RECOMBINANT VACCINE AGAINST COVID-19 IN PARAMYXOVIRUS VIRAL VECTOR

Abstract

The present invention relates to a viral vector capable of generating a cellular immune response against a spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), characterized in that the viral vector is a Newcastle disease virus (NDV) comprising a nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 14 and an exogenous nucleotide sequence encoding antigenic sites of SARS-CoV-2, wherein the exogenous nucleotide sequence comprises a sequence having at least 95% sequence identity with a sequence that translates into the amino acid sequence of SEQ ID NO: 11, wherein the exogenous nucleotide sequence comprises an ectodomain of a SARS-CoV-2 S protein fused with a transmembrane domain and a cytoplasmic domain of an NDV fusion (F) protein, wherein the polybasic cleavage site of the ectodomain of the S protein is mutated to alanine and amino acids 817, 892, 899, 942,986 and 987 of the ectodomain of the S protein of SEQ ID NO: 11 are prolines, such that the viral vector and antigenic sites are stable after at least 3 consecutive passages in chicken embryo.

Description

RECOMBINANT COVID-19 VACCINE IN PARAMYXOVIRUS VIRAL VECTOR FIELD OF INVENTION The present invention relates to techniques used in the prevention and control of coronavirus disease 2019 (COVID-19), and more particularly to a recombinant viral vector vaccine having an inserted exogenous nucleotide sequence encoding proteins with antigenic activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and a pharmaceutically acceptable vehicle, adjuvant and / or excipient. BACKGROUND OF THE INVENTION Coronaviruses (CoV) are a family of viruses that cause the common cold and severe illnesses such as Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS-CoV). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) outbreak, which began in December 2019 in Wuhan, China. On March 11, 2020, the World Health Organization (WHO) declared COVID-19 a pandemic. Currently, there are no medications or vaccines available to treat COVID-19, and a significant number of deaths have been reported, primarily among elderly patients with comorbidities. As of May 4, 2020, more than 3.4 million cases had been recorded worldwide, with over 239,000 deaths, and these numbers continue to rise, mainly in Europe and the United States, countries with a larger proportion of elderly populations who have contracted the infection. To date, the only effective measures to counter the spread of COVID-19 are isolating the population, quarantining infected individuals, suspending most commercial activities and businesses, and providing intensive clinical therapy for patients with severe symptoms. However, the adoption of such containment measures has dramatically impacted the economies of all countries currently battling this pandemic. In the search for a solution to this emerging infectious disease, vector vaccines offer an active (live) vaccine approach that does not involve the whole pathogen. According to information from the WHO (https: / / www.who.int / blueprint / prioritvdiseases / kev-action / novel-coronavirus-landscape-ncov.pdf, accessed May 4, 2020), some institutions and pharmaceutical companies are developing recombinant vaccines against nC7fr ίΠ / 77Ω7 / E / YILI COVID-19 vaccines based on human adenovirus vectors, MVA, VSV, and measles, among others. Previously, several vectored vaccines using these vectors were described for SARS-CoV. However, one group found that ferrets immunized with an MVA / SARS-CoV vaccine developed hepatitis (CZUB, Markus, et al. Evaluation of modified vaccinia virus Ankara based recombinant SARS vaccine in ferrets. Vaccine, 2005, vol. 23, no. 17-18, pp. 2273-2279). SARS-CoV vaccine constructs based on the replication of a defective human adenovirus type 5 expressing a partial or total SARS-CoV spike glycoprotein S have been evaluated for immunogenicity in rats and monkeys (LIU, Ran-Yi, et al. Adenoviral expression of a truncated SI subunit of SARS-CoV spike protein results in specific humoral immune responses against SARS-CoV in rats. Virus research, 2005, vol. 112, no 1-2, p. 24-31.; and GAO, Wentao, et al.Effects of a SARS-associated coronavirus vaccine in monkeys. The Lancet, 2003, vol. 362, no 9399, p. 1895-1896.), but immunization depends on a high dose of vaccine, and safety and protective efficacy have not been demonstrated. An attenuated version of human parainfluenza virus type 3, a common pediatric respiratory pathogen, has also been described to express the SARS-CoV spike glycoprotein S, of which a single intranasal and intratracheal inoculation was shown to be immunogenic and protective against SARS-CoV in a challenge in hamsters and African green monkeys (BISHT, Himani, et al. Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice. Proceedings of the National Academy of Sciences, 2004, vol. 101, no. 17, p. 6641–6646).However, a concern with any vector based on a common pathogen is that the adult population has significant immunity from prior exposure, which will restrict viral vector infection and replication, thus reducing its immunogenicity. Indeed, comparisons of the immunogenicity of vaccines vectored with vaccinia virus and those vectored with human adenovirus type 5 in rodents, non-human primates, and humans demonstrated that pre-existing immunity to the vector greatly reduced the immunogenicity of these vaccines (KANESA-THASAN, Niranjan, et al. Safety and immunogenicity of NYVAC-JEV and ALVAC-JEV attenuated recombinant Japanese encephalitis virus—poxvirus vaccines in vaccinia-nonimmune and vaccinia-immune humans. Vaccine, 2000, vol. 19, no. 4–5, pp. 483–491; SHARPE, Sally, et al.Induction of simian immunodeficiency virus (SlV)-specific CTL in rhesus macaques by vaccination with modified vaccinia virus Ankara expressing SIV transgenes: influence of pre-existing anti-vector immunity. Journal of General Virology, 2001, vol. 82, no 9, p. 2215-2223.; y ZHI, Yan, et al. Efficacy of severe acute respiratory syndrome vaccine based on a nonhuman primate adenovirus in the presence of immunity against human adenovirus. Human gene therapy, 2006, vol. 17, no 5, p. 500-506.). On the other hand, Newcastle disease virus (NDV) has been described as a vector that can potentially be used to develop human vaccine vectors, nC7fr ίΠ / 77Ω7 / E / YILI, as for example in patent documents WO2011059334, US9476033, or US10308913. NDV is a non-segmented, negative-stranded RNA virus of the Paramyxoviridae family, and its natural hosts are birds, making it antigenically distinct from common human pathogens. The group of DiNapoli et al., 2007 (DINAPOLI, Joshua M., et al. Newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens. Proceedings of the National Academy of Sciences, 2007, vol. 104, no 23, p. 9788-9793.A study evaluated a non-dose viral vector (NDV) expressing the SARS-CoV spike glycoprotein (S) as a topical respiratory vaccine vector, targeting SARS-CoV, by direct viral analysis of nasal and lung tissues collected at necropsy during peak SARS-CoV replication. African green monkeys immunized via the respiratory tract with two doses of this vaccine developed SARS-CoV neutralizing antibody titers comparable to the secondary response observed in animals immunized with a different experimental SARS-CoV vaccine and challenged with SARS-CoV. When animals immunized with the NDV expressing the S spike glycoprotein were challenged with a high dose of SARS-CoV, direct viral analysis of lung tissues obtained at necropsy during peak viral replication demonstrated an average reduction of 236, or 1,102-fold, in lung SARS-CoV titers compared to control animals.However, the SARS-CoV spike glycoprotein (S) exhibits significant differences from that of SARS-CoV-2 (WALLS, Alexandra C., et al. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell, 2020). Coronavirus spike (S) glycoproteins promote cell entry and are the primary target of antibodies. According to Wall et al. (2020), the SARS-CoV-2 spike glycoprotein harbors a furin cleavage site at the boundary between the SI / S2 subunits, which is processed during biogenesis and significantly differentiates this virus from SARS-CoV and SARS-related coronaviruses. This is the first time a coronavirus with a polybasic cleavage site for a protease has been described.Furthermore, the spike S glycoprotein has a metastable prefusion conformation, which transforms into a highly stable postfusion conformation, facilitating membrane fusion but making it very difficult to produce recombinantly. Therefore, it is not possible to know or deduce whether a recombinant vectored COVID-19 vaccine based on NDV or any other virus will be effective for the treatment or prevention of COVID-19, nor whether the construction of a viral vector that includes the SARS-CoV-2 spike S glycoprotein will be stable—that is, retain its ability to replicate—after several consecutive passages in cell lines to achieve a viral titer suitable for industrial-scale vaccine production. nC7fr ίΠ / 77Ω7 / Β / YΙΛΙ Furthermore, the most effective way to insert SARS-CoV-2 genes into a recombinant vaccine to produce an immune response that allows the pandemic to be controlled has not been determined, much less in a Newcastle disease virus vector. Therefore, it is absolutely necessary to develop a COVID-19 vaccine that provides a sufficient level of protection for effective control of the current pandemic. OBJECTS OF THE INVENTION Taking into account the defects of the prior art, it is an object of the present invention to provide an effective recombinant paramyxovirus viral vector vaccine against coronavirus disease 2019 (COVID-19). Another object of the present invention is to provide for the use of a recombinant paramyxovirus viral vector vaccine for the control of COVID-19. It is a further object of the present invention to provide a paramyxovirus viral vector construct with an inserted exogenous nucleotide sequence encoding proteins with antigenic activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is stable after being subjected to consecutive passages in a cell line. These and other objectives are achieved through a recombinant COVID-19 vaccine in a paramyxovirus viral vector in accordance with the present invention. BRIEF DESCRIPTION OF THE INVENTION To this end, a recombinant vaccine has been invented comprising a viral vector based on the Newcastle disease virus, which has inserted an exogenous nucleotide sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), capable of generating a cellular immune response, and a pharmaceutically acceptable vehicle, adjuvant and / or excipient. DETAILED DESCRIPTION OF THE INVENTION During the development of the present invention, it has been unexpectedly found that a recombinant vaccine comprising a paramyxovirus viral vector capable of generating a cellular immune response having inserted an exogenous nucleotide sequence encoding for antigenic sites of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), and a pharmaceutically acceptable vehicle, adjuvant and / or excipient, provides adequate protection against coronavirus disease 2019 (COVID-19). nC7fr ίη / 77Π7 / E / YΙΛΙ The viral vector used may be active (live) or inactivated (dead). Inactivation means that the recombinant virus functioning as a viral vector, containing the nucleotide sequence that codes for SARS-CoV-2 antigenic sites, has lost the ability to replicate. Inactivation is achieved through well-known physical or chemical procedures, preferably chemical inactivation with formaldehyde or beta-propiolactone (Office International des Epizooties 2008). Newcastle Disease. OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Office International des Epizooties. France, pp. 576–589). Conversely, an active or live virus is understood to retain its capacity to replicate. Preferably, the viral vector used is a paramyxovirus, selected from any paramyxovirus, including any serotype, genotype, or genetic class, including lentogenic, mesogenic, and velogenic viruses. It is also preferable to use paramyxoviruses that can be reverse genetically modified to remove phenylalanine at position 117 and basic amino acids near position Q114, which confer pathogenicity to paramyxoviruses, or paramyxoviruses belonging to the genus Avulavirus that infect birds, such as Newcastle disease virus (NDV) or Sendai virus. More preferably, the viral vector is NDV, and this viral vector is preferably selected from lentogenic or mesogenic strains, such as LaSota, Bl, QV4, Ulster, Roakin, or Komarov strains. Preferably, the recombinant virus is from the LaSota strain.Even more preferably, the NDV viral vector comprises SEQ ID NO: 6 or SEQ ID NO: 14. With regard to the exogenous nucleotide sequence encoding antigenic sites of SARS-CoV-2, in the present invention the nucleotide sequence used is preferably selected from a sequence encoding the SARS-CoV-2 spike glycoprotein (S) or a sequence encoding a sequence derived therefrom. The SARS-CoV-2 spike glycoprotein (S) comprises two functional subunits responsible for binding to the host cell receptor (subunit S1) and for fusion of the viral and cellular membranes (subunit S2). In a preferred embodiment of the invention, the exogenous nucleotide sequence encoding antigenic sites of SARS-CoV-2 is selected from a sequence encoding the S1 subunit of the SARS-CoV-2 spike glycoprotein, a sequence encoding the S2 subunit of the SARS-CoV-2 spike glycoprotein,a sequence encoding both SI and S2 subunits of the SARS-CoV-2 spike glycoprotein S, a sequence encoding at least a fragment of either the SI or S2 subunits of the SARS-CoV-2 spike glycoprotein S, a sequence with at least 80% identity with the sequence encoding the SI subunit of the SARS-CoV-2 spike glycoprotein S, a sequence with at least 80% identity with the sequence encoding the S2 subunit of the SARS-CoV-2 spike glycoprotein S, a sequence with at least 80% identity with a sequence encoding both SI and S2 subunits of the SARS-CoV-2 spike glycoprotein S, a sequence with at least 80% identity with a sequence encoding at least less a fragment of the SI or S2 subunits of the SARS-CoV-2 spike glycoprotein S,a sequence encoding the two SI and S2 subunits of the SARS-CoV-2 spike S glycoprotein devoid of at least one epitope located between the nucleotides corresponding to amino acids 1 to 460 of the SI sequence, a sequence encoding the SI subunit of the SARS-CoV-2 spike S glycoprotein devoid of at least one epitope located between