Synthetic variants of the rabies virus glycoprotein g for the generation of pseudotyped baculovirus and use thereof in Anti-rabies vaccine formulations

Abstract

The present invention relates to synthetic designs or chimeric proteins for pseudotyping baculovirus (Autographa californica nuclear polyhedrosis virus) with the rabies virus glycoprotein G (gG) on its surface (Bac::gG-FL), which can be used in anti-rabies vaccine formulations. The chimeric protein is designed from a gene cassette containing gene fragments of the ectodomain of the G glycoprotein of the Pasteur strain rabies virus, a linker of 7 amino acids (GGGGSGG), as well as transmembrane (TM) and cytoplasmic (CT) regions of the gp64 baculovirus protein, with the arrangement of the sequences in the designed gene cassette being shown in figure 1.

Description

[0001] SYNTHETIC VARIANTS OF RABIES VIRUS GLYCOPROTEIN G FOR THE GENERATION OF PSEUDOTYPED BACULOVIRUSES AND THEIR USE IN ANTI-RABIES VACCINE FORMULATIONS

[0002] TECHNICAL FIELD

[0003]

[0001] The present invention is framed within the technical field of biotechnology and the pharmaceutical industry for the veterinary field.

[0004] STATE OF THE ART

[0005]

[0002] Rabies is one of the most important zoonotic viral diseases globally. Despite the existence of a preventive vaccination program, it is estimated to cause 55,000 deaths annually in developing countries. This is attributed to inefficient immunization coverage of both the susceptible population and the canine population in cities. It is also attributed to the short-lived immunity generated by currently available vaccines. These difficulties necessitate the development of rabies vaccines based on new technologies that improve the duration of immunity while simultaneously reducing production costs to facilitate their mass distribution.

[0006]

[0003] Over the last few decades, genetic engineering techniques have enabled the development of viral vectors. Thanks to their characteristics, these vectors can be used to introduce genetic material into cells in vivo or to present recombinant antigens on their surface; the latter process is known as pseudotyping. These features have made viral vectors one of the most important technologies for developing next-generation vaccines, as they have proven capable of generating immunity to specific antigens. Baculoviruses stand out among this group of vectors. These viruses infect insects and are harmless to mammals. They are characterized by being produced in eukaryotic cellular machinery, which allows them to express complex proteins with the post-translational modifications necessary for their functionality and folding.

[0007]

[0004] Glycoprotein G (gG) is the only component on the surface of the rabies virus and is responsible for host cell recognition. This makes it the only protein capable of generating immunity against the disease by being the target of neutralizing antibodies against the virus. Furthermore, since gG is arranged on the viral surface as homotrimers, the use of an anchoring system based on fusion to the transmembrane (TM) and cytoplasmic (CT) regions of the baculoviral protein gp64 is the most suitable alternative for baculovirus pseudotyping with gG. However, direct fusion of two heterologous protein domains can decrease the stability of the resulting chimera or generate misfolding.This difficulty could be overcome by using linkers between the fused domains, a strategy that has proven effective for designing chimeric proteins and bispecific antibodies, improving their stability, expression, and functionality. In the context of baculovirus pseudotyping, using a linker between the ectodomain of gG and the transmembrane region of gp64 could improve the independence of these two domains, granting it better immunogenicity compared to other chimeric designs.

[0008]

[0005] In the field of patents, document WO9509249 is known, describing oral rabies vaccines comprising at least 100 pg of an isolated rabies glycoprotein G produced in eukaryotic cells and methods for immunizing mammals against rabies infection comprising the step of oral administration to the mammal. Chimeric proteins and orally active vaccines for immunizing a mammal against a pathogenic infection comprising chimeric proteins and methods for using them are described. Antipathogenic oral vaccines comprising at least 100 pg of an isolated rabies glycoprotein G and an isolated pathogenic antigen or a peptide comprising a neutralizing epitope of a pathogenic antigen and methods for using them are described.

[0009]

[0006] It is also known from document US2014178419 that it relates to virus-like particles (VLPs) comprising rabies virus (RV) glycoproteins (G proteins) and methods for preparing and using them, including immunogenic compositions such as vaccines for the treatment and / or prevention of rabies virus infection. RV VLPs comprise one or more RV glycoproteins (G proteins). RV G proteins may be derived from any suitable RV strain.

