Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/70—Multivalent vaccine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention relates to anatoxins and antibodies for use in the prevention and treatment of coronavirus disease.
• the invention relates to anatoxins and antibodies for the use in the prevention and treatment of coronavirus disease, wherein said anatoxins are toxins produced by a cell in the presence of a coronavirus, such as, for example, SARS-CoV-2, and are capable of inducing an immunogenic response against said toxins.
• a coronavirus such as, for example, SARS-CoV-2
• viruses belonging to the coronavirus category are divided into five classes and many of them cause gastrointestinal diseases in the following animals: pigs, cattle and birds.
• betacoronaviruses are mainly transmitted through the respiratory tract or faecal-oral routes and infect epithelial cells, both respiratory (nasal, tracheal and pulmonary) and intestinal (villi and crypts of the ileum and colon).
• the SarS-CoV2 virus has manifested its power in the past 10 months. To date about 20 million people have been infected worldwide and over eight-hundred thousand have died. Numerous studies have been conducted with the aim of furthering knowledge about this virus in order to understand its mechanism of action and find effective therapies.
• the COVID- 19 virus an RNA virus
• Shine-Dalgarno a further ribosomal binding site, a subsequence of 5'-UGAUCC-3' (TGATCC), invariably exists in mRNA for highly expressed genes. Poorly expressed genes seem to be controlled only by the Shine-Dalgarno base pairing.
• the Shine-Dalgarno (SD) sequence is a sequence of nucleotides present in messenger RNA, made up of AGGAGG, and is an exclusive ribosomal binding site in bacteria and in archaea. It is located between the 8 and 13 bases before the start codon AUG (ATG). Ribosomal subunits are assembled at this site and it is read by the 16S subunit.
• the tRNA can add amino acids in sequence as dictated by the codons by moving downstream of the initial translation site.
• the Shine-Dalgarno sequence is common in bacteria, but rarer in archaea. Protein synthesis by ribosomes takes place on a linear substrate but at non-uniform speeds. A transient ribosome pausing can influence a variety of co-translational processes, including protein targeting and folding. This pausing is influenced by the messenger RNA sequence. Therefore, the redundancy in the genetic code enables the translation of the same protein at different speeds.
• the Applicant has now surprisingly found that the coronavirus classified as Sars-Cov2, and the congeners thereof, induce the cells of the host, bacteria and eukaryotic cells, to produce toxins that act on different types of receptors. Therefore, the Applicant has discovered that the coronavirus behaves above all and first as a phage that causes the release of toxins from bacterial cells.
• the toxins produced are specific, as they reinforce the toxic effect of the virus and explain the multi- systemic signs present both in humans and in animals following a coronavirus infection.
• the toxic effect of the coronavirus- induced toxins explain the symptoms of hyposmia and hypogeusia which are typical of some diseases today classified as neuroimmune diseases, including Parkinson’s disease and Alzheimer’s disease, correlated with acetylcholine mechanisms.
• the toxins produced in the presence of the virus are similar to the toxins of the conotoxin family, characterised by four or five disulphide bridges, distinguished by the cysteine motif C — C-CC — C, and on average have the same tertiary and quaternary forms, though the amino acid sequence of the primary sequence may vary slightly.
• the toxins induced by SARS-CoV-2 exhibit a strong bond with both nicotinic and muscarinic acetylcholine receptors.
• neurotoxins are divided into two categories: short-chain neurotoxins (60-62 amino acids, four disulphide bonds) and long-chain neurotoxins (66-74 or more amino acids, five disulphide bonds).
• the long-chain peptide binds with greater affinity to the alpha 7 nicotinic acetylcholine receptor or through the fifth disulphide bond (D. Servent et al., “Only Snake Curaremimetic Toxins with a Fifth Disulfide Bond Have High Affinity for the Neuronal 7 Nicotinic Receptor”, J. Biol. Chem., vol. 272, pp. 24279-86, Oct. 1997, doi: 10.1074/jbc.272.39.24279.) and the conotoxins found have 4 or 5 disulphide bridges, similar to the ones reported in the literature.
• the family of the three-finger toxins (about 70 3FTxs: cardiotoxic and neurotoxic) are polypeptides composed of 60-74 amino acid residues. They show different functionalities, despite having a conserved structure.
• One distinct structural characteristic is the single fold, consisting of three loops (-stranded), which emerge from a hydrophobic globular core. The four or five disulphide bridges, obligatory between certain Cys, stabilise the three-dimensional structure.
