WO2022149058A1 - Recombinant live immunogenic composition comprising newcastle disease virus (ndv) expressing the s1 subunit and the rbd of the spike protein of sars-cov-2 - Google Patents

Abstract

The present invention relates to the obtainment of two live recombinant or vectored NDV vaccines expressing the S1 subunit and the RBD of the S protein of SARS-CoV-2 which, when applied intranasally in a hamster animal model, generate the production of neutralising antibodies against SARS-CoV-2. The invention also shows that these live vaccines are compatible for combined use with each other to induce the production of neutralising antibodies against SARS-CoV-2 without substantial cross-interference, indicating a synergy between the two vaccines, wherein 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) express the S1 subunit and the RBD of the S protein of SARS-CoV-2 in mammals.

Description

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

TECHNICAL FIELD

[001] The present invention falls within the pharmaceutical industry. The present invention is related to obtaining two vectorized recombinant viruses derived from Newcastle disease virus (NDV) comprising the S1 subunit and the receptor binding domain called RBD (RBD). : Receptor Binding Domain), both from the new Severe Acute Respiratory Syndrome coronavirus -2 (SARS-CoV-2) through the reverse genetics system, and to the corresponding product of a vaccine or recombinant live 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 to immunize, and protect against coronavirus disease 2019 (COVID-19) in mammals and superior human.

BACKGROUND

[002] The present invention was developed based on the use of a viral vector previously elaborated, with the Title N° 9204 of the Registry of Industrial Property “VACCINE THAT COMPRISES RECOMBINANT NEWCASTLE DISEASE VIRUS (NDV) THAT EXPRESSES THE GENE OF THE SPÍCULA S DEL VIRUS DE LA BRONQUITIS INFECCIOSA AVAR (IBV)”, granted by the Directorate of Inventions and New Technologies of INDECOPI certified in Resolution No. 003059-2018/DIN-INDECOPI. STATE OF THE TECHNIQUE

[003] COVID-19 is an infectious disease in humans caused by the highly pathogenic coronavirus, SARS-CoV-2, which emerged in Wuhan and its rapid international spread has posed a serious global public health emergency (Zhou et al. 2020 ; Wu et al. 2020; Zhu et al. 2020). SARS-CoV-2 infected patients show a variety of symptoms including dry cough, fever, headache, tiredness and shortness of breath

(https://www.who.int/es/emergencies/diseases/novel-coronavirus-2019/advice-for-public/q-a-coronaviruses), with an estimated mortality rate of between 3 and 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.

[004] 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 analyzes 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 ordered from 5' to 3' sense: replicase (ORF1a/ORF1b), spike (S), envelope (E), membrane (M), and nucleocapsid ( N). Additionally, seven putative ORFs encoding accessory proteins are interspersed between structural genes (Chan et al. 2020). Most of the proteins encoded by SARS-CoV-2 are similar in length to the corresponding SARS-CoV proteins. Of four structural genes, SARS-CoV-2 shares more than 90% amino acid (aa) identity with SARS-CoV-2. SARS-CoV, except for the S gene, which diverges (H. Zhou et al. 2020; Lu et al. 2020).

[005] Coronaviruses use the S-glycoprotein (comprised of an S1 subunit and an S2 subunit) in the envelope to bind to their cellular receptors. Such binding triggers a cascade of events leading to fusion between the membranes; cellular and viral for cellular entry of the virus. Previous studies with cryo-electron microscopy of the S protein of SARS-CoV and its interaction with the cellular receptor ACE2 (ACE2, English: Angiotensin-Converting Enzyme 2), have shown that the S1 subunit binds to the receptor, then this binding with the receptor induces a dissociation of S1 with ACE2, which induces S2 to go 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 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 without 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 an affinity in the nanomolar range. low, 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).

[006] The S protein of SARS-CoV-2 has a full size of 1273 amino acids, longer than that of SARS-CoV (1255 aa). It is distinct from the S proteins of most members of the Sarbecovirus subgenus of the betacoronavirus genus, and shares sequence similarities of amino acids from 76.7 to 77.0% with SARS-CoV from civets and humans. Regarding RBD, the amino acid similarity between SARS-CoV-2 and SARS-CoV is only 73%. Another specific genomic feature of SARS-CoV-2 is the insertion of four amino acid residues (PRRA) at the junction of the S1 and S2 subunits of the S protein (Andersen et al. 2020). This insertion generates a polybasic cleavage site (RRAR), which allows efficient cleavage by the presence of 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 insertion (PAA) in RmYN02, a recently reported bat-derived coronavirus in Rhinolophus malayanus in China (H. Zhou et al 2020).

[007] On the other hand, the antigenicity of the RBD plays an important role for 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 RBD SARS-CoV-2 S protein induce a potent functional antibody response in immunized mice, rabbits, and non-human primates ( Macaca mulatta) as early as 7 to 14 days after injection of a single dose of vaccine. Sera from immunized animals blocked RBD binding to the ACE2 receptor expressed on the cell surface, and neutralized infection with a pseudovirus SARS-CoV-2 and SARS-CoV-2 in vitro. Notably, vaccination also provided protection in nonhuman primates against an in vivo challenge with SARS-CoV-2 (Yang et al. 2020).

[008] In the future, vaccination could be the most effective method for a strategy of prevention, reduction of morbidity and mortality of COVID-19 in the long term. As of October 2, 2020, -174 vaccine candidates against COVID-19 have been reported and 51 are beginning clinical trials in humans. Therefore, the choice of an ideal vaccine could take longer.

[009] Different platforms for obtaining vaccines against SARS-CoV-2 are underway, 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).

[010] Among the different vaccine development platforms, the vector-viral vaccine has a potential advantage over the other vaccines, because it can be used as live vaccines and inactivated vaccines, which can induce a Th1 and Th2 immune response. Various vaccine viral vectors have been proposed such as: adenovirus, measles virus, Ankara virus, acute vesicular stomatitis and human parainfluenza. However, each viral vector presents some limitations that may or may not be possible to overcome, among them, for example, the immunogenicity of some viral vector vaccines has not been ideal, on the other hand, a defective replication of the adenovirus vaccine vector may not induce a good local immunity, in the case of the vesicular stomatitis virus so far it is questionable, and the human parainfluenza vector may not be effective in adult humans due to their pre-existing immunity.

