Combination therapy for covid-19 vaccination

Abstract

FIELD: biotechnology.SUBSTANCE: combination comprising a selective estrogen receptor modulator (SERM) and a COVID-19 vaccine is described for the prevention or treatment of adverse effects of a COVID-19 vaccine in a subject. The use of the described combination for the prevention of COVID-19 in a subject is disclosed. Also the use of a selective estrogen receptor modulator (SERM) for the prevention or treatment of adverse effects of a COVID-19 vaccine in a subject is disclosed. The use of a pharmaceutical composition containing a selective estrogen receptor modulator (SERM) and pharmaceutically acceptable excipients in combination with a COVID-19 vaccine for the prevention of COVID-19 in a subject is described. The use of a pharmaceutical composition containing selective estrogen receptor modulators (SERMs) and pharmaceutically acceptable excipients for the prevention or treatment of adverse effects of a COVID-19 vaccine in a subject is described.EFFECT: kit of parts for the prevention or treatment of adverse effects of a COVID-19 vaccine in a subject is disclosed, comprising: a) a COVID-19 vaccine; and b) a pharmaceutical composition comprising a selective estrogen receptor modulator (SERM).36 cl, 8 dwg, 6 ex

Description

[0001] Field to which the invention relates

[0002] The present invention relates to a combination of an estrogen receptor modulator and a COVID-19 vaccination.

[0003] Technology Level

[0004] Coronaviruses (Covs) are a large family of enveloped, single-stranded RNA viruses belonging to the family Coronaviridae. The limited number of coronaviruses known to infect humans were previously considered relatively harmless human respiratory pathogens causing mild infections. However, two subtypes of coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), have emerged that cause severe and sometimes fatal respiratory tract infections in humans (Pereira, H.G., 1989, Coronaviridae, In J. S. Porterfield (ed.), Andrewes' Viruses of Vertebrates, 5 thed. pp. 42-57; Holmes KV et al., Fields Virology 1996, 1: 1075-1093). In December 2019, cases of atypical pneumonia occurred in China, and a new coronavirus was later identified as the cause. The World Health Organization (WHO) named this virus SARS-CoV-2 and the associated disease COVID-19.

[0005] The virus spread rapidly worldwide, and on March 11, 2020, the WHO declared SARS-CoV-2 infection a pandemic. Most people infected with COVID-19 experience mild to moderate respiratory illness (fever, fatigue, dry cough, and shortness of breath) and recover without requiring specific treatment. Older adults and those with underlying health conditions, such as cardiovascular disease, diabetes, chronic respiratory disease, and cancer, are more likely to develop severe illness. Furthermore, an analysis of the epidemiological and clinical characteristics and outcomes of patients infected with SARS-CoV-2 found that 15% of people with COVID-19 symptoms develop serious illness, including severe pneumonia, and 5% experience critical illness with life-threatening complications.Critical illness includes acute respiratory distress syndrome (ARDS), sepsis, septic shock, cardiac disease, thromboembolic events such as pulmonary embolism and multiple organ failure.

[0006] There is also growing evidence that those who develop critical illness from COVID-19 are reasonably expected to experience long-term sequelae, such as rare neurological and psychiatric complications. These may include stroke, delirium, anxiety, depression, brain damage or inflammation, and sleep disturbances.

[0007] With the sharp rise in cases and deaths worldwide, and its social and economic impacts, efforts have been focused on developing safe and effective vaccines to help control the spread of the disease and end the COVID-19 pandemic.

[0008] Various vaccines are currently being developed, and some of them have already been approved for the prevention of COVID-19.

[0009] All COVID-19 vaccines and vaccine candidates act by inducing an immune response against viral protein antigens.

[0010] SARS-COV-2 is characterized by four structural proteins, namely, spike (S) protein, envelope (E) protein, membrane protein (M) and nucleocapsid protein (N) (Chen Y et al., J Med Virol 2020, 92: 418-423).

[0011] Currently, the spike (S) protein is used as a target antigen in COVID-19 vaccines. This protein is highly susceptible to attack because it is located on the surface of the virus and is considered an important antigenic determinant capable of inducing a protective immune response (Ou et al., Nat Commun 2020; 11: 1620).

[0012] In addition, it is an essential molecule for viral entry into cells via the cell entry receptor Angiotensin-converting enzyme II (ACE2). The spike protein sequence (UniProt ID: P0DTC2) is 1273 amino acids long and consists of a signal peptide (amino acids 1-13) located at the N-terminus, the S1 subunit (14-685 residues), and the S2 subunit (686-1273 residues); the latter two regions are responsible for receptor binding and membrane fusion, respectively. Specifically, the S1 subunit mediates receptor binding to ACE2 through the receptor-binding domain (RBD), and the S2 subunit is responsible for membrane fusion (Letko et al., Nat Microbiol 2020, 5: 562-569).

[0013] Vaccines and vaccine candidates developed or under development for COVID-19 are mainly divided into two broad categories: traditional inactivated or live attenuated viral vaccines that introduce viral protein antigens into the host, and newer gene-based vaccines such as DNA vaccines and mRNA vaccines.

[0014] DNA and mRNA vaccines deliver genes encoding viral protein antigens for in vivo production in host cells and are considered a superior approach for COVID-19 vaccination compared to traditional vaccine approaches given their improved safety and suitability for mass production.

[0015] All three vaccines approved so far by the EMA are based on this technological approach: the mRNA-based Comirnaty developed by Pfizer and BioNTech, the mRNA-based COVID-19 vaccine developed by Moderna, and the viral vector-based DNA COVID-19 vaccine ChAdOx1-S developed by the University of Oxford and AstraZeneca.

[0016] Administration of these vaccines to a subject induces the production of spike protein in situ in the body, which causes the immune system to initiate an immune response.

[0017] Recent evidence suggests that the SARS-CoV-2 spike protein has potential cellular signaling activity in addition to and independent of its function in promoting viral infection.

