LIVE RECOMBINANT IMMUNOGENIC COMPOSITION COMPRISING NEWCASTLE DISEASE VIRUS (NDV) EXPRESSING THE S1 SUBSUNT AND RBD OF THE SARS-COV-2 SPIKE PROTEIN

Abstract

The present invention relates to obtaining two live recombinant or vectored NDV vaccines that express the S1 and RBD subunits of the SARS-CoV-2 spike protein, which, when administered intranasally in a hamster animal model, generate the production of neutralizing antibodies against SARS-CoV-2. The invention further shows that these live vaccines are compatible for combined use to induce the production of neutralizing antibodies against SARS-CoV-2 without substantial cross-interference, indicating a synergy between the two vaccines, where the recombinant NDV viruses (rNDV-LS1-HN-RBD / SARS-CoV-2 (SEQ ID No. 7) and rNDV-LS1-S1-F / SARS-CoV-2 (SEQ ID No. 13)) successfully express the S1 and RBD subunits of the SARS-CoV-2 spike protein in mammals.

Description

LIVE RECOMBINANT IMMUNOGENIC COMPOSITION COMPRISING NEWCASTLE DISEASE VIRUS (NDV) EXPRESSING THE S1 SUBSUNT AND RBD OF THE SARS-CoV-2 SPIKE PROTEIN TECHNICAL FIELD The present invention falls within the pharmaceutical industry. The present invention relates to obtaining two recombinant vectored viruses derived from Newcastle disease virus (NDV), comprising the S1 subunit and the receptor-binding domain (RBD), both derived from the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), through reverse genetics, and to the corresponding product of a live recombinant vaccine or immunogenic composition obtained from the mixture of the two recombinant viruses: rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2, for immunizing and protecting against coronavirus disease 2019 (COVID-19) in mammals and humans. BACKGROUND The present invention was developed based on the use of a previously prepared viral vector, with Title No. 9204 of the Industrial Property Registry “VACCINE COMPRISING RECOMBINANT NEWCASTLE DISEASE VIRUS (NDV) EXPRESSING THE S SPIKE GENE OF AVIAN INFECTIOUS BRONCHITIS VIRUS (IBV)”, granted by the Directorate of Inventions and New Technologies of INDECOPI certified in Resolution No. 003059-2018 / DININDECOPI. STATE OF THE ART COVID-19 is an infectious disease in humans caused by the highly pathogenic coronavirus SARS-CoV-2, which emerged in Wuhan and whose rapid international spread has posed a serious global public health emergency (Zhou et al. 2020; Wu et al. 2020; Zhu et al. 2020). Patients infected with SARS-CoV-2 exhibit a variety of symptoms, including dry cough, fever, headache, fatigue, and shortness of breath (https: / / www.who.int / es / emergencies / diseases / novelcoronavirus-2019 / advice-for-public / qa-coronaviruses), with an estimated mortality rate ranging from 3% to 5% (Huang et al. 2020; Liu et al. 2020; Wang et al. 2020). Since the initial outbreak in December 2019, SARS-CoV-2 has spread throughout China and to more than 80 countries and areas worldwide. nzzfrnn / zznz / E / YiAi SARS-CoV-2 belongs to the Coronaviridae family and possesses a single-stranded, positive-sense ribonucleic acid (RNA) genome of -29.9 kilobases (kb) in length (Su et al. 2016; H. Zhou et al. 2020). Phylogenetic analyses of coronavirus genomes have revealed that SARS-CoV-2 is a member of the betacoronavirus genus (P. Zhou et al. 2020; Wu et al. 2020; Zhu et al. 2020; Lu et al. 2020), and within that genus, it shares 79% genomic sequence identity with SARS-CoV and 50% with MERS-CoV (Lu et al. 2020). It has six open reading frames (ORFs) that are arranged in the 5' to 3' direction: replicase (ORF1a / ORF1b), spike (S), envelope (E), membrane (M), and nucleocapsid (N). In addition, there are seven putative ORFs that encode accessory proteins that are interspersed between the structural genes (Chan et al. 2020).Most of the proteins encoded by SARS-CoV-2 are of similar length to the corresponding proteins of SARS-CoV. Of four structural genes, SARS-CoV-2 shares more than 90% amino acid (aa) identity with SARS-CoV, except for the S gene, which diverges (H. Zhou et al. 2020; Lu et al. 2020). Coronaviruses utilize the S glycoprotein (comprising an S1 subunit and an S2 subunit) on their envelope to bind to their cellular receptors. This binding triggers a cascade of events leading to fusion between the cellular and viral membranes, allowing viral entry into the cell. Previous cryo-electron microscopy studies of the SARS-CoV S protein and its interaction with the ACE2 cellular receptor (Angiotensin-Converting Enzyme 2) have demonstrated that the S1 subunit binds to the receptor. This binding induces dissociation of S1 from ACE2, which in turn induces S2 to transition from a metastable pre-fusion state to a more stable post-fusion state that is essential for membrane fusion (Gui et al. 2017; Song et al. 2018; Kirchdoerfer et al. 2018; Yuan et al. 2017). Therefore, binding to the ACE2 receptor is an initial and critical step for SARS-CoV to enter target cells.Recent studies have also highlighted the important role of the ACE2 receptor in mediating SARS-CoV-2 entry (P. Zhou et al. 2020; Walls et al. 2020; Letko, Marzi, and Munster 2020; Hoffmann et al. 2020). For example, HeLa cells expressing the ACE2 receptor are susceptible to SARS-CoV-2 infection, while those lacking ACE2 are not (P. Zhou et al. 2020). In vitro binding measurements also showed that the receptor binding domain (RBD) of the SARS-CoV-2 S1 subunit binds to the ACE2 receptor with affinity in the low nanomolar range, indicating that the RBD is a key functional component within the S1 subunit that is responsible for SARS-CoV-2 binding to the ACE2 receptor (Walls et al. 2020; Tian et al. 2020). The SARS-CoV-2 spike protein has a full length of 1273 amino acids, longer than that of SARS-CoV (1255 aa). It is distinct from the spike proteins of most members of the Sarbecovirus subgenus of the betacoronavirus genus and shares 76.7% to 77.0% amino acid sequence similarity with SARS-CoV from civets and humans. Regarding the receptor-binding domain (RBD), the amino acid similarity between SARS-CoV-2 and SARS-CoV is only 73%. Another SARS-CoV-2-specific genomic feature is the insertion of four amino acid residues (PRRA) at the junction of the S1 and nzzfrnn / zznz / E / YiAi subunits. S2 of the S protein (Andersen et al. 2020). This insertion generates a polybasic cleavage site (RRAR), which allows for efficient cleavage by furin and other proteases (Coutard et al. 2020). This S1-S2 cleavage site is not observed in all related viruses belonging to the subgenus Sarbecovirus, except for a similar three-amino-acid (PAA) insertion in RmYN02, a bat-derived coronavirus recently reported in Rhinolophus malayanus in China (H. Zhou et al. 2020). On the other hand, the antigenicity of the RBD plays an important role in the induction of antibodies, which could block the binding of the RBD to the ACE2 receptor, as shown in a study where a recombinant vaccine comprising residues 319–545 of the SARS-CoV-2 spike protein RBD induced a potent functional antibody response in immunized mice, rabbits, and non-human primates (Macaca mulatta) as early as 7 or 14 days after injection of a single vaccine dose. Sera from the immunized animals blocked the binding of the RBD to the ACE2 receptor expressed on the cell surface and neutralized infection with a SARS-CoV-2 pseudovirus and SARS-CoV-2 in vitro. Notably, vaccination also provided protection in non-human primates against in vivo challenge with SARS-CoV-2 (Yang et al. 2020). Looking ahead, vaccination could be the most effective method for a long-term strategy to prevent and reduce COVID-19 morbidity and mortality. As of October 2, 2020, 174 COVID-19 vaccine candidates had been reported, and 51 were beginning