WO2022061439A1 - Use of the infectious bronchitis virus (ibv), vaccine for immunizing mammals against coronavirus and method for immunizing mammals against coronavirus - Google Patents

Use of the infectious bronchitis virus (ibv), vaccine for immunizing mammals against coronavirus and method for immunizing mammals against coronavirus Download PDF

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WO2022061439A1
WO2022061439A1 PCT/BR2021/050410 BR2021050410W WO2022061439A1 WO 2022061439 A1 WO2022061439 A1 WO 2022061439A1 BR 2021050410 W BR2021050410 W BR 2021050410W WO 2022061439 A1 WO2022061439 A1 WO 2022061439A1
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ibv
sars
cov
vaccine
virus
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PCT/BR2021/050410
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French (fr)
Portuguese (pt)
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Maria Izabel Florindo Guedes
Mauricio Fraga Van TILBURG
Ney De Carvalho ALMEIDA
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Fundação Universidade Estadual Do Ceará - Funece
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    • 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
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • IBV INFECTIOUS BRONCHITIS VIRUS
  • VACCINE FOR IMMUNIZATION OF MAMMALIANS AGAINST CORONAVIRUS AND METHOD OF IMMUNIZATION OF MAMMALIANS AGAINST CORONAVIRUS
  • the present invention is located in the fields of biotechnology, immunology and the worldwide effort against COVID-19. More specifically, it discloses a vaccine preparation for medical and public health purposes, based on the use of avian infectious bronchitis virus for the immunization of mammals, including humans, against coronavirus, SARS-CoV-2 and/or related viruses.
  • the avian coronavirus (IBV) causing Infectious Bronchitis of Chickens (IBV) was employed for the preparation of vaccines for the immunization of mammals against SARS-CoV 2/COVID-19, with surprising results.
  • the vaccine against SARS-CoV2 comprises the IBV in any state (live attenuated, killed, with only its isolated or associated molecular structures), and it has been shown to be effective in immunizing mammals against SARS-CoV 2, whether it be humoral or cellular.
  • the results obtained in mammals immunized with the vaccine comprising the IBV showed satisfactory activity of the neutralizing/inhibitory action of the antibodies against the SARS-CoV 2 virus in cell culture, denoting that anti-IBV antibodies surprisingly also work in the protection against COVID-19. .
  • the infectious bronchitis virus was identified in the United States by Schalk and Hawn and characterized as an RNA virus belonging to the Coronaviridae family group III (delta). Said virus has a helical capsid and a viral envelope, and causes a disease, mainly, of respiratory involvement of an acute nature.
  • SARS-CoV-2 severe acute respiratory syndrome 2
  • COVID 19 coronavirus
  • US20040265339A1 , US4500638 etc. from the European and Asian continents (CN1 1 1084150A, KR20200073355A, KR20200043742 A, etc.), from Brazil (PI0407948-5) describe the history of the use of the vaccine against avian infectious bronchitis and its various forms of production and viability in the fight against the referred pathology and always in birds, notably in chickens, but never in mammals.
  • avian infectious bronchitis vaccine virus live and attenuated
  • this approach provides benefits in in relation to dead viruses, without the risks of live viruses that cause disease in humans, even if they are attenuated.
  • the use of animal viruses in human vaccines, especially the infectious bronchitis virus of chickens (IBV), has never been used in mammals for any immunization purpose.
  • the present invention changes this scenario.
  • the present invention solves the problems of the prior art from the use of infectious bronchitis virus of chickens (IBV) for the preparation of a vaccine for immunization against SARS-CoV2 and related viruses in mammals.
  • IBV infectious bronchitis virus of chickens
  • the present invention provides a vaccine comprising at least one infectious bronchitis virus (IBV) and a pharmaceutically acceptable carrier or diluent for immunizing mammals against coronavirus, SARS-CoV-2 and/or related viruses .
  • IBV infectious bronchitis virus
  • the present invention presents the use of infectious bronchitis virus (IBV) for the manufacture of a vaccine for the protection of mammals against coronavirus, SARS-CoV-2 and/or related viruses.
  • IBV infectious bronchitis virus
  • the present invention provides a method of immunizing mammals against coronavirus, SARS-CoV-2 and/or related viruses.
  • Figure 2 light absorbance values in the 450 nm spectrum detected in an ELISA test for the detection of antibodies produced in mice against IBV on day 45 of the experiment.
  • GS subcutaneous group
  • GO oral group
  • GN nasal group
  • CONTR CONTR - control group.
  • FIG. 3 Evidence of viral protein bands with similar molecular weights against both SARS-COV 2 and IBV. Approximately 84kDa, equivalent to the envelope protein S2. In the upper highlighted bands proteins with molecular weights equivalent to Spike proteins (180KDa) of both viruses are identified. Proteins with molecular weights equivalent to spike 1 or S1 proteins (approx. 1 10KDa) are identified in the middle bands and proteins with molecular weights equivalent to spike 2 or S2 proteins (approx. 84KDa) are identified in the lower bands.
  • FIG. 4 Viral serum neutralization test - sample of SARS-Cov 2 infection in monolayer of Vero cells, in violet color. After three days of infection, there was the formation of 27 circular spaces in the monolayer, indicating cellular destruction caused by the SARS-CoV 2 infection.
  • Figure 5 Viral serum neutralization test - negative sample of SARS-Cov 2 in monolayer of Vero cells, in violet color. After three days of infection, there was no formation of circular spaces in the monolayer, indicating no cell destruction due to the absence of SARS-CoV 2 infection - negative control.
  • FIG. 6 Viral seroneutralization test - positive sample against SARS-COV 2 in a monolayer of Vero cells, in violet color, seroneutralized by anti-SARS-CoV2 antibodies produced in a seropositive human. After three days of infection, there was no formation of spaces circular in the monolayer, indicating the non-destruction of cells by viral seroneutralization by SARS-CoV 2 - positive control.
  • FIG. 7 Viral seroneutralization test - positive sample for SARS-CoV 2 in a monolayer of Vero cells, in violet color, seroneutralized by anti-IBV antibodies produced in mice immunized with the vaccine for IBV subcutaneously. After three days of infection, there was the formation of 5 circular spaces in the monolayer, indicating very little cell destruction and the effectiveness of approximately 82% of viral seroneutralization of SARS-CoV 2 by anti-IBV antibodies - Sample.
  • Figure 8 Image of the serum test of mice immunized with IBV orally (28 days group) by western blotting technique in membrane transferred with the heat-inactivated SARS-CoV 2 viral proteins. Surrounded by the highlighted circle in the figure is a protein of molecular weight equivalent to that of the S1 protein (approx. 110KDa).
  • Figure 9 demonstrates a comparative genomic study between the avian infectious bronchitis virus and SARS-CoV 2, through the use of data contained in the National Center for Biotechnology Information (NCBI) by the BLAST (Basic Local Alignment Search Tool) software that showed similarities greater than 50%.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • Figure 10 (a) and (b) Alignment of the sequence of 4 strains of IBV and 4 strains of SARS-CoV-2, with red arrows indicating positions 484 and 501 of the receptor binding domain (RBD) of the SARS-CoV-2.
  • the asterisk indicates the conservation of the residue
  • the colon indicates a substitution by a similar residue and the point indicates a low similarity between the residues
  • FIG 11 (a)-(i) IBV RBD (blue cartoon) bound in different neutralizing antibodies nAbs (surfaces), (j) Comparison of the binding energies of the IBV RBD (SARS-CoV-2) with the antibodies. were used the codes 101 - 1 1 1 as it is still an ongoing research.
  • FIG. 13 Serum quantification of glucose, total cholesterol and triglycerides levels of mice immunized with IBV by subcutaneous, oral and nasal routes.
  • SC Subcutaneous immunization route.
  • Figure 14 Quantification of serum levels of urea, creatinine, aspartate aminotransferase and alanine aminotransferase of mice immunized with IBV by subcutaneous, oral and nasal routes.
  • SC Subcutaneous immunization route.
  • the present invention provides the use of infectious bronchitis virus of chickens for the production of a vaccine for the immunization of mammals against SARS-CoV2.
  • IBV from the English infectious bronchitis virus, denotes the cause of infectious bronchitis in chickens, belongs to the genus Gammacoronavirus, family Coronaviridae.
  • SARS-CoV2 pictured here can also be identified as COVID 19, Coronavirus and related.
  • the present invention describes the use of the vaccine against infectious bronchitis of chickens (IBV) as a model of immunization in mammals against SARS-CoV2.
  • the present invention makes use of the virus against IBV as a way to ensure an antibody response (humoral) and effective in mammals against COVID 19 (Figure 7).
  • a vaccine comprising at least one infectious bronchitis virus (IBV) and a pharmaceutically acceptable carrier or diluent for the immunization of mammals against coronavirus, SARS-CoV-2 and/or related viruses.
  • IBV infectious bronchitis virus
  • the present invention presents the use of infectious bronchitis virus (IBV) for the manufacture of a vaccine for the protection of mammals against coronavirus, SARS-CoV-2 and/or related viruses.
  • IBV infectious bronchitis virus
  • the IBV is live and attenuated.
  • the IBV is from the Massachusetts H120 strain with NCBI reference genetic sequence: NC_001451.1 which comprises a gene encoding the spike protein.
  • the vaccine comprises an adjuvant.
  • the vaccine is administered orally, including sublingually; nasal, for example, by thick drop, sprinkling, inhalation or spray; eyepiece; subcutaneous or intramuscular; with or without the use of adjuvants.
  • the vaccine comprises viral concentration of approximately 400 to approximately 4000 viruses per dose for application to humans.
  • the vaccine comprises producing anti-IBV antibodies that bind to the RBD portion of the spike protein of the virus.
  • the present invention provides a method of immunizing mammals against coronavirus, SARS-CoV-2 and/or related viruses, which comprises administering a vaccine containing at least one infectious bronchitis virus (IBV) and a pharmaceutically acceptable carrier or diluent.
  • a vaccine containing at least one infectious bronchitis virus (IBV) and a pharmaceutically acceptable carrier or diluent.
  • the present invention provides several surprising technical advantages and effects. It provides the use of an already known vaccine against IBV for the immunization in mammals against SARS-CoV2 in an effective and safe way. It facilitates the administration of the vaccine, which can be, among others, intranasally (drops or spray). It also provides self-application, being practical from a logistical point of view, not depending on trained applicators.
  • the vaccine of invention has a low production cost and its production is done with known technology.
  • the surprising new use of infectious bronchitis virus in chickens has been shown to be effective in protecting mammals against SARSCov2 infection. In the present invention, even though the vaccine virus is live and attenuated, it does not affect mammals and therefore does not make them sick, but produces an effective immune response against SarsCov2/COVID19.
  • the virus to be used for the production of the vaccine is selected from the group comprising: Arkansas, Connecticut, Delaware and Massachusetts serotypes, including Ma5 and M41, GA variants, California variants, D207 variants ( D274), D212 (D1466), variant 4/91 (793B), Italian-02, QX, D388, and/or BRI serotypes. All such serotypes are known in the art and readily available to one skilled in the art.
  • the virus used is the prototype of all coronaviruses, that is, a coronavirus of the gamma group, therefore a "relative" of SARS-CoV2.
  • Live and weakened (attenuated) complete virus of the Massachussetts H120 strain with NCBI reference genetic sequence: NC_001451.1 was used.
  • the American Type Culture Collection (ATCC) makes available the prototype of the Massachusetts serotypes - the M41 , code VR-21 , which after successive passages in embryonated eggs or cell cultures can form the H120, as well as the egg-adapted serotype - code VR-22, and of serotypes Arkansas 99 (code VR-841) and Connecticut A5968 (code VR-817).
  • the preparation of the vaccine involves the use of the complete killed virus or portions of this virus, either only using the genetic material that defines the Spike protein or any other that has immunogenic power, or even as a viral vector for express immunogenic proteins of the SARS-CoV2 itself.
  • Virus-like particles [0060]
  • Virus-like particles are comparable to viruses, but do not contain the genetic information of viruses. They correspond to an empty shell that presents a coronavirus protein. The absence of genetic information negates the risk of viral replication and propagation.
  • a virus that is not pathogenic to humans for example an adenovirus
  • a coronavirus protein for example an adenovirus
  • Viral vectors expressing foreign proteins are widely used to induce a strong immune response against various pathogens, including viruses.
  • nucleic acid molecule encoding a protein from a coronavirus, for example the SARS-CoV2 spike protein, optionally formulated with liposomes, is administered to an individual.
  • Nucleic acid is often modified to improve the amount and duration of expression of the protein it encodes.
  • Nanoparticles can be organic based, for example liposomes or polymeric nanoparticles; or inorganic base, such as gold nanoparticles.
  • Antigen presenting cells [0068] Antigen presenting cells
  • antigen presenting cells including macrophages and dendritic cells, are designed to present coronavirus protein fragments on their surfaces to activate immune cells.
  • Adjuvant vaccines [0070] Adjuvant vaccines:
  • SARS-CoV 2 was identified by the RT-PCR technique following the following protocol:
  • the SARS-COV2 virus was extracted from the material using a commercial viral RNA extraction kit (QIAamp Virai RNA Mini Kit - ID: 52906), following the protocol recommended by the manufacturer.
  • the samples were normalized to 100 ng so that the RNA synthesis could be performed using a commercial kit (High-capacity cDNA Reverse Transcription Kit with RNAse inhibitor - Ref: 4374966) and the programming adjustments in the thermocycler with the following parameters: 25°C per 10 minutes, 37°C for 2 hours and 85°C for 5 minutes.
  • the material was diluted to 100 ml with nuclease-free water and then PCR amplification was performed with a commercial kit (GoTaq qPCR Master Mix - Ref: A6002) using the primers indicated by the Center of Disease Control and Prevention (CDC) for diagnosis of the disease (available at: https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primerprobes.html) . This was followed by amplification and electrophoresis in 1% agarose, in which 10x loading buffer was added to the sample at a dilution of 1/5 and the run was performed at 80V; 200 mA; 30 minutes.
  • CDC Center of Disease Control and Prevention
  • the gel was then submerged in 50 ml of TAE solution with the addition of SYBR (SYBR Safe DNA gel strain - Ref: S33102) at a dilution of 1:10,000 for 30 minutes and finally analyzed in a transilluminator.
  • SYBR SYBR Safe DNA gel strain - Ref: S33102
  • the ELISA test was performed according to the steps described below: [0082] First step: 40 female Swiss mice were immunized, divided into 4 groups of 10 individuals each, according to the route of administration used: subcutaneous, oral, intranasal and control, with 100ml doses at a low concentration of 1mg/100ml of vaccine IBV diluted in a dilution solution without decontaminating agents. At this stage, in addition to the first dose, given on day 1, two more boosters were given: on day 15 and on day 28. The immunization of the subcutaneous group was added, in the first dose, with the use of Complete Freund's Adjuvant (FCA).
  • FCA Complete Freund's Adjuvant
  • the use of Incomplete Freund's Adjuvant (FIA) was added and in the last booster (28 days), adjuvants were not used.
  • the dose was directly administered into the oral cavity of the animals, while in the intranasal group, this administration was done by spraying.
  • the control group remained without any contact with the immunizing agent throughout the experimental period.
  • the sera of the animals to obtain the antibodies were collected on days 28, before the second booster and on day 45.
  • the antibodies obtained were destined to the execution of the ELISA technique to compare the titers of IgG, lgG1 and lgG2a and seroneutralization, and kept frozen at -20°C.