[0018] For example, it has been shown that the full-length S1 subunit of the SARS-CoV-2 spike protein can induce proinflammatory responses in vitro in mouse and human macrophages (Shirato et al., Heyon 2021, 7(2) e06187), and can activate cell signaling events in cultured human vascular cells (Suzuki et al., Vascular Pharmacology 137 (2021) 106823).

[0019] In view of the above, it has been suggested that the presence of the spike protein produced by vaccination in the body may interfere with biological processes, thereby impeding the immune response and / or causing side effects in the short and long term.

[0020] Estrogens are a group of steroid hormones that play an important role in the normal development of the female sexual and reproductive system, of which estradiol (E2) is the most potent and abundant.

[0021] These hormones regulate a wide range of physiological functions, such as the development and maintenance of secondary sexual characteristics, metabolism, bone homeostasis, blood salt balance, immune and inflammatory responses, stress responses, and / or neuronal function.

[0022] Imbalances in the levels or activity of these hormones are associated with many pathological processes. In particular, estrogens are implicated in the etiology of breast, endometrial, kidney, and uterine cancer (Okamoto et al., Toxicology Letters 2020, 318: 99-103). Furthermore, decreased estrogen levels are associated with a significant increase in bone resorption, as well as with early and late forms of osteoporosis in postmenopausal women (Riggs BL, J. Clin. Invest 2000, 106, 1203-1204).

[0023] Furthermore, exogenous estrogen administration is associated with dysregulation of multiple aspects of hemostatic and fibrinolytic pathways that contribute to the creation of a prothrombotic environment, with a higher risk of thrombotic events (Abou-Ismail et al., Thrombosis Research 2020, 192: 40-51).

[0024] Estrogen activity is mediated by a nuclear receptor, the estrogen receptor, which functions as a hormone-activated transcription factor. Upon activation by a ligand, this receptor binds to the promoters of target genes and forms a transcriptional complex with a number of interacting proteins, collectively known as coregulators, which can either activate (coactivators (CoA)) or inactivate (corepressors (CoR)) transcriptional activity, thereby causing activation or repression of target gene expression (Patel et al., Pharmacology & Therapeutics 2018, 186: 1-24). The interaction between the nuclear estrogen receptor and coactivators is mediated by the LxxLL motif (where L is leucine and x is any amino acid) contained and shared in coactivator proteins, which is necessary and sufficient for the binding of these proteins to the receptor and for enhancing its transcriptional activity (Patel et al., Pharmacology & Therapeutics 2018, 186: 1-24).

[0025] Essence of the invention

[0026] The present inventors unexpectedly discovered that the SARS-CoV-2 spike protein interferes with estrogen receptor signaling.

[0027] In more detail, the present inventors discovered that the SARS-CoV-2 spike protein contains an LxxLL motif homologous to that of nuclear coactivator 1 (NCOA1), which is capable of binding to the NCOA1 binding domain on the estrogen receptor and thereby activating its transcriptional activity. The present inventors also demonstrated in vitro that the SARS-CoV-2 spike protein exhibits estrogen-like activity under various experimental conditions.

[0028] Therefore, in the context of COVID-19 vaccination, the spike protein produced after vaccination may interfere with estrogen receptor activity and create an imbalance between the physiological and pathological activities mediated by this receptor, thereby causing undesirable serious side effects. Furthermore, due to estrogen's role in regulating the immune response, estrogen receptor activation by the spike protein may impact vaccine efficacy.

[0029] The present inventors also unexpectedly discovered that it is possible to induce a reversal of the spike activity on the estrogen receptor by administering a selective estrogen receptor modulator (SERM).

[0030] Selective estrogen receptor modulators (SERMs) are a group of nonsteroidal compounds that bind to the estrogen receptor (ER) and modulate its activity. SERMs can act as ER agonists or antagonists in a tissue-specific manner.

[0031] It has been proposed that SERMs exert their activity by influencing estrogen receptor signaling by binding to the receptor and inducing distinct conformational changes that affect its ability to interact with coactivators and corepressors.

[0032] Considering the above data, it is expected that the administration of SERM in combination with the administration of COVID-19 vaccine will be beneficial in improving the immune response and reducing the side effects of vaccination.

[0033] Accordingly, a first object of the invention is a combination of a SERM, preferably selected from tamoxifen, raloxifene, 4-hydroxytamoxifen, droloxifene, ospemifene, arzoxifene, toremifene and bazedoxifene, and a COVID-19 vaccine.

[0034] Another object of the invention is the above-mentioned combination for use in the prevention of COVID-19 in a subject.

[0035] Another object of the invention is a SERM for use in combination with a COVID-19 vaccine for the prevention of COVID-19 in a subject.

[0036] Another object of the invention is a SERM for use in the prevention or treatment of adverse effects of a COVID-19 vaccine in a subject.

[0037] Another object of the invention is a pharmaceutical composition comprising a SERM and pharmaceutically acceptable excipients for use in combination with a COVID-19 vaccine for the prevention of COVID-19 in a subject.

[0038] Another object of the invention is a pharmaceutical composition comprising a SERM and pharmaceutically acceptable excipients for use in the prevention of side effects of a COVID-19 vaccine in a subject.

[0039] Another object of the invention is a set of parts comprising:

[0040] a) COVID-19 vaccine; and

[0041] b) a pharmaceutical composition containing an effective amount of a SERM.

[0042] Another object of the invention is a method for preventing COVID-19 in a subject, comprising administering to said subject a COVID-19 vaccine in combination with a SERM.

[0043] Another object of the invention is a method for preventing or treating adverse effects of a COVID-19 vaccine in a subject, comprising administering a SERM to said subject.

[0044] Description of drawings

[0045] Figure 1 shows the estrogen receptor (ER) complexed with the nuclear receptor coactivator. The entire complex is shown in the figure; ER monomers are represented as a gray surface, and NCOA segments are represented by black figures and lines.

[0046] Figure 2 shows the network of the most significant interactions of ER1 and ER2 (both in red spheres). Among the most robust interacting proteins, nuclear receptor coactivators (NCOAs) directly bind to nuclear receptors and stimulate transcriptional activity.

[0047] Figure 3 shows the sequence alignment between the LDX region in the spike (amino acids 818-822) and other LDX domains of the cofactor-receptor.