human clinical trials. Therefore, selecting the ideal vaccine could take some time. Different platforms for obtaining vaccines against SARS-CoV-2 are in execution, including strategies that include: deoxyribonucleic acid (DNA), messenger ribonucleic acid (mRNA) the latter wrapped in a lipid nanoparticle, inactivated viruses, live attenuated viruses, subunit proteins, recombinant viral vectors (Hu et al. 2020). Among the different vaccine development platforms, viral vector vaccines have a potential advantage over others because they can be used as live or inactivated vaccines, which can induce both Th1 and Th2 immune responses. Several viral vaccine vectors have been proposed, such as adenovirus, measles virus, Ankara virus, acute vesicular stomatitis virus, and human parainfluenza virus. However, each viral vector has limitations that may or may not be overcome. For example, the immunogenicity of some viral vector vaccines has not been ideal. Furthermore, defective replication of the adenovirus vaccine vector might not induce good local immunity. In the case of vesicular stomatitis virus, the effectiveness of the vector is currently questionable, and the human parainfluenza vector might not be effective in adults due to their pre-existing immunity. Overcoming all these limitations is Newcastle disease virus (NDV), an avian virus, which has many characteristics that make it suitable as a potential vaccine vector for SARS-CoV-2 (Rohaim and Muñir 2020). NDV is an important pathogen in the poultry industry, belongs to the genus Orthoavulavirus, family Paramyxoviridae, and is formally known as Avian orthoavulavirus 1 [https: / / talk.ictvonline.org / taxonomy]. The NDV has a single-stranded, non-segmented, negative-sense RNA genome. The NDV genome length is 15,186 nucleotides (nt) and contains six genes in the order 3-NP-PM-FHN-L-5', which encode the nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and long polymerase protein (L). It also contains two non-structural proteins, V and W, which are produced during transcription of the P gene (Cattoli et al. 2011; Dimitrov et al. 2016). The NDV genome length must be a multiple of six for efficient RNA replication, thus fulfilling the so-called "rule of six." Each transcriptional unit contains a main ORF flanked by short untranslated regions (UTRs), which are followed by conserved transcription initiation and termination sequences known as the start gene (GS), end gene (GE), and between them are the non-coding intergenic sequences (IGSs), also called untranslated sequences. Virion construction and assembly are associated with three glycoproteins: F, HN, and M. The F and HN glycoproteins are anchored to the virion envelope. The M glycoprotein is located on the inner surface of the viral envelope. The HN and F glycoproteins are incorporated into the virion via the interaction of their cytoplasmic tail domains (DCs) with the M protein (Dolganiuc et al. 2003; Pantua et al. 2006). The HN protein contains the binding and recognition site for the host cell receptor. On the other hand, the F glycoprotein mediates the fusion of the virus with the host cell membrane, thus contributing to the initiation of the NDV replication cycle (Kim et al. 2011; Kumar et al. 2011). Both are capable of generating neutralizing antibodies because they are protective antigens against NDV. nzzfrnn / zznz / E / YiAi The main determinant of virulence in avian NDV is the activation of the F glycoprotein, which occurs through cleavage mediated by cellular endoproteases. In many velogenic and some mesogenic strains, the F cleavage site is rich in basic amino acids and is rapidly olivated by intracellular proteases such as furin, thus allowing replication in a variety of tissues. On the other hand, lentogenic and some mesogenic strains contain few basic residue sites and therefore depend on secretory proteases found primarily in the lungs for cleavage; consequently, their replication is restricted to epithelial surfaces where the protease is present. Its natural host is birds, and it is antigenically distinct from common human pathogens. It is exclusively a cytoplasmic virus and therefore does not integrate its viral genes into the host genome, which enhances its safety profile. The severity of avian disease depends on the pathotype of the NDV strains: lentogenic, mesogenic, and velogenic. Lentogenic strains (LaSota) cause mild or asymptomatic infections that are restricted to the respiratory tract. Viruses of intermediate virulence are called mesogenic, while viruses that cause systemic infection and high mortality are called velogenic. Lentogenic and mesogenic strains are often used as live NDV vaccines for poultry worldwide. Recent studies have highlighted the potential use of NDV as a vaccine vector for both veterinary and / or human use. Reverse genetics has been used in the design and development of several modern vaccines. If foreign gene insertions are required to be expressed on the surface of the NDV, it is important to consider certain construction strategies, one of which is to fuse them with the domains: DC and transmembrane (DTM) (DTM, from the English: Domain Transmembrane) of the F and / or HN protein, another option is that the foreign protein is inserted with its complete CT and DTM. Thus, with the aim of developing a live recombinant immunogenic vaccine or composition for SARS-CoV-2, based on an NDV vector, that can express the S1 subunit and the RBD of the S protein from SARS-CoV-2, the present invention focuses on the development of two recombinant vectored vaccines named: rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2, obtained by reverse genetics using complementary fragments that encompass the complete genomes of rNDV-LS1-S1-F / SARS-CoV-2 and rNDV-LS1nzzfrnn / zznz / E / YiAi HN-RBD / SARS-CoV-2. As well as its application in a single formulation combining both recombinant viruses: rNDV-LS1-S1-F / SARS-CoV-2 and rNDV-LS1-HN-RBD / SARS-CoV-2, in addition to having the intranasal (IN) route of immunization, proving to be a less invasive route of application compared to other vaccines. From the patent field, document CN112011521 is known to refer to a novel coronavirus vaccine candidate using a recombinant Newcastle disease virus vector and its method of construction and application using genetically modified vaccine technology. The candidate is based on the LaSota strain of Newcastle disease virus vaccine, the P gene from LaSota NDV strains, and the C3756T gene mutation (M inserted between the BAMHI site deletion) of the novel coronavirus S gene; the novel nucleotide sequence of the coronavirus S gene is shown in SEQ ID NO. 1. The LaSota strain of attenuated NDV is used as the vector background, and the S gene (full ORF) of SARS-CoV-2 is inserted into the P and M genes. The silent C3756T mutation is introduced into the conserved region of the S gene, thereby deleting the BamHL site. Document CN101629178 also describes a manually combined Newcastle disease virus F gene and recombinant expression vector and an application thereof, wherein the codons of the NDVF OFR gene are all replaced with chicken bias codons, the EcoR V restriction sites and Kozak sequences are added upstream, the Xbal I restriction sites and TGA termination codons are added downstream, and the downstream A bases of the termination codons are changed to G. The resulting genes can be efficiently expressed in eukaryotic cells, and the expression efficiency of the resulting genes is higher than that of wild-type genes. Document CN106047823 describes a bivalent vaccine strain R-H120-Lasota (HN) obtained by replacing a 5a gene of the H120 strain with an HN gene from avian Newcastle disease