Synthetic variants of the glycoprotein g of the rabies virus for the generation of pseudotyped baculoviruses and their use in Anti-rabies vaccine formulations
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- Application Number
- AE202603183
- Authority / Receiving Office
- AE · AE
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2024-07-18
- Filing Date
- 2025-04-25
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Abstract
Description
SYNTHETIC VARIANTS OF THE GLYCOPROTEIN G OF THE RABIES VIRUS FOR THE GENERATION OF PSEUDOTYPED BACULOVIRUSES AND THEIR USE IN ANTI-RABIES VACCINE FORMULATIONS TECHNICAL FIELD [1] The present invention falls within the technical field of biotechnology and the pharmaceutical industry for the veterinary field. STATE OF THE ART [2] Rabies is one of the most important zoonotic viral diseases globally. Although there is a preventive vaccination program against the disease, it is estimated to cause 55,000 deaths per year 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 make it necessary to develop rabies vaccines based on new technologies that improve the duration of immunity and, at the same time, reduce production costs to facilitate mass distribution. [3] Over the last few decades, genetic manipulation techniques have enabled the development of viral vectors, which, thanks to their characteristics, can be used to introduce genetic material into cells in vivo or to present recombinant antigens on their surface, the latter process being known as pseudotyping. These characteristics have made viral vectors one of the most important technologies for the development of new-generation vaccines, as they have been shown to be capable of generating immunity to specific antigens. Within this group of vectors, baculoviruses stand out, a group of viruses that infect insects and are harmless to mammals. These are characterized by being produced in eukaryotic cellular machinery, which allows them to express complex proteins with post-translational modifications necessary for their functionality and folding. [4] Glycoprotein G (gG) is the only component on the surface of the rabies virus and is responsible for recognizing host cells. This makes it the only protein capable of generating immunity against the disease, as it is the target of neutralizing antibodies against the virus. Furthermore, given that gG is arranged in homotrimers on the viral surface, 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 pseudotyping baculovirus with gG. However, the direct fusion of two heterologous protein domains can decrease the stability of the resulting chimera or generate incorrect folding of the same. This difficulty could be solved by using connectors or linkers between the fused domains, a strategy that has proven effective for the design of chimeric proteins and bispecific antibodies, improving their stability, expression, and functionality. In the context of baculovirus pseudotyping, the use of a linker between the ectodomain of gG and the transmembrane region of gp64 could improve the independence of both domains, giving it better immunogenicity compared to other chimeric designs. [5] In the field of patents, document WO9509249 is known, which describes oral rabies vaccines comprising at least 100 µg of an isolated rabies G glycoprotein 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 pathogenic infection comprising chimeric proteins and methods for using them are described. Antipathogenic oral vaccines comprising at least 100 µg of an isolated rabies G glycoprotein and an isolated pathogenic antigen or a peptide comprising a neutralizing epitope of a pathogenic antigen and methods for using them are described. [6] US2014178419 also discloses 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. The RV VLP particles comprise one or more RV glycoproteins (G proteins). RV G proteins may be derived from any suitable RV strain. [7] On the other hand, 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, which includes the steps of: 1) artificially synthesizing the gene of the highly immunogenic rabies virus glycoprotein; The gene shown in SEQ No. 1 is divided 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 plasmid vector pUC18; 2） Construction of a highly immunogenic rabies virus glycoprotein adenovirus expression vector; 3）Obtaining recombinant adenoviruses that express highly immunogenic rabies virus glycoprotein. [8] 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 double 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 and express it on the surface of the virion capsule, and the CMV promoter to drive the rabies virus G protein in lactation. The expression cassette in mammalian cells CMV-RVG-poly (A), which is highly expressed in animal cells, wherein the nucleotide sequence of the rabies virus G protein is shown in sequence table SEQ ID NO: 1. [9] Patent document CN104211817 provides a method for producing the recombinant protein of the rabies virus above, which comprises using a baculovirus / insect cell expression system to produce the recombinant protein: a fusion gene of a rabies virus envelope protein and a core peptide, a rabies virus. The matrix protein gene optimized according to the codon preferences of insect cells, the optimized gene sequences are cloned together into a transfer expression vector with multiple promoters and expression cassettes (e.g., double promoter and double expression cassette) and are located downstream of different promoters. Competent E. coli DH10Bac cells were transformed, and the positive plasmid was extracted into recombinant baculovirus Bacmid plasmid. The recombinant baculovirus is transfected with the recombinant baculovirus Bacmid plasmid to rescue the recombinant baculovirus (e.g., insect cell confluence reached 80%). When transfected, the recombinant baculovirus obtained is inoculated into insect cells, and after harvesting, the supernatant is collected to obtain recombinant rabies virus protein particles.