[0010]

[0007] Furthermore, patent document CN102964433 discloses a highly immunogenic rabies virus glycoprotein, which has good immunogenicity. The rabies virus glycoprotein is characterized by its amino acid sequence as shown in SEQ No. 1 and its DNA sequence as shown in SEQ No. 2. It also refers to a method for preparing the rabies virus glycoprotein that includes the steps of: 1) Artificial synthesis of the gene for the highly immunogenic rabies virus glycoprotein; The gene shown in SEQ No.1) Divide into two parts, a segment A of 1-730 bp and a segment B of 710-1575 bp, which are synthesized respectively, and the synthesized gene segment is cloned into the BamHI site of the pUC18 plasmid vector; 2) Construction of a highly immunogenic rabies virus glycoprotein adenovirus expression vector, 3) Obtaining recombinant adenoviruses expressing highly immunogenic rabies virus glycoprotein.

[0011]

[0008] Patent CN103088063 describes a recombinant pseudotype baculovirus BV-RVG / RVG, deposited in the China Type Culture Collection (CCTCC) under deposit number CCTCC NO: V201022, characterized in that the recombinant virus has dual expression of the rabies virus G protein. The expression cassette contains both the PPH promoter to drive the expression of the rabies virus G protein on the surface of the virus capsid, and the CMV promoter to drive the rabies virus G protein on the lactate surface. The CMV-RVG-poly(A) mammalian cell expression cassette, which is highly expressed in animal cells, in which the nucleotide sequence of the rabies virus G protein is shown in Sequence Table SEQ ID NO: 1.

[0012]

[0009] Patent document CN104211817 provides a method for producing the recombinant rabies virus protein described above, comprising using a baculovirus / insect cell expression system to produce the recombinant protein: a fusion gene of a rabies virus envelope protein and a nuclear peptide, and a rabies virus. The matrix protein gene is optimized according to the preferential codons of the insect cells. The optimized genetic sequences are cloned together into a transfer expression vector with multiple promoters and expression cassettes (e.g., a double promoter and a double expression cassette) and are located downstream of different promoters. Competent E. coli DHI OBac cells are transformed, and the positive plasmid is extracted into the recombinant baculovirus Bacmid plasmid.The recombinant baculovirus is transfected with the Bacmid recombinant baculovirus plasmid to rescue the recombinant baculovirus (e.g., when the insect cells reached 80% cell confluence). The resulting recombinant baculovirus is then inoculated into insect cells, and after collection, the supernatant is harvested to obtain recombinant rabies virus protein particles.

[0013]

[0010] Document JP3139286 refers to obtaining rabies virus G protein by infecting cells with Baculovirus integrated with a gene encoding the rabies virus G protein via exchange recombination instead of a gene encoding a polyhedrin. A gene encoding the rabies virus G protein is integrated downstream of a Baculovirus polyhedrin promoter with a gene encoding the rabies virus G protein via exchange recombination, replacing the polyhedrin gene, to yield a recombinant Baculovirus. Spodoptera frugiperda cells are infected with the recombinant virus and cultured. The rabies virus G protein accumulated in the cells is then isolated and purified. The gene encoding the rabies virus G protein is preferably derived from the RC-HL strain of rabies virus.

[0014]

[0011] From the perspective of scientific documents, the article titled “Rabies-virus-glycoprotein-pseudotyped recombinant baculovirus vaccine confers complete protection against lethal rabies virus challenge in a mouse model” by Qunfeng Wu et al., published in Veterinary Microbiology, is known. This article refers to the fact that the rabies virus has been a constant threat to humans and animals. A novel strategy for generating a rabies virus vaccine based on a pseudotyped baculovirus is developed here. The recombinant baculovirus (BV-RVG / RVG) was pseudotyped with the rabies virus glycoprotein (RVG) and also simultaneously expressed another RVG under the control of the CMV immediate early promoter. In vitro, this RVG pseudotyped baculovirus vector induced syncytium formation in insect cells and showed more efficient gene delivery in mammalian cells.Mice immunized with BV-RVG / RVG developed higher levels of virus-neutralizing antibodies and conferred 100% protection against rabies virus challenge. These data indicate that the RVG pseudotyped baculovirus BV-RVG / RVG can be used as an alternative strategy for developing a safe and effective rabies vaccine.

