RU2847261C1 - Natural antibodies in prevention and therapy - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention refers to biotechnology, immunology and medicine. Disclosed is the use of a mixture of human or humanised natural IgM and/or IgA antibodies that recognize oxidised phospholipids or oxidation-specific epitopes, for the treatment or prevention of a disorder or disease associated with/related to/caused by a deficiency of natural IgM/IgA antibodies in a subject.

EFFECT: invention provides additional agents and methods of treating or preventing disorders or diseases associated with/related to/caused by deficiency of natural IgM/IgA antibodies in a subject.

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Description

The present invention relates to a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes (oxidation-specific epitopes), intended for use in a method of treating or preventing a disorder or disease associated/linked to and/or caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject. Furthermore, the present invention relates to a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method of treating or preventing a disorder or disease associated/linked to and/or caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, wherein said natural IgM and/or IgA antibodies are derived from IgM and/or IgA-enriched plasma pools from healthy individuals. Furthermore, the present invention relates to a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method for treating or preventing a disorder or disease associated with and/or caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, wherein said antibody is a recombinant human monoclonal natural IgM and/or IgA antibody. Furthermore, the present invention relates to a vaccine comprising a compound that induces the formation of natural IgM and/or IgA antibodies, intended for use in a method for reducing or preventing clinical signs or a disease associated with and/or caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, wherein said vaccine comprises a pharmaceutically acceptable carrier or excipient. Furthermore, the present invention relates to said vaccine intended for use in a method for reducing or preventing clinical signs or disease associated with and/or caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, wherein said compound induces a human natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes.

Conventional B lymphocytes, also called B2 cells or follicular (FO) B cells, originate from hematopoietic stem cells and pass through various distinct stages during development until the antigen-challenging cells eventually differentiate into immunoglobulin (antibody)-secreting plasma cells or memory B cells.

In most mammals, the early stages of B2 cell development occur in the bone marrow, while final maturation processes occur in peripheral (secondary) lymphoid organs such as the spleen, lymph nodes, Peyer's patches, etc. The most important distinguishing feature of B lymphocytes is their B-cell antigen receptor (BCR), the specificity of which is not encoded by the germline and, therefore, must be generated individually during early lymphocyte development. The BCR primarily consists of a membrane-anchored variant of immunoglobulin associated with the Ig-α/Ig-β signal transducer and coreceptors such as CD19. Although a B cell expresses immunoglobulin with only one unique specificity, the pool of all lymphocytes together has the ability to recognize virtually any foreign substance.

This enormous diversity is achieved by random recombination of the variable segments (V _H ), diversity segments (D _H ), and joining segments (J _H ) of genes in the heavy chain locus and the V _L and J _L gene segments in the light chain locus, which, after assembly, encode the variable domains of immunoglobulin. There is a large variety of variants for each segment in the vertebrate genome. For example, the human immunoglobulin heavy chain locus contains up to 51 V _H , 27 D _H , and 6 J _H gene segments, plus numerous V _L and J _L gene segments in the light chain locus. Thus, a repertoire of immunoglobulins with approximately 3 × 10 ¹¹ different specificities can be generated by random recombination of these gene segments.

This diversity of combinations can be further increased by incorporating non-matrix-encoded N-nucleotides into the gene segment junctions using an enzyme such as terminal deoxynucleotidyl transferase (TdT), which is specifically expressed early in B2 cell development. Because immunoglobulin gene rearrangement involves an irreversible change in DNA sequence, all progeny of a given activated B cell should inherit the same receptor specificity, including memory B cells, which are the basis for long-term immunity against a specific pathogen.

When a mature B2 cell encounters antigen and is activated, it migrates to the B cell border layer of a T cell in a lymphatic organ, such as a lymph node or spleen, where it presents antigen fragments internalized via MHC class II molecules. When a primed CD4 ^pos follicular helper T cell (T _FH ) expresses a T cell antigen receptor (TCR) specific for the MHC class II/antigen complex presented by the activated B cell, it produces costimulatory molecules such as CD40 ligand and cytokines such as interleukin 4 (IL-4), which are required for full B cell activation. A small fraction of fully activated B cells rapidly differentiates into short-lived plasma cells that secrete antigen-specific IgM antibodies, also known as adaptive or immune IgM. Most activated B cells, together with cognate _T cells, form terminal centers (GCs), which are specialized structures in the lymphatic organ where affinity maturation and recombination, leading to class switching (CSR), occur.

Affinity maturation is a process by which somatic hypermutations (SHMs) are introduced into the variable regions of immunoglobulin genes to alter the affinity of the expressed BCR for a specific antigen. B cells expressing mutant BCRs with reduced affinity for the antigen are counter-selected and die, while B cell clones expressing mutant BCRs with increased affinity can further develop. The simultaneous onset of CSR leads to a genetic rearrangement of the VDJ sequence, which directly shifts the VDJ structure relative to the constant regions of C-μ and C-δ, for example, to C-γ. Consequently, an irreversible deletion of the C-μ or C-δ sequence required for the expression of the IgM and IgD isotypes, respectively, occurs in the genome of the corresponding B cells, resulting in the B cells producing IgG molecules containing γ-HC. Within seven days, the GC reaction yields plasma cells that secrete large quantities of high-affinity IgG antibodies, which account for the majority of serum IgG levels, and memory B cells that express high-affinity IgG BCRs, which rapidly differentiate into IgG-secreting plasma cells upon re-encounter with the same pathogen.

In addition to the normal B2 cells described above, another population of mature B cells, called B1 cells, has been described. B1 cells represent a small subset of B cells and, due to their unique functions, are often referred to as innate-like lymphocytes.

Thus, these cells have several characteristics that distinguish them from B2/FO B cells. Most of the knowledge about B1 cells and their specific functions has been obtained from studies in mice, where they can be easily identified by the expression of a specific set of surface markers. Murine B1 cells typically have an IgM ^hi /IgD ^low /B220 ^low /CD23 ^neg /CD43 ^pos /Mac-1 ^pos phenotype, which differs from the IgM ^low /IgD ^hi /B220 ^hi /CD23 ^pos /CD43 ^neg /Mac-1 ^neg phenotype of naive mature B2 cells. Based on CD5 expression, B1 cells can be further subdivided into CD5 ^pos B1a cells and CD5 ^neg B1b cells. This B cell population is referred to as B1 because it is the first B cell population to emerge during ontogenesis and is present at birth, while the B2 cell population emerges later after birth. Despite extensive research in recent years, the origin of B1 cells remains unclear.

One hypothesis is that B1 cells originate from a distinct line of neonatal progenitor cells in the fetal liver, and that this population is maintained in adults due to its capacity for self-renewal.

The second model proposes that B1 cells continually arise from bone marrow-derived immature B cells that express BCR with corresponding autoreactivity. Current thinking suggests that enhanced BCR signaling through self-antigen recognition is crucial for the development and maintenance of B1 cells.

Regardless of their origin, it is well known that the B1 cell population declines with age. In adult mice, B1 cells are predominantly localized in the peritoneal and pleural cavities and are only occasionally present in the lymph nodes or spleen. However, they can often migrate to sites of infection, where they produce protective IgM antibodies that do not require T cell assistance.

Importantly, and in stark contrast to B2 cells, B1 cells have the unique ability to spontaneously secrete antibodies even in the absence of infection or specific immunization. In fact, even in gnotobiotic mice raised under strict germ-free conditions, B1 cells maintained normal serum IgM levels similar to those in mice raised under conventional conditions, leading to these antibodies being referred to as natural antibodies (Chou, Fogelstrand, et al., J Clin Invest, vol. 119 (5), 2009).

This distinguishes them from immune antibodies, which were virtually absent in mice raised under similar germ-free conditions. It has been established that in mouse serum, approximately 80% of serum IgM are natural antibodies, and the remaining 20% are immune IgM. Furthermore, natural antibodies make up the majority of serum IgA antibodies. Natural antibodies differ from immune antibodies secreted by B2 cells in several respects. For example, natural antibodies share preexisting germline-encoded variable domain sequences, meaning that they are produced using a specific, limited set of D _H -proximal V _H gene segments and in combination with certain LC genes. Natural antibody genes do not have significant somatic hypermutations or N-nucleotide insertions at junctional regions due to the low level of TdT expression in B1 cells.

Natural antibodies bind to a wide variety of structures, such as bacterial cell wall components, viruses (including influenza, vesicular stomatitis virus, and lymphocytic choriomeningitis virus), and autoantigens (including phospholipids, DNA, or misfolded proteins exposed by dying cells). For this reason, natural antibodies are considered autoreactive and polyreactive. Furthermore, natural antibodies play a protective role against fungal and parasitic infections.

A B1 cell population characterized by spontaneous IgM secretion, effective T cell stimulation, and tonic intracellular signaling has been described (Griffin, Holodick, et al., J Exp Med, vol. 208 (1), 2011). Based on these criteria, a small population of B cells present in adult cord blood and peripheral blood expressing a unique CD20 ^pos CD27 ^pos CD43 ^pos CD5 ^pos CD70 ^neg phenotype can be classified as human B1 cells, which are phenotypically distinct from murine B1 cells. Importantly, this B cell population was particularly enriched in cord blood and was found to decline with age. It is noteworthy that although a significant proportion of human B1 cell-derived immunoglobulins bound prototypical natural antibody antigens such as phosphorylcholine and the dsDNA mimetic domain, their V _H and V _L regions do not exhibit the distorted gene repertoires of their murine counterparts, and the V _H genes of human B1 cell immunoglobulins contain N-nucleotide additions in the V _H -D _H and D _H -J _H fusions.

Much of the evidence for the protective role of natural antibodies against certain pathogens comes from in vivo experiments using genetically modified mice whose B cells were unable to secrete IgM or lacked certain B cell populations, such as B1a cells. These experiments demonstrated that mice lacking natural IgM derived from B1 cells experienced significantly increased mortality after influenza virus infection, although B2-derived IgM was still produced in response to the infection. Notably, infected mice could be protected from lethal infection by transferring serum from uninfected wild-type mice containing natural IgM, but not serum from mice lacking soluble IgM. The antiviral activity of natural IgM was further demonstrated using hemagglutinin (HA) inhibition assays using bronchoalveolar lavage (BAL) fluid from uninfected mice. Although nearly 100% neutralizing activity was achieved with IgM-containing BAL fluid, depletion of IgM antibodies reduced HA inhibition by >75%. Thus, it can be suggested that natural IgM provides protection against serious influenza virus infections by neutralizing HA activity and IgM-induced clearance of viral particles. Natural IgM also provides significant protection against serious bacterial infections, such as those caused by Streptococcus pneumoniae, the most common cause of pneumonia. Experiments using transgenic mice have demonstrated that the presence of B1a cells and natural IgM is critical for survival during S. pneumoniae infection, while mice lacking B1a cells and natural IgM antibodies are more susceptible to fatal infection (Haas, Roe et al., Immunity, vol. 23 (1), 2005).

Interestingly, another study demonstrated that transfer of IgG-depleted serum from naive young mice to infected mice lacking natural IgM resulted in a protective effect, whereas serum from aged donor mice had no beneficial effect (Holodick, Vizconde, et al., J Immunol 196 (10), 2016). These data suggest that serum levels of protective natural IgM antibodies decline with age, consistent with the observed decrease in B1a cells in older mice. Overall, it can be concluded that natural IgM and possibly IgA antibodies serve as a first line of defense against invading pathogens, which is important for bridging the time lag required for the production of high-affinity class-switched immune antibodies by FO/B2 cells.

Mice deficient in IgM antibody secretion have also been shown to be more susceptible to developing certain diseases, such as autoimmunity or atherosclerosis, which share the common feature of proinflammatory states independent of infectious agents (Boes, Schmidt et al., Proc Natl Acad Sci USA, vol. 97 (3), 2000) (Lewis, Malik et al., Circulation, vol. 120 (5), 2009). Similar findings have been reported in humans, where patients with an autoimmune disorder such as systemic lupus erythematosus (SLE) have reduced levels of IgM antibodies in their peripheral blood, although the reason for this reduction remains unclear (Senaldi, Ireland et al., Arthritis Rheum, vol. 31 (9), 1988).

The role of natural IgM/IgA in facilitating the clearance of apoptotic cells and cellular waste from the circulation provides a mechanistic explanation for their regulatory properties and their role in chronic inflammatory diseases. The cell membrane of apoptotic cells is susceptible to oxidative damage, leading to the formation of a heterogeneous mixture of oxidized phospholipids and degradation products. Both enzymatic and non-enzymatic processes can lead to the oxidation of phospholipids, primarily polyunsaturated fatty acids such as phosphatidylcholine, a major component of the cell membrane.

Enzymatic reaction mediators such as cytochrome P450, lipoxygenases and cyclooxygenases, and non-enzymatic components including reactive oxygen species (ROS) or free radicals generated as a result of cellular oxygen use in the mitochondria, or environmental factors such as smoking, are involved in the lipid peroxidation reaction of cell membranes, although the exact mechanisms still need to be elucidated (Bochkov, Oskolkova et al., Antioxid Redox Signal, vol. 12 (8), 2010).

Continuous fragmentation of oxidized phospholipids such as oxidized phosphatidylcholine (oxPC), oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine (oxPAPC), oxidized cardiolipin (oxCL), oxidized phosphatidylserine (oxPS), and oxidized phosphatidylethanolamine (oxPE) results in the formation of highly reactive cleavage products such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), which then react with the amino groups of exposed lysine residues of proteins and with neighboring aminophospholipid molecules. Furthermore, 2-(ω-carboxyethyl)pyrrole (CEP) is an adduct between (E)-4-hydroxy-7-oxohept-5-enoic acid, an oxidative moiety of docosahexaenoic acid, and the amino groups of lysines or aminophospholipids. The resulting oxidized lipid-protein adducts and modified lipids with altered structures are neo-autoangiogens, also known as oxidative stress-specific epitopes (OSEs), and represent damage-associated molecular patterns (DAMPs) recognized by a variety of cellular and soluble pattern-recognition receptors (PRRs) of the innate immune system. It has been established that OSEs can be important targets for monoclonal natural antibodies derived from mice, and it has been found that 20% to 30% of natural IgM antibodies in mouse and human sera recognize various OSEs, including MDA and 4-HNE adducts and the phosphocholine headgroup exposed by oxPC or oxPAPC (hereinafter referred to as OSE-specific IgM) (Chou, Fogelstrand et al., J Clin Invest, vol. 119 (5), 2009).

Regardless of the trigger, all apoptotic mammalian cells from any cell source display OSE on their surface, and OSE abundance increases as the apoptotic process progresses. Apoptotic cells are recognized by OSE-specific IgM and possibly IgA antibodies, and their binding induces persistent clearance of cellular debris. Although some soluble PRRs, such as C-reactive protein (CRP), can bind to apoptotic cells, their clearance by macrophages is reduced fourfold in the absence of IgM antibodies, suggesting a critical and non-redundant role for natural IgM in this process. Upon binding to apoptotic cells, the pentameric IgM molecule adopts a mushroom-shaped conformation with a central protrusion in the Fc region, likely to recruit C1q, a protein belonging to the complement system. C1q then cleaves and recruits other serum proteins of the complement cascade, resulting in the deposition of large quantities of cleaved fragments, called iC3b, on the surface of apoptotic cells. Apoptotic cells are thus opsonized by iC3b and can be recognized by dendritic cells and macrophages expressing specific receptors, such as complement receptor 3 (CR3) or CD91, which trigger downstream intracellular signaling processes leading to the engulfment of cellular debris and the production of anti-inflammatory molecules such as IL-10 and TGFβ. Clearance of apoptotic cells and cellular waste products via this mechanism is anti-inflammatory, and therefore, a minimal serum level of OSE-specific IgM is required to maintain normal tissue homeostasis.

However, in some situations, the balance between OSE formation and clearance via innate immune mechanisms is lost due to increased rates of apoptosis or oxidative stress, or impaired immune function, such as decreased serum levels of OSE-specific IgM antibodies. In such situations, apoptotic cells presenting large quantities of OSE accumulate in tissue or the vascular wall, where they release intracellular components that are not normally present in the bloodstream and that have the ability to trigger both innate and adaptive immune responses and therefore participate in chronic inflammatory processes.

Importantly, the fluorolipids present in low-density lipoprotein (LDL) can also be modified by oxidative stress, leading to the formation of OSEs similar to those formed in the membrane of apoptotic cells. The role of OSEs as initiators of chronic inflammatory responses is well known in cardiovascular diseases such as atherosclerosis. These patients typically exhibit high concentrations of various OSE species exposed to oxidized LDL (oxLDL) in the intima of medium- and large-sized arteries, where they associate with cellular PRRs such as SRA-I, SRA-II, SRB-I, CD36, TLR-4, and TLR-6, which are expressed by macrophages located in the subendothelial space. Recognition of oxLDL by the heterotrimeric CD36/TLR-4/TLR-6 receptor complex results in uncontrolled uptake by macrophages, priming of the inflammasome via NFκB signaling, and release of proinflammatory cytokines such as IL-6, TNFα, and IL-1β, chemoattractants such as CCL2 and CXCL8, leading to the recruitment and activation of additional monocytes and T cells to the site of injury and the creation of a proinflammatory environment (Stewart, Stuart et al., Nat Immunol, vol. 11 (2), 2010). Enhanced oxLDL uptake leads to the deposition of cholesterol-rich lipids in intracellular endosomes and the transformation of macrophages into so-called foam cells, which are considered a hallmark of atherosclerotic lesion development. Chronic oxLDL uptake combined with inefficient intracellular cholesterol degradation can lead to the formation of cholesterol crystals, which damage lysosomal membranes. This then activates the NOD/LRR/pyrin domain protein 3 (NLRP3) inflammasome complex, further enhancing IL-1β secretion and the spread of the inflammatory response. Not only macrophages but also endothelial cells can recognize OSE via the cellular PRR LOX-1, leading to oxLDL uptake and cholesterol accumulation in intracellular compartments. Cholesterol release into the cytoplasm due to lysosomal membrane damage induces the endoplasmic reticulum (ER) stress response, leading to apoptosis of the corresponding macrophages and endothelial cells.

This then leads to the development of plaques, which contain an acellular necrotic core filled with cellular debris and lipid mush and covered with a fibrous latticework. When these plaques rupture or break, the released material comes into contact with the circulating blood coagulation system, thereby triggering thrombus formation, which can lead to serious clinical manifestations such as myocardial infarction or stroke.

In healthy individuals, OSEs exposed to oxLDL bind via OSE-specific IgM and possibly IgA antibodies, thereby inhibiting oxLDL binding to cellular PRRs expressed on macrophages. This prevents uncontrolled oxLDL uptake, foam cell formation, and proinflammatory responses. Furthermore, OSE-specific IgM and possibly IgA antibodies limit the accumulation of apoptotic cells in developing lesions by recognizing OSEs on the surface of apoptotic cells and inducing their persistent clearance in a C1q-dependent manner.

According to this explanation, it would be expected that individuals with reduced serum levels of OSE-specific IgM and possibly IgA may be predisposed to developing cardiovascular disease (CVD). In fact, several human studies have demonstrated that plasma levels of OSE-specific IgM inversely correlate with the risk of developing CVD. For example, the concentration of MDA-LDL-specific IgM antibodies inversely correlates with carotid intima-media thickness or the risk of developing stenosis of >50% of the coronary artery diameter (Karvonen, Paivansalo et al., Circulation, vol. 108 (17), 2003) (Tsimikas, Brilakis et al., J Lipid Res, vol. 48 (2), 2007). According to these data, IgM titer to oxPC has been reported to be inversely correlated with the incidence of heart attacks or strokes (Fiskesund, Stegmayr et al., Stroke vol. 41 (4), 2010) (Gronlund, Hallmans et al., Eur J Cardiovasc Prev Rehabil vol. 16 (3), 2009). Thus, these data suggest that OSE-specific IgM antibodies have the ability to reduce the risk of developing CVD, although the mechanisms are not fully understood.

OSE accumulation in disease-related tissues has also been found in patients with other “sterile” diseases, such as “sterile” acute lung injury (ALI), age-related macular degeneration (AMD), multiple sclerosis (MS), or Alzheimer’s disease (AD) (Weismann and Binder, Biochim Biophys Acta, vol. 1818 (10), 2012). Furthermore, OSE accumulate in a wide variety of acute situations induced by pathogenic infections. For example, the avian influenza H5N1 virus leads to ALI in the lungs of infected mice, and lung tissue is characterized by high levels of oxidized phospholipids and OSE (Imai, Kuba et al., Cell, vol. 133 (2), 2008).

The main source of phospholipids in the lung is surfactant, which forms a film at the alveolar fluid-air interface and reduces surface tension. Surfactant contains up to 90% phospholipids, including phospholipids with polyunsaturated fatty acids that can be oxidized. BAL fluid from mice infected with the H5N1 influenza virus contains OSEs, such as oxPAPC and MDA adducts, which stimulate the secretion of large amounts of the proinflammatory cytokine IL-6 by alveolar macrophages via the TLR4-TRIF-TRAF6 signaling pathway. It is noteworthy that the transfer of purified synthetically oxidized surfactant phospholipids into the lungs of uninfected healthy wild-type mice is sufficient to induce robust alveolar macrophage activation and ALI, and IL-6 production in response to oxidized surfactant phospholipids can be reduced by the murine monoclonal antibody EO6, which recognizes the phosphocholine headgroup of oxPC and oxPAPC. Importantly, both phosphocholine-exposed oxPC and MDA adducts were detected in the lung tissue of patients who died from acute respiratory distress syndrome (ARDS), the most severe form of ALI induced by H5N1 and SARS coronavirus (CoV) infections. Furthermore, the formation of oxidized phospholipids and OSE was also detected in the lung tissue of anthrax-infected rhesus macaques, anthrax-infected rabbits, monkeypox-infected cynomolgus monkeys, and Yersinia pestis-infected cynomolgus monkeys. Thus, across multiple species, infection with various lethal pulmonary pathogens, such as the H5N1 influenza virus, SARS-CoV, anthrax, Y. pestis, or monkeypox virus, initiates OSE formation in the lungs. These data demonstrate that oxidative stress and OSE accumulation are important drivers of macrophage activation and ALI across species.

A novel coronavirus, SARS-CoV-2, has now been described, responsible for the ongoing global pandemic outbreak of severe acute respiratory syndrome (SARS-CoV-2) (COVID-19). According to data available as of April 5, 2020, more than 1.4 million people have been infected, with over 65,000 deaths in over 200 countries and territories. The overall mortality rate for SARS-CoV-2 infections is >5%, and the majority of deceased patients have ARDS.

Based on the prior art, there is a need to develop additional means and methods for the effective treatment or prevention of a disorder or disease caused by the SARS-CoV-2 coronavirus.

The present invention is based on the unexpected finding that in individuals infected with SARS-CoV-2, the likelihood of developing severe symptoms and ARDS is significantly dependent on risk factors such as older age and comorbid medical conditions, including CVD, diabetes, respiratory disease, or hypertension. Furthermore, epidemiological data suggest a relatively lesser severity of COVID-19 disease in women compared to men, as well as in individuals who have been vaccinated, for example, against pneumococcus or tuberculosis (e.g., BCG vaccination (w/BCG). Importantly, the core pathological features of ARDS in SARS-CoV-2-infected patients appear to be similar between H5N1 and SARS-CoV infections and are characterized by inflammatory cell accumulation, edema formation, and a marked increase in proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNFα, reminiscent of a cytokine storm profile. This suggests a common feature of eventual immune evasion in infected individuals, regardless of the pathogen.

Thus, the present invention is based on the hypothesis that a possible common feature of host immune deficiency in various infectious diseases, as well as in SARS-CoV-2-infected patients, is the massive formation of oxidized phospholipids and accumulation of OSE in the lungs, which stimulates the production of proinflammatory cytokines in macrophages and thereby initiates the deterioration phase of COVID-19 (and other inflammatory infectious diseases).

In the context of the present invention, high levels of circulating OSE-specific IgM and possibly IgA antibodies provide protection because they bind to oxidized phospholipids and OSE and thereby promote safe clearance via the C1q pathway, accompanied by the production of anti-inflammatory cytokines such as IL-10 and TGFβ by phagocytes, which in turn counteracts the activation of alveolar macrophages and the induction of fatal cytokine storm syndrome and ARDS.

The possible involvement of OSE-specific IgM/IgA antibodies in the pathology of COVID-19 disease is supported by several observations.

First, elderly patients (>60 years) have a significantly higher mortality rate (up to 30%) compared to younger patients (0-29 years), whose mortality rate is below 1%. The higher predisposition of elderly patients to develop severe forms of ALI and ARDS is consistent with data showing that the number of human B1 cells and natural B1-derived antibodies decreases with age (Griffin, Holodick et al., J Exp Med, vol. 208 (1), 2011) (Rodriguez-Zhurbenko, Quach et al., Front Immunol, vol. 10, 2019)).

Second, patients with underlying medical conditions such as CVD have a significantly higher mortality rate (>10%) compared to patients without preexisting conditions. As described above, susceptibility to developing CVD is inversely associated with serum levels of OSE-specific IgM antibodies, which is also a risk factor for the development of severe forms of ALI and ARDS. This correlation is further supported by the fact that men have more severe infection and a higher mortality rate compared to women, which is consistent with the higher serum IgM levels found in women compared to men (Butterworth, McClellan et al., Nature, vol. 214 (5094), 1967) (Palmer, Schulze et al., J Dev Orig Health Dis, vol. 6 (6), 2015).

Taken together, these data indicate an inverse correlation between the serum level of OSE-specific IgM and possibly IgA antibodies and the severity of COVID-19, and, accordingly, from the perspective of the present invention, the administration of OSE-specific IgM and possibly IgA antibodies to COVID-19 patients is a highly effective way to alleviate ALI and ARDS induced by SARS-CoV-2 infection (and other infections) or to protect infected individuals from developing severe symptoms such as ALI and ARDS.

In general, the proinflammatory diseases described here, which are either pathogen-independent chronic conditions or infection-induced acute conditions, share a common characteristic feature such as a high load of oxidized phospholipids and OSE in inflamed tissues resulting from reduced levels of OSE-specific IgM and possibly IgA antibodies.

In seemingly very different diseases, significant accumulation of OSE shifts the balance from the constant clearance of OSE-presenting debris or molecules toward the sensing of OSE by cellular PRRs expressed by macrophages, leading to their activation and the release of large amounts of proinflammatory cytokines such as IL-6. By targeting this inflammatory pathway, administration of OSE-specific IgM and/or IgA antibodies or plasma pools enriched with them to affected patients restores homeostatic conditions according to the present invention, facilitating the clearance of OSE-containing cellular debris or molecules such as oxLDL via C1q-dependent mechanisms, which is accompanied by the production of anti-inflammatory cytokines such as IL-10 and TGFb by phagocyte cells.

The new data obtained in the creation of the present invention are all the more unexpected since it is known to those skilled in the art that oxPL and OSE exhibit anti-inflammatory and protective effects in sepsis and acute injuries.

In fact, as already mentioned above, the anti-inflammatory actions of oxPL and OSE are concentration-dependent and include (1) inhibition of “sterile” acute lung injury induced by inflammatory mediators of viral and bacterial origin (Ma et al., Am J Physiol Lung Cell Mol Physiol. 286 (2004): 808–816; Nonas et al., Am J Respir Crit Care Med. 173 (2006): 1130–1138); (2) inhibition of "aseptic" acute lung injury induced by noxious mechanical ventilation, and therefore it has been suggested that the use of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine (PEIPC)- and 1-palmitoyl-2-(5,6-ethoxycyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC)-like stabilizing compounds may have beneficial effects in other models of "aseptic" lung injury such as ischemia/reperfusion (Nonas et al., Crit Care. 12, 2008, R27); and (3) inhibition of pulmonary vascular leakage and inflammation in secondary acute lung injury caused by acute necrotizing pancreatitis (Li et al., Pancreas. 35, 2007, pp. 27-36).

These anti-inflammatory actions are mediated by enhancing endothelial barrier function (Birukov et al., Circ Res. 95 (2004): 892–901; Birukova et al., Am J Physiol Lung Cell Mol Physiol. 292 (2007): 924–935), inducing signaling pathways that lead to upregulation of anti-inflammatory genes, and inhibiting the expression of proinflammatory genes (Eligini et al., Cardiovasc Res. 55 (2002): 406–415; Ma et al., Am J Physiol Lung Cell Mol Physiol. 286 (2004): 808–816; Otterbein et al., Nat Med. 6 (2000): 422–428; Otterbein et al. al., Nat Med. 9 (2003): 183-190) and by preventing the interaction of proinflammatory bacterial products with host cells (Bochkov et al., Nature. 419 (2002): 77-81; Walton et al., Arterioscler Throm Vase Biol. 23 (2003): 1197-1203). However, for patients suffering from ARDS, which is induced by infection with pulmonary pathogens such as SARS-CoV-2, SARS-CoV, and possibly H5N1 influenza viruses, the present invention hypothesizes several mechanisms involved in the formation of ROS and the occurrence of oxidative stress in the lungs of affected patients when the accumulation of oxPL and OSE reaches concentrations high enough to stimulate the biological actions described herein.

In the context of the general principle, the present invention provides not only additional means and methods for the treatment or prevention of a disorder or disease caused by the SARS-CoV-2 coronavirus, in particular (severe) symptoms such as ALI and ARDS.

Rather, the above general principle applies in the more general terms of the present invention to the treatment or prevention of a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject.

Thus, according to the present invention, a subset of natural IgM and/or IgA antibodies, preferably OSE-specific IgM and/or IgA antibodies, is used to treat or prevent a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject.

From the position of the known prior art, the present invention was based on the task of developing additional means and methods for the treatment or prevention of a disorder or disease associated/linked/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject.

The stated problem is solved with the help of the proposed embodiments of the invention, characterized in the claims.

Thus, the present invention relates to a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method of treating or preventing a disorder or disease associated/linked/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject.

More specifically, the first object of the present invention is a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, wherein said natural IgM and/or IgA are obtained from IgM and/or IgA-enriched plasma pools from healthy individuals.

The second object of the present invention is a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, wherein said antibody is a recombinant human monoclonal natural IgM antibody.

A third object of the present invention is a vaccine comprising a compound that induces the formation of natural IgM and/or IgA antibodies, intended for use in a method for reducing or preventing clinical signs or a disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, wherein said vaccine comprises a pharmaceutically acceptable carrier or excipient. Preferably, the present invention relates to said vaccine intended for use in a method for reducing or preventing clinical signs or a disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, wherein said compound induces a human natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes.

As stated above, it was unexpectedly discovered in the creation of the invention and described in the present application that a subset of natural IgM and/or IgA antibodies can be used to treat or prevent a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, i.e. a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes can be used.

Thus, the present invention relates generally to a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method of treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject.

In the context of the present invention, the term “natural IgM and/or IgA antibody” means an IgM and/or IgA isotype antibody present in the serum of a healthy animal or a healthy human, which is produced by B1 cells regardless of infection or immunization and which has auto- and/or polyreactive antigen-binding properties.

Although the present invention focuses on "natural IgM and/or IgA antibodies", when referring to "natural antibodies", the term refers not only to "natural IgM and/or IgA antibodies", but may also refer to "natural IgG", in particular IgG2.

The human or humanized natural IgM and/or IgA antibody according to the present invention is also characterized in that it recognizes oxidized phospholipids and/or oxidation-specific epitopes and, therefore, belongs to a subgroup of the spectrum of natural IgM and/or IgA in a subject.

Thus, the human or humanized natural IgM and/or IgA antibody of the present invention, which recognizes oxidized phospholipids and/or oxidation-specific epitopes, belongs to a subgroup/subfraction/subpopulation of the natural IgM and/or IgA spectrum in a subject. The terms "subfraction" and "subpopulation" are used below as equivalent to the term "subgroup."

In a preferred embodiment of the invention, the human or humanized natural IgM and/or IgA antibody according to the present invention, which recognizes oxidized phospholipids and/or oxidation-specific epitopes, belongs to a subgroup/subfraction/subpopulation of the spectrum of natural IgM and/or IgA from IgM- and/or IgA-enriched plasma pools of healthy individuals specified in the present description. Said IgM- and/or IgA-enriched plasma pools of healthy individuals are themselves preferably enriched in natural IgM and/or IgA antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes.

Methods for enriching antibodies that specifically recognize oxidized phospholipids and/or oxidation-specific epitopes are known to those skilled in the art and can be used conventionally. For example, antibody affinity purification methods can be used to enrich a sample for specific antibodies. Affinity purification methods based on the antigen-specific affinity of only those antibodies in a sample that bind to a specific antigen molecule (i.e., oxidized phospholipids and/or oxidation-specific epitopes as defined in the present invention) via their specific antigen-binding domains are known to those skilled in the art.

In preferred embodiments of the invention, enrichment with IgM and/or IgA antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes means that:

the concentration of IgM and/or IgA antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes is more than 5% higher than the concentration in the total spectrum of IgM and/or IgA in the subject, preferably higher by more than 10%, 20%, 30%, 40%, 50%, 60% or 70% or even more than 80%, 90%, 95% or 99%.

In other preferred embodiments of the invention, the concentration of IgM and/or IgA antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes is 5%-70% higher than the concentration in the total spectrum of IgM and/or IgA in the subject, preferably 10%-70% higher, 20%-70% higher, 30%-70% higher, 40%-70% higher, 50%-70% higher, or 60%-70% higher.

In other preferred embodiments of the invention, the concentration of IgM and/or IgA antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes is 5%-99% higher than the concentration in the total spectrum of IgM and/or IgA in the subject, preferably higher than 10%-99%, higher than 20%-99%, higher than 30%-99%, higher than 40%-99%, higher than 50%-99%, higher than 60%-99%, higher than 70%-99%, higher than 80%-99% or higher than 90%-99%.

In other preferred embodiments of the invention, the human or humanized natural IgM and/or IgA antibody of the present invention that recognizes oxidized phospholipids and/or oxidation-specific epitopes belongs to a subgroup/subfraction/subpopulation of the total IgM and/or IgA spectrum in an individual (and/or, for example, belongs to a subgroup/subfraction/subpopulation of the total IgM and/or IgA spectrum from IgM and/or IgA-enriched plasma pools from healthy individuals referred to herein), comprises primarily antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes. The term "contains primarily antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes" preferably means that the subgroup/subfraction/subpopulation is substantially pure, i.e., consists primarily of antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes.

Preferably, the term "pure" or "essentially consisting of" means that the composition comprising said human or humanized natural IgM and/or IgA antibody according to the present invention contains more than 30% of antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes in the total spectrum of IgM and/or IgA antibodies in said composition, preferably more than 35%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100%.

In other embodiments of the invention, the term "pure" means that a composition comprising said human or humanized natural IgM and/or IgA antibody according to the present invention comprises from 30% to 100% of antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes in the total spectrum of IgM and/or IgA antibodies in said composition, preferably from 35% to 100%, from 40% to 100%, from 50% to 100%, from 60% to 100%, from 70% to 100%, from 80% to 100%, from 90% to 100% or from 99% to 100%.

The terms "human" and "humanized" antibodies in the context of the present description have the meanings indicated below.

The terms "recognizes," "binds," and "binds to" are used interchangeably in the context of the present invention and refer to the recognition and binding (interaction) of at least two "antigen-interacting sites" with each other. The term "antigen-interacting site" according to the present invention refers to a polypeptide motif, i.e., a portion of the antibody or antigen-binding fragment according to the present invention, that has the ability to specifically interact with a specific antigen or a specific group of oxidized phospholipid antigens and/or oxidation-specific epitopes. It should also be clear that said binding/interaction also refers to "specific recognition." The term "specifically recognizes" according to the present invention means that an antibody has the ability to specifically interact with and/or bind to at least two molecules, each of which is an oxidized phospholipid and/or an oxidation-specific epitope as described herein. Antibodies can recognize, interact with, and/or bind to different epitopes on oxidized phospholipids and/or oxidation-specific epitopes. This term refers to the specificity of the antibody molecule, i.e., its ability to distinguish between specific regions of oxidized phospholipids and/or oxidation-specific epitopes.

The term "specific interaction" in the context of the present invention means that the antibody or antigen-binding fragment thereof proposed in the invention does not cross-react, or substantially does not cross-react, with (poly)peptides of similar structures. Thus, the antibody proposed in the invention specifically binds to/interacts with oxidized phospholipid structures and/or oxidation-specific epitopes.

The term "oxidized phospholipids and/or oxidation-specific epitopes (referred to above and below in this description as "OSE")" in the context of the present invention refers to an immunogenic structure that is created as a result of the reaction of lipid peroxidation present in mammalian cell membranes, lipoproteins such as low-density lipoprotein and high-density lipoprotein, in bacterial cell walls and/or viral envelope membranes, and which can be specifically recognized by an IgM and/or IgA antibody.

The structures of "oxidized phospholipids and/or oxidation-specific epitopes" are well characterized, and the present invention is not limited to particular "oxidized phospholipids and/or oxidation-specific epitopes".

Preferably, the "oxidized phospholipids and/or oxidation-specific epitopes" are oxidized phosphatidylcholine, oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, oxidized phosphatidylethanolamine, and terminal cleavage products such as malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and 2-(ω-carboxyethyl)pyrrole (CEP).

The cross-reactivity of a panel of antibodies under study can be tested, for example, by assessing the binding of said panel of antibodies under standard conditions (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988 and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999) to the oxidized phospholipid and/or oxidation-specific epitope of interest, as well as to several more or less similar (structurally and/or functionally) oxidized phospholipids and/or oxidation-specific epitopes.

Only those constructs (i.e., antibodies, their antigen-binding fragments, etc.) that bind to a specific structure of an oxidized phospholipid and/or an oxidation-specific epitope, such as a specific epitope of an oxidized phospholipid and/or an oxidation-specific epitope, but do not bind or substantially do not bind to any of the other epitopes of the same oxidized phospholipid, are considered specific for the epitope of interest or the oxidized phospholipid and/or the oxidation-specific epitope, and they are selected for additional studies using the method provided herein. These methods may include, among other things, binding studies, blockade studies, and competition studies with structurally and/or functionally closely related molecules. Binding studies also include FACS analysis, surface plasmon resonance (SPR, e.g. using BIAcore®), analytical ultracentrifugation, isometric titration calorimetry, fluorescence anisotropy, fluorescence spectroscopy or radiolabeled ligand binding assays.

Specificity can thus be determined experimentally using methods known in the art and those described herein. Such methods include (but are not limited to) Western blotting, ELISA, RIA, ECL, IRMA, and peptide scanning.

The term "treatment or prevention" in the context of the present invention refers to the treatment or prevention of a deficiency of natural IgM/IgA antibodies.

In this context, "natural IgM/IgA antibody deficiency" refers generally to deficiencies that share the commonality of a lack of sufficient amounts of naturally occurring B1 cell-produced antibodies (primarily IgM and IgA), which contributes (significantly) to the pathogenesis of these conditions. Since NAD is considered a major factor in a wide variety of diseases through a similar, specific mechanism, such as the inability of the innate immune system to effectively remove oxidative stress-specific epitope (OSE) degradation products, it seems possible that NAD modulation strategies in affected patients may represent a valuable new strategy for the prevention and treatment of these conditions. Since this critical event initiates the development of many different diseases that share a common cause, the invention proposes to introduce a new term for this condition, oxidative stress-specific epitope accumulation syndrome (OSEAS).

Thus, as indicated in the first, second and third aspect of the present invention, respectively, NAD can be modulated in 2 ways, namely: I) by administering natural B1 antibody preparations for therapeutic intervention or II) by inducing B1 antibodies for the purpose of preventing disease through active immunization (e.g. BCG, Pneumococcus vaccination).