  • Second stage 40 female Swiss mice were immunized, divided into 3 groups, according to the route of administration used, with equal numbers of individuals: subcutaneous, oral and intranasal, with doses of 100ml in a concentration of 10mg /100ml of vaccine IBV diluted in a dilution solution without decontaminating agents.
  • a further booster was performed on day 15.
  • the use of complete Freund's adjuvant was added to the first dose and to the booster dose (15 days) there was no use was made of adjuvant.
  • the protocol of the previous step was followed.
  • Sera from animals to obtain antibodies were collected on days: 7, 15 (before the booster dose), 21, 28 and 45 days.
  • the antibodies obtained were sent to the ELISA technique for comparisons of IgG titer concentration, western blotting, in addition to serum neutralization and kept at -20°C.
  • mice immunized by the subcutaneous route responded satisfactorily to the production of antibodies against IBV without showing any apparent symptoms of the disease that affects the birds, in both harvests (days 28 and 45), with a higher result in the harvest of the day 45 of the experiment.
  • Electrophoresis showed the presence of a band close to the 80kD marker, similar to the weight obtained by the S2 protein of SARS-CoV 2, the supposed epitope responsible for the infection (figure 3).
  • SARS-Cov 2 obtained: Following identification and confirmation by RT-PCR, it was isolated in a monolayer of vero cells using suitable cell culture media, plus antibiotics and probiotics and conditioned at -80 s C .
  • SARS-CoV 2 seroneutralization by anti-IBV antibodies After viral isolation, where 7.8x104 virus/mL was obtained, seroneutralization was performed (Golden test) of the SARS-CoV 2 sample obtained by isolation in 12-well plates containing a monolayer of Vero cells plus gel ( Figures 4, 5, 6 and 7), and an 82% efficacy was obtained in the neutralization of SARS - CoV 2 by the polyclonal anti-IBV antibodies produced in mice subcutaneously ( Figure 7) (since it was the route that proved to be the most effective for the immunization of mice), possibly through its binding with the virus in the RBD portion of the spike protein.
  • the present invention is characterized by using the chicken coronavirus (IBV) as a model of immunization in mammals against SARS-CoV 2 in a unique and unprecedented way, in order to obtain a significant and effective immunization.
  • IBV chicken coronavirus
  • the virus used in this embodiment is that present in the commercial vaccine against infectious avian bronchitis (IBV).
  • IBV infectious avian bronchitis
  • the production of the vaccine can be carried out according to one of the technologies contained in the state of the art, either with dead, weakened virus or in parts (proteins, genetic material%) in its most different strains or serotypes, it is not limited to the Massachusetts H120 only, due to the similarity between the IBV virus and SARS-CoV2 in their great propensities to form mutations.
  • the present invention relates to the use of a vaccinal virus (IBV) used, until now, only in birds, as a model of use in mammals in the immunization against SARS-CoV 2.
  • the routes of administration for the vaccinal virus, according to the present invention, the routes are: oral, including sublingual; nasal, whether by thick drop, sprinkling, inhalation or spray; ocular or more invasive routes such as subcutaneous or intramuscular, with or without the use of adjuvants.
  • mice reacted with proteins considered specific for SARS-CoV2, more specifically the spike protein 1 . This was evidenced in a Western blot test, verified through detection “spots” on the membrane that contained the SARS-CoV2 virus incorporated ( Figure 8).
  • a total of 11 antibodies complexed to the spike protein of SARS-CoV-2 were retrieved from the Protein Data Bank - PDB, with the aid of Pymol, 2.5 software (https://pymol.Org/2/). Each antibody was renamed and saved in pdb format preserving the light and heavy chains.
  • the antigen selected for study was the spike protein of infectious bronchitis coronavirus - IBV (PDB: 6CV0), region of the RBD.
  • MFCC Molecular Fragmentation with Conjugate Caps
  • the collection was carried out in loco, in a room adapted for this purpose, in conditions necessary for a better convenience for both the collector and the person who would have the sample collected.
  • PPE were used.
  • the puncture site was cleaned with 70% alcohol.
  • puncture coagulation was produced with the aid of appropriate dressings.
  • Approximately 5 ml of blood was collected from each person to be tested.
  • As for PCR collection all samples were collected either via the nasal or pharyngeal route, at the discretion of the individual to be collected.
  • the swabs were disposable and after collection they were collected in a collection solution and properly identified.
  • Vero E6 cells (6 x 10 5 per well) were seeded in 12-well plates and incubated at 37°C in L-15 culture medium with 2% fetal bovine serum and 1% penicillin/streptomycin antibiotics (GIBCO). The following day, 60 PFU of SARS-CoV-2 were incubated with mouse sera diluted in L-15 medium (1:100) and previously heated at 56°C for 30 minutes to inactivate the complement system. The samples and the positive control (virus only) were incubated at 37°C for 1 hour under gentle agitation. The virus-serum mixture was added, in duplicate, to pre-seeded Vero E6 cells at 90-100% confluence.
  • GEBCO penicillin/streptomycin antibiotics
  • Serum levels of glucose, total cholesterol, triglycerides, urea, creatinine, alanine aminotransferase and aspartate aminotransferase were measured using biochemical test kits (BIOCLIN®), as directed by the manufacturer. For this purpose, the absorbances were evaluated through the Synergy 2 (Biotek®) microplate reader.
  • histopathological analyzes were performed on various organs including lung and secondary lymphoid organs (spleen and lymph nodes).
  • the material under study comes from lung samples fixed in buffered formalin, embedded in paraffin, cut with a microtome with a thickness of 4 pm and subjected to automatic staining in Hematoxylin & Eosin. Stained slides are examined by a single pathologist for signs of inflammatory lesion.
  • the paraffin blocks containing representative lung samples are used to remove a cylindrical fragment (donor block) using a 2 mm diameter needle (Quick Ray, UNITMA®) and transfer it to a 60 wells.
  • the receptor block is organized following the sequence: 4 cases of positive controls in the vertices, composed of appropriate tissues indicated in the antibody package insert; added from 28 cases, in duplicate.
  • the basic steps of the immunohistochemical process are:
  • an antigen recovery treatment is applied to expose the epitopes through heat, using antibodies anti-TNF-alpha, IL-1 beta primers.
  • the histological pieces are deparaffinized and then hydrated. After hydration, the pieces are immersed in 0.1 M citrate buffer (pH 6.0) and heated at approximately 100oC for 15 min, followed by cooling at room temperature for 20 min. Then they are washed in PBS. Blocking of endogenous peroxidase is performed with 3% hydrogen peroxide solution for 15 min. After washing with PBS, the primary antibodies are diluted in PBS - 5% BSA and the samples are incubated for 12h. Then the slides are washed with PBS and incubated (30 min) with the biotinylated secondary antibody diluted in 5% PBS-BSA.
  • Th1 profile is evaluated in blood and spleen samples obtained from mice and Syrian hamsters by immunophenotyping by flow cytometry. Briefly, after collection of samples and lysis of red blood cells, antigens for identification of T cells (CD4+ and CD8+), the lymphocytes are labeled with antibodies conjugated to fluorochromes. Markers for antigens such as CD3, CD4, CD8, T-bet and CD1 19 are used to identify subpopulations.
  • region (i) contains practically the three hot spots of interaction between the SARS-CoV-2 RBD with the human ACE2 binding site, according to the interaction energy map in Figure 10 (c) obtained in quantum biochemistry simulations in studies carried out by the group and which are in the preparation phase of the article.
  • these two regions are shown to be potential targets for antibodies that block the cell invasion mechanism by the virus by binding to the SARS-CoV-2 RBD and, therefore, competing with the ACE2 binding site.
  • Table 1 Analysis of biochemical parameters and markers of liver and kidney damage in the sera of people who work in poultry, handling IBV in the processes of vaccination of birds, in order to verify possible damage caused by the IBV. The collections were performed in the morning, without fasting recommendation.
  • AST Aspartate aminotransferase
  • ALT Alanine Aminotransferase
  • the vaccine composition of the present invention for human application comprises viral concentration of approximately 400 to approximately 4000 viruses per dose.

Abstract

The present invention pertains to the fields of biotechnology, immunology and the world effort against COVID-19. Caused by a recently discovered virus from the Coronaviridae family, known as SARS-CoV-2 and the cause of the current coronavirus (COVID-19) pandemic, the disease is characterized by severe acute respiratory syndrome as the main pathological process. In the present invention, avian coronavirus (IBV), which causes infectious bronchitis (IBV) in chickens, a disease with a certain degree of similarity to COVID-19 and which has been studied for some time, was used for preparing vaccines for immunizing mammals against SARS-CoV-2/COVID-19, with surprising results. In the present invention, the vaccine against SARS-CoV-2 comprises IBV in any state (live attenuated, killed, or only isolated or combined molecular structures thereof) and has proven effective in the immunization of mammals against SARS-CoV-2, whether humoral or cellular. The results obtained in mammals immunized with the vaccine comprising IBV have shown satisfactory neutralizing/inhibitory activity of antibodies against the SARS-CoV-2 virus in cell culture, indicating that anti-IBV antibodies surprisingly also protect against COVID-19.

Description

Relatório Descritivo de Patente de Invenção Invention Patent Descriptive Report
USO DE VÍRUS DA BRONQUITE INFECCIOSA (IBV), VACINA PARA IMUNIZAÇÃO DE MAMÍFEROS CONTRA CORONAVÍRUS E MÉTODO DE IMUNIZAÇÃO DE MAMÍFEROS CONTRA CORONAVÍRUS USE OF INFECTIOUS BRONCHITIS VIRUS (IBV), VACCINE FOR IMMUNIZATION OF MAMMALIANS AGAINST CORONAVIRUS AND METHOD OF IMMUNIZATION OF MAMMALIANS AGAINST CORONAVIRUS
Campo da Invenção Field of Invention
[0001] A presente invenção se situa nos campos da biotecnologia, imunologia e do esforço mundial contra a COVID-19. Mais especificamente, revela uma preparação vacinai com fins médicos e de saúde pública, baseada no uso do vírus da bronquite infecciosa aviária para a imunização de mamíferos, inclusive em humanos, contra coronavírus, SARS-CoV-2 e/ou vírus relacionados. Na presente invenção, o coronavírus aviário (IBV) causador da Bronquite Infecciosa das Galinhas (IBV) foi empregado para a preparação de vacinas para a imunização de mamíferos contra o SARS-CoV 2/COVID-19, com resultados surpreendentes. Na presente invenção, a vacina contra o SARS-CoV2 compreende o IBV em qualquer estado (vivo atenuado, morto, com apenas suas estruturas moleculares isoladas ou associadas), e se mostrou eficaz na imunização de mamíferos contra o SARS-CoV 2, seja ela humoral ou celular. Os resultados obtidos em mamíferos imunizados com a vacina compreendendo o IBV mostraram satisfatória atividade da ação neutralizante/inibitória dos anticorpos contra o vírus SARS-CoV 2 em cultivo de células, denotando que anticorpos anti-IBV surpreendentemente funcionam também na proteção contra a COVID-19. [0001] The present invention is located in the fields of biotechnology, immunology and the worldwide effort against COVID-19. More specifically, it discloses a vaccine preparation for medical and public health purposes, based on the use of avian infectious bronchitis virus for the immunization of mammals, including humans, against coronavirus, SARS-CoV-2 and/or related viruses. In the present invention, the avian coronavirus (IBV) causing Infectious Bronchitis of Chickens (IBV) was employed for the preparation of vaccines for the immunization of mammals against SARS-CoV 2/COVID-19, with surprising results. In the present invention, the vaccine against SARS-CoV2 comprises the IBV in any state (live attenuated, killed, with only its isolated or associated molecular structures), and it has been shown to be effective in immunizing mammals against SARS-CoV 2, whether it be humoral or cellular. The results obtained in mammals immunized with the vaccine comprising the IBV showed satisfactory activity of the neutralizing/inhibitory action of the antibodies against the SARS-CoV 2 virus in cell culture, denoting that anti-IBV antibodies surprisingly also work in the protection against COVID-19. .
Antecedentes da Invenção Background of the Invention
[0002] Desde que iniciada no século XVIII, a ideia da prática de imunização, suscitou a possibilidade de os homens lidarem mais facilmente com as diversas enfermidades que assolavam as civilizações, evitando a “possível” grande mortalidade e fazendo com que a humanidade conseguisse a permanência de sua existência. A partir dos experimentos do médico inglês Edward Jenner, que utilizou inoculado de pústulas de varíola bovina em uma criança e confirmou que esta não adoeceu, após ter observado que mulheres que ordenhavam vacas eram mais resistentes (imunes) à infecção causada pelo agente etiológico da varíola humana chegou à conclusão de que essas pessoas se tornavam imunes à varíola. A doença, chamada de cowpox, assemelhava-se à varíola humana pela formação de pústulas. Tal fato se deu a partir da experiência, em 1796. Surgiu aí a primeira vacina, que foi divulgada no ano de 1798 no trabalho intitulado “Um Inquérito sobre as Causas e os Efeitos da Vacina da Varíola". Assim, a partir deste estudo várias buscas por novos achados e técnicas de produção vacinai foram descritos para as mais diversas enfermidades que assolam os seres humanos, animais e plantas. [0002] Since it started in the 18th century, the idea of the practice of immunization, raised the possibility for men to deal more easily with the various diseases that plagued civilizations, avoiding the "possible" great mortality and making humanity achieve the permanence of its existence. Based on the experiments of the English physician Edward Jenner, who used inoculated from cowpox pustules in a child and confirmed that he did not get sick, after observing that women who milked cows were more resistant (immune) to the infection caused by the etiological agent of smallpox, he came to the conclusion that these people became immune to smallpox. The disease, called cowpox, resembled human smallpox in the formation of pustules. This fact was based on the experience in 1796. The first vaccine appeared there, which was published in the year 1798 in the work entitled "An Inquiry into the Causes and Effects of the Smallpox Vaccine". searches for new findings and vaccine production techniques have been described for the most diverse diseases that plague humans, animals and plants.
[0003] Em 1931 , o vírus da Bronquite Infecciosa das Galinhas (em inglês, infectious bronchitis virus (IBV)) foi identificado nos Estados Unidos por Schalk e Hawn e caracterizado como um vírus RNA pertencente à família Coronaviridae grupo III (delta). Referido vírus possui um capsídeo helicoidal e um envelope virai, e provoca uma doença, principalmente, de acometimento respiratório de natureza aguda. [0003] In 1931, the infectious bronchitis virus (IBV) was identified in the United States by Schalk and Hawn and characterized as an RNA virus belonging to the Coronaviridae family group III (delta). Said virus has a helical capsid and a viral envelope, and causes a disease, mainly, of respiratory involvement of an acute nature.
[0004] Identificada pela primeira vez em Wuhan, República Popular da China, em 12 de dezembro de 2019 - com o primeiro caso tendo sido reportado em 31 de dezembro do mesmo ano - a síndrome respiratória aguda grave 2 (SARS- CoV-2) é uma doença respiratória aguda causada por coronavirus (COVID 19) que apresenta certo grau de similaridade na sintomatologia da bronquite infecciosa das galinhas (IBV). [0004] First identified in Wuhan, People's Republic of China, on December 1-2 , 2019 - with the first case reported on December 31 of the same year - severe acute respiratory syndrome 2 (SARS-CoV-2 ) is an acute respiratory disease caused by coronavirus (COVID 19) that presents a certain degree of similarity in the symptoms of infectious bronchitis of chickens (IBV).