[0015]

[0012] Also known is the article entitled “Highly efficient production of rabies virus glycoprotein G ectodomain in Sf9 insect cells” by Targovnik Alexandra et al. from 2019, in which a complete process was developed to produce a large quantity of the rabies virus glycoprotein G (GE) ectodomain in insect cells as a suitable antigen for detecting rabies antibodies. Using the baculovirus expression vector system in Sf9 insect cells combined with a novel chimeric promoter (polh-pSeL), the expression level reached a yield of 4.1 ± 0.3 mg / L of culture, which was significantly higher than that achieved with the standard polh promoter alone. The protein was recovered from cell lysates and easily purified in a single step by metal ion affinity chromatography, with a yield of 95% and a purity of 87%.Finally, GE was successfully used in an assay to detect specific antibodies in serum samples derived from animals vaccinated against rabies. The efficient strategy developed in this work is an interesting method for producing high quantities of this glycoprotein.

[0016]

[0013] In this regard, it is clear that there is an unmet need to provide a pseudotyped baculovirus containing the rabies virus glycoprotein G on its surface that is useful for rabies vaccine formulations.The use of a vaccine formulation with these characteristics offers the following advantages over currently available inactivated vaccines: It eliminates the risk of virulence reversion, as it is a vaccine based on a mammalian-safe vector and contains only the rabies virus gamma (gG); it reduces production costs by being based on an insect cell culture system, potentially improving the accessibility of rabies vaccines; the baculoviral particle possesses intrinsic adjuvant characteristics that enhance the generated immune response; and the use of a linker in the design of the chimeric gamma (gG) improves its incorporation into the baculovirus and the immunity generated compared to previously developed baculovirus vaccine formulations. BRIEF DESCRIPTION OF THE INVENTION.

[0017]

[0014] The present invention is directed to synthetic vahants of the rabies virus glycoprotein G (gG) (Bac::gG-FL) that can be used for baculovirus (Autographa californica nuclear polyhedrosis virus) pseudotyping, and can be used in rabies vaccine formulations.

[0018] DESCRIPTION OF THE FIGURES

[0019]

[0015] Figure 1 shows the arrangement of the ectodomain of the rabies virus gG, as well as the transmembrane (TM) and cytoplasmic (CD) domains used in each chimera. The location of the linker in the gG-FL chimera is also shown.

[0020]

[0016] Figure 2 shows a schematic of the pFastBad ::gG-AN transfer plasmid

[0021]

[0017] Figure 3 shows a schematic of the pFastBad ::gG-FL transfer plasmid

[0022]

[0018] Figure 4 shows a schematic of the pFastBad ::gG-TM transfer plasmid

[0023]

[0019] Figure 5 shows the amplification products of the atfTn7 region of recombinant bacmides containing the gG-AN (3776 bp), gG-FL (3803 bp), and gG-TM (3770 bp) chimeras; as well as the amplification product of the same region in a wild-type (WT) parental baculovirus (300 bp).

[0020] Figure 6 demonstrates the Western blot detection of each of the synthetic gG vahants expressed by the pseudotyped baculoviruses.

[0024]

[0021] Figure 7 shows the replication kinetics of the pseudotyped baculoviruses expressing each of the synthetic gG variants compared to that of a WT baculovirus. Each point represents the average plaque-forming units per mL (PFU / mL) of 3 independent replicates.

[0025]

[0022] Figure 8 demonstrates the ability of synthetic gG vahants to localize to the cell membrane and to be exposed on the surface. This is demonstrated by their detection using indirect immunofluorescence in non-permeabilized cells.

[0026]

[0023] Figure 9 shows the total protein profile on polyachlamide gels of each purified pseudotyped baculovirus. The arrow indicates the band corresponding to the synthetic gG variants.

[0027]

[0024] Figure 10 shows the level of integration of each synthetic gG vahant into the purified baculoviral particles. Values ​​correspond to three independent assays. Values ​​are expressed in normalized relative units (NRUs).

[0028]

[0025] Figure 11 shows the generation of specific antibodies to gG in mice immunized with each pseudotyped baculovirus at 14 and 28 days post-immunization.

