 25 mI total volume) on day 0. Mice were simultaneously treated with either 3 pg, 6 pg, 10 pg of ssON or vehicle (PBS) in further 25 mI total volume via intranasal application. Treatment with ssON (SEQ ID NO:1 ) or PBS was repeated with a total volume of 50 mI and indicated dosage on day 2 after infection. The animals were scored daily and the disease incidence were measured over 5 days after virus inoculation. Untreated infection resulted in a mortality rate of 90 - 100% due to disseminated disease after 5-7 days. The disease severity was determined with the following scoring system, which distinguished in (i) body weight loss, (ii) appearance of animals fur, (iii) appearance of animals eyes and (iv) behavior and other disease symptoms. The score value was a result of a cumulative calculation of all following gradually observations (i) 0 (no weight loss), 1 (< 5% weight loss), 5 (> 5% weight loss), 10 (> 10% weight loss) or 20 (> 20% weight loss) points for weight loss according to initial weight; (ii) 0 (smooth, shiny and clean), 1 (piloerection), 5 (ragged) or 10 (shaggy and dirty) points for fur; (iii) 0 (open and clean), 5 (one secretion clotted eye) or 10 (two secretion clotted eyes) points for eye(s); (iv) 10 points for hump formation, lethargy or coordination disorders; 20 points for respiratory difficulties, paralysis, morbidity or death. Reaching the cumulative score value of 20 over a period of 24 hours, the animal reached humane endpoints and was euthanized. Animals with a single score value of 20 were immediately euthanized.

Viral DNA was isolated from 200 mI of lung homogenates (gentleMACS Octo Dissociator, Miltenyi Biotec) using QIAamp MinElute Virus Spin Kit (Qiagen). Quantitative detection of human HSV-2 DNA was performed on a LightCycler instrument (Roche) by using 5 mI of isolated viral DNA by means of a modified real-time PCR protocol described previously (Burrows etal.

2002 BMC Microbiology 2;12; Stang etal. 2005 J Virol (19):12487-94). Quantification was performed through standard curve method with dilutions from 3 x 10⁶ to 30 copies per reaction. Statistical evaluation of the data was performed by Kruskal-Wallis test (one-way ANOVA) and Dunn’s Pairwise Multiple Comparison Procedures as post hoc test in comparison to vehicle control.

Example 1

Inhibition of HIV-1 by pre-treatment with ssON ssON (SEQ ID NO: 1 inhibit HIV-1 binding, fusion and infection of cells. TZM-bl were treated with ssON or a Ctrl ssON (SEQ ID NO: 6) for 2h followed by infection with R5- (A) or X4 tropic (B) HIV-1 . Shown are average values (± SD) of triplicate measurements from three independent experiments. C)

Either cells or virus was preincubated with ssON for 30 min. Shown are average values (± SD) of triplicate measurements from two independent experiments. D) Either cells were preincubated with ssON for 5, 50 or 250 min and ssON removed followed by adding virus (red lines) or cells were preincubated with virus for 5, 50 or 150 min and virus removed followed by adding ssON (blue lines). Shown are average values (± SD) of triplicate measurements from two independent experiments. E) To determine binding of R5-tropic HIV-1 to TZM-bl cells, cells were incubated with ssON for 2h followed by adding virus for 1 h. After washing of cells, a p24 ELISA was performed with the cell lysates. Shown are average values (± SD) from two independent experiments. F) A blam-vpr fusion assay was performed to determine fusion of HIV-1 to cells. Shown are average values (± SD) from three independent experiments. G) Human monocyte derived dendritic cells (DC) or activated T cells were exposed to HIV-1 _BaL in the presence or absence of ssON and the frequency of p24⁺ cells were analyzed using flow cytometry. Shown are average values (± SD) of 5 different donors.