[0005] Como mostrado em várias revisões científicas, desde meados da década de 50 do século passado, o uso da vacina de vírus vivo atenuado da bronquite infecciosa aviária na prática de imunização de plantéis de galinhas foi o que se mostrou mais eficaz no combate à enfermidade nos animais, principalmente quando se relaciona custo de produção e operação ao efeito protetor. [0005] As shown in several scientific reviews, since the mid-50s of the last century, the use of the live attenuated virus vaccine of avian infectious bronchitis in the practice of immunization of chicken flocks was the most effective in combating disease in animals, especially when the cost of production and operation is related to the protective effect.
[0006] Grande parte das patentes para perspectivas de desenvolvimento vacinai na atualidade é relacionada à utilização de porções das partículas virais capazes de serem reconhecidas em exclusividade pelas células de defesa do organismo invadido, no intuito de que haja produção, por estas células, de anticorpos capazes de neutralizar especificamente o agente etiológico e, por consequência, inibir o surgimento da doença. [0006] Most patents for vaccine development perspectives today are related to the use of portions of viral particles capable of to be recognized exclusively by the defense cells of the invaded organism, in order for these cells to produce antibodies capable of specifically neutralizing the etiological agent and, consequently, inhibiting the onset of the disease.
[0007] Desde o início do século passado que pesquisadores se utilizam de agentes etiológicos de doenças animais, seja em estado morto, seja vivo atenuado (enfraquecido) ou em “porções”, na elaboração de vacinas para o combate a enfermidades em humanos, tal como acontece até hoje com o uso da BCG para tuberculose. Semelhante abordagem é feita no caso das vacinas para o combate à gripe aviária, gripe suína, entre outras. [0007] Since the beginning of the last century, researchers have been using etiological agents of animal diseases, either in a dead state, be it live attenuated (weakened) or in "portions", in the elaboration of vaccines to combat diseases in humans, such as as is the case today with the use of BCG for tuberculosis. A similar approach is taken in the case of vaccines to combat avian flu, swine flu, among others.
[0008] Patentes depositadas em vários escritórios de patentes, incluindo os Estados Unidos da América (US4645665, US20140141043A1 ,[0008] Patents filed with various patent offices, including the United States of America (US4645665, US20140141043A1,
US20040265339A1 , US4500638 etc.), dos continentes europeu e asiático (CN1 1 1084150A, KR20200073355A, KR20200043742 A, etc.), do Brasil (PI0407948-5) descrevem o histórico da utilização da vacina contra bronquite infecciosa aviária e as suas mais diversas formas de produção e viabilização no combate à referida patologia e sempre em aves, notadamente em galinhas, mas nunca em mamíferos. US20040265339A1 , US4500638 etc.), from the European and Asian continents (CN1 1 1084150A, KR20200073355A, KR20200043742 A, etc.), from Brazil (PI0407948-5) describe the history of the use of the vaccine against avian infectious bronchitis and its various forms of production and viability in the fight against the referred pathology and always in birds, notably in chickens, but never in mammals.
[0009] Relatos científicos que remontam o ano de 1968 e subsequentes afirmam que indivíduos que manipulavam aves que foram imunizadas contra IBV apresentaram anticorpos contra o IBV. Como a bronquite infecciosa aviária não pode acometer humanos, já que esta doença das aves não é uma zoonose, nunca foram relatados casos da doença em humanos. [0009] Scientific reports dating back to 1968 and subsequent states that individuals who handled birds that were immunized against IBV had antibodies against IBV. As infectious avian bronchitis cannot affect humans, as this disease of birds is not a zoonosis, cases of the disease in humans have never been reported.
[0010] Diversas vacinas estão sendo desenvolvidas mundo afora na intenção de acabar com ou minimizar os efeitos da pandemia provocada pelo coronavirus da síndrome aguda respiratória do tipo 2 (SARS-CoV 2), sendo a enfermidade designada de doença por coronavirus 19 (COVID 19). Até o momento, todas as iniciativas dos mais diversos laboratórios e nas mais diferentes fases de testes pré-clínicos e clínicos usam o SARS-CoV 2 ou porções do SARS-CoV 2. Espera- se que estas vacinas possam se tornar realidade com a maior brevidade possível no combate a esta grave enfermidade que assola a humanidade. [0010] Several vaccines are being developed around the world with the intention of ending or minimizing the effects of the pandemic caused by the acute respiratory syndrome coronavirus type 2 (SARS-CoV 2), the disease being called coronavirus disease 19 (COVID 19). ). To date, all initiatives from the most diverse laboratories and in the most different phases of pre-clinical and clinical trials use SARS-CoV 2 or portions of SARS-CoV 2. It is hoped that these vaccines can become reality with the greatest possible possible brevity in the fight against this serious disease that plagues humanity.
[0011 ] Entretanto, boa parte dos pesquisadores acredita que o SARS-CoV 2 tem alto poder mutagênico e que tais mutações ocorrem após dois anos da presença de vírus circulante, o que provocaria uma nova “onda” de infecções, com possíveis agravamentos. Nações já anteciparam que indivíduos testados positivamente contra a COVID 19 sofreram nova infecção, a qual se confirmada for, derruba toda a retórica temporal do processo de mutagênese virai. [0011 ] However, most researchers believe that SARS-CoV 2 has high mutagenic power and that such mutations occur after two years of the presence of circulating virus, which would provoke a new “wave” of infections, with possible worsening. Nations have already anticipated that individuals tested positively against COVID 19 have suffered a new infection, which if confirmed, overturns all temporal rhetoric of the viral mutagenesis process.
[0012] Semelhante fenômeno ocorre nas galinhas, as quais, dependendo da localidade em que são criadas, são acometidas pelos mais diferentes sorotipos de IBV, até mesmo mais de um. Porém, adota-se no esquema vacinai das galinhas normalmente uma vacina cujo sorotipo é o mais frequente e que dá a melhor proteção cruzada contra os demais e sempre fazendo reforço vacinai, a fim de deixar ativo o sistema imunológico destas, com níveis de anticorpos garantidores de proteção contra a patologia. [0012] A similar phenomenon occurs in chickens, which, depending on the location where they are raised, are affected by the most different serotypes of IBV, even more than one. However, a vaccine is normally adopted in the chicken vaccination schedule, the serotype of which is the most frequent and which gives the best cross-protection against the others. protection against pathology.
[0013] Portanto, novas vacinas que visam ou que mostrem uma proteção promovida por anticorpos apenas para o SARS-CoV 2 circulante nesta situação pandémica poderão cair em desuso caso seja comprovada a formação dos mais diversos sorotipos virais. [0013] Therefore, new vaccines that target or show protection promoted by antibodies only for the SARS-CoV 2 circulating in this pandemic situation may fall into disuse if the formation of the most diverse viral serotypes is proven.
[0014] A utilização de vírus completo atenuado ou de vírus morto na produção vacinai pressupõe que as células de defesa tenham uma capacidade maior de reconhecimento de diversas “áreas” do agente etiológico, impelindo a possibilidade de proteção cruzada aos mais diversos sorotipos. Acontece que o uso de tais vacinas pode também mostrar-se ineficaz. No caso do uso do vírus morto, o organismo pode não responder a contento. No caso do vírus vivo atenuado, existe a insegurança quanto à possibilidade de provocar a doença, mesmo que de forma mais branda. [0014] The use of attenuated complete virus or killed virus in vaccine production presupposes that the defense cells have a greater ability to recognize different "areas" of the etiological agent, pushing the possibility of cross-protection to the most diverse serotypes. It turns out that the use of such vaccines can also prove to be ineffective. In the case of using the dead virus, the organism may not respond satisfactorily. In the case of the live attenuated virus, there is uncertainty about the possibility of causing the disease, even if in a milder way.
[0015] É um objetivo da presente invenção o uso do vírus vacinai da bronquite infecciosa aviária (vivo e atenuado) como modelo de imunização em mamíferos contra a COVID 19. Uma vez que o vírus da bronquite infecciosa aviária não provoca doença em humanos, esta abordagem proporciona benefícios em relação aos vírus mortos, sem os riscos de vírus vivos que provocam doenças em humanos, ainda que atenuados. O uso de vírus animais em vacinas para humanos, especialmente o vírus da bronquite infecciosa das galinhas (IBV), nunca foi usado em mamíferos para qualquer fim de imunização. A presente invenção muda este cenário. [0015] It is an objective of the present invention to use the avian infectious bronchitis vaccine virus (live and attenuated) as a model of immunization in mammals against COVID 19. Since the avian infectious bronchitis virus does not cause disease in humans, this approach provides benefits in in relation to dead viruses, without the risks of live viruses that cause disease in humans, even if they are attenuated. The use of animal viruses in human vaccines, especially the infectious bronchitis virus of chickens (IBV), has never been used in mammals for any immunization purpose. The present invention changes this scenario.
[0016] Do que se depreende da literatura pesquisada, não foram encontrados documentos antecipando ou sugerindo os ensinamentos da presente invenção, de forma que a solução aqui proposta, além de aplicação industrial, possui novidade e atividade inventiva frente ao estado da técnica. [0016] From what can be seen from the researched literature, no documents were found anticipating or suggesting the teachings of the present invention, so that the solution proposed here, in addition to industrial application, has novelty and inventive step compared to the state of the art.
Sumário da Invenção Summary of the Invention
[0017] A presente invenção resolve os problemas do estado da técnica a partir do uso do vírus da bronquite infecciosa das galinhas (IBV) para a preparação de uma vacina para a imunização contra SARS-CoV2 e vírus correlacionados em mamíferos. [0017] The present invention solves the problems of the prior art from the use of infectious bronchitis virus of chickens (IBV) for the preparation of a vaccine for immunization against SARS-CoV2 and related viruses in mammals.
[0018] Em um primeiro objeto, a presente invenção apresenta uma vacina compreendendo pelo menos um vírus da bronquite infecciosa (IBV) e um veículo ou diluente farmaceuticamente aceitável para a imunização de mamíferos contra coronavirus, SARS-CoV-2 e/ou vírus relacionados. [0018] In a first object, the present invention provides a vaccine comprising at least one infectious bronchitis virus (IBV) and a pharmaceutically acceptable carrier or diluent for immunizing mammals against coronavirus, SARS-CoV-2 and/or related viruses .
[0019] Em um segundo objeto, a presente invenção apresenta o uso de vírus da bronquite infecciosa (IBV) para a fabricação de uma vacina para a proteção de mamíferos contra coronavirus, SARS-CoV-2 e/ou vírus relacionados. [0019] In a second object, the present invention presents the use of infectious bronchitis virus (IBV) for the manufacture of a vaccine for the protection of mammals against coronavirus, SARS-CoV-2 and/or related viruses.
[0020] Em um terceiro objeto, a presente invenção apresenta um método de imunização de mamíferos contra coronavirus, SARS-CoV-2 e/ou vírus relacionados. [0020] In a third object, the present invention provides a method of immunizing mammals against coronavirus, SARS-CoV-2 and/or related viruses.
[0021] Estes e outros objetos da invenção serão imediatamente valorizados pelos versados na arte e serão descritos detalhadamente a seguir. [0021] These and other objects of the invention will be immediately appreciated by those skilled in the art and will be described in detail below.
Breve Descrição das Figuras Brief Description of Figures
[0022] São apresentadas as seguintes figuras: [0023] Figura 1 - valores da absorbância luminosa em espectro 450 nm verificado em teste de ELISA para detecção de anticorpos produzidos em camundongo contra o IBV no dia 28 do experimento. GS - grupo subcutâneo; GO - grupo oral; GN - grupo nasal e CONTR - grupo controle. [0022] The following figures are presented: [0023] Figure 1 - light absorbance values in the 450 nm spectrum verified in an ELISA test for the detection of antibodies produced in mice against IBV on day 28 of the experiment. GS - subcutaneous group; GO - oral group; GN - nasal group and CONTR - control group.
[0024] Figura 2 - valores da absorbância luminosa em espectro 450 nm detectado em teste de ELISA para detecção de anticorpos produzidos em camundongo contra o IBV no dia 45 do experimento. GS - grupo subcutâneo; GO - grupo oral; GN - grupo nasal e CONTR - grupo controle. [0024] Figure 2 - light absorbance values in the 450 nm spectrum detected in an ELISA test for the detection of antibodies produced in mice against IBV on day 45 of the experiment. GS - subcutaneous group; GO - oral group; GN - nasal group and CONTR - control group.
[0025] Figura 3. Evidência de bandas proteicas virais com semelhantes pesos moleculares tanto contra SARS-COV 2 quanto para o IBV. Aproximadamente de 84kDa, equivalente à proteína S2 do envelope. Nas bandas superiores destacadas estão identificadas as proteínas com pesos moleculares equivalentes às proteínas Spike (180KDa) de ambos os vírus. Nas bandas do meio estão identificadas as proteínas com pesos moleculares equivalentes às proteínas spike 1 ou S1 (aprox.1 10KDa) e nas bandas inferiores estão identificadas as proteínas com pesos moleculares equivalentes as proteínas spike 2 ou S2 (aprox. 84KDa). [0025] Figure 3. Evidence of viral protein bands with similar molecular weights against both SARS-COV 2 and IBV. Approximately 84kDa, equivalent to the envelope protein S2. In the upper highlighted bands proteins with molecular weights equivalent to Spike proteins (180KDa) of both viruses are identified. Proteins with molecular weights equivalent to spike 1 or S1 proteins (approx. 1 10KDa) are identified in the middle bands and proteins with molecular weights equivalent to spike 2 or S2 proteins (approx. 84KDa) are identified in the lower bands.
[0026] Figura 4 - Teste de soroneutralização virai - amostra de infecção de SARS-Cov 2 em monocamada de células Vero, em cor violeta. Após três dias de infecção, verificou-se a formação de 27 espaços circulares na monocamada, indicando destruição celular provocada pela infecção por SARS-CoV 2. [0026] Figure 4 - Viral serum neutralization test - sample of SARS-Cov 2 infection in monolayer of Vero cells, in violet color. After three days of infection, there was the formation of 27 circular spaces in the monolayer, indicating cellular destruction caused by the SARS-CoV 2 infection.
[0027] Figura 5 - Teste de soroneutralização virai - amostra negativa de SARS- Cov 2 em monocamada de células Vero, em cor violeta. Após três dias de infecção, verificou-se a não formação de espaços circulares na monocamada, indicando a não destruição celular por ausência de infecção por SARS-CoV 2 - controle negativo. [0027] Figure 5 - Viral serum neutralization test - negative sample of SARS-Cov 2 in monolayer of Vero cells, in violet color. After three days of infection, there was no formation of circular spaces in the monolayer, indicating no cell destruction due to the absence of SARS-CoV 2 infection - negative control.
[0028] Figura 6 - Teste de soroneutralização virai - amostra positiva contra SARS- COV 2 em monocamada de células Vero, em cor violeta, soroneutralizada por Anticorpos anti-SARS-CoV2 produzido em humano soropositivo. Após três dias de infecção, verificou-se a não formação de espaços circulares na monocamada, indicando a não destruição celular por soroneutralização virai por SARS-CoV 2 - controle positivo. [0028] Figure 6 - Viral seroneutralization test - positive sample against SARS-COV 2 in a monolayer of Vero cells, in violet color, seroneutralized by anti-SARS-CoV2 antibodies produced in a seropositive human. After three days of infection, there was no formation of spaces circular in the monolayer, indicating the non-destruction of cells by viral seroneutralization by SARS-CoV 2 - positive control.