virus based on a reverse genetic system constructed from avian infectious bronchitis virus H120. The recombinant vaccine strain can simultaneously protect against strains that counteract the standard toxin of IBV and strains that counteract the standard toxin of Newcastle disease virus and can be used to prevent avian infectious bronchitis virus and avian Newcastle disease virus. It further reports that the vaccine was administered with double doses of SARS virus to experimental animals, and the viral content at the peak of lung replication was significantly lower. nzzfrnn / zznz / E / YiAi Patent WO2000 / 67786 relates to cDNAs for producing infectious, attenuated Newcastle disease virus (NDV), methods for preparing the cDNAs, and the vector containing the cDNA optionally linked to an operable promoter. It also describes methods for preparing the vaccines and methods for using them to prevent or treat Newcastle disease in an avian host. The patent reports nucleotide sequences from the entire NDV genome, including the main region, the terminal region, and the NP region, as well as the proteins encoded by these nucleotide sequences.This patent suggests that these approaches have made it possible to begin characterizing the factors that act on the transcription and replication of several non-segmented negative-strand RNA viruses to recover complete infectious recombinant viruses from full-length cDNA for several non-segmented negative-strand RNA viruses, among which it suggests rabies virus, vesicular stomatitis virus, measles virus, Sendai virus, human respiratory syncytial virus, rinderpest virus and parainfluenza, and suggests including the nucleotide sequences of the complete NP gene, the complete trailer region and the intergenic regions in the NP-P and P. For its part, patent WO2015 / 013178 shows the construction of recombinant NDVs expressing ILTV gB, gC and gD and a schematic diagram representing the full-length antigenome of the LaSota strain of NDV with the insertion of an added gene designed to express the gB of ILTV consisting of the full ORF of gB fused to the last 12 amino acids of the cytoplasmic tail of the F protein of NDV and also suggests methods for immunizing a mammal against a non-avian pathogen. Patent WO2016 / 138160 provides methods for inducing an immune response in a subject to Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The immune response is a protective immune response that inhibits or prevents MERS-CoV infection in the subject. It also provides CoV polypeptides and the nucleic acid molecules that encode them, as well as neutralizing antibodies that bind specifically to the MERS-CoV spike protein and antigen-binding fragments thereof. Peruvian patent 1179-2014 / DIN reports a chimeric viral particle or virus comprising an asymptomatic lentogenic Newcastle disease virus (NDV) with an Intracranial Pathogenicity Index (IPCI) of zero expressing at least one gene from an IBV strain, wherein the gene or at least one of the genes from an IBV strain corresponds to the complete spike (S) gene or part of the spike (S) gene, and wherein said complete S gene or part of the S gene may be located in a non-coding intergenic region, specifically between the P and M genes. It also reports that studies in SARS, a human coronavirus, demonstrate that the S2 domain is capable of generating neutralizing antibodies and that proper folding and homotrimer formation of the nzzfrnn / zznz / E / YiAi S protein requires the presence of both the S1 and S2 domains, because S2 contains specific trimerization sites that participate in the merger process. Furthermore, document EP2251034 provides a negative-sense chimeric RNA virus that enables the immunization of a subject, for example, a bird, against two infectious agents using a single chimeric virus of the invention. In particular, the invention provides chimeric Newcastle disease viruses (NDV) engineered to express and incorporate into their virions a fusion protein comprising the ectodomain of a protein from an infectious agent and the transmembrane and cytoplasmic domains of an NDV protein. Such chimeric viruses induce an immune response against both NDV and the infectious agent. Additionally, the scientific literature includes an article titled “Newcastle disease virus (NDV) expressing the SARS-CoV-2 spike protein as a vaccine candidate.” This article states that the NDV vector vaccine against SARS-CoV-2 described in the study has similar advantages to other viral vector vaccines, but the NDV vector can be amplified in embryonated chicken eggs, allowing for high yields and low cost per dose, thus providing an important option for a cost-effective SARS-CoV-2 vaccine. The article describes a method for rescuing LaSota NDV that expresses the SARS-CoV-2 spike protein. The article “A Newcastle disease virus (NDV) expressing the membrane-anchored Spike 1 protein as an inactivated vaccine against SARS-CoV-2” also indicates that an attenuated recombinant NDV expressing the membrane-anchored SF chimera (NDV-S) was investigated as a candidate for an inactivated vaccine against SARS-CoV-2 with and without adjuvant in mice and hamsters, where it was found that the SF chimera expressed by the NDV vector is very stable with no loss of antigenicity after 3 weeks of storage at 4°C in allantoic fluid. Finally, there is also the article titled “Newcastle disease virus, a virus with a restricted host range, as a vaccine vector for intranasal immunization against emerging pathogens,” which involves Newcastle disease virus (NDV) as a potential vaccine vector against SARS-CoV. Like human parainfluenza virus type 3, NDV is a non-segmented, negative-stranded RNA virus of the Paramyxoviridae family. However, its natural host is birds, and it is antigenically distinct from common human pathogens. Nevertheless, NDV vectors were created where one of the constructs was a recombinant copy of the lentogenic LaSota strain, modified in such a way that the cleavage sequence of its F protein was replaced with that of NDV-BC, resulting in the NDV-VF virus. Based on these last two, vaccine viruses expressing a total length of 1 were constructed.255 aa of the SARS-Conzzfrnn / zznz / E / YiAi S protein. V. It also indicates that the S1 domain contains the receptor-binding site of the S protein as well as major neutralizing epitopes. On the other hand, to date, there is no live NDV vectored vaccine or live recombinant immunogenic NDV vectored composition candidate against SARS-CoV-2 that expresses the S1 subunit or the RBD of the SARS-CoV-2 S, and that in turn provokes a robust humoral and cellular response, in addition to having a non-invasive intranasal (IN) route of application. In addition to having a profitable, economical and cheap production, in embryonated chicken eggs and / or its production in multiple cell lines. In this sense, it is clear that there is a need, not yet met in the state of the art, for a live recombinant vaccine or a live recombinant immunogenic vectored composition that includes the NDV virus that expresses and incorporates into its viral structure the S1 subunit and the RBD of the SARS-CoV-2 S protein, and that, when applied intranasally, presents significant levels of protection against SARS-CoV-2, as well as high titers of neutralizing antibodies against SARS-CoV-2. DESCRIPTION OF THE FIGURES Figure 1. Prediction of the transmembrane (TMD) and cytoplasmic (DC) domains of the hemagglutinin-neuraminidase (HN) protein. The prediction was performed using the amino acid (aa) sequence in the TMHMM Server 2.0 program. Figures 2A-2C. Schematics showing the construction strategy of the new viruses rNDV-LS1HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2. Figure 