• Toxic phospholipases A2 also interact with nicotinic acetylcholine receptors. Almost all of these toxins bind with high affinity to the periphery of nicotinic acetylcholine receptors (AChR) (Denis Serventt ⁇ , Valerie Winckler-Dietricht, Hai-Yan Hu, Pascal Kessler ⁇ , Pascal Drevett, Daniel Bertrand and Andre Menezt, “Only Snake Curaremimetic Toxins with a Fifth Disulfide Bond Have High Affinity for the Neuronal a7 Nicotinic Receptor”,* J. Biol. Chem. 272, 24279-24286).
• AChR nicotinic acetylcholine receptors
• alpha-Cbtx binds to two subtypes of AChR using both common and specific residues.
• the bond with the toxins probably occurs in homologous regions of neuronal and muscle AChR (Ackermann, EJ, Ang, ETH, Kanter, JR, Tsigelny, I. and Taylor, P. (1998) J. Biol Chem. 273, 10958-10964).
• Three-finger toxins block post-synaptic transmission by means of nicotinic receptors (nAARTr).
• nAARTr nicotinic receptors
• Certain toxins possess a base phospholipase unit and possess anticoagulant properties; others induce platelet aggregation, and still others instead inhibit it.
• Many “curarizing toxins” with a three-finger structure recognise a protein that binds to acetylcholine and block the nicotinic receptors in skeletal muscles, causing flaccid paralysis.
• Other toxins recognise the acetylcholine receptors situated on neurons.
• Cardiotoxins damage heart function; they depolarise the membranes of numerous excitable cells and are potent cytotoxins. Overall, the toxins endowed with a three-finger structure exert at least seven different functions. Some toxins of snake venoms do not belong to either of the two previously mentioned structural categories.
• Sarafotoxins are vasoconstrictor peptides with 21 amino acids and two disulphide bridges. Finally, disintegrins all possess the conserved arginine-glycine-aspartic acid sequence and inhibit the aggregation of platelets, of which they recognise one of the surface proteins, glycoprotein lla/lllb, on the surface of platelets and other cells. Proteinase toxins activate prothrombin, as well as the coagulation factor Xa (FXa) (Medical Use of Snake Venom Proteins, CRC Press, Boca Raton, 1990).
• FXa coagulation factor Xa
• aChE and BuChE acetylcholinesterase and butyryl-cholinesterase inhibitors
• aChE and BuChE acetylcholinesterase and butyryl-cholinesterase inhibitors
• a non-hereditary BuChE deficiency can manifest itself during pregnancy, in newborn infants or also in the event of chronic infections, in the event of malnutrition, in liver diseases and neoplasia and in anaemia due to iron deficiency and drugs such as cocaine, morphine, codeine and succinylcholine, organophosphorus (OP) poisoning or an excess of acetylcholinesterase inhibitors (which are administered in cases of PD and AD).
• Inhibiting acetylcholinesterase (aChE) and increasing the availability of acetylcholine in cholinergic synapses improves cholinergic transmission in the pharmacological treatment of Alzheimer’s disease (AD).
• the cumulative incidence of pneumonia is 51.9 per 1000, or 5.19%, in subjects over 65 years old undergoing therapy with the cholinesterase inhibitor galantamine, or rivastigmine for dementia.
• aChE predominates also at a neuronal level
• BuChE is largely localised and secreted by glial cells.
• choline acetyltransferase the speed-limiting enzyme that catalyses the synthesis of ACh.
• About 10%-15% of cholinergic neurons within the human hippocampus and the amygdala seem to express BuChE rather than aChE.
• AChE and BuChE share about 65% amino acid sequence homology despite being encoded by different genes on the human chromosomes 7 (7q22) and 3 (3q26), respectively.
• both bind and cleave Ach to a large and effective degree, albeit with slightly different kinetics, rendering the metabolic products choline and acetic acid identical.
• ACh is involved in the modulation of inflammatory states. Recent data have demonstrated that ACh derived from parasympathetic innervations inhibits the release of tumour necrosis factor (TNF-a) and interleukin 1 (IL-1 ) by macrophages through the activation of nicotinic receptors, which supports the existence of the inflammatory route of an “anticholinergic” (Rosas-Ballina M, Tracey KJ., “Immune System Neurology: Neural Reflexes Regulate Immunity”, Neurone, 2009; 64: 28-32).
• TNF-a tumour necrosis factor
• IL-1 interleukin 1
• the nicotinic and muscarinic receptor subtypes are present both on neurons and on glial cells, where they mediate motor control, memory regulation, thermoregulation (difficult-to-control hyperthermia), the functions of synaptic plasticity as well as auto- and heteroreceptors.
• muscarinic receptors seem to be broadly involved in various functions, such as the contraction of smooth muscle, glandular secretion and the regulation of heart rate (49-50).
