Process for producing an antimalarial vaccine composition, products and use

Abstract

The invention relates to a process for producing an antimalarial vaccine composition containing the recombinant chimeric protein SEQ ID NO: 2, which comprises a conserved region I, targeted by neutralising antibodies, linked to an immunodominant region containing repeats of central amino acids from the different alleles (VK210, P. vivax-like and VK247) of the circumsporozoite protein (PvCSP), predominant on the surface of sporozoites, the infective form of Plasmodium vivax, followed by the C-terminal domain region, target of T lymphocytes, of the PvCSP protein. It also relates to the vaccine composition produced by said process, which is directed at malaria caused by Plasmodium vivax and presents high immunogenicity and protection, with efficacy superior to that of the compositions presented in the prior art. The process revealed in this technology allows the production of a high-purity API (100% by RP-HPLC and 98.8% by SE-HPLC) with a high yield (5,082.5 mg from 3L of purified supernatant).

Description

"PROCESS FOR THE PRODUCTION OF ANTI-MALARIAL VACCINE COMPOSITION, PRODUCTS AND USE"

[0001] The present invention relates to a process for the production of an antimalarial vaccine composition that has as its Active Pharmaceutical Ingredient (API) the recombinant chimeric protein defined by SEQ ID No. 2, which comprises a conserved region I (RI) targeted by neutralizing antibodies, linked to an immunodominant region containing central amino acid repeats of the different alleles (VK210, P.vivax-like and VK247) of the circumsporozoite protein (PvCSP), predominant on the surface of sporozoites, the infective form of Plasmodium vivax, followed by the C-terminal domain region, targeted by T lymphocytes, of the PvCSP protein. The invention also relates to the vaccine composition produced by the process of the present technology. The vaccine composition is directed against malaria caused by Plasmodium vivax, the major cause of the disease in Brazil, and exhibits high immunogenicity (in vitro biological activity = 29.10 x 10 4 U / mg; in vivo biological activity = total IgG titers: 10 6; IgG1: 10 7 ; IgG2c: 10 4 ) and protection (83.3% protection and total reduction of parasitemia, nine days after challenge with Plasmodium berghei Pb / PvVK210), with efficacy superior to that of the compositions presented in the prior art (28.5% protection and a tenfold reduction in parasitemia rate, five days after challenge with Plasmodium berghei Pb / PvVK210). The process disclosed in the present technology allows the production of a high-purity API (100% by RP-HPLC and 98.8% by SE-HPLC) and high yield (5,082.5 mg from 3L of purified supernatant).

[0002] Malaria is a serious global health problem, and arguably the world's deadliest tropical parasitic disease. In 2022, the global number of malaria cases reached 249 million, a figure well above the estimated number of cases before the COVID-19 pandemic, and a an increase of five million compared to 2021 (WHO. World Health Organization. World Malaria Report 2023. Available at: https: / / www.who.int / teams / global-malaria-programme / reports / world-malaria-report-2023. Accessed on April 14, 2024).

[0003] Malaria is caused by parasites of the genus Plasmodium, which are transmitted to people through the bite of infected female mosquitoes of the genus Anopheles, known as "malaria vectors." There are five species of parasites that cause malaria in humans, and two of them, P. falciparum and P. vivax, pose the greatest threat. It is known that mortality induced by P. vivax is very low compared to that of P. falciparum; however, mortality from P. vivax has been increasing significantly due to its resistance to chloroquine. This parasite has caused serious complications, including anemia, acute respiratory distress syndrome, cerebral malaria, malnutrition, and kidney failure in several countries.

[0004] There are various study strategies for malaria vaccination, with formulations targeting the pre-erythrocytic phase of the life cycle aiming to generate antibodies capable of neutralizing sporozoites and preventing them from invading hepatocytes.

[0005] Circumsporozoite protein (CSP) is the main component of the sporozoite surface, being necessary for the invasion of human host liver cells. This protein has 3 clearly defined regions: an extensive central region of species-specific tandem repeat amino acids, flanked by 2 highly conserved regions, the N-terminal region and the C-terminal region.