[0029] Figura 7 - Teste de soroneutralização virai - amostra positiva para SARS-CoV 2 em monocamada de células Vero, em cor violeta, soroneutralizada por Anticorpos anti-IBV produzido em camundongos imunizados com a vacina para IBV por via subcutânea. Após três dias de infecção, verificou-se a formação de 5 espaços circulares na monocamada, indicando a pouquíssima destruição celular e a eficácia de aproximadamente 82% da soroneutralização virai dos SARS-CoV 2 por anticorpos anti-IBV - Amostra. [0029] Figure 7 - Viral seroneutralization test - positive sample for SARS-CoV 2 in a monolayer of Vero cells, in violet color, seroneutralized by anti-IBV antibodies produced in mice immunized with the vaccine for IBV subcutaneously. After three days of infection, there was the formation of 5 circular spaces in the monolayer, indicating very little cell destruction and the effectiveness of approximately 82% of viral seroneutralization of SARS-CoV 2 by anti-IBV antibodies - Sample.
[0030] Figura 8 - Imagem do teste do soro de camundongo imunizado com IBV pela via oral (grupo 28 dias) pela técnica de western blotting em membrana transferida com as proteínas virais do SARS-CoV 2 inativado pelo calor. Envolta do círculo destacado na figura há uma proteína de peso molecular equivalente ao da proteína S1 (aprox. 1 10KDa). [0030] Figure 8 - Image of the serum test of mice immunized with IBV orally (28 days group) by western blotting technique in membrane transferred with the heat-inactivated SARS-CoV 2 viral proteins. Surrounded by the highlighted circle in the figure is a protein of molecular weight equivalent to that of the S1 protein (approx. 110KDa).
[0031] Figura 9 demonstra um estudo genômico comparativo entre o vírus da bronquite infecciosa aviária e o SARS-CoV 2, através do uso de dados contidos no National Center for Biotecnology Information (NCBI) pelo software BLAST (Basic Local Alignment Search Tool) que evidenciou semelhanças superiores a 50%. [0031] Figure 9 demonstrates a comparative genomic study between the avian infectious bronchitis virus and SARS-CoV 2, through the use of data contained in the National Center for Biotechnology Information (NCBI) by the BLAST (Basic Local Alignment Search Tool) software that showed similarities greater than 50%.
[0032] Figura 10 - (a) e (b) Alinhamento da sequência de 4 cepas do IBV e 4 cepas do SARS-CoV-2, com as setas vermelhas indicando as posições 484 e 501 do domínio ligante do receptor (RBD) do SARS-CoV-2. O asterisco indica a conservação do resíduo, os dois pontos indicam uma substituição por um resíduo similar e o ponto indica uma baixa similaridade entre os resíduos, (c) Mapa das energias de interação entre os resíduos ACE2 (eixo horizontal) e os resíduos do RBD do SARS-CoV-2 (eixo vertical), com a escala de cores indicando os valores. [0032] Figure 10 - (a) and (b) Alignment of the sequence of 4 strains of IBV and 4 strains of SARS-CoV-2, with red arrows indicating positions 484 and 501 of the receptor binding domain (RBD) of the SARS-CoV-2. The asterisk indicates the conservation of the residue, the colon indicates a substitution by a similar residue and the point indicates a low similarity between the residues, (c) Map of the interaction energies between the ACE2 residues (horizontal axis) and the RBD residues of SARS-CoV-2 (vertical axis), with the color scale indicating the values.
[0033] Figura 11 - (a)-(i) RBD do IBV (cartoon azul) ligado em diferentes anticorpos neutralizantes nAbs (superfícies), (j) Comparativo das energias de ligação do RBD do IBV (SARS-CoV-2) com os anticorpos. Foram utilizados os códigos de 101 - 1 1 1 por ainda se tratar de uma pesquisa em andamento. [0033] Figure 11 - (a)-(i) IBV RBD (blue cartoon) bound in different neutralizing antibodies nAbs (surfaces), (j) Comparison of the binding energies of the IBV RBD (SARS-CoV-2) with the antibodies. were used the codes 101 - 1 1 1 as it is still an ongoing research.
[0034] Figura 12 - Soroneutralização PRNT do SARS-Cov-2 com soro de pessoas que trabalham em granjas e realizam o processo de imunização das aves: P=trabalhador e C+ =controle e C- = controle. [0034] Figure 12 - PRNT serum neutralization of SARS-Cov-2 with serum from people who work on farms and carry out the process of immunization of birds: P=worker and C+ =control and C- = control.
[0035] Figura 13 - Quantificação sérica dos níveis de glicose, colesterol total e triglicerídeos dos camundongos imunizados com IBV por vias subcutânea, oral e nasal. SC: Via de imunização sub-cutânea. [0035] Figure 13 - Serum quantification of glucose, total cholesterol and triglycerides levels of mice immunized with IBV by subcutaneous, oral and nasal routes. SC: Subcutaneous immunization route.
[0036] Figura 14 - Quantificação sérica dos níveis de ureia, creatinina, aspartato aminotransferase e alanina aminotransferase dos camundongos imunizados com IBV por vias subcutâneas, oral e nasal. SC: Via de imunização sub-cutânea. [0036] Figure 14 - Quantification of serum levels of urea, creatinine, aspartate aminotransferase and alanine aminotransferase of mice immunized with IBV by subcutaneous, oral and nasal routes. SC: Subcutaneous immunization route.
Descrição Detalhada da Invenção Detailed Description of the Invention
[0037] A presente invenção apresenta o uso do vírus da bronquite infecciosa das galinhas para a produção de uma vacina para a imunização de mamíferos contra SARS-CoV2. [0037] The present invention provides the use of infectious bronchitis virus of chickens for the production of a vaccine for the immunization of mammals against SARS-CoV2.
[0038] Os termos e definições do presente pedido são entendidos por: [0038] The terms and definitions of this application are understood by:
[0039] IBV, do inglês infectious bronchitis virus, denota o causador da bronquite infecciosa das galinhas, pertence ao gênero Gammacoronavirus, da família Coronaviridae. [0039] IBV, from the English infectious bronchitis virus, denotes the cause of infectious bronchitis in chickens, belongs to the genus Gammacoronavirus, family Coronaviridae.
[0040] SARS-CoV2 aqui retratada, também pode ser identificado como COVID 19, Coronavirus e relacionados. [0040] SARS-CoV2 pictured here, can also be identified as COVID 19, Coronavirus and related.
[0041] A presente invenção descreve o uso da vacina contra a bronquite infecciosa das galinhas (IBV) como modelo de imunização em mamíferos contra SARS-CoV2. [0041] The present invention describes the use of the vaccine against infectious bronchitis of chickens (IBV) as a model of immunization in mammals against SARS-CoV2.
[0042] A presente invenção faz uso do vírus contra IBV como forma de garantir uma resposta por anticorpos (humoral) e eficaz em mamíferos contra a COVID 19 (Figura 7). [0042] The present invention makes use of the virus against IBV as a way to ensure an antibody response (humoral) and effective in mammals against COVID 19 (Figure 7).
[0043] É, portanto, um objeto da presente invenção, uma vacina compreendendo pelo menos um vírus da bronquite infecciosa (IBV) e um veículo ou diluente farmaceuticamente aceitável para a imunização de mamíferos contra coronavirus, SARS-CoV-2 e/ou vírus relacionados. [0043] It is therefore an object of the present invention, a vaccine comprising at least one infectious bronchitis virus (IBV) and a pharmaceutically acceptable carrier or diluent for the immunization of mammals against coronavirus, SARS-CoV-2 and/or related viruses.
[0044] Em um segundo objeto, a presente invenção apresenta o uso de vírus da bronquite infecciosa (IBV) para a fabricação de uma vacina para a proteção de mamíferos contra coronavirus, SARS-CoV-2 e/ou vírus relacionados. [0044] In a second object, the present invention presents the use of infectious bronchitis virus (IBV) for the manufacture of a vaccine for the protection of mammals against coronavirus, SARS-CoV-2 and/or related viruses.
[0045] Em uma concretização do primeiro ou segundo objeto, o IBV é vivo e atenuado. [0045] In an embodiment of the first or second object, the IBV is live and attenuated.
[0046] Em uma concretização, o IBV é da cepa Massachussetts H120 com sequência genética de referência NCBI: NC_001451 .1 que compreende um gene que codifica para a proteína spike. [0046] In one embodiment, the IBV is from the Massachusetts H120 strain with NCBI reference genetic sequence: NC_001451.1 which comprises a gene encoding the spike protein.
[0047] Em uma concretização a vacina compreende um adjuvante. [0047] In one embodiment the vaccine comprises an adjuvant.
[0048] Em uma concretização, a vacina é administrada via oral, incluindo sublingual; nasal, por exemplo, por gota grossa, aspersão, inalação ou spray; ocular; subcutânea ou intramuscular; com ou sem uso de adjuvantes. [0048] In one embodiment, the vaccine is administered orally, including sublingually; nasal, for example, by thick drop, sprinkling, inhalation or spray; eyepiece; subcutaneous or intramuscular; with or without the use of adjuvants.
[0049] Em uma concretização, a vacina compreende concentração viral de aproximadamente 400 a aproximadamente 4000 vírus por dose para aplicação em humanos. [0049] In one embodiment, the vaccine comprises viral concentration of approximately 400 to approximately 4000 viruses per dose for application to humans.
[0050] Em uma concretização, a vacina compreende a produção de anticorpos anti-IBV que se ligam à porção RBD da proteína spike do vírus. [0050] In one embodiment, the vaccine comprises producing anti-IBV antibodies that bind to the RBD portion of the spike protein of the virus.
[0051] Em um terceiro objeto, a presente invenção apresenta um método de imunização de mamíferos contra coronavirus, SARS-CoV-2 e/ou vírus relacionados, que compreende a administração de vacina contendo pelo menos um vírus da bronquite infecciosa (IBV) e um veículo ou diluente farmaceuticamente aceitável. [0051] In a third object, the present invention provides a method of immunizing mammals against coronavirus, SARS-CoV-2 and/or related viruses, which comprises administering a vaccine containing at least one infectious bronchitis virus (IBV) and a pharmaceutically acceptable carrier or diluent.
[0052] A presente invenção proporciona diversas vantagens e efeitos técnicos surpreendentes. Proporciona o uso de uma vacina já conhecida contra IBV para a imunização em mamíferos contra SARS-CoV2 de forma eficaz e segura. Proporciona facilidade na administração da vacina, que pode ser, entre outras, por via intranasal (gotas ou spray). Proporciona também a auto-aplicação, sendo prática no ponto de vista da logística, não dependendo de aplicadores treinados. A vacina de invenção tem baixo custo de produção e sua produção é feita com tecnologia conhecida. O surpreendente novo uso do vírus da bronquite infecciosa das galinhas se demonstrou eficaz na proteção de mamíferos contra a infecção do SarsCov2. Na presente invenção, mesmo o vírus vacinai sendo vivo e atenuado, não acomete os mamíferos e portanto não os faz adoecer, mas produz uma resposta imunológica eficaz contra o SarsCov2/COVID19. [0052] The present invention provides several surprising technical advantages and effects. It provides the use of an already known vaccine against IBV for the immunization in mammals against SARS-CoV2 in an effective and safe way. It facilitates the administration of the vaccine, which can be, among others, intranasally (drops or spray). It also provides self-application, being practical from a logistical point of view, not depending on trained applicators. The vaccine of invention has a low production cost and its production is done with known technology. The surprising new use of infectious bronchitis virus in chickens has been shown to be effective in protecting mammals against SARSCov2 infection. In the present invention, even though the vaccine virus is live and attenuated, it does not affect mammals and therefore does not make them sick, but produces an effective immune response against SarsCov2/COVID19.
Exemplos Examples
[0053] A invenção será ainda mais detalhada por meio de exemplos descritos a seguir, que têm o intuito somente de exemplificar algumas das inúmeras maneiras de se realizar a invenção sem limitar, contudo, seu escopo. [0053] The invention will be further detailed through the examples described below, which are intended only to exemplify some of the numerous ways of carrying out the invention without limiting, however, its scope.
[0054] Uso do vírus da bronquite infecciosa das galinhas na preparação de vacina contra Sarscov2 [0054] Use of infectious bronchitis virus of chickens in the preparation of vaccine against Sarscov2
[0055] O uso do vírus da bronquite infecciosa das galinhas para preparação de vacina para a imunização de mamíferos foi concretizado e testado através em três vias de aplicação: oral, intranasal e subcutânea. Os resultados foram significantes na produção de uma resposta humoral contra o IBV (Figuras 1 e 2). [0056] Na presente invenção, o vírus a ser utilizado para a produção da vacina é selecionado do grupo que compreende: sorotipos das variedades Arkansas, Connecticut, Delaware e Massachusetts, inclusive a Ma5 e M41 , variantes GA, variantes Califórnia, variantes D207 (D274), D212 (D1466), variante 4/91 (793B), ltalian-02, QX, D388, e/ou sorotipos BRI. Todos esses sorotipos são de conhecimento no estado da técnica e facilmente disponíveis para um técnico no assunto. [0055] The use of the infectious bronchitis virus of chickens to prepare a vaccine for the immunization of mammals was realized and tested through three routes of application: oral, intranasal and subcutaneous. The results were significant in producing a humoral response against IBV (Figures 1 and 2). [0056] In the present invention, the virus to be used for the production of the vaccine is selected from the group comprising: Arkansas, Connecticut, Delaware and Massachusetts serotypes, including Ma5 and M41, GA variants, California variants, D207 variants ( D274), D212 (D1466), variant 4/91 (793B), Italian-02, QX, D388, and/or BRI serotypes. All such serotypes are known in the art and readily available to one skilled in the art.
[0057] Para os experimentos do presente exemplo, o vírus utilizado é protótipo de todos os coronavirus, ou seja, um coronavirus do grupo gama, portanto um “parente” do SARS-CoV2. Foi utilizado o vírus completo vivo e enfraquecido (atenuado) da cepa Massachussetts H120 com sequência genética de referência NCBI: NC_001451.1. A American Type Culture Collection (ATCC) disponibiliza o protótipo dos sorotipos Massachusetts - o M41 , código VR-21 , que após sucessivas passagens em ovos embrionados ou culturas celulares pode vir a formar o H120, como também, do sorotipo adaptado em ovo - código VR-22, e dos sorotipos Arkansas 99 (código VR-841 ) e Connecticut A5968 (código VR- 817). [0057] For the experiments of the present example, the virus used is the prototype of all coronaviruses, that is, a coronavirus of the gamma group, therefore a "relative" of SARS-CoV2. Live and weakened (attenuated) complete virus of the Massachussetts H120 strain with NCBI reference genetic sequence: NC_001451.1 was used. The American Type Culture Collection (ATCC) makes available the prototype of the Massachusetts serotypes - the M41 , code VR-21 , which after successive passages in embryonated eggs or cell cultures can form the H120, as well as the egg-adapted serotype - code VR-22, and of serotypes Arkansas 99 (code VR-841) and Connecticut A5968 (code VR-817).
[0058] Em outra concretização, a preparação da vacina envolve o uso do vírus completo morto ou de porções deste vírus, seja apenas utilizando o material genético que define a proteína Spike ou qualquer outra que tenha poder imunogênico, ou ainda como um vetor virai para expressar proteínas imunogênicas do próprio SARS-CoV2. [0058] In another embodiment, the preparation of the vaccine involves the use of the complete killed virus or portions of this virus, either only using the genetic material that defines the Spike protein or any other that has immunogenic power, or even as a viral vector for express immunogenic proteins of the SARS-CoV2 itself.