[10] For its part, document JP3139286 refers to obtaining rabies virus G protein by infecting cells with Baculovirus integrated with a gene to encode rabies virus G protein through exchange recombination instead of a gene encoding polyhedrin. A gene encoding rabies virus G protein is integrated downstream of a promoter of a gene encoding a baculovirus polyhedrin using a swap recombination instead of the gene encoding the polyhedrin to give a recombinant baculovirus. Spodoptera frugiperda cells are infected with the recombinant virus and cultured. The rabies virus G protein accumulated in the cells is separated and purified. The gene encoding the rabies virus G protein is preferably derived from the RC-HL strain of the rabies virus.
[11] From the perspective of scientific documents, there is an article entitled “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, which refers to the fact that the rabies virus has been a constant threat to humans and animals. Here, a new strategy for generating a vaccine against the rabies virus based on a pseudotyped baculovirus is developed. 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 to develop a safe and effective vaccine against rabies virus.
[12] There is also an 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 amount of the ectodomain of rabies virus glycoprotein G (GE) of the rabies virus in insect cells as an antigen suitable for detecting rabies antibodies. Using the baculovirus expression vector system in Sf9 insect cells combined with a new 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, the 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 amounts of this glycoprotein.
[13] In this regard, there is clearly an unmet need for a pseudotyped baculovirus containing the rabies virus G glycoprotein on its surface that is useful for rabies vaccine formulations. The use of a vaccine formulation with these characteristics offers the following solutions compared to currently available inactivated vaccines: It eliminates the risk of virulence reversion by being a vaccine based on a vector that is harmless to mammals and containing only the gG of the rabies virus; it reduces production costs by being based on an insect cell culture system and, with this, could improve access to rabies vaccines; the baculoviral particle has intrinsic adjuvant characteristics that enhance the immune response generated; and the use of a linker in the design of the chimeric gG improves its incorporation into baculovirus and the immunity generated compared to previously developed baculovirus vaccine proposals. BRIEF DESCRIPTION OF THE INVENTION
[14] The present invention relates to synthetic variants of the glycoprotein G (gG) of the rabies virus (Bac::gG-FL) that can be used for pseudotyping baculovirus (Autographa californica nuclear polyhedrosis virus) and can be used in rabies vaccine formulations. DESCRIPTION OF THE FIGURES
[15] Figure 1 shows the arrangement of the ectodomain of rabies virus gG, as well as the transmembrane (TM) and cytoplasmic (CD) domains used in each chimera. Similarly, the location of the linker in the gG-FL chimera is shown.
[16] Figure 2 shows a diagram of the pFastBac1::gG-AN transfer plasmid.
[17] Figure 3 shows a diagram of the pFastBac1::gG-FL transfer plasmid.
[18] Figure 4 shows a diagram of the pFastBac1::gG-TM transfer plasmid
[19] Figure 5 shows the amplification products of the attTn7 region of the recombinant bacmids containing the chimeras 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).
[20] Figure 6 shows the Western blot detection of each of the synthetic gG variants expressed by the pseudotyped baculoviruses.
[21] 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.
[22] Figure 8 demonstrates the ability of synthetic gG variants to localize to the cell membrane and be exposed on the surface. This was detected by indirect immunofluorescence in non-permeabilized cells.
[23] Figure 9 shows the total protein profile in polyacrylamide gels of each purified pseudotyped baculovirus. The arrow indicates the band corresponding to the synthetic gG variants.
[24] Figure 10 demonstrates the level of integration of each synthetic gG variant in the purified baculoviral particles. The values correspond to three independent assays. Values expressed in normalized relative units (NRU)
[25] Figure 11 shows the generation of gG-specific antibodies in mice immunized with each pseudotyped baculovirus at 14 and 28 days post-immunization.