[0048] Figure 4 shows the best 3D docking hypothesis for spike-ER blind docking. The proteins are shown as images. The ER dimer is shown in white, and the spike protein is shown in black.

[0049] Figure 5 shows the best 3D docking hypothesis for spike-ER motif-based docking. The ER dimer is shown in white, and the spike protein is shown in black.

[0050] Fig. 6 shows the proliferation of MCF-7 cells treated for 24 hours with estradiol (1 nM) (ESTR), spike protein (10 ng / ml) (SPIKE), raloxifene (2 μM) (RAL), a combination of estradiol (1 nM) and spike protein (10 ng / ml) (ESTR+SPIKE), a combination of estradiol (1 nM) and raloxifene (2 μM) (ESTR+RAL), a combination of spike protein (10 ng / ml) and raloxifene (2 μM) (SPIKE+RAL), a combination of estradiol (1 nM), spike protein (10 ng / ml), and raloxifene (2 μM) (ESTR+SPIKE+RAL) measured using the BrdU proliferation assay as described in Example 5. The experiment was performed in three technical replicates. The results represent the mean ± standard deviation for three different experiments (n=3).

[0051] Fig. 7 shows the proliferation of MDA-MB-231 cells treated for 24 hours with estradiol (1 nM) (ESTR), spike protein (10 ng / ml) (SPIKE), raloxifene (2 μM) (RAL), a combination of estradiol (1 nM) and spike protein (10 ng / ml) (ESTR+SPIKE), a combination of estradiol (1 nM) and raloxifene (2 μM) (ESTR+RAL), a combination of spike protein (10 ng / ml) and raloxifene (2 μM) (SPIKE+RAL), a combination of estradiol (1 nM), spike protein (10 ng / ml), and raloxifene (2 μM) (ESTR+SPIKE+RAL) measured using the BrdU proliferation assay, as described in Example 5. The experiment was performed in three technical replicates. The results represent the mean ± standard deviation for three different experiments (n=3).

[0052] Fig. 8 shows the TRAP activity, expressed in U / L, in RAW cells not differentiated into osteoclasts and treated with vehicle (control without RANKL), RAW-OC cells treated for 24 hours with vehicle (control with RANKL), 17β-estradiol (1 nM) (Estradiol), Spike (10 ng / ml) (Spike), Raloxifene (2 μM) (Raloxifene), a combination of Spike (10 ng / ml) and Raloxifene (2 μM) (Spike+Raloxifene), a combination of Spike (10 ng / ml) and 17β-estradiol (1 nM) (Spike+Estradiol), a combination of 17β-estradiol (1 nM) and Raloxifene (2 μM) (Estradiol + Raloxifene), a combination of 17β-estradiol (1 nM), Spike (10 ng / ml) and Raloxifene (2 μM) (Estradiol + Spike + Raloxifene), measured as described in Example 6. Data are presented in U / l. The experiment was performed in six technical replicates. The results represent the mean ± standard deviation of one representative experiment out of three performed.

[0053] Detailed description of the invention

[0054] The first object of the invention is a combination of a selective estrogen receptor modulator (SERM) and a COVID-19 vaccine.

[0055] As discussed above, the inventors of the present invention found that administration of the above combination reduces side effects and improves the immune response compared with administration of the COVID-19 vaccine alone.

[0056] Thus, another object of the present invention is the above-mentioned combination for use in the prevention of COVID-19 in a subject.

[0057] Another object of the invention is a SERM for use in combination with a COVID-19 vaccine for the prevention of COVID-19 in a subject.

[0058] Another object of the invention is a SERM for use in the prevention or treatment of adverse effects of a COVID-19 vaccine in a subject.

[0059] The said subject is preferably a human being.

[0060] The said COVID-19 vaccine is a COVID-19 spike protein-based vaccine.

[0061] The term “spike protein-based vaccine” means any COVID-19 vaccine that uses the SARS-CoV-2 spike protein, its variant, or its immunogenic fragment as the antigen. This definition includes any vaccine that, upon administration to a subject, administers the SARS-CoV-2 spike protein, its variant, or its immunogenic fragment, either as such or linked to inactivated or live attenuated virus. Furthermore, this definition includes any mRNA or DNA vaccine that induces the production of the SARS-CoV-2 spike protein, its variant, or its immunogenic fragment in situ after administration to a human subject.

[0062] The designated COVID-19 vaccine is preferably an mRNA or DNA COVID-19 vaccine.

[0063] In the context of the present invention, “mRNA or DNA vaccine against COVID-19” refers to any vaccine that includes mRNA or DNA, the administration of which to a subject results in the production in the body of such subject of the SARS-CoV-2 spike protein, a variant thereof, or an immunogenic fragment thereof.

[0064] According to the present invention, a "variant" of the SARS-CoV-2 spike protein means a protein whose amino acid sequence differs from the SARS-CoV-2 spike protein sequence in that it contains one or more substitutions, deletions of internal amino acids, or insertions of one or more amino acids. Preferably, said substitutions, deletions, or insertions retain the antigenic properties of the SARS-CoV-2 spike protein. Preferably, said variant has a sequence with an amino acid identity to the SARS-CoV-2 spike protein sequence of at least 90%, 95%, 98%, or 99%.

[0065] According to the present invention, an “immunogenic fragment” of the SARS-CoV-2 spike protein means a protein having an amino acid sequence that corresponds to the N-terminal and / or C-terminal truncated sequence of the SARS-CoV-2 spike protein or a variant thereof, and retaining the antigenic properties of the SARS-CoV-2 spike protein or a variant thereof.

[0066] The said immunogenic fragment of the spike protein preferably has an amino acid sequence that consists of at least 50%, 60%, 70%, 80% or 90% of the amino acid sequence of the full-length SARS-CoV-2 spike protein.

[0067] Preferably, said immunogenic fragment comprises one or more of the following amino acid sequences of the full-length SARS-CoV-2 spike protein (UniProt ID: P0DTC2): from position 232 to 246, from position 233 to 247, from position 471 to 503, from position 604 to 625, from position 817 to 833, from position 818 to 822, from position 891 to 907, from position 897 to 913, from position 1164 to 1191, from position 1182 to 1209.