[011] 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, 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]. [012] NDV has a single-stranded, non-segmented RNA genome of negative polarity. The length of the NDV genome is 15,186 nucleotides (nt) and it contains six genes in the order of 3'-NP-PMF-HN-L-5', which code for the nucleoprotein (NP), phosphoprotein (P), protein matrix (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), long protein polymerase (L), in addition to the presence of two non-structural proteins called V and W that are produced at the time of transcription of the P gene (Cattoli et al. 2011; Dimitrov et al. 2016). The length of the NDV genome must be a multiple of six for efficient RNA replication, thus fulfilling the so-called “rule of six”.

[013] Each transcriptional unit contains a major ORF flanked by short untranslated regions (UTRs), which are followed by conserved transcription initiation and termination sequences known as gene initiation or gene start (GS) (GS, from the English: Gen Start), final gene or Gen End (GE) (GE, from the English: Gene End) and between both are located the intergenic sequences (IGSs) (IGSs, from the English: Intergenic Sequences) non-coding or also called untranslated sequences.

[014] The construction and assembly of the virion is associated with three glycoproteins; F, HN and M. The glycoproteins F and HN are anchored in the envelope of the virion. The M glycoprotein is located on the inner face of the viral envelope. The HN and F glycoproteins are incorporated into the virion via the interaction of their cytoplasmic stems or also called cytoplasmic domains (DC) together with the M protein (Dolganiuc et al. 2003 ; Pantua et al. 2006).

[015] The HN protein contains the binding and recognition site for the host cell receptor. On the other hand, glycoprotein F mediates the fusion of the virus with the host cell membrane, thus contributing at the start of the NDV replicative cycle (Kim et al. 2011; Kumar et al. 2011). Both are capable of generating neutralizing antibodies since they are protective antigens against NDV.

[016] The main determinant of virulence in NDV in birds is the activation of glycoprotein F, which occurs by cellular endoprotease-mediated cleavage. In many velogenic and some mesogenic strains, the F cleavage site is rich in basic amino acids and is rapidly cleaved by intracellular proteases such as furin, thus allowing replication in a variety of tissues. On the other hand, lentegenic and some mesogenic strains contain few basic residue sites that will depend on secretory proteases found mainly in the lung for their cleavage, therefore, their replication restriction is towards epithelial surfaces where the protease is found. .

[017] Its natural host is birds, and it is antigenically distinct from common human pathogens. They are exclusively cytoplasmic viruses and therefore they do not integrate their viral genes into the host genome, which favors and raises their safety profile.

[018] The severity of the avian disease depends on the pathotype of the NDV strains:lentogenic, mesogenic, and velogenic. Thelentogenic strains (LaSota), cause a mild or asymptomatic infection that is restricted to the respiratory tract. Viruses of intermediate virulence are called mesogenic, while viruses that cause systemic infection and high mortality are called velogenic. Lentegenic and mesogenic strains are in many cases used as live NDV vaccines for birds throughout the world.

[019] Recent studies have highlighted the potential use of NDV to be used as a vaccine vector for either veterinary and/or human use. The Reverse genetics system has been used for the design and development of various modern vaccines.

[020] If foreign gene inserts 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 with the domains: DC and transmembrane (DTM) (DTM). : Domain Transmembrane ) of the F and/or HN protein, another option is for the foreign protein to be inserted with its complete CT and DTM.

[021] In this way and in order to develop a vaccine or recombinant live immunogenic composition for SARS-CoV-2, based on an NDV vector, which 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 vectorized recombinant vaccines called: rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS-CoV-2, obtained by the reverse genetics system using complementary fragments spanning the entire genomes of rNDV-LS1-S1-F/SARS-CoV-2 and rNDV-LS1-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 application route compared to other vaccines.

[022] From the field of patents, document CN112011521 is known, which refers to a new strain of coronavirus candidate for a recombinant Newcastle disease virus vector vaccine and its method of construction and application by means of genetically modified vaccine technology. . The candidate belongs to the Newcastle disease virus vaccine LaSota strain as a carrier, the P gene of the LaSota NDV strains, and the gene mutation (C3756T, M is inserted between the deletion of the BAMHI site) of the mutation of the new S gene coronaviruses; the novel nucleotide sequence of the coronavirus S gene shown in SEQ ID NO.1. The LaSota strain of the attenuated strain of NDV is used as the vector background, and the S gene (full ORF) of the SARS-CoV-2 virus is inserted into the P gene and the M gene, and the silent mutation of C3756T is introduced into the conserved region of the S gene, thus removing the BamHI site.

[023] Document CN101629178 describes a hand-combined Newcastle disease virus F gene and a recombinant expression vector and an application thereof, wherein the codons of the NDVF OFR gene are all replaced into NDVF bias codons. chicken, EcoR V restriction sites and Kozak sequences are added upstream, Xbal I restriction sites and TGA stop codons are added downstream, and A bases downstream of the stop codons are changed to G, the obtained genes can be expressed efficiently in eukaryotic cells, and the expression efficiency of the obtained genes is higher than that of wild-type genes.

[024] For its part, document CN 106047823 describes a bivalent vaccine strain R-H120-Lasota (HN) obtained by replacing a 5a gene of the H120 strain with an HN gene of the avian Newcastle disease virus based on a H120 avian infectious bronchitis virus reverse genetic system constructed. The recombinant vaccine strain can simultaneously protect IBV standard toxin counteracting strains and Newcastle disease standard toxin counteracting strains and can be used to prevent avian infectious bronchitis virus and Newcastle disease virus. Poultry Newcastle. It also reports that the vaccine was applied with double doses of the SARS virus in experimental animals and the content of the virus at the peak of pulmonary replication was much lower. [025] WO2000/67786 relates to cDNAs for producing attenuated, infectious Newcastle disease virus (NDV) and to methods of making the cDNAs and to the vector containing the cDNA optionally linked to an operable promoter. Methods of preparing the vaccines and methods of using them to prevent or treat Newcastle disease in an avian host are also described. It reports nucleotide sequences of the entire NDV genome, the leading region, the trailing 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 the characterization of factors acting in the transcription and replication of various non-segmented negative-stranded RNA viruses to recover whole infectious recombinant viruses from full-length cDNAs for various RNA viruses. non-segmented negative-chain virus among which he 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 entire NP gene , the entire trailer region, and the intergenic regions in the NP-P and P.

[026] For its part, patent W02015/013178 teaches the construction of recombinant NDV expressing ILTV gB, gC and gD and a schematic diagram representing the full-length antigenome of the NDV LaSota strain with the insertion of an added gene designed to express the ILTV gB consisting of the entire gB ORF fused to the last 12 amino acids of the cytoplasmic tail of the NDV F protein and also suggests methods of immunizing a mammal against a non-avian pathogen.