• Ipratropium and oxitropium M1 I M3 receptor antagonists
• tiotropium non-selective
• atropine or other muscarinic antagonists deriving from their pharmacokinetics
• the presence of a quaternary ammonium in their chemical formula enables a local effect that reduces their absorption by the bronchi (Goodman & Gilman Manual. Le basi farmacologiche della rick, Laurence, L., Brunton, Randa Hilal-Dandan, et al. 2018).
• coronavirus is better defined as oral-nasal-faecal and has phage-like behaviour.
• the discovery of the production of toxins induced by the virus can explain the severe symptoms induced by the virus in infected individuals.
• the mechanism of the virus induces the release of conotoxins, snake-like phospholipases A2 and proteins like prothrombin (activating the coagulation factor Va), protein like bradykinin- C type natriuretic factor, and proteins belonging to the zinc metalloproteinase family.
• the above-mentioned toxins were detected by mass spectrometry in the plasma of individuals affected by the pathogen and were then reproduced in vitro in a bacterial culture.
• the toxins and neurotoxins found and tested were produced in vitro in a bacterial matrix and through mass spectrometry analysis it was possible to observe that although they show amino acid variables and, in some cases, are transcribed as shorter proteins, they maintain their stimulating power, given the metabolically active nature of the protein transcript.
• toxoids The primary structure of the toxins (“toxoids”) is slightly different in every production cycle, but always manifests the same tertiary and quaternary folds, enabling them to be used, by virtue of denaturation processes, as anatoxins, i.e. as immunogenic molecules. Furthermore, their use makes it possible to replicate in bacterial hosts, based on a design of cDNA deriving from knowledge of their sequence, multiple copies which can then be inoculated into laboratory animals to create hybridomas. From the latter it is possible develop monoclonal and polyclonal antibodies useful for treating both animal and human subjects affected by the disease.
• the above-mentioned denatured toxins which are capable of inducing an immunogenic effect and permitting the stimulation of the immune system for the spontaneous formation of antibodies against SARS-CoV-2 and other coronaviruses.
• sequences of denatured anatoxins or toxoids or toxins according to the present invention can be used as partial or total sequences for the preparation of vaccines for the prevention and treatment of disease induced by coronaviruses, such as Sars-Cov2.
• coronaviruses such as Sars-Cov2.
• the same application can be envisaged for the prevention and treatment of other coronaviruses such as MERS and SARS-CoV.
• a product for use in the prevention and in the treatment of the coronavirus disease said product consisting of: a) one or more immunogenic anatoxins of one or more toxins produced by a cell in the presence of a coronavirus, wherein said one or more immunogenic anatoxins comprise or consist of an immunogenic amino acid sequence equal to the amino acid sequence of said one or more toxins or to immunogenic parts of the amino acid sequence of said one or more toxins, or having a percentage of sequence identity with the amino acid sequence of said one or more toxins, or with immunogenic parts of the amino acid sequence of said one or more toxins, of at least 20%, preferably at least 50%, or at least 60% or at least 70%, or at least 80% or at least 90%, b) a polyclonal or monoclonal antibody against said one or more toxins produced by a cell in the presence of a coronavirus; or c) a pharmaceutical composition comprising or consisting of the product as defined in a) and/or
• the term “one or more toxins produced by a cell in the presence of a coronavirus” means one or more toxins produced naturally or artificially and having an amino acid sequence equal to those of the toxins produced by a cell in the presence of a coronavirus, i.e. by a cell infected by a coronavirus, such as, for example, SARS-CoV-2.
• Said one or more anatoxins can be prepared from one or more toxins directly produced by a cell in the presence of a coronavirus or by said one or more toxins produced synthetically, for example recombinantly.
• the anatoxins can have an immunogenic amino acid sequence with different percentages of identity with respect to the amino acid sequences of the toxins produced by a cell in the presence of a coronavirus.
• the anatoxins can comprise or consist in the whole or partial amino acid sequences of the amino acid sequences of the toxins.
• the anatoxins can comprise an amino acid sequence that is an immunogenic part or portion of the amino acid sequence of the toxins or which has a percentage of identity, as defined above, with the immunogenic part or portion of the amino acid sequence of the toxins such as to maintain the immunogenicity thereof.
• said one or more toxins are selected from the group consisting of toxins capable of binding an acetylcholine receptor and/or acetylcholinesterase, phospholipase A2 toxins, prothrombin-activating toxins, toxins having the function of bradykinin-C type natriuretic factor and toxins having the function of zinc metalloproteinase.
• said one or more toxins When said one or more toxins are capable of binding an acetylcholine receptor and/or acetylcholinesterase, said one or more toxins comprise a cysteine motif capable of binding an acetylcholine receptor, for example a cysteine motif of a toxin selected from the group consisting of a conotoxin capable of binding an acetylcholine receptor, a three-finger toxin capable of binding an acetylcholine receptor, such as, for example, the bungoratoxins.