[0068] The said SERM is preferably selected from tamoxifen, raloxifene, 4-hydroxytamoxifen, droloxifene, ospemifene, arzoxifene, toremifene and bazedoxifene.

[0069] A particularly preferred SERM according to the invention is raloxifene.

[0070] As discussed above, the use of SERM in combination with the COVID-19 vaccine according to the invention prevents the side effects resulting from the interference of the spike protein produced in the host body after administration of the COVID-19 vaccine with the physiological function of the estrogen receptor and improves the response of the immune system to the vaccine.

[0071] These findings lead to improved therapies to prevent COVID-19.

[0072] Preferably, the said side effects are undesirable effects resulting from overactivation of the estrogen receptor.

[0073] Thus, administration of the SERM according to the invention is particularly advantageous for subjects who may experience more severe side effects as a result of COVID-19 vaccine administration and the associated increase in estrogen receptor activity. These include, for example, subjects diagnosed with ER-positive breast cancer or any pathological condition that alters the physiological balance of hemostasis, such as a bleeding disorder.

[0074] According to one embodiment, said subject is a subject diagnosed with ER-positive breast cancer.

[0075] According to another embodiment, said subject is a subject diagnosed with a pathological condition that alters the physiological balance of hemostasis.

[0076] Administration of SERM in combination with the COVID-19 vaccine according to the invention is also advantageous to all other categories of subjects to avoid side effects and improve the immune response to the COVID-19 vaccine.

[0077] Thus, according to one embodiment, said subject is a subject who has not been diagnosed with ER-positive breast cancer.

[0078] According to another embodiment, said subject is a subject who has not been diagnosed with a pathological condition that alters the physiological balance of hemostasis.

[0079] According to another embodiment, said subject is a subject who has not been diagnosed with ER-positive breast cancer, a pathological condition that alters the physiological balance of hemostasis.

[0080] According to all the objects of the invention, the SERM can be administered simultaneously with or separately from the COVID-19 vaccine.

[0081] It is preferably administered separately from the COVID-19 vaccine and according to a different administration schedule.

[0082] Preferably, the SERM is administered to a subject who has received, is receiving, or is to receive a COVID-19 vaccine, preferably once or twice a day, for a period of time starting two weeks after the first dose of the vaccine is administered and ending at least two weeks after the last dose of the vaccine is administered, more preferably at least one month after the last dose of the vaccine is administered.

[0083] The frequency of administration and duration of treatment will vary depending on the dose of SERM used and / or the type of COVID-19 vaccine.

[0084] According to one embodiment, the SERM is administered at a dose and frequency of administration that is typically used to treat other pathologies.

[0085] More preferably, in order to determine the correct dose and administration schedule, the estrogen levels of the subject to receive the COVID-19 vaccine are measured before vaccination, and treatment with SERMs is planned based on the patient's baseline levels.

[0086] SERM is preferably administered to a subject who has received, is receiving, or is to receive a COVID-19 vaccine in the form of a pharmaceutical composition.

[0087] Accordingly, another object of the present invention is a pharmaceutical composition comprising a SERM as described above and pharmaceutically acceptable excipients, for use in combination with a COVID-19 vaccine for the prevention of COVID-19 in a subject, as described previously.

[0088] Another object of the invention is a pharmaceutical composition comprising a SERM as described above and pharmaceutically acceptable excipients, for use in the prevention or treatment of side effects of a COVID-19 vaccine in a subject as described above.

[0089] Preferably, the pharmaceutical composition of the present invention is prepared in suitable dosage forms containing an effective amount of SERM and pharmaceutically acceptable excipients.

[0090] Administration of the pharmaceutical composition of the present invention to a subject is carried out in accordance with known methods and may include oral administration, parenteral administration, preferably selected from intravenous, intraperitoneal, intramuscular, intraarterial, subcutaneous administration, topical administration, buccal administration or rectal administration (suppositories).

[0091] According to the present invention, the phrase "effective amount" means a dosage of a compound or composition sufficient to significantly achieve a desired response.

[0092] The vaccine and SERM may also be present as a kit of parts.

[0093] Accordingly, another object of the present invention is a kit of parts comprising:

[0094] a) a COVID-19 vaccine as described above, and

[0095] b) a pharmaceutical composition containing a SERM as described above.

[0096] The route of administration of the SERM or pharmaceutical composition for use according to the present invention depends on the specific compound used or contained in the pharmaceutical composition and corresponds to known methods of administering the compound, which are typically systemic administration, preferably by oral, parenteral or inhalation routes. The term "parenteral" in the context of the present application includes intravenous, intraperitoneal, intracerebral, intrathecal, intracranial, intramuscular, intra-articular, intrasynovial, intrasternal, intraocular, intra-arterial, subcutaneous, intradermal injection or infusion.

[0097] The composition of the present invention can be formulated into dosage forms for oral, inhalation or injection use, such as tablets, capsules, powders, solutions, suspensions and emulsions.

[0098] The pharmaceutically acceptable excipient according to the present invention includes any possible solvents, diluents, or other auxiliary dissolving, dispersing or suspending agents, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, suitable for the desired specific dosage form.

[0099] Some examples of substances that can serve as pharmaceutically acceptable excipients include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil; sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; Isotonic saline solution; sterilized water; Ringer's solution; buffered saline solution; dextrose solution; maltodextrin solution; ethyl alcohol; and phosphate buffer solutions.

[0100] Furthermore, the composition of the present invention can be formulated into dosage forms for inhalation or injection, such as solutions, suspensions and emulsions, by further adding diluents, dispersants and surfactants.

[0101] Also, the composition of the present invention can be suitably formulated using appropriate methods known in the art or the method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton Pa.

[0102] The term "pharmaceutically acceptable" is intended to define, without any limitation, any substance suitable for the preparation of a pharmaceutical composition intended for administration to a living being.