the nucleotides corresponding to amino acids 1 to 460 of the SI sequence, or a sequence encoding the two SI and S2 subunits of the SARS-CoV-2 spike S glycoprotein stabilized in its prefusion form by the inclusion of at least two proline substitutions in the S2 subunit. In a preferred embodiment, the epitope located between the nucleotides corresponding to amino acids 1 to 460 of the SI sequence is selected from the amino acid sequences SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In another preferred embodiment,The exogenous nucleotide sequence encoding antigenic sites of SARS-CoV-2 is selected from a sequence with at least 80% identity to any of the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In an additional preferred embodiment, the sequence encoding the two SI and S2 subunits of the SARS-CoV-2 spike glycoprotein S, stabilized in its prefusion form by the inclusion of at least two proline substitutions in the S2 subunit, is selected from a sequence with at least 80% identity to any sequence translating to any of the amino acid sequences SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. The exogenous nucleotide sequence encoding SARS-CoV-2 antigenic sites of the vaccine of the present invention can be prepared by chemical synthesis of the nucleotide sequence of interest for subsequent insertion into the NDV viral vector. The insertion of the exogenous nucleotide sequence is performed using standard molecular biology cloning techniques and can be inserted into any of the intergenic regions of the NDV genome. The infectious clone thus produced is transfected into a cell culture for the generation of the recombinant virus or origin virus. The virus is replicated through consecutive passages in any suitable growth system, such as SPF chicken embryos, or commercial cell lines or cell lines specifically designed to grow viruses, until the viral concentration required to achieve an antigenic response is reached, preferably between 10⁶⁰ and 10¹⁰ EID₅₀% / mL. It is preferable that the virus be stable after at least three consecutive passages in the growth system once retrieved from cell culture, so that stable production at an industrial scale can be achieved.For virus isolation, the virus is removed from the nC7fr ίΠ / 77Ω7 / Β / YILI system suitable for its growth and separated from cellular or other components, typically by well-known clarification procedures such as filtration, ultrafiltration, gradient centrifugation, ultracentrifugation and column chromatography, and may be further purified as desired using well-known procedures, e.g., plate assays. In the active vaccine form, the vaccine consists of a naturally lentogenic live vaccine virus or one attenuated using procedures known to the prior art. In the inactivated vaccine form, once the viral concentration required to elicit an antigenic response has been reached, the virus is inactivated. Preferably, inactivation is carried out using physical or chemical processes known to the prior art, preferably chemical inactivation with formaldehyde, beta-propiolactone, or binary ethyleneimine (BE L). Pharmaceutically acceptable vehicles for the vaccines of the present invention are preferably aqueous solutions or emulsions. More particularly, aqueous solutions are preferred for live virus vaccines, and preferably the vehicle used for inactivated vaccines is compatible with an immunoadjuvant used to enhance the immune response of the inactivated vaccine. In a further embodiment in which the vaccine is inactivated, it is preferably accompanied by a pharmaceutically acceptable adjuvant. In the embodiment using an adjuvant, squalene-based adjuvants are preferred, preferably those designated MF-59®, AddaVax®, or AS03®, TLR-9 receptor agonists such as CpG-1018, or cationic lipids such as R-DOTAP. Regarding vaccine administration, it can be done intramuscularly, intranasally, subcutaneously, by spray, or nebulization, using the appropriate methods and techniques for each case and depending on whether it is an active or inactivated vaccine. Preferably, the vaccine is administered at least once intramuscularly and / or intranasally. In a particularly preferred modality, the vaccine is administered at least twice to generate a higher immune response, either by maintaining the same route of administration or by changing the route, with a virus concentration preferably between 10⁶⁰ and 10⁸ × 5⁵ EID₅₀% / mL per dose, depending on the volume of vaccine administered according to the selected route. Preferably, the vaccine is administered twice intramuscularly in either its active or inactivated form, twice intranasally in its active form, or once intranasally followed by once intramuscularly.The administration of the vaccines in the twice-administered modality can be carried out within a period of 7 to 35 days between the first and second administration, preferably within a period of 14 to nC7fr ίΠ / 77Ω7 / B / YILI days between the first and second administration, and more preferably the first time is administered in its active form intranasally and the second time intramuscularly, either in its active or inactivated form. In another aspect of the present invention, it has been found that it is possible to administer intranasally a dose of an active virus comprising antigenic sites of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), preferably the recombinant paramyxovirus of the present invention, followed by a second intramuscular dose of a SARS-CoV-2 antigen, achieving a highly effective immune response. Preferably, the antigen of the second dose is the same active virus as the first dose, but once immunization has been performed intranasally, a person skilled in the art may infer that it is possible to administer any other SARS-CoV-2 antigen intramuscularly. Even more preferably, the antigen of the second dose is the same virus as the first dose in its inactivated form. Preferably, the vaccine of the present invention is formulated with a volume of 0.5 ml per dose containing the appropriate concentration of the virus for intramuscular administration, either in its active or inactivated form. In the intranasal administration modality, the preferred volume per dose is 0.2 ml. The vaccine, in accordance with the principles of the present invention, additionally, does not cause adverse events in mammals. The present invention will be better understood from the following examples, which are presented for illustrative purposes only to allow a full understanding of the preferred embodiments of the present invention, without implying that there are no other unillustrated embodiments that can be put into practice based on the detailed description above. EXAMPLE 1 NDV Vector Generation La Sota To clone the RNA genome of the NDV strain LaSota and thus generate a viral vector in the form of plasmid DNA designated pLS11801140 (SEQ ID NO: 6), total viral RNA was first extracted from the NDV strain LaSota using the triazololysis method. From the purified RNA, cDNA (complementary DNA) of the viral genome was synthesized using the previously purified total RNA as a template. To clone all the genes of the NDV genome (15,183 base pairs (bp)), seven fragments with overlapping ends and cohesive restriction sites were amplified