[0059] Algumas outras tecnologias conhecidas pelo estado da técnica e que podem ser utilizadas para a produção de vacinas são descritas a seguir: [0059] Some other technologies known by the state of the art and that can be used for the production of vaccines are described below:
[0060] Partículas semelhante ao vírus: [0060] Virus-like particles:
[0061] Partículas semelhantes ao vírus são comparáveis aos vírus, mas não contêm a informação genética dos vírus. Elas correspondem a um invólucro vazio que apresenta uma proteína de coronavirus. A ausência de informação genética anula o risco de replicação virai e propagação. [0061] Virus-like particles are comparable to viruses, but do not contain the genetic information of viruses. They correspond to an empty shell that presents a coronavirus protein. The absence of genetic information negates the risk of viral replication and propagation.
[0062] Vetores virais como vacinas: [0062] Viral vectors as vaccines:
[0063] Nesta categoria de vacinas, um vírus que não é patogênico ao homem, por exemplo um adenovirus, é modificado para expressar uma proteína de coronavirus. Vetores virais expressando proteína estranhas são amplamente usados para induzir uma forte resposta imune contra vários patógenos, incluindo vírus. [0063] In this category of vaccines, a virus that is not pathogenic to humans, for example an adenovirus, is modified to express a coronavirus protein. Viral vectors expressing foreign proteins are widely used to induce a strong immune response against various pathogens, including viruses.
[0064] Vacinas baseadas em ácido nucleico: [0064] Nucleic acid based vaccines:
[0065] Nesta categoria de vacinas, uma molécula de ácido nucleico codificando uma proteína de um coronavirus, por exemplo a proteína spike de SARS-CoV2, opcionalmente formulada com lipossomas, é administrada a um indivíduo. O ácido nucleico é frequentemente modificado para melhorar a quantidade e duração da expressão da proteína que ele codifica. [0065] In this category of vaccines, a nucleic acid molecule encoding a protein from a coronavirus, for example the SARS-CoV2 spike protein, optionally formulated with liposomes, is administered to an individual. Nucleic acid is often modified to improve the amount and duration of expression of the protein it encodes.
[0066] Nanopartículas: [0066] Nanoparticles:
[0067] Nesta categoria de vacinas, partículas que são formuladas com uma proteína de coronavirus ou um ácido nucleico codificando a proteína são usadas. Tais partículas são prontamente internalizadas nas células imunes. Nanopartículas podem ser a base orgânica, por exemplo lipossomas ou nanopartículas poliméricas; ou a base inorgânica, tais como nanopartículas de ouro. [0067] In this category of vaccines, particles that are formulated with a coronavirus protein or a nucleic acid encoding the protein are used. Such particles are readily internalized into immune cells. Nanoparticles can be organic based, for example liposomes or polymeric nanoparticles; or inorganic base, such as gold nanoparticles.
[0068] Células apresentadoras de antíqeno: [0068] Antigen presenting cells:
[0069] Nesta categoria de vacinas, células apresentadoras de antígeno (APC), incluindo macrófagos e células dendríticas, são projetadas para apresentar fragmentos de proteína de coronavirus em suas superfícies para ativar as células imunes. [0069] In this category of vaccines, antigen presenting cells (APC), including macrophages and dendritic cells, are designed to present coronavirus protein fragments on their surfaces to activate immune cells.
[0070] Vacinas adjuvantes: [0070] Adjuvant vaccines:
[0071] Nesta categoria de vacinas, moléculas adicionais estão presentes na formulação de modo a potencializar a resposta imune às proteínas do vírus. [0071] In this category of vaccines, additional molecules are present in the formulation in order to potentiate the immune response to the virus proteins.
[0072] Semelhança entre o coronavirus aviário e SARS-CoV 2 [0072] Similarity between avian coronavirus and SARS-CoV 2
[0073] Mesmo sendo de grupos de coronavirus diferentes - IBV pertence ao gênero Gamacoronavirus e SARS-CoV 2 ao gênero Betacoronavirus - ambos são “parentes”, pertencentes à família Coronaviridae. A presente invenção mostra de forma surpreendente, que há uma semelhança de proteína estrutural entre eles, evidenciada por uma proteína de peso molecular de aproximadamente 84kDa, sugestiva de ser a S2 (spike 2) (figura 3), mediante o teste de eletroforese descrito mais abaixo. [0073] Even though they are from different coronavirus groups - IBV belongs to the genus Gamacoronavirus and SARS-CoV 2 to the genus Betacoronavirus - both are "relatives", belonging to the Coronaviridae family. The present invention surprisingly shows that there is a structural protein similarity between them, evidenced by a protein with a molecular weight of approximately 84kDa, suggestive of being S2 (spike 2) (figure 3), by means of the electrophoresis test described above. below.
[0074] O SARS-CoV 2 foi identificado pela técnica da RT-PCR seguindo o seguinte protocolo: [0074] SARS-CoV 2 was identified by the RT-PCR technique following the following protocol:
[0075] O vírus SARS-COV2 foi extraído do material por meio de kit comercial de extração de RNA Virai (QIAamp Virai RNA Mini Kit - ID: 52906), sendo seguido o protocolo recomendado pelo fabricante. As amostras foram normalizadas para 100 ng para que fosse realizada a síntese do RNA por kit comercial (High- capacity cDNA Reverse Transcription Kit with RNAse inhibitor - Ref: 4374966) e os ajustes da programação no termociclador com os seguintes parâmetros: 25°C por 10 minutos, 37°C por 2 horas e 85°C por 5 minutos. Após a síntese, o material foi diluído para 100 ml_ com água livre de nuclease e realizada então a amplificação por PCR com kit comercial (GoTaq qPCR Master Mix - Ref: A6002) utilizando os primers indicados pelo Center of Disease Control and Prevention (CDC) para diagnóstico da doença (disponibilizado em: https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primerprobes.html). Seguiu-se à amplificação e a realização de uma eletroforese em agarose 1 %, na qual se adicionou na amostra o tampão loading buffer 10x na diluição de 1/5 e realizou-se a corrida na programação de 80V; 200 mA; 30 minutos. O gel então foi submerso em 50 ml_ de solução TAE com adição de SYBR (SYBR Safe DNA gel strain - Ref: S33102) na diluição de 1 :10,000 por 30 minutos e por fim analisado em transiluminador. [0075] The SARS-COV2 virus was extracted from the material using a commercial viral RNA extraction kit (QIAamp Virai RNA Mini Kit - ID: 52906), following the protocol recommended by the manufacturer. The samples were normalized to 100 ng so that the RNA synthesis could be performed using a commercial kit (High-capacity cDNA Reverse Transcription Kit with RNAse inhibitor - Ref: 4374966) and the programming adjustments in the thermocycler with the following parameters: 25°C per 10 minutes, 37°C for 2 hours and 85°C for 5 minutes. After synthesis, the material was diluted to 100 ml with nuclease-free water and then PCR amplification was performed with a commercial kit (GoTaq qPCR Master Mix - Ref: A6002) using the primers indicated by the Center of Disease Control and Prevention (CDC) for diagnosis of the disease (available at: https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primerprobes.html) . This was followed by amplification and electrophoresis in 1% agarose, in which 10x loading buffer was added to the sample at a dilution of 1/5 and the run was performed at 80V; 200 mA; 30 minutes. The gel was then submerged in 50 ml of TAE solution with the addition of SYBR (SYBR Safe DNA gel strain - Ref: S33102) at a dilution of 1:10,000 for 30 minutes and finally analyzed in a transilluminator.
[0076] Para comparação básica entre os dois coronavirus de grupos diferentes, resolveu-se analisar as soluções virais em gel de poliacrilamida (PAGE), contendo dodecil sulfato de sódio (SDS) em gel de concentração a 4% e o gel de separação a 12% de acordo com o método descrito por Laemmli (1970), usando a coloração com azul de coomassie. [0076] For a basic comparison between the two coronaviruses of different groups, it was decided to analyze the viral solutions in polyacrylamide gel (PAGE), containing sodium dodecyl sulfate (SDS) in a 4% concentration gel and the separation gel at 12% according to the method described by Laemmli (1970), using coomassie blue staining.
[0077] Imunização em mamíferos [0077] Immunization in mammals
[0078] Para a realização da imunização em mamíferos, utilizou-se como modelo camundongos da linhagem Swiss, fêmeas, com 22 dias de vida, divididos em 4 grupos de 10 indivíduos cada. Os grupos avaliados eram: controle negativo, imunização por via oral, imunização por via nasal e imunização por via subcutânea. As imunizações ocorreram nos dias 1 , 21 e 35 da obtenção dos animais (início do experimento). As colheitas de sangue para obtenção do soro ocorreram nos dias 28 e 45 do início do experimento. Os animais foram mantidos em caixas de contenção apropriadas para manejo de camundongos e em tamanho apropriado para a população (10 animais) de cada caixa, em condições favoráveis (temperatura, umidade e luz) ao crescimento e bem-estar destes, com alimentação balanceada nutricionalmente peletizada própria para roedores e água potável, ambos ad libitum. [0078] To carry out the immunization in mammals, female Swiss mice, 22 days old, divided into 4 groups of 10 individuals each, were used as a model. The groups evaluated were: negative control, oral immunization, nasal immunization and subcutaneous immunization. Immunizations took place on days 1, 21 and 35 of obtaining the animals (beginning of the experiment). Blood collections to obtain serum occurred on days 28 and 45 of the beginning of the experiment. The animals were kept in containment boxes suitable for handling mice and in an appropriate size for the population (10 animals) of each box, under favorable conditions (temperature, humidity and light) for their growth and well-being, with a nutritionally balanced diet. pellets suitable for rodents and drinking water, both ad libitum.
[0079] Obtenção dos anticorpos e comparação das proteínas entre os vírus [0080] Com a obtenção do sangue através de coleta retro-orbital, separou-se o soro dos animais e foram feitos testes imunoenzimáticos à base de peroxidase (ELISA) e, em paralelo, eletroforese para comparação dos pesos moleculares das proteínas de superfície e estruturais do IBV vacinai com o SARS-CoV 2 já mencionados na literatura. [0079] Obtaining the antibodies and comparing the proteins between the viruses [0080] With the collection of blood through retro-orbital collection, the serum of the animals was separated and immunoenzymatic tests based on peroxidase were performed (ELISA) and, in parallel, electrophoresis to compare the molecular weights of surface and structural proteins of IBV vaccine with SARS-CoV 2 already mentioned in the literature.
[0081] O teste de ELISA foi realizado conforme as etapas descritas abaixo: [0082] Primeira etapa: foram imunizados 40 camundongos fêmeas da linhagem swiss, divididas em 4 grupos de 10 indivíduos cada, de acordo com a via de administração utilizada: subcutâneo, oral, intranasal e controle, com doses de 100ml em uma concentração baixa de 1 mg/100ml de IBV vacinai diluído em solução de diluição sem agentes de descontaminação. Nesta etapa, além da primeira dose, feita no dia 1 , foram feitos mais dois reforços: no dia 15 e no dia 28. A imunização do grupo subcutâneo teve acrescida, na primeira dose, o uso de Adjuvante Completo de Freund (FCA). Já na primeira dose de reforço (15 dias), foi acrescido o uso de Adjuvante Incompleto de Freund (FIA) e no último reforço (28 dias), não foi feito o uso de adjuvantes. No grupo oral, administrou- se diretamente a dose na cavidade bucal dos animais, enquanto no grupo intranasal, essa administração foi feita por borrifação em spray. O grupo controle manteve-se sem qualquer contato com o imunizante durante todo o período experimental. Os soros dos animais para obtenção dos anticorpos foram colhidos nos dias 28, antes do segundo reforço e no dia 45. Os anticorpos obtidos foram destinados à execução da técnica de ELISA para comparativos de títulos de IgG, lgG1 e lgG2a e soroneutralização, e mantidos congelados a - 20°C. [0081] The ELISA test was performed according to the steps described below: [0082] First step: 40 female Swiss mice were immunized, divided into 4 groups of 10 individuals each, according to the route of administration used: subcutaneous, oral, intranasal and control, with 100ml doses at a low concentration of 1mg/100ml of vaccine IBV diluted in a dilution solution without decontaminating agents. At this stage, in addition to the first dose, given on day 1, two more boosters were given: on day 15 and on day 28. The immunization of the subcutaneous group was added, in the first dose, with the use of Complete Freund's Adjuvant (FCA). In the first booster dose (15 days), the use of Incomplete Freund's Adjuvant (FIA) was added and in the last booster (28 days), adjuvants were not used. In the oral group, the dose was directly administered into the oral cavity of the animals, while in the intranasal group, this administration was done by spraying. The control group remained without any contact with the immunizing agent throughout the experimental period. The sera of the animals to obtain the antibodies were collected on days 28, before the second booster and on day 45. The antibodies obtained were destined to the execution of the ELISA technique to compare the titers of IgG, lgG1 and lgG2a and seroneutralization, and kept frozen at -20°C.
[0083] Segunda etapa: foram imunizadas 40 camundongos fêmeas da linhagem swiss, divididas em 3 grupos, de acordo com a via de administração utilizada, com números iguais de indivíduos: subcutâneo, oral e intranasal, com doses de 100ml em uma concentração de 10mg/100ml de IBV vacinai diluído em solução de diluição sem agentes de descontaminação. Nesta etapa, além da primeira dose, feita no dia 1 , foi feita mais um reforço no dia 15. Apenas na imunização do grupo subcutâneo teve acrescida na primeira dose o uso de adjuvante completo de Freund e na dose de reforço (15 dias) não foi feito o uso de adjuvante. Em relação à forma como foram administradas nas outras duas vias, seguiu-se o protocolo da etapa anterior. Os soros dos animais para obtenção dos anticorpos foram colhidos nos dias: 7, 15 (antes da dose de reforço), 21 , 28 e 45 dias. Os anticorpos obtidos foram destinados à técnica de ELISA para comparativos de concentração de títulos de IgG, western blotting, além da soroneutralização e mantidos à -20°C. [0083] Second stage: 40 female Swiss mice were immunized, divided into 3 groups, according to the route of administration used, with equal numbers of individuals: subcutaneous, oral and intranasal, with doses of 100ml in a concentration of 10mg /100ml of vaccine IBV diluted in a dilution solution without decontaminating agents. At this stage, in addition to the first dose, performed on day 1, a further booster was performed on day 15. Only in the immunization of the subcutaneous group, the use of complete Freund's adjuvant was added to the first dose and to the booster dose (15 days) there was no use was made of adjuvant. Regarding the way they were administered in the other two routes, the protocol of the previous step was followed. Sera from animals to obtain antibodies were collected on days: 7, 15 (before the booster dose), 21, 28 and 45 days. The antibodies obtained were sent to the ELISA technique for comparisons of IgG titer concentration, western blotting, in addition to serum neutralization and kept at -20°C.
[0084] No teste de western blotting, foi utilizado o vírus completo de SARS-CoV 2 inativado pelo calor. As proteínas virais foram submetidas à eletroforese em gel de poliacrilamida a 4-12% (SDS-PAGE), como descrito anteriormente, e depois transferida para uma membrana de nitrocelulose conforme Towbin et al. (1979). A membrana foi imersa em tampão PBS-molico (5%) contendo os anticorpos produzidos em camundongos contra o IBV vacinai. Para a revelação, foram utilizados anticorpos anti-lgG de camundongo ligado à peroxidase, produzido em cabra da Sigma. [0084] In the western blotting test, the heat-inactivated complete SARS-CoV 2 virus was used. The viral proteins were subjected to 4-12% polyacrylamide gel electrophoresis (SDS-PAGE), as described above, and then transferred to a nitrocellulose membrane as Towbin et al. (1979). The membrane was immersed in PBS-molic buffer (5%) containing the antibodies produced in mice against the vaccine IBV. For the development, anti-mouse IgG antibodies linked to peroxidase, produced in Sigma goat, were used.