• a cysteine motif capable of binding an acetylcholine receptor for example a cysteine motif of a toxin selected from the group consisting of a conotoxin capable of binding an acetylcholine receptor, a three-finger toxin capable of binding an acetylcholine receptor, such as, for example, the bungoratoxins.
• said one or more anatoxins can comprise a cysteine motif of the toxins capable of binding an acetylcholine receptor, for example a cysteine motif of a toxin selected from the group consisting of a conotoxin capable of binding an acetylcholine receptor, a three-finger toxin capable of binding an acetylcholine receptor, such as, for example, the bungoratoxins.
• a cysteine motif of the toxins capable of binding an acetylcholine receptor for example a cysteine motif of a toxin selected from the group consisting of a conotoxin capable of binding an acetylcholine receptor, a three-finger toxin capable of binding an acetylcholine receptor, such as, for example, the bungoratoxins.
• the coronavirus can be selected from the group consisting of SARS-CoV-2, SARS-CoV, virus causing coronavirus Middle East respiratory syndrome or MERS-CoV, preferably SARS-CoV-2.
• the cell can be selected from the group consisting of a bacterial cell, for example of the bacteria of the gut microbiota or of the oral-nasal-faecal route of a subject infected by coronavirus, a cell of a subject infected by coronavirus, a eukaryotic cell present in a subject infected by coronavirus.
• the above-mentioned product can be used both in humans and in animals.
• said one or more anatoxins comprise or consist of an amino acid sequence or of an immunogenic portion thereof, wherein said amino acid sequence is selected from the group consisting of (in the list below the UniProt and Swiss Prot ID of the toxins present in fragments identified in the bacterial cultures and in the plasma of subject affected by the virus SARS-CoV-2 are also shown):
• PAHLLVLAAV CISLSGASSI APQPLNLIQF GNMIQCTIPG SSPLLDYADY GCYCGRGGSG TPVDK (SEQ ID NQ:10);
• Q9PUG8 MYPAHLLVLLAVCVSLLGASNIPLPSLDFEQFGKMIQCTIPCEESC
• AVCNCDRAAAICFARFPYNKNYWSINTEIHCR (SEQ ID N0:17);
• HLVAI SEQ ID NO:25
• GKFMYILHGCPFQPWC SEQ ID NO:29
• GKFMNVLRRSGCPWEP (SEQ ID NO:32);
• VQPGTKCG EGMVCGFGECIGLETALGINQ (SEQ ID NO:44);
• CKQLDEDCGYGYSCCEDL (SEQ ID NO:52); TVVSINLDHAFDGRNAAANNKATDLMARTVRRFCSDPPCRISNPE SCGW (SEQ ID NO:53);
• VLATTGVSFTLDRASDGGNAVAKKSDVTARFNWRCCLIPACRRN HKKFC SEQ ID NO:55
• RDVCELPFEEGPCFAAIR (SEQ ID NO:56);
• ALGEGDGQAVAGDRNPSEARSTHEHFLQRLIRLIHGSDCQPCGQ YVCCP (SEQ ID NO:58);
• PAHLLVLAAV CISLSGASSI APQPLNLIQF GNMIQCTIPG SSPLLDYADY GCYCGRGG SEQ ID NO:59
• PPKRDTLRNLLKIGTRGQGGCVPPGGGRCKANQACTKGGNPGT CGFQ SEQ ID NO:72;
• VLVAAVLLSAQVMVQGDGDQPADRDAVPRDDNPGGTIGKFMYIL HGCPFQP (SEQ ID NO:76);
• LEQKWLAPAAPPLEQKWLAPDAPPM (SEQ ID NQ:80); ESPAGGTTAFREELSPGPEAASGPAAPHRLPKSKGASATSAASR PMRD (SEQ ID NO:81);
• the anatoxins according to the present invention can be obtained, for example, by denaturation of said one or more toxins produced by a cell in the presence of a coronavirus, i.e. by a cell that is infected by a coronavirus.
• the product can be a vaccine or an antidote.
• the vaccine according to the present invention is useful against the symptoms of the neurotoxins released following contagion with the virus SARS-CoV-2 and enables prompt intervention in the treatment, prevention and resolution of the pathology both in humans and in animals.
• the sequences can also be obtained by cloning of a cDNA and subsequent production in a bacterial culture, or with other methods.
• the study is further confirmed by the presence of the toxins and neurotoxins found, tested and produced in vitro in a bacterial matrix. Through mass spectrometry analysis it was possible to observe that although the toxins show amino acid variables and, in some cases, are transcribed as shorter proteins, they maintain their stimulating power, given the metabolically active nature of the protein transcript. This fact provides a clear explanation also of the reason why the hyperimmune plasma of individuals who have recovered works in treating the pathology.