[0029]

[0026] Figure 12 shows the antibody titers generated 41 days post-immunization in mice immunized with each pseudotyped baculovirus. A: specific antibody titers using the Platelia® Rabies ELISA kit, values ​​expressed in equivalent units per mL (EU / mL). B: neutralizing antibody titers using the rapid fluorescent focus inhibition test (RFFIT), values ​​expressed in international units per mL (IU / mL).

[0030]

[0027] Figure 13 shows the percentage of survival to a challenge assay against the rabies virus awarded by each pseudotyped baculovirus with the different synthetic variants or synthetic designs of gG.

[0031]

[0028] Figure 14 shows the generation of specific antibodies to the rabies virus in pigs after 2 immunizations via intramuscular and subcutaneous injection with Farvac RAB. The dotted line represents the minimum positive seroconversion value established by the kit.

[0032]

[0029] Figure 15 shows the generation of rabies virus-specific antibodies in 3-month-old dogs immunized with different doses of Farvac RAB. The dotted line represents the minimum positive seroconversion value established by the kit.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034]

[0030] In a first aspect, the present invention relates to a synthetic vahant or synthetic design of the rabies virus glycoprotein G (gG-FL), which consists of genetic fragments from the ectodomain of the Pasteur variant rabies virus glycoprotein G, a 7-amino-acid linker sequence (GGGGSGG), and transmembrane (TM) and cytoplasmic (CT) regions of the baculoviral protein gp64, wherein the arrangement of the sequences in the designed gene cassette is shown in Figure 1. This synthetic vahant can be used to generate pseudotyped baculoviruses using standard genetic manipulation techniques for its expression and localization on the baculoviral surface. The baculovirus pseudotyped with the synthetic gG-FL vahant can therefore be used in rabies vaccine formulations.

[0035]

[0031] In this sense, the present invention refers to a synthetic variant of the rabies virus glycoprotein G, which is used to generate a pseudotyped baculovirus containing the rabies virus glycoprotein G on its surface for anti-rabies vaccine formulations, wherein the gene cassette described in the present invention is integrated into the baculovirus genome by standard genetic manipulation procedures and its expression results in a variant of the synthetic glycoprotein G.

[0036]

[0032] During its expression, synthetic or chimeric gG (gG-FL) undergoes a secretion process from the cytoplasm to the extracellular region, directed by the signal peptide (SS) gp64. Upon reaching the cell membrane, the TM and CT regions of gp64 allow the synthetic or chimeric gG to anchor to viral egress regions, resulting in the incorporation and exposure of the gG ectodomain on the surface of the generated baculovirus particle. Furthermore, the inclusion of the linker sequence grants freedom of movement and independence to the gG and gp64 domains, stabilizing the chimeric protein anchored to the viral surface and, at the same time, improving its immunogenicity compared to other synthetic designs (gG-AN and gG-TM).

[0037]

[0033] Accordingly, the recombinant baculovirus containing the gene cassette designed according to the present invention in its genome will, in turn, be a vector containing the synthetic or chimeric version of glycoprotein G (gG-FL) on its surface and is identified as “Bac::gG-FL”. Thus, Bac::gG-FL can be produced in Sf-9 cells or any other suitable insect cell line using standard cell culture methods.

[0038]

[0034] By presenting glycoprotein G, the most immunogenic protein of the rabies virus, Bac::gG-FL can be used to immunize animals and generate an immune response against the rabies virus. Vaccine formulations composed of Bac::gG-FL (Farvac Rab) have been evaluated in culture supernatants or purified by density gradient centrifugation, along with oil-based or aluminum salt-based adjuvants. Administered intramuscularly, subcutaneously, and intraperitoneally, the formulation has been shown to stimulate the production of neutralizing and rabies-specific antibodies in mice, pigs, and puppies. Furthermore, titration of the produced antibodies demonstrates that the amount of antibodies generated in the animals exceeds 0.5 IU / mL. Similarly, potency assays have shown that each dose provides 4.65 IU / mL of protection.

[0039]

[0035] In a second aspect, the present invention relates to the process for obtaining and evaluating a pseudotyped baculovirus containing the synthetic vahant of chimeric rabies virus glycoprotein G (gG-FL) on its surface for anti-rabies vaccine formulations.