[027] 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. Also provides CoV polypeptides and acid molecules nucleic encoding them and neutralizing antibodies that specifically bind to the S protein of MERS-CoV and antigen-binding fragments thereof.

[028] The Peruvian patent 1179-2014/DIN reports a viral particle or chimeric virus that comprises an asymptomatic,lentogenic Newcastle disease virus (NDV) with values of the Intracranial Pathogenicity Index (IPIC) equal to zero that expresses at least a gene from a virus strain (IBV), wherein the gene or at least one of the genes from an IBV strain corresponds to the entire spike (S) gene or part of the spike (S) gene, where said entire 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, show that the S2 domain is capable of generating neutralizing antibodies and that a correct folding and formation of the homotrimer of the S protein requires the presence of the S1 and S2 domains, since S2 contains specific trimerization sites that in turn participate in the fusion process.

[029] On the other hand, EP2251034 provides a negative carrier chimeric RNA virus that allows a subject, for example, a bird, to be immunized against two infectious agents using a single chimeric virus of the invention. In particular, the invention provides chimeric Newcastle disease viruses (NDV) designed to express and incorporate into their virions a fusion protein comprising the ectodomain of an infectious agent protein and the transmembrane and cytoplasmic domain of an NDV protein. . Such chimeric viruses induce an immune response against NDV and the infectious agent.

[030] Additionally, in the scientific literature, the article entitled "Newcastle disease virus (NDV) expressing the SARS-1 CoV-2 spike protein as a candidate vaccine" teaches that The NDV vector vaccine against SARS-CoV-2 described in this study has advantages similar to those of other viral vector vaccines, but the NDV vector can be amplified in embryonated chicken eggs, which allows high yields and low costs per dose and providing an important option for a cost-effective vaccine against SARS-CoV-2. Teaches a method for the rescue of NDV LaSota expressing the Spike protein from SARS-CoV-2.

[031] It is also known from the article “A Newcastle disease virus (NDV) expressing membrane-anchored Spike 1 protein as an inactivated vaccine against SARS-CoV-2” and indicates that an attenuated recombinant NDV was investigated expressing the membrane-anchored S-F chimera (NDV-S) 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 S-F chimera expressed by the NDV vector is very stable without loss of antigenicity after 3 weeks of storage at 4°C in allantoic fluid.

[032] Finally, the article entitled "Newcastle disease virus, a restricted host range virus, as a vaccine vector for intranasal immunization against emerging pathogens" is also known, which involves the 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 family Paramyxoviridae. However, its natural host is birds, and it is antigenically distinct from common human pathogens. However, NDV vectors were made in which one of the constructs was a recombinant copy of thelentogenic LaSota strain that was modified in such a way that the cleavage sequence of its F protein was replaced with that of NDV-BC, which resulted in the NDV-VF virus and based on these last two vaccine viruses are constructed that express the total length of 1,255 aa of the S protein of the SARS-Co-V. It also indicates that the S1 domain contains the S protein receptor-binding site as one of the major neutralizing epitopes.

[033] On the other hand, to date, there is no candidate live NDV vectored vaccine or recombinant live NDV vectored immunogenic composition against SARS-CoV-2 that expresses the S1 subunit or the RBD of the S of SARS-CoV-2. 2, and which in turn provokes a robust humoral and cellular response, in addition to having a non-invasive intranasal (IN) application route.

[034] In addition to having a profitable, economical and cheap production, in embryonated chicken eggs and/or its production in multiple cell lines.

[035] In this sense, it is clear that there is a need, not yet satisfied in the state of the art, in terms of obtaining a recombinant live vaccine or vectorized recombinant live immunogenic composition that comprises the NDV virus that expresses and incorporates into its viral structure, the S1 subunit and the RBD of the SARS-CoV-2 protein S, and that when applied intranasally, they present significant levels of protection against SARS-CoV-2, as well as high titers of neutralizing antibodies against SARS-CoV-2.

DESCRIPTION OF THE FIGURES

[036] Figure 1. Prediction of domains; transmembrane (DTM) and cytoplasmic (DC) protein Hemagglutinin-neuraminidase (HN). The prediction was made using the amino acid sequence (aa) in the TMHMM Server 2.0 program.

[037] Figure 2. Scheme showing the construction strategy of the new viruses rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS-CoV-2. [038] Figure 3. Plasmid map pNDV-LS1-HN-RBD/SARS-CoV-2 (20315 bp).

[039] 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- is shown schematically. 2 (16182 bp) (SEQ ID NO: 7). Two successive subclonings were performed to build the rNDV-LS1-HN-RBD/SARS-CoV-2 construct. The NDV-LS1 plasmid (19319 bp) was linearized between the BbvCI cutting sites (third position of NDV) to insert the 1013 bp fragment (SEQ ID NO: 1) which includes the BbvCI sites, generating the NDV-LS1 plasmid -HN-RBD/SARS-CoV-2 (20315 bp).

[040] Figure 5. Prediction of Fusion protein DTMs and DCs (F). The prediction was made using the amino acid sequence (aa) in the TMHMM Server 2.0 program.

[041] Figure 6. Plasmid map pNDV-LS1-S1-F/SARS-CoV-2 (21743 bp).

[042] Figure 7. The subcloning strategy of the S1-F/SARS-CoV-2 gene (2441 bp) (SEQ ID NO: 8) generated by the rNDV-LS1-S1-F/SARS virus is shown schematically. -CoV-2 (17610 bp) (SEQ ID NO: 13). Two successive subclonings were performed to construct the rNDV-LS1-S1-F/SARS-CoV-2 virus construct. Plasmid pNDV-LS1 (19319 bp) was linearized between the BbvCI cutting 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 plasmid -LS1-S1- F/SARS-CoV-2 (21743 bp).

[043] Figure 8. Verification of the expression and incorporation of S1-F and HN-RBD in the virion, by Western blot assay. [044] Figure 9. Detection of the genetic sequence of the S1-F and HN-RBD inserts by RT-PCR.

[045] Figure 10. Immunofluorescence Assay.

[046] Figure 11. Assay of cell binding and internalization 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 cellular receptor ACE2.

[047] Figure 12. In vitro growth properties of rNDV-LSI-HN-RBD/SARS-CoV-2, rNDV-LS1-S1-F/SARS-CoV-2, and rNDV-LS1 viruses in the cell line DF-1.