3. Map of plasmid pNDV-LS1-HN-RBD / SARS-CoV-2 (20315 bp). Figure 4. The subcloning strategy of the HN-RBD / SARS-CoV-2 gene (SEQ ID NO: 1) that generates the recombinant virus rNDV-LS1-HN-RBD / SARS-CoV-2 (16182 bp) (SEQ ID NO: 7) is shown schematically. Two successive subclonings were performed to construct the rNDVLS1-HN-RBD / SARS-CoV-2 construct. The NDV-LS1 plasmid (19319 bp) was linearized between the BbvCI cleavage sites (third position of NDV) to insert the 1013 bp fragment (SEQ ID NO: 1) which includes the BbvCI sites, generating the NDV-LS1-HN-RBD / SARS-CoV-2 plasmid (20315 bp). nzzfrnn / zznz / E / YiAi Figure 5. Prediction of the DTM and DC of the Fusion protein (F). The prediction was made using the amino acid (aa) sequence in the TMHMM Server 2.0 program. Figure 6. Map of plasmid pNDV-LS1-S1-F / SARS-CoV-2 (21743 bp). Figure 7. The subcloning strategy of the S1-F / SARS-CoV-2 gene (2441 bp) (SEQ ID NO: 8) that generates the rNDV-LS1-S1-F / SARS-CoV-2 virus (17610 bp) (SEQ ID NO: 13) is shown schematically. Two successive subclonings were performed to construct the rNDV-LS1S1-F / SARS-CoV-2 virus construct. The pNDV-LS1 plasmid (19319 bp) was linearized between the BbvCI cleavage sites (third position of the NDV genome) to insert the 2441 bp fragment (SEQ ID NO: 8) which includes the BbvCI sites, generating the NDV-LS1-S1-F / SARS-CoV-2 plasmid (21743 bp). Figures 8A-8B. Verification of the expression and incorporation of S1-F and HN-RBD in the virion, by Western blot assay. Figures 9A-9B. Detection of the genetic sequence of the S1-F and HN-RBD inserts by RT-PCR. Figure 10. Immunofluorescence Assay. Figure 11. Cell binding and internalization assay by efficiency to the ACE2 receptor expressed in Vero E6 cells. A. Graphical representation of S1-F and HN-RBD binding to the ACE2 receptor. B. Percentage of binding (%) between rNDV-LS1-HN-RBD / SARS-CoV-2 (10.2%) and rNDV-LS1-S1-F / SARS-CoV-2 (40.4%) to the ACE2 cell receptor. Figure 12. In vitro growth properties of the rNDV-LS1-HN-RBD / SARS-CoV-2, rNDV-LS1S1-F / SARS-CoV-2, and rNDV-LS1 viruses in the DF-1 cell line. Figure 13. Immunization and challenge schedule in hamsters with rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2. Figure 14. Evaluation of neutralizing antibodies using a surrogate neutralization test (TNs). nzzfrnn / zznz / E / YiAi Figure 15. Evaluation of SARS-CoV-2 anti-RBD IgG levels by indirect ELISA. DESCRIPTION OF THE SEQUENCE LIST SEQ ID NO: 1= Nucleotide sequence comprising the design of the HN-RBD / SARS-CoV-2 gene (1013 bp). SEQ ID NO: 2= Nucleotide sequence comprising the hemagglutinin-neuraminid (HN) gene (1734 bp) of the pNDV-LS1 plasmid (7535-9268 nt). SEQ ID NO: 3 = Amino acid sequence comprising the HN protein (577 aa) of the pNDVLS1 plasmid. SEQ ID NO: 4 = Nucleotide sequence comprising the cytoplasmic (DO) and transmembrane (DTM) domains of the HN protein (144 bp) of the pNDV-LS1 plasmid (7534-7678 nt). SEQ ID NO: 5 = Nucleotide sequence comprising the SARS-CoV-2 Spike (S) gene (3822 bp) (21563-25384 nt) with GenBank accession number- MN908947.3. SEQ ID NO: 6 = Nucleotide sequence comprising the RBD domain (636 bp) of the SARS-CoV-2 S (990-1623 nt) with GenBank accession number MN908947.3 for the design of the HN-RBD / SARSCoV-2 gene. SEQ ID NO: 7= Nucleotide sequence comprising the complete genome of the recombinant virus rNDV-LS1-HN-RBD / SARS-CoV-2 (16182 bp). SEQ ID NO: 8= Nucleotide sequence comprising the design of the S1-F / SARS-CoV-2 gene (2441 bp) optimized for expression in Gallus Gallos. SEQ ID NO: 9 = Nucleotide sequence comprising the Fusion (F) gene (1662 bp) of the pNDV-LS1 plasmid (5667-7328 nt). SEQ ID NO: 10= Amino acid sequence comprising the F protein (553 aa) of the pNDV-LS1 plasmid (5667-7328 nt). ηζζ^ηη / ζζηζ / Β / γίΛΐ SEQ ID NO: 11= Nucleotide sequence comprising the DC and DTM domains of the F protein (162 bp) of the pNDV-LS1 plasmid (7166-7328 nt), optimized for expression in Gallus Gallus. SEQ ID NO: 12= Nucleotide sequence comprising the S1 subunit of the S gene (2043 bp) of SARS-CoV-2 with the accession number in GenBank-MN908947.3. SEQ ID NO: 13= Nucleotide sequence comprising the complete genome of the recombinant virus rNDV-LS1-S1-F / SARS-CoV-2 (17610 bp). DESCRIPTION OF THE INVENTION The present invention reports, in a first aspect thereof, a live recombinant vectored vaccine or live recombinant immunogenic composition comprising recombinant Newcastle disease virus (NDV) expressing the S1 subunit and the RBD of the SARS-CoV-2 S protein with significant high levels of protection and high immunization titers, wherein the vaccine or immunogenic composition comprises recombinant viruses named rNDV-LS1-HNRBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2, the construction of which is described later in the invention. Accordingly, in a second aspect of the present invention, it contemplates a recombinant virus rNDV-LS1-HN-RBD / SARS-CoV-2 characterized by SEQ ID No. 7 expressing the S1 subunit and the RBD of the SARS-CoV-2 S protein. Additionally, in a third aspect of the present invention, it contemplates a recombinant virus rNDV-LS1-S1-F / SARS-CoV-2 characterized by SEQ ID No. 13 that expresses the S1 subunit and the RBD of the SARS-CoV-2 S protein. Furthermore, in a fourth aspect of the present invention, it contemplates a synergistic combination of a recombinant rNDV-LS1-HN-RBD / SARS-CoV-2 virus characterized by SEQ ID No. 7 and a recombinant rNDV-LS1-S1-F / SARS-CoV-2 virus characterized by SEQ ID No. 13 in combination in the same recombinant live immunogenic composition or live vaccine that express the S1 subunit and the RBD of the SARS-CoV-2 S protein. The present invention further comprises the use of a recombinant virus rNDV-LS1-HN-RBD / SARS-CoV-2 characterized by SEQ ID No. 7 and a recombinant virus rNDV-LS1-S1-F / SARS-CoV-2 characterized by SEQ ID No. 13 for the manufacture of a recombinant immunogenic composition or live recombinant vaccine for the treatment of SARS-CoV-2. Additionally, the present invention comprises a method for controlling infection caused by SARS-CoV-2 by administering to mammals the recombinant live immunogenic composition or recombinant live vaccine comprising a recombinant virus rNDV-LS1-HNnzzfrnn / zznz / E / YiAi RBD / SARS-CoV-2 characterized by SEQ ID No. 7 and a recombinant virus rNDV-LS1-S1-F / SARS-CoV-2 characterized by SEQ ID No. 13 in combination in the same recombinant live immunogenic composition or live vaccine. In this regard, the live recombinant immunogenic composition or vaccine according to the present invention, comprising Newcastle disease virus (NDV) expressing the S1 subunit and the RBD of the SARS-CoV-2 spike protein, may further comprise a pharmaceutically acceptable adjuvant and / or excipient or vehicle, wherein pharmaceutically acceptable adjuvants are defined as substances that enhance specific immune responses to antigens by modulating the activity of immune cells. Examples of adjuvants that may be employed in the present invention for the live recombinant vaccine include, but are not limited to, saponins, agonist antibodies for costimulatory molecules, Freund's adjuvant, muramyl dipeptide (MPD), bacterial DNA (oligo CpG), lipopolysaccharides (LPS), MPL (Mozilla Public License) and synthetic derivatives, lipopeptides, and liposomes, among others. The adjuvant is an immunomodulator.In one embodiment of the invention, other preferred adjuvants may be squalene, Quillaja saponaria, and surfactants. Vaccine compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous vaccine preparation, preferably isotonic with the recipient's blood. These vaccines can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a