Without getting into a specific theory, what is common to all NAD is that there is a continuous fragmentation of oxidized phospholipids such as oxidized phosphatidylcholine, oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, and oxidized phosphatidylethanolamine, resulting in the formation of highly reactive breakdown products such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), which then react with the amino groups exposed on lysine residues of proteins and with neighboring aminophospholipid molecules. Furthermore, 2-(ω-carboxyethyl)pyrrole (CEP) is an adduct between (E)-4-hydroxy-7-oxohept-5-enoic acid, an oxidative moiety of docosahexaenoic acid, and amino groups of lysines or aminophospholipids. The resulting oxidized lipid-protein adducts and modified lipids with altered structures are neo-autoangiogens, also called oxidation-specific epitopes (OSEs) (i.e., herein collectively referred to as "oxidized phospholipids and/or oxidation-specific epitopes"), and they are damage-associated molecular patterns (DAMPs) recognized by a variety of cellular and soluble pattern recognition receptors (PRRs) of the innate immune system. Apoptotic cells are recognized by the IgM antibodies proposed in the present invention, which are specific for an oxidized phospholipid and/or an oxidation-specific epitope, and their binding induces the persistent clearance of cellular debris. Upon binding to apoptotic cells, the pentameric IgM molecule adopts a mushroom-shaped conformation with a central protruding region in the Fc region, likely to recruit C1q, a protein belonging to the complement system. C1q then cleaves and recruits other serum proteins of the complement cascade, resulting in the deposition of large quantities of cleaved fragments, called iC3b, on the surface of apoptotic cells. Thus, apoptotic cells are opsonized by iC3b and can be recognized by dendritic cells and macrophages expressing specific receptors, such as complement receptor 3 (CR3) or CD91, which trigger downstream intracellular signaling processes leading to the engulfment of cellular debris and the production of anti-inflammatory molecules such as IL-10 and TGFβ. Clearance of apoptotic cells and cellular waste products via this mechanism is anti-inflammatory, and therefore, a minimal serum level of OSE-specific IgM is required to maintain normal tissue homeostasis.

Thus, according to the present invention, high levels of circulating IgM antibodies specific for an “oxidized phospholipid and/or oxidation-specific epitope” have a protective effect because they bind to oxidized phospholipids and OSE and thereby promote safe clearance using the C1q pathway, which is accompanied by the production of anti-inflammatory cytokines such as IL-10 and TGFβ by phagocytes, which in turn counteracts the activation of alveolar macrophages and the induction of fatal cytokine storm syndrome.

As will be described in more detail below, the term "treatment" and/or "prevention" etc. in the context of the present description generally means achieving the desired pharmacological and/or physiological effect. Thus, in the context of the present invention, treatment may refer to the treatment of (acute) states of a certain disease, but may also refer to prophylactic treatment in terms of complete or partial prevention of the disease or its symptom. Preferably, the term "treatment" means therapeutic treatment in terms of partial or complete cure of the disease and/or side effect and/or symptom associated with the disease. The term "acute (condition)" in this regard means that the subject has symptoms of the disease. In other words, the subject to be treated actually needs treatment, and the term "acute treatment" in the context of the present invention refers to measures taken for the acute treatment of the disease after the onset of the disease or an outbreak of the disease. Treatment may also be prophylactic or preventative treatment, i.e. measures taken to prevent disease, such as to prevent infection and/or the onset of illness.

The antibody for use in the present invention is a humanized or human antibody, i.e., a fully human antibody. In another preferred embodiment, the antibody for use in the present invention is a murine antibody.

In a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof intended for use according to the present invention is a monoclonal or polyclonal antibody. In another preferred embodiment of the invention, the antibody or antigen-binding fragment thereof intended for use according to the present invention is a humanized or human antibody, namely a fully human antibody. In another preferred embodiment of the invention, the antibody or antigen-binding fragment thereof intended for use according to the present invention is a murine antibody.

In the context of the present invention, and as will be described in more detail below, the term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies have the advantage that they can be synthesized using a hybridoma culture that is substantially free of contamination by other immunoglobulins. The adjective "monoclonal" indicates that the antibody is from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. As noted above, monoclonal antibodies for use in the present invention can be produced using the hybridoma method described in Kohler, Nature 256, 1975, p. 495.

The term "polyclonal antibody," as used herein, refers to an antibody produced among or in the presence of one or more other non-identical antibodies. Generally, polyclonal antibodies are produced from a B lymphocyte in the presence of several other B lymphocytes that produce non-identical antibodies. Polyclonal antibodies are typically obtained directly from the body of an immunized animal.

The term "human antibody" or "fully human antibody" as used herein refers to an antibody that comprises protein sequences of only human immunoglobulin. A fully human antibody may contain murine carbohydrate chains if it is produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Similarly, a "mouse antibody" or "mouse antibody" refers to an antibody that comprises protein sequences of only mouse immunoglobulin. Alternatively, a "fully human antibody" may contain rat carbohydrate chains if it is produced in rats, in rat cells, or in hybridomas derived from rat cells. Similarly, a "rat antibody" refers to an antibody that comprises protein sequences of only rat immunoglobulin. Similarly, a "rabbit antibody" refers to an antibody that comprises protein sequences of only rabbit immunoglobulin. Similarly, a "rodent antibody" refers to an antibody that contains protein sequences of only rodent immunoglobulin. Fully human antibodies can also be produced, for example, using phage display, which is a widely used screening technology by which fully human antibodies can be produced and screened. Furthermore, human antibodies produced using phage display technology can be used in the context of the present invention. Phage display methods are described, for example, in US 5,403,484, US 5,969,108 and US 5,885,793. Other technologies that can be used to create fully human antibodies include a modification of mouse hybridoma technology. Transgenic mice are created that contain a human immunoglobulin locus that replaces their own mouse genes (see, for example, US 5,877,397).

The term "chimeric antibodies" refers to an antibody that comprises a variable region as provided herein fused or chimerized with a region of an antibody (e.g., a constant region) from another species, such as a human or a non-human animal (e.g., mouse, horse, rabbit, dog, cow, chicken).

In some aspects described in the context of the present invention, the term "antibody" also refers to recombinant human antibodies, homologous antibodies, and heterohybrid antibodies. The term "recombinant human antibody" includes all antibodies having human sequences that are produced, expressed, created, or isolated using recombinant techniques, such as antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes; antibodies obtained as a result of expression using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial library of human antibodies; or antibodies produced, expressed, created, or isolated using any other method that involves splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences. However, such antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, somatic mutagenesis in vivo), and as a result, the amino acid sequences of the VH and VL regions of the recombinant antibodies represent sequences that, although of human germline origin and related to human germline VH and VL sequences, may not naturally occur in the human germline antibody population in vivo.

The term "heterologous antibody" is used to refer to a transgenic non-human organism that produces the antibody. This term refers to an antibody that has an amino acid sequence or coding nucleotide sequence that corresponds to a sequence present in an organism that is not a transgenic non-human animal, and is typically different from the transgenic non-human animal.

The term "heterohybrid antibody" refers to an antibody that contains light and heavy chains from different organisms. For example, an antibody containing a human heavy chain associated with a murine light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric and humanized antibodies.

The term "antibody" also refers to humanized antibodies. "Humanized" forms of non-human (e.g., mouse or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain a minimal sequence derived from a non-human immunoglobulin.

Humanized antibodies are often human immunoglobulins (the recipient antibody) in which residues from the complementarity-determining region (CDR) of the recipient are replaced with residues from the CDR of a non-human species (the donor antibody), such as mouse, rat, or rabbit, that have the desired specificity, affinity, and potency. In some cases, residues from the Fv framework region of the human immunoglobulin are replaced with the corresponding residues of a non-human antibody. In addition, a humanized antibody may contain residues that are not present in either the recipient antibody or the imported CDR or framework sequences. These modifications are performed to further refine and optimize the antibody's performance. In general, a humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to regions of a non-human immunoglobulin, and all or substantially all of the FR regions have the consensus sequence of a human immunoglobulin. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. For further details, see Jones, Nature 321 (1986), pp. 522-525; Reichmann, Nature 332 (1998), pp. 323-327; and Presta, Curr Op Struct Biol 2 (1992), pp. 593-596.

A popular method for humanizing antibodies involves CDR grafting, in which a functional antigen-binding site from a non-human "donor" antibody is grafted onto a human "acceptor" antibody. CDR grafting methods are known in the art and are described, for example, in US 5,225,539, US 5,693,761, and US 6,407,213. Another similar method is to produce humanized antibodies from transgenic animals genetically engineered to contain one or more humanized immunoglobulin loci that are capable of gene rearrangement and gene conversion (see, for example, US 7,129,084).

Thus, in the context of the present invention, the term "antibody" refers to complete immunoglobulin molecules, as well as to parts of said immunoglobulin molecules (i.e., "antigen-binding fragment thereof"). In addition, the term refers, as described above, to modified and/or altered antibody molecules. The term also refers to antibodies obtained/synthesized recombinantly or synthetically. The term also refers to intact antibodies, as well as to antibody fragments, such as separated light and heavy chains, Fab, Fv, Fab', Fab'-SH, F(ab')2. The term "antibody" also includes (but is not limited to) fully human antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies, and antibody constructs such as single-chain Fv (scFv) or antibody-fusion proteins.

"Single-chain Fv" or "scFv" antibody fragments in the context of the present invention have the V _H and V _L domains of the antibody, wherein said domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the V _H and V _L domains, which provides the ability of the scFv to form the structure required for antigen binding. Technologies used to produce single-chain antibodies are described, for example, by Pluckthun in: The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore, eds., Springer-Verlag, NY, 1994, pp. 269-315.

For the purposes of this description, a "Fab fragment" consists of one light chain and the _CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The "Fc" region contains two fragments of the heavy chain, each containing the _CH2 and _CH3 domains of the antibody or containing the _CH2 , _CH3 , and _CH4 domains of the antibody. The two fragments of the heavy chain are held together by two or more disulfide bonds and by hydrophobic interactions between the _CH3 domains.

A "Fab fragment" contains one light chain and a portion of one heavy chain that contains the V _H domain and the _CH 1 domain, as well as a region between the _CH 1 and _CH 2 domains through which an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab') ₂ molecule.

The "F(ab') ₂ fragment" contains two light chains and two heavy chains, each containing a portion of the constant region between the _CH1 and _CH2 domains, which can form an interchain disulfide bond between the two heavy chains. Thus, the P(ab') ₂ fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

The "Fv region" contains the variable regions from both the heavy and light chains, but does not contain the constant regions.

The antibodies, antibody constructs, antibody fragments, antibody derivatives (all Ig-derived) used according to the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example by amino acid deletion(s), insertion(s), substitution(s), addition(s) and/or recombination(s) and/or any other modification(s) known in the art, individually or in combination. Methods for introducing said modifications into the DNA sequence encoding the amino acid sequence of the immunoglobulin chain are well known to those skilled in the art; see, for example, Sambrook, 1989, loc. cit. The term "Ig-derived domain" refers in particular to (poly)peptide constructs comprising at least one CDR. Fragments or derivatives of the above Ig-derived domains mean (poly)peptides that are parts of the above antibody molecules and/or that have been modified using chemical/biochemical or molecular biology methods. The corresponding methods are known in the art and are described, inter alia, in laboratory manuals (see Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd ed., 1989 and 3rd ed., 2001; Gerhardt et al., Methods for General and Molecular Bacteriology, ASM Press, 1994; Lefkovits, Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press, 1997; Golemis, Protein-Protein Interactions: A Molecular Cloning Manual, Cold Spring Harbor Laboratory Press, 2002).

The antibody in the context of the present invention intended for the above and below mentioned use is not particularly limited if it is "an IgM and/or IgA antibody recognizing oxidized phospholipids and/or oxidation-specific epitopes".

Thus, the antibody may be any antibody that specifically binds to or specifically recognizes or interacts with an oxidized phospholipid and/or an oxidation-specific epitope, i.e., a domain or antigen, preferably a surface antigen of an oxidized phospholipid and/or an oxidation-specific epitope. A person skilled in the art can easily generate said antibody directed against a given domain (i.e., an antigen, preferably a surface antigen of an oxidized phospholipid and/or an oxidation-specific epitope), and determine whether the corresponding antibody has the ability to detect/bind to a given domain, antigen, preferably a surface antigen of an oxidized phospholipid and/or an oxidation-specific epitope.

IgA antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes can induce anti-inflammatory or pro-inflammatory actions, which depend on the IgA subclass and the glycosylation pattern of the antibody. It is known in the art that IgA2 in immune complexes exerts pro-inflammatory effects on macrophages and neutrophils through binding to the Fc-alpha receptor, while IgA1 lacks these properties. Furthermore, anti-inflammatory IgA1 contains a higher amount of terminal sialic acid compared to IgA2, and removal of sialic acid increases the pro-inflammatory capacity of IgA1. In the sera of healthy individuals, IgA1 predominates over IgA2 (ratio 9:1), while a shift towards the IgA2 subclass is associated with inflammation and disease activity (Steffen et al., Nat Commun. vol. 11(1), 2020). Thus, the natural IgA antibody in the context of the present invention is preferably an anti-inflammatory IgA1 antibody and is preferably characterized by a high sialic acid content.

According to the first aspect of the invention, the above-mentioned human or humanized natural IgM and/or IgA antibody, which recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, according to the present invention is a natural IgM and/or IgA, which is derived from IgM and/or IgA-enriched plasma pools from healthy individuals.

As will be further described in more detail below, in said first subject matter of the invention, the human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes may be represented by both natural IgM and/or IgA and preparations enriched with natural IgM and/or IgA.

Thus, in a preferred embodiment of the invention, the IgM and/or IgA enriched plasma pools from healthy individuals comprise a product which is an IgM and/or IgA enriched "intravenous normal immunoglobulin" (IVIG).

Intravenous normal immunoglobulin (IVIG) products contain a high dose of pooled immunoglobulin G (IgG) and are derived from the plasma of a large pool of donor blood. IVIG was initially used to treat infectious diseases. Since the early 1980s, evidence has emerged that IVIG can also modulate the immune system.

In some immune-mediated diseases (such as idiopathic thrombocytopenic purpura, Kawasaki syndrome, Guillain-Barré syndrome, severe dermatomyositis, graft-versus-host disease, and septic shock), IVIG has been shown to be effective. Although the exact mechanism of action of IVIG in autoimmune and immune-regulatory disorders remains poorly understood, IVIG can block Fc receptors on inflammatory cells, neutralize autoantibodies, modulate cytokine production by leukocytes, and block complement activation (Ott et al., J Allergy Clin Immunol. 108, 2001; Kazatchkine et al., Int Rev Immunol 5, 1989; Basta et al., J Clin Invest 84, 1989).

Using in vitro models such as lectin stimulation and the mixed lymphocyte response (MLR), it has been demonstrated that marketed IVIG preparations influence immune cell activation and proliferation through one of their mechanisms of action (Nachbaur et al., Immunology 90(212), 1997; Andersson et al., Immunol Rev. 139(21), 1994; Amran et al., Clin Immunol Immunopathol 73(180), 1994).

Furthermore, it has been suggested that cytokine modulation by IVIG may, at least in part, account for the beneficial effects observed in human bone marrow or solid organ transplant recipients receiving IVIG (Sullivan et al., N Engl J Med 323, 1990; Peraldi et al., Transplant 62(1670), 1996).

The two main uses of IVIG are replacement therapy for primary or acquired antibody deficiency disorders and therapeutic modulation of the immune system in patients with autoimmune or inflammatory conditions.

Pentaglobin® is a marketed IVIG specifically enriched with IgM and IgA. The active ingredients of Pentaglobin® are human plasma proteins (50 mg/ml), of which at least 95% are immunoglobulins, including immunoglobulin G (IgG) 38 mg (76%), immunoglobulin M (IgM) 6 mg (12%), and immunoglobulin A (IgA) 6 mg (12%). A more detailed distribution of IgG subclasses is known and corresponds to approximately 63% (IgG1), 26% (IgG2), 4% (IgG3), and 7% (IgG4). The specified content of toxin-binding and neutralizing antibodies to various gram-positive and gram-negative bacteria, such as Escherichia coli, Pseudomonas, and Klebsiella, is particularly suitable for the treatment of serious bacterial infections and for immunoglobulin replacement in immunocompromised patients. Pentaglobin® is currently the only immunoglobulin-containing product approved for this purpose. A meta-analysis of studies evaluating the efficacy of adjuvant therapy for sepsis and septic shock demonstrated that the use of immunoglobulins significantly reduces the risk of mortality in patients with sepsis. This effect is significantly more intense with IgM-enriched immunoglobulin than with standard immunoglobulins.

Pentaglobin® has been used for a wide range of off-label indications. Viral heart disease, also known as myocarditis, is a heart condition caused by a virus. The virus attacks the heart muscle, causing inflammation and disruption of the electrical pathways that ensure the heart beats properly. Treatment with Pentaglobin® has been reported to be highly effective in reducing myocardial inflammation. Adenovirus infection was eradicated better than Parvo B19 infection (Maisch JACC 2016, abstract 1346, Maisch, 2018 Mar 23, Circulation. 2010; 122 A20154).

In November 2002, severe acute respiratory syndrome (SARS) spread across all continents within a few weeks, and a novel coronavirus (SARS-CoV) was identified as the causative agent. Noh et al. from the University of Hong Kong administered Pentaglobin® to a cohort of 12 patients with severe SARS who had not responded acceptably to corticosteroid-based therapy (Noh et al., Int. J. Tuberc. Lung Dis. 8(10), 2004). After initiation of treatment with Pentaglobin®, significant improvements in radiographic parameters and oxygen requirements were observed, leading to uneventful recovery in ten patients after treatment. It was suggested that the inhibitory effect on cytokine release is an important mechanism of action in the treatment of SARS using Pentaglobin®.

Due to its multimeric structure, IgM exhibits higher opsonization activity, greater agglutination strength, greater phagocytic activity, and higher specific complement activation compared to monomeric IgG. These structural advantages of IgM have a decisive impact on the clinical status of patients.

Without being bound by a particular theory, according to preferred embodiments of the invention, it was found that intravenous administration of Pentaglobin® to patients infected with pulmonary pathogens such as SARS-CoV-2, SARS-CoV, MERS-CoV, influenza virus, anthrax pathogen or other pathogens that can induce severe ALI and ARDS, had a protective effect that was dependent on the presence of OSE-specific IgM and/or IgA antibodies.

Thus, according to a preferred embodiment of the invention, a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes is obtained from Pentaglobin®, which is, however, enriched in human or humanized natural IgM and/or IgA antibody compared to a Pentaglobin® preparation containing 76% immunoglobulin G (IgG), 12% immunoglobulin M (IgM) and 12% immunoglobulin A (IgA) of total plasma proteins.

Thus, in preferred embodiments of the invention, natural IgM and/or IgA are obtained from IgM and/or IgA-enriched plasma pools from healthy individuals in which:

IgM accounts for more than 12% of total plasma proteins, preferably more than 13%, 15%, 20%, 30%, 40% or 50%.

In other preferred embodiments of the invention, IgM accounts for 13% to 15% of total plasma proteins, preferably 13% to 20%, 13% to 30%, 13% to 40%, or 13% to 50%.

IgA accounts for more than 12% of total plasma proteins, preferably more than 13%, 15%, 20%, 30%, 40% or 50%.

In other preferred embodiments of the invention, IgA accounts for 13% to 15% of the total plasma proteins, preferably 13% to 20%, 13% to 30%, 13% to 40%, or 13% to 50%.

In another preferred embodiment of the invention, IgM and/or IgA enriched plasma pools from healthy individuals, representing an "intravenous normal immunoglobulin" (IVIG) product with higher IgM and/or IgA content, are marketed under the name IVIG-Trimodulin.

Trimodulin is an IgM concentrate derived from human plasma with high levels of IgG, IgM, and IgA, currently developed for the treatment of severe community-acquired pneumonia (sCAP). Trimodulin (an IgM concentrate) acts through a wide variety of mechanisms, interfering with pathophysiological processes that could otherwise lead to severe respiratory failure, severe sepsis, multiple organ failure, and ultimately death. In addition to neutralizing bacterial endotoxins and exotoxins, IgM enhances pathogen recognition by certain immune cells and enhances their destruction.

Specifically, trimodulin is a natural multivalent antibody preparation derived from human plasma for intravenous administration. Trimodulin contains 23% IgM, 21% IgA, and 56% IgG immunoglobulins. Thus, the active ingredients of trimodulin are IgM, which accounts for 23% of total plasma proteins, IgA, which accounts for 21%, and IgG, which accounts for 56%.

In a preferred embodiment of the invention, a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes is obtained from trimodulin, which is enriched in human or humanized natural IgM and/or IgA antibody, compared to a trimodulin preparation containing 56% immunoglobulin G (IgG), 23% immunoglobulin M (IgM) and 21% immunoglobulin A (IgA) of total plasma proteins.

Thus, in preferred embodiments of the invention, natural IgM and/or IgA are obtained from IgM and/or IgA-enriched plasma pools from healthy individuals in which:

IgM accounts for more than 23% of total plasma proteins, preferably more than 24%, 25%, 30%, 40%, or 50%.

In other preferred embodiments of the invention, IgM accounts for 24% to 27% of total plasma proteins, preferably 24% to 30%, 24% to 40%, 24% to 50%, or 24% to 60%.

IgA accounts for more than 21% of total plasma proteins, preferably more than 23%, 25%, 30%, 40% or 50%.

In other preferred embodiments of the invention, IgA accounts for 22% to 25% of total plasma proteins, preferably 22% to 30%, 22% to 40%, 22% to 50%, or 22% to 60%.

Thus, the above-mentioned human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, according to the present invention, is a natural IgM and/or IgA that is derived from IgM and/or IgA-enriched plasma pools from healthy individuals, wherein several pathways leading to an increase in the protective effect can be used to achieve said enrichment:

1) Plasma can be obtained from the serum of a young woman no older than 29 years of age, since the serum of this population group contains the highest concentration of OSE-specific antibodies.

2) The concentration of IgM and/or IgA antibodies can be varied from 12% in the standard composition to higher concentrations indicated above using conventional methods for enrichment of IgM and/or IgA antibodies known to those skilled in the art.

3) Although the concentration of OSE-specific antibodies in the product is unknown, the product composition can be further enriched with OSE-specific IgM and IgA antibodies using conventional enrichment methods with the corresponding specific antibodies known to those skilled in the art.

Thus, as indicated above, the human or humanized natural IgM and/or IgA antibody according to the present invention, which recognizes oxidized phospholipids and/or oxidation-specific epitopes, preferably belongs to a subgroup/subfraction/subpopulation of the spectrum of natural IgM and/or IgA of an individual, which is enriched in purified antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes. Accordingly, with regard to the said enrichment and/or purification for antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes, the same applies, mutatis mutandis, to the said first aspect of the present invention, set out above in the context of a more general description of the present invention.

In another preferred embodiment of the invention, the above-mentioned human or humanized natural IgM and/or IgA antibody, which recognizes oxidized phospholipids and/or oxidation-specific epitopes, enriched in human or humanized natural IgM and/or IgA antibody, is obtained from the plasma of subjects vaccinated with the vaccine specified in the third aspect of the present invention, which will be further described below. In a preferred embodiment of the invention, said vaccine is a Pneumococcus or bacillus Calmette-Guérin (BCG)-based vaccine. In another preferred embodiment of the invention, said subject is a young woman no older than 25 years of age.

In a preferred embodiment of the invention, said disorder or disease associated/linked/caused by a deficiency of natural IgM/IgA antibodies (NAD) as specified in the first aspect of the present invention is an infectious, neurodegenerative, metabolic, autoimmune or cardiovascular disease associated with a deficiency of natural antibodies.

In fact, with respect to autoimmune diseases, the present invention unexpectedly found (see Example 22 and Fig. 3) that autoimmune IgG antibodies were formed in patients with severe COVID-19. These data confirm that the absence of natural antibodies (NAs) according to the present invention can lead to the production of autoimmune antibodies during severe COVID-19. The presence of these autoimmune antibodies indicates recurrent or prolonged symptoms of COVID-19 disease, confirming that sufficient levels of natural antibodies, the use of (monoclonal) natural IgM or IgA, or preparations enriched with natural antibodies (e.g., Pentaglobin®), according to the present invention can prevent the formation of or reduce the levels of autoimmune antibodies.

In another preferred embodiment of the invention, a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes is intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, wherein said disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) is the viral infectious disease COVID-19 caused by the SARS-CoV-2 r-coronavirus.

The term "SARS-CoV-2" refers to viruses whose amino acid sequences expressed by open reading frames are at least 70% identical to the reference sequence deposited in Genebank under the number NC_045512.2.

More specifically, the term "SARS-CoV-2" refers to viruses whose sequences are at least 70% identical to the complete genomic sequence deposited in Genebank under accession number NC_045512.2 (SEQ ID NO:17).

In a more preferred embodiment of the invention, the term "SARS-VoV-2" refers to the genomic sequence of SEQ ID NO: 17, which is at least n% identical to the above sequence, where n is an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

With regard to determining sequence identity, the following condition should be applied: when the sequences being compared are not of the same length, the degree of identity refers either to the percentage of nucleic acid residues in the shorter sequence that are identical to the nucleic acid residues in the longer sequence, or to the percentage of nucleic acid residues in the longer sequence that are identical to the nucleic acid residues in the shorter sequence. Preferably, it refers to the percentage of nucleic acid residues in the shorter sequence that are identical to the nucleic acid residues in the longer sequence. The degree of sequence identity can be determined by methods well known in the art, using suitable computer algorithms such as CLUSTAL.

When using the Clustal-based analysis method to decide whether a particular sequence is, for example, at least 60% identical to a reference sequence, the default parameters can be used or the following parameters, which are preferred, can be used: matrix: Blosum 30; gap opening penalty: 10.0; gap widening penalty: 0.05; (divergence delay 40; gap separation distance: 8 when comparing amino acid sequences. When comparing nucleotide sequences, the gap widening penalty is preferably set to 5.0.

Preferably, the degree of identity is calculated over the entire length of the sequence.

In another preferred embodiment of the invention, a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes is intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, wherein said disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) is long COVID-19 (long COVID, post-COVID syndrome).

Long COVID, also known as post-acute sequelae of SARS-CoV-2 infection, post-acute sequelae of COVID-19 (PASC), chronic COVID syndrome (CCS), and long-term COVID, is a theoretical condition hypothesized to be characterized by long-term sequelae that persist beyond the typical recovery period from coronavirus disease 2019 (COVID-19). Commonly reported symptoms include fatigue, headaches, shortness of breath, anosmia (loss of smell), muscle weakness, low-grade fever, cognitive dysfunction, sleep disturbances, intermittent fevers, gastrointestinal symptoms, anxiety, and/or depression.

In preferred embodiments of the invention, in the context of the present description, long COVID is distinguished from acute COVID-19, which is defined by signs and symptoms within the first 4 weeks after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Thus, the term “long COVID (long COVID, post-COVID syndrome)” in the context of the present invention, in preferred embodiments of the invention, refers to ongoing symptomatic COVID-19 with manifestations persisting for 4 to 12 weeks after onset and/or post-COVID-19 syndrome, the manifestations of which persist for 12 or more weeks after onset.

In a preferred embodiment of the invention, a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, as provided in the present invention, is an antibody that has the ability to inhibit the spread of a virus from an infected cell to an adjacent second uninfected cell (cell-to-cell spread).

Cell-to-cell dissemination is the ability of a virus to spread to an adjacent second uninfected cell without releasing cell-free particles.

To determine whether an antibody has the ability to inhibit the spread of a virus from an infected cell to an adjacent second uninfected cell (cell-to-cell spread), methods well known to those skilled in the art can be used.

For example, the following assay can be used: Vero cells grown to confluence on glass coverslips in 24-well tissue culture plates are infected with a constant amount of herpes simplex virus type 1 (HSV-1) at 400 TCID ₅₀ /well for 4 h at 37°C. One median cytopathogenic dose (1 TCID ₅₀ ) is the amount of a cytopathogenic agent, such as a virus, that can cause a cytopathogenic effect in 50% of the inoculated cells in a culture. The viral inoculum is then removed, the cells are washed twice with PBS and further incubated for 2 days at 7°C in 1 ml of DMEM, 2% FCS, Pen/Strep containing excess of either natural OSE-specific IgM and/or IgA antibodies, which may be from plasma pools or may be monoclonal antibodies, or polyclonal antiviral control serum as a positive control to prevent virus shedding via the supernatant. Viral antigens or virus-infected cells are detected using fluorescently labeled antiviral serum.

Preferably, an antibody is considered to inhibit cell-to-cell spread if, in the above assay, less than 20% of neighboring cells are infected, preferably if less than 15%, less than 10%, less than 5%, more preferably less than 3%, and most preferably less than 1% of neighboring cells are infected.

In a preferred embodiment of the invention, a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, as provided in the present invention, is an antibody that has the ability to neutralize a virus infection, preferably thereby preventing infection of target cells.

Neutralization is the ability of agents to prevent the virus from infecting uninfected cells.

To determine whether an antibody has the ability to neutralize the virus, thereby preventing infection of uninfected cells, methods well known to those skilled in the art can be used.

For example, the following analysis can be used:

Herpes simplex virus type 1 (HSV-1) at 100 TCID ₅₀ is incubated for 1 h at room temperature with serial dilutions of either natural OSE-specific IgM and/or IgA antibodies, which may be derived from plasma pools or may be monoclonal antibodies, or with a polyclonal antiviral control serum as a positive control, or with DMEM, 2% FCS, Pen/Strep as a negative control. One median cytopathogenic dose (1 TCID ₅₀ ) is the amount of a cytopathogenic agent, such as a virus, that can cause a cytopathogenic effect in 50% of the inoculated cells in a culture.

Vero cells grown to 80% confluence in 96-well tissue culture plates are infected for 2 days at 37°C by adding 100 µl of DMEM, 2% FCS, Pen/Strep containing the virus-antibody/plasma inoculum, positive control, or negative controls. The cells are further incubated for 2 days at 37°C. The cytopathic effects induced by virus infection are assessed by counting wells containing plaques and determining the infectivity of the virus-antibody/plasma inoculum as TCID ₅₀ /ml of inoculum. Preferably, the antibody is considered to neutralize a virus infection if the infectivity is less than 20%, preferably the infectivity is less than 15%, less than 10%, less than 5%, more preferably less than 3%, and most preferably less than 1%, compared to the negative control.

In a preferred embodiment of the invention, a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, as provided in the present invention, is an antibody that has anti-inflammatory activity, preferably has the ability to:

reduce the accumulation of free oxidized phospholipids, preferably in infected lungs,

clear lung tissue from cellular debris,

stimulate the secretion of IL-10 and/or TGFβ; and/or

neutralize pro-inflammatory immune responses triggered by cytokines.

To determine whether an antibody has anti-inflammatory activity, it is preferably able to:

reduce the accumulation of free oxidized phospholipids,

preferably in infected lungs,

clear lung tissue from cellular debris,

stimulate the secretion of IL-10 and/or TGFβ; and/or

To neutralize proinflammatory immune responses triggered by cytokines, methods well known to those skilled in the art can be used. For example, the ability of monoclonal or polyclonal IgM and/or IgA antibodies to bind to oxidized phospholipids or oxidation-specific epitopes can be assessed using ELISA methods and antigens, such as oxidized phospholipids and/or oxidation-specific epitopes, cross-linked to a carrier protein such as BSA. The ability of monoclonal or polyclonal IgM and/or IgA antibodies to bind to apoptotic cells can be tested using flow cytometry-based assays or immunofluorescence microscopy using fluorescently labeled primary or secondary antibodies. To test C1q recruitment, apoptotic cells can be incubated with monoclonal or polyclonal IgM and/or IgA antibodies in the presence of mouse or human serum, followed by staining with fluorescently labeled C1q-specific antibodies and flow cytometric analysis. To test phagocytosis, equal numbers of labeled apoptotic cells and phagocytes are incubated in the presence of mouse or human serum and with or without OSE-specific monoclonal or polyclonal IgM and/or IgA antibodies. IgM- and/or IgA-dependent phagocytosis can then be assessed using flow cytometry. The anti-inflammatory activity of OSE-specific IgM and/or IgA antibodies can be tested using murine or human macrophages or monocytes incubated with oxidized phospholipids or oxidation-specific epitopes present on LDL, a carrier protein such as BSA, or bronchoalveolar lavage fluid obtained from mice infected with pulmonary pathogens such as influenza virus, with or without OSE-specific IgM and/or IgA antibodies. The resulting activation of macrophages or monocytes can be tested by quantifying the concentration of secreted proinflammatory cytokines, such as IL-6, using methods such as ELISA. The protective effect in the presence of OSE-specific IgM and/or IgA antibodies can be tested by intravenous injection of antibodies into mice infected with a lethal dose of H5N1 influenza virus or other viral pathogens. The protective effect in the presence of OSE-specific IgM and/or IgA antibodies can be tested by intravenous injection of Idlr-/- or apoe-/- antibodies into atherosclerosis-prone mice.

In another preferred embodiment of the invention, a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes is intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject in whom said disorder or disease,

associated/linked/caused by deficiency of natural antibodies IgM/IgA (NAD), is an inflammatory disease or viral infectious disease.

The present invention is not limited to a specific inflammatory disease or viral infectious disease. A common characteristic of the list of diseases exemplified in this application is the accumulation of oxidative stress-specific epitopes (OSEs) from phospholipid membranes that are not sufficiently removed from the body. Because this critical event initiates the development of many different diseases with a common cause, the invention proposes the introduction of a new term for this condition: oxidative stress-specific epitope accumulation syndrome (OSEAS).

Thus, in a preferred embodiment of the invention, said inflammatory disease is selected from the group consisting of infectious diseases mediated by respiratory viruses, preferably COVID-19, influenza, MERS-COV or SARS-COV; infectious diseases caused by bacterial infections that are mediated by gram-positive or gram-negative pathogens, fungi or parasites; and "sterile" inflammatory diseases, preferably cardiovascular diseases, atherosclerosis, coronary heart disease, heart attack and stroke, metabolic disorders such as diabetes mellitus, neurodegenerative diseases, preferably Alzheimer's disease, and autoimmune diseases, preferably systemic lupus erythematosus or multiple sclerosis.

In fact, with respect to autoimmune diseases, the present invention unexpectedly found (see Example 22 and Fig. 3) that IgG-type autoimmune antibodies were formed in patients with severe COVID-19. These data confirm that the absence of natural antibodies (NAs) according to the present invention can lead to the production of autoimmune antibodies during severe COVID-19. The presence of these autoimmune antibodies indicates recurrent or prolonged symptoms of COVID-19, confirming that sufficient levels of natural antibodies, the use of (monoclonal) natural IgM or IgA, or preparations enriched with natural antibodies (e.g., Pentaglobin®) according to the present invention can prevent the formation of or reduce the level of autoimmune antibodies.

Furthermore, in another preferred embodiment of the invention, said viral infectious disease is selected from the group consisting of infections caused by coronaviruses (preferably SARS-CoV, SARS-CoV-2, MERS); influenza viruses, parainfluenza viruses, respiratory syncytial viruses (RSV), rhinoviruses, adenoviruses, enteroviruses, human metapneumoviruses, herpesviruses, preferably HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7, HHV-8.

Another preferred embodiment of the present invention is a pharmaceutical composition comprising an effective amount of a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention, and at least one pharmaceutically acceptable excipient.

The terms "pharmaceutical composition" and "pharmaceutically acceptable excipient" as used in the present invention are described in more detail below in the context of the second aspect of the present invention, and they apply mutatis mutandis to the first aspect of the present invention when referring to the terms "pharmaceutical composition" and "pharmaceutically acceptable excipient".

According to a second aspect of the invention, the above-mentioned human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes is intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, according to the present invention, is not IgM and/or IgA derived from a naturally occurring source of the type, for example, the above-described enriched IgM and/or IgA plasma pools from healthy individuals.

In contrast, according to said second aspect of the invention, said human or humanized natural IgM and/or IgA antibody, which recognizes oxidized phospholipids and/or oxidation-specific epitopes, is intended for use in a method for treating or preventing a disorder or disease,

associated/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, according to the present invention, is a recombinant human natural IgM or IgA antibody.

In fact, fully human monoclonal OSE-specific IgM or IgA isotype antibodies can protect against pathogen-induced ALI and ARDS in accordance with the above concept of the present invention, and thus may have beneficial effects when administered to patients with “sterile” infectious diseases such as atherosclerosis, SLE, MS, AMD and AD.

To isolate fully human OSE-specific IgM antibodies, single human B1 cells were sorted and purified according to the CD20 ^pos CD27 ^pos CD43 ^pos CD5 ^pos CD70 ^neg phenotype, the V _H and V _L genes were amplified from single cells, and the resulting recombinant antibodies were tested for reactivity.

Two further clones described below (designated "Clone 1" and "Clone 2"; see, in particular, Example 23) have binding reactivity with antigens typical of natural antibodies, including DNA, oxidized phospholipids such as oxCL and oxPC, oxLDL, MDA-LDL, influenza virus, lipopolysaccharide (LPS), and misfolded oligomers of amyloid-β peptide. Thus, these two clones of monoclonal natural IgM antibodies are considered promising candidates for protecting patients with infectious, metabolic, cardiovascular, and neurodegenerative diseases associated with OSEAS, and diseases including infectious diseases that mediate ALI and ARDS, such as those caused by SARS-CoV-2, SARS-CoV, MERS-CoV, influenza virus, anthrax, or other, possibly as yet unknown, pulmonary pathogens that mediate severe forms of ALI and ARDS. Furthermore, as will be described in more detail below, these two clones of monoclonal natural IgM antibodies can be used to protect people from the development of other sterile chronic inflammatory diseases mediated by OSEAS, such as atherosclerosis, SLE, DM II, MS, AMD, and AD.

However, the present invention is not limited to the two natural antibodies mentioned. More precisely, the present invention relates in the said second aspect to the above-mentioned human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, according to the present invention, wherein the said antibody in a more general sense is a recombinant human monoclonal natural IgM or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes.

As noted above, the term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies have the advantage that they can be synthesized using a hybridoma culture that is substantially uncontaminated by other immunoglobulins. The adjective "monoclonal" indicates that the antibody is from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. As noted above, monoclonal antibodies for use in the present invention can be produced using the hybridoma method described in Kohler, Nature 256, 1975, p. 495.

The term "recombinant" generally refers to a compound that consists of elements that do not occur naturally in the specified combination.

In a preferred embodiment of the invention, the human recombinant monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention, is an antibody that recognizes said oxidized phospholipids and/or oxidation-specific epitopes described above.

Preferably, the antibody binds in a polyreactive manner to said oxidized phospholipids and/or oxidation-specific epitopes, preferably, for example, to oxLDL.

In other preferred embodiments of the invention, said antibody binds to oxidized cardiolipin; viral particles (such as, for example, influenza virus particles); proinflammatory cytokines (for example, TNFα) and/or misfolded proteins.

Preferably, said antibody binds to misfolded proteins characteristic of neurodegenerative diseases, such as, for example, "oligomeric amyloid-β peptides."

In a further preferred embodiment of the invention, said antibody binds to oxidized phospholipids and/or oxidation-specific epitopes present in the plasma membrane of mammalian cells, in circulating lipoproteins, in the membrane surrounding viruses, or in the cell wall of bacteria, fungi or parasites, in apoptotic cells, cellular debris, oxidized LDL, viruses, preferably influenza viruses or SARS coronaviruses, or microbes, preferably Staphylococcus pneumoniae; with misfolded proteins that accumulate in neurodegenerative diseases, preferably such as amyloid-β oligomeric peptides.

In a preferred embodiment of the invention, the recombinant human monoclonal natural IgM and/or IgA antibody recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, malondialdehyde-, 4-hydroxynonenal- and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or amyloid-β oligomeric peptide.

In another preferred embodiment of the invention, the recombinant human monoclonal natural IgM and/or IgA antibody is characterized not only by the fact that it recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, malondialdehyde-, 4-hydroxynonenal- and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or amyloid-β oligomeric peptide. The recombinant human monoclonal natural IgM and/or IgA antibody of the present invention may also be characterized in that it recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, but not to phosphorylcholine of unoxidized phosphatidylcholine and unoxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, to oxidized cardiolipin, but not to unoxidized cardiolipin, to oxidized phosphatidylserine, but not to unoxidized phosphatidylserine, to malondialdehyde-, 4-hydroxynonenal- and/or 2-(ω-carboxyethyl)pyrrole-modified proteins, but not to native proteins, and/or to amyloid-β oligomeric peptide, but not to with monomeric amyloid-β peptide.

Assays for determining whether an antibody binds to the above structures are known in the art and, for example, may be the assays described above in this description.

In a preferred embodiment of the invention, a recombinant human monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention,

comprises complementarity determining regions: V _H CDR1, which comprises SEQ ID NO: 1, V _H CDR2, which comprises SEQ ID NO: 2, V _H CDR3, which comprises SEQ ID NO: 3, V _L CDR1, which comprises SEQ ID NO: 4, V _L CDR2, which comprises SEQ ID NO: 5, and V _L CDR3, which comprises SEQ ID NO: 6, wherein said antibody recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, to oxidized cardiolipin, to oxidized phosphatidylserine, to malondialdehyde-, 4-hydroxynonenal- and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or to amyloid-β oligomeric peptide; or

contains the complementarity determining regions V _H CDR1, which contains SEQ ID NO: 9, V _H CDR2, which contains SEQ ID NO: 10, V _H CDR3, which contains SEQ ID NO: 11, V _L CDRI, which contains SEQ ID NO: 12, V _L CDR2, which contains SEQ ID NO: 13, and V _L CDR3, which contains SEQ ID NO: 14, wherein said antibody recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, with oxidized cardiolipin, with oxidized phosphatidylserine, with malondialdehyde-, 4-hydroxynonenal- and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or with an amyloid-β oligomeric peptide.