[0040]

[0036] The process for obtaining pseudotyped baculovirus containing the synthetic vahant of chimeric rabies virus glycoprotein G on its surface may comprise the following steps: a) Design and synthesis of the gene cassettes of the synthetic or chimeric gG; b) Obtaining pseudotyped baculoviruses with the synthetic vahants of gG; c) Evaluation of the incorporation and surface exposure levels of the synthetic or chimeric gG vahants in the pseudotyped baculoviruses; and d) Immunological evaluation of the pseudotyped baculoviruses.

[0037] In step a), the sequences comprising the synthetic vahants of the chimeric gG are selected, and transfer plasmids are constructed to integrate the resulting gene cassettes into the baculovirus genome. For this purpose, the amino acid sequences of the gG of the rabies virus vahante Pasteur were used (accession number in GenBank: AAA47218).1) and the baculovirus gp64 protein (Genbank accession number: NP_054158.1) were used to design the expression cassettes shown in Figure 1. After codon optimization for expression in Spodoptera frugiperda, the nucleotide sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 were obtained for the synthetic vahant or chimeric sequences gG-AN, gG-TM, and gG-FL, respectively. These sequences encode the expression of the amino acid sequences SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. The chimeric cassettes were chemically synthesized and cloned into the pUC57 plasmid. The cassettes were then subcloned into the pFastBad transfer plasmid by digestion with the BamHI and Hindlll enzymes. This process resulted in the plasmids pFastBad ::gG-AN (Figure 2), pFastBad ::gG-TM (Figure 3) and pFastBad ::gG-FL (Figure 4), whose sequences are SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0041]

[0038] In step b), the plasmids pFastBad ::gG-AN, pFastBad gG-TM, and pFastBad ::gG-FL are used to generate recombinant baculoviruses, where the chimeric expression cassettes are integrated into the baculovirus genome using transposition techniques. After a selection process of recombinant genomes, these are extracted and purified using standard DNA purification techniques. The purified genomes are subsequently analyzed by PCR to confirm the integration of the gene cassettes, where the amplification products for the recombinant genomes containing the gG-AN, gG-TM, and gG-FL chimeras are 3776 bp, 3770 bp, and 3803 bp, respectively; while in the parental genome where there has been no transposition, the product is 300 bp (Figure 5).

[0039] Purified recombinant baculovirus genomes are used to transfect Sf-9 insect cells using a lipotransfectant.Once inside the cells, the modified genomes are able to generate complete and infectious baculoviral particles due to the insect's cellular machinery. At 72 hours post-transfection, and after the appearance of cytopathic effects, the culture supernatant is recovered and analyzed by PCR. The size of the PCR products for each recombinant baculoviral particle is expected to be the same as that obtained when analyzing the genomes in step b). Similarly, the supernatants are analyzed by Western blot under non-reducing conditions to confirm the expression of the synthetic chimeric variants gG-AN, gG-TM, and gG-FL using a monoclonal antibody specific to glycoprotein G (Santa Cruz Biotech). The size of the detected protein is expected to be approximately 45 kilodaltons (kDa) (Figure 6).Thus, the supernatants that meet the aforementioned evaluation criteria are selected, corresponding to the pseudotyped baculoviruses with the synthetic variants gG-AN, gG-TM and gG-FL in passage 0 (P0), which are identified from this point on as Bac::gG-AN, Bac::gG-TM and Bac::gG-FL, respectively.

[0042]

[0040] Once the P0 of each pseudotyped baculovirus has been established, a P1 is generated to amplify the viral stock. This is done by infecting Sf-9 cell cultures at a density of 2x10 6 cells / mL in a volume of no less than 60 mL, the cultures are then incubated at 28°C for 48 hours. After this time, the cells are separated from the culture by centrifugation and the resulting supernatant is titrated by plate assay. The resulting P1s are used to perform subsequent evaluations and for the scale-up process in medium- or large-scale production.

[0043]

[0041] A first step in evaluating pseudotyped baculoviruses is determining the replication capacity of each. To do this, infections are performed with each pseudotyped baculovirus and a wild-type (WT) baculovirus at the same multiplicity of infection (MOI) and incubated for up to 96 hours at 28°C with shaking. Every 24 hours, an aliquot is taken for titration of each virus. This demonstrates that Bac::gG-AN and Bac::gG-FL maintain the same replication capacity as the parental WT baculovirus, reaching titers above 1 x 10⁻⁶. 8 PFU / mL from 48 hours post-infection (Figure 7). However, Bac::gG-TM shows inefficient replication, reaching maximum titers of 7x10 7 PFU / mL at all evaluation times.