by PCR. Fragment 1(F1) spans from nucleotide (nt) 1-1755, nC7fr ίη / 77P7 / E / YΙΛΙ F2 ranges from nt 1-3321, F3 comprises from nt 1755-6580, F4 ranges from 6,151-10,210, F5 spans from nt 7,381-11,351, F6 ranges from 11,351-14,995 and F7 comprises from nt 14,701-15,186. The assembly of the 7 fragments was carried out within the cloning vector called pLS11801140 (SEQ ID NO: 6) using standard ligation techniques, which allowed the reconstruction of the NDV LaSota genome, which after cloning contains a unique SacII restriction site, between the P and M genes, which serves for the cloning of any gene of interest in this viral region of the vector. In addition, another vector called pLS11801140_L289A (SEQ ID NO: 14) was generated, for which the same process described above for pLS11801140 was followed, but including the amino acid L289A in the F gene of the NDV genome. EXAMPLE 2 Cloning of various exogenous SARS-CoV-2 nucleotide sequences within the SacII site of the DNDVLS11801140 vector. To clone various exogenous nucleotide sequences derived from the SARS-CoV-2 spike S glycoprotein, the following 6 versions of the SARS-CoV-2 spike S glycoprotein gene were assembled in InSHico using Vector NTi® software based on the Wuhan-Hu-1 strain (accession number NC_045512.2): Spike S1 / S2 SARS-CoV-2: Sequence of the spike glycoprotein S of SARS-CoV-2 (with SI and S2 subunits) without modifications (SEQ ID NO: 1). Spike SI SARS-CoV-2 / TMCyto: Sequence of the SI subunit of the SARS-CoV-2 spike S glycoprotein fused to the transmembrane and cytoplasmic (TMCyto) sequence of the NDV F gene (SEQ ID NO: 2). Spike S1 / S2 SARS-CoV-2 / TMCyto: Sequence of the ectodomain of the spike S glycoprotein of SARS-CoV-2 fused to the transmembrane and cytoplasmic sequences (TMCyto) of the F gene of NDV (SEQ ID NO: 3). Spike S1 / S2 SARS-CoV-2 / PreF: Sequence of the ectodomain of the SARS-CoV-2 spike glycoprotein S fused to the transmembrane and cytoplasmic (TMCyto) sequence of the NDV F gene, modified so that the NDV F protein acquires the Pre-Fusion conformation. The cleavage site of the spike glycoprotein S was mutated from RRAR to A and 2 mutations were introduced to Proline at amino acids K986P and V987P (SEQ ID NO: 4). Spike S1 / S2 SARS-CoV-2 / PreF / -ADE: Sequence of the ectodomain of the spike S glycoprotein of SARS-CoV-2 fused to the transmembrane and cytoplasmic (TMCyto) sequences of the F gene of NDV, modified so that the F protein of nC7fr ίΠ / 77Ω7 / Β / ΥΙΛΙ NDV acquires the pre-fusion conformation and avoids antibody-dependent amplification of infection (ADE). The cleavage site of the spike glycoprotein S was mutated from RRAR to A, and two proline mutations were introduced at amino acids K986P and V987P. The epitope deletion corresponding to amino acids located at positions 363 to 368 was synthetically introduced (SEQ ID NO: 5). Spike S1 / S2 SARS-CoV-2 / Hexapro: SARS-CoV-2 spike glycoprotein S ectodomain sequence stabilized in its prefusion form and four additional prolines distributed in the synthetic gene to give greater stability to the Spike protein expressed by NDV (SEQ ID NO: 11). The above sequences were initially cloned independently into a pUC vector. The pUC inserts were then sub-cloned using standard genetic engineering techniques into the SacII restriction site, located between the P and M genes of the NDV LaSota genome contained in plasmid pLS11801140 (SEQ ID NO: 6). Plasmid pLS11801140 (SEQ ID NO: 6) also contains all the transcription and translation signal sequences so that each of the five gene versions could be transcribed and translated, thus generating six different versions of the SARS-CoV-2 spike glycoprotein (S). The cloning process resulted in six NDV cDNA (complementary DNA) clones, named respectively: pNDVLS / Spike S1 / S2 SARS-CoV-2. pNDVLS / Spike SI SARS-CoV-2 / TMCyto. pNDVLS / Spike S1 / S2 SARS-CoV-2 / TMCyto. pNDVLS / Spike S1 / S2 SARS-CoV-2 / PreF. pNDVLS / Spike S1 / S2 SARS-CoV-2 / PreF / -ADE. pNDVLS / Spike S1 / S2 SARS-CoV-2 / Hexapro. Each of the generated plasmids was characterized by PCR to detect the presence of each version of the SARS-CoV-2 spike glycoprotein (S glycoprotein). They were also characterized by restriction enzyme digestion, yielding the expected restriction patterns. The stability and sequence of the PCR product for each version of the SARS-CoV-2 spike glycoprotein (S glycoprotein) were confirmed by sequencing. nC7h ίΠ / 77Ω7 / E / YΙΛΙ EXAMPLE 4 Generation of recombinant viruses Each of the plasmids generated in the previous example was chemically transformed and then propagated independently in E.coH for 16 hours under continuous shaking at 37 °C. The DNA from each clone was purified using standard molecular biology procedures. Ten micrograms (µg) of purified DNA were used in transfection experiments using lipofectamine in Hep2 and A-549 cells. Forty-eight hours after transfection, each of the recombinant viruses generated from the six transfections was recovered from the supernatant and used in viral propagation assays in specific pathogen-free (SPF) embryonated chicken eggs for subsequent vaccine preparation. EXAMPLE 5 Propagation of recombinant viruses From the production eggs, embryonated SPF chicken eggs were inoculated with the predetermined infectious dose for each of the recombinant viruses prepared in the previous example. The embryos were incubated at 37°C for 48 hours, with daily monitoring of mortality. After this period, the live embryos were refrigerated overnight, preferably for 24 hours, and the amnioallantoic fluid (AAF) was collected under aseptic conditions and clarified by centrifugation. The AAF was used to characterize the generation of recombinant viruses recovered from the E. co / iy cell culture by hemagglutination and by RT-PCR, using specific primers to amplify the sequence located between the P and M genes, thus demonstrating the presence of the various versions of the SARS-CoV-2 spike glycoprotein (S) cloned in each of the recovered recombinant viruses.Once identity was established by RT-PCR, the stability of the various inserts was determined by sequencing each one. The following 6 recombinant viruses were generated from transfection and propagation assays in SPF chicken embryonated eggs: rNDVLS / Spike S1 / S2 SARS-CoV-2. rNDVLS / Spike YES SARS-CoV-2 / TMCyto. rNDVLS / Spike S1 / S2 SARS-CoV-2 / TMCyto. rNDVLS / Spike S1 / S2 SARS-CoV-2 / PreF. rNDVLS / Spike S1 / S2 SARS-CoV-2 / PreF / -ADE. rNDVLS / Spike S1 / S2 SARS-CoV-2 / Hexapro. nC7b ίΠ / 77Ω7 / Β / YΙΛΙ EXAMPLE 6 Development of active and inactivated vaccines against COVID-19. The viruses prepared in the previous example were purified from FAA as previously described in the prior art (SANTRY, Lisa A., et al. Production and purification of high-titer Newcastle disease virus for use in preclinical mouse models of cancer. Molecular Therapy-Methods & Clinical Development, 2018, vol. 9, p. 181-191.; and NESTOLA, Piergiuseppe, et al. Improved virus purification processes for vaccines and gene therapy. Biotechnology and bioengineering, 2015, vol. 112, no. 5, p. 843-857.). The live vaccines were prepared for intramuscular and intranasal administration in aqueous solution. For this purpose, the active pharmaceutical ingredient (API) was mixed with a stabilizing solution (TPG) to obtain three vaccines with four different concentrations depending on the volume required for administration: a minimum of 10⁷⁰EIEP₅₀% / mL per dose, a minimum of 10⁷⁵EIEP₅₀% / mL per dose, a minimum of 10⁸⁰EIEP₅₀% / mL per dose, and a minimum of 10⁸⁵EIEP₅₀% / mL per dose. Table 1 shows the composition of IL of the TPG stabilizing solution. nC71? ίη / 77Π7 / E / YΙΛΙ Table 1 Component Quantity Trehalose Dihydrate 75.0 g Sodium Phosphate Dibasic 1.30 g Potassium Phosphate Monobasic 0.50 g Monosodium Glutamate 0.90 g Water for Injection 1,000 mL Similarly, viruses purified using the same technique as for live vaccines were inactivated by chemical inactivation with a 10% formaldehyde solution in PBS added dropwise, and an oil-water emulsion was prepared as an adjuvant for testing in pigs. The oil phase comprised 25% of the formulation, the internal aqueous phase 25%, and the external aqueous phase 50%. For the preparation of the aqueous phase, sterile purified water and surfactants of the Span 80 and Tween 80 type were used. For the preparation of the oil phase, mineral oil and surfactants of the Span 80 and Tween 80 type were used. Thus, four vaccines with four different concentrations were obtained: providing a minimum of 107 · 0 DIEP50% / mL per dose, providing a minimum of 107 · 5 DIEP50% / mL per dose, providing a minimum of 1080 DIEP50% / mL per dose, and providing a minimum of 108 · 5 DIEP50% / mL per dose. To prepare the emulsion, the aqueous phase was slowly added to the oil phase under constant stirring. A homogenizer was used to achieve the specified particle size. EXAMPLE 7 Stability tests of the structures in consecutive passes Example 7A - Stabilization of the S (spike) protein with two prolines Two of the constructs made in accordance with Example 5 were subjected to consecutive passages in SPF embryos as described in Example 5, and the recovered viruses were tested to confirm their stability and identity, particularly with regard to the viral titer achieved and the permanence and integrity of the inserted SARS-CoV-2 antigen. The construct designated rNDVLS / Spike S1 / S2 SARS-CoV-2 / PreF in Example 5 comprises the ectodomain of the gene, which will be fused to the transmembrane cytoplasmic (TMC or TMCyto) region of the F (Fusion) gene of Newcastle disease virus. This fusion ensures that the Spike protein encoded by this chimeric gene (ectodomain + TMCyto) is incorporated into the Newcastle capsid and exposed on the viral surface as the main antigen. The nucleotide sequence of the chimeric gene in this version has been optimized for the use of human codons. The furin (F) cleavage site was removed, and two prolines were introduced into the sequence to ensure the pre-fusion structure of the final protein. According to the literature and previous studies based on the SARS-CoV virus, this structure with two prolines is capable of stabilizing the structure of the Spike protein for the generation of antibodies with the correct conformation to neutralize the SARS-CoV-2 virus: Once generated, the source virus was characterized by RT-PCR to confirm the presence of the cloned Spike gene within the NDV genome. The identity and stability of the Spike gene within the Newcastle disease virus genome were also confirmed by sequencing. The expression of the Spike protein by the source virus was also confirmed by immunoperoxidase assay. This origin virus was propagated by two consecutive passages in a 10-day-old SPF chicken embryo in order to increase the titer and generate the Master Seed and one more passage in a chicken embryo to generate the Production Seed from which an experimental vaccine was formulated. RT-PCR characterization tests of the master seed, production seed, and the generated experimental vaccine yielded positive results, amplifying the band corresponding to the inserted Spike gene. However, when the recombinant viruses from each nC7fr ίΠ / 77Ω7 / E / YILI passage were sequenced, three mutations were identified in the Spike gene. One transcription stop codon was located in the coding sequence of subunit 2, and two more mutations were found in the carboxy-terminal region. When immunoperoxidase analysis was performed to detect Spike protein expression in the master seed, production seed, and experimental vaccine, a gradual decrease in expression was observed. With each additional passage, less protein was detected by the anti-Spike antibody, to the point where the experimental vaccine showed almost no Spike protein. These results indicated that the Spike gene could be detected by RT-PCR and remained inserted within the vector; however, with each passage in the chicken embryo, the gene's stability was compromised. Even so, since the master seed had a good Spike expression result by immunoperoxidase, this material was used to formulate the vaccine used in the Preclinical Trial in Pigs. However, analysis of the sera of vaccinated pigs at 0 and 21 days of age indicated that the Spike protein, expressed by the recombinant virus of the rNDVLS / Spike S1 / S2 SARS-CoV-2 / PreF version of example 5, did not induce specific IgG antibodies, nor specific neutralizing antibodies against SARS-CoV-2. This result clearly indicated that, despite the structure designed with two prolines in the sequence, the generation of the Spike protein was compromised, resulting in the expression of Spike protein with a three-dimensional structure unsuitable for induction of neutralizing antibodies, contrary to what was expected. Example 7 B - Stabilization of the S protein (spike) with 6 prolines The Spike gene of the rNDVLS / Spike S1 / S2 SARS-CoV-2 / Hexapro version retains the ectodomain of the Spike gene fused to the transmembrane cytoplasmic (TMC or TMCyto) region of the F (Fusion) gene of Newcastle disease virus. The nucleotide sequence of the chimeric gene has been optimized for human codon usage. The furin (F) cleavage site was removed, and six prolines were introduced into the sequence to ensure the hexapro structure of the final protein. The same processing methodology was applied to generate the Hexapro origin virus and the subsequent master seed, production seed, and experimental vaccine. With this design, the same tests performed according to Example 7A—RT-PCR, sequencing, immunoperoxidase, and SDS-PAGE (Coomassie)—yielded positive results for the identity and stability of the chimeric Hexa-Pro Spike protein, unlike the construction of the same Example 7A. The recombinant virus rNDVLS / Spike S1 / S2 SARS-CoV-2 / Hexapro of example 5, was used in Pre-Clinical trials in SPF pigs, with positive results for the detection of IgG antibody and neutralizing antibodies against SARS-CoV-2. nC7fr ίΠ / 77Ω7 / E / YΙΛΙ EXAMPLE 8 Study to evaluate the level of safety and immunogenicity produced in pigs by the active vaccine against COVID-19 nC7fr ίΠ / 77Ω7 / E / YΙΛΙ A study was conducted to evaluate the safety and immunogenicity of the vaccine in accordance with the principles of the present invention in SPF pigs. For this study, a virus was designed using the plasmid pLS11801140_L289A (SEQ ID NO: 14) generated in example 1 with the Spike S1 / S2 SARS-CoV-2 / Hexapro version, following the process described above in examples 2 