Assays for determining whether an antibody binds to the above structures are known in the art and, for example, may be the assays described above in this description.

Monoclonal antibodies having the above-described structure, namely SEQ ID NOs: 1-6 and SEQ ID NOs: 9-14, respectively, are further characterized in Example 23 below.

SEQ ID NO: 1-6 are derived from "Clone 1".

SEQ ID NO: 9-14 are derived from "Clone 2".

In preferred embodiments of the invention, the monoclonal antibodies described above with reference to SEQ ID NOs: 1-6 and SEQ ID NOs: 9-14, respectively, are not monospecific.

In preferred embodiments of the invention, the monoclonal antibodies described above with reference to SEQ ID NOs: 1-6 and SEQ ID NOs: 9-14, respectively, are bispecific or multispecific.

"Bispecific" in this context means that the monoclonal antibody binds specifically to two different antigens and/or antigen epitopes.

“Multispecific” in this context means that the monoclonal antibody specifically binds to more than two different antigens and/or antigen epitopes, preferably three, four, or five different antigens and/or antigen epitopes.

In a more preferred embodiment of the invention, said distinct antigen and/or antigen epitope is a danger-associated molecular pattern (DAMP).

Damage-associated molecular patterns (DAMPs) are known in the field as intracellular molecules that are a component of the innate immune response and are released from damaged or dead cells as a result of injury or pathogen infection. They are also referred to as danger-associated molecular patterns, damage signals, and alarm signals because they serve as a warning signal to the body, alerting it to any damage or infection of its cells. DAMPs are endogenous danger signals released into the extracellular space in response to cellular damage due to injury or pathogen infection.

In a particularly preferred embodiment of the invention, said danger-associated molecular pattern (DAMP) is selected from the group consisting of OxLDL, MDA-LDL, MDA-BSA, PC-BSA and DNA.

Thus, in a particularly preferred embodiment of the invention, the monoclonal antibodies described above with reference to SEQ ID NOs: 1-6 and SEQ ID NOs: 9-14, respectively, bind to at least two danger-associated molecular patterns (DAMPs) selected from the group consisting of OxLDL, MDA-LDL, MDA-BSA, PC-BSA and DNA.

In another particularly preferred embodiment of the invention, the monoclonal antibodies described above with reference to SEQ ID NOs: 1-6 (corresponding to Clone 1) bind to the danger-associated molecular patterns (DAMPs) of OxLDL, MDA-LDL, MDA-BSA, PC-BSA, and DNA. They are thus multispecific according to the present invention.

In another, even more preferred embodiment of the invention, the monoclonal antibodies described above with reference to SEQ ID NOs: 9-14 (corresponding to Clone 2) bind to the danger-associated molecular patterns (DAMPs) of oxLDL and DNA. They are thus bispecific according to the present invention.

The term "CDR" as used herein refers to the "complementarity-determining region," which is well known in the art. CDRs are portions of immunoglobulins that determine the specificity of these molecules and mediate contact with a specific ligand. CDRs are the most variable part of the molecule and contribute to the diversity of these molecules. There are three CDRs, CDR1, CDR2, and CDR3, in each V domain. CDR-H denotes the CDR of the variable heavy chain, and CDR-L refers to the CDR of the variable light chain. VH denotes the variable heavy chain, and VL denotes the variable light chain. CDRs derived from the Ig region can be determined according to the principles described in Kabat, "Sequences of Proteins of Immunological Interest," 5th ed. NIH Publication No. 91-3242 U.S. Department of Health and Human Services 1991; Chothia, J. Mol. Biol. 196, 1987, cc. 901-917 or Chothia, Nature 342, 1989, cc. 877-883.

Thus, in accordance with the basic concepts, the antibody molecule specified in the description can be selected from the group consisting of a full-length IgA or IgM or from multivalent F(ab)-, Fab'-SH-, Fv-, Fab'-, F(ab')2- fragments of an IgG-class antibody, a chimeric antibody, an antibody with grafted CDRs, a fully human antibody, a bivalent antibody construct, an antibody-fused protein, a synthetic antibody, a bivalent single-chain antibody, a trivalent single-chain antibody, and a multivalent single-chain antibody.

In a further preferred embodiment of the invention, the recombinant human monoclonal natural antibody is IgM and/or IgA.

As noted above, "humanization approaches" are well known in the art and have primarily been described for antibody molecules, such as Ig-derived molecules. The term "humanized" refers to humanized forms of non-human (e.g., murine) antibodies or fragments thereof (such as Fv, Fab, Fab', F(ab'), scFv, or other antigen-binding partial antibody sequences) that contain some portion of a sequence derived from a non-human antibody. Humanized antibodies include human immunoglobulins in which residues from the complementarity-determining regions (CDRs) of human immunoglobulin are replaced by residues from the CDRs of antibodies from non-human species, such as mice, rats, or rabbits, that exhibit the desired binding specificity, affinity, and potency. In general, a humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to regions of a non-human immunoglobulin and all or substantially all of the FR regions are regions of a human immunoglobulin consensus sequence. A humanized antibody may optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically from a human immunoglobulin; see, inter alia, Jones et al., Nature 321 (1986), pp. 522-525, Presta, Curr. Op. Struct. Biol. 2 (1992), pp. 593-596. Methods for humanizing non-human antibodies are well known in the art. Typically, a humanized antibody has one or more amino acids introduced into it from a non-human source while still retaining the original binding activity of the antibody. Methods for humanizing antibodies/antibody molecules are further described in detail by Jones et al., Nature 321 (1986):522-525; Reichmann et al., Nature 332 (1988):323-327; and Verhoeyen et al., Science 239 (1988):1534-1536. Specific examples of humanized antibodies, such as anti-EpCAM antibodies, are known in the art (see, e.g., LoBuglio, Proceedings of the American Society of Clinical Oncology Abstract, 1997:1562; and Khor, Proceedings of the American Society of Clinical Oncology Abstract, 1997:847).

Thus, according to the present invention, there are provided antibody molecules or antigen-binding fragments thereof that are humanized and that can be successfully used in pharmaceutical compositions.

Furthermore, in a preferred embodiment of the invention, a recombinant human monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention,

comprises an amino acid sequence that is at least 70% identical to the amino acid residues present at positions 1-25, 34-50, 59-96 and 116-126 in the sequence SEQ ID NO: 7, and at positions 1-25, 35-51, 55-90 and 101-110 in the sequence SEQ ID NO: 8, wherein said antibody recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-

arachidonoylphosphatidylcholine, with oxidized cardiolipin, with oxidized phosphatidylserine, with malondialdehyde-, 4-hydroxynonenal-, and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or amyloid-β oligomeric peptide; or

contains an amino acid sequence that is at least 70% identical to the amino acid residues present at positions 1-25, 34-50, 59-96 and 116-126 in the sequence SEQ ID NO: 15, and at positions 1-26, 34-50, 54-89 and 99-108 in the sequence SEQ ID NO: 16, wherein said antibody recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, with oxidized cardiolipin, with oxidized phosphatidylserine, with malondialdehyde-, 4-hydroxynonenal- and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or amyloid-β oligomeric peptide.

Monoclonal antibodies having the structure described above with reference to SEQ ID NOs: 1-6 and SEQ ID NOs: 9-14, respectively, are further characterized in Example 23 below.

SEQ ID NO: 1-6 are derived from "Clone 1".

SEQ ID NO: 9-14 are derived from "Clone 2".

In preferred embodiments of the invention, the monoclonal antibodies described above and below with reference to SEQ ID NOs: 7-8 and SEQ ID NOs: 15-16, respectively, are not monospecific.

In preferred embodiments of the invention, the monoclonal antibodies described above with reference to SEQ ID NOs: 1-6 and SEQ ID NOs: 9-14, respectively, are bispecific or multispecific.

"Bispecific" in this context means that the monoclonal antibody binds specifically to two different antigens and/or antigen epitopes.

“Multispecific” in this context means that the monoclonal antibody specifically binds to more than two different antigens and/or antigen epitopes, preferably three, four, or five different antigens and/or antigen epitopes.

In a more preferred embodiment of the invention, said distinct antigen and/or antigen epitope is a danger-associated molecular pattern (DAMP) as already described above.

In a most preferred embodiment of the invention, said danger-associated molecular pattern is selected from the group consisting of OxLDL, MDA-LDL, MDA-BSA, PC-BSA and DNA.

Thus, in a particularly preferred embodiment of the invention, the monoclonal antibodies described above and below with reference to SEQ ID NOs: 7-8 and SEQ ID NOs: 15-16, respectively, bind to at least two danger-associated molecular patterns (DAMPs) selected from the group consisting of OxLDL, MDA-LDL, MDA-BSA, PC-BSA and DNA.

In another particularly preferred embodiment of the invention, the monoclonal antibodies described above and below with reference to SEQ ID NO: 7-8 (corresponding to "Clone 1") bind to the danger-associated molecular pattern (DAMP), OxLDL, MDA-LDL, MDA-BSA, PC-BSA, and DNA. Thus, it is multispecific according to the present invention.

In another particularly preferred embodiment of the invention, the monoclonal antibodies described above and below with reference to SEQ ID NOs: 15-16 (corresponding to "Clone 2") bind to the danger-associated molecular pattern (DAMP), OxLDL, and DNA. Thus, it is bispecific according to the present invention.

In a preferred embodiment of the invention, a recombinant human monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention, comprises or consists of a VH domain (heavy chain variable region) and a VL domain (light chain variable region),

i.e., the amino acid sequence of the variable region of the heavy chain of the antibody as presented in SEQ ID NO:7, the amino acid sequence of the variable region of the light chain of the antibody as presented in SEQ ID NO:8, wherein said antibody recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, malondialdehyde-, 4-hydroxynonenal- and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or amyloid-β oligomeric peptide; or

the amino acid sequence of the variable region of the heavy chain of the antibody, which is presented in SEQ ID NO: 15, the amino acid sequence of the variable region of the light chain of the antibody, which is presented in SEQ ID NO: 16, wherein said antibody recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, to oxidized cardiolipin, to oxidized phosphatidylserine, to malondialdehyde-, 4-hydroxynonenal- and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or amyloid-β oligomeric peptide.

Assays for determining whether an antibody binds to the above structures are known in the art and, for example, may be the assays described above in this description.

However, the antibody used in the present invention is not limited to the variable regions of the heavy and light chains specifically mentioned, but may also be an antibody or an antigen-binding fragment thereof that comprises or consists of a VH domain and a VL domain, the sequences of which are at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 75%, 70%, 65%, 60%, 55% or 50% identical to the sequences of SEQ ID NOs: 7 and 8, respectively, the sequences of SEQ ID NOs: 15 and 16, respectively,

if the antibody has the ability to recognize and bind to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, malondialdehyde-, 4-hydroxynonenal- and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or amyloid-β oligomeric peptide.

In addition, the antibody or antigen-binding fragment thereof is a molecule that comprises VH and VL domains having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions relative to the sequences of SEQ ID NO: 7 and 8 or SEQ ID NO: 15 and 16. In addition, the antibody or antigen-binding fragment thereof is an antigen-binding fragment selected from the group consisting of Fab, Fab', Fab'-SH, FV, scFV, F(ab')2 and a dimeric antibody.

To decide whether an amino acid sequence has a certain degree of identity with the sequences of SEQ ID NOs: 7, 8, 15 and 16, a person skilled in the art can use means and methods well known in the art, such as alignment, either manually or using computer programs known to those skilled in the art. Said alignment can be performed, for example, using means and methods known to those skilled in the art, for example, using a computer algorithm such as the Lipman-Pearson method (Science, 227, 1985, p. 1435) or the CLUSTAL algorithm. Preferably, when using said alignment, maximum homology is assigned to conserved amino acid residues present in the amino acid sequences. In a preferred embodiment of the invention, ClustalW2 is used to compare amino acid sequences. In the case of pairwise comparisons/alignments, the following parameters are preferably selected: protein weight matrix: BLOSUM 62; open gaps: 10; Gap expansion: 0.1. In case of multiple comparisons/alignments, the following parameters are preferred: Protein weight matrix: BLOSUM 62; Open Gaps: 10; Gap expansion: 0.2; Gap size: 5; No gap end.

According to the present invention, the term "identical" or "percent identity" in the context of two or more nucleotide or amino acid sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of the same amino acid residues or nucleotides (e.g., 60% or 65% identity, preferably 70-95% identity, more preferably at least 95% identity with the nucleotide sequences or with the amino acid sequences described above that have the ability to bind to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, to oxidized cardiolipin, to oxidized phosphatidylserine, to malondialdehyde-, 4-hydroxynonenal- and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or amyloid-β oligomeric peptide, when compared and aligned to achieve maximum correspondence in the comparison window or in the designated region, measured using a sequence comparison algorithm known in the art or by manual alignment and visual inspection. Sequences that are identical, for example, by 60%-95% or more, are considered as substantially identical sequences. The said definition can also be applied to the complement of the test sequence. Preferably, the described identity is considered for a region consisting of at least about 15-25 amino acids or nucleotides, more preferably for a region consisting of at least about 50-100 amino acids or nucleotides. It is well known to those skilled in the art how to determine identity between/for sequences, using, for example, algorithms such as those based on the CLUSTALW computer program (Thompson, Nucl. Acids Res. 2, 1994, pp. 4673-4680) or FASTDB (Brutlag, Comp. App. Biosci. 6, 1990, pp. 237-245), which are known in this field.

Although the FASTDB algorithm generally does not consider internal, inappropriate deletions or additions in sequences, i.e., gaps, in its calculations, this can be manually corrected to avoid overestimation of % identity. However, the CLUSTALW algorithm does take gaps into account when calculating sequence identity. BLAST and BLAST 2.0 algorithms are also available for those skilled in the art (Altschul, Nucl. Acids Res. 25 (1997): 3389–3402; Altschul, J. Mol. Et. 36 (1993): 290–300; Altschul, J. Mol. Biol. 215 (1990): 403–410). The BLASTN program for nucleotide sequences uses a wordlength (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands as default parameters. For amino acid sequences, the BLASTP program uses a wordlength (W) of 3 and an expectation (E) of 10 as default parameters. The BLOSUM62 scoring matrix (Henikoff, PNAS 89, 1989, p. 10915) uses alignments (B) of 50, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

Preferably, the amino acid substitution(s) are(are) "conservative substitution(s)," a term referring to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), as a result of which the changes often may not result in a change in the biological activity of the protein. Those skilled in the art will appreciate that, in general, single amino acid substitutions in non-critical regions of a polypeptide do not significantly alter biological activity (see, e.g., Watson, Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., 4th ed., 1987, p. 224). Furthermore, substitutions of structurally or functionally similar amino acids are less likely to impair biological activity. In the context of the present invention, the binding compounds/antibodies of the present invention comprise polypeptide chains whose sequences include up to 0 (no change), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more conservative amino acid substitutions compared to the specific amino acid sequences indicated in the description, for example, SEQ ID NO: 9 (which corresponds to the variable region of the heavy chain of the antibody) and SEQ ID NO: 10 (which corresponds to the variable region of the light chain of the antibody). In the context of the present description, the phrase "up to X" conservative amino acid substitutions includes 0 substitutions and any number of substitutions up to 10, and includes 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 substitutions.

The said example substitutions are preferably carried out in accordance with those indicated below in Table 1:

Furthermore, in a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof,

intended/intended for use according to the present invention, comprises an amino acid sequence that is at least 70% identical to the amino acid residues present at positions 1-25, 34-50, 59-96 and 116-126 in the sequence SEQ ID NO: 7, and at positions 1-25, 35-51, 55-90 and 101-110 in the sequence SEQ ID NO: 8, wherein said antibody recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, to oxidized cardiolipin, to oxidized phosphatidylserine, to proteins modified with malondialdehyde, 4-hydroxynonenal and 2-(ω-carboxyethyl)pyrrole and/or an oligomeric peptide amyloid-β.

Furthermore, in a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof,

intended/intended for use according to the present invention, comprises an amino acid sequence that is at least 70% identical to the amino acid residues present at positions 1-25, 34-50, 59-96 and 116-126 in the sequence SEQ ID NO: 15, and at positions 1-26, 34-50, 54-89 and 99-108 in the sequence SEQ ID NO: 16, wherein said antibody recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, to oxidized cardiolipin, to oxidized phosphatidylserine, to proteins modified with malondialdehyde, 4-hydroxynonenal and 2-(ω-carboxyethyl)pyrrole and/or an oligomeric peptide amyloid-β.

Furthermore, in a preferred embodiment of the invention, the antibody for use according to the present invention comprises an amino acid sequence that is at least 75%, at least 80%, more preferably at least 85%, at least 90%, even more preferably at least 95% and most preferably 98% identical to the entire framework sequence compared to the amino acid residues present at positions 1-25, 34-50, 59-96 and 116-126 in SEQ ID NO: 7, and at positions 1-25, 35-51, 55-90 and 101-110 in SEQ ID NO: 8.

The said antibodies can be used for the medical purposes specified in the present invention if the antibody or its antigen-binding fragment binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, to oxidized cardiolipin, to oxidized phosphatidylserine, to malondialdehyde-modified, 4-hydroxynonenal-modified and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or amyloid-β oligomeric peptide, as specified above and below in the present description.

Thus, in a preferred embodiment of the invention, an antibody for use according to the present invention comprises an amino acid sequence that has the above-mentioned variable regions of the light and heavy chains (i.e., the above-mentioned CDRs, i.e., V _H CDR1 comprising SEQ ID NO: 1, V _H CDR2 comprising SEQ ID NO: 2, V _H CDR3 comprising SEQ ID NO: 3, V _L CDR1 comprising SEQ ID NO: 4, V _L CDR2 comprising SEQ ID NO: 5, and V _L CDR3 comprising SEQ ID NO: 6), wherein the amino acid sequence has variability in the framework region, as a result of which the entire sequence of the framework regions is identical by at least 75%, at least 80%, more preferably by at least 85%, at least 90%, even more preferably by at least 95% and most preferably by 98% or even by 99 or 100% compared to the amino acid residues present at positions 1-25, 34-50, 59-96 and 116-126 in SEQ ID NO: 7, and at positions 1-25, 35-51, 55-90 and 101-110 in SEQ ID NO: 8.

Furthermore, in a preferred embodiment of the invention, the antibody for use according to the present invention comprises an amino acid sequence that is at least 75%, at least 80%, more preferably at least 85%, at least 90%, even more preferably at least 95% and most preferably 98% identical to the entire framework sequence compared to the amino acid residues present at positions 1-25, 34-50, 59-96 and 116-126 in SEQ ID NO: 15, and at positions 1-26, 34-50, 54-89 and 99-108 in SEQ ID NO: 16.

The said antibodies can be used for the medical purposes specified in the present invention if the antibody or its antigen-binding fragment binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, to oxidized cardiolipin, to oxidized phosphatidylserine, to malondialdehyde-modified, 4-hydroxynonenal-modified and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or amyloid-β oligomeric peptide, as specified above and below in the present description.

Thus, in a preferred embodiment of the invention, an antibody for use according to the present invention comprises an amino acid sequence that has the above-mentioned variable regions of the light and heavy chains (i.e., the above-mentioned CDRs, i.e., V _H CDR1 comprising SEQ ID NO: 9, V _H CDR2 comprising SEQ ID NO: 10, V _H CDR3 comprising SEQ ID NO: 11, V _L CDR1 comprising SEQ ID NO: 12, V _L CDR2 comprising SEQ ID NO: 13 and V _L CDR3 comprising SEQ ID NO: 14), wherein the amino acid sequence has variability in the framework region, as a result of which the entire sequence of the framework regions is identical by at least 75%, at least 80%, more preferably by at least 85%, at least 90%, even more preferably by at least 95% and most preferably by 98% or even by 99 or 100% compared to the amino acid residues present at positions 1-25, 34-50, 59-96 and 116-126 in SEQ ID NO: 15, and at positions 1-26, 34-50, 54-89 and 99-108 in SEQ ID NO: 16.

In this context, a polypeptide has "at least X% sequence identity" in the framework regions of SEQ ID NO: 7 or 8 (or SEQ ID NO: 15 or 16) if SEQ ID NO: 7 or SEQ ID NO: 8 (or SEQ ID NO: 15 or 16) are aligned with the best-matching sequence of the polypeptide of interest, and the amino acid identity between the two aligned sequences is at least X% at positions 1-25, 34-50, 59-96, and 116-126 in SEQ ID NO: 7 (or SEQ ID NO: 15) and at positions 1-25, 35-51, 55-90, and 101-110 in SEQ ID NO: 8 (or SEQ ID NO: 16). As noted above, this comparative analysis of the primary structure of amino acid sequences can be performed using, for example, publicly available homology software programs, such as the BLAST program available on the homepage of the National Center for Biotechnology Information (NCBI), using the default parameters specified therein. Furthermore, methods for calculating the percent sequence identity of sets of amino acid sequences or nucleotide sequences are known in the art.

Furthermore, in a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof proposed/proposed for use according to the present invention comprises a V _H having SEQ ID NO: 7 and a V _L having SEQ ID NO: 8.

Furthermore, in a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof proposed/proposed for use according to the present invention comprises a V _H having SEQ ID NO: 7 and a V _L having SEQ ID NO: 8, wherein said antibody recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, malondialdehyde, 4-hydroxynonenal and 2-(ω-carboxyethyl)pyrrole modified proteins and/or amyloid-β oligomeric peptide.

Furthermore, in a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof proposed/proposed for use according to the present invention comprises a V _H having SEQ ID NO: 15 and a V L having SEQ ID NO: 16.

Furthermore, in a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof proposed/proposed for use according to the present invention comprises a V _H having SEQ ID NO: 15 and a V l having SEQ ID NO: 16, wherein said antibody recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, to oxidized cardiolipin, to oxidized phosphatidylserine, to malondialdehyde, 4-hydroxynonenal and 2-(ω-carboxyethyl)pyrrole modified proteins and/or amyloid-β oligomeric peptide.

The term "recognizes and binds to" a certain structure as defined above may be applied, mutatis mutandis, to the antibodies defined above that "recognize and bind to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, malondialdehyde-modified, 4-hydroxynonenal-modified, and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or amyloid-β oligomeric peptide."

In a preferred embodiment of the invention, an antibody that "recognizes and binds to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, malondialdehyde-, 4-hydroxynonenal-, and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or amyloid-β oligomeric peptide" has a dissociation constant KD of no more than 0.5×10 ^-3 nM, no more than 1.0×10 ^-3 M, no more than 0.5×10 ^-4 M, no more than 1.0×10 ^-4 M, no more than 5×10 ^-4 M, no more than 1×10 ^-5 M, no more than 3×10 ^-5 M, no more than 5.0× ^10-5 M, no more than 1.0× ^10-6 M, preferably no more than 0.5× ^10-7 M, more preferably no more than 1.0× ^10-7 M, even more preferably no more than 1.0× ^10-8 M and most preferably no more than 1.0× ^10-9 M. KD is a dissociation constant as a measure of the tendency of a complex to reversibly dissociate into its components (i.e., the affinity of an antibody for an antigen) and is the inverse of the association constant. The above values relate to binding to a single binding site of the antibody. KD can be calculated using the Scatchard equation, and methods for determining KD are well known in the art.

In the present case, since the antibody is preferably an IgM antibody consisting of five antibodies (and is accordingly present in the form of a pentamer which has ten binding sites), its avidity is actually higher and it has a dissociation constant KD of not more than 0.5×10 ^-3 nM, not more than 1.0×10 ^-3 M, not more than 0.5×10 ^-4 M, not more than 1.0×10 ^-4 M, not more than 5×10 ^-4 M, not more than 1×10 ^-5 M, not more than 3×10 ^-5 M, not more than 5.0×10 ^-5 M, not more than 1.0×10 ^-6 M, preferably not more than 0.5×10 ^-7 M, more preferably not more than 1.0×10 ^-7 M, even more preferably not more than 1.0×10 ^-8 M and most preferably not more than 1.0×10 ^-9 M.

Avidity is a measure of the combined strength of multiple affinities of individual noncovalent binding interactions, such as that between an antibody and its antigen. Thus, avidity differs from affinity, which describes the strength of a single interaction.

The recombinant human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes intended for use according to the present invention is not limited to the specific antibody(ies) mentioned above, but may be any recombinant human monoclonal natural IgM antibody, if it recognizes and binds to oxidized phospholipids and/or oxidation-specific epitopes.

With the ordinary skill of a person skilled in the art and using conventional methods, one skilled in the art, based on the description of the present invention, can readily derive relevant epitopes (as well as functional fragments) from the structure of oxidized phospholipids and/or oxidation-specific epitopes, which can be used to create antibodies of the polyclonal and monoclonal type. However, one skilled in the art can also readily produce engineered antibodies, such as CDR-grafted antibodies or humanized and fully human antibodies, etc.

As noted above, monoclonal antibodies are most preferred in the context of the present invention. Any technology that allows for the production of antibodies using continuous cell line cultures can be used to produce monoclonal antibodies. Examples of these technologies include the hybridoma method, the trioma method, the human B-cell hybridoma method, and the EBV hybridoma method, all designed to produce human monoclonal antibodies (Shepherd and Dean, Monoclonal Antibodies, A Oxford University Press, 2000; Goding and Goding, Monoclonal Antibodies Principles and Practice - Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, Academic Pr Inc, USA).

Antibodies (or derivatives) can also be produced using peptidomics. Furthermore, technologies described for producing single-chain antibodies (see, inter alia, U.S. Patent 4,946,778) can be adapted to produce single-chain antibodies that specifically recognize an antigen. Furthermore, transgenic animals can be used to express humanized antibodies to the desired antigen.

The present invention also relates to the production of specific antibodies against oxidized phospholipids and/or oxidation-specific epitopes. The basis for said production is, for example, immunization of animals such as mice. However, according to the present invention, other animals are also used to produce antibodies/antisera. For example, monoclonal and polyclonal antibodies can be produced using rabbits, mice, goats, donkeys, etc. The amount of the specific antibody produced can be determined using ELISA, which is also presented below in the present description. Additional methods for producing antibodies are well known in the art (see, for example, Harlow and Lane, "Antibodies, A Laboratory Manual," CSH Press, Cold Spring Harbor, 1988).

As a second example, the basis of said production is the sorting of single cells of human B cells and the expression cloning of the genes of immunoglobulin heavy and light chains. In this example, single human B cells can be sorted based on the expression of specific surface markers, such as CD20 ^pos CD27 ^pos CD43 ^pos CD70 ^neg , to identify B1 cells, or based on the binding of fluorescently labeled specific antigens, such as phosphorylcholine adducts or MDA. As a third example, said production is based on the sorting of human B cells based on the expression of specific surface markers, such as CD20 ^pos CD27 ^pos CD43 ^pos CD70 ^neg , to identify B1 cells, or based on the binding of fluorescently labeled specific antigens, such as phosphorylcholine adducts or MDA. Sorted B cells can be transduced with the EBV virus to immortalize the B cells, followed by single-cell cloning using methods such as single-cell FACS sorting or limiting dilution. The supernatant obtained from the resulting B cell lines can be tested for antigen specificity, and the immunoglobulin heavy and light chain genes can be cloned from B clones expressing antibodies of the desired specificity. As a fourth example, this production is based on antibody selection from combinatorial phage antibody libraries. In this example, antigens such as phosphorylcholine adducts or MDA can be used to select specific antibodies.

As used herein, the term "specifically binds" refers to a binding reaction that allows the detection of the presence of desired oxidized phospholipids and/or oxidation-specific epitopes and antibodies in the presence of a heterogeneous population of proteins and other biological substances.

Thus, under certain assay conditions, specific antibodies and the corresponding oxidized phospholipids and/or oxidation-specific epitopes bind to each other and do not bind in significant amounts to other components present in the sample.

Specific binding to the target analyte under such conditions may require a binding moiety selected based on its specificity for the particular target analyte. A wide variety of immunoassay formats can be used to select antibodies that have specific reactivity to a particular antigen. For example, solid-phase ELISA immunoassays are commonly used to select monoclonal antibodies that have specific immunoreactivity with the analyte (see, e.g., Shepherd and Dean, Monoclonal Antibodies, Oxford University Press, 2000, and/or Howard and Bethell, Basic Methods in Antibody Production and Characterization, Crc. Pr. Inc., 2000, for descriptions of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Generally, the specific or selective response should be at least twice the background signal-to-noise ratio and, more specifically, more than 10-100 times the background signal. Those skilled in the art readily understand how to obtain and create specific binding molecules directed against new polypeptides. They can be easily used for specific binding assays to avoid unwanted cross-reactivity; for example, polyclonal antibodies can be easily purified and selected using known methods (see Shepherd and Dean, loc. cit.).

The term "antibody or antigen-binding fragment thereof" according to the invention means that the antibody molecule or antigen-binding fragment thereof has the ability to specifically recognize or specifically interact with and/or bind to at least a partial structure of the said oxidized phospholipid and/or oxidation-specific epitope. This term refers to the specificity of the antibody molecule, i.e. its ability to distinguish specific regions of the oxidized phospholipid and/or oxidation-specific epitope. Thus, the specificity can be determined experimentally by methods known in the art and by the methods disclosed and described in the present application. Such methods include (but are not limited to) Western blotting, ELISA, RIA, ECL, IRMA assays and peptide scanning. These methods known in the art also include determining KD values.

In a preferred embodiment of the invention relating to a recombinant human monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention in accordance with the second aspect of the present invention, said disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) is an infectious, neurodegenerative, metabolic, autoimmune or cardiovascular disease associated with a deficiency of natural antibodies.

The same applies, mutatis mutandis, to the said infectious, neurodegenerative, metabolic, autoimmune or cardiovascular disease associated with a deficiency of natural antibodies as indicated above in the context of the first aspect of the present invention.

In fact, with respect to autoimmune diseases, it was unexpectedly discovered (see Example 22 and Fig. 3) that autoimmune IgG antibodies are formed in patients with severe COVID-19. These data confirm that the absence of natural antibodies (NAs) according to the present invention can lead to the production of autoimmune antibodies during severe COVID-19. The presence of these autoimmune antibodies indicates recurrent or prolonged symptoms of COVID-19, confirming that sufficient levels of natural antibodies, the use of (monoclonal) natural IgM or IgA, or preparations enriched with natural antibodies (e.g., Pentaglobin®), according to the present invention can prevent the formation of or reduce the level of autoimmune antibodies.

In a preferred embodiment of the invention relating to a recombinant human monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention in accordance with the second aspect of the present invention, said disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) is the viral infectious disease COVID-19 caused by the SARS-CoV-2 r-coronavirus.

In another preferred embodiment of the invention, relating to a recombinant human monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention in accordance with the second aspect of the present invention, said disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) is long-term COVID.

The same applies, mutatis mutandis, to the said viral infectious disease COVID-19 caused by the β-coronavirus SARS-CoV-2 and long-term COVID-19, respectively, as indicated above in the context of the first subject matter of the present invention.

In a preferred embodiment of the invention, relating to a recombinant human monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention in accordance with the second aspect of the present invention, said antibody has the ability to neutralize a virus and/or inhibit the spread of a virus from an infected cell to an adjacent second uninfected cell (cell-to-cell spread).

The same applies, mutatis mutandis, to the said ability to inhibit the spread of a virus from an infected cell to an adjacent second uninfected cell (cell-to-cell spread) as set out above in the context of the first aspect of the present invention.

In a preferred embodiment of the invention, relating to a recombinant human monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention in accordance with the second aspect of the present invention, said antibody has the ability to neutralize virus infection and thereby prevent infection of target cells.

The same applies, mutatis mutandis, to the said ability to “neutralize a virus” and/or “neutralize infection by a virus and thereby prevent infection of target cells” as is indicated above in the context of the first object of the present invention.

In a preferred embodiment of the invention, relating to a recombinant human monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention in accordance with the second aspect of the present invention, said antibody has anti-inflammatory activity, preferably the ability to:

reduce the accumulation of free oxidized phospholipids, preferably in infected lungs,

clear lung tissue from cellular debris and/or

stimulate the secretion of IL-10 and/or TGFβ.

Said anti-inflammatory activity, preferably said ability to: reduce the accumulation of free oxidized phospholipids, preferably in infected lungs, clear lung tissue of cellular debris and/or stimulate the secretion of IL-10 and/or TGFβ; and/or neutralize pro-inflammatory cytokines, applicable mutatis mutandis, is the same as indicated above in the context of the first object of the present invention.

In a preferred embodiment of the invention relating to a recombinant human monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention in accordance with the second aspect of the present invention, said disorder or disease associated with a deficiency of natural IgM/IgA antibodies (NAD) is an inflammatory disease or a viral infectious disease.

To the said disorder or disease associated with a deficiency of natural IgM/IgA antibodies (NAD), which is an inflammatory disease or a viral infectious disease, the same applies, mutatis mutandis, as indicated above in the context of the first aspect of the present invention.

In a preferred embodiment of the invention relating to a recombinant human monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention in accordance with the second aspect of the present invention, said inflammatory disease is selected from the group consisting of infectious diseases mediated by respiratory viruses, preferably COVID-19, influenza, MERS-COV or SARS-COV; infectious diseases caused by bacterial infections that are mediated by gram-positive or gram-negative pathogens, fungi or parasites; and "sterile" diseases, preferably cardiovascular diseases, atherosclerosis, coronary heart disease, heart attack and stroke, metabolic disorders such as diabetes mellitus, neurodegenerative diseases, preferably Alzheimer's disease, and autoimmune diseases, preferably systemic lupus erythematosus or multiple sclerosis.

To said inflammatory diseases selected from the group consisting of infectious diseases mediated by respiratory viruses, preferably COVID19, influenza, MERS-COV or SARS-COV; infectious diseases caused by bacterial infections that are mediated by gram-positive or gram-negative pathogens, fungi or parasites; and "sterile" diseases, preferably cardiovascular diseases, atherosclerosis, coronary heart disease, heart attack and stroke, metabolic disorders such as diabetes mellitus, neurodegenerative diseases, preferably Alzheimer's disease, and autoimmune diseases, preferably systemic lupus erythematosus or multiple sclerosis, the same applies, mutatis mutandis, as specified above in the context of the first aspect of the present invention.

In a preferred embodiment of the invention relating to a recombinant human monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention in accordance with the second aspect of the present invention, said viral infectious disease is selected from the group consisting of infections caused by coronaviruses (preferably SARS-CoV, SARS-CoV-2, MERS); influenza viruses, parainfluenza viruses, respiratory syncytial viruses (RSV), rhinoviruses, adenoviruses, enteroviruses, human metapneumoviruses, herpesviruses, preferably HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7, HHV-8.

To said viral disease selected from the group consisting of infections caused by coronaviruses (preferably SARS-CoV, SARS-CoV-2, MERS); influenza viruses, parainfluenza viruses, respiratory syncytial viruses (RSV), rhinoviruses, adenoviruses, enteroviruses, human metapneumoviruses, herpesviruses, preferably HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7, HHV-8, the same applies, mutatis mutandis, as specified above in the context of the first aspect of the present invention.

The above antibodies are particularly valuable for medical applications.

Thus, in a preferred embodiment of the invention, the present invention relates to a pharmaceutical composition comprising an effective amount of a recombinant human monoclonal natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use according to the present invention as defined above, and at least one pharmaceutically acceptable excipient.

The term "treatment or prevention" and the like in the context of the present invention generally refers to the production of a desired pharmacological and/or physiological effect. Thus, in the context of the present invention, the term "treatment" may refer to the treatment of (acute) states of a particular disease, but may also refer to prophylactic treatment in terms of complete or partial prevention of the disease or its symptom. Preferably, the term "treatment" refers to a therapeutic effect in terms of partial or complete cure of the disease and/or adverse effects and/or symptoms inherent in the disease. In this context, "acute" (condition) means that the subject exhibits symptoms of the disease. In other words, the subject to be treated actually needs treatment, and the term "acute treatment" in the context of the present invention refers to measures taken to actually treat the disease after the onset of the disease or an outbreak of the disease. Treatment may also be prophylactic or preventative treatment, i.e. measures taken to prevent disease, such as to prevent infection and/or the onset of illness.

The pharmaceutical composition of the present invention can be administered using a wide variety of delivery methods known to those skilled in the art. Administration can be systemic, topical, oral, or via aerosols, including (but not limited to) tablets, needle injections, inhalers, creams, foams, gels, lotions, and ointments.

An excipient or carrier is an inactive substance included in a formulation along with the active substance, i.e., the antibody mentioned above in the present invention, to create bulking compositions containing effective active substances. Excipients are often referred to as "bulking agents," "fillers," or "diluents." Bulking allows for easy and precise distribution of the drug substance during the manufacture of the dosage form. They can also serve various therapeutic-enhancing purposes, such as facilitating the absorption or solubility of the drug, or other pharmacokinetic purposes. Excipients can also be useful during the manufacturing process by facilitating the handling of the corresponding active substance, for example, by improving powder flowability or reducing sticking, in addition to promoting in vitro stability, such as preventing denaturation during the expected shelf life. The selection of appropriate excipients also depends on the route of administration and dosage form, as well as the active substance and other factors.

Thus, a pharmaceutical composition containing an effective amount of the above-described antibody according to the present invention may be in solid, liquid or gaseous form and may, inter alia, be in the form of powder(s), tablet(s), solution(s) or aerosol(s). Preferably, said pharmaceutical composition optionally contains a pharmaceutically acceptable carrier and/or diluent.

The pharmaceutical compositions can be administered to a subject in a suitable dose. Suitable compositions can be administered by various routes, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal, or intrabronchial administration. Particularly preferred is administration by injection and/or delivery, such as to a site in the pulmonary artery or directly to the lung. The compositions according to the invention can also be administered directly to the target site, such as by biolistic delivery to an external or internal target site, such as the lung. The dosage regimen should be determined by the attending physician and clinical factors. As is well known in medicine, dosages for any particular patient depend on many factors, including the patient's size, body surface area, age, the specific compound being administered, gender, time and route of administration, general health, and other medications being administered concurrently. The protein pharmaceutically active substance may be present in amounts ranging from 1 ng to 10 mg/kg of body weight per dose; however, doses below or above this approximate range are possible, particularly taking into account the factors mentioned above. If the regimen is a continuous infusion, a range of 1 mcg to 10 mg of substance per kg of body weight per minute may also be used.

Examples of suitable pharmaceutical carriers, excipients, and/or diluents are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be prepared using well-known conventional methods. Such pharmaceutical compositions can be administered to a subject in an acceptable dose, i.e., in an "effective amount," which can be readily determined by one of skill in the art using methods known in the art. The dosage regimen should be determined by the attending physician and clinical factors. As is well known in medicine, dosages for any particular patient depend on many factors, including the patient's size, body surface area, age, the specific compound being administered, gender, time and route of administration, general health, and other medications being administered concurrently.

Thus, the antibody of the present invention described above is preferably administered in an effective amount. The term "effective amount" refers to an amount sufficient to induce a detectable therapeutic response in the subject to whom the pharmaceutical composition is to be administered. Accordingly, the content of the antibody of the present invention in the pharmaceutical composition is not limited if it is necessary for the above-mentioned treatment, but is preferably from 0.0000001 to 10% by weight, based on the total composition. In addition, the antibody described herein is preferably used in a carrier. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the carrier to impart isotonicity to the composition. Examples of the carrier include, but are not limited to, saline, Ringen's solution, and dextrose solution. Suitable excipients, carriers or stabilizers are preferably non-toxic at the dosages and concentrations employed and include, but are not limited to, buffers such as citrate, phosphate and other organic acids; salt-forming counterions such as sodium and potassium; low molecular weight polypeptides (>10 amino acid residues); proteins such as serum albumin or gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as histidine, glutamine, lysine, asparagine, arginine or glycine; carbohydrates including glucose, mannose or dextrins; monosaccharides; disaccharides; other sugars such as sucrose, mannitol, trehalose or sorbitol; chelating agents such as EDTA; nonionic surfactants such as Tween, pluronic or polyethyleneglycol; antioxidants, including methionine, ascorbic acid, and tocopherol; and/or preservatives, such as

octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and meta-cresol). Suitable carriers and their forms are described more fully in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., 1985.