[0006] Mosquirix™, the most advanced malaria vaccine currently available, is based on the circumsporozoite protein of P. falciparum (PfCSP). Also called RTS, S, it is a vaccine. This recombinant vaccine, whose IFA is produced in Saccharomyces cerevisiae, comprises the conserved C-terminal domain and the central repeat domain of PfCSP in combination with the hepatitis B virus surface antigen (HBsAg). However, this vaccine is not effective against P. vivax (EMA. European Medicines Agency. Assessment report: Mosquirix™. 2015. Available at: https: / / www.ema.europa.eu / en / opinion-medicine-use-outside-EU / human / mosquirix#opinion-details Accessed on: April 14, 2024).

[0007] Plasmodium vivax is predominant in the Americas and Asia. In Brazil in 2019, it is estimated that 43,000 people live in areas at risk for malaria, and the Plasmodium vivax parasite is the main cause of the disease in the country, responsible for approximately 86% of registered cases (WHO, 2020). Therefore, the development of a vaccine against malaria caused by P. vivax becomes very important, primarily for use in Asia and the Americas. (WHO. World Health Organization.) World Malaria Report 2020: 20 years of global progress and challenges. Available at: https: / / www.who.int / publications / i / item / 9789240015791 Accessed on April 14. 2024).

[0008] Unlike the central region of PfCSP, the central region of the P. vivax circumsporozoite protein (PvCSP) exhibits three allelic variants: VK-210, P. vivax-like, and VK-247. This allelic variability is conserved by evolutionary pressure, as another immune escape strategy of the parasite, and all three identified variants have a worldwide distribution.

[0009] Several studies based on PvCSP have been conducted to develop vaccines against malaria caused by P. vivax. In this regard, two known vaccines can be mentioned: VMP001 / AS01 B and Rv21 / Matrix-M. The active pharmaceutical ingredient (API) of the first vaccine was expressed. in E. coli and has nine VK-210 repeats and one VK-247 repeat fused to the conserved N- and C-terminal regions of PvCSP. The adjuvant of this vaccine is AS01 B, a liposomal formulation composed of 50 pg of monophosphorylated lipid A (MPL) and 50 pg of Quillaja saponaria fraction 21 (QS-21). However, VMP001 / AS01B does not induce sterile protection against P. vivax (BENNETT JW, YADAVA A, TOSH D, SATTABONGKOT J, KOMISAR J, WARE LA, et al. Phase 1 / 2a trial of Plasmodium vivax malaria vaccine candidate VMP001 / AS01B in malaria-naive adults: safety, immunogenicity, and efficacy. PLoS Negl Trap Dis. 10, no. 2, p. e0004423, 2016). Furthermore, its production in E. coli requires many purification steps, which can hinder and increase the costs of its industrial production. The second vaccine mentioned, Rv21 / Matrix-M, contains IFA expressed in Pichia pastoris, which comprises the central repeats of the VK-210 and VK-247 variants and the C-terminal sequence of PvCSP fused to the hepatitis B antigen (HbsAg), with four more amino acids added to the C-terminal in the form of virus-like particles (VLPs). Its adjuvant, Matrix-M, consists of a mixture of two different populations of nanoparticles, 85% Matrix-A and 15% Matrix-C, which contain different fractions of saponins that have complementary activities (SALMAN, A., MONTOYA-DÍAZ, E., WEST, H. ET AL. Rational development of a protective P. vivax vaccine evaluated with transgenic rodent parasite challenge models. Sci Rep, v. 7, p. 46482, 2017; STERTMAN L, PALM AKE, ZARNEGAR B, CAROW B et al.The Matrix-M™ adjuvant: A critical component of vaccines for the 21st century. Hum Vaccin Immunother. v. 19, n. 1, p. 2189885, 2023). However, both vaccine formulations mentioned offer limited protection against two variants of P. vivax (VK-210 and VK-247), and do not offer protection against P. vivax-like.