[048] Figure 13. Hamster immunization and challenge scheme with rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS-CoV-2.

[049] Figure 14. Evaluation of neutralizing antibodies by surrogate neutralization test (NTs).

[050] Figure 15. Evaluation of SARS-CoV-2 anti-RBD IgG levels by indirect ELISA.

DESCRIPTION OF THE LISTING OF SEQUENCES

[051] SEQ ID NO: 1= Nucleotide sequence that comprises the design of the HN-RBD/SARS-CoV-2 gene (1013 bp).

[052] SEQ ID NO: 2= Nucleotide sequence comprising the hemagglutinin-neuraminides (HN) gene (1734 bp) of the plasmid pNDV-LS1 (7535-9268 nt).

[053] SEQ ID NO: 3 = Sequence of amino acids that comprise the HN protein (577 aa) of the plasmid pNDV-LS1. [054] SEQ ID NO: 4 = Nucleotide sequence comprising the domains: cytoplasmic (DC) and transmembrane (DTM) of the HN protein (144 bp) of the plasmid pNDV-LS1 (7534-7678 nt).

[055] SEQ ID NO: 5 = Nucleotide sequence comprising the Spike (S) gene (3822 bp) of SARS-CoV-2 (21563-25384 nt) with access number GenBank- MN908947.3.

[056] SEQ ID NO: 6 = Nucleotide sequence comprising the RBD domain (636 bp) of S SARS-CoV-2 (990-1623 nt) with access number GenBank- MN908947.3 for HN gene design -RBD/SARS-CoV-2.

[057] SEQ ID NO: 7= Nucleotide sequence comprising the complete genome of the recombinant virus rNDV-LS1-HN-RBD/SARS-CoV-2 (16182 bp).

[058] SEQ ID NO: 8= Nucleotide sequence comprising the design of the S1-F/SARS-CoV-2 gene (2441 bp) optimized for its expression in Gallus Gallus.

[059] SEQ ID NO: 9 = Nucleotide sequence comprising the Fusion (F) gene (1662 bp) of plasmid pNDV-LS1 (5667-7328 nt).

[060] SEQ ID NO: 10 = Sequence of amino acids that comprise the F protein (553 aa) of the plasmid pNDV-LS1 (5667-7328 nt).

[061] SEQ ID NO: 11= Nucleotide sequence comprising domains: DC and DTM of protein F (162 bp) of plasmid pNDV-LS1 (7166-7328 nt), optimized for expression in Gallus Gallus.

[062] SEQ ID NO: 12= Nucleotide sequence comprising the S1 subunit of the S gene (2043 bp) of SARS-CoV-2 with access number in GenBank-MN908947.3. [063] 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

[064] The present invention reports in a first aspect thereof, a recombinant vectorized live vaccine or recombinant live immunogenic composition comprising the recombinant Newcastle disease virus (NDV) that expresses the S1 subunit and the RBD of the S protein of SARS-CoV-2 with significant high levels of protection and high immunization titers, where the vaccine or immunogenic composition comprises the recombinant viruses called rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1- F/SARS-CoV-2 whose construction is described later in the invention.

[065] Consequently, 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 that expresses the S1 subunit and the RBD of SARS-CoV-2 protein S.

[066] 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 S protein of SARS-CoV-2.

[067] Even more, in a fourth aspect of the present invention, it contemplates a synergistic combination 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 in combination in the same recombinant live immunogenic composition or live vaccine that express the S1 subunit and the RBD of the S protein of SARS-CoV- two. [068] 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.

[069] Additionally, the present invention comprises a method for controlling the 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-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 in combination in the same recombinant live immunogenic composition or vaccine live.

[070] In this sense, the recombinant live immunogenic composition or vaccine according to the present invention comprising the Newcastle disease virus (NDV) expressing the S1 subunit and the RBD of the Spike protein of SARS-CoV-2 it may further comprise a pharmaceutically acceptable adjuvant and/or excipient(s), wherein pharmaceutically acceptable adjuvants are defined as substances that enhance antigen-specific immune responses by modulating immune cell activity. Examples of adjuvants that can be used in the present invention for the recombinant live vaccine include, but are not limited to saponins, agonist antibodies to co-stimulatory molecules, Freund's adjuvant, muramyl dipeptide (MPD), bacterial DNA (oligo CpG), lipo- polysaccharides (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. [071] Vaccine compositions suitable for parenteral administration conveniently comprise a sterile aqueous or nonaqueous vaccine preparation, which is preferably isotonic with the blood of the recipient. These vaccines can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be used including synthetic mono- or diglycerides. Also, fatty acids such as oleic acid can be used in the preparation of injectables. A suitable carrier formulation can be found for subcutaneous, intravenous, intramuscular, oral administrations. In the case of live recombinant vaccines according to the present invention, sterile water, yeasts, starches, gelatin, albumin, sucrose, lactose, sodium glutamate and glycine in pharmaceutically acceptable amounts can be used as adjuvants, vehicles and/or diluents.

[072] In another embodiment of the invention, the vaccine or recombinant live immunogenic composition according to the present invention, comprises a recombinant virus rNDV-LS1-HN-RBD/SARS-CoV-2 characterized by SEQ ID No. 7, a recombinant virus rNDV-LS1-S1-F/SARS-CoV-2 characterized by SEQ ID No. 13 or a mixture thereof, sterile water and optionally, adjuvants such as squalene, quillaja saponaria and surfactants.

[073] Recombinant live immunogenic compositions or vaccines according to the present invention may also optionally contain suitable preservatives, such as: chloride benzalkonium; chlorobutanol, parabens and thiomerosal, among others; inactivating agents such as formaldehyde, glutaraldehyde, propiolactone and beta-propiolactone are used in parts per million (ppm) or parts per billion (ppb) amounts.

[074] Other protocols for administration of vaccine compositions will be known to one of ordinary skill in the art, in which the number of doses, schedule of injections, injection sites, mode of administration, and the like may vary according to recommended practice. Administration of the vaccine compositions to mammals other than humans (eg, for testing purposes or for 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 primate, bovine, equine, porcine, ovine, feline, and rodent.

[075] Vaccine compositions according to the present invention may also optionally contain suitable preservatives, such as: benzalkonium chloride; chlorobutanol, parabens and thiomerosal, 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.