• the use of the sequences found for the purpose of treating living beings affected by the virus shows to be a safe method.
• the method proposed in example 1 does not imply any problems tied, as in hyperimmune plasma, to Rh factors, or ABO blood compatibility or HLA immune compatibility.
• the use according to the present invention does not imply the use of vaccines with other viruses as adjuvants.
• the denaturation of the toxins can be carried out by means of known methods such as chemical denaturation, for example by treatment with chemical substances, including urea, guanidinium chloride and guanidinium thiocyanate. Denaturation can also be obtained by treatment with formaldehyde. As an alternative to chemical denaturation, the denaturation of the toxins can be carried out by means of thermal denaturation, both hot and cold.
• the present invention further relates to the product as defined above for use against poisoning by one or more toxins of an amino acid sequence from SEQ ID NO:1 to SEQ ID NO:85, for example as a consequence of a bite or sting or contact with animals, ingestion of foods, inhalation of gas or vapours containing said toxins.
• FIG. 1 shows a diagram of the percentage of viral replication in a bacterial culture medium up to 30 days
• FIG. 2 shows a diagram of the frequency, in log-e values, of the toxins in the faecal samples A, B, C: sample “A” mixture of bacteria and virus Sars-Cov2, a SARS-CoV-2-negative multi-bacterial sample “B” inoculated with the supernatant and virus of sample “A”, sample “C” only a bacterial fraction derived from sample “A” after the collection and centrifugation of a replicate thereof.
• FIG. 36 shows the increase in time- and dose-dependent immunisation in groups of mice with 3 different toxoids (SEQ ID NO:11 , 68, 52).
• FIG. 37 shows the increase in antibodies in mice groups 2,3,4 for the three toxoids used (SEQ ID NO: 11 top line, SEQ ID NO: 52 middle line).
• FIG. 38 shows the assay of plasma pseudocholinesterase.
• FIG. 40 shows the ratio of toxins that are produced by a culture of bacteria with SARS-CoV-2 and by a culture of bacteria and bovine coronavirus B-CoV.
• FIG. 41 shows the TEM image, at the 11 th day, of amniotic liquid of a fertilised egg, inoculated at the 7th day with a supernatant of bacterial culture containing bovine betacoronavirus (B-CoV). Newly formed viral particles are visible around dark agglomerates.
• B-CoV bovine betacoronavirus
• EXAMPLE 1 Identification of the toxins produced by cells in the presence of SARS-CoV-2, preparation of the denatured toxins and study on the effectiveness of the denatured toxins according to the present invention
• SAN 1ST Surface- Activated Chemical lonisation/Electrospray-NIST technology was used to obtain and compare the proteomic profiles.
• An LC Ultimate 3000 UPLC system (ThermoFisher) was used to obtain the separation of the analytes for each sample prior to MS analysis.
• a C-18 reversed-phase LC column was used (50 x 2.1 mm; particle size 5 pm; pore size 100 A; Phenomenex [Torrance, CA OR San Jose], USA).
• the eluent flow was 0.25 mL / min and the injection volume was 15 pL.
• the mobile phases were: A) 0.2% formic acid (v I v) (HCOOH) and B) acetonitrile (CH3CN).
• the elution gradient was: 2% (v / v) B between 0 and 2 min; from 2 to 30% of B between 2 and 7 min; from 30 to 80% between 7 and 9 min; 80% B between 9 and 12 min; 80-2% B between 12 and 12.1 min; the column was reequilibrated with 2% B between 12.1 and 17 min.
• Mass spectrometry the samples of plasma were analysed with an HCT ion trap mass spectrometer (Bremen, Germany) coupled to a surface-activated chemical ionisation (SACI) I ESI source and operated in the positive and negative ion mode.
• SACI surface-activated chemical ionisation
• the full scan spectra were acquired in the 40-3500 m/z interval for the non-targeted metabolomic/proteomic analysis to detect the analytes.
• the same m/z interval was maintained both for the discovery studies and for the studies on selective biomarkers in order to standardise the instrument response throughout the whole SAN 1ST study, principally in terms of scan speed.
• the parameters of the ion source were: ESI capillary voltage: 1500 V, SACI surface voltage: 47 V, drying gas: 2 L I min, nebulising gas: 80 psi, temperature: 40 °C.
• the tandem mass spectrometry (MS I MS) experiments on plasma samples were performed with collision-induced dissociation using helium as the collision gas.
• An ion trap was used to isolate and fragment the precursor ions (isolation windows ⁇ 0.3 m/z; collision energy: 30% of its maximum value, which was 5 V from peak to peak), and an Orbitrap mass analyzer was used to obtain fragments with an extremely accurate m/z ratio (resolution 15,000; m/z error ⁇ 10 ppm).