The progress of therapeutic treatment can be monitored by periodic evaluation. The antibody of the present invention or the pharmaceutical composition of the invention may be in the form of sterile aqueous or non-aqueous solutions, suspensions and emulsions, as well as creams and suppositories. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents and inert gases, etc. In addition, the pharmaceutical composition of the invention may contain other agents depending on the intended use of the pharmaceutical composition. The said agents may be, for example, polyoxyethylene sorbitan monolaurate, which is sold under the trade name Tween, propylene glycol, EDTA, citrate, sucrose, as well as other agents suitable for the intended use of the pharmaceutical composition, which are well known to a person skilled in the art.

According to the present invention, the term "pharmaceutical composition" refers to a composition intended for administration to a patient, preferably a sick human being.

The invention also relates to a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject as described above in this description.

The same applies, mutatis mutandis, to the said preferred variant of the method of treatment as is indicated above in the context of an antibody or pharmaceutical composition intended for the said use.

In the present invention, in a preferred embodiment, the subject is a mammal, such as a dog, cat, pig, cow, sheep, horse, rodent, such as a rat, mouse, and guinea pig, or a primate, such as a gorilla, chimpanzee, and human. In the most preferred embodiment, the subject is a human.

In the present invention, in a preferred embodiment of the above-mentioned first and second objects of the invention

The said human or humanized natural IgM and/or IgA antibody, which recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject as described above in the present description, can be administered in combination with an immunomodulator. Preferably, the said combination therapy is characterized by synergistic effects on the treatment proposed in the present invention.

The same applies, mutatis mutandis, to the said preferred variants of combination therapy as is indicated above in the context of the first and/or second objects of the present invention and/or the antibody or pharmaceutical composition intended for the above-mentioned use.

The term "combination" as used herein refers to a combination of a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject as described above in this description, and an immunomodulator as described below in this description.

A preferred embodiment of the invention provides for simultaneous administration. However, the combination also provides for sequential administration of the two components, i.e., the human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes mentioned above, and the immunomodulator mentioned below. Thus, one component can be administered before, simultaneously with, or after the other component of the combination, or vice versa.

Thus, the term "in combination" in the context of the present description is not limited to the period of time between the administration of the human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes specified above and the immunomodulator specified below. Thus, when the two components are not administered simultaneously/in parallel, the administrations may be separated from each other by 1 min, 5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h or 72 h or any suitable period of time easily determined by a person skilled in the art and/or specified in the present description. In a preferred embodiment of the invention, when the two components are not administered simultaneously/in parallel, the administrations may be separated from each other by 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours or 72 hours, or any suitable time interval easily determined by one of skill in the art and/or indicated herein.

Immunomodulators are well known to those skilled in the art and the term generally refers to an agent or drug that has the ability to modulate the behavior and/or function of specific immune cells or other cell types, such as endothelial cells, of the host.

Immunomodulators are known to act at various levels of the immune system. Therefore, various types of agents or drugs have been developed that selectively either inhibit or enhance specific populations and subpopulations of immunocompetent cells, i.e., lymphocytes, macrophages, neutrophils, natural killer (NK) cells, and cytotoxic T lymphocytes (CTLs).

More specifically, in preferred embodiments of the invention, said immunomodulator, immunomodulatory agent, or drug may have immunostimulatory (proinflammatory) effects on the immune response. Thus, in preferred embodiments of the invention, the immunomodulator, immunomodulatory agent, or drug may be an immunostimulant.

Alternatively, in other preferred embodiments of the invention, said immunomodulator, immunomodulatory agent or drug may have immunosuppressive (anti-inflammatory) effects on the immune response.

Immunomodulators are well known to those skilled in the art, and the immunomodulator proposed in the present invention is not limited to specific immunomodulators. Rather, one skilled in the art can select a suitable immunomodulator as long as it exhibits the aforementioned functional capabilities.

In preferred embodiments of the invention, the immunomodulatory drugs can be either small molecules or biological substances. The nature of the immunomodulatory drug, which is a small molecule or biological substance, is not limited. In preferred embodiments of the invention, the immunomodulatory drugs can be selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CD40 (agonist), CD40 ligand, anti-GM-CSF, anti-CSF-1R, anti-CTLA-4, anti-IL-6, anti-IL-6R, anti-CCL2, anti-CCL5, anti-CCR5 (antagonist), anti-CCR2 (antagonist). Examples of immunomodulatory agents include, but are not limited to, antibodies that bind to cytokines such as IL-6 or their specific receptors such as IL-6R and thereby neutralize the proinflammatory effects of the cytokine on other cell types, including immune cells.

Other examples of immunomodulatory agents include, but are not limited to, antibodies that bind to chemokines such as CCL2 and CCL5 or their specific receptors such as CCR2 and CCR5, and thereby neutralize the chemotactic effects of chemokines on other cell types, including immune cells.

Further examples of immunomodulatory agents include (but are not limited to) synthetic molecules such as maraviroc, which bind to cytokine receptors such as CCR5 and thereby neutralize the chemotactic effects of chemokines on other cell types, including immune cells.

One skilled in the art can select an appropriate immunomodulator suitable for modulating the behavior and/or function of specific immune cells or other cell types, such as endothelial cells, according to the present invention and according to the information provided above.

For example, immunomodulators can be selected from the group consisting of antibodies that bind to cytokines (preferably IL-6), antibodies that bind to a specific cytokine receptor (preferably IL-6R), antibodies that bind to chemokine(s) (preferably CCL2 and CCL5), antibodies that bind to specific receptor(s) of chemokine(s) (preferably CCR2 and CCR5), synthetic molecules (preferably maraviroc).

In a more preferred embodiment of the invention, the immunomodulator is maraviroc.

Maraviroc belongs to the class of CCR5 receptor antagonists and is used as an antiretroviral drug for the treatment of HIV infection. It is also classified as an entry inhibitor. It also reduces graft-versus-host disease in patients undergoing allogeneic bone marrow transplantation for leukemia. Maraviroc is an entry inhibitor. Specifically, maraviroc is a negative allosteric modulator of the CCR5 receptor, which is present on the surface of some human cells. The chemokine receptor CCR5 is a crucial coreceptor for most HIV strains and is necessary for viral entry into the host cell. The drug binds to CCR5, thereby blocking the association of the HIV gp120 protein with the receptor. As a result, HIV is unable to penetrate human macrophages and T cells.

In another also more preferred embodiment of the invention, the immunomodulator is dexamethasone.

Dexamethasone is a low-cost, medically available corticosteroid used in various inflammatory diseases due to its anti-inflammatory and immunosuppressant properties. Thus, dexamethasone is an anti-inflammatory and immunosuppressant immunomodulator. A preliminary report from the RECOVERY trial conducted at the University of Oxford (UK) demonstrated that dexamethasone reduced the mortality rate by approximately one-third in ventilated patients with severe COVID-19. This was likely due to its anti-inflammatory and immunosuppressant effects, which are most noticeable in patients with severe disease, as they are characterized by strong proinflammatory profiles. In contrast, dexamethasone had no beneficial effect on COVID-19 patients who did not require ventilation; in fact, it may even increase the mortality rate. Dexamethasone treatment was found to induce ACE expression, leading to the production of angiotensin II. Furthermore, angiotensin II receptor type 1 expression was shown to be increased by dexamethasone, as dexamethasone is likely involved in proinflammatory signaling via angiotensin II and in increasing oxidative stress and the accumulation of oxidized phospholipids/OSE. Although in severe COVID-19, the immunosuppressive and anti-inflammatory effects of dexamethasone likely outweigh this proinflammatory effect, in milder cases of COVID-19, where patients do not require ventilation, the role of dexamethasone-induced angiotensin II oxidative stress becomes more prominent, as reflected in the observed increase in mortality.

Preferably, combination therapy using dexamethasone with oxidized phospholipid/OSE-specific natural IgM/IgA antibodies provided in the present invention has a synergistic effect and increases the effectiveness of treatment even in patients with milder cases of COVID-19 who do not require ventilation.

In the present invention, in a preferred embodiment of the above-mentioned first and second aspects of the invention, said human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject as specified above in the present description, can be administered in combination with an antiviral compound, preferably an antiviral compound such as:

remdesivir;

favipiravir;

camostat mesylate;

nafomostate mesylate;

umifenovir; and/or

more potent neo-minophage C.

Preferably, such combination therapy has a synergistic effect on the treatment provided by the present invention.

The same applies, mutatis mutandis, to the said preferred variants of combination therapy as is indicated above in the context of the first and/or second objects of the present invention and/or the antibody or pharmaceutical composition intended for the above-mentioned use.

To the said concept of "combination" and "combination therapy" in the context of a combination with an antiviral agent, preferably with remdesivir; favipiravir; camostat mesylate; nafostat mesylate; umifenovir; and/or a more potent neo-minophagen C, and preferred variants of the said combination, the same applies, mutatis mutandis, as indicated above in the context of combination therapy with an immunomodulator.

Thus, as discussed above, the present invention is based on a common feature of host immune deficiency: various infectious diseases trigger massive formation and accumulation of oxidized phospholipids and OSE in the lungs, including in SARS-CoV-2-infected patients. This triggers the production of proinflammatory cytokines in macrophages, thereby initiating the deterioration phase of COVID-19 (and other infectious diseases). In the context of the present invention, high levels of circulating OSE-specific IgM and possibly IgA antibodies provide protection by binding to oxidized phospholipids and OSE, thereby promoting their safe clearance, which in turn counteracts the induction of the fatal cytokine storm syndrome and ARDS.

Thus, as noted above, it was unexpectedly found in the creation of the invention that a subset of natural IgM and/or IgA antibodies can be used to treat or prevent a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, i.e., to use a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes. According to the above, the present invention is based on the unexpectedly found fact that the stimulatory effect of oxidized phospholipids (oxPL) and oxidation-specific epitopes (OSE) on the activation of macrophages and the secretion of proinflammatory cytokines, such as IL-6, is involved in the initiation of the fatal cytokine storm syndrome and in the development of acute lung injury and ARDS in patients with severe COVID-19.

The above data form the basis for the above-mentioned first and second objects of the present invention, which relate to the fact that a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes can be used to treat or prevent a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject.

Since in the said embodiments of the present invention it is established that the reduction of oxidized phospholipids and/or oxidation-specific epitopes by using a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes for the treatment or prevention of a disorder or disease,

associated with/related to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, then, without being bound by theory, this unexpectedly discovered fact opens up the possibility of using combination therapy using compounds that stimulate the production of natural IgM/IgA antibodies or that contain natural IgM/IgA antibodies (e.g., using plasma or serum preparations enriched with natural IgM/IgA antibodies) or that contain recombinant IgM/IgA monoclonal antibodies specific for oxidized phospholipids or OSE, in combination with compounds that have a direct effect on pathogens, in particular, in the case of SARS-CoV-2 (but not limited to them), antiviral compounds, preferably such as remdesivir, favipiravir (Avigan), camostat mesylate, nafostat mesylate, umifenovir (Arbidol) and more neo-minophagen C (SNMC), creating a synergistic therapeutic effect by 1) eliminating the source of increased oxidized phospholipid/OSE production, i.e., viral replication, and 2) reducing accumulated products such as oxidized phospholipid/OSE through oxidized phospholipid/OSE-specific natural IgM/IgA antibodies.

Antiviral compounds are well known to those skilled in the art and generally refer to an agent, drug, or compound that has the ability to inhibit the development and/or reproduction of viruses. Thus, the term "antiviral compounds" refers to an agent, drug, or compound used to treat a viral infection. Most antiviral agents target specific viruses, but a broad-spectrum antiviral agent is effective against a broad range of viruses. Unlike most antibiotics, antiviral drugs do not destroy their target pathogen; rather, they inhibit its development. Thus, antiviral compounds in the context of the present invention are agents that kill a virus and/or suppress the virus's ability to replicate, thereby inhibiting the virus's ability to replicate and/or multiply.

Antiviral compounds are well known to those skilled in the art, and the antiviral compound proposed in the present invention is not limited to a particular antiviral compound. Rather, one skilled in the art can select a suitable antiviral compound as long as it exhibits the aforementioned functional capabilities.

Regarding the method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject according to the present invention, one skilled in the art can select an appropriate antiviral compound suitable for inhibiting viral development in accordance with the present invention. For example, the antiviral compound can be selected from the group consisting of antiviral compounds belonging to the class of direct-acting drugs and those belonging to the class of indirect-acting drugs.

Thus, a person skilled in the art knows that antiviral compounds are generally divided into two main classes based on their mechanism of action, namely, the aforementioned direct-acting antiviral compounds and indirect-acting antiviral compounds.

Direct-acting antiviral compounds are further subdivided into entry inhibitors, protease inhibitors, replication inhibitors, virus production inhibitors, virus release inhibitors, and virus maturation inhibitors depending on the stage they act on in the virus replication process.

Indirect-acting antiviral compounds exert their function by acting on cellular factors that modulate the cell so that viral replication is inhibited.

In preferred embodiments of the invention, the antiviral compound is selected from the group consisting of remdesivir; favipiravir; camostat mesylate; nafostat mesylate; umifenovir and the more potent neo-minophagen C (SNMC).

The above-mentioned remdesivir and favipiravir belong to the class of direct-acting antiviral compounds (they affect viral replication), camostat mesylate, nafostat mesylate and the more potent neo-minophagen C (SNMC) belong to the class of indirect-acting antiviral compounds, and umifenovir has a dual function.

Remdesivir (also known as GS-5734) is a monophosphoramidate prodrug of an adenosine analogue. It is a direct-acting antiviral agent with a broad antiviral spectrum, including filoviruses, paramyxoviruses, pneumoviruses, and coronaviruses. Remdesivir inhibits viral replication by blocking RNA-dependent RNA polymerase, causing premature, delayed chain termination during viral RNA synthesis. It has been clinically studied as a therapeutic agent against Ebola virus infection and was found to be well-tolerated, although less effective than monoclonal antibody-based therapies. Remdesivir is a potent inhibitor of SARS-CoV-2 replication in vitro and has demonstrated clinical benefits in rhesus macaques infected with SARS-CoV-2. A Phase 3 SIMPLE clinical trial conducted in patients with moderate COVID-19 found that patients treated with remdesivir for 5 days were 65% more likely to experience clinical improvement than those in the standard-of-care group. Clinical improvement was also demonstrated in the ACTT-1 trial conducted by the National Institute of Allergy and Infectious Diseases (NIAID) in hospitalized patients with varying degrees of disease severity. Remdesivir is approved under an emergency use authorization (EUA) for the treatment of patients with COVID-19.

Favipiravir (Avigan) is a prodrug that is metabolized to the antiviral product favipiravir-ribofuranosyl-5'-triphosphate. It blocks viral replication through chain termination during RNA-dependent RNA polymerase-mediated viral genome replication, using a mechanism similar to remdesivir. Thus, favipiravir also has broad-spectrum activity against several RNA viruses. Favipiravir was approved in 2014 in Japan for the treatment of new or emerging influenza viruses and has since been approved in China and Russia. Favipiravir likely blocks SARS-CoV-2 replication and has shown some beneficial effects in clinical trials. It has since been approved for use under the compassionate use program in Japan for the treatment of COVID-19.

Preferably, combination therapy comprising the use of natural IgM/IgA antibodies and the direct-acting antiviral drugs remdesivir or favipiravir has a synergistic effect by reducing the virus replication-induced accumulation of oxidized phospholipids/OSE in combination with improved clearance of accumulated oxidation products by oxidized phospholipid/OSE-specific natural IgM/IgA antibodies.

Camostat mesylate was developed in Japan as a protease inhibitor in the 1980s and was used to treat acute symptoms of chronic pancreatitis and postoperative reflux esophagitis. Camostat mesylate exhibits activity against transmembrane serine protease 2 (TMPRSS2), which has been shown to be required for efficient entry of SARS-CoV-1 and SARS-CoV-2 into lung cells.

Similar to camostat mesylate, the serine protease inhibitor nafomostat mesylate, approved for clinical use in Japan, also blocks TMPRSS2 and has been shown to block SARS-CoV-2 infection in vitro. Both drugs block TMPRSS2 activity, thereby preventing cleavage of the viral S protein, a prerequisite for viral entry into the target host cell. According to the present invention, a combination of TMPRSS2-inhibiting drugs that inhibit viral replication, such as camostat mesylate or nafomostat mesylate, with oxidized phospholipid/OSE-specific natural IgM/IgA antibodies, preferably exhibits synergistic therapeutic effects.

Because nafostat mesylate also inhibits/antagonizes the accumulation of receptor for advanced glycation end products (RAGE) ligands, its combination with oxidized phospholipid/OSE-specific natural IgM/IgA antibodies preferably has several synergistic effects by 1) reducing viral replication (via TMPRSS2 blockade), 2) decreasing the amount of RAGE ligands, and 3) clearing oxidation products via oxidized phospholipid/OSE-specific natural IgM/IgA antibodies.

Umifenovir (Arbidol) is a dual-function direct-acting/host-targeting virological agent approved in Russia and China for the treatment of respiratory viral infections, including influenza A and B. Umifenovir has been shown to effectively inhibit SARS-CoV-2 replication in vitro and is hypothesized to have antiviral activity in vivo. Umifenovir blocks viral membrane fusion with the target host cell membrane, thereby inhibiting viral entry, but its dual activity may also interfere with viral production and/or release. It is a hydrophobic molecule with the ability to form aromatic stacking interactions with specific amino acid residues, which likely contributes to its direct-acting antiviral activity, for example, by binding to viral glycoproteins important for viral entry. Furthermore, umifenovir has lipid binding properties and is believed to exert antiviral activity by directly binding to the viral lipid bilayer, as well as by directly binding to the plasma membrane of target cells, preventing viral uptake by endocytosis. Of particular note is the fact that umifenovir has antioxidant potential and, compared to the antioxidant Trolox, exhibits a more prolonged antioxidant activity in vitro.

Preferably, umifenovir exhibits synergistic effects in combination with oxidized phospholipid/OSE-specific natural IgM/IgA antibodies through two mechanisms: 1) it prevents viral replication and 2) reduces oxidative stress as a result of its antioxidant properties.

More potent neo-minophagen C (SNMC) is a glycyrrhizin-containing drug approved in Japan for the treatment of chronic liver diseases. Glycyrrhizin (GL) is a triterpene present in the roots and rhizomes of licorice (Glycyrrhiza glabra) and has been shown to have anti-inflammatory, antioxidant, and antiviral activities. Licorice extract has been shown to inhibit LDL oxidation and may have antioxidant activity. In an ex vivo study, LDL isolated from normolipidemic subjects who received oral licorice was found to be more resistant to oxidation than LDL isolated before licorice supplementation. In patients infected with the hepatitis C virus, long-term treatment with SNMC reduced the development of liver cirrhosis and hepatocellular carcinoma. Furthermore, GL has been shown to effectively block SARS-CoV-1 replication in vitro. GL is metabolized to systemically active glycyrrhizic acid (GA), which inhibits 11-beta-hydroxysteroid dehydrogenase (llbHSD), and both GL and GA have demonstrated antiviral activity. llbHSD inhibition can result in cortisol-mediated activation of mineralocorticoid receptors in aldosterone-specific peripheral tissues, including the lung, kidney, and nasal and endothelial cells, resembling the activity of high aldosterone levels. Aldosterone infusion in animal models resulted in decreased expression of the SARS-CoV-1 and -2 ACE2 receptors in the kidney, suggesting that GL/GA-induced llbHSD inhibition may also reduce ACE2 expression in aldosterone-specific tissue, thereby reducing viral entry and dissemination. It has also been suggested that GA inhibits the transcriptional expression of the protease TMPRSS2, a protein that, along with ACE2, is required for the effective entry of SARS-CoV-2 into cells. Importantly, although GL/GA can reduce protective ACE2 levels, which reduces angiotensin II levels, both GL and GA have anti-inflammatory effects through antagonism of toll-like receptor 4 (TLR4), and GL has been shown to inhibit ligand binding to RAGE.

Thus, GL and GA exert antiviral effects through down-regulation of the receptor (ACE2) and protease (TMPRSS2) required for efficient viral entry, as well as through anti-inflammatory actions that manifest as down-modulation of proinflammatory cytokine production and response to them.

Thus, preferably, the mentioned reduction in viral replication and reduction in oxidized phospholipids/OSE production, as well as the anti-inflammatory and antioxidant actions characteristic of SNMC, in combination with the removal of oxidized products by oxidized phospholipids/OSE-specific natural IgM/IgA antibodies, result in synergistic therapeutic actions.

As noted above, it was unexpectedly discovered in the creation of the invention that a subset of natural IgM and/or IgA antibodies can be used to treat or prevent a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, i.e., a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes. According to the above, the present invention is based on the unexpectedly discovered fact that the stimulatory effect of oxidized phospholipids (oxPL) and oxidation-specific epitopes (OSE) on macrophage activation and secretion of proinflammatory cytokines, such as IL-6, is involved in the initiation of the fatal cytokine storm syndrome and in the development of acute lung injury and ARDS in patients with severe COVID-19.

The said data form the basis of the above-mentioned first and second objects of the present invention, concerning the fact that a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes can be used to treat or prevent a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject.

Since, according to the said embodiments of the present invention, it has been established that the reduction of the amount of oxidized phospholipids and/or oxidation-specific epitopes using a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes can be used to treat or prevent a disorder or disease associated with/related to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, then, without going into theory, the said unexpectedly established fact opens up the possibility of modulating other functions of immune cells that are normally important for an effective antimicrobial immune response, ultimately counteracting, in general, the formation of oxidized phospholipids and/or oxidation-specific epitopes.

This is explained as follows.

For example, oxPARS administration was found to render mice highly susceptible to E. coli peritonitis, as evidenced by increased mortality and enhanced bacterial growth and dissemination. When studied under these experimental conditions, oxPARS potently inhibited the phagocytic capacity of neutrophils and macrophages, while stimulating the ability of macrophages to release large amounts of IL-6 (Kparr et al., J Immunol. 178 (2007): 993–1001). These data suggest that oxPLs formed during inflammatory responses may be involved in Gram-negative sepsis-associated death by impairing the phagocytic capacity of macrophages while simultaneously stimulating the secretion of proinflammatory cytokines such as IL-6. Furthermore, oxPLs have been shown to negatively regulate dendritic cell (DC) function. Maturation of DCs induced by pathogen-derived signals, such as those induced by TLR ligands, is a critical step in initiating the adaptive immune response required to clear infection. oxPLs, which are produced during infection, apoptosis, and tissue injury, have been shown to influence DC activation and maturation by blocking TLR3- and TLR4-mediated induction of costimulatory molecules such as CD40, CD80, CD83, and CD86 and secretion of the cytokines IL-12 and TNF. As a result, oxPAPC significantly reduces the costimulatory activity of TLR ligand-activated DCs, as evidenced by a reduced ability to induce proliferation and effector cytokine production by antigen-specific T cells (Bluml et al., J Immunol. 175 (2005): 501–508). In addition to the inhibitory effects of oxPL on T cell function by blocking DC maturation, oxPL has also been shown to directly inhibit T cell effector functions. Primary human T cells stimulated in the presence of different classes of oxPL, but not their native, non-oxidized counterparts, exhibited impaired proliferative capacity, upregulated activation markers such as MHC-II, CD25, and CD69, secreted Th1 effector cytokines such as IFNγ, IL-2, and IL-10, and displayed cytotoxic activity against antigen-positive target cells (Seyerl et al., Eur J Immunol. 38 (2008):778–787).

Based on the entire set of data obtained, without going into theory, it became clear during the development of the invention that oxPL and OSE formed in the lungs of patients with COVID-19 and patients infected with other dangerous pulmonary pathogens, such as SARS-CoV and H5N1, inhibit important functions of immune cells, including the phagocytic properties of macrophages and neutrophils, the maturation of DCs and their costimulatory activity, the development of Th1-type responses and the effector phase of pathogen-specific T-cell cytotoxicity, and thereby further contribute to the spread of the virus, the development of severe pneumonia and acute lung failure.

As detailed above, in order to influence the said inhibitory effects of oxPL and OSE on important immune cell functions, the administration of OSE-specific IgM and/or IgA isotype antibodies or plasma pools enriched therewith to affected patients restores the antiviral immune response and pathogen clearance according to the present invention.

The findings of the present invention are all the more surprising since it is known to those skilled in the art that oxPL and OSE exhibit anti-inflammatory and protective effects in sepsis and acute injury.

In fact, as noted above, the anti-inflammatory actions of oxPL and OSE are concentration-dependent and include (1) inhibition of “sterile” acute lung injury induced by inflammatory mediators of viral and bacterial origin (Ma et al., Am J Physiol Lung Cell Mol Physiol. 286 (2004): 808–816; Nonas et al., Am J Respir Crit Care Med. 173 (2006): 1130–1138); (2) inhibition of "aseptic" acute lung injury induced by noxious mechanical ventilation, and therefore it has been suggested that the use of stabilizing compounds such as 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine (PEIPC) and 1-palmitoyl-2-(5,6-ethoxycyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC) may have beneficial effects in other "aseptic" modes of lung injury such as ischemia/reperfusion (Nonas et al., Crit Care. 12, 2008, R27); and (3) inhibition of pulmonary vascular leakage and inflammation in secondary acute lung injury induced by acute necrotizing pancreatitis (Li et al., Pancreas. 35, 2007, pp. 27-36).

These anti-inflammatory actions are mediated by enhanced endothelial barrier function (Birukov et al., 2004 Circ Res. 95:892–901; Birukova et al., 2007 Am J Physiol Lung Cell Mol Physiol. 292:924–935), induction of signaling pathways leading to upregulation of anti-inflammatory genes, and inhibition of proinflammatory gene expression (Eligini et al., 2002 Cardiovasc Res. 55:406–415; Ma et al., 2004 Am J Physiol Lung Cell Mol Physiol. 286:808–816; Otterbein et al., 2000 Nat Med. 6:422–428; Otterbein et al., Nat Med. 9 (2003): 183-190), and by preventing the interaction of proinflammatory bacterial products with host cells (Bochkov et al., Nature. 419 (2002): 77-81; Walton et al., Arterioscler Throm Vase Biol. 23 (2003): 1197-1203). However, we hypothesize that in patients with ARDS caused by infections associated with pulmonary pathogens such as SARS-CoV-2, SARS-CoV, and possibly H5N1 influenza viruses, multiple mechanisms involved in ROS formation and oxidative stress are converged in the lungs of affected patients, where oxPL and OSE accumulate at concentrations high enough to potentiate the biological actions described herein.

In addition to the lack of sufficient quantities of oxPL and OSE-specific natural antibodies of the IgM and possibly IgA isotype, which leads to ineffective anti-inflammatory clearance of oxPL and OSE formed in inflamed tissues, as described above in the present invention without going into theory, this unexpectedly established fact opens up the possibility of modulating other functions of immune cells that are normally important for an effective antimicrobial immune response, ultimately counteracting, in general, the formation of oxidized phospholipids and/or oxidation-specific epitopes.

More specifically, based on the unexpectedly obtained data obtained in the present invention, it becomes apparent that several other mechanisms may be involved in the massive formation of ROS and the accumulation of oxPL and OSE to proinflammatory concentrations in disorders or diseases associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject.

These mechanisms include (but are not limited to):

(a) virus-induced downregulation of the expression of the surface receptor angiotensin-converting enzyme 2 (ACE2);

(b) accumulation of ligands for the receptor for advanced glycation end products (RAGE, also referred to as AGER) and, as a result, enhanced RAGE signaling; and

(c) depletion of protective alveolar macrophages CD169+CD206+ expressing the macrophage receptor with collagen structure (MARCO).

Thus, it is quite clear that these mechanisms provide opportunities for additional use of a human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, according to the present invention, thereby further counteracting the creation of oxidized phospholipids and/or oxidation-specific epitopes.

These additional mechanisms and possible ways of (additional) counteracting the creation of oxidized phospholipids and/or oxidation-specific epitopes are illustrated in Fig. 2A and Fig. 2B, respectively.

Thus, in a preferred embodiment of the above first and second objects of the invention,

the said human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes, intended for use in a method for treating or preventing a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject, should be used in combination with:

(a) an angiotensin-converting enzyme (ACE) inhibitor/antagonist, an angiotensin II receptor type 1 (AT1R) inhibitor/antagonist and/or a compound that modulates ACE2 receptor expression; and/or agonists

Ang(1-7), AT2R and/or MAS receptor agonists;

(b) a compound that inhibits/antagonizes/neutralizes ligands for the receptor for advanced glycation end products (RAGE) and/or a RAGE inhibitor/antagonist; and/or

(c) granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or a compound that increases the phagocytic activity of alveolar macrophages (AM), preferably with azithromycin.

Preferably, said combination therapy has a synergistic effect with respect to the treatment proposed in the present invention.

The same applies, mutatis mutandis, to the said preferred variants of combination therapy as is indicated above in the context of the first and/or second objects of the present invention and/or the antibody or pharmaceutical composition intended for the above-mentioned use.

Combination therapy with:

(a) an angiotensin-converting enzyme (ACE) inhibitor/antagonist, an angiotensin II receptor type 1 (AT1R) inhibitor/antagonist and/or a compound that modulates ACE2 receptor expression; and/or agonists

Ang(1-7), AT2R and/or MAS receptor agonists;

(b) a compound that inhibits/antagonizes/neutralizes ligands for the receptor for advanced glycation end products (RAGE) and/or a RAGE inhibitor/antagonist; and/or

(c) granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or a compound that increases the phagocytic activity of alveolar macrophages (AM), preferably with azithromycin,

described in more detail below.

(a) Angiotensin-converting enzyme (ACE) inhibitor/antagonist, angiotensin II receptor type 1 (AT1R) inhibitor/antagonist and/or a compound that modulates ACE2 receptor expression; and/or Ang(1-7), AT2R and/or MAS receptor agonists

The ACE2 receptor has been identified as a functional receptor for SARS-CoV-2 and SARS-CoV entry into host target cells. ACE2 is expressed at high levels in lung tissue, primarily in alveolar type II (AT2) epithelial cells, at moderate levels in bronchial and tracheal epithelial cells, and at low levels in cardiac, renal, and small intestinal epithelial cells. Furthermore, ACE2 expression is normally upregulated in response to viral infection by type I and II interferons in primary human upper respiratory tract epithelial cells.

However, in disorders or diseases associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD), particularly SARS-CoV-2, infection is distinguished by its failure to induce a significant interferon response. Thus, without getting into theory, it is clear that this explains the generally lower ACE2 levels.

ACE2 shares high homology with ACE, and both receptors play distinct key roles in regulating the renin-angiotensin system, which controls arterial blood pressure, electrolyte homeostasis, and cardiovascular regulation and remodeling. ACE cleaves angiotensin I (Ang-I) to form angiotensin II (Ang-II), while ACE2 inactivates Ang-II by cleaving it to form Ang(1-7) and is a negative regulator of the system. Ang-II binds to two downstream receptors, angiotensin II receptor 1 (AT1R) and angiotensin II receptor 2 (AT2R), and Ang(1-7) binds to the MAS receptor (MAS1 pro-oncogene, GPCR). Signaling through AT1R leads to vasoconstriction, hypertrophy, fibrosis, and inflammation, while AT2R and MAS receptors promote vasodilation, antifibrotic, and anti-inflammatory functions.

Using genetically modified mice, it was demonstrated that ACE2 and AT2R protect mice from severe acute lung injury induced by acid aspiration or sepsis, while other components of the renin-angiotensin system, including ACE, Ang-II, and AT1R, enhance disease pathogenesis, induce pulmonary vascular permeability and pulmonary edema, and impair lung function (Imai et al., Nature. 2005, 436, pp. 112–116).

In light of the above, it is clear that downregulation of ACE2 cell surface expression due to its use as an entry receptor for SARS-CoV-2 and SARS-CoV contributes to lung injury pathology in addition to the deleterious effects mediated by viral replication.

Thus, the administration of an angiotensin-converting enzyme (ACE) inhibitor/antagonist, an angiotensin II receptor 1 (AT1R) inhibitor/antagonist and/or a compound that modulates (i.e. increases or decreases) the expression of the ACE2 receptor; and/or Ang(1-7), AT2R agonists and/or MAS receptor agonists in combination therapy according to the above-mentioned first and/or second objects of the present invention and/or an antibody or pharmaceutical composition intended for the above-mentioned use, represent additional ways of counteracting the creation of oxidized phospholipids and/or oxidation-specific epitopes.

In the context of the present invention, the term "modulated (i.e. increased or decreased) expression" preferably means that the expression and/or activity of the ACE2 receptor in a given cell and/or a given organism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and most preferably at least 90% or 100% higher (or lower) in the presence of the corresponding compound than in the corresponding cell and/or the corresponding organism in the absence of the corresponding compound. In even more preferred embodiments of the invention, the increase (or decrease) in expression may be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments of the invention, the expression is at least 10-fold, more preferably at least 100-fold, and even more preferably at least 1000-fold higher (or lower) than in the corresponding cell and/or the corresponding organism in the absence of the corresponding compound.

The term "reduced" expression of the ACE2 receptor also refers to a situation in which the corresponding cell and/or the corresponding organism does not express the corresponding ACE2 receptor, i.e., the situation refers to zero corresponding expression. The term "increased" expression of the ACE2 receptor refers to a situation in which the ACE2 receptor is overexpressed and the concentration of the overexpressed ACE2 receptor preferably constitutes at least 5%, 10%, 20%, 30%, or 40% of the total protein content of the cell and/or organism.

Methods for measuring the expression level of said ACE2 receptor in a cell and/or organism are well known to those skilled in the art. In one embodiment, the expression level is measured by measuring the amount of the corresponding protein. Such methods are well known to those skilled in the art and include Western blotting, ELISA, etc. In another embodiment, the expression level is measured by measuring the amount of the corresponding RNA. Such methods are well known to those skilled in the art and include, for example, Northern blotting.

In fact, intraperitoneal administration of purified SARS-CoV spike (S) protein, which is the major viral protein responsible for ACE2 binding, was found to result in S protein accumulation in the lungs of mice subjected to acid aspiration. This induces downregulation of ACE2 expression, thereby increasing pathological changes in the lung parenchyma, assessed by increased lung compliance, vascular permeability, and edema formation. Importantly, Ang-II levels were significantly increased in the lung tissue of these mice due to S protein-induced ACE2 downregulation, and blockade of AT1R signaling with the specific inhibitor losartan significantly reduced the severity of acute lung injury and pulmonary edema (Kuba et al., Nat Med. 11 (2005): 875-879). Similarly, ACE2 expression was downregulated in lung homogenates from SARS-CoV-infected mice compared to uninfected controls. Corroborating these findings, a small number of studies found that plasma Ang-II levels were significantly elevated and linearly related to viral load and the severity of lung injury in COVID-19 patients (Liu et al., Sci China Life Sci. 63, 2020, pp. 364–374). Notably, elevated Ang-II levels were also found in patients infected with H5N1, the influenza A virus that has a mortality rate of up to 70% due to the induction of ARDS and respiratory failure. Additional analyses in mice demonstrated that infection with H5N1, but not H1N1, virus downregulated ACE2 expression in lung tissue, and serum Ang-II levels were significantly increased in animals with severe ARDS (Zhou et al., Nat Comm. 5:, 2014, p. 594). These results were unexpected because H5N1 influenza viruses have not been reported to use ACE2 as an entry receptor into infected host cells, and this may explain why only H5N1, but not H1N1, influenza virus induced ROS formation and oxPL accumulation in the lung tissue of infected mice, worsening the pathology of lung injury (Imai et al., Cell. 133, 2008, pp. 235–249).

The crucial role of ACE2 in regulating lung injury was further confirmed using ACE2-deficient mice, where ACE2 deficiency was shown to increase the severity of acute lung injury caused by acid aspiration, sepsis, or H5N1, while administration of soluble recombinant human ACE2 had a protective effect. Conversely, genetic deletion of ACE protected mice from severe lung injury.

Given that ACE2 is a key negative regulatory factor in the severity of pulmonary edema and acute respiratory failure, the present invention proposes that ACE2 downregulation and elevated Ang-II levels are a common molecular mechanism involved in the pathology of various pathogen-induced pulmonary diseases. Notably, Ang-II is an effective stimulator of expression and activation.

nicotinamide adenine dinucleotide phosphate oxidase (NAD(P)H) in various cell types, and one of the main actions of AT1R activation is the formation of ROS.

Thus, the present invention hypothesizes that elevated Ang-II concentrations found in patients infected with SARS-CoV-2, SARS-CoV, or H5N1 continuously stimulate ROS production through AT1R activation, which in turn activates the peroxidation of phospholipids present in cell membranes and surfactant, producing oxPL and OSE, which accumulate in the lungs and likely other inflamed tissues of infected patients. In situations in which the anti-inflammatory clearance of oxPL and OSE is insufficient, for example, due to decreased serum levels of oxPL and OSE-specific natural IgM and possibly IgA antibodies, as described herein, oxPL and OSE accumulate to concentrations sufficient to enhance the biological effects described herein.

Thus, it can be hypothesized that the administration of oxPL and OSE-specific natural IgM and possibly IgA antibodies or plasma pools enriched with them, in combination with drugs that specifically inhibit and/or reduce the expression of ACE receptors and/or AT1R, and/or increase ACE2 expression or increase ACE2-induced Ang-II metabolism, will have an additive or even synergistic effect on the formation of oxPL and OSE, and on the proinflammatory and immunomodulatory functions of oxPL and OSE in patients with COVID-19 and patients infected with other dangerous pulmonary pathogens, such as SARS-CoV and H5N1.

Thus, the administration of an angiotensin-converting enzyme (ACE) inhibitor/antagonist, an angiotensin II type 1 receptor (AT1R) inhibitor/antagonist and/or a compound that modulates the expression of the ACE2 receptor; and/or Ang(1-7), AT2R agonists and/or MAS receptor agonists in combination therapy according to the above-mentioned first and/or second objects of the present invention and/or an antibody or pharmaceutical composition intended for the above-mentioned use, represent additional ways of counteracting the creation of oxidized phospholipids and/or oxidation-specific epitopes.

A person skilled in the art can select an appropriate angiotensin-converting enzyme (ACE) inhibitor/antagonist, angiotensin II receptor 1 (AT1R) inhibitor/antagonist and/or a compound that modulates (i.e. increases or decreases) the expression of the ACE2 receptor that has the desired property according to the present invention and as described above.

For example, angiotensin-converting enzyme (ACE) inhibitors/antagonists and/or angiotensin II receptor type 1 (AT1R) inhibitors/antagonists can be selected from the group consisting of ramipril, lisinopril, olmesartan, telmisartan, losartan and azilsartan.

Examples of compounds that modulate (i.e. increase or decrease) ACE2 receptor expression can be selected from the group consisting of thiazolidinediones and ibuprofen.

Compounds that modulate (i.e., increase or decrease) the expression of the ACE2 receptor are well known to those skilled in the art, and the compounds that modulate (i.e., increase or decrease) the expression of the ACE2 receptor according to the present invention are not limited to specific compounds. Thus, a person skilled in the art can select a suitable compound that modulates (i.e., increases or decreases) the expression of the ACE2 receptor if it has the functional properties described above.

In fact, drugs that inhibit/antagonize ACE receptors (e.g., ramipril, lisinopril) or AT1R (e.g., olmesartan, telmisartan, losartan, azilsartan) are well known in the art and are widely used clinically for the control of acute and chronic hypertension, the treatment of left ventricular dysfunction and heart failure, the prevention of strokes, and the prevention and treatment of nephropathy in patients with hypertension or diabetes. Similarly, ACE2 expression levels can be increased by thiazolidinediones or ibuprofen, and upregulation of ACE2 expression reduces Ang-II levels, which are involved in reducing the accumulation of oxPL and OSE. Thus, the present invention suggests that drugs that upregulate ACE2 expression can be used in combination therapy with natural IgM and/or IgA antibodies targeting oxPL or OSE in the late stage of COVID-19, when the viral infection has been eliminated.

Because patients with hypertension and cardiovascular disease are likely treated with ACE and/or AT1R inhibitors, and this patient population has a high mortality rate following SARS-CoV-2 infection, concern has been raised about whether the use of such drugs could worsen morbidity and mortality from COVID-19. This hypothesis is based on data showing that multiple AT1R inhibitors (e.g., olmesartan, telmisartan, losartan, azilsartan) increased ACE2 mRNA expression and protein levels in animal models of cardiovascular disease, which in turn could facilitate SARS-CoV-2 entry into host cells and viral replication.