[076] The immunogenic compositions or vaccine according to the present invention may be administered by any conventional route, including injection, oral, inhalation intranasal aerosol, more preferably intranasally. I. Design and construction of plasmids containing the complete genome of the new recombinant viruses

[077] Using the reverse genetics system, it was possible to develop a new vaccine for SARS-CoV-2, based on an NDV vector, which can express the S1 subunit and RBD genes from the SARS S protein -CoV-2. The present invention focuses on the development of two recombinant viruses called rNDV-LS1-HN-R B D/SA RS-Co V-2 and rNDV-LS1-S1-F/SARS-CoV-2, as well as their application in a single Recombinant live immunogenic composition or combination formulation of both vaccines or immunogenic compositions (rLS1-HN-RBD/SARS-CoV-2 + rNDV-LS1-S1-F/SARS-CoV-2).

DETAILED DESCRIPTION OF THE INVENTION

[078] To achieve this, 2 versions of the virus have been designed, where the S1 and RBD subunit-based sequences of the S protein from the new strain of SARS-CoV-2 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, they were designed to be cloned in different ways in the plasmid pNDV-LS1 (19319 bp ), which contains the complete NDV genome of the LaSota vaccine strain (lentogenic pathotype) (15186 bp) which has been previously recovered and described (Izquierdo-Lara et al. 2019).

[079] The recombinant plasmids obtained were named; pNDV-LSI-HN-RBD/SARS-CoV-2 and pN DV-LS1 -S1 -F/SARS-CoV-2.

1) pNDV-LS1-HN-RBD/SARS-CoV-2

1. Genetic map of the plasmid pNDV-LS1-HN-RBD/SARS-CoV-2

[080] The first step consisted in the design of the HN-RBD/SARS-CoV-2 gene (see SEQ ID NO: 1). This gene consisted of an ORF corresponding to the gene HN which contains the DTM and DC of the HN protein (144 bp) of the plasmid pNDV-LS1 (see SEQ ID NO: 1, 2, and 4) and the ectodomain of the RBD protein (636 bp) (see SEQ ID NO : 6) of SARS-CoV-2, with the aim of expressing the RBD on the surface of the virion (see Figure 2C). The sequence corresponding to the DTM and CT of the HN protein was predicted with 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 surface of the virion. The RBD sequence selected for this design was 636 bp, which was 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

[081] 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 consists in that the length of the virus genome must be a multiple of 6, this because it seems that the packaging of the genome occurs every 6 nucleotides ( Peeters et al. 2000), for which a Thymine "t" nucleotide was added followed by the stop codon of the HN-RBD/SARS-CoV-2 gene (see SEQ ID NO: 1). A flaw in the number of nucleotides in the NDV genome can severely alter viral replication.

[082] This HN-RBD/SARS-CoV-2 gene sequence was chemically synthesized and then cloned into the pUC57 cloning plasmid by GenScript (Piscataway, NJ, USA), said sequence was designed to be flanked at both ends by the unique restriction site called BbvCI (NEB, New England BioLabs, Ipswich, MA, USA) which contains the "CCTCAGC" sequence, is located in the intergenic region between the P and M genes of pNDV-LS1 (19319 bp) (see Figure 4). This plasmid was then purified and extracted using QIAGEN Plasmid Midi Kit (100) (QIAGEN, Valencia, CA, USA), following the manufacturer's instructions.

[083] pNDV-LS1 (19319 bp), was digested by this 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 the NDV as described above, thus obtaining the linearized plasmid. The second step consisted in the insertion of the synthetic gene HN-RBD/SARS-CoV-2 (1013 bp) (SEQ ID NO: 1) in the plasmid pNDV-LS1 (19319 bp), generating the new plasmid called pNDV-LS1- HN-RBD/SARS-CoV-2 (20315 bp) (see Figures 3 and 4). Thus obtaining the recombinant virus called rNDV-LS1-HN-RBD/SARS-CoV-2 (16182 bp) (see SEQ ID NO: 7).

[084] pNDV-LS1 (19319 bp), was digested by this 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 the NDV as described above, thus obtaining the linearized plasmid. The second step consisted in the insertion of the synthetic gene HN-RBD/SARS-CoV-2 (1013 bp) (SEQ ID NO: 1) in the plasmid pNDV-LS1 (19319 bp), generating the new plasmid called pNDV-LS1- HN-RBD/SARS-CoV-2 (20315 bp) (see Figures 3 and 4). Thus obtaining the recombinant virus called rNDV-LS1-HN-RBD/SARS-CoV-2 (16182 bp) (see SEQ ID NO: 7).

2. Genetic map of the plasmid pNDV-LS1-S1-F/SARS-CoV-2

[085] The first step consisted in 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 "atg" nucleotides, then fused with the ectodomain of the S1 subunit of the S of SARS-CoV-2, and with the fragment that encodes the DTM and CT of the protein F (162 bp) of the NDV (see SEQ ID NO: 8, 9 and 11), with the aim that S1 is expressed on the surface of the virion (see Figure 2C).

[086] The sequence corresponding to the TM and CT of the F protein was predicted with the TMHMM Server 2.0 program (see Figure 5).

[087] The new gene was named S1-F/SARS-CoV-2 (2441 bp) (see SEQ ID NO: 8), and having the N-terminal region of the F protein should be able to expose the protein of the S1 subunit on the surface of the virion. 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

[088] The new gene was named S1-F/SARS-CoV-2 (2441 bp) (see SEQ ID NO: 8), and having the N-terminal region of the F protein should be able to expose the protein of the S1 subunit on the surface of the virion. 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.

[089] 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 consists in that the length of the virus genome must be a multiple of 6, this because it seems that the packaging of the genome occurs every 6 nucleotides ( Peeters et al. 2000) for which the "tgac" nucleotides were added followed by the stop condom of the S1-F/SARS-CoV-2 gene (see SEQ ID NO: 8). A flaw in the number of nucleotides in the NDV genome can severely alter viral replication. [090] 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), said sequence was designed to that is flanked at both ends by the unique restriction site called BbvCI (NEB, New England BioLabs, Ipswich, MA, USA) which contains the "CCTCAGC" sequence. which is 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.

[091] Plasmid pNDV-LS1 (19319 bp) was digested by this 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 from NDV as mentioned above, thus obtaining the linearized plasmid. The second step consisted in the insertion of the synthetic gene S1-F/SARS-CoV-2 (2441 bp) (see SEQ ID NO: 8) in the plasmid pNDV-LS1 (19319 bp), generating the new plasmid called pNDV-LS1 -S1- F/SARS-CoV-2 (21743 bp) (see Figure 8). Thus obtaining the virus called rNDV-LS1-S1-F/SARS-CoV-2 (17610 bp) (see SEQ ID NO: 13).