• the replication of the virus SARS-CoV-2 within a mixed bacterial culture was obtained using the “Brogna-Petrillo” method (application no. 102020000022519 of 24/03/2020).
• a mixed sample “A” of bacteria and the virus Sars-Cov2 was placed on a multi-potent culture medium for many bacterial species, including anaerobic ones.
• the concentrations of viral loads were measured with Luminex technology (S. A. Dunbar, “Applications of Luminex® xMAPTM technology for rapid, high-throughput multiplexed nucleic acid detection”, Clin. Chim. Acta Int. J. Clin. Chem., vol. 363, n. 1 , pp. 71-82, Jan.
• a SARS-CoV-2-negative multi-bacterial sample “B” was inoculated with the supernatant and virus of sample “A”, following centrifugation at 2400 G for 10 minutes.
• Sample “B” was specifically selected to be predisposed to inoculation, from faeces of a donor subject, negative for every virus. The cultures were conducted for 30 days.
• MAIdi-biotyper analyses were performed in order to better identify the bacterial replicative motility and try to identify the bacterial family that lends itself most to being an ideal substrate for viral replication (B. Karolski, L. O. B. Cardoso, L. H. Gracioso, C. A. O. Nascimento, and E. A. Perpetuo, “MALDI-Biotyper as a tool to identify polymer producer bacteria”, J. Microbiol. Methods, vol. 153, pp. 127-132, 2018, doi: 10.1016/j.mimet.2O18.09.01 ).
• the diagram shown in figure 1 reveals that, at 30 days, in sample “B”, negative for the presence of eukaryotic cells, a Sars-Cov2 viral replication of 1100% was present and in sample “A” that replication reached the threshold of 500%.
• the analysis revealed an exponential increase in viral replication in the bacterial culture of the two samples. Measurements were performed every day to assess the viral replication, using the Luminex technology system (Dunbar, 2006).
• the amino acid motif was identified in the protein sequences found, C — C-CC-C- of the conotoxins, sequences of snakelike phospholipases A2 and proteins like prothrombin (activating the coagulation factor Va), protein like bradykinin-C type natriuretic factor, and proteins belonging to the zinc metalloproteinase family.
• the control cases were negative for said proteins.
• the toxins were analysed using a mass spectrometry technique called SACI-CIMS.2.
• SACI-CIMS.2 a mass spectrometry technique
• the latter is based on an advanced approach that allows analytic signals to be filtered out from the instrument bottom (CIMS technology: Ionic mobility). In this manner the selectivity and sensitivity of the system increase exponentially.
• Figures 3-35 show the mass spectrometry graphs for some detected amino acid sequences which exemplify the numerous sequences of the toxins detected in the samples. All of the detected amino acid sequences showed a correspondence with the known toxins listed below or with fragments thereof. Identification of the toxins was achieved using the proteins stored in the UniProtKB.3 database and correspondence with the following known toxins were found:
• D2DGD8 LVLAIVLILM LVSLSTGAEE SGQEISMVGP
• RTVRRFCSDPPCRISNPESCGWEP SEQ ID N0:4
• ASKFVWSSCTLTAFANFSKTTSAACLKDTYRKQ (SEQ ID NO:23);
• VPPGGGRCKANQACTKGGNPGTCGFQYDLCLCLRN SEQ ID NO:24
• HLVAI SEQ ID NO:25
• CDAWCGSRGKKLSFGCAATCPKVNPGIDIECCSTDNCNPHPKLRP SEQ ID NO:27
• GKFMYILHGCPFQPWC SEQ ID NO:29
• GKFMNVLRRSGCPWEP (SEQ ID NO:32);
• A8YPR6 MFVSRLAASGLLLLSLLALSLDGKPLPQRQPHHIQPMEQKWLAPD APPLEQKWLAPDAPPLEQKWLAPAAPPLEQKWLAPDAPPMEQKWLAP DAPPMEQKWLAPDAPPMEQKWLAPDAAPLEQKWLA PDAPPMEQKWLAPDAPPMEQKWQPQIPSLMEQRQLSSGGTTALRQEL SPRAEAASGPAVVGGGGGGGSKAALALPKPPKAKGAAAATSRLMR DLRPDGKQASQKWGRLVDHDHDHHHHPGSSVGGGGGGGGGGAR RLKGLAKKGVAKGCFGLKLDRIGSMSGLGC (SEQ ID NO:33);
• amino acid sequences of the toxins detected in the samples showed a variable percentage of identity with the sequences SEQ ID NO:1 -53 or with fragments thereof.
• sequences of the toxins whose diagrams are shown in figures 3-35 showed a variable identity with the following amino acid sequences:
• the analysis was performed using an Ultimate 3000 HPLC system (Thermofisher, USA).