[0085] Na análise imunoenzimática verificou-se que o grupo subcutâneo dos camundongos gerou anticorpos muito acima do cutoff em relação aos demais grupos para IBV (FIGURAS 1 e 2). Assim, verificou-se que os camundongos imunizados pela via subcutânea responderam satisfatoriamente à produção de anticorpos contra IBV sem apresentar qualquer sintomatologia aparente da doença que acomete as aves, em ambas as colheitas (dias 28 e 45), tendo um resultado maior na colheita do dia 45 do experimento. Na eletroforese, verificou- se a presença de uma banda próxima ao marcador de 80kD, semelhante ao peso obtido pela proteína S2 do SARS-CoV 2, o suposto epítopo responsável pela infecção (figura 3). [0085] In the immunoenzymatic analysis, it was found that the subcutaneous group of mice generated antibodies far above the cutoff in relation to the other groups for IBV (FIGURES 1 and 2). Thus, it was verified that the mice immunized by the subcutaneous route responded satisfactorily to the production of antibodies against IBV without showing any apparent symptoms of the disease that affects the birds, in both harvests (days 28 and 45), with a higher result in the harvest of the day 45 of the experiment. Electrophoresis showed the presence of a band close to the 80kD marker, similar to the weight obtained by the S2 protein of SARS-CoV 2, the supposed epitope responsible for the infection (figure 3).
[0086] Isolamento do SARS-Cov 2 obtido: Seguinte a identificação e confirmação por RT-PCR, este foi isolado em monocamada de células vero utilizando-se meios de cultivo celular adequados, acrescidos de antibióticos e probióticos e acondicionada a -80sC. [0086] Isolation of SARS-Cov 2 obtained: Following identification and confirmation by RT-PCR, it was isolated in a monolayer of vero cells using suitable cell culture media, plus antibiotics and probiotics and conditioned at -80 s C .
[0087] Soroneutralização do SARS-CoV 2 por anticorpos anti-IBV: Após, o isolamento virai, onde se obteve 7,8x104 vírus/mL, fez-se a soroneutralização (Golden test) da amostra de SARS-CoV 2 obtidas através do isolamento em placas de 12 poços contendo monocamada de células Vero acrescida de gel (Figuras 4, 5, 6 e 7), e se obteve 82% de eficácia na neutralização do SARS- CoV 2 pelos anticorpos policlonais anti-IBV produzidos em camundongos por via subcutânea (Figura 7) (uma vez que foi a via que se mostrou mais eficaz para a imunização dos camundongos), possivelmente através de sua ligação com o vírus na porção RBD da proteína spike. [0087] SARS-CoV 2 seroneutralization by anti-IBV antibodies: After viral isolation, where 7.8x104 virus/mL was obtained, seroneutralization was performed (Golden test) of the SARS-CoV 2 sample obtained by isolation in 12-well plates containing a monolayer of Vero cells plus gel (Figures 4, 5, 6 and 7), and an 82% efficacy was obtained in the neutralization of SARS - CoV 2 by the polyclonal anti-IBV antibodies produced in mice subcutaneously (Figure 7) (since it was the route that proved to be the most effective for the immunization of mice), possibly through its binding with the virus in the RBD portion of the spike protein.
[0088] Novos testes de soroneutralização virai em células vero E6 foram refeitos, dessa vez com uma concentração virai dez vezes maior e se obteve resultados próximos a 95%, após 30 dias da imunização e 15 dias do reforço, quando administrada a vacina por via oral/nasal. Verificamos que após 45 dias da imunização e 30 do reforço, os títulos de anticorpos neutralizantes ainda eram considerados satisfatórios, isto é, superiores a 80%. [0088] New viral seroneutralization tests in vero E6 cells were redone, this time with a viral concentration ten times higher and results were obtained close to 95%, after 30 days of immunization and 15 days of booster, when the vaccine was administered via the oral/nasal. We found that after 45 days of immunization and 30 days of booster, neutralizing antibody titers were still considered satisfactory, that is, greater than 80%.
[0089] A presente invenção é caracterizada por usar o coronavirus das galinhas (IBV) como modelo de imunização em mamíferos contra o SARS-CoV 2 de forma ímpar e inédita, de modo a obter uma imunização significativa e eficaz. [0089] The present invention is characterized by using the chicken coronavirus (IBV) as a model of immunization in mammals against SARS-CoV 2 in a unique and unprecedented way, in order to obtain a significant and effective immunization.
[0090] Por se tratar de um vírus não zoonótico e que, mesmo sendo de outra espécie animal, mostrou possibilidade de induzir uma resposta imunológica em mamíferos, inclusive nos seres humanos. O vírus utilizado nesta concretização é aquele presente na vacina comercial contra bronquite infecciosa aviária (IBV). O isolamento, a titulação viral do IBV e todo o processo de produção obedeceram às normas constantes na Instrução Normativa n°7, de 10 de março de 2006 do Ministério da Agricultura, Pecuária e Abastecimento do Brasil (MAPA, 2006), não sofrendo nenhuma modificação ou alteração, apenas para verificar se este vírus, por sua segurança e eficácia, poderá ser utilizado como vírus vacinai contra a COVID-19. [0090] Because it is a non-zoonotic virus and that, even being from another animal species, showed the possibility of inducing an immune response in mammals, including humans. The virus used in this embodiment is that present in the commercial vaccine against infectious avian bronchitis (IBV). The isolation, viral titration of the IBV and the entire production process followed the norms contained in Normative Instruction No. modification or alteration, only to verify if this virus, due to its safety and efficacy, can be used as a vaccine virus against COVID-19.
[0091] A produção da vacina pode ser feita de acordo com uma das tecnologias constantes no estado da técnica, seja com vírus morto, enfraquecido ou em partes (proteínas, material genético...) nas suas mais diferentes cepas ou sorotipos, não se limitando apenas a Massachussetts H120, devido à semelhança entre o vírus IBV e o SARS-CoV2 nas suas grandes propensões de formar mutações. [0091] The production of the vaccine can be carried out according to one of the technologies contained in the state of the art, either with dead, weakened virus or in parts (proteins, genetic material...) in its most different strains or serotypes, it is not limited to the Massachusetts H120 only, due to the similarity between the IBV virus and SARS-CoV2 in their great propensities to form mutations.
[0092] Métodos de uso. A presente invenção refere-se à utilização de um vírus de caraterística vacinai (IBV) utilizado, até hoje, apenas em aves, como modelo de uso em mamíferos na imunização contra o SARS-CoV 2. As vias de administração para o vírus vacinai, de acordo com a presente invenção, são as vias: oral, incluindo sublingual; nasal, seja por gota grossa, aspersão, inalação ou spray; ocular ou por vias mais invasivas como subcutânea ou intramuscular, com ou sem uso de adjuvantes. [0092] Usage methods. The present invention relates to the use of a vaccinal virus (IBV) used, until now, only in birds, as a model of use in mammals in the immunization against SARS-CoV 2. The routes of administration for the vaccinal virus, according to the present invention, the routes are: oral, including sublingual; nasal, whether by thick drop, sprinkling, inhalation or spray; ocular or more invasive routes such as subcutaneous or intramuscular, with or without the use of adjuvants.
[0093] Além dos testes acima relatados, que foram testes de detecção de títulos de anticorpos e o considerado teste de ouro (soroneutralização), o qual verifica se esses anticorpos produzidos têm ação para soroneutralizar o vírus, foram também realizados estudos de similaridade entre os vírus da bronquite infecciosa aviária e o SARS-CoV 2. Isso foi feito através da viabilização de um perfil eletroforético das proteínas virais totais, e também pelo estudo genômico comparativo entre os vírus através do uso de dados contidos no National Center for Biotecnology Information (NCBI) através do software BLAST (Basic Local Alignment Search Tool) que evidenciou semelhanças superiores a 50% (Figura 9). [0093] In addition to the tests reported above, which were antibody titer detection tests and the gold test (seroneutralization), which checks whether these antibodies produced have an action to seroneutralize the virus, similarity studies were also carried out between the avian infectious bronchitis virus and SARS-CoV 2. This was done through the feasibility of an electrophoretic profile of the total viral proteins, and also by the comparative genomic study between the viruses through the use of data contained in the National Center for Biotechnology Information (NCBI ) through the BLAST (Basic Local Alignment Search Tool) software, which showed similarities greater than 50% (Figure 9).
[0094] De forma surpreendente, os anticorpos anti-IBV produzidos em camundongos reagiram com proteínas consideradas específicas para o SARS- CoV2, mais especificamente a proteína spike 1 . Isso foi evidenciado em teste de Western blot, verificado através de “manchas” de detecção na membrana que continha incorporada o vírus SARS-CoV2 (Figura 8). [0094] Surprisingly, the anti-IBV antibodies produced in mice reacted with proteins considered specific for SARS-CoV2, more specifically the spike protein 1 . This was evidenced in a Western blot test, verified through detection “spots” on the membrane that contained the SARS-CoV2 virus incorporated (Figure 8).
[0095] A viabilização da técnica de desafiar animais foi realizada, isto é, os animais foram imunizados com a vacina e, após 15 dias do reforço, foram submetidos a uma infecção pelo SARS-CoV2 para acompanhamento de possíveis sinais e sintomas da doença, caso se desenvolva. Este acompanhamento também revela tipos de células que são acionadas/ativas no desencadeamento da resposta imunológica. [0096] Adicionalmente, foi realizado teste in silico para a identificação de regiões semelhantes na porção RBD da proteína spike de ambos os vírus e que nesta região do SARS-CoV2, o anticorpo anti-IBV tem o poder de se ligar, porém ainda falta o resultado de quão forte é esta interação. [0095] The feasibility of the technique of challenging animals was carried out, that is, the animals were immunized with the vaccine and, after 15 days of the booster, they were subjected to an infection by SARS-CoV2 to monitor possible signs and symptoms of the disease, if it develops. This tracking also reveals cell types that are triggered/active in triggering the immune response. [0096] Additionally, an in silico test was performed to identify similar regions in the RBD portion of the spike protein of both viruses and that in this region of SARS-CoV2, the anti-IBV antibody has the power to bind, but it still lacks the result of how strong this interaction is.
[0097] Para o teste in silico, realizou-se a análise de biologia estrutural e computacional dos vírus IBV e SARS-CoV-2: [0097] For the in silico test, the structural and computational biology analysis of the IBV and SARS-CoV-2 viruses was performed:
[0098] Alinhamento de múltipla sequência [0098] Multiple Sequence Alignment
[0099] Para analisar as sequências e identificar as semelhanças entre as proteínas spike de SARS-COV-2 (South African - B.1.351 : ID: 7LYQ_1 ; India - B.1.617: ID QUX03874.1 ; Britanica - B.1.1.7: ID P0DTC2 e com as deleções relatadas del69-70, del144, e mutações N501Y, A570D, D614G, P681 H, T716I, S982A e D1 1 18H, R1 185L; Brazil - P1 : ID P0DTC2 com alterações L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y e T1027I), SARS-CoV-1 (PDB ID: 6WAQ) e IBV H120 Massachusetts (ID: CCQ71744.1 ; ID: AGW24533.1 ; ID: ALJ77588.1 ), e a cepa vacinai (ID: AAA46228.1 ) - (Bijlenga et al., 2004) foram recuperados do banco de dados GenBank NCBI (National Center for Biotechnology Information) e executado no ClustalW (Hung e Weng, 2016). Os dados foram posteriormente analisados no programa BioEdit v.7.2.6.1 (http://www.mbio.ncsu.edu/bioedit/bioedit.html). [0099] To analyze the sequences and identify the similarities between the SARS-COV-2 spike proteins (South African - B.1.351 : ID: 7LYQ_1 ; India - B.1.617 : ID QUX03874.1 ; Britanica - B.1.1 . 7: ID P0DTC2 and with the reported deletions del69-70, del144, and mutations N501Y, A570D, D614G, P681 H, T716I, S982A and D1 118H, R1 185L; Brazil - P1 : ID P0DTC2 with changes L18F, T20N, P26S , D138Y, R190S, K417T, E484K, N501Y, H655Y and T1027I), SARS-CoV-1 (PDB ID: 6WAQ) and IBV H120 Massachusetts (ID: CCQ71744.1 ; ID: AGW24533.1 ; ID: ALJ77588.1 ) , and the vaccine strain (ID: AAA46228.1) - (Bijlenga et al., 2004) were retrieved from the GenBank NCBI (National Center for Biotechnology Information) database and run in ClustalW (Hung and Weng, 2016). The data were later analyzed using the BioEdit v.7.2.6.1 program (http://www.mbio.ncsu.edu/bioedit/bioedit.html).