The results (Fig. 1 ) show a marked and highly significant reduction in viral infection of both CCR5 and CXCR4 co-receptor using HIV-1 in a dose- dependent manner. The IC50 was 15 nM for CCR5 and 11 ,4 nM for the CXCR4 HIV-1 . However, the Ctrl-ssON composed of 15 nucleotides did not show any significant inhibition of HIV-1 . The data further shows that ssON inhibits HIV-1 binding to the cell surface and the subsequent fusion event (F). The example also shows that ssON is able to inhibit HIV-1 infection in two different types of primary human target cells; dendritic cells and activated T cells (G). Example 2

Drug sensibility assays of the combinations of ssON with WG-am or VQ-am

TZM.bl (A and B) and peripheral blood mononuclear cells (PBMCs) (C and D) were treated with ssON (SEQ ID NO:1 ) (1 nM to 1 mM) and WG-am or VQ-am (1 mM to 1 mM) and then infected with HIV-1 , JRFL (A and C) or NL4.3 (B and D) strains. Non-infected cells were used as infectivity control and defined as 100% of infection. The Log (Concentration) refers to nM concentration for ssON and mM concentration for dipeptides. As shown in Figure 2, there was a dose-dependent inhibition of HIV-1 in TZM.bl and

PBMCs using ssON. The data further shows useful combination with another treatment composed of dipeptides.

Example 3 ssON inhibit HIV-1 replication independent of the subtype and co-receptor usage

TZM.bl cells were treated with WG-am alone (1 mM to 1 mM), ssON (SEQ ID NO:1 ) alone (1 nM to 1 mM) and with the combination (A-G) and then infected with ART treated patient derived chimeric viruses. N represents the number of viral isolates corresponding to each subtype. Non-infected cells were used as infectivity control. The Log (Concentration) makes reference to nM concentration for ssON and mM concentration for dipeptides. The inhibitory potential of ssON and WG-am alone as well as the combination ssON/WG-am towards a panel of patient derived HIV-1 comprising a wide variety of subtypes and drug resistance patterns, defined on the analysis of the pol sequence. The evaluation of the agents against chimeric viruses (n=30) derived from ART- treated patients showed that 1mM WG-am alone and 1mM ssON alone significantly inhibited the replication of all viruses, independent on the subtype, but they did not completely abrogate the replication (Figs. 3A and 3G). In contrast WG-am:ssON (1 mM: 1 mM) inhibited all isolates >95% (Fig. 3J) due to a high synergism. Geno2pheno software reported that 16 of 30 patient-derived HIV-1 strains were CCR5 tropic, 12 of 30 were CXCR4 tropic and the remaining two strains were not possible to predict with certainty.

Example 4 ssON exhibit antiviral activity against various pseudotyped particles

HEK293T cells were treated with ssON (SEQ ID NO:1 ) for 2h followed by infection with pseudotyped particles harboring influenza (A), ebola (B), vesicular stomatitis virus (VSV) (C), murine leukemia virus (MLV) (D), rabies (E) lassa (F), lyssa (G) or SARS-CoV-2 (H) glycoproteins. Shown are average values (± SD) of triplicate measurements from two (F,G) to three (A-E, H) independent experiments. The results (Fig. 4) show reduction in viral infection of different viruses and the concentration of ssON resulting in a 50% reduction in infectivity (IC50) is shown for each virus. The data show differential effectiveness in vitro depending on the viral family.

Example 5 ssON exhibits antiviral activity against HSV-2 and measles virus but not against Zika, encephalomvocarditis virus (EMCV) and SARS-CoV-2

Vero E6 (A,C), TZM-bl (B), HEK293T (D) or Caco-2 cells were treated with ssON (SEQ ID NO:1 ) for 2h followed by infection with the individual virus. Shown are average values (± SD) of triplicate measurements from two (C, E) or three (A, B, D) independent experiments. The results (Fig. 5) show reduction in viral infection of HSV-2 (IC50 74,2nM) and measles virus (IC50 108,3 nM). However, no activity was detected against zika virus, EMCV or SARS-CoV-2.

Example 6 ssON reduces viral load of HSV-2 in vivo in mice

Mice were inoculated with HSV-2 (1x10⁴ PFU) intranasal with or without ssON (3pg, 6pg or 10pg/ mouse) and second treatment with ssON (SEQ ID NO:1) was provided day 2. Mice were weighed every day (A) and the clinical score was measured daily (B). Mice were sacrificed day 5 after virus inoculation and the viral load in the lungs were measured by PCR. There was a significant reduction in HSV-2 in the group treated with the highest dose ssON (1 Opg) (lower panels).