[0103] Dosage forms may also contain other traditional ingredients, such as: preservatives, stabilizers, surfactants, buffers, osmotic regulators, emulsifiers, sweeteners, colorants, flavors, etc.

[0104] The dosage forms of the pharmaceutical composition of the present invention can be obtained by methods known to a pharmaceutical chemist, and they include mixing, granulating, pressing, dissolving, sterilization, and the like.

[0105] Another object of the invention is a method for preventing COVID-19 in a subject, comprising administering to said subject a COVID-19 vaccine in combination with a SERM.

[0106] Another object of the invention is a method for preventing or treating adverse effects of a COVID-19 vaccine in a subject, comprising administering a SERM to said subject.

[0107] Experimental part

[0108] Examples 1-4

[0109] Materials and Methods

[0110] 1. Interactome analysis

[0111] The STRING database [Szklarczyk D et al., Nucleic Acids Res. 2021, 49: D605-D612, doi: 10.1093 / nar / gkaa1074], which combines all known and predicted associations between proteins, including both physical interactions and functional associations, was used to analyze functional associations between biomolecules. Each protein-protein interaction is marked with a “score.” This score does not indicate the strength or specificity of the interaction, but only its reliability. All scores range from 0 to 1, where 1 corresponds to the highest possible reliability.

[0112] 2. Selecting a 3D model

[0113] A 3D model of the spike was created based on PDB 6VYB, reverted to its wild-type form and fully glycosylated. Asymmetric glycosylation of the three protomers was inferred based on published glycoanalytic data for N-glycans and O-glycans (Casalini L et al., ACS Cent. Sci. 2020, 6: 1722–1734, https: / / doi.org / 10.1021 / acscentsci.0c01056). The Amber14SB force field (Maier JA et al., J. Chem. Theory Comput. 2015, 11:3696–3713, https: / / doi.org / 10.1021 / acs.jctc.5b00255) was used for protein modeling, and the GLYCAM06j-1 version of the GLYCAM06 force field (Kirschner KN et al., J. Comput. Chem. 2008, 29:622–655, https: / / doi.org / 10.1002 / jcc.20820) was used for carbohydrate moieties. The resulting structure was used as a starting point for Molecular Docking (MD) modeling. Topology files were generated with the GROMACS pdb2gmx tool using the amber99sb force field (Lindorff-Larsen K et al., Proteins 2010, 78:1950-1958, https: / / dx.doi.org / 10.1002 / prot.22711). The protein was placed in a triclinic box extending up to 15 Å from the solute and immersed in TIP3P water molecules (Jorgensen WL et al., J Chem Phys 1983, 79:926-935, https: / / dx.doi.org / 10.1063 / 1.445869). Counterions were added to neutralize the net charge using the genion GROMACS tool. After energy minimization, the system was relaxed for 5 nsec by applying 1000 kJ mol positional restraints. -1 nm -2to the protein atoms. After this step, unrestricted MD simulations were performed for 1 microsecond with a time step of 2 femtoseconds using the GROMACS 2018.3 simulation package (Galileo and Marconi-100 supercomputer, CINECA, Bologna, Italy) (Abraham MJ et al., SoftwareX 2015, 1:19–25, https: / / dx.doi.org / 10.1016 / j.softx.2015.06.001). The relationship between temperature and V-scaling was used to maintain a constant temperature at 300 K (26.85°C) (Bussi G et al., J Chem Phys, 2007 126:014101. https: / / dx.doi.org / 10.1063 / 1.2408420). To process long-range electrostatic interactions, the Ewald particle-mesh method was used (Darden T et al., J. Chem. Phys 1993, 98:10089, https: / / doi.org / 10.1063 / 1.464397). The first 5 ns of each trajectory were excluded from the analysis. The trajectory obtained after 1 μs of MD simulation was clustered to obtain representative structures.In particular, the structure used for the docking studies was the first centroid of the first cluster obtained from the MD experiment.

[0114] For the estrogen receptor, the XRAY PDB model with code 3OLL, containing estradiol and nuclear receptor coactivator 1, was used (Möcklinghoff S et al., ChemBioChem 2010, 11:2251-2254, https: / / doi.org / 10.1002 / cbic.201000532).

[0115] 3. Protein-protein docking procedure

[0116] Two separate protein inputs were provided: one for the receptor and one for the ligand. Specifically, the spike protein and ER were used as the receptor and ligand, respectively. HDOCK then performed docking to test putative binding modes using an FFT-based search, followed by protein-protein interaction evaluation. Finally, the top 100 predicted complex structures were presented, and the top ten hypotheses were visually inspected to confirm the reliability of the calculation. The entire workflow is described (Yan Y et al., Nat Protoc 2020, 15:1829–1852, https: / / doi.org / 10.1038 / s41596-020-0312-x).

[0117] Given the lack of structural information on the binding mechanism between the spike protein and the nuclear estrogen receptor (ER), the first screening involved identifying proteins known to interact with estrogen receptors. After identifying these ER-interacting proteins, the second step involved examining any sequence similarities between the spike protein and ER effector proteins.

[0118] Example 1

[0119] Nuclear receptor coactivators and the LxxLL motif

[0120] Many transcription factors and cofactors exhibit a common structural motif that mediates interactions with effector proteins. The motif involved in these protein-protein interactions is called LxxLL (LDX), and it is associated with various aspects of transcriptional regulation (Plevin MJ et al., Trends Biochem Sci 2005, 30:66-69. https: / / dx.doi.org / 10.1016 / j.tibs.2004.12.001). The LxxLL sequence was initially identified in proteins that bind the activation function-2 (AF-2) region of the ligand-binding domains (LBDs) of the nuclear receptor. These motifs are fundamental in the regulation of nuclear receptors by many nuclear receptor binding proteins, including coactivators (NCOA-1, 2, and 3) (Heery DM et al., Nature 1997, 387:733-736, https: / / doi.org / 10.1038 / 42750).

[0121] Experimental and structural evidence showing interaction between ER and NCOA allowed us to focus on the LxxLL motif and, based on this, sequence mapping of the spike protein was performed in search of structural motifs and homologous parts of the spike that could mimic the interaction between ER and its nuclear coactivators (Fig. 1).