to 6. The vaccine was formulated in four doses of 10⁷⁰EI₅₀E ... Table 3 Vaccine Route Application Volume Number of Applications Group Swine IN IM 1080 DIEP50% / mL active virus XX 2.0 mL 2 (0 and 21 days) 1 8 107·5 DIEP50% / mL active virus XX 2.0 mL 2 (0 and 21 days) 2 6 107 0 DIEP50% / mL active virus XX 2.0 mL 2 (0 and 21 days) 3 6 108·5 DIEP50% / mL active virus XX 1.0 mL 2 (0 and 21 days) 4 6 108 0 DIEP50% / mL active virus XX 1.0 mL 2 (0 and 21 days) 5 6 107·5 DIEP50% / mL active virus XX 1.0 mL 2 (0 and 21 days) 6 6 107 0 DIEP50% / mL active virus XX 1.0 mL 2 (0 and 21 days) 7 6 107·5 DIEP50% / mL active virus XX 2.0 mL / 1.0 mL 2 (0 days) 8 6 107·5 DIEP50% / mL active virus XX 2.0 mL / 1.0 mL 2 (0 and 21 days) 9 6 108 0 DIEP50% / mL active virus XX 1.0 mL 2 (0 and 21 days) 10 6 where: IN = Intranasal, IM = Intramuscular, X = 1 dose A total of 62 SPF pigs of similar age and body weight (3-4 weeks old) were used in the study in its different experimental groups. The animals were randomly placed in isolation pens according to their weight. No relevant adverse reactions were observed in any of the animals. The animals were observed for clinical signs throughout the study period. The clinical signs monitored were abnormal respiration, abnormal posture, and rectal temperature each morning. For animal welfare reasons, the animals were observed more than once a day. In the clinical report, only in group 10 (inactivated vaccine), one piglet in group 10 (inactivated vaccine) exhibited an adverse reaction 30 seconds after vaccination, presenting with salivation, depression, and muscle tremors. The piglet was attended to immediately, rinsed with cold water, and its response was assessed. Five minutes after the adverse reaction, the pig showed no serious clinical manifestations; it remained depressed for one hour and then returned to normal. This same pig did not show any adverse post-vaccination reaction after the second vaccination. No clinical signs were observed in the daily checks of any of the piglets in any of the groups throughout the entire trial. This indicates that the vaccines used, with different titers and routes of administration, were safe and met the safety standards. To determine viral load, samples were taken (nasal swabs on day 0 pre-vaccination, day 1 post-first vaccination, and day 1 post-second vaccination) to assess the presence of the vaccine and were evaluated based on the genetic material load of the vaccine virus. Genetic load was also assessed at sacrifice in lung tissue samples and determined by RT-PCRtrq against the vector virus (NDV), detecting the insert encoding the SARS-CoV-2 spike protein within the same vector. All samples were negative for the detection of genetic material against the vector virus (NDV), both in the baseline sampling and 24 hours after the first vaccination. For the evaluation of antibodies against the SARS-CoV-2 Spike, a commercial ELISA kit (GenScript), authorized by the FDA, was used; which detects in a non-functional way neutralizing antibodies against the RBD of the SARS-CoV-2 virus. The degree of immunogenicity induced by vaccination was assessed by the production of neutralizing antibodies (GenScript cPass) against the SARS-CoV-2 RBD protein. Serological samples were taken on days 0, 21, and 28 post-first vaccination. The results for the groups on day 35 from the first vaccination are shown in Table 4 below. nC7b ίη / ΖΖΠΖ / Β / ΥΙΛΙ Table 4 Group ELISA - Virus Serum Neutralization ° / o of inhibition Average Titer (ELISA-VSN) Average (+) (-) (%) Positive Group 1 8 0 100 79.29 1:190 (loq2 = 7.57) Group 2 4 2 66.66 55.52 - Group 3 5 1 83.33 59.98 - Group 4 6 0 100 95.39 - Group 5 6 0 100 92.10 1:1,667 (loq2 = 10.70) Group 6 6 0 100 92.85 1:700 (log2 = 9.45) Group 7 6 0 100 91.06 1:200 (loq2 = 7.64) Group 8 4 1 80 32.06 - Group 9 6 0 100 94.90 1:1,100 (loq2 = 10.10) Group 10 6 0 100 96.04 1:1,800 (loq2 = 10.81) Cx(+) ​​Hum 2 0 100 94.21 1:600 (loq2 = 9.22 Cx (+) Kit NA NA NA 94.42 1=900 (log2=9.81) It should be noted that to compare the results, sera from a patient sick with SARS-CoV-2 who suffered the disease in parallel with the performance of the test were included, 5 identified as Cx (+), and it could be observed that for several groups the average titers were even higher than those of the convalescent patient. Additionally, for Group 1, which received two intranasal vaccines, the same test was performed using oral fluids to detect the possibility of local immunity. A synergistic effect was observed in Group 9, which received the first dose intranasally and the second dose intramuscularly. While comparable results are not available, the positive antibody levels in oral fluids for Group 1 on days 28 and 35 suggest the possibility of preventing SARS-CoV-2 infection via the primary route of infection (upper respiratory mucosa) when two doses are administered intranasally. Similarly, fourteen days after the second application, day 35 post first vaccination, all surviving animals were humanely slaughtered and samples of lung, lymph nodes, liver, kidney and spleen were collected to determine the presence of the vaccine virus by RT-PCRtrq, as well as for histopathology to evaluate possible lung lesions using the planimetry technique and the changes of the lung at the macro and microscopic level, present in the lung for the intranasal route and in the area of ​​the intramuscular application. Following the humane slaughter of the pigs and the performance of necropsies, it was found that the lungs of all the animals showed no lesions suggestive of viral infection and therefore of the active vaccine used. At the site of intramuscular vaccine administration, no active or chronic inflammatory processes were detected, nor were areas of fibrosis or abscesses present; indicating that the intranasal or intramuscular administration of the vaccine did not cause lung or tissue lesions at the injection site. It can be observed from this example that, in accordance with the principles of the present invention, it is possible to obtain a stable recombinant virus for large-scale industrial production, which can demonstrate its safety and immunogenicity in a mammalian animal model through various routes of administration in its active or inactivated form. This same example demonstrates that it is possible to administer an intranasal dose of an active virus containing SARS-CoV-2 antigenic sites, as tested in Example 8, followed by a second intramuscular dose of the same recombinant virus. From this experiment, a skilled technician can infer that it is possible to administer any other SARS-CoV-2 antigen intramuscularly to achieve protection, since the intranasal administration of the vaccine in a first dose was sufficient to stimulate a systemic response to the viral antigen administered intramuscularly, which could be achieved by administering a different vaccine. Therefore, even though specific embodiments of the invention have been illustrated and described, it should be emphasized that numerous modifications are possible, such as the virus used as a viral vector and the exogenous viral sequence employed. Therefore, the present invention shall not be considered restricted except as required by prior art and the appended claims.