[26] Figure 12 shows the antibody titers generated 41 days post-immunization in mice immunized with each pseudotyped baculovirus A: titration of specific antibodies using the Platelia® Rabies ELISA kit, values expressed in equivalent units per mL (EU / mL). B: neutralizing antibody titration using the rapid fluorescent focus inhibition test (RFFIT), values expressed in international units per mL (IU / mL).
[27] Figure 13 shows the percentage of survival in a rabies virus challenge test granted by each pseudotyped baculovirus with the different synthetic variants or synthetic designs of gG.
[28] Figure 14 shows the generation of rabies virus-specific antibodies in pigs after two intramuscular and subcutaneous immunizations with Farvac RAB. The dotted line represents the minimum positive seroconversion value established by the kit.
[29] 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. DETAILED DESCRIPTION OF THE INVENTION
[30] In a first aspect, the present invention relates to a synthetic variant or synthetic design of the rabies virus glycoprotein G (gG-FL), which consists of the genetic fragments of the ectodomain of the Pasteur variant rabies virus glycoprotein G, a 7-amino acid linker sequence (GGGGSGG), as well as transmembrane (TM) and cytoplasmic (CT) regions of the baculoviral protein gp64, where the arrangement of the sequences in the designed gene cassette is shown in Figure 1. This synthetic variant 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 variant can therefore be used in rabies vaccine formulations.
[31] In this regard, the present invention refers to a synthetic variant of the G glycoprotein of the rabies virus, which is used to generate a pseudotyped baculovirus containing the G glycoprotein of the rabies virus 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 synthetic glycoprotein G variant.
[32] During its expression, synthetic or chimeric gG (gG-FL) undergoes a secretion process from the cytoplasm to the extracellular region directed by the gp64 signal peptide (SS). Upon reaching the cell membrane, the TM and CT regions of gp64 allow the synthetic or chimeric gG to anchor in viral exit regions, resulting in the incorporation and exposure of the gG ectodomain on the surface of the generated baculovirus particle. In addition, the inclusion of the linker sequence or connecting sequence gives freedom of movement and independence to the gG and gp64 domains, stabilizing the chimeric protein anchored on the viral surface and, at the same time, improving its immunogenicity compared to other synthetic designs (gG-AN and gG-TM).
[33] Consequently, 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 under standard cell culture methods.
[34] 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) in culture supernatants or purified by density gradient, together with oil-based or aluminum salt-based adjuvants, have been evaluated. Through intramuscular, subcutaneous, and intraperitoneal administration, the formulation has been shown to stimulate the production of neutralizing antibodies specific to the rabies virus in mice, pigs, and puppies. In addition, titration of the antibodies produced shows that the amount of antibodies generated in animals exceeds 0.5 IU / mL. Similarly, determination of the degree of protection in a potency assay shows that each dose has 4.65 IU / mL.
[35] In a second aspect, the present invention relates to the process for obtaining and evaluating a pseudotyped baculovirus containing the synthetic variant of chimeric rabies virus glycoprotein G (gG-FL) on its surface for anti-rabies vaccine formulations.
[36] The process for obtaining the pseudotyped baculovirus containing the synthetic variant 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 synthetic gG variants;c) Evaluation of the levels of incorporation and surface exposure of the synthetic or chimeric gG variants in the pseudotyped baculoviruses; andd) Immunological evaluation of the pseudotyped baculoviruses.
[37] In step a), the sequences comprising the synthetic variants of the chimeric gG are selected and the transfer plasmids are constructed to integrate the resulting genetic cassettes into the baculovirus genome. For this purpose, the amino acid sequences of the gG of the Pasteur variant rabies virus (GenBank accession number: AAA47218.1) and of the gp64 protein of baculovirus (GenBank accession number: NP_054158.1) were used to design the expression cassettes shown in Figure 1. After a process of codon optimization for expression in Spodoptera frugiperda, nucleotide sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 were obtained for the synthetic variants or chimeras gG-AN, gG-TM, and gG-FL, respectively. These sequences encode the expression of amino acid sequences SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. The chimeric cassettes are chemically synthesized and cloned into the pUC57 plasmid. The cassettes are then subcloned into the pFastBac1 transfer plasmid by digestion with the enzymes BamHI and HindIII. This process resulted in the plasmids pFastBac1::gG-AN (Figure 2), pFastBac1::gG-TM (Figure 3), and pFastBac1::gG-FL (Figure 4), whose sequences are SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.