To analyze the effects of ACE and AT1R inhibitors on the clinical course of COVID-19, a retrospective, single-center analysis of 112 patients was conducted. This analysis demonstrated that the use of these drugs had no effect on the mortality rate of COVID-19 patients with cardiovascular disease. Mice pre-treated with olmesartan showed a reduction in the incidence of acute pulmonary edema and lung injury induced by acid aspiration and the SARS-CoV S protein, consistent with the putative protective effect of losartan on the severity of lung injury.

To determine whether treatment of patients with COVID-19 with renin-angiotensin modulators results in beneficial or harmful effects, multicenter, double-blind, placebo-controlled, randomized trials are currently underway to examine the effects of losartan on mortality and hospitalization in COVID-19 patients requiring hospitalization (NCT04312009) and those not requiring hospitalization (NCT04311177). Because recombinant human ACE2 exhibited beneficial effects in animal models of acid aspiration-induced acute lung injury, SARS-CoV S protein, or H5N1 infection, its use in the treatment of patients with COVID-19 was tested in a randomized, controlled pilot clinical trial (NCT04287686). However, this study was terminated on March 17, 2020, for reasons that remain unknown.

Based on the molecular mechanisms involved in the pathology of SARS-CoV-2, SARS-CoV, and H5N1 influenza virus-induced lung injury described in the present invention, the present invention suggests that oxPL and OSE-specific IgM and possibly IgA antibodies or plasma pools enriched therein can be combined with human recombinant ACE2.

Preferably, said combination therapy has a synergistic effect with respect to the treatment proposed in the present invention, i.e. by affecting the formation of oxPL and OSE in inflamed tissues and the pro-inflammatory and immunomodulatory functions of oxPL and OSE in patients with COVID-19.

Thus, in a preferred embodiment of the invention, the administration of ACE2, preferably human ACE2 and more preferably human recombinant ACE2, in combination therapy according to the above-mentioned first and/or second aspects of the present invention and/or an antibody or pharmaceutical composition intended for the above-mentioned use, represent additional ways of counteracting the creation of oxidized phospholipids and/or oxidation-specific epitopes.

Thus, the therapeutic use of recombinant ACE2 is described, for example, by Monteil et al., Cell. 181, 2020, pp. 1–9; Imai et al., Nature. 436, 2005, pp. 112–116; Zhou et al., Nat Comm. 5, 2014, p. 3594 and Khan et al., Critical Care. 21, 2017, p. 234.

Human ACE2 is known to those skilled in the art and has the amino acid sequence shown in SEQ ID NO: 18.

However, the present invention is not limited to the administration of a particular human ACE2 having the amino acid sequence presented in SEQ ID NO: 18, as provided in the present invention, but also the use of ACE2 that has an amino acid sequence that is at least 70% identical to SEQ ID NO: 18, wherein ACE2 has the ability to cleave angiotensin-II to Ang(1-7).

In a more preferred embodiment of the invention, ACE2 comprises an amino acid sequence that is at least n% identical to SEQ ID NO: 18 above, wherein n is an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, and has the ability to cleave angiotensin II to Ang(1-7).

The same applies to the above definition of sequence identity.

Assays for determining the activity of ACE2 (and its variants) in cleaving angiotensin II to Ang(1-7) are known in the art.

As described above, ACE2 converts Ang-II to Ang(1-7), which activates AT2R and the MAS receptor, reducing inflammation, thrombosis, pulmonary injury, and fibrosis. Ang(1-7) has also been shown to prevent NAD(P)H oxidase (NOX) activation and exhibit antioxidant activity. MAS receptor activation has been proposed as a sarcopenia therapy by Biophytis, the company that developed BIO101, a small molecule targeting the MAS receptor. Phase 2/3 clinical trials using BIO101 in patients with COVID-19 are currently ongoing.

Thus, in a preferred embodiment of the invention, the administration of Ang(1-7), AT2R agonists and/or MAS receptor agonists in a combination therapy according to the above-mentioned first and/or second aspects of the present invention and/or an antibody or pharmaceutical composition intended for the above-mentioned use represent additional ways to counteract the creation of oxidized phospholipids and/or oxidation-specific epitopes, thereby reducing the accumulation of oxPL and OSE and the pro-inflammatory or immunomodulatory functions of oxPL and OSE in a subject having a disorder or disease associated/linked/caused by a deficiency of natural IgM/IgA antibodies (NAD), preferably in a patient with COVID-19.

Preferably, said combination therapy has a synergistic effect with respect to the treatment proposed in the present invention, i.e. by affecting the formation of oxPL and OSE in inflamed tissues and the proinflammatory and immunomodulatory functions of oxPL and OSE in a subject having a disorder or disease,

associated/linked/caused by deficiency of natural IgM/IgA antibodies (NAD), preferably in a patient with COVID-19.

Angiotensin(1-7) (or Ang(1-7)) is known to those skilled in the art as an active heptapeptide of the renin-angiotensin system (RAS), which has the peptide sequence Asp-Arg-Val-Tyr-Ile-His-Pro). Angiotensin(1-7) is a vasodilator that plays important roles in cardiovascular organs such as the heart, blood vessels, and kidneys, whose functions are often opposite to those attributed to the main effector component of the RAS, angiotensin II (Ang II). Ang(1-7) has been shown to have antioxidant and anti-inflammatory effects. Ang(1-7) plays a protective role in cardiomyocytes of spontaneously hypertensive rats by increasing the expression of nitric oxide synthase enzymes in endothelial and neuronal cells, leading to increased nitric oxide production. Ultimately, Ang(1-7) exerts antiarrhythmogenic effects in animal models. In blood vessels, Ang(1-7) induces the release of vasodilators such as prostanoids and nitric oxide.

A person skilled in the art can select a suitable AT2R or MAS receptor agonist that has the property required according to the present invention and as described above.

For example, Ang(1-7) or AT2R or MAS receptor agonists can be selected from the group consisting of:

AT2R-aroHHCTOB: Ang(1-7), peptide agonists such as CG42112A and dKcAng(1-7) (LP2-3), 3-amino acid substituted angiotensin II gamma repeat mimetics incorporated into angiotensin II; small molecule agonists such as Compound 21; and agonist monoclonal antibodies; MAS receptor agonists: Ang(1-7), peptide agonists such as TXA127 (Ang(1-7)), cyclic Ang(1-7), Ang(1-6)-O-Ser-Glc-NH ₂ (PNA5), hydroxypropyl-β-cyclodextrin (HPβCD)/Ang(1-7), CGEN-856 and CGEN-857; small molecule agonists such as in Sarconeos (BIO101) and AVE0991; and agonistic monoclonal antibodies.

(b) A compound that inhibits/antagonizes/neutralizes ligands for the receptor for advanced glycation end products (RAGE) and/or RAGE inhibitors/antagonists

The receptor for advanced glycation end products (RAGE) is a proinflammatory PRR implicated in the pathogenesis of numerous inflammatory diseases. RAGE exists in the body in two main forms: membrane-bound RAGE (mRAGE) and soluble RAGE (sRAGE). mRAGE exhibits signaling activity in response to ligand binding, while sRAGE functions as a decoy receptor that sequesters RAGE ligands and thereby counteracts mRAGE signaling and inflammatory responses. In adult human tissues, RAGE is constitutively expressed at baseline in the lungs, where it is primarily localized to the basement membrane of alveolar epithelial cells type 1 (ATI). In addition to expression in the pulmonary epithelium, RAGE expression is also found in vascular smooth muscle cells, airway smooth muscle cells, endothelial cells, neurons, and immune cells such as macrophages, DCs, eosinophils, T cells, and B cells. However, many cells and tissues induce RAGE expression only when they are activated to do so, for example, due to the local availability of RAGE ligands. RAGE can bind to numerous different endogenous ligands, which are classified as DAMPs, including advanced glycation end products (AGEs), S100 proteins/calgranulins, high-mobility group protein 1 (HMGB1), DNA or RNA, OSE, and phosphatidylserine.

AGEs are formed by the non-enzymatic Maillard reaction between the carbonyl group on an aldose sugar (usually glucose) and amino groups on proteins or phospholipids. Elevated AGE levels are found in patients with diabetes due to high blood glucose levels. Importantly, age and oxidative stress also increase AGE levels. S100 proteins are small calcium-binding proteins localized to sites of inflammation, where they are released by activated inflammatory cells. Numerous S100 proteins can activate RAGE in various tissues, initiating an inflammatory response. HMGB1 is a nuclear protein normally involved in chromatin remodeling; however, it can also be passively released from damaged cells as a proinflammatory alarmin. Furthermore, neutrophils, macrophages, natural killer cells, and DCs can actively secrete HMGB1, which is often associated with DNA. Neutrophils secrete NETs (neutrophil extracellular traps), which consist of decondensed DNA bound by histones and HMGB1.

NETosis, i.e., NET secretion by neutrophils, is likely increased during severe COVID-19, and serum concentrations of NETosis products (e.g., cell-free DNA) correlate with COVID-19 severity (Middleton et al., Blood. 2020, doi: 10.1182/blood.2020007008). Through electrostatic interactions between the positive charge on RAGE and the negative charges on the nucleic acid backbone, RAGE also directly binds to DNA or RNA, facilitating their uptake by the cell to enhance inflammatory responses. RAGE activation induces sustained nuclear factor kappa B (NFκB) signaling, and a major byproduct of RAGE signaling is excessive ROS formation, which are also involved in NFκB activation and enhance other inflammatory mechanisms such as increased expression of vascular cell adhesion molecule 1 (VCAM-1), oxPL and OSE formation, or cell apoptosis.

The presence of RAGE ligands in the extracellular environment has been shown to upregulate RAGE expression. This occurs because NFkB can directly bind to the RAGE gene, increasing RAGE expression and further amplifying inflammatory signaling cascades. Importantly, RAGE ligands are not cleaved or altered, preventing additional signaling when bound to RAGE and transmitting signals through RAGE. Thus, increased RAGE signaling leads to the production of more RAGE ligands, such as AGEs and OSEs, due to ROS formation and oxidative stress. As the ligands accumulate, they continually amplify the inflammatory response, accumulating in the inflamed area, thereby causing chronic pathological inflammation in various respiratory diseases.

The central role of RAGE in lung injury pathology is supported by data showing that RAGE-deficient mice are protected from hyperoxia-induced acute lung injury and associated death, suggesting that intact RAGE signaling exacerbates lung inflammation and respiratory failure. RAGE-deficient mice are also partially protected from lung injury induced by challenge with Gram-negative (E. coli) or Gram-positive (S. pneumoniae) bacteria.

In humans, systemic and alveolar levels of HMGB1, S100A12, and sRAGE from damaged ATI cells, as well as elevated sRAGE levels in ARDS patients and plasma levels, correlate with severe lung injury and increased mortality. The destructive positive feedback loop between RAGE, NFkB, ROS, and the production of increased RAGE ligands may be attenuated by sRAGE release either from damaged cells through alternative splicing events or by proteolytic cleavage of mRAGE by ADAM 10 or matrix metalloproteinase 9. Notably, mRAGE has also been shown in mice to bind to Mac-1 integrin (aMb2, CD11b/CD18) on leukocytes, facilitating their recruitment to inflamed tissue. Given that murine B1 cells constitutively express Mac-1 and that influenza virus infection leads to the recruitment of B1 cells to lung tissue, where protective natural IgM antibodies are secreted, it is plausible that B1 cell recruitment to inflamed tissue may be initiated by activated RAGE. Furthermore, it is well established that many RAGE ligands, such as AGE, OSE, DNA/RNA, and phosphatidylserine, are targets of B1-derived natural antibodies in mice and humans. Taken together, these data suggest that RAGE plays a pivotal role in proinflammatory immune responses in inflamed tissues, which may be further exacerbated in situations where effective clearance of RAGE ligands, such as AGE, DNA/RNA, OSE, and apoptotic cells, cannot occur, for example, due to reduced levels of natural IgM and possibly IgA antibodies.

Thus, the present invention hypothesizes that RAGE ligands drive the pathogenesis and ARDS in patients with severe COVID-19 who have evidence of increased oxidative stress, AGE accumulation, due to advanced age and comorbidities such as diabetes, and reduced levels of natural IgM and/or IgA antibodies, which normally promotes the anti-inflammatory clearance of RAGE ligands.

Thus, in a preferred embodiment of the invention, the administration of a compound that inhibits/antagonizes/neutralizes ligands for the receptor for advanced glycation end products (RAGE) in a combination therapy according to the above-mentioned first and/or second aspects of the present invention and/or an antibody or pharmaceutical composition intended for the above-mentioned use represents an additional way to counteract the creation of oxidized phospholipids and/or oxidation-specific epitopes, thereby reducing the accumulation of oxPL and OSE and the pro-inflammatory or immunomodulatory functions of oxPL and OSE in a subject having a disorder or disease associated/linked/caused by a deficiency of natural IgM/IgA antibodies (NAD), preferably in a patient with COVID-19.

Preferably, said combination therapy has a synergistic effect with respect to the treatment proposed in the present invention, i.e. by affecting the formation of oxPL and OSE in inflamed tissues and the proinflammatory and immunomodulatory functions of oxPL and OSE in a subject having a disorder or disease,

associated/linked/caused by deficiency of natural IgM/IgA antibodies (NAD), preferably in a patient with COVID-19.

A person skilled in the art can select a suitable compound that inhibits/antagonizes/neutralizes ligands for the receptor for advanced glycation end products (RAGE) that has the property required according to the present invention and as described above.

For example, compounds that inhibit/antagonize/neutralize ligands for the receptor for advanced glycation end products (RAGE) can be selected from the group consisting of nafostat mesylate (NM), gabexate mesylate (GM), sivelestat, atorvastatin, simvastatin, methotrexate (MTX), alagebrium (ALT-711), SYI-2074 (ALT-2074), and paquinimod (ABR-215757).

Other examples of compounds that inhibit/antagonize/neutralize ligands for the receptor for advanced glycation end products (RAGE) are compounds that can be selected from the group consisting of small molecules such as glycyrrhizin (GL), carbenoxolone (CBX), tanshinone I, tanshinone IIa, cryptotanshinone, sodium sulfonate-derivatized tanshinone IIa (TSNIIA-SS), epigallocatechin-3-gallate (EGCG), quertecin, lycopene, nafostat mesylate (NM), gabexate mesylate (GM), sivelestat, atorvastatin, simvastatin, ethyl pyruvate (EP) and derivatives thereof, methotrexate (MTX), alagebrium (ALT-711), SYI-2074 (ALT-2074), cromolyn, paquinimod (ABR-215757) and tasquinimod (ABR-215050); recombinant proteins such as box A protein (a truncated N-terminal domain of HMGB1) and box A-acidic tail fusion protein; peptides such as S100P-derived peptides, carnosine, homocarnosine, anserine, glutathione; and monoclonal antibodies specific for proteins, lipids, and other molecules modified with carboxymethyl lysine (CML), proteins, lipids, or other molecules modified with carboxyethyl lysine (CEL), proteins, lipids, or other molecules modified with glucose, HMGB1, and S100 proteins.

Furthermore, accumulation of RAGE ligands may be involved in the production of long-acting autoimmune IgG, which explains the prolonged symptoms in some recovering COVID-19 patients.

In fact, the present invention unexpectedly revealed (see Example 22 and Fig. 3) that autoimmune antibodies are produced in patients with severe COVID-19. These data confirm that the absence of natural antibodies (NAs) can lead to the production of autoimmune antibodies in severe COVID-19. The presence of these autoimmune antibodies indicates recurrent or prolonged symptoms of COVID-19, confirming that sufficient levels of natural antibodies, administration of monoclonal natural IgM or IgA, or preparations enriched with natural antibodies (e.g., Pentaglobin®) according to the present invention can prevent the formation of or reduce levels of autoimmune antibodies.

In this scenario, it can be hypothesized that administration of oxPL and OSE-specific IgM and possibly IgA antibodies or plasma pools enriched with them counteracts the destructive inflammatory immune response by promoting the clearance of RAGE ligands from the body and thus represents an important mechanism involved in the resolution of the self-amplifying proinflammatory signaling cascade driven by RAGE. Furthermore, the present invention hypothesizes that the administration of oxPL and OSE-specific IgM and possibly IgA antibodies or plasma pools enriched therefor, in combination with selective RAGE inhibitors or antagonists or in combination with antibodies specific for RAGE ligands, such as anti-HMGB1 antibodies, has a synergistic effect by affecting the formation of oxPL and OSE and other RAGE ligands in inflamed tissues and the proinflammatory and immunomodulatory functions of oxPL, OSE and other RAGE ligands in patients with COVID-19.

Thus, in a preferred embodiment of the invention, the administration of a RAGE inhibitor/antagonist in a combination therapy according to the above-mentioned first and/or second aspects of the present invention and/or an antibody or pharmaceutical composition intended for the above-mentioned use represent additional ways to counteract the creation of oxidized phospholipids and/or oxidation-specific epitopes, thereby reducing the accumulation of oxPL and OSE and the pro-inflammatory or immunomodulatory functions of oxPL and OSE in a subject having a disorder or disease associated with/related to/caused by a deficiency of natural IgM/IgA antibodies (NAD), preferably in a patient with COVID-19.

Preferably, said combination therapy has a synergistic effect with respect to the treatment proposed in the present invention, i.e. by affecting the formation of oxPL and OSE in inflamed tissues and the proinflammatory and immunomodulatory functions of oxPL and OSE in a subject having a disorder or disease,

associated/linked/caused by deficiency of natural IgM/IgA antibodies (NAD), preferably in a patient with COVID-19.

One skilled in the art can select a suitable RAGE inhibitor/antagonist that has the property required according to the present invention and as described above.

As examples, RAGE inhibitors/antagonists can be selected from the group consisting of small molecules such as TTP488 (azeliragon) and its derivatives, FPS-ZM1; antagonist RAGE-specific peptides; and antagonist RAGE-specific monoclonal antibodies.

(c) Granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or a compound that enhances the phagocytic activity of alveolar macrophages (AM), preferably azithromycin

The lung contains a large number of macrophages, including two main populations of interstitial (IM) and alveolar macrophages (AM), which are located in different anatomical compartments.

AM typically express the major transcription factor peroxisome proliferator-activated receptor γ (PPARγ), a critical regulator of lipid metabolism that is induced by granulocyte-macrophage colony-stimulating factor (GM-CSF). TGFβ is another cytokine required for AM development and homeostasis. Unlike GM-CSF, which is secreted by alveolar epithelial cells type 2 (AT2), TGFβ is produced by AM itself, maintaining its homeostasis.

Murine AMs originate from fetal monocytes, seed the lung, and differentiate into mature AMs after birth under the influence of GM-CSF, TGFβ, and PPARγ. Accumulating evidence supports the concept that AMs are maintained in the lung at steady state through their capacity for self-renewal under the critical influence of GM-CSF and PPARγ, which are conserved between mice and humans. AMs are localized in the alveolar lumen, where gas exchange occurs at the alveolar-capillary membrane, and their strategic position allows them to clear the airways of pathogens, apoptotic cells, and other airborne particles through phagocytosis, which is essential for maintaining vital oxygen uptake. AMs express several factors that enhance immune tolerance, such as TGFβ, as well as inhibitory receptors, limiting their proinflammatory activity under steady state conditions.

The primary function of AM is their ability to catabolize surfactant. Deficiencies in GM-CSF or PPARγ, and consequently AM deficiency, lead to inflammatory lung disease due to impaired surfactant metabolism. The ability of AM to catabolize surfactant is based on the expression of phagocytic receptors such as SR-AI and the macrophage receptor for collagen-like structures (MARCO), which bind to oxPLs present in surfactant and facilitate their entry into intracellular compartments where lipids are used for energy metabolism via fatty acid oxidation. In contrast, IM and monocyte-derived macrophages do not express MARCO under steady-state conditions or express it at significantly lower levels, and they do not utilize lipids for energy metabolism, relying on glycolysis. The importance of MARCO for AM-mediated clearance of oxPLs present in surfactant was demonstrated using genetically modified mice lacking MARCO expression. In these mice, ozone exposure resulted in excessive production of various classes of oxPLs in the surfactant, which contributed to an inflammatory environment and acute lung injury, while wild-type MARCO-expressing mice were protected from severe lung injury induced by ozone instillation. The study also demonstrated that intratracheal instillation of oxPLs induced a significant neutrophil influx in MARCO-deficient mice but had no effect on wild-type mice, consistent with increased oxPL uptake by normal AM compared to MARCO-deficient AM (Dahl et al., J Clin Invest. 117 (2007): 757–764).

These results therefore suggest that AMs potentially internalize proinflammatory oxPL via MARCO without triggering the typical deleterious lung response mediated by proinflammatory macrophages or monocyte-derived neutrophils. These data also suggest that reduced AM levels in the lungs contribute to the development of severe lung injury and ARDS in individuals induced by infections with pulmonary pathogens such as SARS-CoV-2, SARS-CoV, or the H5N1 influenza virus.

In fact, a single-cell RNA sequencing analysis of immune cells in the BAL fluid of patients with varying degrees of COVID-19 severity and healthy donors revealed that classical MARCO-positive AMs were depleted in the lungs of severely ill patients, while AMs were abundant in the BAL fluid of patients with mild COVID-19 and healthy donors. BAL fluid from patients with severe COVID-19 contained higher relative levels of proinflammatory macrophages and monocyte-derived neutrophils and lower relative levels of DCs and effector T cells compared to patients with mild disease. Furthermore, pulmonary macrophages from patients with severe COVID-19 expressed significantly higher levels of inflammatory cytokines and chemokines such as IL-6, IL-1β IL-8, TNFα, CCL2, CCL3, CCL4, and CCL7 compared with macrophages contained in the BAL fluid of patients with mild COVID-19.

Thus, the absence of AMs contributes to the creation of a proinflammatory environment in the lungs of patients with severe COVID-19, responsible for the development of fatal ARDS, although the mechanism of AM depletion remains unclear. Since AMs are located in close proximity to AT1 and AT2 pneumocytes and express low levels of ACE2 receptors, it is possible that AMs are directly infected by SARS-CoV-2, causing their depletion, as observed in patients with severe COVID-19. In support of this hypothesis, human coronavirus E229 has been shown to infect AMs, leading to the secretion of proinflammatory cytokines such as CCL4, CCL5, and TNFalpha. Similarly, diffuse alveolar injury was associated with SARS-CoV-1 infection of AT2 pneumocytes and AM in the lung of a 73-year-old man 7 days after hospitalization (Shieh et al., Hum Pathol. 36, 2005, pp. 303–309). AT2 pneumocytes are the major surfactant producers, and the authors hypothesized that infection of this cell type initiates increased surfactant accumulation with higher levels of oxPL and OSE as a result of enhanced oxidative stress and impaired clearance mechanisms.

Since SARS-CoV-2 uses the same entry receptor (ACE2) and the same cellular proteases (furin, TMPRSS2) for S protein-mediated viral entry, it is highly likely that SARS-CoV-2 infects both AT2 pneumocytes and AM, which could likely contribute to increased surfactant accumulation with inflammatory levels of oxPL and OSE, cellular debris, and AM depletion as a result of their inflammation.

In support of direct SARS-CoV-2 infection, subcapsular macrophage populations from CD 169+ lymph nodes and the splenic marginal zone express the SARS-CoV-2 entry receptor ACE2 and these macrophages were found to contain the SARS-CoV-2 nucleoprotein (NP) (Feng et al., 2020, medRxiv, doi: https://doi.org/10.1101/2020.03.27.20045427).

Alternatively, mouse studies have demonstrated that AM numbers were reduced by several forms of tissue injury, such as ionizing radiation, viral infection, and LPS-induced lung injury. This suggests that damage to the pulmonary epithelium may lead to a reduction in integrin-dependent TGFβ activation in AM, thereby causing a decrease in AM numbers. In the case of COVID-19, it is possible that reduced AT2 cell numbers due to SARS-CoV-2 replication impairs local GM-CSF production, which in turn leads to AM depletion.

Alternatively, mouse experiments have demonstrated that with increasing age, AM naturally depletes, leading to a decrease in their numbers in older mice. It has also been suggested that the contribution of HPSC-derived monocytes to the AM compartment steadily increases with age. The proliferative properties of AM and the clearance of apoptotic neutrophils by AM are impaired in older mice. Furthermore, AM from older mice are resistant to IFNγ, resulting in reduced killing of phagocytosed pathogens.

Thus, it is highly likely that age-related changes in pulmonary macrophages contribute to a state of weakened host defenses, coupled with monocytic and neutrophilic inflammation, which causes excessive tissue damage in the lungs. Notably, mouse studies have shown that B1 cells residing in the pleural space are recruited to the lungs in response to infection, where they give rise to a population of so-called innate response-activating (IRA) B cells, which produce large amounts of IL-3 and GM-CSF, the latter of which induces increased secretion of natural IgM antibodies via an autocrine mechanism. The importance of IRA-B cells in the clearance of infection was demonstrated using mice lacking IRA-B cells due to B cell-restricted GM-CSF deficiency, as these mice were particularly susceptible to bacterial sepsis, as evidenced by a significantly higher mortality rate compared to control mice, associated with marked inflammation, induction of cytokine release syndrome, and more severe bacteremia, leading to septic shock, multiple organ failure, and death.

In humans, GM-CSF-producing IRA-B cells with a CD5+CD19+CD20+IgM+IgD+ phenotype have also been detected in the tonsils, functioning as a first line of defense against upper respiratory tract infections. Given that B1 cell numbers decline with age in mice and humans, the absence of GM-CSF-producing IRA-B cells in the lungs of infected individuals not only results from reduced levels of protective natural IgM and/or IgA antibodies but may also contribute to AM depletion and the pathogenesis of lung injury. In support of this model, the present study found that low plasma IL-3 levels were associated with increased severity and mortality in SARS-CoV-2 infection, demonstrating that IRA-B cells were absent from severely ill COVID-19 patients.

Thus, the present invention hypothesized that administration of GM-CSF to patients with COVID-19 may have beneficial effects in terms of restoring the AM population and the production of protective natural IgM and possibly IgA antibodies by B1 and/or IRA B cells.

Thus, in a preferred embodiment of the invention, the use of GM-CSF in a combination therapy according to the above-mentioned first and/or second aspects of the present invention and/or an antibody or pharmaceutical composition intended for the above-mentioned use represents an additional way to counteract the creation of oxidized phospholipids and/or oxidation-specific epitopes, thereby reducing the accumulation of oxPL and OSE and the proinflammatory or immunomodulatory functions of oxPL and OSE in a subject having a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD), preferably in a patient with COVID-19.

Preferably, said combination therapy has a synergistic effect with respect to the treatment proposed in the present invention, i.e. by affecting the formation of oxPL and OSE in inflamed tissues and the proinflammatory and immunomodulatory functions of oxPL and OSE in a subject having a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD), preferably in a patient with COVID-19.

GM-CSF is known to those skilled in the art and has the amino acid sequence shown in SEQ ID NO: 19.

However, the present invention is not limited to the administration of a specific GM-CSF having the amino acid sequence presented in SEQ ID NO: 19 in the present invention, but also includes the administration of GM-CSF comprising an amino acid sequence at least 70% identical to SEQ ID NO: 19, wherein said GM-CSF has the ability to induce proliferation of TF-1 cells.

In a more preferred embodiment of the invention, GM-CSF comprises an amino acid sequence that is at least n% identical to the above sequence of SEQ ID NO: 19, wherein n is an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein said GM-CSF has the ability to induce TF-1 cell proliferation.

The above information applies to the determination of sequence identity.

Assays for determining the activity of GM-CSF (and its variants) in inducing TF-1 cell proliferation are known in the art.

Additionally, treatment of 1,438 hospitalized patients with COVID-19 with azithromycin was found to have a beneficial effect. The hazard ratio for patients treated with azithromycin was 0.56 [95% CI, 0.26-1.21]. The odds of death were slightly reduced for patients treated with azithromycin (10.0% [95% CI, 5.9%-14.0%]) compared with untreated patients (12.7% [95% CI, 8.3%-17.1%]) (Rosenberg et al., JAMA. 323, 2020, pp. 2493-2502). Clearly, further studies are required, but these results are of great interest because azithromycin has been shown to enhance the phagocytosis of apoptotic bronchial epithelial cells by alveolar macrophages (Hodge et al., Eur Respir J. 28, 2006, pp. 486-495, Hodge et al., European Respiratory Journal, 2006).

Thus, in a preferred embodiment of the invention, the use of a compound that enhances the phagocytic activity of alveolar macrophages (AM), preferably azithromycin, in a combination therapy according to the above-mentioned first and/or second aspects of the present invention and/or an antibody or pharmaceutical composition intended for the above-mentioned use, represents an additional way to counteract the creation of oxidized phospholipids and/or oxidation-specific epitopes, thereby reducing the accumulation of oxPL and OSE and the proinflammatory or immunomodulatory functions of oxPL and OSE in a subject having a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD), preferably in a patient with COVID-19.

Preferably, said combination therapy has a synergistic effect with respect to the treatment proposed in the present invention, i.e. by affecting the formation of oxPL and OSE in inflamed tissues and the proinflammatory and immunomodulatory functions of oxPL and OSE in a subject having a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD), preferably in a patient with COVID-19.

A person skilled in the art can select a suitable compound that increases the phagocytic activity of alveolar macrophages (AM), which has the property required according to the present invention and as described above.

A third object of the present invention is a vaccine comprising a compound that induces the production of natural IgM and/or IgA antibodies, intended for use in a method for reducing or preventing clinical signs or diseases,

associated/linked/caused by deficiency of natural IgM/IgA antibodies (NAD) in a subject, wherein said vaccine contains a pharmaceutically acceptable carrier or excipient.

A vaccine typically refers to an "immunogenic composition" that contains at least one agent that resembles a pathogenic virus or microorganism. Thus, a vaccine is typically a biological preparation that typically provides active, acquired immunity to a specific infectious disease. A vaccine is often prepared from weakened or killed forms of the microbe, its toxins, or one of its surface proteins. The agent stimulates the body's immune system to recognize the agent as a threat, destroy it, and further recognize and destroy any microorganisms or viruses related to this agent that it may encounter in the future.

A vaccine or its immunogenic component typically elicits an immunological response (cellular or humoral immune response) in the host to the composition.

However, the term "vaccine" in the context of the specific aspects of the present invention refers to a pharmaceutical composition that (predominantly) does not induce an immunological response in an animal or subject, but contains a compound that induces the production of natural IgM and/or IgA antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes.

Epidemiological data suggest that, in some cases, a population vaccinated against a specific pathogen may acquire cross-immunity to other unrelated pathogens. A striking current example is COVID-19. Observational data suggest that in populations with previous vaccination, for example, against Pneumococcus pneumoniae or Bacillus tuberculosis, COVID-19 may be both less frequent and less severe. Although potential cross-immunity based on prior vaccination in these populations has been discussed, there is currently no convincing explanation for the actual cause of such protection. The authors hypothesized that the superior protection against COVID-19, for example, in individuals vaccinated with BCG or pneumococcus, is due to vaccine-induced OSE-specific antibodies that provide cross-immune protection against unrelated pathogens. Thus, vaccines that have the ability to induce not only specific immune responses against the pathogen used for vaccination, but also OSE-specific antibodies, may represent a new class of reagents for active immunotherapeutic interventions.

Without going into theory, vaccination strategies aimed at inducing endogenous production of OSE-specific IgM or IgA antibodies could be used to protect healthy individuals from chronic “sterile” inflammatory diseases or pathogen-induced severe forms of ALI and ARDS.

In mice, vaccination with Mycobacterium tuberculosis lipids or bacille Calmette-Guérin (BCG) vaccine stimulated B1 cells to produce natural IgM antibodies with specificities for the phosphocholine headgroup of phosphatidylcholine and cardiolipin (Russo and Mariano, Immunobiology, vol. 215 (12), 2010) (Ordonez, Savage et al., Immunology, 2018). Similarly, immunization of atherosclerosis-prone mice with inactivated pneumococcal extracts resulted in the formation of high levels of

Phosphocholine-specific IgM antibodies that had atheroprotective functions because they blocked the uptake of oxLDL by macrophages (Binder, Horkko et al., Nat Med, vol. 9 (6), 2003). These studies demonstrated molecular mimicry between OSE and epitopes within bacterial cell wall components such as M. tuberculosis and S. pneumoniae and showed that vaccination of humans with BCG or Pneumovax23 can induce immune responses that protect against chronic inflammatory diseases such as atherosclerosis, SLE, MS, and AD, but also against the severe course of pathogen-induced ALI and ARDS. It is noteworthy that pretreatment with the BCG vaccine may likely protect people from fatal COVID-19, since the mortality rate was reduced in newly vaccinated individuals compared with unvaccinated individuals.

The vaccine may additionally contain components typical of the pharmaceutical compositions mentioned above. However, the immunologically active component of the vaccine may comprise complete viral particles or complete bacteria, either in their original form or as attenuated particles or attenuated bacteria.

In another form, the immunologically active component of the vaccine may contain the corresponding elements of organisms (subunit vaccines), where said elements are obtained either by destruction of the entire particle or bacteria or cultures of growing cells containing such particles, and optionally subsequent purification steps to obtain the desired structure(s), or by synthetic processes involving appropriate manipulations using a suitable system based, for example, on bacteria, insects, mammals or other species, plus optionally subsequent isolation and purification procedures, or by inducing synthetic processes in an animal requiring vaccination by directly incorporating genetic material using acceptable pharmaceutical compositions (polynucleotide vaccination). The vaccine may contain one or more of the elements described above. The term "vaccine" in the context of specific aspects of the present invention refers to a modified live attenuated vaccine. In other specific aspects of the present invention, the vaccine may, inter alia, be a live vaccine, a live attenuated vaccine, an inactivated vaccine or a conjugate vaccine.

Various physical and chemical inactivation methods are known in the art. The term "inactivated" refers to previously virulent or non-virulent viruses or bacteria that have been irradiated (ultraviolet (UV) radiation, X-rays, electron beams, or gamma radiation), heated (e.g., for 30 minutes to several hours at 55°C to 65°C, e.g., 3 hours at 56°C), or chemically treated to inactivate or destroy the virus or bacteria while maintaining their immunogenicity.

More specifically, the term "inactivated" in relation to a virus means that the virus does not have the ability to replicate in vivo or in vitro.

For example, the term "inactivated" may refer to a virus that has been propagated in vitro and then inactivated using chemical or physical means, rendering it incapable of replication. In another example, the term "inactivated" may refer to viruses and/or bacteria that have been propagated and then inactivated using chemical or physical means, resulting in the formation of a viral suspension, fragments, or viral components that can be used as a vaccine component. As used herein, the terms "inactivated," "killed," or "KV" are used interchangeably.

The term "live vaccine" refers to a vaccine containing a live virus, specifically the active component of a live virus

Optionally, one or more pharmaceutically acceptable carriers or excipients mentioned herein include any and all solvents, dispersion media, coating agents, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. In some aspects of the invention, and particularly those relating to lyophilized immunogenic compositions, stabilizing agents include stabilizers for lyophilization or freeze-drying.

In the context of the present description, the terms "vaccine" and "vaccine composition" are used interchangeably and refer in particular to a composition that elicits a protective immune response in a subject treated with the composition.

Regarding the concept “for use in a method of reducing or preventing clinical signs or diseases,

associated/linked to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject”, the same as indicated above in the context of the first and/or second objects of the present invention applies to the vaccine, mutatis mutandis.

In a preferred embodiment of the invention, a vaccine for use in a method for reducing or preventing clinical signs or diseases associated with/related to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject comprises a compound that induces a human natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes.

With regard to the concept of “human or humanized natural IgM and/or IgA antibody that recognizes oxidized phospholipids and/or oxidation-specific epitopes”, the same applies to the vaccine, mutatis mutandis, as indicated above in the context of the first and/or second objects of the present invention.

In a preferred embodiment of the invention, a vaccine for use in a method for reducing or preventing clinical signs or diseases associated with/related to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject is a bacille Calmette-Guérin (BCG) vaccine.

In a preferred embodiment of the invention, said Bacille Calmette-Guérin vaccine comprises an attenuated Mycobacterium bovis bacterium.

In another preferred embodiment of the invention, a vaccine for use in a method for reducing or preventing clinical signs or diseases associated with/related to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject is a Pneumococcus tuberculosis vaccine.

In a preferred embodiment of the invention, the Pneumococcus tuberculosis vaccine is the Pneumovax®23 vaccine or the Prevenar®13 vaccine.

In a preferred embodiment of the invention, said pneumococcal vaccine comprises polysaccharide epitopes from several strains of pneumococci.

In a preferred embodiment of the invention, a vaccine for use in a method for reducing or preventing clinical signs or diseases associated with/related to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject is a vaccine having the ability to:

stimulate the production of natural IgM specific for oxidized phospholipids and/or oxidation-specific epitopes;

reduce the accumulation of free oxidized phospholipids, preferably in infected lungs;

reduce the accumulation of oxidized phospholipids and/or oxidation-specific epitopes on LDL, preferably in atherosclerotic lesions;

stimulate the secretion of IL-10 and/or TGFβ, preferably by alveolar macrophages; and/or

reduce the accumulation of misfolded proteins such as amyloid-β oligomers, preferably in brain tissue,

and/or neutralize proinflammatory cytokines.

With regard to the concepts of “stimulating the production of natural IgM, reducing the accumulation of free oxidized phospholipids, preferably in infected lungs, stimulating the secretion of IL-10 and/or TGFβ, preferably by alveolar macrophages; and/or neutralizing proinflammatory cytokines”, the same applies to the vaccine, mutatis mutandis, as indicated above in the context of the first and/or second objects of the present invention.

In a preferred embodiment of the vaccine described above, the clinical signs or disease associated with natural IgM/IgA antibody deficiency (NAD) is an infectious disease.

More preferably, the infectious disease in question is a viral infectious disease.

Even more preferably, said infectious disease is selected from the group consisting of infections caused by coronaviruses, preferably SARS-CoV, SARS-CoV-2, MERS; influenza viruses, parainfluenza viruses, respiratory syncytial viruses (RSV), rhinoviruses, adenoviruses, enteroviruses, human metapneumoviruses, herpesviruses, preferably HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7, HHV-8.

In a more preferred embodiment of the invention, a vaccine for use in a method for reducing or preventing clinical signs or diseases associated with/related to/caused by a deficiency of natural IgM/IgA antibodies (NAD) in a subject is a vaccine for a viral infectious disease that is COVID-19 caused by the SARS-CoV2 r-coronavirus.

With regard to the term “the viral infectious disease is COVID-19 caused by the β-coronavirus SARS-CoV2”, the same applies to the vaccine, mutatis mutandis, as indicated above in the context of the first and/or second objects of the present invention.

In a preferred embodiment of the invention, the agent or compound, preferably the antigen, contained in the vaccine of the present invention may also be a recombinant or synthetically created/synthesized agent.

Thus, in a preferred embodiment of the invention, the vaccine is a vaccine in which at least some of the antigens contained therein are obtained by recombinant methods.

Other aspects and advantages of the invention will be described in the following examples, which are provided for illustrative purposes and do not limit the invention in any way. Each publication, patent, patent application, or other document mentioned in this application is hereby incorporated by reference in its entirety.

The drawings show:

Fig. 1 - Change in clinical parameters in patients with COVID-19 treated with Pentaglobin®

Five patients with exacerbation of COVID-19 pneumonia were treated with Pentaglobin®. "P" denotes the days on which patients received Pentaglobin® (filled circles). Clinical parameters such as IL-6 (A), CRP (B), PCT (C), and mean 24-hour partial pressure of carbon dioxide ( _pCO2 ) in blood (standard deviations denoted by whiskers) (D) were quantified at different time points. The presence (+) or absence (-) of SARS-CoV-2 in bronchoalveolar lavage (BAL) fluids was monitored.

Fig. 2 Oxidative stress is responsible for excessive inflammation in patients with COVID-19

Illustration of the mechanisms leading to ROS formation and oxPL and OSE accumulation (Fig. 2A). Manipulation of these mechanisms allows counteracting OSE formation (Fig. 2B).

Fig. 3 - Detection of antinuclear autoimmune antibodies in the serum of patients with COVID-19

HD: Healthy donor serum; COV: COVID-19 patient serum; positive control (from the Kallestad HEp2 kit).

Fig. 4 - SARS-CoV-2-infected lung cells have elevated oxPL levels

A) Lung adenocarcinoma cells (Calu-3) were infected with SARS-CoV-2 for three days and then immunostained with mouse monoclonal natural IgM E06, which detects the phosphorylcholine headgroup of oxPL. Cells were counterstained with an antibody to the SARS-CoV-2 NC protein to demonstrate infection. DAPI staining allowed visualization of cell nuclei. Upon magnification, E06 counterstaining of rounded, apoptotic SARS-CoV-2 NC-positive cells was detected. B) Quantification of staining intensity in four independent images was performed using ImageJ software.