[0044]

[0042] In step c), it is verified that the synthetic variants of gG are correctly translocated to the surface of cell membranes, and the incorporation levels of the synthetic variants in the pseudotyped baculoviruses are compared. For the first step, Sf-9 cells are infected in 6-well arrays at an MOI of 3 and incubated at 28°C for 72 hours. After incubation, the cells are fixed with a 4% paraformaldehyde solution, which preserves the structural characteristics of the cells and proteins for microscopic observation. Then, an indirect immunofluorescence procedure is performed using a monoclonal antibody against the rabies virus glycoprotein G (Santa Cruz Biotech). Since membrane permeabilization was not performed, the antibody only has access to structures located on the cell surface, and therefore, gG detection will only occur in these regions.Thus, Figure 8 shows that all synthetic variants are detected in the cell membrane regions, demonstrating that they are all correctly expressed and localized on the cell surface.

[0045]

[0043] Additionally, baculoviruses are purified by sucrose density gradient centrifugation and ultracentrifugation from previously infected culture supernatants. Total protein is extracted from each pseudotyped baculovirus using Laemmli buffer, and then 7 pg of total protein from each sample is loaded onto polyacrylamide gels, separated electrophoretically, and finally stained using Coomassie staining (Figure 9). Subsequently, the resulting protein profile is digitized and processed using image analysis software, where the densitometric value of the band corresponding to the synthetic gG variants and of the entire sample lane is obtained. These data are used to normalize the values ​​and estimate the amount of gG in the protein profile of each baculovirus, allowing for comparison of the degree of incorporation of each synthetic vahant in the pseudotyped baculoviruses.Thus, as shown in Figure 10, the incorporation levels of gG in the baculoviral particle of Bac::gG-FL are significantly higher than the incorporation in Bac::gG-TM, while maintaining similar levels with respect to Bac::gG-AN.

[0046]

[0044] In step d), in vivo immunological evaluation is performed to establish the degree of immunogenicity of each pseudotyped baculovirus and, consequently, of the synthetic gG vahant molecules carried on their surface. For this purpose, immunization assays are performed in BALB / c mice aged 4 to 6 weeks, which are immunized intraperitoneally with the corresponding pseudotyped baculoviruses previously purified using a sucrose gradient in PBS. The dose and mouse grouping are described in the following table:

[0047] Table No. 1 Groups established for the immunological evaluation of pseudotyped baculoviruses.

[0045] All groups are administered the corresponding antigen on day 0, as well as a booster with the same dose on day 14 post-immunization (pi). In addition, pre-immune blood samples are taken from each mouse, as well as on days 14, 28, and 41 pi, for the detection and titration of specific antibodies to gG in equivalent units (EU / mL) and neutralizing antibodies to the rabies virus in international units (IU / mL).

[0048]

[0046] All groups immunized with the pseudotyped baculoviruses generated specific antibodies to gG after the first immunization, which remained until 28 days post-immunization (Pi) (Figure 11). However, when performing antibody titration on day 41 Pi, it was observed that Bac::gG-FL stimulated the generation of higher titers of specific and neutralizing antibodies compared to Bac::gG-AN and Bac::gG-TM, the latter being the one that generated the fewest antibodies of the three (Figure 12).

[0049]

[0047] To complement the immunogenicity data, a rabies virus challenge assay was performed. Female BALB / c mice weighing 20–25 g were used and randomly assigned to be immunized with baculoviruses pseudotyped with each synthetic gG variant. The number of mice and the arrangement of each group are detailed in the following table:

[0050] Table No. 2 Groups established for the challenge trial against the rabies virus.

[0051]

[0048] Each group received 0.5 mL of the corresponding antigen intraperitoneally, as well as a booster with the same dose on day 14 post-immunization (pi). At 28 days pi, all mice received 50 lethal doses (ID_50) of the CVS rabies challenge virus via intracranial administration. The mice were then monitored daily until day 16 post-challenge to record mortality. At the end of the evaluation, the group immunized with the baculovirus pseudotyped with the synthetic vahant gG-FL (Bac::gG-FL) showed a higher percentage of survival to challenge compared to the other groups evaluated (Figure 13), corresponding to the greater quantity of previously generated neutralizing and specific antibodies.