• the column used was a Phenomenex Kinetex PFP 50x4.1 mm 2.6 pm.
• the analyses were performed using a two-phase gradient: Phase A (H20+0.2% formic acid (HCOOH)) and Phase B (acetonitrile CH3CN).
• Phase A H20+0.2% formic acid (HCOOH)
• Phase B acetonitrile CH3CN
• Data acquisition took place with the aid of an HCT ULTRA mass spectrometer (Bruker, Bremen, Italy) provided with a partially octopolar field on the analyser.
• the ionisation source used was a SAC I.
• the protein fraction was extracted from the bacterial culture in the following manner:centrifugation of the supernatant, collection of the lightest fraction and filtration over a 0.22 pm bacteriological membrane. Extract in the absence of a denaturation process: Desalinisation. Precipitation with CH3CN solvent 1 :3. Resuspension with NH4HCO3 50 mmol buffer, pH 7.8 (refolding conditions). Stabiliser: 1% sorbic acid (antibacterial).
• the toxins were rendered immunogenic in the following manner: collection after filtration over a 0.22 pm bacteriological membrane. 8M urea was used at room temperature. Heating was avoided to prevent cyanylation of the lysins. Protein precipitation with CH3CN added in a 1 :3 ratio. Precipitate reconstituted with a 50 pm NH4HCO3 solution. Denaturing detergent: SDS. Stabiliser: 1% sorbic acid (antibacterial).
• the denaturation of the toxic preparations obtained from the supernatant of the bacterial cultures can also be carried out with conventional methods, including chemical denaturation: for example by treatment with chemical substances, including urea, guanidinium chloride and guanidinium thiocyanate.
• the protein molecule-denaturing property of these substances is due to their ability to transiently bind, through weak bonds, such as, for example, hydrogen bonds, the amino acid residues constituting the protein. Binding with a chaotropic agent, preferably thermodynamically rather than intramolecularly with other residues, or intermolecularly with water molecules, ensures that the protein is no longer capable of maintaining its three-dimensional structure and becomes denatured.
• the denaturation of the toxins can be carried out by thermal denaturation, both hot and cold. Hot denaturation must be rapid and not progressive in order to prevent a new recompacting of the protein/toxin.
• a further possible method is denaturation with formaldehyde: inactivation is achieved by means of Ramon’s method, developed 100 years ago but still used.
• the toxin is treated with a 4% o formaldehyde solution for about 30 days at a temperature of 37 °C, until reaching completed detoxification.
• the anatoxin is immunogenic (“formalinized diphtheria toxoid (anatoxin)”, am. j. public health n. y. n 1912, vol. 15, n. 12, pp. 1092-1093, Dec. 1925).
• the last step was simple inoculation into animal samples, wild-type mice, not genetically modified and not sacrificed.
• the overexpression of the ACE2 receptor highly discussed in the literature was not taken into consideration, deliberately and specifically, for the simple reason that the toxins were tested for the purpose of assessing whether their presence is associated with “SARS-CoV-2 virus-like” clinical effects and symptoms.
• the observations were fever, objective muscular weakness (understood as difficulty in moving), interstitial pneumonia (chest X-ray) and acts of dyspnoea/coughing, and an increase in the D-dimer.
• a limited area of the abdomen about 21 -2 cm2 was shaved. Prior to immunisation, the skin was hydrated for several minutes and then dried with dry fabric.
• the W-T group with the antidote was immunised with a volume of 30pL of the anatoxic solution prepared.
• the toxic pool was instead administered daily to the non-immunised W-T group for 5 days.
• a lethal toxic dose that of the tetanus toxin was taken as reference, i.e. ⁇ 2.5ng/kg (WHO 2010).
• WHO 2010 a lethal toxic dose
• a daily dose was administered until the appearance of symptoms, whereas in the immunised W-T mice administration began 4 weeks earlier to wait for the antibody concentration to increase.
• “Covid-19 like” symptoms appeared in the non-immunised group and upon cessation of the administration of the toxic pool, the anatoxic pool was administered and a regression of the symptoms was observed.
• no symptoms appeared at the time of administration of the toxic pool and up to 14 days thereafter.
• there is very ample literature regarding the safe use of anatoxins against toxins in order to immunise subjects.
• EXAMPLE 2 Study on mice concerning the effectiveness of antibodies according to the present invention against the toxins produced by cells in the presence of SARS-CoV-2.
• the toxins/proteins obtained, as indicated in example 1 , from bacterial cultures that replicate coronavirus were suitably ultracentrifuged with a refrigerated centrifuge and purified with a method analogous to the one described in example 1.