[0122] Example 2

[0123] Network analysis of estrogen receptor binding protein

[0124] The network of the most significant interactions of ER1 and ER2 (both in spheres) (Fig. 2) highlights that, among the most robust interacting proteins with ER1 and ER2, nuclear receptor coactivators (NCOAs) directly bind nuclear receptors and stimulate transcriptional activity in a hormone-dependent manner. Many experimental data related to complexes between ER and NCOAs, which are deposited in the Protein Data Bank, support these results and can be found in the Uniprot database (ER1: https: / / www.uniprot.org / uniprot / P03372#structure; ER2: https: / / www.uniprot.org / uniprot / Q92731#structure).

[0125] By combining the sequence alignment between NACO and spike with three-dimensional analysis of the viral protein, it was determined that the spike protein contains a sequence homologous to LxxLL, also structurally well defined (in fact, this region suggests alpha-helix conformations), in the outer zone, which could, in principle, act as the site of the ER interaction region (Fig. 3, amino acids 818-828 of the SARS-CoV-2 spike proteins). Different domains of LDX are associated with specific cofactor-receptor interactions, and the LDX1 spike resembles the NCOA1 of LDX4 and belongs to the Class III domain (Savkur RS et al., J Pept Res 2004, 63:207-212, https: / / dx.doi.org / 10.1111 / j.1399-3011.2004.00126.x)

[0126] Example 3

[0127] Blind docking of spike protein-ER

[0128] It was experimentally confirmed that ER does not bind the receptor-binding domain (RBD) of the viral protein (data not shown). To confirm and validate the in silico prediction, the ability of ER to interact in a region other than the viral RBD was assessed in a blind docking mode between the two proteins using the HDOCK server [http: / / hdock.phys.hust.edu.cn / ]. The blind docking approach does not take into account any structural deviations and is completely unsupervised. The best binding hypothesis found provides evidence for the high affinity of ER for the lateral region of the spike protein, which belongs to the so-called “fusion peptide portion” (Fig. 4).

[0129] Example 4

[0130] Spike protein-ER motif-directed docking

[0131] The structural information about the ER residues recognized by NCOA was used to conduct a docking study of ER on the spike by optimizing protein-protein interactions. Using protein structures and residue constraints as input, the HDOCK server generated a suitable model. Given the known structural information about the interaction between ER and NCOA, a second docking study was performed, which considered the ER residues that guarantee interaction with NCOA, thus guiding the molecular docking procedure. The best binding hypothesis obtained as a result of the directed docking study is shown in Figure 5, where ER binding to the spike region containing motifs homologous to the LxxLL pattern is highlighted (Figure 5).

[0132] Example 5

[0133] Induction of cancer cell proliferation

[0134] MCF-7 and MDA-MB-231 cells were used in this experiment. MCF-7 is a hormone-dependent invasive ductal carcinoma cell line (both estrogen receptor and progesterone receptor-positive), and MDA-MB-231 is a human breast cancer epithelial cell line that lacks the estrogen receptor.

[0135] Cells were obtained from ATCC and grown in DMEM without phenol red supplemented with 10% fetal bovine serum (FBS), penicillin / streptomycin, at 37°C in an atmosphere of 5% CO2 and 95% humidity. For each assay, cells were seeded at a density of 10 4 cells / cm 2 Before treatment, to reduce estrogen levels in FBS and avoid any interference, cells were cultured for 24 hours in a medium containing 5% serum treated with dextran-coated activated carbon.

[0136] The cells were then treated for 24 hours with estradiol (1 nM) (ESTR), spike protein (10 ng / ml) (SPIKE), raloxifene (2 μM) (RAL), a combination of 17β-estradiol (1 nM) and spike protein (10 ng / ml) (ESTR+SPIKE), a combination of 17β-estradiol (1 nM) and raloxifene (2 μM) (ESTR+RAL), a combination of spike protein (10 ng / ml) and raloxifene (2 μM) (SPIKE+RAL), a combination of 17β-estradiol (1 nM), spike protein (10 ng / ml) and raloxifene (2 μM) (ESTR+SPIKE+RAL).

[0137] Next, a BrdU proliferation assay was performed to measure cell proliferation. This assay involves the incorporation of BrdU into the DNA of dividing cells cultured in microtiter plates using the cell layer as a solid phase. The BrdU cell proliferation ELISA kit (Abcam, ab126556) was used according to the manufacturer's instructions. BrdU was added to the wells for 24 hours, and then the cells were fixed using fixation solution. The cells were then washed and incubated with an anti-BrdU detection antibody for 1 hour at room temperature. After incubation, the cells were washed and incubated with goat anti-mouse antibodies conjugated to horseradish peroxidase for 30 minutes at room temperature. For detection, the chromogenic substrate tetramethylbenzidine (TMB) was added and the colored product was detected using a spectrophotometer (450 / 550 nm).

[0138] The results obtained are shown in Fig. 6 for MCF7 cells and in Fig. 7 for MDA-MB-231 cells. As can be seen, the SARS-CoV-2 spike protein exhibits proliferation-inducing activity in breast cancer cells, which is dependent on the presence of the estrogen receptor. In fact, in MCF-7 cells, treatment with 17β-estradiol or spike leads to a similar increase in cell proliferation, whereas in MDA-MB-231 cells, 17β-estradiol and spike, either individually or in combination, do not significantly affect cell proliferation. The proliferation-promoting activity of both 17β-estradiol and spike is blocked by treatment with the SERM raloxifene. These results clearly demonstrate that spike acts on cells by activating the estrogen receptor and that it is capable of inducing the proliferation of ER-positive cancer cells.

[0139] Example 6

[0140] TRAP activity by ELISA in RAW-OC

[0141] To further demonstrate that the spike activates the estrogen receptor, the effect of 17β-estradiol and SARS-CoV-2 spike protein on TRAP activity was compared in an osteoclast cell model.

[0142] The mouse monocytic cell line RAW 264.7, which differentiates into osteoclasts under the influence of recombinant RANKL, was used.