parenterally acceptable, non-toxic diluent or solvent, for example, a solution in 1,3-butanediol. Acceptable vehicles and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. Any soft fixed oil, including synthetic mono- or diglycerides, can be used for this purpose.Furthermore, fatty acids such as oleic acid can be used in the preparation of injectables. A suitable vehicle formulation can be found for subcutaneous, intravenous, intramuscular, and oral administration. For live recombinant vaccines according to the present invention, adjuvants, vehicles, and / or diluents may include sterile water, yeasts, starches, gelatin, albumin, sucrose, lactose, sodium glutamate, and glycine in pharmaceutically acceptable quantities. In another embodiment of the invention, the live recombinant vaccine or immunogenic composition according to the present invention comprises a recombinant rNDV-LS1-HN-RBD / SARS-CoV-2 virus characterized by SEQ ID No. 7, a recombinant rNDV-LS1-S1-F / SARS-CoV-2 virus characterized by SEQ ID No. 13 or a mixture thereof, sterile water and optionally, adjuvants such as squalene, quillaja saponaria and surfactants. The recombinant live immunogenic compositions or vaccines according to the present invention may also optionally contain suitable preservatives, such as: benzalkonium chloride; chlorobutanol, parabens and thimerosal, among others; inactivating agents such as formaldehyde, glutaraldehyde, propiolactone and beta-propiolactone are used in quantities of parts per million (ppm) or parts per billion (ppb). A person skilled in the art will be familiar with other protocols for administering vaccine compositions, in which the dosage, injection schedule, injection sites, method of administration, and similar aspects may vary according to recommended practice. Administration of vaccine compositions to mammals other than humans (e.g., for testing or veterinary therapeutic purposes) is carried out under substantially the same conditions as described above. A subject, as used herein, is a mammal, preferably a human, and includes primates, cattle, horses, pigs, sheep, cats, and rodents. The vaccine compositions according to the present invention may also optionally contain suitable preservatives, such as: benzalkonium chloride; chlorobutanol, parabens and thimerosal, among others; inactivating agents such as formaldehyde, glutaraldehyde, propiolactone and beta-propiolactone are used in an amount at the parts per million (ppm) or parts per billion (ppb) level. The immunogenic compositions or vaccine according to the present invention can be administered by any conventional route, including injection, oral, intranasal aerosol, or inhalation, most preferably by intranasal route. Design and construction of plasmids containing the complete genome of new recombinant viruses Using reverse genetics, a new vaccine for SARS-CoV-2 was developed, based on an NDV vector, capable of expressing the S1 subunit and RBD genes from the SARS-CoV-2 spike protein. The present invention focuses on the development of two recombinant viruses named rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2, as well as their application in a single recombinant live immunogenic composition or formulation combining both vaccines or immunogenic compositions (rLS1-HN-RBD / SARS-CoV-2 + rNDV-LS1-S1-F / SARS-CoV-2). DETAILED DESCRIPTION OF THE INVENTION To achieve this, two versions of the virus have been designed, where the sequences based on the S1 subunit and RBD of the S protein of the new SARS-CoV-2 strain isolated in Wuhan-Hu-1-China was used in this study (GenBank accession number- MN908947.3) https: / / www.ncbi.nlm.nih.gov / nuccore / MN908947.3, were designed to be cloned in different ways into the pNDV-LS1 plasmid (19319 bp), which contains the complete NDV genome of the LaSota vaccine strain (lentogenic pathotype) (15186 bp) which has been recovered and described previously (Izquierdo-Lara et al. 2019). The recombinant plasmids obtained were named; pNDV-LS1-HN-RBD / SARS-CoV-2 and pNDVLS1-S1-F / SARS-CoV-2. 1) pNDV-LS1-HN-RBD / SARS-CoV-2 Genetic map of the pNDV-LS1-HN-RBD / SARS-CoV-2 plasmid The first step involved designing the HN-RBD / SARS-CoV-2 gene (see SEQ ID NO: 1). This gene consisted of an ORF corresponding to the HN gene, which contains the DTM and DC of the HN protein (144 bp) from the pNDV-LS1 plasmid (see SEQ ID NO: 1, 2, and 4) and the ectodomain of the RBD protein (636 bp) (see SEQ ID NO: 6) from SARS-CoV-2, with the aim of expressing the RBD on the virion surface (see Figure 2C). The sequence corresponding to the DTM and CT of the HN protein was predicted using the TMHMM Server 2.0 program (see Figure 1). The new gene was named HN-RBD / SARS-CoV-2, which, by containing the N-terminal region of the HN protein, should be able to expose the HN-RBD / SARS-CoV-2 protein on the virion surface. The RBD sequence selected for this design was 636 bp, taken from the strain isolated in Wuhan-Hu-1, China (GenBank Accession Number: MN908947.3) (990-1623 nt) (see SEQ ID NO: 6). https: / / www.ncbi.nlm.nih.gov / nuccore / MN908947.3 Following this HN-RBD / SARS-CoV-2 sequence (see SEQ ID NO: 6), the upstream and downstream sequences of the NDV M protein were added to the ends. The final construction of each plasmid had to comply with the "rule of six," which states that the length of the viral genome must be a multiple of 6, since it appears that genome packaging occurs every 6 nucleotides (Peeters et al. 2000). To achieve this, a thymine nucleotide "t" was added, followed by the stop codon of the HN-RBD / SARS-CoV-2 gene (see SEQ ID NO: 1). A defect in the number of nucleotides in the NDV genome can severely disrupt viral replication. nzzfrnn / zznz / E / YiAi This HN-RBD / SARS-CoV-2 gene sequence was chemically synthesized and subsequently cloned into the pUC57 cloning plasmid by GenScript (Piscataway, NJ, USA). This sequence was designed to be flanked at both ends by the unique restriction site BbvCI (NEB, New England BioLabs, Ipswich, MA, USA), which contains the “CCTCAGC” sequence and is located in the intergenic region between the P and M genes of pNDV-LS1 (19,319 bp) (see Figure 4). This plasmid was then purified and extracted using the QIAGEN Plasmid Midi Kit (100) (QIAGEN, Valencia, CA, USA), following the manufacturer's instructions. The pNDV-LS1 (19319 bp) was digested by the same single-cut enzyme BbvCI (NEB, New England BioLabs, Ipswich, MA, USA), which is located in the intergenic region between the P and M genes of NDV as previously described, thus obtaining the linearized plasmid. The second step consisted of inserting the synthetic gene HN-RBD / SARS-CoV-2 (1013 bp) (SEQ ID NO: 1) into the pNDV-LS1 plasmid (19319 bp), generating the new plasmid called pNDV-LS1-HN-RBD / SARS-CoV-2 (20315 bp) (see Figures 3 and 4). This resulted in the recombinant virus called rNDV-LS1-HN-RBD / SARS-CoV-2 (16182 bp) (see SEQ ID NO: 7). The pNDV-LS1 (19319 bp) was digested by the same single-cut enzyme BbvCI (NEB, New England BioLabs, Ipswich, MA, USA), which is located in the intergenic region between the P and M genes of NDV as previously described, thus obtaining the linearized plasmid. The second step consisted of inserting the synthetic gene HN-RBD / SARS-CoV-2 (1013 bp) (SEQ ID NO: 1) into the pNDV-LS1 plasmid (19319 bp), generating the new plasmid called pNDV-LS1-HN-RBD / SARS-CoV-2 (20315 bp) (see Figures 3 and 4). This resulted in the recombinant virus called rNDV-LS1-HN-RBD / SARS-CoV-2 (16182 bp) (see SEQ ID NO: 7). Genetic map of plasmid pNDV-LS1-S1-F / SARS-CoV-2 The first step consisted of the design of the S1-F / SARS-CoV-2 gene (2441 bp) (see SEQ ID NO: 8). This gene