[0100] Docking molecular (anticorpo - antigeno IBV) [0100] Molecular docking (antibody - IBV antigen)
[0101] Um total de 1 1 anticorpos complexados à proteína spike de SARS-CoV- 2 foram recuperados do Protein Data Bank - PDB, com auxílio do software Pymol, 2.5 software (https://pymol.Org/2/). Cada anticorpo foi renomeado e salvo no formato pdb preservando as cadeias leves e pesadas. O antigeno selecionado para estudo foi a proteína spike do coronavirus da bronquite infecciosa - IBV (PDB: 6CV0), região do RBD. Posteriormente, a determinação das afinidades do anticorpo - antigeno foi por meio do acoplamento molecular com servidor PatchDock disponível em https://bioinfo3d.cs.tau.ac.il/PatchDock/php.php (Schneidman-Duhovny et al., 2005). Dez complexos de maior afinidade em termos de interações eletrostáticas e energia de ligação livre foram refinados usando o servidor FireDock, http://bioinfo3d.cs.tau.ac.il/FireDock/php.php (Mashiach et al., 2008). Por fim, para estimar a energia de interação dos anticorpos complexado ao antígeno em termos de AG (Xue et al., 2016), o servidor PROtein binDIng enerGY prediction (PRODIGY) (https://bianca.science.uu.nl/prodigy/) foi aplicado nos ensaios. [0102] Bioquímica quântica [0101] A total of 11 antibodies complexed to the spike protein of SARS-CoV-2 were retrieved from the Protein Data Bank - PDB, with the aid of Pymol, 2.5 software (https://pymol.Org/2/). Each antibody was renamed and saved in pdb format preserving the light and heavy chains. The antigen selected for study was the spike protein of infectious bronchitis coronavirus - IBV (PDB: 6CV0), region of the RBD. Subsequently, the determination of antibody-antigen affinities was done through molecular coupling with the PatchDock server available at https://bioinfo3d.cs.tau.ac.il/PatchDock/php.php (Schneidman-Duhovny et al., 2005) . Ten complexes with the highest affinity in terms of electrostatic interactions and free binding power were refined using the FireDock server, http://bioinfo3d.cs.tau.ac.il/FireDock/php.php (Mashiach et al., 2008). Finally, to estimate the interaction energy of the antibodies complexed to the antigen in terms of AG (Xue et al., 2016), the PROtein binDIng enerGY prediction (PRODIGY) server (https://bianca.science.uu.nl/prodigy) /) was applied in the trials. [0102] Quantum Biochemistry
[0103] Considerando a impossibilidade de se resolver analiticamente a equação de Schrodinger para sistemas de muitos elétrons, faz-se necessário empregar métodos computacionais que otimizem o uso dos recursos de processamento, e ao mesmo tempo calculem, com boa precisão, as energias de sistemas multieletrônicos. Dentre os métodos desenvolvidos ao longo dos últimos anos, destacamos o Molecular Fragmentation with Conjugate Caps (MFCC), que calcula a energia de interação entre proteína-proteína e proteína-ligante a partir da decomposição da proteína em fragmentos formados por resíduos de aminoácidos (Zhang e Zhang, 2003a, 2003b). Nas extremidades desses fragmentos, “capas” (caps) são utilizadas para simular o meio externo. As “capas” escolhidas são os resíduos de aminoácidos imediatamente anterior e posterior ao resíduo de interesse. Ademais, átomos de hidrogênio são adicionados para completar a valência daqueles que tiveram as suas ligações “cortadas”. Uma descrição mais detalhada pode ser encontrada em outros trabalhos do grupo (Amaral et al., 2020; da Costa et al., 2012; Morais et al., 2020). Os métodos quânticos utilizados pela técnica de MFCC para o cálculo quântico da energia de interação individual de cada aminoácido presente no sítio de ligação são realizados utilizando-se a Teoria do Funcional da Densidade (DFT, do inglês Density Functional Theory), por meio do software (Delley, 2000, 1990). [0103] Considering the impossibility of analytically solving the Schrodinger equation for systems with many electrons, it is necessary to employ computational methods that optimize the use of processing resources, and at the same time calculate, with good precision, the energies of systems electronics. Among the methods developed over the last few years, we highlight the Molecular Fragmentation with Conjugate Caps (MFCC), which calculates the interaction energy between protein-protein and protein-ligand from the decomposition of the protein into fragments formed by amino acid residues (Zhang and Zhang, 2003a, 2003b). At the ends of these fragments, “caps” are used to simulate the external environment. The "covers" chosen are the amino acid residues immediately preceding and following the residue of interest. Furthermore, hydrogen atoms are added to complete the valence of those that have had their bonds “cut”. A more detailed description can be found in other works by the group (Amaral et al., 2020; da Costa et al., 2012; Morais et al., 2020). The quantum methods used by the MFCC technique for the quantum calculation of the individual interaction energy of each amino acid present at the binding site are performed using the Density Functional Theory (DFT), using the software (Delley, 2000, 1990).
[0104] Ainda, foi feito um levantamento sorológico para detecção de anticorpos anti-IBV em indivíduos que trabalham imunizando aves na indústria avícola, para que estes sejam submetidos a testes de soroneutralização virai. [0104] Also, a serological survey was carried out to detect anti-IBV antibodies in individuals who work immunizing birds in the poultry industry, so that they are subjected to viral seroneutralization tests.
[0105] Coleta de soro e análise dos anticorpos de trabalhadores que manipulam a vacina comercial em indústria avícola. [0105] Serum collection and analysis of antibodies from workers who handle the commercial vaccine in the poultry industry.
[0106] Foram coletadas amostras (N = 10) de soro de pessoas que trabalham com o processo de vacinação em duas Granjas: Granja Serjal (2 amostras) e Granja Santa Lúcia (8 amostras). Todos são funcionários que trabalham diretamente com a imunização das aves. Essa imunização é feita a cada 2-3 meses. Leva-se cinco dias de imunização, isto é, em cinco dias é feita a imunização de todos os galpões. Após a autorização da granja, os funcionários foram apresentados à equipe de coleta e responderam um questionário que compreendia perguntas como idade, gênero, tempo de trabalho com imunização, se portador de comorbidade, se teve COVID19, se foi vacinado contra a COVID19. Todos os participantes responderam um questionário. Finalizado esta etapa foram submetidos a coleta por swab nasofaríngeo para PCR e punção venosa para coleta de soro. [0106] Serum samples (N = 10) were collected from people who work with the vaccination process in two farms: Granja Serjal (2 samples) and Granja Santa Lúcia (8 samples). All are employees who work directly with the immunization of birds. This immunization is done every 2-3 months. It takes five days of immunization, that is, in five days all the sheds are immunized. After authorization from the farm, the employees were introduced to the collection team and answered a questionnaire that included questions such as age, gender, working time with immunization, if they had comorbidity, if they had COVID19, if they were vaccinated against COVID19. All participants answered a questionnaire. After this step, they were submitted to nasopharyngeal swab collection for PCR and venipuncture for serum collection.
[0107] A coleta foi feita in loco, em uma sala adaptada para tal fim, em condições necessárias para uma melhor comodidade tanto para quem coletava quanto para a pessoa que teria a amostra coletada. EPIs foram utilizados. A higienização do local da punção foi feita com álcool a 70%. Utilizou-se tubos tipo vacutainer e contendo heparina e gel separador, além de agulhas descartáveis. Após a coleta, a coagulação da punção foi produzida através do auxílio de curativos apropriados. Foram colhidos aproximadamente 5ml de sangue de cada pessoa a ser testada. Quanto a coleta para PCR, todas as amostras foram coletadas ou por via nasal ou faríngea, a critério da melhor comodidade do indivíduo a ser coletado. Os swabs eram descartáveis e após a coleta eram coletados em uma solução de coleta e devidamente identificados. [0107] The collection was carried out in loco, in a room adapted for this purpose, in conditions necessary for a better convenience for both the collector and the person who would have the sample collected. PPE were used. The puncture site was cleaned with 70% alcohol. Vacutainer-type tubes containing heparin and separator gel, in addition to disposable needles, were used. After collection, puncture coagulation was produced with the aid of appropriate dressings. Approximately 5 ml of blood was collected from each person to be tested. As for PCR collection, all samples were collected either via the nasal or pharyngeal route, at the discretion of the individual to be collected. The swabs were disposable and after collection they were collected in a collection solution and properly identified.
[0108] As amostras foram transportadas em isopor com gelo para o Laboratório de Biotecnologia e Biologia Molecular para a realização dos testes. [0108] The samples were transported in styrofoam with ice to the Biotechnology and Molecular Biology Laboratory for testing.
[0109] Teste de neutralização por redução de placa (PRNT) com soros de pessoas que trabalham com o IBV (cepa Massachutsetts H120) [0109] Plaque Reduction Neutralization Test (PRNT) with sera from people working with IBV (Massachutsetts strain H120)
[0110] Células Vero E6 (6 x 105 por poço) foram semeadas em placas de 12 poços e incubadas a 37°C em meio de cultura L-15 com 2% de soro fetal bovino e 1 % de antibióticos penicilina/estreptomicina (GIBCO). No dia seguinte, 60 PFU do SARS-CoV-2 foram incubados com os soros dos camundongos diluídos em meio L-15 (1 :100) e previamente aquecidos a 56°C por 30 minutos para inativação do sistema complemento. As amostras e o controle positivo (apenas o vírus) permaneceram incubados a 37°C por 1 hora sob leve agitação. A mistura de vírus-soro foi adicionada, em duplicata, às células Vero E6 pré-semeadas com confluência de 90-100%. Após 2 h de incubação a 37°C e agitando a cada 15 minutos as placas, a mistura foi removida e 1 ml do overlay contento 1 ,5% de carboximetilcelulose em L-15 contendo 2% de FBS e 1 % de antibióticos penicilina/estreptomicina (GIBCO) foram adicionados em cada poço. Após 3 dias de incubação a 37°C, 1 ml de formaldeído a 3,65% em PBS foi adicionado nos poços. Decorrido o tempo de 1 hora, a mistura foi removida e as células coradas com 0,5% cristal violeta. As placas foram lavadas com água para remoção do excesso de corante, fotografadas e contadas utilizando o programa Imaged®. A percentagem de neutralização foi calculada utilizando a fórmula: [0110] Vero E6 cells (6 x 10 5 per well) were seeded in 12-well plates and incubated at 37°C in L-15 culture medium with 2% fetal bovine serum and 1% penicillin/streptomycin antibiotics (GIBCO). The following day, 60 PFU of SARS-CoV-2 were incubated with mouse sera diluted in L-15 medium (1:100) and previously heated at 56°C for 30 minutes to inactivate the complement system. The samples and the positive control (virus only) were incubated at 37°C for 1 hour under gentle agitation. The virus-serum mixture was added, in duplicate, to pre-seeded Vero E6 cells at 90-100% confluence. After 2 h of incubation at 37°C and shaking the plates every 15 minutes, the mixture was removed and 1 ml of the overlay containing 1.5% carboxymethylcellulose in L-15 containing 2% FBS and 1% penicillin/antibiotics streptomycin (GIBCO) were added to each well. After 3 days of incubation at 37°C, 1 ml of 3.65% formaldehyde in PBS was added to the wells. After 1 hour, the mixture was removed and the cells stained with 0.5% crystal violet. The plates were washed with water to remove excess dye, photographed and counted using the Imaged® program. The percentage of neutralization was calculated using the formula:
[(amostra x 100) / controle positivo] -100 [(sample x 100) / positive control] -100
[0111] Western Blotting com anticorpos induzidos pela via oral [0111] Western blotting with orally induced antibodies
[0112] No teste de western blotting, foram utilizados o vírus completo SARS- CoV 2 inativado pelo calor. As proteínas virais foram submetidas à eletroforese em gel de poliacrilamida a 4-12% (SDS-PAGE), como descrito anteriormente, e depois transferida para uma membrana de nitrocelulose conforme Towbin et al. (1979). A membrana foi imersa em tampão PBS-molico (5%) contendo os anticorpos produzidos em camundongos contra o IBV vacinai pelas vias oral e intranasal. Foram utilizados anticorpos anti-lgG de camundongo ligado à peroxidase, produzido em cabra da Sigma para revelação. [0112] In the western blotting test, the heat-inactivated SARS-CoV 2 complete virus was used. Viral proteins were subjected to 4-12% polyacrylamide gel electrophoresis (SDS-PAGE), as described above, and then transferred to a nitrocellulose membrane as Towbin et al. (1979). The membrane was immersed in a PBS-molic buffer (5%) containing the antibodies produced in mice against IBV vaccine by the oral and intranasal routes. Peroxidase-produced goat anti-mouse IgG antibodies from Sigma were used for development.
[0113] Análises bioquímicas camundongos imunizados com IBV [0113] Biochemical analysis of mice immunized with IBV
[0114] Os níveis séricos de glicose, colesterol total, triglicerídeos, uréia, creatinina, alanina aminotransferase e aspartato aminotransferase foram mensurados através dos kits de testes bioquímicos (BIOCLIN®), conforme orientação do fabricante. Para esta finalidade, as absorbâncias foram avaliadas através do leitor de microplacas aparelho Sinergy 2 (Biotek®). [0114] Serum levels of glucose, total cholesterol, triglycerides, urea, creatinine, alanine aminotransferase and aspartate aminotransferase were measured using biochemical test kits (BIOCLIN®), as directed by the manufacturer. For this purpose, the absorbances were evaluated through the Synergy 2 (Biotek®) microplate reader.
[0115] A resposta imune celular foi analisada por citometria de fluxo para avaliação de fenótipo de ativação celular, assim como proliferação de linfócitos T e produção de citocinas, após estimulação com antígenos do SARS-Cov-2. [0115] Cellular immune response was analyzed by flow cytometry to assess cell activation phenotype, as well as T lymphocyte proliferation and cytokine production, after stimulation with SARS-Cov-2 antigens.
[0116] Além disso, foram feitas análises histopatológicas de vários órgãos incluindo pulmão e órgãos linfóides secundários (baço e linfonodos). [0116] In addition, histopathological analyzes were performed on various organs including lung and secondary lymphoid organs (spleen and lymph nodes).
[0117] Histologia (etapa em desenvolvimento) [0117] Histology (development stage)
[0118] O material em estudo é proveniente de amostras de pulmão fixados em formalina tamponada, incluídos em parafina, cortados ao micrótomo com espessura de 4 pm e submetidos à coloração automática em Hematoxilina & Eosina. As lâminas coradas são examinadas por um único patologista quanto à existência de sinais de lesão inflamatória. [0118] The material under study comes from lung samples fixed in buffered formalin, embedded in paraffin, cut with a microtome with a thickness of 4 pm and subjected to automatic staining in Hematoxylin & Eosin. Stained slides are examined by a single pathologist for signs of inflammatory lesion.
[0119] Montagem do Tissue Microarrav [0119] Tissue Microarrav Assembly
[0120] Os blocos de parafina contendo amostras representativas de pulmão são utilizados para a retirada de um fragmento cilíndrico (bloco doador) mediante uso de uma agulha de 2 mm de diâmetro (Quick Ray, UNITMA®) e transferência para um bloco receptor de 60 poços. O bloco receptor é organizado seguindo a sequência: 4 casos de controles positivos nos vértices, compostos por tecidos apropriados indicados na bula dos anticorpos; adicionados de 28 casos, em duplicata. As etapas básicas do processo imunohistoquímico são: [0120] The paraffin blocks containing representative lung samples are used to remove a cylindrical fragment (donor block) using a 2 mm diameter needle (Quick Ray, UNITMA®) and transfer it to a 60 wells. The receptor block is organized following the sequence: 4 cases of positive controls in the vertices, composed of appropriate tissues indicated in the antibody package insert; added from 28 cases, in duplicate. The basic steps of the immunohistochemical process are:
1 . Fixação e inclusão dos tecidos; 1 . Fixation and inclusion of tissues;
2. Corte e montagem das secções de tecido; 2. Cutting and assembling the tissue sections;
3. Desparafinização e reidratação dos cortes; 3. Deparaffinization and rehydration of the cuts;
4. Recuperação antigênica; 4. Antigen recovery;
5. Coloração imunoistoquímica; 5. Immunohistochemical staining;
6. Contra-coloração, se desejado; 6. Counterstain, if desired;
7. Desidratação e estabilização com montagem; 7. Dehydration and stabilization with mounting;
8. Observação ao microscópio. 8. Microscope observation.
[0121] Devido ao processo de fixação, é aplicado um tratamento de recuperação antigênica para expor os epítopos através do calor, sendo utilizados anticorpos primários anti-TNF-alfa, IL-1 beta. [0121] Due to the fixation process, an antigen recovery treatment is applied to expose the epitopes through heat, using antibodies anti-TNF-alpha, IL-1 beta primers.
[0122] Imunohistoquimica [0122] Immunohistochemistry
[0123] Resumidamente, as peças histológicas são desparafinizadas e em seguida hidratadas. Após a hidratação as peças são imersas em tampão citrato 0,1 M (pH 6.0) e aquecidas a aproximadamente 100oC durante 15 min, seguido de resfriamento em temperatura ambiente durante 20 min. Em seguida são lavadas em PBS. O bloqueio da peroxidase endógena é será realizado com solução peróxido de hidrogênio 3% durante 15 min. Após lavagem com PBS, os anticorpos primários são diluídos em PBS - BSA 5% e as amostras são incubadas por 12h. Em seguida, as lâminas são lavadas com PBS e incubadas (30 min) com o anticorpo secundário biotinilado diluído em PBS-BSA 5%. Após a remoção do anticorpo biotinilado, com lavagem em PBS, as lâminas são incubadas por 30 min com o complexo ABC (Horseradish Peroxidase Standard). As lâminas são, então, lavadas em PBS e incubadas com DAB/peróxido (2 min) para revelação da reação. Após remoção do DAB com água destilada, é feita a contra-coloração com hematoxilina de Harry’s, desidratação e montagem das lâminas. [0123] Briefly, the histological pieces are deparaffinized and then hydrated. After hydration, the pieces are immersed in 0.1 M citrate buffer (pH 6.0) and heated at approximately 100oC for 15 min, followed by cooling at room temperature for 20 min. Then they are washed in PBS. Blocking of endogenous peroxidase is performed with 3% hydrogen peroxide solution for 15 min. After washing with PBS, the primary antibodies are diluted in PBS - 5% BSA and the samples are incubated for 12h. Then the slides are washed with PBS and incubated (30 min) with the biotinylated secondary antibody diluted in 5% PBS-BSA. After removing the biotinylated antibody, washing in PBS, the slides are incubated for 30 min with the ABC complex (Horseradish Peroxidase Standard). Slides are then washed in PBS and incubated with DAB/peroxide (2 min) to reveal the reaction. After removing the DAB with distilled water, counterstaining with Harry's hematoxylin, dehydration and mounting of the slides are performed.