Fig. 5 - Increased oxidative stress in the serum of patients with COVID-19 compared to healthy donors

Malondialdehyde (MDA) is one of the most widely used biomarkers of lipid peroxidation. MDA concentrations were determined in serum from 8 hospitalized patients with severe COVID-19, 20 outpatients with moderate COVID-19, and 10 healthy donors using a commercially available kit (Lipid Peroxidation (MDA) Assay Kit, Abeam) and according to the manufacturer's instructions. Statistical significance was calculated using Student's t-test. **** P < 0.0001.

Fig. 6 - Significantly elevated serum oxLDL levels in patients with COVID-19

OxLDL levels were measured in serum obtained from COVID-19 patients (COV, n=28) and healthy donors (HD, n=10). OxLDL levels were measured using an oxidized LDL ELISA kit (catalog no. 10-1143-01, Mercodia) according to the manufacturer's instructions. Statistical significance was calculated using Student's t-test. **** P < 0.0001.

Fig. 7 - Elevated levels of oxLDL IgG and IgA autoantibodies in serum from patients with severe COVID-19 compared to serum from healthy donors or patients with moderate cases of COVID-19.

Plates were coated with oxLDL, and the coated plates were incubated with diluted serum (1:20). After washing, bound serum-derived antibodies to oxLDL were detected by adding horseradish peroxidase-conjugated anti-human IgG or anti-human IgA antibodies. Optical density (OD) was read at 450 nm after substrate addition. COV, serum from COVID-19 patients (severe disease, n=8; moderate disease, n=20); HD, serum from healthy donors (n=10). Statistical significance was calculated using Student's t-test. **** P<0.0001.

Fig. 8 - OxPL-specific monoclonal antibodies compete with IgG from the serum of patients with COVID-19 for binding to oxLDL

ELISA plates were coated with oxLDL and preincubated with either assay buffer, mouse monoclonal antibodies E06, 509, or a combination of both. Serum samples from hospitalized patients with severe COVID-19 (n=8) were then added to the coated and preincubated wells, and IgG antibody binding was determined using an HRP-conjugated anti-human IgG secondary antibody. IgG binding to oxLDL was determined by OD values at 450 nm, and data were expressed as the percentage inhibition of IgG binding to oxLDL in wells preincubated with the indicated monoclonal antibodies compared to wells preincubated with assay buffer.

Each serum sample was tested in triplicate and statistical significance was calculated using the one-tailed Mann-Whitney test. *** P < 0.001.

Fig. 9 Serum from patients with COVID-19 contains elevated levels of IgG and IgA antibodies to oxidation-specific epitopes

ELISA plates were coated with 10 μg/mL of the indicated antigens. Coated and blocked wells were incubated with human serum samples obtained from hospitalized COVID-19 patients (COV; n=8) or healthy donors (HD; n=8) diluted 1:20, and bound antibodies were identified using HRP-conjugated isotype-specific secondary antibodies. Each serum sample was tested in triplicate, and antibody binding was expressed as optical density (OD) values. Statistical significance of the difference in antibody binding to COV versus HD sera was calculated using Student's t-test. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Fig. 10 - IgM fraction from pentaglobin binds to apoptotic cells displaying oxPL

HEK293T cells were treated overnight with various concentrations _{of H2O2 and} the following day immunostained with either mouse monoclonal IgM E06, which binds to the phosphorylcholine headgroup of oxPL, or pentaglobin, and then stained with species-specific APC-labeled IgM antibodies. Control cells were incubated with secondary antibodies only. Apoptotic cells were identified as Sytox-positive.

Fig. 11 - The IgM fraction from Pentaglobin® binds to SARS-CoV-2 infected cells

Lung adenocarcinoma cells (Calu-3) were infected with SARS-CoV-2 for three days and then immunostained using pentaglobin and the SARS-CoV-2 NC protein. FITC-labeled anti-human IgM was used to detect IgM binding to pentaglobin-stained cells. DAPI staining allowed visualization of cell nuclei. Magnification revealed signals indicating staining of SARS-CoV-2 NC-positive cells with pentaglobin-derived IgM. Quantitative analysis of the mean staining intensities of three independent images, obtained using ImageJ software, is shown on the right, with standard deviations indicated by the whiskers. Student's t-test was used to assess statistical significance.

Fig. 12 - Pentaglobin® contains antibodies that bind to oxLDL

ELISA plates were coated with oxLDL and incubated with the indicated concentrations of pentaglobin. Antibody binding to oxLDL was detected using HRP-conjugated isotype-specific antibodies. (A) Antibody binding was expressed as OD450 values versus Pentaglobin® concentration. (B) Quantification of antibody isotypes in pentaglobin that bound oxLDL was determined based on the ratio of OD450 values of individual isotypes to all isotypes. (C) Binding of individual isotypes to oxLDL was normalized relative to the concentration of the corresponding isotype in pentaglobin.

Fig. 13 - Pentaglobin contains antibodies that bind to oxidation-specific epitopes

ELISA plates were coated with phosphorylcholine-BSA (PC-BSA), malondialdehyde-BSA (MDA-BSA), 4-hydroxynonenal-BSA (HNE-BSA), or unconjugated bovine serum albumin (BSA) and incubated with the indicated concentrations of Pentaglobin®. Antibody binding to the antigen used for sensitization was detected using HRP-conjugated isotype-specific antibodies recognizing IgG, IgM, and IgA isotypes (anti-IgG/M/A) or specific for IgM (anti-IgM). Antibody binding was expressed as OD 450 nm values depending on the Pentaglobin® concentration.

Examples

Example 1: Demonstration of binding of engineered nlgM to oxidized lipids

Commercially available ELISA kits (e.g., MDA-BSA-coated ELISA plate (DEIACP15) or anti-ox-LDL ELISA (DEIA081J) from Creative Diagnostics) were used to assess the binding of the generated OSE-specific IgM and/or IgA antibodies to oxidized phospholipids. Avanti Lipid Snoopers® ELISA strips coated with oxidized phosphatidylcholine were used to analyze the binding of IgM and/or IgA antibodies. IgM and/or IgA antibodies against oxidized cardiolipin or oxidized phosphatidylserine were quantified using a well-established ELISA method (Frostegård Su et al., PLoS One, Vol. 9 (12), 2014).

Binding to oxidized lipids was further studied by inducing cell apoptosis in cell culture and staining with generated OSE-specific IgM and/or IgA antibodies, Pentaglobin®, or serum from BCG/Pneumovax-vaccinated individuals. Flow cytometry was used to specifically detect apoptotic cells (e.g., using the annexation marker V), and nlgM binding was monitored using commercially available secondary anti-IgM and anti-IgA antibodies.

Example 2: Demonstration of binding of OSE-specific IgM and/or IgA antibodies to virus-infected cells

Viral infection of cells triggers a multitude of events that facilitate viral replication, including changes in the lipid composition of cell membranes. For example, various viruses induce the translocation of phosphatidylserines to the outer surface of the cell membrane, where they are prone to oxidation. This process normally occurs during cellular apoptosis and reflects the "eat me" signal to phagocytes. This induced change in the lipid composition of cell membranes is incorporated into viral membranes during viral budding. Thus, some viruses use this as a "mask" to improve uptake by phagocytic cells, which recognize the virus as an apoptotic body, potentially facilitating infection and viral spread. Changes in the lipid composition of viral membranes, as well as viral surface glycoproteins, can directly affect nIgM binding.

The resulting monoclonal OSE-specific IgM and/or IgA antibodies were studied for binding to cells infected with herpes simplex virus type 1 or 2 or to cells expressing viral surface glycoproteins (e.g., glycoprotein S of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Env of human immunodeficiency virus (HIV-1), glycoprotein G of vesicular stomatitis virus (VSV), glycoproteins gB, gD, gH, gL, gl, or gE of HSV-1/2). In these assays, the binding affinity (Kd) was determined to cells expressing the corresponding viral glycoproteins and to cells that did not express the protein, which served as a negative control.

Example 3: Demonstration of direct antiviral activity of Pentaglobin®, OSE-specific IgM and/or IgA antibodies and serum from BCG/Pneumovax-vaccinated individuals in vitro and in vivo

Various in vitro and in vivo models were used to study the direct antiviral effects of Pentaglobin®, OSE-specific IgM and/or IgA monoclonal antibodies, and sera from BCG/Pneumovax-vaccinated individuals on virus infection and dissemination.

Prototype viruses from various viral families were studied. These included Herpesviridae (HSV-1/2), Rabdoviridae (recombinant VSV with a GFP reporter, ryVSVdeltaOEP), as well as murine leukemia virus (MLV), and HIV-1-based vectors pseudotyped with various glycoproteins, including VSV-G, HIV-1 Env, or SARS-CoV-2 glycoprotein S.

It is likely that enveloped viruses contain oxidized phospholipids and OSEs in their membranes, which can be bound by OSE-specific IgM and/or IgA antibodies. Direct effects on cell-free viral infection were studied by incubating serial dilutions of pre- and post-BCG/Pneumovax sera with monoclonal OSE-specific IgM and/or IgA antibodies or Pentaglobin® with viruses or viral vectors and then infecting appropriate target cell lines, such as the African green monkey Vero cell line or the human embryonic kidney HEK293T cell line. Depending on the virus or vector used, infection was determined either by plaque formation (HSV-1 or HSV-2) or by the delivery of reporter genes upon virus or viral vector infection (e.g., GFP or luciferase).

For HSV-1 and HSV-2, the effects of pre- and post-vaccination BCG/Pneumovax serum, Pentaglobin®, and monoclonal OSE-specific IgM and/or IgA antibodies on cell-to-cell virus spread were measured by infecting target cells with virus and then administering serial dilutions of the above-mentioned products containing OSE-specific IgM and/or IgA antibodies.

Immunodeficient and immunocompetent mice were infected intravaginally with HSV-1 or HSV-2 and injected either before infection (prophylactic approach) or after infection (therapeutic approach) with various doses of OSE-specific monoclonal IgM and/or IgA antibodies, Pentaglobin®, or serum from BCG/Pneumovax-vaccinated individuals.

Survival and damage progression were monitored at various time points. Viral replication was measured using quantitative PCR. In these experiments, Pentaglobin® served as a positive control, as the high HSV seroprevalence likely implies the presence of virus-neutralizing IgG.

Natural IgM is also known to protect against primary cutaneous HSV-1 infections (Deshpande, Kumaraguru et al., Cell Immunol 202(2), 2000). We investigated whether intravenous prophylactic or therapeutic injection of OSE-specific IgM and/or IgA antibodies or sera from BCG/Pneumovax-vaccinated individuals could protect against skin lesions induced by HSV-1 infection.

We also studied whether topical treatment with compounds containing the aforementioned OSE-specific IgM and/or IgA antibodies could prevent the development of lesions following primary cutaneous infection with HSV-1. Because the HSV-1 seroprevalence in the human population is approximately 80%, only serum from HSV-1/2 seronegative (naive) individuals was used in the development of the invention.

Example 4: Analysis of nlgM binding to specific leukocyte surface proteins

IgM-ALA (nlgM leukocyte-binding antigen) has been shown to have beneficial effects in heart and kidney transplant recipients (Lobo, Schlegel et al., J Immunol 180(3), 2008) (Lobo, Front Immunol 7, 2016). Low or absent IgM-ALA levels are associated with increased inflammation, graft-versus-host disease, and graft rejection. IgM-ALA has been shown to bind to (i) certain costimulatory receptors such as CD4, CD86, CD40, and PD1, and (ii) chemokine receptors. However, these polyreactive IgM-ALA autoantibodies exhibit some specificity, as they do not randomly bind to glycoproteins on other cellular receptors, such as CD8, CD80, CD40L, PDL1, CD28, CD1d, and HLA. IgM-ALA has also been shown to bind to TcR, CD3, and CD45.

Using ELISA and flow cytometry, we examined whether the identified and selected monoclonal nIgMs bind to the aforementioned leukocyte costimulatory receptors. Prevention of proinflammatory responses by these nIgMs can be assessed by incubating primary blood monocytes or purified leukocyte populations with nIgMs under stimulating conditions, followed by multiplex analysis of secreted cytokines using developed flow cytometry-based methods (Biolegend).

Example 5: Anti-inflammatory effects of Pentaglobin® or OSE-specific monoclonal IgM and/or IgA antibodies or serum from BCG/Pneumovax-vaccinated individuals against acute respiratory distress syndrome (ARDS) in vivo

Infection with SARS-CoV-2 results in the development of coronavirus disease (COVID-19), which can manifest as subclinical or moderate symptoms, as well as severe symptoms characteristic of acute respiratory syndrome (ARDS). Two preclinical mouse models of ARDS resulting from direct lung injury have been described (D'Alessio, Methods Mol Biol, vol. 1809, 2018). They used intratracheal instillation of LPS ("sterile" inflammation) or Streptococcus pneumoniae to mimic human pneumonia. These models were chosen because they are highly reproducible, induce persistent neutrophilic alveolitis and alveolar-capillary membrane destruction, are easily titrated (degrees of pulmonary inflammation), and allow assessment of both the early and resolving phases of acute lung injury (ALI).

Mice with ARDS were treated with Pentaglobin®, monoclonal nlgM, or serum from BCG/Pneumovax-vaccinated individuals or control serum from unvaccinated individuals. Treatment progression, i.e., improvement of ARDS, was monitored.

Example b: Generation of OSE-specific IgM and/or IgA antibody libraries from BCG/Pneumovax-vaccinated SARS-CoV-2-seropositive individuals with moderate/asymptomatic SARS-CoV-2 infection

BCG/Pneumovax-vaccinated individuals have been shown to have an advantage in fighting severe infections caused by immunologically invisible pathogens due to increased levels of natural antibodies.

The spectrum of natural IgM has been shown to change with age in terms of both expression level and diversity (Rodriguez-Zhurbenko, Quach et al., Front Immunol, vol. 10, 2019). This observation suggests that the spectrum of nlgM in young individuals may have enhanced antipathogenic properties.

To evaluate this, phage display libraries of natural IgM and/or IgA antibodies were generated from young BCG/Pneumovax-vaccinated SARS-CoV-2-seropositive individuals with a moderate/asymptomatic phase of SARS-CoV-2 infection. Several IgM and/or IgA monoclonal antibodies were cloned from these natural antibody libraries. These monoclonal antibodies exhibit low-affinity polyreactivity with natural antibody-specific antigens and viral glycoprotein binding, and possess virus-neutralizing activity. The IgM and/or IgA monoclonal antibodies were tested individually and in combination (pools) to evaluate enhanced virus-neutralizing activity when administered as a pool.

Example 7. Large-scale population analysis of serum concentrations of OSE-specific IgM and/or IgA antibodies and correlation with BCG/Pneumovax vaccination status

To demonstrate that the BCG/Pneumovax vaccine induces higher levels of nIgM antibody protection, nIgM concentrations were analyzed and compared between the sera of individuals not vaccinated with BCG and/or Pneumovax and those of individuals vaccinated with BCG and/or Pneumovax. Serum levels of OSE-specific IgM and/or IgA were determined using the ELISA methods described above. Serum pools from vaccinated and unvaccinated individuals were compared for virus-neutralizing activity using the virus assays described above.

Example 8. Determination of the in vivo effects of Pentaglobin®, monoclonal OSE-specific IgM and/or IgA, and serum from BCG-Pneumovax-vaccinated individuals on atherosclerosis

The two most commonly used mouse models of atherosclerosis are the apoE-/- model and the ldlr-/- model. These two models were used to study whether injection of Pentaglobin®, monoclonal nlgM, or serum from BCG/Pneumovax-vaccinated individuals could reduce atherosclerosis.

Example 9. SARS-CoV-2-infected lung cells have elevated oxPL levels

As described above, pathogens such as SARS-CoV-2, SARS-CoV, or the H5N1 influenza virus trigger elevated levels of lipid peroxidation in cell membranes, leading to the production of oxidized phospholipids (oxPL), which arise from oxidative damage. It is likely that oxPL is produced by SARS-CoV-2-infected cells as a result of cellular stress responses.

Indeed, oxPL accumulation was detected on the surface of SARS-CoV-2-infected lung adenocarcinoma (Calu-3) cells, as determined by the increased staining intensity of infected cells with mouse natural IgM E06, which detects the phosphorylcholine headgroup of oxPL but not native PL (Fig. 4). These data suggest that SARS-CoV-2-infected cells represent an important source of oxPL, which can accumulate to proinflammatory concentrations in the absence of effective clearance, for example, by natural IgM antibodies.

Example 10. Increased oxidative stress in the serum of patients with COVID-19

Lipids containing polyunsaturated fatty acids are particularly susceptible to oxidative attack, typically by reactive oxygen species (ROS), which leads to a chain reaction with the production of oxPL and end products such as malondialdehyde (MDA), which are further implicated in the pathology of pathogen-induced inflammation in infected tissue. OxPL and aldehydes such as MDA are not localized exclusively to the tissue in which they were formed under the influence of ROS and cellular stress responses, but also enter the bloodstream and are subsequently released by cells during oxidative stress.

Since increased oxPL formation by SARS-CoV-2-infected cells has been demonstrated (see above), it is suggested that the level of degradation products such as MDA may be elevated in the serum of COVID-19 patients.

In fact, significantly elevated MDA concentrations were detected in the serum of hospitalized COVID-19 patients, and high serum MDA concentrations differentiated patients with severe disease from patients with moderate disease or healthy donors (Fig. 5). High MDA levels in the serum of patients with severe COVID-19 likely result from impaired clearance mechanisms involved in the neutralization and degradation of oxPL and OSE, leading to the accumulation of oxPL and OSE to concentrations high enough to induce proinflammatory immune responses that contribute to the hyperinflammation observed in patients with severe COVID-19.

Example 11. Significantly elevated oxLDL levels in the serum of patients with COVID-19

Human low-density lipoprotein (LDL) is a key lipid-protein complex in the blood and is a crucial component of the metabolism responsible for lipid transport in the body. OxPL and aldehydes such as MDA in the bloodstream can induce oxidative modifications of circulating LDL and other lipoproteins (Parthasarathy et al., Methods Mol Biol, vol. 610, 2010, p. 403).

LDL oxidation is a complex process in which both lipids and proteins undergo oxidative changes and form complex products. For example, peroxidized lipids are broken down to form aldehydes such as MDA, which covalently modify the amino groups of lysine residues in LDL apolipoprotein B100 (apoB100). This not only leads to the formation of Schiff bases, which modify the charges on amino acids, but also leads to proteolysis of the apoB100 protein and the formation of both intra- and intermolecular cross-links between proteolyzed apoB100, resulting in extensive alterations in the protein's composition and structure. Thus, unlike native LDL, oxidized LDL (oxLDL) particles carry numerous new antigenic determinants that are recognized by receptors of the innate and adaptive immune system and vascular wall cells, thereby playing a key pathogenic role in cardiovascular disease (CVD). In CVD, the proinflammatory functions of oxLDL are mediated by dendritic cells and macrophages, which bind oxLDL with high affinity via phagocytic receptors. This leads to uncontrolled oxLDL uptake and macrophage conversion into foam cells, a defining characteristic of fatty streaks and atherosclerotic lesions. Furthermore, T cell activation has been linked to modified LDL, as oxLDL-derived peptides have been shown to be recognized by T cells (Stemme et al., Proc Natl Acad Sci, vol. 92, 1995, p. 3893).

In the present invention, it was unexpectedly found that patients with severe COVID-19 had high serum MDA concentrations (Fig. 5), indicating that circulating lipoproteins such as LDL may be oxidatively modified by aldehydes released from SARS-CoV-2-infected cells.

Consistent with this, significantly elevated oxLDL levels were detected in the serum of COVID-19 patients compared to serum from healthy donors (Fig. 6). Notably, the murine monoclonal antibody 4E6, which was used to identify oxLDL in this experiment, binds to a conformational epitope in the apoB-100 fragment of LDL that results from the substitution of at least 60 lysine residues of apoB-100 with aldehydes. This number of substituted lysines corresponds to the minimum amount required for phagocytic receptor-mediated uptake of oxLDL by macrophages.

Thus, the obtained results demonstrated three new and unexpected discoveries:

1) large numbers of oxPL and OSE-bearing structures, such as SARS-CoV-2-infected cells and cellular debris, are formed in the lungs and possibly other infected tissues of COVID-19 patients;

2) aldehydes such as MDA are released into the serum of COVID-19 patients, for example, by SARS-CoV-2-infected lung cells, where they induce oxidative modifications of circulating lipoproteins such as LDL; and

3) Circulating oxidized LDL particles in COVID-19 patients require high binding affinity for phagocytic receptors expressed by innate immune cells and therefore have the potential to induce systemic proinflammatory responses similar to those described for CVD when efficient clearance does not occur.

Example 12. Elevated levels of anti-oxLDL IgG and IgA autoantibodies in the serum of patients with severe COVID-19

For diseases associated with elevated oxLDL levels, such as atherosclerosis and other CVDs, diabetes mellitus, systemic lupus erythematosus, or rheumatoid arthritis, it is known that up to 90% of oxLDL is captured by autoantibodies, forming oxLDL immune complexes, the levels of which often correlate with disease severity.

Autoantibodies that form complexes with oxLDL in disease states are primarily of the IgG1 and IgG3 isotypes, both of which are pro-arterogenic and can induce pro-inflammatory responses through their interaction with Fc-gamma receptors expressed by innate immune cells such as macrophages (Mironova et al.; Arterioscler Thromb Vase Biol. 16(2), Feb 1996, pp. 222–229) (Virella et al., Clin Diagn Lab Immunol, v. 10, 2003, p. 499). oxLDL-IgG immune complexes thus induce more potent pro-inflammatory responses than free oxLDL because the immune complexes recruit Fc-gamma receptors in addition to phagocytic receptors. Fc-gamma receptor-mediated activation of the NLRP3 inflammasome is involved in the secretion of proinflammatory cytokines, such as IL-1b and IL-6, from innate immune cells in response to oxLDL-IgG immune complexes (Rhoads et al., J Immunol; 198(5), 1 Mar 2017 pp. 2105-2114).

Because severe COVID-19 patients were unexpectedly found to have significantly elevated serum oxLDL levels compared to moderate patients or healthy donors, we investigated whether these patients had elevated titers of anti-oxLDL IgG autoantibodies that could form proinflammatory immune complexes.

Significantly elevated levels of anti-oxLDL IgG autoantibodies in the sera of COVID-19 patients compared to the sera of healthy donors fully supported the concept presented herein and correlated with disease severity (Fig. 7).

Since the serum IgA antibody fraction, primarily the IgA2 subtype, can induce proinflammatory responses through the binding of IgA2-containing immune complexes to macrophages and neutrophils expressing the Fc-alpha receptor, anti-oxLDL IgA autoantibody levels were also compared between patients with severe COVID-19, patients with moderate COVID-19, and healthy donors. It is noteworthy that anti-oxLDL IgA autoantibody levels were found to be specifically and significantly increased in patients with severe COVID-19 compared with those with moderate COVID-19 or healthy donors (Fig. 7).

These unexpected findings support the role of pathogenic oxLDL-IgG and oxLDL-IgA immune complexes in patients with severe COVID-19, in whom they initiate systemic hyperinflammatory responses.

Example 13. OxPL-specific monoclonal antibodies compete with IgG from COVID-19 patient serum for binding to oxLDL

oxLDL particles are characterized by a wide variety of immunogenic determinants that have not yet been studied in detail and that can be bound by antibodies.

To demonstrate that anti-oxLDL antibodies in the sera of COVID-19 patients indeed bind to oxPL, an oxPL masking assay was performed using two mouse monoclonal antibodies that bind to different classes of oxPL:

The IgM E06 antibody binds to the phosphorylcholine head group exposed to oxidized phosphatidylcholine (oxPC), while the IgM 509 antibody binds to oxidized phosphatidylethanolamine (oxPE) but does not bind to its native non-oxidized analog (Bochkov et al., Biomark Med., vol. 10 (8), 2016, pp. 797-810).

ELISA plates were coated with oxLDL, and the coated wells were pre-incubated with the indicated monoclonal antibodies, individually or in combination, to block oxPC and/or oxPE exposed to oxLDL. Serum samples from COVID-19 patients were then added, and IgG binding to oxLDL was determined using an HRP-conjugated anti-human IgG secondary antibody.

The data demonstrated that blockade of either oxPC or oxPE alone on oxLDL by pre-incubation with one of these two monoclonal antibodies did not result in detectable inhibition of IgG binding to oxLDL.

However, when both classes of oxPL were blocked simultaneously, significant inhibition of IgG antibody binding to oxLDL particles in the sera of COVID-19 patients, averaging -20% (Fig. 8), was detected. These data suggest that anti-oxLDL IgG autoantibodies in COVID-19 patients do exhibit specificity for oxPL and likely contain additional clones that recognize OSEs other than oxPC and oxPE.

Example 14. Serum from patients with COVID-19 contains elevated levels of IgG and IgA antibodies to oxidative-specific epitopes

OxPLs such as oxPC and oxPE may be mainly located on oxLDL particles in the early oxidation phase, while lipid peroxidation end products such as malondialdehyde or 4-hydroxynonenal dominate on LDL particles with an increased degree of oxidation.

To demonstrate that anti-oxLDL antibodies in the sera of COVID-19 patients bind to different types of OSEs exposed on oxLDL particles, ELISA plates were coated with phosphorylcholine (PC), malondialdehyde (MDA), and 4-hydroxynonenal (HNE), mimicking early- and late-stage oxidative stress-specific epitopes. The coated wells were incubated with serum from

hospitalized patients with severe COVID-19 or healthy donor serum were tested, and antibody binding was identified using isotype-specific HRP-conjugated secondary antibodies.

It was found that the serum of healthy donors contained IgG and IgA antibodies that bound to PC, while the levels of anti-MDA and anti-HNE IgG and IgA antibodies were low or undetectable, respectively (Fig. 9).

Since PC is an immunodominant determinant of pneumococcal cell wall polysaccharides, the presence of anti-PC IgG and IgA antibodies in healthy donors may be associated with previous pneumococcal infection or vaccination. However, and this is a significant difference, significantly higher levels of IgG and IgA antibodies binding to MDA and HNE were detected in the sera of patients with severe COVID-19 compared to the sera of healthy donors (Fig. 9), indicating that the major fraction of anti-oxLDL IgG and IgA autoantibodies in the sera of COVID-19 patients indeed bound to oxidative-specific epitopes exposed by LDL.

These new data support the existence of the following mechanism that is involved in the pathogenesis of severe COVID-19:

1) oxPL and OSE are formed in patients with COVID-19;

2) efficient clearance of oxPL and OSE cannot occur in the COVID-19 patient population due to the deficiency of natural antibodies, leading to the accumulation of oxPL and OSE in SARS-CoV-2-infected cells, apoptotic cells and lipoproteins, which initiates autoimmune responses and the formation of IgG and IgA autoantibodies;

3) Anti-oxPL and anti-OSE IgG and IgA autoantibodies bind to oxPL and OSE-bearing structures, such as on LDL particles or infected cells, and mediate pro-inflammatory immune responses as they simultaneously engage phagocytic receptors and Fc-gamma receptors expressed by innate immune cells, thereby leading to the hyperinflammatory state found in patients with severe COVID-19.

Example 15. The IgM fraction from pentaglobin binds to apoptotic cells displaying oxPL

As described above, COVID-19 patients develop oxidative stress, and increased oxPL expression has been detected in the membranes of SARS-CoV-2-infected cells and circulating lipoprotein particles. These structures have the ability to induce IgG and IgA autoantibody responses and are likely involved in the pathogenesis of severe COVID-19 if they are not effectively cleared.

In mice, natural IgM antibodies and other soluble pattern recognition receptors (PRRs) of the innate immune system bind to oxPL- and OSE-bearing structures and thereby promote their safe and anti-inflammatory clearance.

Thus, the novel concept outlined herein is that treatment of patients with severe COVID-19 using oxPL-specific natural IgM antibodies neutralizes the pro-inflammatory actions of oxPL, facilitates their safe clearance, and thereby prevents the induction of destructive autoimmune responses.

Pentaglobin® is an infusion-based human immunoglobulin preparation enriched with IgM and IgA antibodies and approved for the treatment of patients with severe bacterial infections and sepsis, as well as immunocompromised patients lacking endogenous immunoglobulins. Because the immunoglobulins in Pentaglobin® are composed of pooled serum antibodies obtained from thousands of healthy donors, the present invention demonstrated that the IgM pool, and to a lesser extent the IgA and IgG fractions, primarily contain natural antibodies that recognize various types of oxPL and OSE.

To assess the presence of oxPL-specific IgM antibodies, Pentaglobin® was used to stain human cells under oxidative stress that display different types of oxPL and OSE in their plasma membrane, and IgM binding was identified using fluorescently labeled secondary antibodies.

Oxidative stress was induced by incubating cells with varying concentrations _{of H2O2 ,} an agent that efficiently initiates lipid peroxidation, and oxPL formation was monitored by staining treated cells with mouse natural IgM E06, which recognizes oxPL-exposed phosphorylcholine headgroups. In fact, the majority of _{H2O2 -} treated cells stained positively with E06, and this was dependent on the induction of apoptosis, indicating that treated cells exposed a significant amount of oxPL on their surface (Fig. 10). When pentaglobin was used for staining, it was found that a significant proportion of cells treated _with 100 μM _H2O2 were bound by the IgM fraction in the preparation, and IgM binding was positively correlated with an increased rate of apoptosis and, consequently, oxPL presentation. Thus, these results provided new evidence that Pentaglobin® does contain a fraction of natural IgM antibodies that bind to oxPL exposed by apoptotic cells.

Example 16. The IgM fraction of Pentaglobin® binds to SARS-CoV-2-infected cells

As described above, it was found that lung adenocarcinoma cells (Calu-3) infected with SARS-CoV-2 express large amounts of oxPL in their membranes, which are bound by the mouse natural IgM antibody E06 (Fig. 4). Furthermore, as also demonstrated above, antibodies within Pentaglobin® were detected that bind to oxPL exposed by cells under conditions of oxidative stress (Fig. 10).

To determine whether Pentaglobin contains antibodies that bind to SARS-CoV-2-infected lung cells, such as oxPL displayed on the plasma membrane of infected cells, Calu-3-infected cells were stained with Pentaglobin®, and IgM binding was detected by incubation with a fluorescently labeled secondary antibody to human IgM. These data suggest that Pentaglobin® contains an IgM fraction that binds to infected cells, and that the majority of these antibodies likely bind to oxPL displayed on the plasma membrane (Fig. 11).

Example 17. Pentaglobin® contains antibodies that bind to oxLDL

OxPLs are not only exposed on apoptotic cell membranes, but can also be present on circulating lipoproteins such as LDL, where they constitute the major pathogenic component of oxLDL.

In fact, large amounts of oxPL have been detected in the plasma membrane of SARS-CoV-2-infected cells and in circulating oxLDL in the serum of COVID-19 patients. Since Pentaglobin® was unexpectedly found to contain natural IgM antibodies that bind to apoptotic cells expressing large amounts of oxPL, we investigated whether Pentaglobin® also contains antibodies that bind to oxLDL. For this purpose, ELISA plates were coated with oxLDL, the coated wells were incubated with varying concentrations of Pentaglobin®, and antibody binding was detected using isotype-specific secondary antibodies.

In fact, Pentaglobin® was found to contain antibodies that bind to oxLDL in a concentration-dependent manner (Fig. 12A). When isotype-specific secondary antibodies were used for identification, it was found that the majority of oxLDL-binding antibodies in Pentaglobin were IgG (-45%), followed in descending order by IgM (-35%) and IgA (-19%) (Fig. 12B). However, given that the immunoglobulins in Pentaglobin® consisted of 78% IgG, 6% IgM, and 6% IgA isotypes, the ratio of oxLDL isotype binding was standardized relative to their compositional ratio, and it was found that the majority of oxLDL-binding antibodies were actually of the IgM isotype (-61%), followed in descending order by IgA (-33%) and IgG (-6%) (Fig. 12B).

For uninfected cells, it was found that -80% of the IgM pool and -50% of serum IgA originate from B1 cells, indicating that IgM and IgA represent the most common isotypes of natural antibodies (Meyer-Bahlburg, Ann N Y Acad Sci, Vol. 1362, 20-15, pp. 122-131). These data thus confirm that the majority of oxLDL-binding IgM and IgA antibodies in Pentaglobin® are human natural antibodies.

Example 18. Pentaglobin® contains antibodies that bind to oxidation-specific epitopes

To demonstrate that anti-oxLDL antibodies in Pentaglobin indeed bind to OSEs exposed by oxidized LDL particles, ELISA plates were coated with different classes of OSEs, including phosphorylcholine (PC), malondialdehyde (MDA), and 4-hydroxynonenal (HNE), which are well-known targets of natural antibodies.

Sensitized wells were incubated with four serial dilutions of Pentaglobin®, and antibody binding was detected using HRP-conjugated secondary antibodies. The results demonstrated that Pentaglobin® does contain antibodies that bind to all classes of OSE tested, and that anti-RS antibodies represent the most prominent OSE-binding fraction, followed in descending order by anti-HNE antibodies and a lower level of anti-MDA antibodies.

It is noteworthy that when an IgM-specific secondary antibody was used to specifically identify IgM binding, a similar binding pattern was found for all classes of OSE tested, indicating that the OSE-binding antibodies in Pentaglobin are primarily IgM (Fig. 13). High titers of anti-RS IgM are typically present in the sera of healthy donors and are most likely the result of previous bacterial infections or immunization with pneumococcal extracts (e.g., Pneumovaxx23) (Nishinarita, Med. Microbiol. Immunol., Vol. 179, 1990, pp. 205-214).

In support of the above, it was found that immunization of mice with pneumococcus induced high levels of PC-specific natural IgM antibodies, which provided protection against atherosclerosis due to molecular mimicry between S. pneumoniae and oxLDL (Binder, Nat. Med., vol. 9, 2003, pp. 736-743).

Taken together, these results demonstrate that Pentaglobin contains naturally occurring antibodies of predominantly IgM isotype that bind to different classes of OSE, and the invention suggests that these antibodies may provide protection against oxPL-induced proinflammatory responses in patients with severe COVID-19.

Example 19. Pentaglobin® contains antibodies that block the binding of IgG and IgA to oxLDL from the serum of patients with COVID-19

The above data suggest that severe COVID-19 is accompanied by the emergence of oxPL- and OSE-specific IgG and IgA autoantibodies that form immune complexes with oxidation-modified particles such as circulating lipoproteins, which are effective “driving forces” of the hyperinflammatory state found in severely ill COVID-19 patients.

The above data support the novel concept that neutralization of oxPL and OSE by natural IgM antibodies neutralizes their proinflammatory potential in COVID-19 patients through several mechanisms:

1) natural IgM blocks the binding of oxidatively modified particles, such as oxLDL, to phagocytic receptors on cells of the innate immune system;

2) natural IgM blocks the binding of IgG and IgA autoantibodies to oxidation-modified particles such as oxLDL, thereby preventing the formation of proinflammatory IgG and IgA-containing immune complexes;

3) natural IgM facilitates safe and anti-inflammatory clearance of oxPL-exposing structures such as cellular debris, thereby preventing the accumulation of oxPL and OSE and the production of IgG and IgA autoantibodies.

As described above, it was unexpectedly found in the creation of the invention that neutralization of two classes of oxPL on oxLDL with monoclonal mouse IgM antibodies significantly inhibited the binding of IgG autoantibodies from the serum of COVID-19 patients to oxLDL, and that Pentaglobin® contains a significant fraction of oxPL-specific human natural antibodies, primarily in its IgM pool, which suggests that Pentaglobin® can inhibit the binding of IgG and IgA autoantibodies present in the serum of COVID-19 patients to oxLDL, similar to what occurs in the case of oxPL-specific mouse monoclonal IgM antibodies.

In fact, it was found that pre-incubation of oxLDL-sensitized wells with Pentaglobin® significantly inhibited the binding of autoantibodies from COVID-19 patient sera to oxLDL by >20%, and this effect was observed for both IgG and IgA autoantibodies (Fig. 14).

These new findings support the potential of Pentaglobin® for the treatment of critically ill COVID-19 patients, as the invention unexpectedly demonstrated its ability to neutralize various classes of oxPL and OSE and prevent the formation of immune complexes containing IgG and IgA autoantibodies, thereby demonstrating its potential to attenuate hyperinflammatory immune responses and promote rapid clinical improvement.

Example 20. Clinical intervention strategy for NAD modulation in COVID-19

COVID-19 is a disease that requires extremely high medical attention for both treatment and prevention. NAD modulation is expected to be a valuable intervention strategy for both treatment and prevention of COVID-19.

Clinical trials of therapeutic effect

a. In a small study of up to 10 patients with severe COVID-19 infection who required mechanical ventilation or were medically judged to require mechanical ventilation within 1-4 hours due to rapid deterioration of lung function, the natural antibody-containing Pentaglobin® was administered at a dose of 5 ml/kg at a rate of 28 ml/h for 3 consecutive days.

b. A controlled phase II clinical trial will then begin in a group of up to 50 patients at two centers. This study will systematically evaluate the potential NAD-modulating activity of Pentaglobin® and correlate it with critical clinical outcome data.

c. A large, multinational, multicenter Phase III trial is then conducted. In addition, new plasma products enriched with natural antibodies (i.e., from female donors aged <25 years) are developed for subsequent approved trials, preferably by the time the first available interim data from the Phase III trial is available.

Then, recombinant monoclonal natural antibodies are simultaneously developed to replace the NAD-modulating Pentaglobin® for the treatment of COVID-19 and other NAD-associated diseases.

Clinical trials of prophylactic action

Preclinical data suggest potential NAD-modulating actions of currently available drugs such as BCG or Pneumovax.

Following successful preclinical confirmation in vitro and in appropriate animal models, trials are underway to induce natural antibodies through vaccination with the appropriate compounds. For some of these compounds (e.g., BCG), protection against acquired severe COVID-19 has been postulated based on epidemiological data. However, the induction of natural antibodies as a potentially important protective tool has not yet been demonstrated. Therefore, NAD modulation is being systematically analyzed in the exploratory portions of these trials, and correlations with clinical outcome data are being sought.

Example 21. Clinical treatment of COVID-19 using Pentaglobin®

Five patients with very severe COVID-19, four of whom required invasive mechanical ventilation, were given pentaglobin.

Patient 1:

A man aged 59 years (No. 1).

Concomitant diseases: Bechterew's disease (patient with morphine-dependent chronic pain), osteoporosis, hypertension.

History of illness: On March 28, 2020, presentation to a local hospital with abdominal pain and rapid deterioration of general condition, positive swab for SARS-CoV-2 on the same day. Rapid development of respiratory failure, worsening of the disease course and the need for intubation and invasive mechanical ventilation on April 4. Transfer to the UKHD ICU on April 14, requiring catecholamines (norepinephrine) at the time of admission. Chest CT on April 14 showed features of typical COVID-19-associated pneumonia, suspected pre-existing pulmonary fibrosis. On April 16, administration of 10 g pentaglobin, initiation of acyclovir therapy after a positive BAL test for HSV-1. Cardiopulmonary stabilization, then transition to dual-chamber ventilation (BIPAP), tracheostomy. On April 22, an attempt at spontaneous breathing was made, but was still poorly tolerated with tachypnea and abnormal breathing mechanics.

Patient 2:

A man aged 80 years (No. 2).

Associated diseases: Bechterew's disease, stage 3 coronary artery disease, acute myocardial infarction due to RCA (right coronary artery) occlusion, acute fracture of the 7th cervical vertebra after collapse.

History of illness: Respiratory infection for 1 week, resistant to antibiotics. On April 2, the patient was found unconscious at home with a laceration of the head. An ambulance was called. Upon arrival, the patient's consciousness was significantly reduced, unresponsive, _SO2 87% at 12 L _O2 /min, and he was transported to the hospital emergency department. Diagnosis: a swab for SARS-2 was positive. CT scan showed central pulmonary artery embolism, typical signs of lung disease in COVID-19. Fever was 38.5°C. ECG showed signs of myocardial infarction (RCA), dilation and stenting of the RCA and successful recanalization, intubation was required for intervention, and he was dependent on catecholamines. During treatment, the lung situation significantly worsened. According to CT scan data from April 12, COVID-19 pulmonary infiltrates significantly worsened with the onset of consolidation. Between April 13 and April 15, 2020, 22.5 g of pentaglobin was administered daily. On April 16, there was a temporary deterioration in continence parameters, but an improvement in diuresis (hemodialysis was initiated), followed by slow clinical stabilization of the kidneys and lungs. On April 20, intermittent CPAP (continuous positive airway pressure) was initiated with a slow decrease in PEEP (positive end-expiratory pressure) over the following days. Attempts to periodically suspend artificial ventilation were successful, but oxygenation was still unsatisfactory. On April 22, three consecutive negative tests for SARS-2 resulted in transfer to the cardiac intensive care unit (ICU) for further cardiopulmonary stabilization and planning of subsequent surgery for a vertebral fracture.