consisted of the “creation of an ORF”, comprised of the nucleotides “atg” subsequently fused with the ectodomain of the S1 subunit of the SARS-CoV-2 S, and with the fragment that encodes the DTM and CT of the F protein (162 bp) of the NDV (see SEQ ID NO: 8, 9 and 11), with the objective that the S1 is expressed on the surface of the virion (see Figure 2C). The sequence corresponding to the TM and CT of the F protein was predicted with the TMHMM Server 2.0 program (see Figure 5). nzzfrnn / zznz / E / YiAi The new gene was named S1-F / SARS-CoV-2 (2441 bp) (see SEQ ID NO: 8), and because it contains the N-terminal region of the F protein, it should be able to expose the S1 subunit protein on the virion surface. The S1 sequence selected for this design was 2043 bp, which was taken from the strain isolated in Wuhan-Hu-1, China (GenBank Accession Number: MN908947.3) (987-1563 nt) (see SEQ ID NO: 12). https: / / www.ncbi.nlm.nih.gov / nuccore / MN908947.3 The new gene was named S1-F / SARS-CoV-2 (2441 bp) (see SEQ ID NO: 8), and because it contains the N-terminal region of the F protein, it should be able to expose the S1 subunit protein on the virion surface. The S1 sequence selected for this design was 2043 bp, which was taken from the strain isolated in Wuhan-Hu-1, China (GenBank Accession Number: MN908947.3) (987-1563 nt) (see SEQ ID NO: 12). https: / / www.ncbi.nlm.nih.gov / nuccore / MN908947.3 Following this S1-F / SARS-CoV-2 sequence (see SEQ ID NO: 8), the upstream and downstream sequences of the NDV M protein were added to the ends. The final construction of each plasmid had to comply with the "rule of six," which states that the length of the viral genome must be a multiple of 6. This is because it appears that genome packaging occurs every 6 nucleotides (Peeters et al. 2000). To achieve this, the nucleotides "tgac" were added, followed by the stop codon of the S1-F / SARS-CoV-2 gene (see SEQ ID NO: 8). A defect in the number of nucleotides in the NDV genome can severely disrupt viral replication. This S1-F / SARS-CoV-2 gene sequence was chemically synthesized, optimized for Gallus gallus, and then cloned into the pUC57 cloning plasmid by GenScript (Piscataway, NJ, USA). This sequence was designed to be flanked at both ends by the unique restriction site BbvCI (NEB, New England BioLabs, Ipswich, MA, USA), which contains the “CCTCAGC” sequence located in the intergenic region between the P and M genes of NDV. This plasmid was purified and extracted using the QIAGEN Plasmid Midi Kit (100) (QIAGEN, Valencia, CA, USA), following the manufacturer's instructions. The pNDV-LS1 plasmid (19319 bp) was digested by the same single-cut enzyme BbvCI (NEB, New England BioLabs, Ipswich, MA, USA), which is located in the intergenic region between the P and M genes of NDV, as previously mentioned, thus obtaining the linearized plasmid. The second step consisted of inserting the synthetic gene S1-F / SARS-CoV-2 (2441 bp) (see SEQ ID NO: 8) into the pNDV-LS1 plasmid (19319 bp), generating the new plasmid called pNDV-LS1-S1-F / SARS-CoV-2 (21743 bp) (see Figures 8A-8B). This resulted in the virus called rNDV-LS1-S1-F / SARS-CoV-2 (17610 bp) (see SEQ ID NO: 13). nzzfrnn / zznz / E / YiAi Recovery of rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2 viruses The new recombinant viruses rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2 were recovered by co-transfecting with the support plasmids containing the genes that synthesize the RNP complex, pCI-L, pCI-N and pCI-P together with the pNDV-LS1-HN-RBD plasmid and the pNDVLS1-S1-F plasmid in Vero cells as described in a previous publication (Chumbe et al. 2017). η77πηη / 77η7 / Β / γΐΛΐ II. Pathogenicity of rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1F / SARS-CoV-2 viruses To evaluate the pathogenicity and determine the biological characteristics of the viruses, different indices were used, such as: Intracerebral Pathogenicity Index (ICPI), Mean Death Time (MDT), both tests were evaluated in 1 day old chickens (Charles River Laboratories), unlike the mean infectious dose (EIDso / mL) which was evaluated in embryonated eggs of 9 to 10 days of age (Charles River Laboratories), the analysis of the cleavage site of the Fusion gene of the rNDV-LS1-HN-RBD / SARSCoV-2 and rNDV-LS1-S1-F / SARS-CoV-2 viruses, showed no changes at the nucleotide and amino acid level, maintaining the cleavage site of both viruses similar to rNDV-LS1 (Table 1). Table 1. Pathogenicity and biological characteristics of the rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDVLS1-S1-F / SARS-CoV-2 viruses ICP Virus | rNDV-LS1-S1- F / SARS-CoV-2 0.0 > 168 h 10 100 2io 112GRQGRL117 The intracerebral pathogenicity index (ICPI) was evaluated in 1-day-old birds. The maximum score for a virulent strain is 2.0, while the score for the Lentogenic strain is 0.0. Mean time to death (MTD) was calculated using 10-day-old embryonated eggs. A value of 60 hours corresponds to velogenic strains, values ​​between 60 and 90 hours correspond to mesogenic strains, and ontogenic strains have a value greater than 90 hours. III. Verification of the HN-RBD and S1-F expression To evaluate the expression of the HN-RBD and S1-F proteins, Vero E6 cells were infected with the recombinant viruses rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2 at a multiplicity of infection (MOI) of 1. After 48 hours of infection, the cells were collected and used. On the other hand, to verify the incorporation of the HN-RBD and S1-F proteins in the rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2 viruses, the viral particles were concentrated by ultracentrifugation using an ultracentrifuge (Ultracentrifuge, Beckam, Culter). These viral particles were obtained from a harvest of allantoic fluid from pathogen-free (SPF) embryonic chicken eggs (Charles River Laboratories) infected with the new recombinant viruses, and were subsequently partially purified in 30% sucrose.The Western blot assay was performed using partially purified viruses from allantoic fluid and infected cells, using a rabbit antibody specific for the SARS-CoV-2 RBD protein (Sino biological, Cat: 4059-2-T62) 2 / 5000 and a rabbit Anti IgG secondary antibody conjugated to HRP (Cat. A01827) 2 / 5000. The results from the Western blot assay showed that the antibodies reacted with the Used from cells infected with the rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2 viruses, detecting a band with a molecular weight of ~30 kDa and ~90 kDa respectively that would correspond to HN-RBD and S1-F, on the other hand, no reactivity was observed with the Used infected with the parental virus (see Figure 8B). In another Western blot assay with the purified viruses rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1S1-F / SARS-CoV-2, the results showed a band with a molecular weight of ~30 kDa and ~90 kDa, respectively, corresponding to HN-RBD and S1-F. Furthermore, no reactivity was observed with the purified parental virus. These results would confirm the incorporation of HN-RBD and S1-F into the viral structure, since the RBD insert was fused to the DC and DTM of the NDV HN protein. In the case of the S1 insert, it was fused to the DC and DTM of the NDV F protein (see Figure 8A). nzzfrnn / zznz / E / YiAi V. Immunofluorescence assay To examine the expression of the HN-RBD and S1-F proteins by the recombinant viruses rNDVLS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2, Vero-E6 cells were infected with the recombinant viruses at an MOI of 0.5. After 48 hours of infection, the cells were fixed with 4% paraformaldehyde for 25 minutes, then the monolayer was washed three times with Dulbecco's phosphate-buffered saline (DPBS) and permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature. The monolayer was then washed three times with DPBS and incubated with rabbit antibody specific against SARS-CoV-2 RBD protein (1:200) (cat, 40592-T62), and chicken antiserum specific against Newcastle disease virus (1:200) (Charles River) for 1.5 hours at room temperature.Subsequently, the monolayer was incubated with Alexa Fluor 594 (1:250) conjugated goat anti-rabbit IgG secondary antibodies (Abcam) and Alexa Fluor 488 (1:1000) conjugated goat anti-chicken IgY secondary antibodies (ab150169, Abcam) for 1 hour at room temperature. Finally, the cell nuclei were stained with DAPI mounting medium (ab104139, Abcam) for 5 minutes. The results were observed using an ObserverAI fluorescence microscope (Cari Zeiss, Germany). Digital images were taken at 400x magnification and processed using an AxioCam MRc5 camera (Cari Zeiss, Germany). Immunofluorescence results in cells infected with the recombinant viruses rNDV-LS1HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2 were positive when they reacted with rabbit antibodies specific to the SARS-CoV-2 RBD protein and chicken antiserum specific to NDV, thus demonstrating the presence of NDV proteins and the expression of the SARS-CoV-2 RBD and S1 proteins. On the other hand, in cells infected with the parental NDV, reactivity was only observed with chicken antiserum specific to NDV (see Figure 10). VI. Cell binding and internalization assay by efficiency to the ACE2 receptor expressed in Vero E6 cells To determine the binding efficiency between the ACE2 receptor and the RBD or S1 subunit of the recombinant viruses rNDV-S1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2, respectively, Vero E6 cells expressing the ACE2 receptor on the cell membrane were collected and washed with DPBS supplemented with 5% fetal bovine serum (FBS). A concentration of 1 x 10⁶ cells was then blocked with DPBS containing 5% normal mouse serum for 30 minutes and subsequently incubated with the recombinant viruses (previously purified with 25% sucrose) at final concentrations of 0.05 and 0.2 pg for 30 minutes at 37 °C. To eliminate residual viral particles that did not bind to the ACE2 receptor of Vero E6 cells during the incubation time, the cells were washed with DPBS nzzfrnn / zznz / E / YiAi supplemented with 5% SFB twice.Subsequently, the cells were labeled with rabbit anti-SARS S1 monoclonal antibodies (1:200) (Sino Biological) for 1 hour at room temperature, followed by the secondary antibody conjugated to Alexa Fluor 488 goat anti-rabbit IgG (1:200). Finally, the cells were analyzed using a FACS Canto II flow cytometer (BD), and the data obtained were analyzed using FlowJo v.10.6 software (BD). The results showed an ACE2 receptor binding reactivity of -10% and -40% in the cells analyzed with the recombinant viruses rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2, respectively. This demonstrates the presence of the RBD and the S1 subunit of SARS-CoV-2 in the envelope structure of the rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1F / SARS-CoV-2 viruses, respectively (see Figure 11). This was expected, since the RBD insert was fused to the DTM and DC of the NDV HN protein. The S1 insert was fused to the DTM and DC of the NDV F protein. These construction designs were made with the purpose of having the RBD and S1 expressed and incorporated into the viral envelope of recombinant viruses and having affinity for the ACE2 receptor. Vile. In vitro growth properties of rNDV-LS1-HN-RBD / SARS-CoV-2 and rN D V-LS1 -S1 -F / S ARS-CoV-2 viruses DF-1 cells (DF-1, from the English: Chicken Embryo Fibroblast), were seeded at a confluence of 70% in a 12-well plate, then 18 hours post-seeding they were infected with the viruses rNDV-LS1-HN-RBD / SARS-CoV-2, rNDV-LS1-S1-F / SARS-CoV-2 and rNDV-LS1 at a MOI of 1. The cells were maintained in DMEM containing 1% FBS and 10% allantoic fluid (AF) with 5% CO2 at 37°C. Supernatants were collected at 12, 24, 36, 48, 60, 72, 84, 96, and 108 hours post-infection (hpi), clarified at 500 rpm for 5 minutes at 4°C to remove cellular debris. Supernatants were quantified by plate assay in DF-1 cells, as previously described (Chumbe et al. 2017). Five days post-incubation, the cells were fixed with 3.2% paraformaldehyde for 6 hours at room temperature, and then stained with 0.2% crystal violet. The titers obtained by plate assay were calculated and reported as plaque-forming units (PFU). This experiment was repeated at three different times. nzzfrnn / zznz / E / YiAi The results showed that both viruses: rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2 showed growth properties similar to the parental virus rNDV-LS1; however, rNDVLS1 showed slightly elevated titers at 24 and 36 hpi compared to rNDV-LS1-HNRBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2, but not significant (see Figure 12). The cytopathic effect (CPE), or syncytium formation, of the rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2 viruses showed the typical CPE of NDV. This may explain why the insertion of both foreign genes did not alter their biological properties. VIII. Immunization in the Hamster Animal Model To evaluate the safety, immunogenicity, and efficacy of the vaccines, forty-eight five-week-old hamsters, both male and female, were used in this study. The experiments were conducted following protocols previously approved by the National Institute of Health (INS) of Peru in conjunction with FARVET SAC. The hamsters were randomly divided into four groups (12 hamsters / group). The hamsters were inoculated intranasally (IN) with 40 µL (2 x 10⁶ PFU / hamster) of the following viruses: rNDV-LS1-HNRBD / SARS-CoV-2 (Group #1), rNDV-LS1-S1-F / SARS-CoV-2 (Group #2), a mixture of both rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2 (Group #3), and a non-immunized control group (Group #4). An IN booster immunization with 40 µL (2 x 10⁶ PFU / hamster) of each vaccine was administered to all groups 15 days post-vaccination (DPV). At 15 and 30 DPV, blood samples were collected to measure the antibody titers generated by the vaccines: anti-IgG antibodies by in-house ELISA-SARS-CoV2 (ELISA: enzyme-linked immunosorbent assay) and to measure the circulating neutralizing antibodies produced by the vaccines, a surrogate neutralization test (TNs) anti-RBD-SARS-CoV2 from a commercial kit (GenScript, Piscataway, NJ, USA) (Cat. No. L00847) was performed. nzzfrnn / zznz / E / YiAi IX. Evaluation of Anti-RBD Neutralizing Antibodies using a Surrogate Neutralization Test (TNs) Forty-eight serum samples were analyzed to evaluate neutralizing antibody titers against SARS-CoV-2. Neutralization assays were performed using the Surrogate Neutralization Test (TNs) Kit (GenScript, Piscataway, NJ, USA) (Cat. No. L00847). This test uses purified proteins, unlike the conventional virus neutralization test (TNc): 1) the RBD of the SARS-CoV-2 spike protein and 2) the ACE2 cell receptor, thus masking the interaction of the virus with the host in an ELISA plate. When the RBD-ACE2 interaction does not occur, neutralization is indicated, meaning the presence of specific neutralizing antibodies in the serum. The advantage of this TNs is the ease with which it can be developed in Biosafety Level 2 (BSL-2) laboratories without any difficulty, following the manufacturer's instructions exactly. The plates were analyzed using an Epoch 2 microplate reader (Bioteck, USA) at 450 nm. The cutoff for positive and negative results for SARS-CoV-2 neutralizing antibodies was interpreted as the inhibition rate. The cutoff was interpreted as follows: positive result >20% (neutralizing antibodies detected), negative result <20% (neutralizing