[0143] More details, RAW264.7 (mouse macrophages ATCC, USA) were cultured according to the manufacturer's protocol. Then 1.5×10 5 cells / cm 2were seeded in 24-well plates and supplemented with mouse receptor activator of nuclear factor kB ligand (RANKL, MilteNY Biotec, Germany) at a final concentration of 35 ng / ml to initiate osteoclast (OC) development (day 0) as described previously (Collin-Osdoby and Osdoby 2012, PMID: 22130930). On day 3, cells were examined microscopically and fresh medium containing RANKL was added. On day 6, the RAW-OC population predominated. Cells were treated with 17-β-estradiol (1 nM), spike (10 ng / ml), raloxifene (2 μM), and their combination for 24 h. After 24 hours of treatment, cells were collected into sterile tubes and resuspended in PBS (pH 7.4) to a concentration of approximately 1 million cells / mL. The cells were then subjected to repeated freeze-thaw cycles to release internal components. Meanwhile, the kit reagents were brought to room temperature. Tartrate-resistant acid phosphatase (TRAP) activity was measured using an enzyme-linked immunosorbent assay purchased from Myobiosource (catalog number MBS1601167).The standard curve, reagents, and samples were prepared according to the manufacturer's protocol. Briefly, 50 µL of standard were added to the standard wells and 40 µL to the sample wells, followed by the addition of 10 µL of anti-TRAP antibody to the sample wells and 50 µL of streptavidin-HRP to the sample and standard wells. The plate was sealed with a plate sealer, mixed well on a rocking platform, and incubated for 1 hour at 37°C. The plate was washed five times with wash buffer, and 50 µL of substrate A solution and 50 µL of substrate B solution were added to each well and incubated for 10 minutes at 37°C in the dark. Finally, 50 µl of stop solution was added to each well and the optical density was immediately determined using a microplate reader set to a wavelength of 450 nm.

[0144] The results obtained are shown in Fig. 8. As can be seen, treatment of cells with either estradiol or spike leads to a significant decrease in TRAP activity, and this effect is blocked by co-treatment with the SERM raloxifene. These results further demonstrate that spike acts by activating the estrogen receptor.

[0145] --->

[0146] SEQUENCE LIST

[0147] <110> DOMPE' FARMACHEUTICHI SPA.

[0148] <120> Combination Therapy for COVID-19 Vaccination

[0149] <130> 252EP

[0150] <160> 9

[0151] <170> BiSSAP 1.3.6

[0152] <210> 1

[0153] <211> 17

[0154] <212> Protein

[0155] <213> Artificial sequence

[0156] <220>

[0157] <223> SPIKELMX1 aa positions 812 - 828

[0158] <400> 1

[0159] Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr

[0160] 1 5 10 15

[0161] Leu

[0162] <210> 2

[0163] <211> 17

[0164] <212> Protein

[0165] <213> Artificial sequence

[0166] <220>

[0167] <223> NCOA1LXD1 aa positions 627 - 643

[0168] <400> 2

[0169] Ser Gln Thr Ser His Lys Leu Val Gln Leu Leu Thr Thr Thr Ala Glu

[0170] 1 5 10 15

[0171] Gln

[0172] <210> 3

[0173] <211> 17

[0174] <212> Protein

[0175] <213> Artificial sequence

[0176] <220>

[0177] <223> NCOA2LDX1 aa positions 635 - 651

[0178] <400> 3

[0179] Ser Lys Gly Gln Thr Lys Leu Leu Gln Leu Leu Thr Thr Lys Ser Asp

[0180] 1 5 10 15

[0181] Gln

[0182] <210> 4

[0183] <211> 17

[0184] <212> Protein

[0185] <213> Artificial sequence

[0186] <220>

[0187] <223> NCOA1LXD2 aa positions 684 - 700

[0188] <400> 4

[0189] Thr Glu Arg His Lys Ile Leu His Arg Leu Leu Gln Glu Gly Ser Pro

[0190] 1 5 10 15

[0191] Ser

[0192] <210> 5

[0193] <211> 17

[0194] <212> Protein

[0195] <213> Artificial sequence

[0196] <220>

[0197] <223> NCOA2LDX2 aa positions 684 - 700

[0198] <400> 5

[0199] Lys Glu Lys His Lys Ile Leu His Arg Leu Leu Gln Asp Ser Ser Ser

[0200] 1 5 10 15

[0201] Pro

[0202] <210> 6

[0203] <211> 17

[0204] <212> Protein

[0205] <213> Artificial sequence

[0206] <220>

[0207] <223> NCOA1LXD3 aa positions 743 - 759

[0208] <400> 6

[0209] Ser Lys Asp His Gln Leu Leu Arg Tyr Leu Leu Asp Lys Asp Glu Lys

[0210] 1 5 10 15

[0211] Asp

[0212] <210> 7

[0213] <211> 17

[0214] <212> Protein

[0215] <213> Artificial sequence

[0216] <220>

[0217] <223> NCOA2LDX3 aa positions 739 - 755

[0218] <400> 7

[0219] Lys Lys Glu Asn Ala Leu Leu Arg Tyr Leu Leu Asp Lys Asp Asp Thr

[0220] 1 5 10 15

[0221] Lys

[0222] <210> 8

[0223] <211> 17

[0224] <212> Protein

[0225] <213> Artificial sequence

[0226] <220>

[0227] <223> NCOA1LXD4 aa positions 907 - 923

[0228] <400> 8

[0229] Gln Cys Ile Ser Ser Gln Leu Asp Glu Leu Leu Cys Pro Pro Thr Thr

[0230] 1 5 10 15

[0231] Val

[0232] <210> 9

[0233] <211> 17

[0234] <212> Protein

[0235] <213> Artificial sequence

[0236] <220>

[0237] <223> NCOA2LDX4 aa positions 1073 - 1089

[0238] <400> 9

[0239] Pro Ser Asp Glu Gly Ala Leu Leu Asp Gln Leu Tyr Leu Ala Leu Arg

[0240] 1 5 10 15

[0241] Asn

[0242] <---

Claims

1. A combination for the prevention or treatment of adverse effects of a COVID-19 vaccine in a subject, comprising a selective estrogen receptor modulator (SERM) and a COVID-19 vaccine.