I. Recovery of rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS-CoV-2 viruses

[092] 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 support plasmids containing the genes that synthesize the complex RNP, pCI-L, pCI-N and pCI-P together with the plasmid pNDV-LS1-HN-RBD and pNDV-LS1-S1-F in Vero cells as described in a previous publication (Chumbe et al 2017). II. Pathogenicity of rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS-CoV-2 viruses

[093] To evaluate the pathogenicity and determine the biological characteristics of viruses, different indices were used, such as: Intracerebral Pathogenicity Index (ICPI), mean death time (MDT, English: Mean Death Time), both tests were evaluated in 1-day-old chickens (Charles River Laboratories), unlike the mean infective dose (EIDso/mL) that was evaluated in 9- to 10-day-old embryonated eggs (Charles River Laboratories). Charles River), analysis of the cleavage site of the Fusion gene of the viruses rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS-CoV-2, showed no changes at the level of nucleotides and amino acids, keeping 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 rNDV-LS1-S1-F/SARS-CoV-2 viruses.

Intracerebral Pathogenicity Index (ICPI) was evaluated in 1-day-old birds. The maximum score of a virulent strain is 2.0, while the score of the Lentogenic strain is 0.0.

Mean death time (MDT) was elaborated in 10-day-old embryonated eggs. The value of 60 hours corresponds to velogenic strains, values between 60 and 90 hours correspond to mesogenic strains, andlentogenic have a value greater than 90 hours. III. Verification of HN-RBD and S1-F expression

[094] To evaluate the expression of 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, cells were harvested 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, concentrated the viral particles by ultracentrifugation using an ultracentrifuge (Ultracentrifuga, Beckam, Culter), these viral particles were obtained from a harvest from allantoic fluid of embryonated pathogen-free chicken eggs (SPF) (SPF, English: Specific- Pathogen-Freé) (Charles River Laboratories) infected with the new recombinant viruses were then partially purified in 30% sucrose. Western blot assay was carried out using viruses partially purified from allantoic fluid and used from infected cells, using a rabbit antibody specific for the RBD protein of SARS-CoV-2 (Sino biologycal, Cat: 4059-2-T62 ) 2/5000 and a secondary antibody Anti rabbit IgG conjugated to HRP (Cat. A01827) 2/5000.

[095] The results from the Western blot assay showed that the antibodies reacted with the lysate of cells infected with the viruses rNDV-LS1-HN-RB D/SA RS- Co V-2 and rNDV-LS1-S1- F/SARS-CoV-2, 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 Usados infected with the virus parent (see Figure 8B).

[096] In another western blot assay with the purified viruses rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS-CoV-2, the results showed a band with a molecular weight ~30kDa and ~90kDa respectively that would correspond to HN-RBD and S1-F, on the other hand, no reactivity with the purified parental virus was observed. These results would confirm the incorporation of HN-RBD and S1-F in the viral structure, since in the construction of the RBD insert it was fused to the DC and DTM of the NDV HN protein. In the case of the S1 insert, it was fused with the DC and DTM of the NDV F protein (see Figure 8A).

IV. Detection of the genetic sequence of the HN-RBD and S1-F inserts by RT-PCR

[097] The RNA of the recombinant viruses rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS-CoV-2, was extracted from allantoic fluid using the QlAamp MinElute Virus kit Spin (Qiagen, Germany), according to the manufacturer's instructions, with some modifications. 200 µl of sample was added to a tube containing 25 ml of Proteinase K and homogenized for 15 seconds. Then, 200 pL of buffer AL was added, previously mixed with 6.2 pL of carrier (1 pg/pL), homogenized for 15 seconds and incubated at 56°C for 1 hour. Lysed samples were placed in the QIAcube Connect (Qiagen, Germany) for automated RNA extraction. RNA quantity and quality were determined using a Biophotometer plus (Eppendorf, Germany).

[098] Complementary DNA (cDNA) was generated from 5 pL of total RNA in a reaction volume of 20 pL, containing 4 pL of ProtoScript II 5X buffer (New England Biolabs, USA), 2 pL of Dithiothreitol 0.1 M, 1 pL of 10 mM dNTP, 0.2 pL of 40 U/pL RNase Inhibitor (New England Biolabs, USA), 1 pL of 200 U/pL ProtoScript II reverse transcriptase (New England Biolabs, USA) , 2 pL of random primer mix (60 mM) and 4.8 pL of nuclease-free water. The reaction was conducted at 42°C for 60 minutes, with a denaturation and inactivation step at 65°C for 5 minutes and 20 minutes, respectively. The cDNA obtained was used as a template for the polymerase chain reaction (PCR). from English: Polymerase Chain Reactor). Amplification was carried out in a total volume of 20 pL, using 10 pL of high-fidelity 2X Master mix Q5 (New England Biolabs, USA), 0.36 pL of primers NDV-3LS1-2020-F1 (5 ^' -GATCATGTCACGCCCAATGC-3 ^' ) and NDV-3LS1-2020-R1 ( ^5' -GCATCGCAGCGGAAAGTAAC-3 ^' ), respectively, 7.28 pL of nuclease-free water (Ambion, USA) and 2 pL of total cDNA. The thermal cycling protocol comprised an initial denaturation step at 98 °C for 30 seconds, followed by 35 cycles of 98 °C for 10 seconds, 72 °C for 20 seconds, 72 °C for 30 seconds for detection of HN- RBD and for 40 seconds for the detection of S1-F. The final extension was performed at 72 °C for 2 min. The PCR products were analyzed by electrophoresis in 1% agarose gels and visualized with a CCD camera (Azure Biosystems, USA).

[099] The RT-PCR results detected a band with a size of -3028 bp in the rNDV-LS1-S1-F/SARS-CoV-2 virus sample, which would correspond to the S1-F insert (see Figure 9B) . On the other hand, a -1600 bp band was detected in the rNDV-LS1-HN-RBD/SARS-CoV-2 virus sample that would correspond to the HN-RBD insert (see Figure 9A). In addition, the RT-PCR products that were sequenced (Macrogen, Seoul, Korea), showed that the genetic sequences of the HN-RBD and S1-F inserts were intact and without alterations (Data not shown).