[38] In step b), the plasmids pFastBac1::gG-AN, pFastBac1::gG-TM, and pFastBac1::gG-FL are used to generate recombinant baculoviruses, where the chimeric expression cassettes are integrated into the baculovirus genome using transposition techniques. After a process of selecting 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 no transposition has occurred, the product is 300 bp (Figure 5).
[39] 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 cell machinery. At 72 hours post-transfection and after the appearance of cytopathic effects, the culture supernatant is recovered and analyzed by PCR, where the size of 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 above evaluation criteria are selected, corresponding to the pseudotyped baculoviruses with the synthetic variants gG-AN, gG-TM, and gG-FL in a passage 0 (P0), which are identified from this point as Bac::gG-AN, Bac::gG-TM, and Bac::gG-FL, respectively.
[40] 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 2x106 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 for further evaluation and for the scaling-up process to medium or large-scale production.
[41] The first phase in evaluating pseudotyped baculoviruses is to determine the replication capacity of each one. To do this, infections are performed with each pseudotyped baculovirus and a WT baculovirus at the same multiplicity of infection (MOI) and incubated for up to 96 hours at 28°C with agitation, where every 24 hours an aliquot is taken to perform the titration of each one. This demonstrates that Bac::gG-AN and Bac::gG-FL maintain the same replication capacity as the parental WT baculovirus, reaching titers above 1x108 PFU / mL 48 hours post-infection (Figure 7). However, Bac::gG-TM shows inefficient replication, reaching maximum titers of 7x107 PFU / mL at all evaluation times.
[42] In stage c), it is verified that the synthetic gG variants are correctly translocated to the surface of the cell membranes, and the levels of incorporation of the synthetic variants in the pseudotyped baculoviruses are compared. For the first point, Sf-9 cells are infected in 6-well plates at an MOI of 3 and incubated at 28°C for 72 hours. After the incubation period, the cells are fixed with a 4% paraformaldehyde solution, which preserves the structural characteristics of the cells and proteins for observation under a microscope. Next, an indirect immunofluorescence procedure is performed using a monoclonal antibody against the G glycoprotein of the rabies virus (Santa Cruz Biotech). Since no membrane permeabilization process was 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.
[43] Additionally, baculoviruses are purified using sucrose density gradient and ultracentrifugation from previously infected culture supernatants. Total proteins are extracted from each pseudotyped baculovirus with Laemmli buffer, and then 7 µg of total proteins from each sample are loaded onto polyacrylamide gels, separated electrophoretically, and finally stained with Coomassie blue (Figure 9). The resulting protein profile is then digitized and processed in an image analysis program, where the densitometric value of the band corresponding to the synthetic gG variants and the entire lane of the sample is obtained. These data are used to normalize the values and estimate the amount of gG in the protein profile of each baculovirus, allowing comparison of the degree of incorporation of each synthetic variant in the pseudotyped baculoviruses. Thus, Figure 10 shows that the levels of gG incorporation in the Bac::gG-FL baculoviral particle are significantly higher than the incorporation in Bac::gG-TM, while maintaining similar levels to Bac::gG-AN.
[44] In step d), an in vivo immunological evaluation is performed to establish the degree of immunogenicity of each pseudotyped baculovirus and, therefore, of the synthetic gG variants carried by each of them on their surface. To this end, immunization trials are carried out on 4- to 6-week-old BALB / c mice, which are immunized intraperitoneally with the corresponding pseudotyped baculoviruses previously purified using a sucrose gradient and in PBS. The dose and grouping of the mice is described in the following table: Table No. 1 Groups established for the immunological evaluation of pseudotyped baculoviruses.GroupNumber of miceAntigenDose15Bac::gG-AN5x108 PFU / mouse25Bac::gG-TM5x108 PFU / mouse35Bac::gG-FL5x108 PFU / mouse45PBS-
[45] All groups are administered the corresponding antigen on day 0, as well as a booster with the same dose on day 14 post-immunization (p.i.). In addition, pre-immune blood samples are taken from each mouse, as well as at 14, 28, and 41 days p.i. for the detection and titration of gG-specific antibodies in equivalent units (EU / mL) and rabies virus-neutralizing antibodies in international units (IU / mL).