Sequence listing

Table I. Primary structure of oligonucleotides. All sequences are written 5’ to 3’. All oligonucleotides were fully phosphorothioated (except SEQ ID NOs: 2, 22, 23) and consist of deoxynucleotides (except SEQ ID NO: 8).

5

Claims

1. A single-stranded oligonucleotide (ssON) with broad-spectrum antiviral activity for use in the treatment and/or prophylaxis of an infection caused by a virus selected from the group consisting of Human immunodeficiency virus type 1 (HIV-1), Ebola virus, rabies virus, Lassa virus, Lyssa virus, Herpes simplex virus (HSV), measles virus, vesicular stomatitis virus (VSV), and murine leukemia virus (MLV) in a subject in need thereof, wherein: (a) the length of said ssON is between 25-40 nucleotides;

(b) either (i) at least 90% of the internucleotide linkages in said ssON are phosphorothioate internucleotide linkages; or (ii) said ssON comprises at least four phosphorothioate internucleotide linkages and/or at least four 2’-0-methyl modifications; and (c) said ssON does not contain any CpG motifs.

2. The ssON for use according to claim 1 , wherein said ssON comprises at least six phosphorothioate internucleotide linkages and/or at least six 2’- O-methyl modifications.

3. The ssON for use according to claim 1 or 2, wherein all internucleotide linkages in said ssON are phosphorothioate internucleotide linkages.

4. The ssON for use according to any one of claims 1 to 3, wherein the length of said ssON is at least from 25 and at least to 35 nucleotides.

5. The ssON for use according to any one of claims 1 to 3, wherein the length of said ssON is at least from 35 and at least to 40 nucleotides.

6. The ssON for use according to any one of claims 1 to 5, wherein not more than 15 consecutive nucleotides in said ssON are complementary with any human mRNA sequence.

7. The ssON for use according to any one of claims 1 to 6, wherein said ssON is not self-complementary.

8. The ssON for use according to any one of claims 1 to 7, wherein the monosaccharides in said ssON are chosen from the group consisting of 2’-deoxyribose and 2’-0-methylribose.

9. The ssON for use according to claim 8, wherein said ssON comprises or consists of a polynucleotide having a sequence sharing at least 76% sequence identity with a sequence of any one of SEQ ID NOs: 1 -4, 7-8, 13-27, or a complementary sequence thereof.

10. The ssON for use according to claim 8, wherein said ssON comprises the sequence shown as SEQ ID NO: 1 , or a complementary sequence thereof, wherein up to ten nucleotides are replaced by another nucleotide.

11. The ssON for use according to any one of claims 1 -10, wherein said ssON prevents binding of the virus to the cell surface and/or subsequent fusion of the virus to the cell membrane.

12. The ssON for use according to any one of claims 1-11 , wherein the concentration of said ssON required to cause a 50% reduction in infectivity (ICso) of the virus is at most 1 x 10^~5M, such as at most 7.5 x

10⁶M, such as at most 5 x 10⁶M, such as at most 2.5 x 10⁶M, such as at most 2 x 10⁶M, such as at most 1.5 x 10⁶M, such as at most 1 x 10 6M, such as at most 0.5 x 10⁶M.

13. The ssON for use according to any one of claims 1 to 12, wherein said infection is caused by a virus selected from the group consisting of HIV- 1 , HSV, Ebola virus and rabies virus.

14. The ssON for use according to any one of claims 1 to 13, wherein said infection is caused by HIV-1 , such as a multi-resistant HIV-1.

15. The ssON for use according to any one of claims 1 to 13, wherein said infection is caused by HSV, such as HSV-2.

16. The ssON for use according to any one of claims 1 to 13, wherein said infection is caused by Ebola virus.

17. The ssON for use according to any one of claims 1 to 16, wherein said ssON is administered as a monotherapy.

18. The ssON for use according to any one of claims 1 to 16, wherein the subject is administered an additional therapeutic agent.

19. The ssON for use according to claim 18, wherein said ssON and said additional therapeutic agent are formulated as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

20. The ssON for use according to any one of claims 1 to 19, wherein the subject is a human.