V. Immunofluorescence Assay

[100] To examine the expression of HN-RBD and S1-F proteins by the recombinant viruses rNDV-LS1-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, cells were fixed with 4% paraformaldehyde for 25 min, then the monolayer was washed three times with Dulbecco's phosphate-buffered saline (DPBS) and permeabilized with 0.1% of Triton X-100 for 15 minutes at room temperature. It was then washed three times with DPBS, and the monolayer was incubated with the rabbit antibody specific against the RBD protein of SARS-CoV-2 (1:200) (cat, 40592-T62), and a chicken antiserum specific against the virus. Newcastle disease (1:200) (Charles River) for 1.5 hours at room temperature. Subsequently, the monolayer was incubated with the secondary antibodies goat anti-rabbit IgG conjugated to Alexa Fluor 594 (1:250) (Abcam), and goat anti-chicken IgY conjugated to Alexa Fluor 488 (1:1000) (ab150169, Abcam) for 1 hour at room temperature. Finally, cell nuclei were stained with DAPI mounting medium (ab104139, Abcam) for 5 minutes. Results were observed using an ObserverAI fluorescence microscope (Cari Zeiss, Germany). Digital images were taken at 400x magnification and processed with the AxioCam MRc5 camera (Cari Zeiss, Germany).

[101] Immunofluorescence results in cells infected with the recombinant viruses rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS-CoV-2 were positive, when they reacted with the antibodies specific rabbit antiserum against the RBD protein of SARS-CoV-2 and the chicken antiserum specific against NDV, thus demonstrating the presence of NDV proteins and the expression of the RBD and S1 of SARS-CoV-2. On the other hand, in cells infected with the parental NDV, reactivity was only observed with the NDV-specific chicken antiserum (see Figure 10).

SAW. Assay of binding and cellular internalization by efficiency to the ACE2 receptor expressed in Vero E6 cells

[102] To determine the binding efficiency between the ACE2 receptor and the RBD or the 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 harvested and washed with DPBS supplemented with 5% fetal bovine serum (SFB). Then one concentration of 1x 10 ⁶ cells were blocked with DPBS with 5% normal mouse serum for 30 minutes, and then incubated with the presence of recombinant viruses (previously purified with 25% sucrose) with final concentrations of 0.05 and 0.2 pg for 30 minutes at 37°C. In order to remove residual viral particles that did not bind to the ACE2 receptor from Vero E6 cells during the incubation time, cells were washed with DPBS supplemented with 5% FBS twice. Subsequently, they were labeled with rabbit anti-SARS S1 monoclonal antibodies (1:200) (Sino biological) for 1 hour at room temperature, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (1:200). ). Finally, the cells were analyzed with the help of the FACS Canto II flow cytometer (BD), and the data obtained were analyzed using the FlowJo v.10.6 software (BD).

[103] The results showed ACE2 receptor binding reactivity of ~10% and ~40% of the cells tested with the recombinant viruses rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F /SARS-CoV-2 respectively. Thus demonstrating the presence of the RBD and the SARS-CoV-2 S1 subunit in the envelope structure of the rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS viruses. -CoV-2 respectively (see Figure 11). This was expected, because in the construction of the RBD insert it was fused to the DTM and DC of the NDV HN protein. In the case of the S1 insert, it was fused with the DTM and DC of the NDV F protein. These construction designs were made so that the RBD and S1 were expressed and incorporated into the viral envelope of the recombinant viruses and had affinity for the ACE2 receptor.

Vile. In vitro growth properties of rNDV-LSI-HN-RBD/SARS-CoV-2 and rN DV-LS 1 -S 1 -F/SARS-CoV-2 viruses [104] DF-1 cells (DF-1, English: Chicken Embryo Fibroblasf), were seeded at 70% confluence in a 12-well plate, after 18 hours of post seeding they were infected with rNDV- LSI-HN-RBD/SARS-CoV-2, rN DV-LS1 -S1 -F/SARS-CoV-2, and rNDV-LS1 at an MOI of 1.

[105] Cells were maintained in DMEM containing 1% FBS and 10% allantoic fluid (AF) with 5% CO2 at 37°C.

[106] Supernatants were collected at 12, 24, 36, 48, 60, 72, 84, 96 and 108 hours post infection (h.p.i), clarified at 500 rpm for 5 minutes at 4°C to remove cell debris. . Supernatants were quantified by plaque assay on DF-1 cells, as previously described (Chumbe et al. 2017).

[107] Five days post incubation, cells were fixed with 3.2% paraformaldehyde for 6 hours at room temperature, and then stained with 0.2% crystal violet.

[108] Titers obtained by plaque assay were calculated and reported as plaque-forming units (PFU). This experiment was repeated at three different times.

[109] 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, without However, rNDV-LS1 showed slightly elevated titers at 24 and 36 hpi compared to rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS-CoV-2, but not significant (see Figure 12).

[110] The cytopathic effect (CPE) or the formation of syncytia of the viruses rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1 -F/SARS-CoV-2, showed the typical CPE of the NDV. This may explain why the insertion of both foreign genes, did not alter their biological properties of rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS-CoV-2.

VII. Immunization in Hamster animal model

[111] To assess the safety, immunogenicity, and efficacy of the vaccines, forty-eight 5-week-old male and female hamsters were used in this study. The experiments were developed following the protocols previously approved by the National Institute of Health (INS) of Peru in conjunction with FARVET S.A.C.

[112] Hamsters were randomly divided into four groups (12 hamsters/group). Hamsters were inoculated intranasally (IN) with 40mI (2x10 ⁶ PFU/hamster) of the viruses rNDV-LS1-HN-RBD/SARS-CoV- 2 (Group #1), rN DV- LS 1 -S 1 - 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 #4). An IN immunization booster with 40mI (2x10 ⁶ PFU/hamster) of each vaccine was applied to all groups 15 days post-vaccination (DPV).

[113] At 15 and 30 DPV, blood samples were collected to measure the titers of antibodies generated by the vaccines: anti-IgG antibodies by ELISA-SARS-CoV-2 in house (ELISA, English: enzyme-linked immunosorbent assay) and to measure the circulating neutralizing antibodies produced by the vaccines, an anti-RBD-SARS-CoV-2 surrogate neutralization test (TNs) was performed from a commercial kit (GenScript, Piscataway, NJ, USA) (Cat. No. L00847).