Claims

1. - A viral vector capable of generating a cellular immune response, characterized in that the viral vector is a paramyxovirus comprising an exogenous nucleotide sequence encoding for antigenic sites of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) modified to provide stability to said viral vector and the antigenic sites after at least 3 consecutive passages in chicken embryo.

2. The viral vector according to claim 1, further characterized in that the viral vector used can be used active or inactivated.

3. The viral vector according to claim 1, further characterized in that the paramyxovirus has had the phenylalanine removed from position 117 and the basic amino acids from the position near position Q114 that give the pathogenicity removed. 4 - The viral vector according to claim 1, further characterized in that the paramyxovirus is selected from Newcastle disease virus and Sendai virus.

5. The viral vector according to claim 4, further characterized in that the paramyxovirus is Newcastle disease virus (NDV).

6. The viral vector according to claim 5, further characterized in that the NDV is selected from the LaSota, Bl, QV4, Ulster, Roakin and Komarov strains. 7.- The viral vector according to claim 6, further characterized in that the NDV comprises SEQ ID NO: 6 or SEQ ID NO:

14. 8 - The viral vector according to claim 1, further characterized in that the exogenous nucleotide sequence is selected from a sequence encoding the spike glycoprotein S of SARS-CoV-2 or a sequence encoding a sequence derived therefrom.

9. The viral vector according to claim 8, further characterized in that the exogenous nucleotide sequence is selected from a sequence encoding the SI subunit of the SARS-CoV-2 spike S glycoprotein; a sequence encoding the S2 subunit of the SARS-CoV-2 spike S glycoprotein; a sequence encoding both the SI and S2 subunits of the SARS-CoV-2 spike S glycoprotein; a sequence encoding at least a fragment of either the SI or S2 subunits of the SARS-CoV-2 spike S glycoprotein; a sequence having at least 80% identity with the sequence encoding the SI subunit of the SARS-CoV-2 spike S glycoprotein; a sequence with an identity of nC7fr ίη / 77Π7 / E / YΙΛΙ at least 80% with the sequence encoding the S2 subunit of the SARS-CoV-2 spike S glycoprotein;a sequence with at least 80% identity to the sequence encoding the two SI and S2 subunits of the SARS-CoV-2 spike glycoprotein S; a sequence with at least 80% identity to a sequence encoding at least a fragment of the SI or S2 subunits of the SARS-CoV-2 spike glycoprotein S; a sequence encoding the two SI and S2 subunits of the SARS-CoV-2 spike glycoprotein S devoid of at least one epitope located between nucleotides corresponding to amino acids 1 to 460 of the SI sequence; a sequence encoding the SI subunit of the SARS-CoV-2 spike glycoprotein S devoid of at least one epitope located between nucleotides corresponding to amino acids 1 to 460 of the SI sequence;or a sequence encoding the two SI and S2 subunits of the SARS-CoV-2 spike glycoprotein S stabilized in its prefusion form by the inclusion of at least two proline substitutions in the S2 subunit; 10. The viral vector according to claim 9, further characterized in that the exogenous nucleotide sequence is selected from a sequence having at least 80% identity with any of the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; or from a sequence having at least 80% identity with any sequence that translates into any of the amino acid sequences SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO:

13. 11.- The viral vector according to claim 9, further characterized in that the epitope located between the nucleotides corresponding to amino acids 1 to 460 of the SI sequence is selected from the amino acid sequences SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO:

10. 12.- A vaccine against coronavirus disease 2019 (COVID-19), characterized in that it comprises a paramyxovirus viral vector comprising an exogenous nucleotide sequence encoding for antigenic sites of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) modified to provide stability to said viral vector and the antigenic sites after at least 3 consecutive passages in chicken embryo, and a pharmaceutically acceptable vehicle, adjuvant and / or excipient.

13. The vaccine according to claim 12, further characterized in that the paramyxovirus is live or inactivated.

14. The vaccine according to claim 12, further characterized in that the paramyxovirus has been deprived of phenylalanine at position 117 and the basic amino acids at the position near position Q114 that confer pathogenicity to paramyxoviruses. nC7fr ίΩ / 77Ω7 / Β / YΙΛΙ 15. The vaccine according to claim 13, further characterized in that the paramyxovirus is selected from Newcastle disease virus and Sendai virus.

16. The vaccine according to claim 13, further characterized in that the paramyxovirus is Newcastle disease virus (NDV).

17. The vaccine according to claim 16, further characterized in that the NDV is selected from the LaSota, Bl, QV4, Ulster, Roakin and Komarov strains.

18. The vaccine according to claim 16, further characterized in that the NDV is selected from SEQ ID NO: 6 or SEQ ID NO:

14.

19. The vaccine according to claim 12, further characterized in that the exogenous nucleotide sequence is selected from a sequence encoding the SARS-CoV-2 spike glycoprotein S or a sequence encoding a sequence derived therefrom.