[0052]

[0049] Since the synthetic vahant gG-FL gives stability and greater immunogenic potential to pseudotyped baculoviruses (Bac::gG-FL), this vahant is used for the vaccine formulation or immunogenic composition Farvac RAB.

[0053] EXAMPLES OF THE BEST WAY TO CARRY OUT THE INVENTION

[0054] Example 1: Preparation of a vaccine formulation or immunogenic composition composed of Bac::gG-FL (Farvac RAB)

[0055]

[0050] Sf-9 cell cultures are infected with Bac::gG-FL at an MOI of 0.4 and incubated at 28°C. 48 hours post-infection, the cells are separated by centrifugation, and the resulting supernatant, containing fresh Bac::gG-FL baculovirus particles, is concentrated by ultracentrifugation and purified using a sucrose gradient. The purified baculovirus or the culture supernatant can be used to immunize companion or farm animals at a dose between 10 7 and 109 PFU directly and / or in combination with oil-based or aluminum salt-based adjuvants. Example 2: Immunization of pigs with Farvac RAB

[0056]

[0051] Twelve-week-old pigs were immunized by administering Farvac RAB subcutaneously and intramuscularly, as well as sterile PBS. The pigs were individually identified and grouped according to the following table:

[0057] Table No. 3 Groups established for immunization in pigs

[0058]

[0052] All pigs received a booster at 14 days post-immunization (pi). Similarly, pre-immune blood samples were taken, as well as samples taken at 14 and 28 days pi. ELISA results showed a slight generation of specific antibodies after the first immunization. However, after the booster, all animals generated antibodies above the seroconversion value established by the positive control of the Platelia® Rabies kit (Figure 14).

[0059] Example 3: Immunization of puppies with Farvac RAB

[0060]

[0053] The immunogenic formulation or composition Farvac RAB was administered subcutaneously to 3-month-old dogs at concentrations of 10 7 , 10 8 and 10 9PFU / dose. Simultaneously, two groups were immunized with an inactivated rabies vaccine or with PBS as controls. Groups of 3 individuals were established according to the following: Table No. 4 Groups established for immunization in pigs

[0061]

[0054] All dogs received a single dose, and partial blood draws were performed at 0 and 25 days post-immunization. This showed that the dogs generated specific antibodies after administration of a single dose of Farvac RAB. Reactivity levels in the ELISA were directly proportional to the administered dose, with those of 10 9 PFU and 10 7 PFUs generated the highest and lowest average reactivity, respectively (Figure 15). Furthermore, it was observed that the group immunized with the 10 dose 9 PFU and the inactivated vaccine had the same degree of immunogenicity.

[0062] Example 4: Farvac RAB potency assay

[0063]

[0055] A challenge test was performed under the potency protocol established by the US National Institutes of Health (NIH). A total of 126 female BALB / c mice weighing between 10 and 14 grams were used. These mice were randomly assigned to be immunized with Farvac RAB at different dilutions. Mice were also assigned to be immunized with an international reference vaccine, prepared at a concentration of 1 international unit per mL (IU / mL), from which serial dilutions were made. Unvaccinated mice were also included as controls for disease development. The arrangement and dosage for each group are detailed in the following table:

[0064] Table No. 5 Groups established for the power test

[0065]

[0056] All mice in the reference vaccine group and the present invention group were immunized intraperitoneally with 0.5 mL of their respective vaccine dilution. Seven days post-immunization, all groups received a booster dose at the same dose and via the same route of administration. On day 14 post-inoculation (pi), challenge was performed by intracranial administration of approximately 30 pL of CVS strain rabies virus at a dose of 25 median lethal doses (LD50) per mouse. In the control group, the challenge was performed in the same manner, but with doses of 25, 2.5, and 0.25 LD50 / mouse. Mice were examined one hour post-inoculation to determine if death had occurred due to procedural trauma. Continuous monitoring was then performed for 14 days to assess the development of signs and mortality caused by the disease.

[0066]

[0057] Thus, the clinical signs of the disease began to be evident from day 6 post-challenge in all groups, and therefore, the deaths recorded from day 8 post-challenge onward were considered to be caused by the rabies virus. In this way, on the last day of evaluation, the survival rates obtained using the Reed and Muench method in the vaccine groups according to the present invention were higher than those of the reference vaccine in all dilutions evaluated (Table 2). This was reflected in the mean protective dose (MPD) calculated for each vaccine, being 88.26 and 18.99 for Farvac RAB and the reference vaccine, respectively (Table 5). Dividing both MPD values, it was established that Farvac RAB had a total protective value of 4.65 IU / mL.