• Three toxins (selected as non-limiting examples among the many tested combinations), namely SEQ ID NO:52, SEQ ID NO:68 and SEQ ID NO:11 , were extracted by means of the sizeexclusion chromatography technique (acronym S.E.C. - also known as molecular sieve chromatography - a chromatographic method in which molecules in solution are separated based on their size, and in some cases molecular weight, by the bands obtained).
• the toxins can also be extracted by means of polyacrylamide gel electrophoresis. The concentration used was measured with the Bradford method (spectrophotometric method for protein quantification) in 8pg/ml.
• the proteins were inoculated at increasing doses in 15 selected mice (mouse strain- Balb/C): one toxin per group made up of 5 mice. The maximum dose reached was 40 ng/ml. Another five mice were used as the control group and they received sterile water subcutaneously. The mice were immunised at increasing doses until day 21 (figures 36-37). Polyclonal antibodies were recovered for each toxin by means of the affinity chromatography technique (dose recovered 3 pg/ml for each antibody).
• mice were inoculated with the above-mentioned toxins (study mice) and 10 mice were used as controls, in order to verify the functionality of the mixture obtained with the three polyclonal antibodies.
• the study mice received a toxic dose of a mixture of the three toxins, at a mean concentration for the mixture of 60ng/ml.
• the mixture of polyclonal antibodies (1 to 100 dilution in sterile water of the dose recovered for each antibody against its own toxin) was inoculated (mean time 36 hours after toxicological exposure).
• the study mice resumed their activity normally, whereas the control mice, which had received the same toxic dose of toxins but had been treated with the placebo (sterile water), did not resume their activity immediately.
• EXAMPLE 3 Study on volunteer patients to assess the preventive effectiveness of the toxins according to the invention against SARS-CoV- 2.
• the mixture of the three toxins mentioned in example 2 (0.5 ng/Kg in 1 ml of sterile saline solution) was administered to 10 healthy adult volunteer subjects (study group) with a negative SARS-CoV-2 molecular test. Another 10 volunteers received sterile saline solution (control group). 3 subjects in the study group and 5 in the control group showed reddening at the injection site.
• mice received repeated doses - intramuscularly - of polyclonal antibodies obtained from the group of immunised mice described in example 2 for up to 21 days.
• 3 anti-polyclonal antibody antibodies were obtained with the same methods described in example 2.
• These antibodies were used in the assay by means of the ELISA immunohistochemical technique to verify the presence or absence of the antibodies against the mixture of the three toxins present in the 10-volunteer study group and control group.
• the blood chemistry analyses showed only slight alterations of plasma pseudocholinesterase, equal to 1 .5 times less than the mean value (figure 38).
• the study group showed an antibody response towards the toxins (figure 39). Over 6 months of observation, it was found that the subjects in the study group never developed symptoms caused by SARS-CoV-2, despite the fact that 7 subjects out of 10 had been living together in the meantime with family members who were symptomatic and positive to SARS-CoV-2.
• EXAMPLE 4 Study on mice concerning the effectiveness of antibodies according to the present invention against the toxins produced by cells in the presence of B-CoV.
• the B-CoV was made to replicate simultaneously with the production of proteins of the bacteria in fertilised chicken eggs monitored after candling.
• a sample of bovine coronavirus was inoculated into the bacterial culture and a pool of toxins similar to those described in the present patent application was obtained (figure 40).
• the supernatant of the bacterial culture with B-CoV and several bacteria were introduced into some eggs (figure 41 ).
• the liquid of the amniotic cavity was drawn.
• TEM, mass spectrometry and rt-PCR tests revealed viral replication and a toxicological increase.
• protein denaturation, and reduction of the viral load the serum obtained was used to carry out the same procedure as in example 2.
• the sample of study animals had an increase in the concentrations of anti-B-CoV antibodies and antibodies against the 3 toxins of example 2. It was chosen to evaluate only these three toxins for purposes of non-exhaustive demonstration. Analogous procedures were carried out to derive the toxins produced by bacteria originating from the gut of cattle. Unlike in example 2, the whole toxic pool was used to immunise the mice. Only some of the antibodies were analysed. The example reproduced is identical to example 2 and the results are similar.
• EXAMPLE 5 Study on mice concerning the effectiveness of antibodies according to the present invention against the toxins produced by cells in the presence of SARS-CoV-2.
• the same example 2 was repeated with the other toxins described in the present patent application and comparable data were obtained.
• the toxins divided into three groups (Table 2), were tested making sure that at least one toxin of each of the three groups was present in every sample included in the study: that is, a toxin with the group C — C-CC--C- typical of conotoxins or three-finger toxins, plus a protein with a sequence similar to snake-like phospholipase A2, plus a toxin with sequence similar to proteins like prothrombin (activating the coagulation factor Va), protein like bradykinin-C type natriuretic factor or the proteins belonging to the zinc metalloproteinase or neurotoxin family.

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