antibodies not detected). rNDV-LS1-HN-RBD / SARS-CoV-2 (Group #1), rNDV-LS1-S1-F / SARS-CoV-2 (Group #2), the mixture of both rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2 (Group #3), and a non-immunized control group (Group #3). #4). The results of the sera collected from the hamsters indicated the early circulation of neutralizing antibodies from the first 15 days post immunization in the groups: # 2 (rNDVLS1-S1-F / SARS-CoV-2) and #3 (rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2) showed an inhibition >60%, while group #1 (rNDV-LS1-HN-RBD / SARS-CoV-2) showed an inhibition of 30%, unlike group #4 (non-immunized control group) which showed an inhibition rate <20%.On the other hand, as expected, these antibody titers increased, so that 30 days after immunization it was detected that groups #2 (rNDV-LS1-S1-F / SARS-CoV-2) presented an inhibition rate between 70 to 80%, group #3 (rNDV-LS1-HN-RBD / SARS-CoV2 and rNDV-LS1-S1-F / SARS-CoV-2) presented an inhibition rate between 80 to 100%, while group #1 (rNDV-LS1-HN-RBD / SARS-CoV-2) presented an inhibition of 35%, unlike group #4 (non-immunized control group) which presented an inhibition rate <5%. The results of this study nzzfrnn / zznz / E / YiAi demonstrate that there is a synergy between the recombinant viruses rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2 when they are present in the same recombinant live immunogenic composition or recombinant live vaccine according to the present invention. Based on these results, we hypothesize that animals in groups 1, 2, and 3 exhibit protection within the first 15 days post-immunization, and that this protection is boosted 30 days post-immunization, generating neutralizing antibody titers with up to 100% inhibition compared to non-immunized animals. These results demonstrate the vaccines' efficacy due to their rapid ability to generate a humoral response. X. Evaluation of Anti-SARS-CoV-2 Neutralizing Antibodies using a Conventional Neutralization Test (TNc) All neutralization assays were performed at the Biosafety Level 3 (BSL3) Laboratory of the National Institute of Health (INS). Collected sera were inactivated at 56°C for 60 minutes prior to use. Vero E6 cells were maintained in 1X DMEM culture medium supplemented with 5% FBS and pre-seeded 18–24 hours prior to use in a 24-well cell culture plate. Following inactivation, all sera from each group were pooled, and serial two-fold dilutions were performed, starting with a 1:20 dilution. The sera were then mixed with SARS-CoV-2 virus (serum-virus) and incubated for 1 hour at 37°C. Ten percent of the mixture was then transferred to each well containing the pre-seeded Vero E6 cell culture monolayer.The cells were incubated for 4 days at 37°C, then fixed with 10% paraformaldehyde for 6 hours and stained with 2% crystal violet for 15 minutes, then washed and dried at room temperature. The limiting dilution was determined by the appearance of SARS-CoV-2 virus plaques or extracellular matrix (ECM) on the cells. The results obtained showed that hamsters immunized with both viruses rNDV-LS1-HNRBD / SARS-CoV-2 + rNDV-LS1-S1-F / SARS-CoV-2 reached a neutralization titer of 1 / 40 at 100%, while the group immunized with rNDV-LS1-HN-RBD / SARS-CoV-2 had a titer of 1 / 20 at 100%, and only rNDV-LS1-S1-F / SARS-CoV-2 reached a titer of 1 / 160 at 100%, which indicates that rNDV-LS1-S1-F / SARS-CoV-2 has caused a greater number of neutralizing antibodies, this is possibly due to the fact that the S1 region presents other important antigenic epitope regions of the S of SARS-CoV-2 (see results Table 2). Table 2. Seroneutralization measures against SARS-CoV-2, obtained from a pool of viruses from viruses infected with rNDV-LS1-HN-RBD / SARS-CoV-2 and rNDV-LS1-S1-F / SARS-CoV-2 nzzfrnn / zznz / E / YiAi Recombinant Viruses Seroneutralization Rate - SARS-CoV-2 Titre 100% 1 / 20 1 / 40 1 / 80 1 / 160 1 / 320 1 / 640 Group #1: rNDV-LS1-HN- RBD / SARS-CoV-2 78.4 67.6 37.8 37.8 16.2 10.8 <1 / 20 Group #2: rNDV-LS1-S1- F / SARS-CoV-2 100.0 100.0 100.0 100.0 97.3 94.6 1 / 160 Group #3 rNDV-LS1-S1- F / SARS-CoV-2 + rNDV-LS1-HN-RBD / SARS- CoV-2 100.0 100.0 75.7 62.2 37.8 21.6 <1 / 40 XI. Evaluation of IgG Anti-RBD of SARS-CoV-2 by indirect ELISA The presence of anti-RBD IgG in vaccinated hamsters was assessed using an in-house indirect ELISA. Microplates were fixed with 1 µg / ml of purified RBD protein diluted in carbonate buffer, pH 9, overnight. The plates were washed with phosphate-tween buffer (PBS-T) and blocked with 3% milk (PBS-T and 3% milk) (Difeo Skim Milk 232100) for 2 hours with shaking at room temperature. After blocking, the plates were washed with PBS-T. Serum was added at a 1:100 dilution for hamster serum, and the plates were incubated for 1 hour at 37°C. 100 pL of HRP-conjugated anti-hamster IgG (1:28000) (Abcam 6892) was used as the secondary antibody. The samples were washed and the substrate 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase was added. After 15 minutes, the reaction was stopped with 2 N H2SO4 and the absorbance was measured at 450 nm. The pre-immune and mock sera from each treatment were used as negative controls. 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Claims

1. A live recombinant immunogenic composition comprising recombinant Newcastle disease virus (NDV) expressing the S1 subunit and RBD of the SARS-CoV-2 S protein, characterized in that it comprises a recombinant virus designated rNDV-LS1HN-RBD / SARS-CoV-2 of SEQ ID No. 7, a recombinant virus designated rNDV-LS1-S1F / SARS-CoV-2 of SEQ ID No. 13, or a mixture thereof.

2. The recombinant live immunogenic composition according to claim 1 characterized in that it comprises the recombinant virus designated rNDV-LS1-HN-RBD / SARSCoV-2 of SEQ ID No.

7.

3. The recombinant live immunogenic composition according to claim 1 characterized in that it comprises the recombinant virus designated rNDV-LS1-S1-F / SARSCoV-2 of SEQ ID No.

13.

4. The live recombinant immunogenic composition according to claim 1 characterized in that it comprises the recombinant virus rNDV-LS1-HN-RBD / SARS-CoV-2 of SEQ ID No. 7 and the recombinant virus rNDV-LS1-S1-F / SARS-CoV-2 of SEQ ID No. 13 in combination in the same immunogenic composition.

5. The live recombinant immunogenic composition according to any of the preceding claims, characterized in that it further comprises pharmaceutically acceptable adjuvants, excipients or vehicles.

6. The recombinant live immunogenic composition according to claim 5, characterized in that the adjuvants are selected from the group consisting of saponins, agonist antibodies for costimulatory molecules, Freund's adjuvant, muramyl dipeptide (MDP), bacterial DNA (oligo CpG), lipopolysaccharides (LPS), MPL (Monophosphoryl lipid A) and synthetic derivatives, lipopeptides, liposomes, squalene, Quillaja, and surfactants.

7. The recombinant live immunogenic composition according to claim 5, characterized in that the pharmaceutically acceptable vehicle is selected from the group consisting of sterile water, Ringer's solution, and isotonic sodium chloride solution.

8. The recombinant live immunogenic composition according to any of the preceding claims, characterized in that it is an intranasal composition. nzzfrnn / zznz / E / YiAi 9. A method for controlling SARS-CoV-2 infection by administering to mammals a live recombinant immunogenic composition or live recombinant vaccine comprising a recombinant rNDV-LS1-HN-RBD / SARS-CoV-2 virus characterized by SEQ ID No. 7 and a recombinant rNDV-LS1-S1-F / SARS-CoV-2 virus characterized by SEQ 5 ID No. 13 in combination in the same live recombinant immunogenic composition or live vaccine.