[0124] Imunofenotipagem [0124] Immunophenotyping
[0125] O perfil Th1 é avaliado em amostras de sangue e de baço obtidos de camundongo e de hamster sírio através de imunofenotipagem por citometria de fluxo. Resumidamente, após a coleta das amostras e lise das hemácias, antígenos para identificação de células T (CD4+ e CD8+), os linfócitos são marcados com anticorpos conjugados a fluorocromos. Marcadores para antígenos como CD3, CD4, CD8, T -bet e CD1 19 são utilizados para identificação de subpopulações. Após a permeabilização da membrana plasmática e incubação das células com os anticorpos por 30 min a 4SC no escuro, as células são lavadas com tampão de FACS, ressuspensas e dez mil eventos, excluindo debris e doublets, são adquiridos no citômetro de fluxo FACS Verse. Os percentuais de células CD8+ e CD4+, bem como de CD4+/T-bet+/CD1 19+, são analisados através de gates criados levando em consideração os tubos dos controles isotípicos no software FlowJo, LCC (v.10). RESULTADOS OBTIDOS NA FASE PRÉ-CLÍNICA [0125] The Th1 profile is evaluated in blood and spleen samples obtained from mice and Syrian hamsters by immunophenotyping by flow cytometry. Briefly, after collection of samples and lysis of red blood cells, antigens for identification of T cells (CD4+ and CD8+), the lymphocytes are labeled with antibodies conjugated to fluorochromes. Markers for antigens such as CD3, CD4, CD8, T-bet and CD1 19 are used to identify subpopulations. After permeabilization of the plasma membrane and incubation of the cells with the antibodies for 30 min at 4 ° C in the dark, the cells are washed with FACS buffer, resuspended and ten thousand events, excluding debris and doublets, are acquired on the FACS flow cytometer. See if. The percentages of CD8+ and CD4+ cells, as well as CD4+/T-bet+/CD1 19+, are analyzed through gates created taking into account the tubes of the isotypic controls in FlowJo software, LCC (v.10). RESULTS OBTAINED IN THE PRE-CLINICAL PHASE
[0126] Biologia computacional [0126] Computational biology
[0127] Na análise do sequenciamento dos dois coranavírus (IBV e SARS-Cov- 2), os domínios RBD do IBV para as quatro cepas estudadas são completamente conservados entre si, como pode ser visto na Figura 10(a-b). Por outro lado, na mesma Figura, são destacadas duas mutações que ocorrem entre as diferentes cepas do SARS-CoV-2 localizadas nas posições 484 e 501 na região do RBD da proteína do vírus. A comparação entre os dois vírus mostra duas regiões: (i) 454- 502: 5 resíduos conservados, 7 com alta similaridade e 8 com alguma similaridade; (ii) 503-561 : 13 resíduos conservados, 8 com alta similaridade e 9 com alguma similaridade. É importante destacar que a região (i) contém praticamente os três hot spots de interação entre o RBD do SARS-CoV-2 com o sítio de ligação da ACE2 humana, conforme o mapa das energias de interação na Figura 10 (c) obtido em simulações de bioquímica quântica em estudos realizados pelo grupo e que estão na fase de preparação do artigo. De modo que essas duas regiões se mostram como potenciais alvos dos anticorpos que bloqueiam o mecanismo de invasão da célula pelo vírus ao se ligar no RBD do SARS-CoV-2 e, portanto, competindo com o sítio de ligação da ACE2. [0127] In the sequencing analysis of the two coranaviruses (IBV and SARS-Cov-2), the RBD domains of the IBV for the four strains studied are completely conserved with each other, as can be seen in Figure 10(a-b). On the other hand, in the same Figure, two mutations that occur between the different strains of SARS-CoV-2 located at positions 484 and 501 in the RBD region of the virus protein are highlighted. The comparison between the two viruses shows two regions: (i) 454-502: 5 conserved residues, 7 with high similarity and 8 with some similarity; (ii) 503-561 : 13 conserved residues, 8 with high similarity and 9 with some similarity. It is important to highlight that region (i) contains practically the three hot spots of interaction between the SARS-CoV-2 RBD with the human ACE2 binding site, according to the interaction energy map in Figure 10 (c) obtained in quantum biochemistry simulations in studies carried out by the group and which are in the preparation phase of the article. Thus, these two regions are shown to be potential targets for antibodies that block the cell invasion mechanism by the virus by binding to the SARS-CoV-2 RBD and, therefore, competing with the ACE2 binding site.
[0128] Em análises preliminares da interação do RBD do IBV com anticorpos neutralizantes (nAbs) ativos contra o SARS-CoV-2 (Figura 1 1 (a-i)), foram obtidas as energias de interações significativas, sempre na região do RBD. Das estruturas analisadas, da estrutura 101 a 1 1 1 , somente a estrutura 106 e a estrutura 1 11 não se ligaram na região do RBD e, portanto, foram descartadas. Além disso, merecem atenção as estruturas 106, 108 e 101 por seus valores de energia de interação mais elevados. No momento, estão em andamento cálculos de Dinâmica Molecular que proporcionam uma melhor compreensão dos sítios de ligação, a partir dos quais teremos uma base mais sólida para a realização dos cálculos de Bioquímica Quântica para o detalhamento das interações resíduo a resíduo dentro do arcabouço da Mecânica Quântica para esses sistemas. [0128] In preliminary analyzes of the interaction of the IBV RBD with neutralizing antibodies (nAbs) active against SARS-CoV-2 (Figure 1 1 (ai)), the energies of significant interactions were obtained, always in the region of the RBD. Of the analyzed structures, from structure 101 to 111, only structure 106 and structure 111 did not bind in the RBD region and, therefore, were discarded. Furthermore, structures 106, 108 and 101 deserve attention for their higher interaction energy values. Molecular Dynamics calculations are currently underway that provide a better understanding of the binding sites, from which we will have a more solid basis for performing Quantum Biochemistry calculations for the detailing of residue-to-residue interactions within the framework of Mechanics Quantum for these systems.
[0129] Soroneutralização de trabalhadores das granjas [0129] Seroneutralization of farm workers
[0130] Na Figura 12 é possível observar os resultados da soroneutralização de cada trabalhador comparado aos controles positivo e negativo. [0130] In Figure 12 it is possible to observe the results of serum neutralization of each worker compared to positive and negative controls.
[0131 ] Perfil hematológico e bioquímico de camundongos Swiss no final do experimento de imunização com o IBV [0131] Hematological and biochemical profile of Swiss mice at the end of the IBV immunization experiment
[0132] A quantificação dos marcadores bioquímicos consiste em uma importante ferramenta de avaliação dos padrões de normalidade experimental (Amorim, 2003). [0132] The quantification of biochemical markers is an important tool for evaluating patterns of experimental normality (Amorim, 2003).
[0133] Conforme pode ser observado na Figura 13, os níveis séricos de glicose (13A), colesterol total (13B) e triglicerídeos (13C) dos camundongos imunizados com IBV não apresentaram diferenças significativas quando comparados aos referidos níveis dos grupos controles (p > 0,05). [0133] As can be seen in Figure 13, the serum levels of glucose (13A), total cholesterol (13B) and triglycerides (13C) of mice immunized with IBV showed no significant differences when compared to the aforementioned levels of the control groups (p > 0.05).
[0134] Sendo assim, a imunização com IBV não foi capaz de alterar as concentrações séricas dos biomarcadores citados frente ao grupo de referência, após o final do experimento. [0134] Therefore, immunization with IBV was not able to change the serum concentrations of the biomarkers cited in relation to the reference group, after the end of the experiment.
[0135] Além dos dados supracitados, outros biomarcadores dos camundongos imunizados com IBV foram avaliados e também se destacaram positivamente (Figura 14). Os níveis séricos de ureia (14A), creatinina (14B), aspartato aminotransferase (14C) e alanina aminotransferase (14D) foram quantificados e mostraram-se similares frente aos seus respectivos grupos controles, sem apresentar redução ou aumento significativo entre eles (p > 0.05). [0135] In addition to the aforementioned data, other biomarkers of IBV-immunized mice were evaluated and also stood out positively (Figure 14). Serum levels of urea (14A), creatinine (14B), aspartate aminotransferase (14C) and alanine aminotransferase (14D) were quantified and were similar to their respective control groups, with no significant decrease or increase between them (p > 0.05).
[0136] Assim como nos dados anteriores, esses grupos de biomarcadores dos perfis renal e hepático apresentam similaridades com os animais do grupo controle, indicando, desta forma, padrão de normalidade no presente teste e sinaliza para a ratificação da segurança vacinai para as fases posteriores (T abela 1 )-[0136] As in the previous data, these groups of biomarkers of the renal and hepatic profiles present similarities with the animals of the control group, thus indicating a pattern of normality in the present test and signals for the ratification of the vaccine safety for the later phases (Table 1)-
[0137] Tabela 1 : Análise dos parâmetros bioquímicos e marcadores de danos hepático e renal nos soros de pessoas que trabalham em avicultura, manuseando o IBV nos processos de vacinação das aves, com o objetivo de verificar possíveis danos causados pelo IBV. As coletas foram realizadas no período da manhã, sem recomendação de jejum.
Figure imgf000028_0001
[0137] Table 1: Analysis of biochemical parameters and markers of liver and kidney damage in the sera of people who work in poultry, handling IBV in the processes of vaccination of birds, in order to verify possible damage caused by the IBV. The collections were performed in the morning, without fasting recommendation.
Figure imgf000028_0001
Legenda: AST, Aspartato aminotransferase; ALT, Alanina aminotransferase Legend: AST, Aspartate aminotransferase; ALT, Alanine Aminotransferase
[0138] Outro fator importante que merece ser destacado são os três tipos de vias de imunização concretizados na presente invenção. Os resultados não apresentaram distinções significativas, indicando que as rotas experimentais das imunizações que foram adotadas não interferem nos parâmetros essenciais, conforme apresentado previamente. [0138] Another important factor that deserves to be highlighted are the three types of immunization routes embodied in the present invention. The results did not present significant distinctions, indicating that the experimental routes of the immunizations that were adopted do not interfere in the essential parameters, as previously presented.
[0139] A composição vacinai da presente invenção para aplicação em humanos compreende concentração viral de aproximadamente 400 a aproximadamente 4000 vírus por dose. [0139] The vaccine composition of the present invention for human application comprises viral concentration of approximately 400 to approximately 4000 viruses per dose.
[0140] Os versados na arte valorizarão os conhecimentos aqui apresentados e poderão reproduzir a invenção nas modalidades apresentadas e em outras variantes e alternativas, abrangidas pelo escopo das reivindicações a seguir. [0140] Those skilled in the art will value the knowledge presented here and may reproduce the invention in the modalities presented and in other variants and alternatives, covered by the scope of the following claims.

Claims

27 Reivindicações 27 Claims
1 . Uso do vírus da bronquite infecciosa (IBV) caracterizado por ser para a fabricação de uma vacina para a imunização de mamíferos contra coronavirus, SARS-CoV-2 e/ou vírus relacionados. 1 . Use of infectious bronchitis virus (IBV) characterized by being for the manufacture of a vaccine for the immunization of mammals against coronavirus, SARS-CoV-2 and/or related viruses.
2. Uso de acordo com a reivindicação 1 caracterizado pelo fato de que o IBV é da cepa Massachussetts H120. 2. Use according to claim 1, characterized in that the IBV is from the Massachussetts H120 strain.
3. Uso de acordo com a reivindicação 1 ou 2 caracterizado pelo fato de que fato de que IBV é vivo e atenuado. 3. Use according to claim 1 or 2 characterized by the fact that IBV is live and attenuated.
4. Vacina para a imunização de mamíferos contra coronavirus, SARS- CoV-2 e/ou vírus relacionados caracterizada por compreender pelo menos um vírus da bronquite infecciosa (IBV) e um veículo ou diluente farmaceuticamente aceitável. 4. Vaccine for immunizing mammals against coronavirus, SARS-CoV-2 and/or related viruses characterized in that it comprises at least one infectious bronchitis virus (IBV) and a pharmaceutically acceptable carrier or diluent.
5. Vacina compreendendo pelo menos um vírus da bronquite infecciosa (IBV) e um veículo ou diluente farmaceuticamente aceitável caracterizada por ser para uso na imunização em mamíferos contra coronavirus. 5. A vaccine comprising at least one infectious bronchitis virus (IBV) and a pharmaceutically acceptable carrier or diluent characterized for use in immunizing mammals against coronaviruses.
6. Vacina de acordo com a reivindicação 5 caracterizada pelo fato do coronavirus ser o SARS-CoV-2. 6. Vaccine according to claim 5, characterized in that the coronavirus is SARS-CoV-2.
7. Vacina de acordo com qualquer uma das reivindicações 4 a 6 caracterizada pelo fato do IBV ser vivo e atenuado. 7. Vaccine according to any one of claims 4 to 6, characterized in that the IBV is live and attenuated.
8. Vacina de acordo com qualquer uma das reivindicações 4 a 7 caracterizada pelo fato do IBV ser da cepa Massachussetts H120. 8. Vaccine according to any one of claims 4 to 7, characterized in that the IBV is from the Massachussetts H120 strain.
9. Vacina de acordo com qualquer uma das reivindicações 4 a 8 caracterizada pelo fato de adicionalmente compreender um adjuvante. 9. Vaccine according to any one of claims 4 to 8, characterized in that it additionally comprises an adjuvant.
10. Vacina de acordo com qualquer uma das reivindicações 4 a 9 caracterizada por se apresentar em forma de administração por via oral ou sublingual; nasal; ocular; subcutânea ou intramuscular. Vaccine according to any one of claims 4 to 9, characterized in that it is in the form of oral or sublingual administration; nasal; eyepiece; subcutaneous or intramuscular.
1 1. Vacina de acordo com qualquer uma das reivindicações 4 a 10 caraterizada por compreender concentração viral de 400 a 4000 vírus por dose para aplicação em humanos. A vaccine according to any one of claims 4 to 10 characterized in that it comprises a viral concentration of 400 to 4000 viruses per dose for application in humans.
12. Método de imunização de mamíferos contra coronavirus, SARS-CoV- 2 e/ou vírus relacionados caracterizado por compreender a administração de um vírus da bronquite infecciosa (IBV) e um veículo ou diluente farmaceuticamente aceitável. 12. A method of immunizing mammals against coronavirus, SARS-CoV-2 and/or related viruses comprising administering an infectious bronchitis virus (IBV) and a pharmaceutically acceptable carrier or diluent.
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