[0067] 10 Table No. 6 Calculation of the average protective dose of Farvac RAB in the potency test.

[0068]

[0058] Similarly, the control groups showed mortality rates directly proportional to the administered viral doses of 25 (88.24%), 15, 2.5 (63.64%), and 0.25 (7.69%) LD50. These values ​​establish that the dose administered to the mice immunized with both vaccines was 17.5 LD50 (Table 6), meeting the assay requirement of between 5-50 LD50 / mouse. Table 7 Calculation of the median lethal dose of the CVS rabies virus used in the challenge

[0069]

[0059] These results demonstrate that the Farvac RAB vaccine exceeds the minimum.

[0070] 5 protection required by QMS of 1 IU / mL per dose.

[0071] List of sequences

[0072] SEQ ID NO. 1 Nucleotide sequence of the synthetic vahant gG-AN

[0073] 10 SEQ ID NO. 2 Nucleotide sequence of the synthetic vahante gG-TM

[0074] SEQ ID NO. 3 Nucleotide sequence of the synthetic vahant gG-FL

[0075] SEQ D NO. 4 Amino acid sequence of the synthetic vahante gG-AN

[0076] SEQ D NO. 5 Amino acid sequence of the synthetic vahante gG-TM

[0077] SEQ D NO. 6 Amino acid sequence of the synthetic vahante gG-FL

[0078] 15 SEQ ID NO. 7 Nucleotide sequence of the pFastBad ::gG-AN plasmid

[0079] SEQ ID NO. 8 Nucleotide sequence of the pFastBad ::gG-TM plasmid

[0080] SEQ ID NO. 9 Nucleotide sequence of the pFastBad ::gG-FL plasmid

[0081] 20

Claims

CLAIMS 1. A synthetic vahant of the rabies virus glycoprotein G (gG) characterized in that it contains genetic fragments of the ectodomain of the rabies virus glycoprotein G, a 7-amino-acid connective sequence (GGGGGSGG), as well as transmembrane (TM) and cytoplasmic (CT) regions of the baculoviral protein gp64.

2. An immunogenic composition characterized in that it comprises a pseudotyped baculovirus with a chimeric synthetic gG variant of claim 1.

3. A process for obtaining a pseudotyped baculovirus with a synthetic variant of the rabies virus glycoprotein G on its surface, characterized in that it comprises the following steps: a) Design and synthesis of the chimeric gene cassettes; b) Obtaining pseudotyped baculoviruses with the synthetic variants of gG; c) Evaluation of the incorporation and surface exposure levels of the synthetic or chimeric variants of gG in the pseudotyped baculoviruses; and d) immunological evaluation of the pseudotyped baculoviruses.

4. The process for obtaining a pseudotyped baculovirus according to claim 3, characterized in that in step a) the sequences comprising the synthetic variants of the chimeric G glycoproteins are selected and the transfer plasmids are constructed to integrate the resulting genetic cassettes into the baculovirus genome.

5. The process for obtaining a pseudotyped baculovirus according to claim 3, characterized in that in step b) the plasmids pFastBad ::gG-AN, pFastBac::Gg-TM and pFastBad ::gG-FL are used to integrate chimeric expression cassettes into the genome of Autographa californica nuclear polyhedrosis virus using standard transposition and / or genetic recombination techniques.

6. The process for obtaining a pseudotyped baculovirus according to claim 3, characterized in that in step b) the purified recombinant genomes transfect Sf-9 insect cells by means of a lipotransfectant.

7. The process for obtaining a pseudotyped baculovirus according to claim 3, characterized in that in step c) it is verified that the synthetic variants or chimeras of gG are correctly translocated on the surface of the cell membranes and the incorporation levels of the synthetic variants or chimeras in the pseudotyped baculoviruses are compared.

8. The process for obtaining a pseudotyped baculovirus according to claim 3, characterized in that in step d) the immunological evaluation is carried out in vivo to establish the degree of immunogenicity and protection provided by each pseudotyped baculovirus and the synthetic variants or chimeric gG that each of them carries on its surface.

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