[46] All groups immunized with pseudotyped baculoviruses generate gG-specific antibodies after the first immunization, which are maintained up to 28 days p.i. (Figure 11). However, when antibody titers were measured on day 41 p.i., 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 generating the fewest antibodies of the three (Figure 12).
[47] To complement the immunogenicity data, a challenge test against the rabies virus was performed. Female BALB / c mice weighing 20-25 g were used and randomly assigned to be immunized with pseudotyped baculoviruses carrying each synthetic gG variant. The number of mice and the arrangement of each group are detailed in the following table: Table No. 2 Groups established for the rabies virus challenge assay.GroupNo. of miceAntigenVaccine doseRabies virus dose113Bac::gG-AN5x108 PFU / mouse50 DL50 / mouse213Bac::gG-TM313Bac::gG-FL410PBS-
[48] 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 (p.i.). On day 28 p.i., all mice received 50 lethal doses (LD50) of the CVS rabies virus challenge 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 pseudotyped baculovirus with the synthetic gG-FL variant (Bac::gG-FL) had a higher survival rate upon challenge than the other groups evaluated (Figure 13), corresponding to the higher amount of neutralizing and specific antibodies generated previously.
[49] Since the synthetic gG-FL variant gives pseudotyped baculoviruses (Bac::gG-FL) stability and greater immunogenic potential, this variant is used for the Farvac RAB vaccine formulation or immunogenic composition. EXAMPLES OF THE BEST WAY TO CARRY OUT THE INVENTION Example 1: Preparation of a vaccine formulation or immunogenic composition composed of Bac::gG-FL (Farvac RAB)
[50] Sf-9 cell cultures are infected with Bac::gG-FL at an MOI of 0.4 and incubated at 28°C. Forty-eight hours post-infection, the cells are separated by centrifugation and the resulting supernatant, containing new Bac::gG-FL baculovirus particles, is concentrated by ultracentrifugation and purified by sucrose gradient. The purified baculovirus or the culture supernatant can be used to immunize companion or farm animals at a dose between 107 and 109 PFU directly and / or in combination with oil-based or aluminum salt-based adjuvants. Example 2: Immunization of pigs with Farvac RAB
[51] 12-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: Table No. 3 Groups established for immunization in pigsGroup Number of pigsVaccineRoute12Farvac RABIntramuscular22Farvac RABSubcutaneous32Sterile PBSIntramuscular
[52] All pigs received a booster 14 days p.i. Similarly, pre-immune blood samples were taken, as well as samples 14 and 28 days p.i. ELISA results showed a slight generation of specific antibodies after the first immunization. However, after the booster was administered, all animals generated antibodies above the seroconversion value established by the positive control of the Platelia® Rabies kit (Figure 14). Example 3: Immunization of puppies with Farvac RAB
[53] The Farvac RAB immunogenic formulation or composition was administered subcutaneously to 3-month-old dogs at concentrations of 107, 108, and 109 PFU / dose. Simultaneously, two groups were immunized with an inactivated rabies vaccine or PBS as controls. Groups of 3 individuals were established according to the following: Table No. 4 Groups established for immunization in pigsGroup Number of dogsDoseRoute13Farvac RAB 107Subcutaneous23Farvac RAB 108Subcutaneous33Farvac RAB 109Subcutaneous43Inactivated vaccineSubcutaneous53PBSSubcutaneous
[54] All dogs received a single dose and partial blood draws were performed at 0 and 25 days post-immunization. In this way, it was observed that the dogs generated specific antibodies after administration of a single dose of Farvac RAB. The reactivity levels in the ELISA were directly proportional to the dose administered, with 109 PFU and 107 PFU generating the highest and lowest average reactivity, respectively (Figure 15). In addition, it was observed that the group immunized with the 109 PFU dose and the inactivated vaccine had the same degree of immunogenicity. Example 4: Farvac RAB potency assay
[55] 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 for this purpose. They were randomly grouped to be immunized with Farvac RAB at different dilutions. At the same time, mice were prepared to be immunized with an international reference vaccine, which was prepared at a concentration of 1 international unit per mL (IU / mL) and used to make serial dilutions. Mice that did not receive any vaccination were also prepared as controls for the development of the disease. The arrangement and dosage for each group is detailed in the following table: Table No. 5 Groups established for the power testGroupVaccine dilutionChallenge dose (rabies virus strain CVS)TotalVaccine of the current invention (Farvac RAB)1:55-50 DL50161:25161:12516 Reference vaccine1:55-50 DL50161:25161:12516Control-25 DL5016-2.5 DL5016-0.25 DL5016
[56] All mice in the reference vaccine and present invention groups were immunized intraperitoneally with 0.5 mL of the dilution of their respective vaccine. Seven days after immunization, a booster was administered to all groups at the same dose and by the same route of administration. Fourteen days post-immunization, the challenge was performed by intracranial administration of approximately 30 µL of the 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. The mice were examined one hour after inoculation to determine whether death had occurred due to trauma from the procedure. They were then monitored continuously for 14 days to assess the onset of signs and mortality caused by the disease.