IX. Evaluation of Neutralizing Anti-RBD Antibodies using a Surrogate Neutralization Test (TNs) [114] Forty-eight serum samples were tested for neutralizing antibody titers against SARS-CoV-2. Neutralization test assays were developed 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) SARS-CoV-2 protein S RBD and 2) the cellular receptor ACE2, thus disguising the interaction of the virus with the host in an ELISA plate. When the interaction of "RBD - ACE2" does not occur, "neutralization" occurs, that is, the existence of a blockade by specific neutralizing antibodies in the serum.

[115] The advantage of these TNs is the ease of developing it in Biosafety Level 2 Laboratories (BSL-2) without any difficulty, exactly following the manufacturer's instructions. Plates were analyzed using an Epoch 2 microplate reader (Bioteck, USA) at 450 nm.

[116] The cut-off limit of positive and negative neutralizing antibodies for SARS-CoV-2 was interpreted as the rate of inhibition. The cut-off interpretation of results was interpreted as follows: positive result >20% (neutralizing antibodies detected), negative result <20% (neutralizing antibodies not detected).

[117] 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 #4).

[118] The results of sera collected from hamsters indicated the early circulation of neutralizing antibodies from the first 15 days post immunization in groups: # 2 (rNDV-LS1-S1- F/SARS-CoV-2) and #3 (rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS-CoV-2) showed ³60% inhibition, while group # 1 (rNDV-LS1-HN-RBD/SARS-CoV-2) presented an inhibition of 30%, unlike group #4 (non-immunized control group) that presented an inhibition rate of <20%. On the other hand, as expected, these antibody titers were increasing, so that 30 days after immunization, it was detected that the groups: # 2 (rNDV-LS1-S1-F/SARS-CoV-2) presented a inhibition rate between 70 to 80%, group #3 (rNDV- LSI-HN-RBD/SARS-CoV-2 and rN DV-LS1 -S1 -F/SARS-CoV-2) presented an inhibition rate between 80 at 100%, while group #1 (rNDV-LS1-HN-RBD/SARS-CoV-2) presented an inhibition of 35%, unlike group #4 (non-immunized control group) that presented an inhibition rate < 5%. The results of this study 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 live immunogenic composition. recombinant or live recombinant vaccine according to the present invention.

[119] With these results obtained, we hypothesize that the animals of groups #1, #2 and #3 present protection in the first 15 days after immunization, and that this is re-potentiated 30 days after immunization (post booster), generate neutralizing antibody titers up to 100% inhibition compared to non-immunized animals. These results determine the efficacy of vaccines due to their rapid ability to generate a humoral response.

X. Evaluation of Neutralizing Anti SARS-CoV-2 Antibodies by means of a Conventional Neutralization Test (TNc)

[120] All neutralization assays were developed at the Biosafety Level 3 Laboratory (BSL-3) of the National Institute of Health (INS). The collected sera were inactivated at 56°C for 60 minutes prior to use. Vero E6 cells were maintained in the medium of 1X DMEM culture supplemented with 5% FBS and pre-seeded 18 to 24 hours, in a 24-well cell culture plate. Following inactivation, mixtures of all the corresponding "pool" sera of each group were made, then serial dilutions of two times were made, starting from the 1:20 dilution. Then the sera were mixed with the SARS-CoV virus -2 (serum-virus) and incubated for 1 hour at 37°C, and then 10 pL of the mixture was transferred to each well containing the previously seeded Vero E6 cell culture monolayer.The cells were incubated for 4 days at 37°C, then they were 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 plaques or CPE of the virus. SARS-CoV-2 on cells.

[121] The results obtained showed that hamsters immunized with both viruses rNDV-LS1-HN-RBD/SARS-CoV-2 + rNDV-LS1-S1-F/SARS-CoV-2 reached a neutralization titer of 1/40 at 100%, while the group that were immunized with rNDV-LS1-HN-RBD/SARS-CoV-2 had a titer of 1/20 at 100%, and only the rNDV-LS1-S1-F/SARS-CoV- 2 reached a titer of 1/160 to 100%, which indicates that the rNDV-LS1-S1-F/SARS-CoV-2 has caused a greater amount of neutralizing antibodies, this is possibly due to the fact that the S1 region presents other regions important antigenic epitopes of SARS-CoV-2 S (see results Table 2).

Table 2. Percentages of serum neutralization against SARS-CoV-2, obtained from a pool of sera from hamsters immunized with rNDV-LS1-HN-RBD/SARS-CoV-2 and rNDV-LS1-S1-F/SARS-CoV- two

XI. Evaluation of SARS-CoV-2 Anti-RBD IgG by indirect ELISA

[122] The presence of anti-RBD IgG in vaccinated hamsters was assessed by in-house indirect ELISA. The microplates were fixed with 1ug/ml of purified RBD protein diluted in carbonate buffer, pH 9, overnight. The plates were washed with phosphate buffer and tween (PBS-T) and blocked with 3% milk (PBS-T and 3% milk) (Difeo Skim Milk 232100), shaking for 2 hours at room temperature. After blocking, plates were washed with PBS-T. Serum was added at 1:100 dilutions for Hamster serum and incubated for 1 hour at 37°C. As secondary antibody, 100 pL of Anti-Hamster IgG (1:28000) (Abcam 6892) conjugated with HRP was used. Washes were performed and the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added. After 15 minutes, the reaction was stopped with 2N H ₂ S0 ₄ and the absorbance was read at 450 nm. Pre-immune and mock sera from each treatment were used as negative control.

[123] The results obtained showed that 30 days after immunization it was detected that groups: # 2 (rNDV-LS1-S1-F/SARS-CoV-2) presented a higher OD of 3.5, while group # 3 (rNDV-LS1- HN-RBD/SARS-CoV-2 + rNDV-LS1-S1-F/SARS-CoV-2) presented an OD of 1.8, while group #1 (rNDV-LS1-HN-RBD/ SARS-CoV-2) presented an OD of 1.5, unlike group #4 (non-immunized control group) that presented an OD of 0.02. [124] These results indicate the ability of the vaccine to generate antibodies against SARS-CoV-2 (see Figure 15).

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Claims

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

3. The recombinant live immunogenic composition according to claim 1, characterized in that it comprises the recombinant virus called rNDV-LS1-S1-F/SARS-CoV-2 of SEQ ID No. 13.

4. The recombinant live 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 recombinant live immunogenic composition according to any of the preceding claims, characterized in that it also 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, agonistic antibodies to 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.

9. A method for controlling the infection caused by SARS-CoV-2 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 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.