2. The combination according to claim 1, wherein said COVID-19 vaccine is a COVID-19 vaccine in which the SARS-CoV-2 spike protein, a variant thereof, or an immunogenic fragment thereof is used as an antigen.

3. The combination according to claim 2, wherein said COVID-19 vaccine is an mRNA or DNA vaccine.

4. The combination according to any one of claims 1-3, wherein said selective estrogen receptor modulators (SERMs) are selected from tamoxifen, raloxifene, 4-hydroxytamoxifen, droloxifene, ospemifene, arzoxifene, toremifene and bazedoxifene.

5. The combination of any one of claims 1-4, wherein said subject is a subject diagnosed with ER-positive breast cancer.

6. The combination of any one of claims 1-4, wherein said subject is a subject diagnosed with a pathological condition that alters the physiological balance of hemostasis.

7. Use of the combination according to paragraph 1 for the prevention of COVID-19 in a subject.

8. The use according to claim 7, wherein said COVID-19 vaccine is a COVID-19 vaccine in which the SARS-CoV-2 spike protein, a variant thereof, or an immunogenic fragment thereof is used as an antigen.

9. The use according to claim 8, wherein said COVID-19 vaccine is an mRNA or DNA vaccine.

10. The use according to any one of claims 7-9, wherein said selective estrogen receptor modulators (SERMs) are selected from tamoxifen, raloxifene, 4-hydroxytamoxifen, droloxifene, ospemifene, arzoxifene, toremifene and bazedoxifene.

11. The use according to any one of claims 7-10, wherein said subject is a subject diagnosed with ER-positive breast cancer.

12. The use according to any one of paragraphs 7-10, wherein said subject is a subject diagnosed with a pathological condition that alters the physiological balance of hemostasis.

13. Use of a selective estrogen receptor modulator (SERM) for the prevention or treatment of adverse effects of a COVID-19 vaccine in a subject.

14. The use according to claim 13, wherein said COVID-19 vaccine is a COVID-19 vaccine in which the SARS-CoV-2 spike protein, a variant thereof, or an immunogenic fragment thereof is used as an antigen.

15. The use according to claim 14, wherein said COVID-19 vaccine is an mRNA or DNA vaccine.

16. The use according to any one of claims 13-15, wherein said selective estrogen receptor modulators (SERMs) are selected from tamoxifen, raloxifene, 4-hydroxytamoxifen, droloxifene, ospemifene, arzoxifene, toremifene and bazedoxifene.

17. The use according to any one of claims 13-16, wherein said subject is a subject diagnosed with ER-positive breast cancer.

18. The use according to any one of paragraphs 13-16, wherein said subject is a subject diagnosed with a pathological condition that alters the physiological balance of hemostasis.

19. Use of a pharmaceutical composition comprising a selective estrogen receptor modulator (SERM) and pharmaceutically acceptable excipients in combination with a COVID-19 vaccine for the prevention of COVID-19 in a subject.

20. The use according to claim 19, wherein said COVID-19 vaccine is a COVID-19 vaccine in which the SARS-CoV-2 spike protein, a variant thereof, or an immunogenic fragment thereof is used as an antigen.

21. The use according to claim 20, wherein said COVID-19 vaccine is an mRNA or DNA vaccine.

22. The use according to any one of claims 19-21, wherein said selective estrogen receptor modulators (SERMs) are selected from tamoxifen, raloxifene, 4-hydroxytamoxifen, droloxifene, ospemifene, arzoxifene, toremifene and bazedoxifene.

23. The use according to any one of claims 19-22, wherein said subject is a subject diagnosed with ER-positive breast cancer.

24. The use according to any one of paragraphs 19-22, wherein said subject is a subject diagnosed with a pathological condition that alters the physiological balance of hemostasis.

25. Use of a pharmaceutical composition comprising selective estrogen receptor modulators (SERMs) and pharmaceutically acceptable excipients for the prevention or treatment of adverse effects of a COVID-19 vaccine in a subject.

26. The use according to claim 25, wherein said COVID-19 vaccine is a COVID-19 vaccine in which the SARS-CoV-2 spike protein, a variant thereof, or an immunogenic fragment thereof is used as an antigen.

27. The use according to claim 26, wherein said COVID-19 vaccine is an mRNA or DNA vaccine.

28. The use according to any one of claims 25-27, wherein said selective estrogen receptor modulators (SERMs) are selected from tamoxifen, raloxifene, 4-hydroxytamoxifen, droloxifene, ospemifene, arzoxifene, toremifene and bazedoxifene.

29. The use according to any one of claims 25-28, wherein said subject is a subject diagnosed with ER-positive breast cancer.

30. The use according to any one of paragraphs 25-28, wherein said subject is a subject diagnosed with a pathological condition that alters the physiological balance of hemostasis.

31. A kit of parts for the prevention or treatment of adverse effects of a COVID-19 vaccine in a subject, comprising: a) a vaccine against COVID-19; and b) a pharmaceutical composition containing a selective estrogen receptor modulator (SERM).

32. A kit of parts according to claim 31, wherein said COVID-19 vaccine is a COVID-19 vaccine in which the SARSCoV-2 spike protein, a variant thereof, or an immunogenic fragment thereof is used as an antigen.

33. A kit of parts according to claim 32, wherein said COVID-19 vaccine is an mRNA or DNA vaccine.

34. The kit of parts according to any one of claims 31-33, wherein said selective estrogen receptor modulators (SERMs) are selected from tamoxifen, raloxifene, 4-hydroxytamoxifen, droloxifene, ospemifene, arzoxifene, toremifene and bazedoxifene.

35. The kit of parts according to any one of claims 31-34, wherein said subject is a subject diagnosed with ER-positive breast cancer.

36. A kit of parts according to any one of paragraphs 31-34, wherein said subject is a subject who has been diagnosed with a pathological condition that alters physiological balance of hemostasis.

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