Patient 3:

Woman aged 62 years (No. 3).

Associated diseases: Obesity, Klippel-Trenaunay syndrome.

History of the disease: On March 29, 2020, admission to a local hospital with fever and shortness of breath for 5 days and deterioration of the general condition, a positive swab for SARS-CoV-2 on the same day. On April 8, rapid development of respiratory failure, worsening and the need for intubation and invasive BIPAP ventilation. Transfer to the intensive care unit of another local hospital (Schwäbisch Hall) and subsequent transfer to the intensive care unit of UKHD on April 13, temporarily requiring low-dose catecholamine. A chest CT scan on April 13 showed features of typical COVID-19-associated pneumonia. A free-floating thrombus was detected in a venous vessel, full-dose heparinization. On April 14 and 15, 10 g of pentaglobin was administered daily. Rapid improvement in cardiac function thereafter, weaning from the apparatus and extubation without any complications. Initiation of acyclovir therapy after a positive HSV-1 test in tracheal fluid on April 19. Negative SARS-CoV-2 swab on April 20. Readmission to the local hospital, 1 L/min _O2 via the nose.

Patient 4:

A 76 year old man (No. 4).

Comorbidities: type II diabetes mellitus, insulin-dependent, hypertension, dyslipidemia, severe stage 3 coronary heart disease with bradycardiac atrial fibrillation, NSTEMI with high-grade LCX stenosis and RCA occlusion.

History of the disease: Onset of fever, shortness of breath, and rapid deterioration of general condition on April 3. The patient was admitted to the local hospital on April 13. A positive SARS-CoV2 test was acquired from a family member the previous day. Rapid deterioration of cardiopulmonary status and the need for intubation and invasive intubation began on April 14. Subsequent development of elevated troponin levels and acute renal failure. Transfer from the UKHD ICU on April 15. Immediate coronary angiography revealed an occlusion of the RCA with sufficient collaterals and a proximal stenosis of the LCX. Stenting of the RCA and LCX was successfully performed in a single session, requiring catecholamines and hemodialysis. A CT scan on April 19 revealed typical features of COVID-19-related lung disease, with a suspected aortic ulcer of unknown origin. A significant deterioration in the pulmonary condition was observed. A CT scan on April 12 revealed significant worsening of COVID-19 pulmonary infiltrates with the onset of consolidation. On April 16, the patient received 10 g of pentaglobin. Episodes of bradycardic atrial fibrillation developed, improving after clonidine was discontinued. Cardiopulmonary stabilization was achieved. A second 10 g pentaglobin injection was administered on April 19. The last episode of fever occurred on April 20, with a temperature of 38.8°C and a decrease in sedative effects. On April 21, an attempt was made to wean the patient off the ventilator; cardiopulmonary stabilization was achieved on April 22.

Patient 5:

A 62 year old man (No. 5).

Concomitant diseases: none.

Medical history: March 29, 2020. History of contact with a person infected with COVID-19. On April 1, dry cough, fever, chills, and deterioration of general condition, admission to a local hospital, a positive SARS-CoV-2 swab test on April 13. Subsequently, a persistent increase in _O2 requirement and the development of respiratory failure. On April 15, transfer to the IMC department.

Patient was admitted to the UKHD due to deteriorating respiratory function. A throat swab was negative for SARS-CoV-2 at the UKHD. A chest CT scan from the same day showed signs of typical COVID-19-associated pneumonia. High-flow oxygen therapy (HFOT, Optiflow®) was administered from April 15 to April 21. On April 16, steroids and 10 g of pentaglobin were administered. Subsequently, pulmonary stabilization allowed for a gradual reduction in mechanical oxygenation and discontinuation of HFOT on April 21. Sputum testing for SARS-CoV-2 was negative on April 17. Importantly, TNT remained consistently high without clinical symptoms or ECG changes. Patient was re-transported to the local hospital in good condition, with 1 L/min of nasal _O2 .

The following tables show the clinical data determined for Patient 1, Patient 2, Patient 3, Patient 4, and Patient 5, respectively, over time.

Figure 1 summarizes the above data for five patients with worsening COVID-19-associated pneumonia treated with Pentaglobin® (P). Figure 1 shows the specific concentrations of clinical parameters such as IL-6 (see Figure 1A), CRP (see Figure 1B), PCT (see Figure 1C), and mean 24-hour partial pressure of carbon dioxide in the blood _pCO2 (see Figure 1D), respectively. CRP, PCT, and IL-6 are the main markers of inflammation. “P”: Pentaglobin® administration. In addition, Figure 1 indicates the controlled presence (+) or absence (-) of SARS-CoV in bronchoalveolar lavages (BAL).

Overall, a study of 5 patients with very severe COVID-19, four of whom required invasive mechanical ventilation, found that pentaglobin administration improved clinical outcomes in almost all cases.

It is important to note that since pentaglobin was not administered in the context of a controlled clinical trial, the doses administered ranged from 10 g on one day to 22 g daily for three consecutive days. Overall, only one patient received the approved dose for the treatment of bacterial sepsis, 5 ml/kg for three consecutive days. However, in most patients, interleukin-6 (IL-6) levels and other inflammatory markers decreased significantly, along with significant improvements in clinical status and, most importantly, ventilation parameters.

It is important to note that all patients received the CCR 5 inhibitor maraviroc as an additional experimental therapy. Although the improvement in inflammatory parameters may be at least partially due to maraviroc, there is clear evidence of a marviroc-independent temporal relationship between pentaglobin administration, reduction in inflammation (I1-6), and clinical improvement. Since proinflammatory, macrophage-mediated effects of maraviroc have also been previously identified, it is also possible that maraviroc may even counteract the anti-inflammatory effects of pentaglobin in at least some cases.

Taken together, the data from this preliminary study demonstrate significant improvement in severe COVID-19 disease, thereby supporting the concept of successful treatment of NAD-related disease. A controlled clinical trial confirming the pentaglobin-mediated therapeutic principle is required.

Example 22. Detection of antinuclear autoimmune antibodies in the serum of patients with COVID-19

HEp2 slides (Kallestad), commonly used to detect antinuclear autoimmune antibodies (ANA) in serum, were incubated with serum from three patients with severe COVID-19 (COV #6, COV #7, COV #8) treated in the intensive care unit or with serum from four healthy donors (HD #1, HD #2, HD #3, HD #4). The results are shown in Fig. 3.

In Fig. 3, the top row shows the data for serum diluted in a ratio of 1:5, the bottom row shows the data for serum diluted in a ratio of 1:10.

The ANA-containing solution included in the test kit was used as a positive control. The total IgG concentration in each undiluted serum is shown below.

The data demonstrate a significantly stronger staining signal in the sera of COVID-19 patients, indicating the presence of ANA in these sera. In contrast, control sera from healthy donors showed either no staining at all or very weak staining, indicating the absence of AHA.

These data support the idea that the absence of natural antibodies (NAs) may be a result of autoimmune antibody production in severe COVID-19. The presence of these autoimmune antibodies is evidence of recurrent or prolonged COVID-19 symptoms, suggesting that sufficient levels of natural antibodies, such as monoclonal natural IgM or IgA, or from natural antibody-enriched preparations (e.g., Pentaglobin®) as described herein, can prevent the formation of autoimmune antibodies or reduce their levels.

Summary of examples 1-22

The data presented above in this description further support the model underlying the present invention and can be explained by the unusual property of pulmonary pathogens such as SARS-CoV-2, SARS, or the H5N1 influenza virus to induce excessive formation of oxPL and OSE in accordance with the mechanisms indicated in this description.

Viral replication and the resulting accumulation of oxPL and OSE in the lungs of infected patients initiate immune responses involving the recruitment and local activation of monocytes, T cells, and B cells, including cells that produce protective virus-specific IgG and oxPL-specific IgM antibodies. Some B cell clones that produce IgM antibodies against oxPL and OSE can induce aberrant class switching from IgM to IgA or IgG, for example, by presenting antigenic oxidative-specific peptides or viral peptides to T cells. These class-switched B cells then produce IgA or IgG autoantibodies that bind to oxPL and OSE exposed to many different oxidatively modified structures, including apoptotic cells and oxLDL. Thus, significantly elevated levels of IgG and IgA autoantibodies, which effectively bind to oxLDL, were detected in the serum of patients with severe COVID-19. These autoantibodies likely form oxLDL-IgG and oxLDL-IgA immune complexes. OxPL-specific natural IgM antibodies protect against proinflammatory IgG and IgA autoantibodies in various ways, such as by blocking oxPL binding to phagocytic receptors, preventing the formation of pathogenic IgG and IgA immune complexes, and by facilitating the safe clearance of oxPL-exposing structures. However, under conditions where the balance between oxLDL formation and neutralization is disrupted, such as when individuals with low levels of endogenous natural IgM and possibly IgAl antibodies are infected with SARS-CoV-2, the production of proinflammatory autoantibody isotypes such as IgG and IgA2 goes out of control, and newly formed IgG- and IgA2-containing oxLDL immune complexes are eventually deposited in various locations throughout the body, such as in the walls of blood vessels, joints, or glomerular capillaries, where they induce potent inflammatory responses through phagocytic receptor-, Fc-gamma-, and Fc-alpha-mediated activation of dendritic cells, macrophages, and neutrophils.

These autoimmune responses become the primary driving force behind the systemic hyperinflammatory state observed in the late phase of severe COVID-19, when the virus is no longer detectable. Over the long term, this ultimately culminates in lupus-like autoimmune manifestations such as arthritis, vascular injury, acute kidney injury, induction of a procoagulant state, and multiorgan damage, possibly contributing to the phenomenon known as long-COVID. Therefore, individuals with reduced levels of oxPL- and OSE-specific natural IgM antibodies and possibly IgA1, which could otherwise neutralize the proinflammatory functions of oxPL-exposing structures, are particularly prone to developing multiorgan hyperinflammatory phenomena driven by oxPL-IgG or oxPL-IgA2 immune complexes.

This concept further confirms that treatment of patients with severe COVID-19 or long-COVID patients with persistent proinflammatory autoimmune states with IgM antibodies, IgG2 or IgG4 antibodies, IgG antibodies modified to reduce Fc effector functions, or their antigen-binding fragments recognizing oxPL and OSE results in a significant reduction in the hyperinflammatory state by neutralizing oxPL- and OSE-exposing structures, thereby preventing the formation of pathogenic oxPL-IgG and oxPL-IgA-containing immune complexes and facilitating their safe clearance.

Example 23. Two monoclonal antibodies that bind to different danger-associated molecular patterns (DAMPs), including OSE and DNA

Herein, two monoclonal antibodies are characterized that bind to different danger-associated molecular patterns (DAMPs), including OSE and DNA.

The structures of these antibodies are described above with reference to SEQ ID NOs: 1-6 (corresponding to "Clone 1") and SEQ ID NOs: 9-14 (corresponding to "Clone 2"), respectively (and also with reference to SEQ ID NOs: 7-8 (corresponding to "Clone 1") and SEQ ID NOs: 15-16 (corresponding to "Clone 2"), respectively).

The antibodies belong to the IgM isotype and were isolated by sorting single human B cells with the phenotype CD5 ^pos CD20 ^pos CD27 ^pos CD43 ^pos CD70 ^neg .

Human B cells with this phenotype have been shown to be the human counterpart of murine B1 cells (Griffin, Holodick et al., J Exp Med, vol. 208, 2011, (1)). Thus, the two monoclonal antibodies described herein possess the characteristics of natural antibodies.

To evaluate the specificity of the monoclonal antibodies isolated by the method described herein, these antibodies were expressed as fully human IgM molecules and their antigen-binding properties were assessed. Two clones were identified that bind to at least two antigens, which are well-known targets for murine and human natural antibodies. The table below summarizes the binding analysis results for the two clones presented herein.

Each clone of mouse or human monoclonal antibodies with the characteristics of natural antibodies known in the art had a pronounced specificity for a clearly defined epitope.

For example, some of the OSE-specific IgM monoclonal antibodies isolated from apoE-deficient mice (including clone E06, which was used in some of the experiments presented herein) had unique binding specificities for the phosphorylcholine (PC) headgroup exposed by oxLDL, oxPL such as 2-(5-oxovaleryl)phosphatidylcholine (POVPC), PC-protein adducts, or PC-containing polysaccharides, but not for MDA, while other clones specifically bound MDA-modified LDL and MDA-protein adducts but did not bind to PC epitopes (Shaw et al., J Clin Invest, vol. 105(12), 2000).

The murine IgM isotype 509 monoclonal antibody used in some of the experiments presented herein was specific for oxidized phosphatidylethanolamine (PE), but not for oxidized phosphatidylcholine, oxidized phosphatidylserine, oxidized phosphatidic acid, or other native non-oxidized analogs (Bochkov et al., Biomark Med., Vol. 10 (8), 2016).

The monoclonal antibody LA25 had exceptional specificity for the OSE malondialdehyde-acetaldehyde (MAA), but not the structurally related OSE MDA (WO/2018/049083, PCT/US2017/050566).

Thus, in creating the invention, it was unexpectedly found that the monoclonal antibodies "Clone 1" and "Clone 2", characterized above, bound to at least two DAMP epitopes, including oxidized LDL, MDA-protein adducts, PC-protein adducts and DNA.

Each of these epitopes has been shown to be involved in chronic and acute proinflammatory diseases and may be a target for natural antibodies.

Because of this unique feature, the monoclonal antibodies described herein are particularly suitable for use in the treatment of patients suffering from inflammatory conditions associated with natural antibody deficiency, such as, for example, acute pathogen-induced inflammation, acute lung injury, atherosclerosis, and many

Other conditions. In such patients, oxidative stress and innate immune responses generate multiple forms of DAMPs, including oxPL, cleavage products such as MDA, and DNA derived from apoptotic cells or neutrophil extracellular traps (NETs), all of which have potent pro-inflammatory effects in the absence of effective clearance from the bloodstream, for example, by natural antibodies. It is also desirable for monoclonal antibodies used to treat these patients to recognize and neutralize the maximum possible amounts of DAMPs and OSEs to achieve anti-inflammatory and beneficial effects, and it was demonstrated in the invention that only a combination of both monoclonal antibodies E06 and 509 had the ability to significantly inhibit the binding of autoreactive IgG antibodies to oxLDL in the serum of patients with COVID-19. Thus, the unique characteristics of the monoclonal antibodies described herein provide important advantages over monospecific natural antibodies known in the art.

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SEQUENCE LIST

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Gly Tyr Thr Phe Thr Ser Tyr Asp

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<223> 2cVH CDR3 of human monoclonal OSE-specific lgM clone#1

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<223> VH of the human monoclonal OSE-specific lgM clone#1

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

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<223> VL of the human monoclonal OSE-specific lgM clone#1

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Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

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Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

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<223> 2cVL CDR3 of human monoclonal OSE-specific lgM clone#2

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Gln Gln Tyr Gly Ser Ser Arg Gly Thr

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<210> 15

<211> 126

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<223> VH of the human monoclonal OSE-specific lgM clone#2

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

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Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Lys Ala Leu Ala Lys Gln Trp Leu Val Gln Gly Val Arg Gly Ala

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Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

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<210> 16

<211> 108

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<223> VL of the human monoclonal OSE-specific lgM clone#2

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Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

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Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

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Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Arg

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Gly Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

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isolate Wuhan-Hu-1, complete genome