[57] Thus, clinical signs of the disease began to be evident from day 6 post-challenge in all groups, so deaths recorded from day 8 post-challenge were considered to be caused by the rabies virus. Thus, 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 No. 2). This was reflected in the mean protective dose (DP50) calculated for each vaccine, which was 88.26 and 18.99 for Farvac RAB and the reference vaccine, respectively (Table No. 5). Dividing both DP50 values, it was established that Farvac RAB had a total protective value of 4.65 IU / mL. Table No. 6 Calculation of the average protective dose of Farvac RAB in the power test.VACCINE ACCORDING TO THE CURRENT INVENTION (Farvac RAB)DoseTotal miceLive miceDead miceCumulative liveCumulative dead% survivalDP501_51615134197.1488.261_25159619773.081_12516106101343.48 REFERENCE VACCINEDoseTotal miceLive miceDead miceCumulative liveCumulative dead% survivalDL501_51612421484.0018.991_25157891242.861_125152132257.41
[58] Similarly, the control groups showed mortality rates directly proportional to the viral doses administered of 25 (88.24%), 2.5 (63.64%), and 0.25 (7.69%) LD50. These values establish that the dose administered to mice immunized with both vaccines was 17.5 LD50 (Table No. 6), complying with the trial requirement of between 5-50 LD50 / mouse. Table No. 7 Calculation of the mean lethal dose of the CVS rabies virus used in the challengeCONTROLEstimated CVS doseTotal miceLive miceDead miceCumulative liveCumulative dead% survivalDL5025102821588.2417.532.58264763.640.259811217.69
[59] These results demonstrate that the Farvac RAB vaccine exceeds the minimum protection required by the WHO of 1 IU / mL per dose. Sequence listing SEQ ID NO. 1 Nucleotide sequence of the synthetic variant gG-ANSEQ ID NO. 2 Nucleotide sequence of the synthetic variant gG-TMSEQ ID NO. 3 Nucleotide sequence of the synthetic variant gG-FLSEQ D NO. 4 Amino acid sequence of the synthetic variant gG-ANSEQ D NO. 5 Amino acid sequence of the synthetic variant gG-TMSEQ D NO. 6 Amino acid sequence of the synthetic variant gG-FLSEQ ID NO. 7 Nucleotide sequence of the plasmid pFastBac1::gG-ANSEQ ID NO. 8 Nucleotide sequence of the plasmid pFastBac1::gG-TMSEQ ID NO. 9 Nucleotide sequence of the plasmid pFastBac1::gG-FL
Claims
1. A synthetic variant of rabies virus glycoprotein G (gG) characterized in that it contains the genetic fragments of the ectodomain of rabies virus glycoprotein G, a connecting sequence of 7 amino acids (GGGGSGG), 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 synthetic chimeric gG variant according to 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 chimeric gene cassettes;b) Obtaining pseudotyped baculoviruses with synthetic variants of gG;c) Evaluation of the levels of incorporation and surface exposure of the synthetic variants or chimeras of gG in the pseudotyped baculoviruses; andd) 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 gene 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 pFastBac1::gG-AN, pFastBac::Gg-TM, and pFastBac1::gG-FL are used to integrate the 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 using 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 to the surface of the cell membranes and the levels of incorporation 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 performed in vivo to establish the degree of immunogenicity and protection provided by each pseudotyped baculovirus and the synthetic variants or chimeras of gG that each of them carries on its surface.