20. The vaccine according to claim 19, further characterized in that the exogenous nucleotide sequence is selected from a sequence encoding the SI subunit of the SARS-CoV-2 spike S glycoprotein; a sequence encoding the S2 subunit of the SARS-CoV-2 spike S glycoprotein; a sequence encoding both the SI and S2 subunits of the SARS-CoV-2 spike S glycoprotein; a sequence encoding at least a fragment of either the SI or S2 subunit of the SARS-CoV-2 spike S glycoprotein; a sequence having at least 80% identity with the sequence encoding the SI subunit of the SARS-CoV-2 spike S glycoprotein; a sequence having at least 80% identity with the sequence encoding the S2 subunit of the SARS-CoV-2 spike S glycoprotein;a sequence with at least 80% identity to the sequence encoding the two SI and S2 subunits of the SARS-CoV-2 spike glycoprotein S; a sequence with at least 80% identity to a sequence encoding at least a fragment of the SI or S2 subunits of the SARS-CoV-2 spike glycoprotein S; a sequence encoding the two SI and S2 subunits of the SARS-CoV-2 spike glycoprotein S devoid of at least one epitope located between nucleotides corresponding to amino acids 1 to 460 of the SI sequence; a sequence encoding the SI subunit of the SARS-CoV-2 spike glycoprotein S devoid of at least one epitope located between nucleotides corresponding to amino acids 1 to 460 of the SI sequence;or a sequence encoding the two SI and S2 subunits of the SARS-CoV-2 spike glycoprotein S stabilized in its prefusion form by the inclusion of at least two proline substitutions in the S2 subunit; 21. The vaccine according to claim 19, further characterized in that the exogenous nucleotide sequence is selected from a sequence having an identity of at least 80% with any of the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; or from a sequence having an identity of at least 80% with any sequence that translates into any of the amino acid sequences SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO:

13.

22. The vaccine according to claim 19, further characterized in that the epitope located between the nucleotides corresponding to amino acids 1 to 460 of the SI sequence is selected from the amino acid sequences SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO:

10.

23. The vaccine according to claim 12, further characterized in that the pharmaceutically acceptable vehicles are preferably aqueous solutions or emulsions.

24. The vaccine according to claim 23, further characterized in that the pharmaceutically acceptable vehicle is selected from a water-oil, oil-water and water-oil-water emulsion.

25. The vaccine according to claim 24, further characterized in that the pharmaceutically acceptable vehicle is a water-oil-water emulsion. 26.- The vaccine according to claim 12, further characterized in that it comprises the virus in a concentration between 1060 and 10100 DIEP50% / mL per dose of a determined volume according to the route of administration.

27. The vaccine according to claim 26, further characterized in that the concentration of active virus required to achieve the antigenic response is between 106 0 and 108·5 DIEP50% / mL per dose.

28. The vaccine according to claim 26, further characterized in that the volume per dose is from 0.2 to 2 mL 29. The vaccine according to claim 12, further characterized in that it is adapted to be administered by intramuscular, intranasal, subcutaneous, spray, or nebulization route.

30. The vaccine according to claim 29, further characterized in that it is adapted to be administered intramuscularly or intranasally.

31. A paramyxovirus comprising an exogenous nucleotide sequence encoding for antigenic sites of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) for use in the treatment or prevention of COVID-19 disease caused by said coronavirus.

32. The paramyxovirus of claim 31, which is adapted to be administered at a dose of between 1060 and 1010 0 DIEP50% / mL in a volume between 0.2 and 2 ml. nC7fr ίΩ / 77Ω7 / B / YILI 33. The paramyxovirus of claim 32, which is adapted to be administered at a dose between 1065 and 108·5 DIEP50% / mL.

34. The paramyxovirus of claim 33, which is adapted to be administered by intramuscular, intranasal, subcutaneous, spray, or nedulization.

35. The paramyxovirus of claim 33, which is adapted to be administered intranasally at a dose between 107·5 and 108·5 DIEP50% / mL.

36. The paramyxovirus of claim 33, which is adapted to be administered intramuscularly with a dose between 1070 and 108·5 DIEP50% / mL.

37. The paramyxovirus of any of claims 34 to 36, which is adapted to be administered in two doses.

38. The paramyxovirus of claim 37, which is adapted to be administered with a difference of 7 to 35 days between the first dose and the second dose.

39. The paramyxovirus of claim 38, which is adapted to be administered with a difference of 21 to 28 days between the first dose and the second dose.

40. The paramyxovirus according to claim 37, which is adapted to be administered in a first dose by the intranasal route and a second dose by the intramuscular route. 41.- The paramyxovirus according to claim 31, which is adapted to generate mucosal immunity against SARS-CoV-2 infection.

42. The use of a paramyxovirus comprising an exogenous nucleotide sequence encoding for antigenic sites of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to prepare a vaccine for COVID-19 disease caused by said coronavirus. 43.- The use of the paramyxovirus according to claim 42, to be administered at a dose of between 106 0 and 10100 DIEP50% / mL in a volume between 0.2 and 2 ml. 44.- The use of the paramyxovirus according to claim 43, to be administered at a dose between 106·5 and 108·5 DIEP50% / mL.

45. The use of the paramyxovirus according to claim 43 for administration by intramuscular, intranasal, subcutaneous, spray, or nebulization route.

46. ​​The use of the paramyxovirus according to claim 44, to be administered intranasally at a dose between 107·5 and 108·5 DIEP50% / mL. 47.- The use of the paramyxovirus according to claim 44, to be administered intramuscularly with a dose between 1070 and 108·5 DIEP50% / mL.

48. The use of the paramyxovirus according to any of claims 45 to 47, for administration in two doses. nC7fr ίΩ / 77Ω7 / Β / YILI 49. The use of the paramyxovirus according to claim 48, to be administered with a difference of 7 to 35 days between the first dose and the second dose.

50. The use of the paramyxovirus according to claim 49, to be administered 21 to 28 days apart between the first dose and the second dose.

51. The use of the paramyxovirus according to claim 48, wherein the first dose is administered intranasally and the second dose intramuscularly.

52. The use of the paramyxovirus according to claim 42, wherein the paramyxovirus generates mucosal immunity against SARS-CoV-2 infection.

53. An active virus comprising antigenic sites of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is adapted for use in the treatment or prevention of COVID-19 disease caused by said coronavirus in a first dose by intranasal route, followed by a second dose by intramuscular route of a SARS-CoV-2 antigen.

54. The active virus according to claim 53, wherein the antigen of the second dose is the same active virus as the first dose. 55,- The active virus according to claim 54, wherein the antigen of the second dose is in its inactivated form.

56. The use of an active virus comprising antigenic sites of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for the preparation of a vaccine for the treatment or prevention of COVID-19 disease caused by said coronavirus in a first dose by intranasal route, followed by a second dose by intramuscular route of a SARS-CoV-2 antigen. 57.- Use in accordance with claim 56, wherein the antigen of the second dose is the same active virus as the first dose.

58. Use in accordance with claim 57, wherein the antigen of the second dose is in its inactivated form.