<400> 17

attaaaggtt tataccttcc caggtaacaa accaaccaac tttcgatctc ttgtagatct 60

gttctctaaa cgaactttaa aatctgtgtg gctgtcactc ggctgcatgc ttagtgcact 120

cacgcagtat aattaataac taattactgt cgttgacagg acacgagtaa ctcgtctatc 180

ttctgcaggc tgcttacggt ttcgtccgtg ttgcagccga tcatcagcac atctaggttt 240

cgtccgggtg tgaccgaaag gtaagatgga gagccttgtc cctggtttca acgagaaaac 300

acacgtccaa ctcagtttgc ctgttttaca ggttcgcgac gtgctcgtac gtggctttgg 360

agactccgtg gaggaggtct tatcagaggc acgtcaacat cttaaagatg gcacttgtgg 420

cttagtagaa gttgaaaaag gcgttttgcc tcaacttgaa cagccctatg tgttcatcaa 480

acgttcggat gctcgaactg cacctcatgg tcatgttatg gttgagctgg tagcagaact 540

cgaaggcatt cagtacggtc gtagtggtga gacacttggt gtccttgtcc ctcatgtggg 600

cgaaatacca gtggcttacc gcaaggttct tcttcgtaag aacggtaata aaggagctgg 660

tggccatagt tacggcgccg atctaaagtc atttgactta ggcgacgagc ttggcactga 720

tccttatgaa gattttcaag aaaactggaa cactaaacat agcagtggtg ttacccgtga 780

actcatgcgt gagcttaacg gaggggcata cactcgctat gtcgataaca acttctgtgg 840

ccctgatggc taccctcttg agtgcattaa agaccttcta gcacgtgctg gtaaagcttc 900

atgcactttg tccgaacaac tggactttat tgacactaag aggggtgtat actgctgccg 960

tgaacatgag catgaaattg cttggtacac ggaacgttct gaaaagagct atgaattgca 1020

gacacctttt gaaattaaat tggcaaagaa atttgacacc ttcaatgggg aatgtccaaa 1080

ttttgtattt cccttaaatt ccataatcaa gactattcaa ccaagggttg aaaagaaaaa 1140

gcttgatggc tttatgggta gaattcgatc tgtctatcca gttgcgtcac caaatgaatg 1200

caaccaaatg tgcctttcaa ctctcatgaa gtgtgatcat tgtggtgaaa cttcatggca 1260

gacgggcgat tttgttaaag ccacttgcga attttgtggc actgagaatt tgactaaaga 1320

aggtgccact acttgtggtt acttacccca aaatgctgtt gttaaaattt attgtccagc 1380

atgtcacaat tcagaagtag gacctgagca tagtcttgcc gaataccata atgaatctgg 1440

cttgaaaacc attcttcgta agggtggtcg cactattgcc tttggaggct gtgtgttctc 1500

ttatgttggt tgccataaca agtgtgccta ttgggttcca cgtgctagcg ctaacatagg 1560

ttgtaaccat acaggtgttg ttggagaagg ttccgaaggt cttaatgaca accttcttga 1620

aatactccaa aaagagaaag tcaacatcaa tattgttggt gactttaaac ttaatgaaga 1680

gatcgccatt attttggcat ctttttctgc ttccacaagt gcttttgtgg aaactgtgaa 1740

aggtttggat tataaagcat tcaaacaaat tgttgaatcc tgtggtaatt ttaaagttac 1800

aaaaggaaaa gctaaaaaag gtgcctggaa tattggtgaa cagaaatcaa tactgagtcc 1860

tctttatgca tttgcatcag aggctgctcg tgttgtacga tcaattttct cccgcactct 1920

tgaaactgct caaaattctg tgcgtgtttt acagaaggcc gctataacaa tactagatgg 1980

aatttcacag tattcactga gactcattga tgctatgatg ttcacatctg atttggctac 2040

taacaatcta gttgtaatgg cctacattac aggtggtgtt gttcagttga cttcgcagtg 2100

gctaactaac atctttggca ctgtttatga aaaactcaaa cccgtccttg attggcttga 2160

agagaagttt aaggaaggtg tagagtttct tagagacggt tgggaaattg ttaaatttat 2220

ctcaacctgt gcttgtgaaa ttgtcggtgg acaaattgtc acctgtgcaa aggaaattaa 2280

ggagagtgtt cagacattct ttaagcttgt aaataaattt ttggctttgt gtgctgactc 2340

tatcattatt ggtggagcta aacttaaagc cttgaattta ggtgaaacat ttgtcacgca 2400

ctcaaaggga ttgtacagaa agtgtgttaa atccagagaa gaaactggcc tactcatgcc 2460

tctaaaagcc ccaaaagaaa ttatcttctt agagggagaa acacttccca cagaagtgtt 2520

aacagaggaa gttgtcttga aaactggtga tttacaacca ttagaacaac ctactagtga 2580

agctgttgaa gctccattgg ttggtacacc agtttgtatt aacgggctta tgttgctcga 2640

aatcaaagac acagaaaagt actngtgccct tgcacctaat atgatggtaa caaacaatac 2700

cttcacactc aaaggcggtg caccaacaaa ggttactttt ggtgatgaca ctgtgataga 2760

agtgcaaggt tacaagagtg tgaatatcac ttttgaactt gatgaaagga ttgataaagt 2820

acttaatgag aagtgctctg cctatacagt tgaactcggt acagaagtaa atgagttcgc 2880

ctgtgttgtg gcagatgctg tcataaaaac tttgcaacca gtatctgaat tacttacacc 2940

actgggcatt gatttagatg agtggagtat ggctacatac tacttatttg atgagtctgg 3000

tgagtttaaa ttggcttcac atatgtattg ttctttctac cctccagatg aggatgaaga 3060

agaaggtgat tgtgaagaag aagagtttga gccatcaact caatatgagt atggtactga 3120

agatgattac caaggtaaac ctttggaatt tggtgccact tctgctgctc ttcaacctga 3180

agaagagcaa gaagaagatt ggttagatga tgatagtcaa caaactgttg gtcaacaaga 3240

cggcagtgag gacaatcaga caactactat tcaaacaatt gttgaggttc aacctcaatt 3300

agagatggaa cttacaccag ttgttcagac tattgaagtg aatagtttta gtggttattt 3360

aaaacttact gacaatgtat acattaaaaa tgcagacatt gtggaagaag ctaaaaaggt 3420

aaaaccaaca gtggttgtta atgcagccaa tgtttacctt aaacatggag gaggtgttgc 3480

aggagcctta aataaggcta ctaacaatgc catgcaagtt gaatctgatg attacatagc 3540

tactaatgga ccacttaaag tgggtggtag ttgtgtttta agcggacaca atcttgctaa 3600

acactgtctt catgttgtcg gcccaaatgt taacaaaggt gaagacattc aacttcttaa 3660

gagtgcttat gaaaatttta atcagcacga agttctactt gcaccattat tatcagctgg 3720

tatttttggt gctgacccta tacattcttt aagagtttgt gtagatactg ttcgcacaaa 3780

tgtctactta gctgtctttg ataaaaatct ctatgacaaa cttgtttcaa gctttttgga 3840

aatgaagagt gaaaagcaag ttgaacaaaa gatcgctgag attcctaaag aggaagttaa 3900

gccatttata actgaaagta aaccttcagt tgaacagaga aaacaagatg ataagaaaat 3960

caaagcttgt gttgaagaag ttacaacaac tctggaagaa actaagttcc tcacagaaaa 4020

cttgttactt tatattgaca ttaatggcaa tcttcatcca gattctgcca ctcttgttag 4080

tgacattgac atcactttct taaagaaaga tgctccatat atagtgggtg atgttgttca 4140

agaggtgtt ttaactgctg tggttatacc tactaaaaag gctggtggca ctactgaaat 4200

gctagcgaaa gctttgagaa aagtgccaac agacaattat ataaccactt acccgggtca 4260

gggtttaaat ggttacactg tagaggaggc aaagacagtg cttaaaaagt gtaaaagtgc 4320

cttttacatt ctaccatcta ttatctctaa tgagaagcaa gaaattcttg gaactgtttc 4380

ttggaatttg cgagaaatgc ttgcacatgc agaagaaaca cgcaaattaa tgcctgtctg 4440

tgtggaaact aaagccatag tttcaactat acagcgtaaa tataagggta ttaaaataca 4500

agaggtgtg gttgattatg gtgctagatt ttacttttac accagtaaaa caactgtagc 4560

gtcacttatc aacacactta acgatctaaa tgaaactctt gttacaatgc cacttggcta 4620

tgtaacacat ggcttaaatt tggaagaagc tgctcggtat atgagatctc tcaaagtgcc 4680

agctacagtt tctgtttctt cacctgatgc tgttacagcg tataatggtt atcttacttc 4740

ttcttctaaa acacctgaag aacattttat tgaaaccatc tcacttgctg gttcctataa 4800

agattggtcc tattctggac aatctacaca actaggtata gaatttctta agagaggtga 4860

taaaagtgta tattacacta gtaatcctac cacattccac ctagatggtg aagttatcac 4920

ctttgacaat cttaagacac ttctttcttt gagagaagtg aggactatta aggtgtttac 4980

aacagtagac aacattaacc tccacacgca agttgtggac atgtcaatga catatggaca 5040

acagtttggt ccaacttatt tggatggagc tgatgttact aaaataaaac ctcataattc 5100

acatgaaggt aaaacatttt atgttttacc taatgatgac actctacgtg ttgaggcttt 5160

tgagtactac cacacaactg atcctagttt tctgggtagg tacatgtcag cattaaatca 5220

cactaaaaag tggaaatacc cacaagttaa tggtttaact tctattaaat gggcagataa 5280

caactgttat cttgccactg cattgttaac actccaacaa atagagttga agtttaatcc 5340

acctgctcta caagatgctt attacagagc aagggctggt gaagctgcta acttttgtgc 5400

acttatctta gcctactgta ataagacagt aggtgagtta ggtgatgtta gagaaacaat 5460

gagttacttg tttcaacatg ccaatttaga ttcttgcaaa agagtcttga acgtggtgtg 5520

taaaacttgt ggacaacagc agacaaccct taagggtgta gaagctgtta tgtacatggg 5580

cacactttct tatgaacaat ttaagaaagg tgttcagata ccttgtacgt gtggtaaaca 5640

agctacaaaa tatctagtac aacaggagtc accttttgtt atgatgtcag caccacctgc 5700

tcagtatgaa cttaagcatg gtacatttac ttgtgctagt gagtacactg gtaattacca 5760

gtgtggtcac tataaacata taacttctaa agaaactttg tattgcatag acggtgcttt 5820

acttacaaag tcctcagaat acaaaggtcc tattacggat gttttctaca aagaaaacag 5880

ttacacaaca accataaaac cagttactta taaattggat ggtgttgttt gtacagaaat 5940

tgaccctaag ttggacaatt attataagaa agacaattct tatttcacag agcaaccaat 6000

tgatcttgta ccaaaccaac catatccaaa cgcaagcttc gataatttta agtttgtatg 6060

tgataatatc aaatttgctg atgatttaaa ccagttaact ggttataaga aacctgcttc 6120

aagagagctt aaagttacat ttttccctga cttaaatggt gatgtggtgg ctattgatta 6180

taaacactac acaccctctt ttaagaaagg agctaaattg ttacataaac ctattgtttg 6240

gcatgttaac aatgcaacta ataaagccac gtataaacca aatacctggt gtatacgttg 6300

tctttggagc acaaaaccag ttgaaacatc aaattcgttt gatgtactga agtcagagga 6360

cgcgcaggga atggataatc ttgcctgcga agatctaaaa ccagtctctg aagaagtagt 6420

ggaaaatcct accatacaga aagacgttct tgagtgtaat gtgaaaacta ccgaagttgt 6480

aggagacatt atacttaaac cagcaaataa tagtttaaaa attacagaag aggttggcca 6540

cacagatcta atggctgctt atgtagacaa ttctagtctt actattaaga aacctaatga 6600

attatctaga gtattaggtt tgaaaaccct tgctactcat ggtttagctg ctgttaatag 6660

tgtcccttgg gatactatag ctaattatgc taagcctttt cttaacaaag ttgttagtac 6720

aactactaac atagttacac ggtgtttaaa ccgtgtttgt actaattata tgccttattt 6780

ctttacttta ttgctacaat tgtgtacttt tactagaagt acaaattcta gaattaaagc 6840

atctatgccg actactatag caaagaatac tgttaagagt gtcggtaaat tttgtctaga 6900

ggcttcattt aattatttga agtcacctaa tttttctaaa ctgataaata ttataatttg 6960

gtttttacta ttaagtgttt gcctaggttc tttaatctac tcaaccgctg ctttaggtgt 7020

tttaatgtct aatttaggca tgccttctta ctgtactggt tacagagaag gctatttgaa 7080

ctctactaat gtcactattg caacctactg tactggttct ataccttgta gtgtttgtct 7140

tagtggttta gattctttag acacctatcc ttctttagaa actatacaaa ttaccatttc 7200

atcttttaaa tgggatttaa ctgcttttgg cttagttgca gagtggtttt tggcatatat 7260

tcttttcact aggtttttct atgtacttgg attggctgca atcatgcaat tgtttttcag 7320

ctattttgca gtacatttta ttagtaattc ttggcttatg tggttaataa ttaatcttgt 7380

acaaatggcc ccgatttcag ctatggttag aatgtacatc ttctttgcat cattttatta 7440

tgtatggaaa agttatgtgc atgttgtaga cggttgtaat tcatcaactt gtatgatgtg 7500

ttacaaacgt aatagagcaa caagagtcga atgtacaact attgttaatg gtgttagaag 7560

gtccttttat gtctatgcta atggaggtaa aggcttttgc aaactacaca attggaattg 7620

tgttaattgt gatacattct gtgctggtag tacatttatt agtgatgaag ttgcgagaga 7680

cttgtcacta cagtttaaaa gaccaataaa tcctactgac cagtcttctt acatcgttga 7740

tagtgttaca gtgaagaatg gttccatcca tctttacttt gataaagctg gtcaaaagac 7800

ttatgaaaga cattctctct ctcattttgt taacttagac aacctgagag ctaataacac 7860

taaaggttca ttgcctatta atgttatagt ttttgatggt aaatcaaaat gtgaagaatc 7920

atctgcaaaa tcagcgtctg tttactacag tcagcttatg tgtcaaccta tactgttact 7980

agatcaggca ttagtgtctg atgttggtga tagtgcggaa gttgcagtta aaatgtttga 8040

tgcttacgtt aatacgtttt catcaacttt taacgtacca atggaaaaac tcaaaacact 8100

agttgcaact gcagaagctg aacttgcaaa gaatgtgtcc ttagacaatg tcttatctac 8160

ttttatttca gcagctcggc aagggtttgt tgattcagat gtagaaacta aagatgttgt 8220

tgaatgtctt aaattgtcac atcaatctga catagaagtt actggcgata gttgtaataa 8280

ctatatgctc acctataaca aagttgaaaa catgacaccc cgtgaccttg gtgcttgtat 8340

tgactgtagt gcgcgtcata ttaatgcgca ggtagcaaaa agtcacaaca ttgctttgat 8400

atggaacgtt aaagatttca tgtcattgtc tgaacaacta cgaaaacaaa tacgtagtgc 8460

tgctaaaaag aataacttac cttttaagtt gacatgtgca actactagac aagttgttaa 8520

tgttgtaaca acaaagatag cacttaaggg tggtaaaatt gttaataatt ggttgaagca 8580

gttaattaaa gttacacttg tgttcctttt tgttgctgct attttctatt taataacacc 8640

tgttcatgtc atgtctaaac atactgactt ttcaagtgaa atcataggat acaaggctat 8700

tgatggtggt gtcactcgtg acatagcatc tacagatact tgttttgcta acaaacatgc 8760

tgattttgac acatggttta gccagcgtgg tggtagttat actaatgaca aagcttgccc 8820

attgattgct gcagtcataa caagagaagt gggttttgtc gtgcctggtt tgcctggcac 8880

gatattacgc acaactaatg gtgacttttt gcatttctta cctagagttt ttagtgcagt 8940

tggtaacatc tgttacacac catcaaaact tatagagtac actgactttg caacatcagc 9000

ttgtgttttg gctgctgaat gtacaatttt taaagatgct tctggtaagc cagtaccata 9060

ttgttatgat accaatgtac tagaaggttc tgttgcttat gaaagtttac gccctgacac 9120

acgttatgtg ctcatggatg gctctattat tcaatttcct aacacctacc ttgaaggttc 9180

tgttagagtg gtaacaactt ttgattctga gtactgtagg cacggcactt gtgaaagatc 9240

agaagctggt gtttgtgtat ctactagtgg tagatgggta cttaacaatg attattacag 9300

atctttacca ggagttttct gtggtgtaga tgctgtaaat ttacttacta atatgtttac 9360

accactaatt caacctattg gtgctttgga catatcagca tctatagtag ctggtggtat 9420

tgtagctatc gtagtaacat gccttgccta ctattttatg aggtttagaa gagcttttgg 9480

tgaatacagt catgtagttg cctttaatac tttactattc cttatgtcat tcactgtact 9540

ctgtttaaca ccagtttact cattcttacc tggtgtttat tctgttattt acttgtactt 9600

gacattttat cttactaatg atgtttcttt tttagcacat attcagtgga tggttatgtt 9660

cacaccttta gtacctttct ggataacaat tgcttatatc atttgtattt ccacaaagca 9720

tttctattgg ttctttagta attacctaaa gagacgtgta gtctttaatg gtgtttcctt 9780

tagtactttt gaagaagctg cgctgtgcac ctttttgtta aataaagaaa tgtatctaaa 9840

gttgcgtagt gatgtgctat tacctcttac gcaatataat agatacttag ctctttataa 9900

taagtacaag tattttagtg gagcaatgga tacaactagc tacagagaag ctgcttgttg 9960

tcatctcgca aaggctctca atgacttcag taactcaggt tctgatgttc tttaccaacc 10020

accacaaacc tctatcacct cagctgtttt gcagagtggt tttagaaaaa tggcattccc 10080

atctggtaaa gttgagggtt gtatggtaca agtaacttgt ggtacaacta cacttaacgg 10140

tctttggctt gatgacgtag tttactgtcc aagacatgtg atctgcacct ctgaagacat 10200

gcttaaccct aattatgaag atttactcat tcgtaagtct aatcataatt tcttggtaca 10260

ggctggtaat gttcaactca gggttattgg acattctatg caaaattgtg tacttaagct 10320

taaggttgat acagccaatc ctaagacacc taagtataag tttgttcgca ttcaaccagg 10380

acagactttt tcagtgttag cttgttacaa tggttcacca tctggtgttt accaatgtgc 10440

tatgaggccc aatttcacta ttaagggttc attccttaat ggttcatgtg gtagtgttgg 10500

ttttaacata gattatgact gtgtctcttt ttgttacatg caccatatgg aattaccaac 10560

tggagttcat gctggcacag acttagaagg taacttttat ggaccttttg ttgacaggca 10620

aacagcacaa gcagctggta cggacacaac tattacagtt aatgttttag cttggttgta 10680

cgctgctgtt ataaatggag acaggtggtt tctcaatcga tttaccacaa ctcttaatga 10740

ctttaacctt gtggctatga agtacaatta tgaacctcta acacaagacc atgttgacat 10800

actaggacct ctttctgctc aaactggaat tgccgtttta gatatgtgtg cttcattaaa 10860

agaattactg caaaatggta tgaatggacg taccatattg ggtagtgctt tattagaaga 10920

tgaatttaca ccttttgatg ttgttagaca atgctcaggt gttactttcc aaagtgcagt 10980

gaaaagaaca atcaagggta cacaccactg gttgttactc acaattttga cttcactttt 11040

agttttagtc cagagtactc aatggtcttt gttctttttt ttgtatgaaa atgccttttt 11100

accttttgct atgggtatta ttgctatgtc tgcttttgca atgatgtttg tcaaacataa 11160

gcatgcattt ctctgtttgt ttttgttacc ttctcttgcc actgtagctt attttaatat 11220

ggtctatatg cctgctagtt gggtgatgcg tattatgaca tggttggata tggttgatac 11280

tagtttgtct ggttttaagc taaaagactg tgttatgtat gcatcagctg tagtgttact 11340

aatccttatg acagcaagaa ctgtgtatga tgatggtgct aggagagtgt ggacacttat 11400

gaatgtcttg acactcgttt ataaagttta ttatggtaat gctttagatc aagccatttc 11460

catgtgggct cttataatct ctgttacttc taactactca ggtgtagtta caactgtcat 11520

gtttttggcc agaggtattg tttttatgtg tgttgagtat tgccctattt tcttcataac 11580

tggtaataca cttcagtgta taatgctagt ttattgtttc ttaggctatt tttgtacttg 11640

ttactttggc ctcttttgtt tactcaaccg ctactttaga ctgactcttg gtgtttatga 11700

ttacttagtt tctacacagg agtttagata tatgaattca cagggactac tccacccaa 11760

gaatagcata gatgccttca aactcaacat taaattgttg ggtgttggtg gcaaaccttg 11820

tatcaaagta gccactgtac agtctaaaat gtcagatgta aagtgcacat cagtagtctt 11880

actctcagtt ttgcaacaac tcagagtaga atcatcatct aaattgtggg ctcaatgtgt 11940

ccagttacac aatgacattc tcttagctaa agatactact gaagcctttg aaaaaatggt 12000

ttcactactt tctgttttgc tttccatgca gggtgctgta gacataaaca agctttgtga 12060

agaaatgctg gacaacaggg caaccttaca agctatagcc tcagagttta gttcccttcc 12120

atcatatgca gcttttgcta ctgctcaaga agcttatgag caggctgttg ctaatggtga 12180

ttctgaagtt gttcttaaaa agttgaagaa gtctttgaat gtggctaaat ctgaatttga 12240

ccgtgatgca gccatgcaac gtaagttgga aaagatggct gatcaagcta tgacccaaat 12300

gtataaacag gctagatctg aggacaagag ggcaaaagtt actagtgcta tgcagacaat 12360

gcttttcact atgcttagaa agttggataa tgatgcactc aacaacatta tcaacaatgc 12420

aagagatggt tgtgttccct tgaacataat acctcttaca acagcagcca aactaatggt 12480

tgtcatacca gactataaca catataaaaa tacgtgtgat ggtacaacat ttacttatgc 12540

atcagcattg tgggaaatcc aacaggttgt agatgcagat agtaaaattg ttcaacttag 12600

tgaaattagt atggacaatt cacctaattt agcatggcct cttattgtaa cagctttaag 12660

ggccaattct gctgtcaaat tacagaataa tgagcttagt cctgttgcac tacgacagat 12720

gtcttgtgct gccggtacta cacaaactgc ttgcactgat gacaatgcgt tagcttacta 12780

caacacaaca aagggaggta ggtttgtact tgcactgtta tccgatttac aggatttgaa 12840

atgggctaga ttccctaaga gtgatggaac tggtactatc tatacagaac tggaaccacc 12900

ttgtaggttt gttacagaca cacctaaagg tcctaaagtg aagtatttat actttattaa 12960

aggattaaac aacctaaata gaggtatggt acttggtagt ttagctgcca cagtacgtct 13020

acaagctggt aatgcaacag aagtgcctgc caattcaact gtattatctt tctgtgcttt 13080

tgctgtagat gctgctaaag cttacaaaga ttatctagct agtgggggac aaccaatcac 13140

taattgtgtt aagatgttgt gtacacacac tggtactggt caggcaataa cagttacacc 13200

ggaagccaat atggatcaag aatcctttgg tggtgcatcg tgttgtctgt actgccgttg 13260

ccacatagat catccaaatc ctaaaggatt ttgtgactta aaaggtaagt atgtacaaat 13320

acctacaact tgtgctaatg accctgtggg ttttacactt aaaaacacag tctgtaccgt 13380

ctgcggtatg tggaaaggtt atggctgtag ttgtgatcaa ctccgcgaac ccatgcttca 13440

gtcagctgat gcacaatcgt ttttaaacgg gtttgcggtg taagtgcagc ccgtcttaca 13500

ccgtgcggca caggcactag tactgatgtc gtatacaggg cttttgacat ctacaatgat 13560

aaagtagctg gttttgctaa attcctaaaa actaattgtt gtcgcttcca agaaaaggac 13620

gaagatgaca atttaattga ttcttacttt gtagttaaga gacacacttt ctctaactac 13680

caacatgaag aaacaattta taatttactt aaggattgtc cagctgttgc taaacatgac 13740

ttctttaagt ttagaataga cggtgacatg gtaccacata tatcacgtca acgtcttact 13800

aaatacacaa tggcagacct cgtctatgct ttaaggcatt ttgatgaagg taattgtgac 13860

acattaaaag aaatacttgt cacatacaat tgttgtgatg atgattattt caataaaaag 13920

gactggtatg attttgtaga aaacccagat atattacgcg tatacgccaa cttaggtgaa 13980

cgtgtacgcc aagctttgtt aaaaacagta caattctgtg atgccatgcg aaatgctggt 14040

attgttggtg tactgacatt agataatcaa gatctcaatg gtaactggta tgatttcggt 14100

gatttcatac aaaccacgcc aggtagtgga gttcctgttg tagattctta ttattcattg 14160

ttaatgccta tattaacctt gaccagggct ttaactgcag agtcacatgt tgacactgac 14220

ttaacaaagc cttacattaa gtgggatttg ttaaaatatg acttcacgga agagaggtta 14280

aaactctttg accgttattt taaatattgg gatcagacat accacccaaa ttgtgttaac 14340

tgtttggatg acagatgcat tctgcattgt gcaaacttta atgttttatt ctctacagtg 14400

ttcccaccta caagttttgg accactagtg agaaaaatat ttgttgatgg tgttccattt 14460

gtagtttcaa ctggatacca cttcagagag ctaggtgttg tacataatca ggatgtaaac 14520

ttacatagct ctagacttag ttttaaggaa ttacttgtgt atgctgctga ccctgctatg 14580

cacgctgctt ctggtaatct attactagat aaacgcacta cgtgcttttc agtagctgca 14640

cttactaaca atgttgcttt tcaaactgtc aaacccggta attttaacaa agacttctat 14700

gactttgctg tgtctaaggg tttctttaag gaaggaagtt ctgttgaatt aaaacacttc 14760

ttctttgctc aggatggtaa tgctgctatc agcgattatg actactatcg ttataatcta 14820

ccaacaatgt gtgatatcag acaactacta tttgtagttg aagttgttga taagtacttt 14880

gattgttacg atggtggctg tattaatgct aaccaagtca tcgtcaacaa cctagacaaa 14940

tcagctggtt ttccattaa taaatggggt aaggctagac tttattatga ttcaatgagt 15000

tatgaggatc aagatgcact tttcgcatat acaaaacgta atgtcatccc tactataact 15060

caaatgaatc ttaagtatgc cattagtgca aagaatagag ctcgcaccgt agctggtgtc 15120

tctatctgta gtactatgac caatagacag tttcatcaaa aattattgaa atcaatagcc 15180

gccactagag gagctactgt agtaattgga acaagcaaat tctatggtgg ttggcacaac 15240

atgttaaaaa ctgtttatag tgatgtagaa aaccctcacc ttatgggttg ggattatcct 15300

aaatgtgata gagccatgcc taacatgctt agaattatgg cctcacttgt tcttgctcgc 15360

aaacatacaa cgtgttgtag cttgtcacac cgtttctata gattagctaa tgagtgtgct 15420

caagtattga gtgaaatggt catgtgtggc ggttcactat atgttaaacc aggtggaacc 15480

tcatcaggag atgccacaac tgcttatgct aatagtgttt ttaacatttg tcaagctgtc 15540

acggccaatg ttaatgcact tttatctact gatggtaaca aaattgccga taagtatgtc 15600

cgcaatttac aacacagact ttatgagtgt ctctatagaa atagagatgt tgacacagac 15660

tttgtgaatg agttttacgc atatttgcgt aaacatttct caatgatgat actctctgac 15720

gatgctgttg tgtgtttcaa tagcacttat gcatctcaag gtctagtggc tagcataaag 15780

aactttaagt cagttcttta ttatcaaaac aatgttttta tgtctgaagc aaaatgttgg 15840

actgagactg accttactaa aggacctcat gaattttgct ctcaacatac aatgctagtt 15900

aaacagggtg atgattatgt gtaccttcct tacccagatc catcaagaat cctaggggcc 15960

ggctgttttg tagatgatat cgtaaaaaca gatggtacac ttatgattga acggttcgtg 16020

tctttagcta tagatgctta cccacttact aaacatccta atcaggagta tgctgatgtc 16080

tttcatttgt acttacaata cataagaaag ctacatgatg agttaacagg acacatgtta 16140

gacatgtatt ctgttatgct tactaatgat aacacttcaa ggtattggga acctgagttt 16200

tatgaggcta tgtacacacc gcatacagtc ttacaggctg ttggggcttg tgttctttgc 16260

aattcacaga cttcattaag atgtggtgct tgcatacgta gaccattctt atgttgtaaa 16320

tgctgttacg accatgtcat atcaacatca cataaattag tcttgtctgt taatccgtat 16380

gtttgcaatg ctccaggttg tgatgtcaca gatgtgactc aactttactt aggaggtatg 16440

agctattatt gtaaatcaca taaaccaccc attagttttc cattgtgtgc taatggacaa 16500

gtttttggtt tatataaaaa tacatgtgtt ggtagcgata atgttactga ctttaatgca 16560

attgcaacat gtgactggac aaatgctggt gattacattt tagctaacac ctgtactgaa 16620

agactcaagc tttttgcagc agaaacgctc aaagctactg aggagacatt taaactgtct 16680

tatggtattg ctactgtacg tgaagtgctg tctgacagag aattacatct ttcatgggaa 16740

gttggtaaac ctagaccacc acttaaccga aattatgtct ttactggtta tcgtgtaact 16800

aaaaacagta aagtacaaat aggagagtac acctttgaaa aaggtgacta tggtgatgct 16860

gttgtttacc gaggtacaac aacttacaaa ttaaatgttg gtgattattt tgtgctgaca 16920

tcacatacag taatgccatt aagtgcacct acactagtgc cacaagagca ctatgttaga 16980

attactggct tatacccaac actcaatatc tcagatgagt tttctagcaa tgttgcaaat 17040

tatcaaaagg ttggtatgca aaagtattct acactccagg gaccacctgg tactggtaag 17100

agtcattttg ctattggcct agctctctac tacccttctg ctcgcatagt gtatacagct 17160

tgctctcatg ccgctgttga tgcactatgt gagaaggcat taaaatattt gcctatagat 17220

aaatgtagta gaattatacc tgcacgtgct cgtgtagagt gttttgataa attcaaagtg 17280

aattcaacat tagaacagta tgtcttttgt actgtaaatg cattgcctga gacgacagca 17340

gatatagttg tctttgatga aatttcaatg gccacaaatt atgatttgag tgttgtcaat 17400

gccagattac gtgctaagca ctatgtgtac attggcgacc ctgctcaatt acctgcacca 17460

cgcacattgc taactaaggg cacactagaa ccagaatatt tcaattcagt gtgtagactt 17520

atgaaaacta taggtccaga catgttcctc ggaacttgtc ggcgttgtcc tgctgaaatt 17580

gttgacactg tgagtgcttt ggtttatgat aataagctta aagcacataa agacaaatca 17640

gctcaatgct ttaaaatgtt ttataagggt gttatcacgc atgatgtttc atctgcaatt 17700

aacaggccac aaataggcgt ggtaagagaa ttccttacac gtaaccctgc ttggagaaaa 17760

gctgtcttta tttcacctta taattcacag aatgctgtag cctcaaagat tttgggacta 17820

ccaactcaaa ctgttgattc atcacagggc tcagaatatg actatgtcat attcactcaa 17880

accactgaaa cagctcactc ttgtaatgta aacagatta atgttgctat taccagagca 17940

aaagtaggca tactttgcat aatgtctgat agagaccttt atgacaagtt gcaatttaca 18000

agtcttgaaa ttccacgtag gaatgtggca actttacaag ctgaaaatgt aacaggactc 18060

tttaaagatt gtagtaaggt aatcactggg ttacatccta cacaggcacc tacacacctc 18120

agtgttgaca ctaaattcaa aactgaaggt ttatgtgttg acatacctgg catacctaag 18180

gacatgacct atagaagact catctctatg atgggtttta aaatgaatta tcaagttaat 18240

ggttacccta acatgtttat cacccgcgaa gaagctataa gacatgtacg tgcatggatt 18300

ggcttcgatg tcgaggggtg tcatgctact agagaagctg ttggtaccaa tttaccttta 18360

cagctaggtt tttctacagg tgttaaccta gttgctgtac ctacaggtta tgttgataca 18420

cctaataata cagattttc cagagttagt gctaaaccac cgcctggaga tcaatttaaa 18480

cacctcatac cacttatgta caaaggactt ccttggaatg tagtgcgtat aaagattgta 18540

caaatgttaa gtgacacact taaaaatctc tctgacagag tcgtatttgt cttatgggca 18600

catggctttg agttgacatc tatgaagtat tttgtgaaaa taggacctga gcgcacctgt 18660

tgtctatgtg atagacgtgc cacatgcttt tccactgctt cagacactta tgcctgttgg 18720

catcattcta ttggatttga ttacgtctat aatccgttta tgattgatgt tcaacaatgg 18780

ggttttacag gtaacctaca aagcaaccat gatctgtatt gtcaagtcca tggtaatgca 18840

catgtagcta gttgtgatgc aatcatgact aggtgtctag ctgtccacga gtgctttgtt 18900

aagcgtgttg actggactat tgaatatcct ataattggtg atgaactgaa gattaatgcg 18960

gcttgtagaa aggttcaaca catggttgtt aaagctgcat tattagcaga caaattccca 19020

gttcttcacg acattggtaa ccctaaagct attaagtgtg tacctcaagc tgatgtagaa 19080

tggaagttct atgatgcaca gccttgtagt gacaaagctt ataaaataga agaattattc 19140

tattcttatg ccacacattc tgacaaattc acagatggtg tatgcctatt ttggaattgc 19200

aatgtcgata gatatcctgc taattccatt gtttgtagat ttgacactag agtgctatct 19260

aaccttaact tgcctggttg tgatggtggc agtttgtatg taaataaaca tgcattccac 19320

acaccagctt ttgataaaag tgcttttgtt aatttaaaac aattaccatt tttctattac 19380

tctgacagtc catgtgagtc tcatggaaaa caagtagtgt cagatataga ttatgtacca 19440

ctaaagtctg ctacgtgtat aacacgttgc aatttaggtg gtgctgtctg tagacatcat 19500

gctaatgagt acagattgta tctcgatgct tataacatga tgatctcagc tggctttagc 19560

ttgtgggttt acaaacaatt tgatacttat aacctctgga acacttttac aagacttcag 19620

agtttagaaa atgtggcttt taatgttgta aataagggac actttgatgg acaacagggt 19680

gaagtaccag tttctatcat taataacact gtttacacaa aagttgatgg tgttgatgta 19740

gaattgtttg aaaataaaac aacattacct gttaatgtag catttgagct ttgggctaag 19800

cgcaacatta aaccagtacc agaggtgaaa atactcaata atttgggtgt ggacattgct 19860

gctaatactg tgatctggga ctacaaaaga gatgctccag cacatatatc tactattggt 19920

gtttgttcta tgactgacat agccaagaaa ccaactgaaa cgatttgtgc accactcact 19980

gtcttttttg atggtagagt tgatggtcaa gtagacttat ttagaaatgc ccgtaatggt 20040

gttcttatta cagaaggtag tgttaaaggt ttacaaccat ctgtaggtcc caaacaagct 20100

agtcttaatg gagtcacatt aattggagaa gccgtaaaaa cacagttcaa ttattataag 20160

aaagttgatg gtgttgtcca acaattacct gaaacttact ttactcagag tagaaattta 20220

caagaattta aacccaggag tcaaatggaa attgatttct tagaattagc tatggatgaa 20280

ttcattgaac ggtataaatt agaaggctat gccttcgaac atatcgttta tggagatttt 20340

agtcatagtc agttaggtgg tttacatcta ctgattggac tagctaaacg ttttaaggaa 20400

tcaccttttg aattagaaga ttttattcct atggacagta cagttaaaaa ctatttcata 20460

acagatgcgc aaacaggttc atctaagtgt gtgtgttctg ttattgattt attacttgat 20520

gattttgttg aaataataaa atcccaagat ttatctgtag tttctaaggt tgtcaaagtg 20580

actattgact atacagaaat ttcatttatg ctttggtgta aagatggcca tgtagaaaca 20640

ttttacccaa aattacaatc tagtcaagcg tggcaaccgg gtgttgctat gcctaatctt 20700

tacaaaatgc aaagaatgct attagaaaag tgtgaccttc aaaattatgg tgatagtgca 20760

acattaccta aaggcataat gatgaatgtc gcaaaatata ctcaactgtg tcaatattta 20820

aacacattaa cattagctgt accctataat atgagagtta tacattttgg tgctggttct 20880

gataaaggag ttgcaccagg tacagctgtt ttaagacagt ggttgcctac gggtacgctg 20940

cttgtcgatt cagatcttaa tgactttgtc tctgatgcag attcaacttt gattggtgat 21000

tgtgcaactg tacatacagc taataaatgg gatctcatta ttagtgatat gtacgaccct 21060

aagactaaaa atgttacaaa agaaaatgac tctaaagagg gttttttcac ttacatttgt 21120

gggtttatac aacaaaagct agctcttgga ggttccgtgg ctataaagat aacagaacat 21180

tcttggaatg ctgatcttta taagctcatg ggacacttcg catggtggac agcctttgtt 21240

actaatgtga atgcgtcatc atctgaagca tttttaattg gatgtaatta tcttggcaaa 21300

ccacgcgaac aaatagatgg ttatgtcatg catgcaaatt acatattttg gaggaataca 21360

aatccaattc agttgtcttc ctattcttta tttgacatga gtaaatttcc ccttaaatta 21420

aggggtactg ctgttatgtc tttaaaagaa ggtcaaatca atgatatgat tttatctctt 21480

cttagtaaag gtagacttat aattagagaa aacaacagag ttgttatttc tagtgatgtt 21540

cttgttaaca actaaacgaa caatgtttgt ttttcttgtt ttattgccac tagtctctag 21600

tcagtgtgtt aatcttacaa ccagaactca attaccccct gcatacacta attctttcac 21660

acgtggtgtt tattaccctg acaaagtttt cagatcctca gttttacatt caactcagga 21720

cttgttctta cctttctttt ccaatgttac ttggttccat gctatacatg tctctgggac 21780

caatggtact aagaggtttg ataaccctgt cctaccattt aatgatggtg tttattttgc 21840

ttccactgag aagtctaaca taataagagg ctggattttt ggtactactt tagattcgaa 21900

gacccagtcc ctacttattg ttaataacgc tactaatgtt gttattaaag tctgtgaatt 21960

tcaattttgt aatgatccat ttttgggtgt ttattaccac aaaaacaaca aaagttggat 22020

ggaaagtgag ttcagagttt attctagtgc gaataattgc acttttgaat atgtctctca 22080

gccttttctt atggaccttg aaggaaaaca gggtaatttc aaaaatctta gggaatttgt 22140

gtttaagaat attgatggtt attttaaaat atattctaag cacacgccta ttaatttagt 22200

gcgtgatctc cctcagggtt tttcggcttt agaaccattg gtagatttgc caataggtat 22260

taacatcact aggtttcaaa ctttacttgc tttacataga agttatttga ctcctggtga 22320

ttcttcttca ggttggacag ctggtgctgc agcttattat gtgggttatc ttcaacctag 22380

gacttttcta ttaaaatata atgaaaatgg aaccattaca gatgctgtag actgtgcact 22440

tgaccctctc tcagaaacaa agtgtacgtt gaaatccttc actgtagaaa aaggaatcta 22500

tcaaacttct aactttagag tccaaccaac agaatctatt gttagatttc ctaatattac 22560

aaacttgtgc ccttttggtg aagtttttaa cgccaccaga tttgcatctg tttatgcttg 22620

gaacaggaag agaatcagca actngtgttgc tgattattct gtcctatata attccgcatc 22680

attttccact tttaagtgtt atggagtgtc tcctactaaa ttaaatgatc tctgctttac 22740

taatgtctat gcagattcat ttgtaattag aggtgatgaa gtcagacaaa tcgctccagg 22800

gcaaactgga aagattgctg attataatta taaattacca gatgatttta caggctgcgt 22860

tatagcttgg aattctaaca atcttgattc taaggttggt ggtaattata attacctgta 22920

tagattgttt aggaagtcta atctcaaacc ttttgagaga gatatttcaa ctgaaatcta 22980

tcaggccggt agcacacctt gtaatggtgt tgaaggtttt aattgttact ttcctttaca 23040

atcatatggt ttccaaccca ctaatggtgt tggttaccaa ccacacag tagtagtact 23100

ttcttttgaa cttctacatg caccagcaac tgtttgtgga cctaaaaagt ctactaattt 23160

ggttaaaaac aaatgtgtca atttcaactt caatggttta acaggcacag gtgttcttac 23220

tgagtctaac aaaaagtttc tgcctttcca acaatttggc agagacattg ctgacactac 23280

tgatgctgtc cgtgatccac agacacttga gattcttgac attacaccat gttcttttgg 23340

tggtgtcagt gttataacac caggaacaaa tacttctaac caggttgctg ttctttatca 23400

ggatgttaac tgcacagaag tccctgttgc tattcatgca gatcaactta ctcctacttg 23460

gcgtgtttat tctacaggtt ctaatgtttt tcaaacacgt gcaggctgtt taataggggc 23520

tgaacatgtc aacaactcat atgagtgtga catacccatt ggtgcaggta tatgcgctag 23580

ttatcagact cagactaatt ctcctcggcg ggcacgtagt gtagctagtc aatccatcat 23640

tgcctacact atgtcacttg gtgcagaaaa ttcagttgct tactctaata actctattgc 23700

catacccaca aattttacta ttagtgttac cacagaaatt ctaccagtgt ctatgaccaa 23760

gacatcagta gattgtacaa tgtacatttg tggtgattca actgaatgca gcaatctttt 23820

gttgcaatat ggcagttttt gtacacaatt aaaccgtgct ttaactggaa tagctgttga 23880

acaagacaaa aacacccaag aagtttttgc acaagtcaaa caaatttaca aaacaccacc 23940

aattaaagat tttggtggtt ttaatttttc acaaatatta cgatccat caaaaccaag 24000

caagaggtca tttattgaag atctactttt caacaaagtg acacttgcag atgctggctt 24060

catcaaacaa tatggtgatt gccttggtga tattgctgct agagacctca tttgtgcaca 24120

aaagtttaac ggccttactg ttttgccacc tttgctcaca gatgaaatga ttgctcaata 24180

cacttctgca ctgttagcgg gtacaatcac ttctggttgg acctttggtg caggtgctgc 24240

attacaaata ccatttgcta tgcaaatggc ttataggttt aatggtattg gagttacaca 24300

gaatgttctc tatgagaacc aaaaattgat tgccaaccaa tttaatagtg ctattggcaa 24360

aattcaagac tcactttctt ccacagcaag tgcacttgga aaacttcaag atgtggtcaa 24420

ccaaaatgca caagctttaa acacgcttgt taaacaactt agctccaatt ttggtgcaat 24480

ttcaagtgtt ttaaatgata tcctttcacg tcttgacaaa gttgaggctg aagtgcaaat 24540

tgataggttg atcacaggca gacttcaaag tttgcagaca tatgtgactc aacaattaat 24600

tagagctgca gaaatcagag cttctgctaa tcttgctgct actaaaatgt cagagtgtgt 24660

acttggacaa tcaaaaagag ttgatttttg tggaaagggc tatcatctta tgtccttccc 24720

tcagtcagca cctcatggtg tagtcttctt gcatgtgact tatgtccctg cacaagaaaa 24780

gaacttcaca actgctcctg ccatttgtca tgatggaaaa gcacactttc ctcgtgaagg 24840

tgtctttgtt tcaaatggca cacactggtt tgtaacacaa aggaattttt atgaaccaca 24900

aatcattact acagacaaca catttgtgtc tggtaactgt gatgttgtaa taggaattgt 24960

caacaacaca gtttatgatc ctttgcaacc tgaattagac tcattcaagg aggagttaga 25020

taaatatttt aagaatcata catcaccaga tgttgattta ggtgacatct ctggcattaa 25080

tgcttcagtt gtaaacattc aaaaagaaat tgaccgcctc aatgaggttg ccaagaattt 25140

aaatgaatct ctcatcgatc tccaagaact tggaaagtat gagcagtata taaaatggcc 25200

atggtacatt tggctaggtt ttatagctgg cttgattgcc atagtaatgg tgacaattat 25260

gctttgctgt atgaccagtt gctgtagttg tctcaagggc tgttgttctt gtggatcctg 25320

ctgcaaattt gatgaagacg actctgagcc agtgctcaaa ggagtcaaat tacattacac 25380

ataaacgaac ttatggattt gtttatgaga atcttcacaa ttggaactgt aactttgaag 25440

caaggtgaaa tcaaggatgc tactccttca gattttgttc gcgctactgc aacgataccg 25500

atacaagcct cactcccttt cggatggctt attgttggcg ttgcacttct tgctgttttt 25560

cagagcgctt ccaaaatcat aaccctcaaa aagagatggc aactagcact ctccaagggt 25620

gttcactttg tttgcaactt gctgttgttg tttgtaacag tttactcaca ccttttgctc 25680

gttgctgctg gccttgaagc cccttttctc tatctttatg ctttagtcta cttcttgcag 25740

agtataaact ttgtaagaat aataatgagg ctttggcttt gctggaaatg ccgttccaaa 25800

aacccattac tttatgatgc caactatttt ctttgctggc atactaattg ttacgactat 25860

tgtatacctt acaatagtgt aacttcttca attgtcatta cttcaggtga tggcacaaca 25920

agtcctattt ctgaacatga ctaccagatt ggtggttata ctgaaaaatg ggaatctgga 25980

gtaaaagact gtgttgtatt acacagttac ttcacttcag actattacca gctgtactca 26040

actcaattga gtacagacac tggtgttgaa catgttacct tcttcatcta caataaaatt 26100

gttgatgagc ctgaagaaca tgtccaaatt cacacaatcg acggttcatc cggagttgtt 26160

aatccagtaa tggaaccaat ttatgatgaa ccgacgacga ctactagcgt gcctttgtaa 26220

gcacaagctg atgagtacga acttatgtac tcattcgttt cggaagagac aggtacgtta 26280

atagttaata gcgtacttct ttttcttgct ttcgtggtat tcttgctagt tacactagcc 26340

atccttactg cgcttcgatt gtgtgcgtac tgctgcaata ttgttaacgt gagtcttgta 26400

aaaccttctt tttacgttta ctctcgtgtt aaaaatctga attcttctag agttcctgat 26460

cttctggtct aaacgaacta aatattatat tagtttttct gtttggaact ttaattttag 26520

ccatggcaga ttccaacggt actattaccg ttgaagagct taaaaagctc cttgaacaat 26580

ggaacctagt aataggtttc ctattcctta catggatttg tcttctacaa tttgcctatg 26640

ccaacaggaa taggtttttg tatataatta agttaatttt cctctggctg ttatggccag 26700

taactttagc ttgttttgtg cttgctgctg tttacagaat aaattggatc accggtggaa 26760

ttgctatcgc aatggcttgt cttgtaggct tgatgtggct cagctacttc attgcttctt 26820

tcagactgtt tgcgcgtacg cgttccatgt ggtcattcaa tccagaaact aacattcttc 26880

tcaacgtgcc actccatggc actattctga ccagaccgct tctagaaagt gaactcgtaa 26940

tcggagctgt gatccttcgt ggacatcttc gtattgctgg acaccatcta ggacgctgtg 27000

acatcaagga cctgcctaaa gaaatcactg ttgctacatc acgaacgctt tcttattaca 27060

aattgggagc ttcgcagcgt gtagcaggtg actcaggttt tgctgcatac agtcgctaca 27120

ggattggcaa ctataaatta aacacagacc attccagtag cagtgacaat attgctttgc 27180

ttgtacagta agtgacaaca gatgtttcat ctcgttgact ttcaggttac tatagcagag 27240

atattactaa ttattatgag gacttttaaa gtttccatt ggaatcttga ttacatcata 27300

aacctcataa ttaaaaattt atctaagtca ctaactgaga ataaatattc tcaattagat 27360

gaagagcaac caatggagat tgattaaacg aacatgaaaa ttattctttt cttggcactg 27420

ataacactcg ctacttgtga gctttatcac taccaagagt gtgttagagg tacaacagta 27480

cttttaaaag aaccttgctc ttctggaaca tacgagggca attcaccatt tcatcctcta 27540

gctgataaca aatttgcact gacttgcttt agcactcaat ttgcttttgc ttgtcctgac 27600

ggcgtaaaac acgtctatca gttacgtgcc agatcagttt cacctaaact gttcatcaga 27660

caagaggaag ttcaagaact ttactctcca atttttctta ttgttgcggc aatagtgttt 27720

ataacacttt gcttcacact caaaagaaag acagaatgat tgaactttca ttaattgact 27780

tctatttgtg ctttttagcc tttctgctat tccttgtttt aattatgctt attatctttt 27840

ggttctcact tgaactgcaa gacataatg aaacttgtca cgcctaaacg aacatgaaat 27900

ttcttgtttt cttaggaatc atcacaactg tagctgcatt tcaccaagaa tgtagtttac 27960

agtcatgtac tcaacatcaa ccatatgtag ttgatgaccc gtgtcctatt cacttctatt 28020

ctaaatggta tattagagta ggagctagaa aatcagcacc tttaattgaa ttgtgcgtgg 28080

atgaggctgg ttctaaatca cccattcagt acatcgatat cggtaattat acagtttcct 28140

gtttaccttt tacaattaat tgccaggaac ctaaattggg tagtcttgta gtgcgttgtt 28200

cgttctatga agacttttta gagtatcatg acgttcgtgt tgttttagat ttcatctaaa 28260

cgaacaaact aaaatgtctg ataatggacc ccaaaatcag cgaaatgcac cccgcattac 28320

gtttggtgga ccctcagatt caactggcag taaccagaat ggagaacgca gtggggcgcg 28380

atcaaaacaa cgtcggcccc aaggtttacc caataatact gcgtcttggt tcaccgctct 28440

cactcaacat ggcaaggaag accttaaatt ccctcgagga caaggcgttc caattaacac 28500

caatagcagt ccagatgacc aaattggcta ctaccgaaga gctaccagac gaattcgtgg 28560

tggtgacggt aaaatgaaag atctcagtcc aagatggtat ttctactacc taggaactgg 28620

gccagaagct ggacttccct atggtgctaa caaagacggc atcatatggg ttgcaactga 28680

gggagccttg aatacaccaa aagatcacat tggcacccgc aatcctgcta acaatgctgc 28740

aatcgtgcta caacttcctc aaggaacaac attgccaaaa ggcttctacg cagaagggag 28800

cagaggcggc agtcaagcct cttctcgttc ctcatcacgt agtcgcaaca gttcaagaaa 28860

ttcaactcca ggcagcagta ggggaacttc tcctgctaga atggctggca atggcggtga 28920

tgctgctctt gctttgctgc tgcttgacag attgaaccag cttgagagca aaatgtctgg 28980

taaaggccaa caacaacaag gccaaactgt cactaagaaa tctgctgctg aggcttctaa 29040

gaagcctcgg caaaaacgta ctgccactaa agcatacaat gtaacacaag ctttcggcag 29100

acgtggtcca gaacaaaccc aaggaaattt tggggaccag gaactaatca gacaaggaac 29160

tgattacaaa cattggccgc aaattgcaca atttgccccc agcgcttcag cgttcttcgg 29220

aatgtcgcgc attggcatgg aagtcacacc ttcgggaacg tggttgacct acacaggtgc 29280

catcaaattg gatgacaaag atccaaattt caaagatcaa gtcattttgc tgaataagca 29340

tattgacgca tacaaaacat tcccaccaac agagcctaaa aaggacaaaa agaagaaggc 29400

tgatgaaact caagccttac cgcagagaca gaagaaacag caaactgtga ctcttcttcc 29460

tgctgcagat ttggatgatt tctccaaaca attgcaacaa tccatgagca gtgctgactc 29520

aactcaggcc taaactcatg cagaccacac aaggcagatg ggctatataa acgttttcgc 29580

ttttccgttt acgatatata gtctactctt gtgcagaatg aattctcgta actacatagc 29640

acaagtagat gtagttaact ttaatctcac atagcaatct ttaatcagtg tgtaacatta 29700

gggaggactt gaaagagcca ccacattttc accgaggcca cgcggagtac gatcgagtgt 29760

acagtgaaca atgctaggga gagctgccta tatggaagag ccctaatgtg taaaattaat 29820

tttagtagtg ctatccccat gtgattttaa tagcttctta ggagaatgac aaaaaaaaaa 29880

aaaaaaaaaa aaaaaaaaaa aaa 29903

<210> 18

<211> 723

<212> PRT

<213> Artificial Sequence

<220>

<223> human Angiotensin-Converting-Enzyme 2 (ACE2)

<400> 18

Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn

1 5 10 15

His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn

20 25 30

Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala

35 40 45

Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln

50 55 60

Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu

65 70 75 80

Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser

85 90 95

Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr

100 105 110

Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu

115 120 125

Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg

130 135 140

Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg

145 150 155 160

Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala

165 170 175

Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val

180 185 190

Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp

195 200 205

Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His

210 215 220

Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser

225 230 235 240

Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg

245 250 255

Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro

260 265 270

Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln

275 280 285

Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro

290 295 300

Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly

305 310 315 320

Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys

325 330 335

Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe

340 345 350

Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr

355 360 365

Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His

370 375 380

Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His

385 390 395 400

Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu

405 410 415

Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr

420 425 430

Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys

435 440 445

Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys

450 455 460

Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr

465 470 475 480

Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile

485 490 495

Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu

500 505 510

Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser

515 520 525

Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly

530 535 540

Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys

545 550 555 560

Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr

565 570 575

Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp

580 585 590

Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys

595 600 605

Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr

610 615 620

Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys

625 630 635 640

Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala

645 650 655

Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys

660 665 670

Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg

675 680 685

Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser

690 695 700

Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro

705 710 715 720

Pro Val Ser

<210> 19

<211> 128

<212> PRT

<213> Artificial Sequence

<220>

<223> human GM-CSF

<400> 19

Met Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His

1 5 10 15

Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp

20 25 30

Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe

35 40 45

Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys

50 55 60

Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met

65 70 75 80

Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser

85 90 95

Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys

100 105 110

Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu

115 120 125

<---

Claims (51)

1. The use of a mixture of human or humanized natural IgM and/or IgA antibodies that recognize oxidized phospholipids or oxidation-specific epitopes for the treatment or prevention of a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies in a subject, said disorder or disease being selected from the group consisting of infections caused by a deficiency of natural antibodies, a neurodegenerative, metabolic, autoimmune or cardiovascular disease, wherein said human natural IgM and/or IgA antibody belongs to a subgroup of IgM and/or IgA antibodies of the subject's antibody spectrum, said subgroup essentially consisting of antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes, wherein said human natural IgM and/or IgA antibodies contain more than 35% of antibodies that recognize oxidized phospholipids and/or oxidation-specific epitopes, from the total number of IgM and/or IgA antibodies. 2. The use according to claim 1, wherein said human natural IgM and/or IgA antibodies refer to a subgroup of IgM and/or IgA antibodies obtained from IgM and/or IgA-enriched plasma pools from healthy individuals, said subgroup consisting essentially of antibodies that recognize oxidized phospholipids or oxidation-specific epitopes. 3. The use according to claim 1 or 2, wherein said disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies is the viral infectious disease COVID-19 caused by the β-coronavirus SARS-CoV2, or long-term COVID-19 (long-COVID-19). 4. The use according to any one of claims 1-3, wherein said mixture has the ability to inhibit the spread of a virus from an infected cell to an adjacent second uninfected cell. 5. Use according to any one of paragraphs 1-4, wherein said mixture has anti-inflammatory activity, preferably the ability to: reduce the accumulation of free oxidized phospholipids, preferably in infected lungs; clear lung tissue from cellular debris, stimulate the secretion of IL-10 and/or TGFb; and/or neutralize proinflammatory immune responses initiated by cytokines. 6. The use according to any one of claims 1-5, wherein said disorder or disease associated with a deficiency of natural IgM/IgA antibodies is an inflammatory disease or a viral infectious disease. 7. The use according to claim 6, wherein said inflammatory disease is selected from the group consisting of infectious diseases mediated by respiratory viruses, preferably COVID-19, long-COVID-19, influenza, MERS-COVID or SARS-COV; infectious diseases caused by bacterial infections that are mediated by gram-positive or gram-negative pathogens, fungi or parasites; and "sterile" diseases, preferably cardiovascular diseases, atherosclerosis, coronary heart disease, heart attack and stroke, metabolic disorders such as diabetes mellitus, neurodegenerative diseases, preferably Alzheimer's disease, and autoimmune diseases, preferably systemic lupus erythematosus or multiple sclerosis. 8. The use according to claim 6, wherein said viral infectious disease is selected from the group consisting of infections caused by coronaviruses, preferably SARS-CoV, SARS-CoV-2, MERS; influenza viruses, parainfluenza viruses, respiratory syncytial viruses (RSV), rhinoviruses, adenoviruses, enteroviruses, human metapneumoviruses, herpesviruses, preferably HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7, HHV-8. 9. A pharmaceutical composition for the treatment or prevention of a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies in a subject, comprising an effective amount of a mixture of human or humanized natural IgM and/or IgA antibodies, as defined in any of claims 1 to 8, and at least one pharmaceutically acceptable excipient. 10. The use according to claim 1, wherein said mixture of antibodies is a mixture of recombinant human monoclonal natural antibodies IgM or IgA. 11. The use according to claim 10, wherein said recombinant antibodies recognize and bind to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, malondialdehyde-modified, 4-hydroxynonenal-modified and 2-(ω-carboxyethyl)pyrrole-modified proteins and/or amyloid-β oligomeric peptide. 12. The use according to claim 10 or 11, wherein said recombinant antibodies comprise the complementarity determining regions V _H CDR1 comprising SEQ ID NO: 1, V _H CDR2 comprising SEQ ID NO: 2, V _H CDR3 comprising SEQ ID NO: 3, V _L CDR1 comprising SEQ ID NO: 4, V _L CDR2 comprising SEQ ID NO: 5, and V L CDR3 comprising SEQ ID NO: 6, wherein said antibodies recognize and bind to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, malondialdehyde, 4-hydroxynonenal and 2-(ω-carboxyethyl)pyrrole modified proteins and/or an oligomeric peptide amyloid-β; or wherein said antibodies comprise complementarity determining regions V _H CDR1 comprising SEQ ID NO: 9, V _H CDR2 comprising SEQ ID NO: 10, V _H CDR3 comprising SEQ ID NO: 11, V _L CDR1 comprising SEQ ID NO: 12, V _L CDR2 comprising SEQ ID NO: 13, and V _L CDR3 comprising SEQ ID NO: 14, wherein said antibodies recognize and bind to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, with oxidized cardiolipin, with oxidized phosphatidylserine, with malondialdehyde, 4-hydroxynonenal and 2-(ω-carboxyethyl)pyrrole modified proteins and/or amyloid-β oligomeric peptide. 13. Use according to any of paragraphs. 10-12, wherein said recombinant antibodies comprise an amino acid sequence that is at least 70% identical to the amino acid residues located at positions 1-25, 34-50, 59-96 and 116-126 of the sequence SEQ ID NO: 7 and at positions 1-25, 35-51, 55-90 and 101-110 of the sequence SEQ ID NO: 8, wherein said antibodies recognize and bind to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, with oxidized cardiolipin, with oxidized phosphatidylserine, with malondialdehyde, 4-hydroxynonenal and 2-(ω-carboxyethyl)pyrrole modified proteins and/or an oligomeric peptide amyloid-β; or wherein said antibodies comprise an amino acid sequence that is at least 70% identical to the amino acid residues located at positions 1-25, 34-50, 59-96 and 116-126 of the sequence SEQ ID NO: 15 and at positions 1-26, 34-50, 54-89 and 99-108 of the sequence SEQ ID NO: 16, wherein said antibodies recognize and bind to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, with oxidized cardiolipin, with oxidized phosphatidylserine, with malondialdehyde, 4-hydroxynonenal and 2-(ω-carboxyethyl)pyrrole modified proteins and/or amyloid-β oligomeric peptide. 14. The use according to any one of claims 10-13, wherein said recombinant antibodies comprise a V _H having SEQ ID NO: 7 and a V _L having SEQ ID NO: 8, wherein said antibodies recognize and bind to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, malondialdehyde, 4-hydroxynonenal and 2-(ω-carboxyethyl)pyrrole modified proteins and/or amyloid-β oligomeric peptide; or wherein said antibodies comprise V _H having SEQ ID NO: 15 and V _L having SEQ ID NO: 16, wherein said antibodies recognize and bind to phosphorylcholine, exposed oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoylphosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, malondialdehyde, 4-hydroxynonenal and 2-(ω-carboxyethyl)pyrrole modified proteins and/or amyloid-β oligomeric peptide. 15. The use according to any one of claims 10-14, wherein said disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies is a neurodegenerative or cardiovascular disease. 16. The use according to any one of paragraphs 10-15, wherein said disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies is the viral infectious disease COVID-19 caused by the β-coronavirus SARS-CoV-2, or is long-term COVID-19. 17. The use according to any one of claims 10-16, wherein said antibodies have the ability to inhibit the spread of a virus from an infected cell to an adjacent second uninfected cell. 18. The use according to any one of paragraphs 10-17, wherein said recombinant antibodies have anti-inflammatory activity, preferably the ability to: reduce the accumulation of free oxidized phospholipids, preferably in infected lungs; clear lung tissue from cellular debris, stimulate the secretion of IL-10 and/or TGFβ; and/or neutralize proinflammatory cytokines. 19. The use according to any one of claims 10-18, wherein said disorder or disease associated with a deficiency of natural IgM/IgA antibodies is an inflammatory disease or a viral infectious disease. 20. The use according to claim 19, wherein said inflammatory disease is selected from the group consisting of infectious diseases mediated by respiratory viruses, preferably COVID-19, long-acting COVID-19, influenza, MERS-COV or SARS-COV; infectious diseases caused by bacterial infections that are mediated by gram-positive or gram-negative pathogens, fungi or parasites; and "sterile" diseases, preferably cardiovascular diseases, atherosclerosis, coronary heart disease, heart attack and stroke, metabolic disorders such as diabetes mellitus, neurodegenerative diseases, preferably Alzheimer's disease, and autoimmune diseases, preferably systemic lupus erythematosus or multiple sclerosis. 21. The use according to claim 19, wherein said viral infectious disease is selected from the group consisting of infections caused by coronaviruses, preferably SARS-CoV, SARS-CoV-2, MERS; influenza viruses, parainfluenza viruses, respiratory syncytial viruses (RSV), rhinoviruses, adenoviruses, enteroviruses, human metapneumoviruses, herpesviruses, preferably HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7, HHV-8. 22. A pharmaceutical composition for the treatment or prevention of a disorder or disease associated with/linked to/caused by a deficiency of natural IgM/IgA antibodies in a subject, comprising an effective amount of a mixture of human or humanized natural IgM and/or IgA antibodies, as defined in any of paragraphs 10-21 and at least one pharmaceutically acceptable excipient. 23. The use according to any of paragraphs 1-8 and 10-21, or the pharmaceutical composition according to paragraph 9 or 22, wherein said mixture or pharmaceutical composition can be used in combination with: (a) an angiotensin-converting enzyme (ACE) inhibitor/antagonist, an angiotensin II receptor 1 (AT1R) inhibitor/antagonist and/or a compound that modulates ACE2 receptor expression; and/or Ang(1-7), AT2R and/or MAS receptor agonists; (b) a compound that inhibits/antagonizes/neutralizes ligands for the receptor for advanced glycation end products (RAGE) and/or a RAGE inhibitor/antagonist; and/or (c) granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or a compound that increases the phagocytic activity of alveolar macrophages (AM), preferably with azithromycin. 24. The use according to any of paragraphs 1-8 and 10-21, or the pharmaceutical composition according to paragraph 9 or 22, where the mixture or pharmaceutical composition can be used in combination with: an antiviral compound, preferably an antiviral compound such as: remdesivir; favipiravir; camostat mesylate; nafomostate mesylate; umifenovir; and/or a drug containing glycyrrhizin. 25. The use according to any of paragraphs 1-8 and 10-21 or the pharmaceutical composition according to paragraph 9 or 22, wherein the mixture or pharmaceutical composition can be used in combination with an immunomodulator, wherein preferably said immunomodulator is: anti-PD-1, anti-PD-L1, anti-CD40, CD40 ligand, anti-CSF-1R, anti-CTLA-4; and/or an antibody that binds to cytokines (preferably IL-6), an antibody that binds to its specific cytokine receptor (preferably IL-6R), an antibody that binds to chemokine(s) (preferably CCL2 and CCL5), an antibody that binds to its specific chemokine receptor(s) (preferably CCR2 and CCR5), and/or synthetic molecules (preferably maraviroc); and/or dexamethasone.