[0010] There are studies that consider the three allelic variants of PvCSP (VK-210, P. vivax-like and VK-247) in vaccine compositions, in the form of chimeric proteins produced from expression in E. coli or Pichia pastoris (CAMARGO, TM, DE FREITAS, EO, GIMENEZ, AM ET AL. Primer booster vaccination with recombinant protein and adenovirus vector expressing Plasmodium vivax circumsporozoite protein (CSP) partially protects mice against Pb / Pv sporozoite challenge. L, NUSSENZWEIG RS, NUSSENZWEIG V, AMINO R, RODRIGUES MM, SOARES IS. Vaccine Containing the Three Allelic Variants of Plasmodium vivax Circumsporozoite Antigen Induces Protection in Mice after Challenge with a Transgenic Rodent Malaria Parasite.1275, 2017; TEIXEIRA LH, TARARAM CA, LASARO MO, CAMACHO AG, ERSCHING J, LEAL MT, HERRERA S, BRUNA-ROMERO O, SOARES IS, NUSSENZWEIG RS, ERTL HC, NUSSENZWEIG V, RODRIGUES MM. Immunogenicity of a prime-boost vaccine containing the circumsporozoite proteins of Plasmodium vivax in rodents. Infect Immun. v. 82, n. 2, p. 793-807, 2014). The proteins described by Camargo et al., 2017; Gimenez et al., 2017 and Teixeira et al., 2014, differ in relation to the amino acid sequence of the protein of the present invention, by possessing a histidine tail. Furthermore, the vaccine compositions presented in these articles, as well as the protein purification methods, are also different from those described here. The adjuvants used in the compositions presented by these authors were Poly-IC (InvivoGen) and Montanide ISA720 (Seppic). In the purification process, Camargo et al., 2017, used agarose resin, affinity chromatography, and an anion exchange column. Gimenez et al., 2017, initially used a column of. Affinity (HisTrap FF) coupled to an ÄKTA prime plus FPLC chromatograph (GE Healthcare, Chicago, IL, USA) and in a second purification step, anion exchange chromatography was used on a Resource Q column coupled to the same chromatograph.

[0011] Document WO2013142278 describes the development of vaccine compositions against P. vivax comprising recombinant proteins, such as IFAs, one of which consists of the fusion of the central repeat regions of different PvCSP alleles (VK-210, P. vivax-like, and VK-247). However, in the technology presented in that document, expression occurs in bacteria, preferentially E. coli. The production and purification of recombinant proteins expressed in bacteria are very complex processes. Generally, insoluble proteins are formed, requiring additional purification steps and high concentrations of urea to overcome solubility problems. At the end of the process, it is also necessary to ensure the removal of urea and residual bacterial endotoxins in the protein (TEIXEIRA LH, TARARAM CA, LASARO MO, CAMACHO AG, ERSCHING J, LEAL MT, HERRERA S, BRUNA-ROMERO O, SOARES IS, NUSSENZWEIG RS, ERTL HC, NUSSENZWEIG V, RODRIGUES MM.Immunogenicity of a prime-boost vaccine containing the circumsporozoite proteins of Plasmodium vivax in rodents. Infect Immun. v. 82, n. 2, p. 793-807, 2014). The amino acid sequence of the protein described in the aforementioned document differs from that of the protein of the present invention only by the presence of a histidine tail. The histidine tail present in vaccine APIs can cause nonspecific immune reactions, since these free histidines can generate bioactive amines such as histamines, which can be responsible for adverse events (BROSNAN ME, BROSNAN JT. Histidine Metabolism and Function. J. Nutr. v. 150, suppl. 1, p. 2570S-2575S, 2020; TSAFACK PB,). TSOPMO A. Effects of bioactive molecules on the concentration of biogenic amines in foods and biological systems. Heliyon. v. 8, no. 9, p. e10456, 2022.

[0012] Patent application BR 10 2022 005915 2 refers to the development of recombinant proteins of allelic variants of the central domain of PvCSP, which are used in the preparation of immunological compositions capable of preventing malaria caused by Plasmodium vivax. In this document, the chimeric proteins do not have a histidine tail at the N- or C-terminal ends. For the purification of these proteins, a protocol was established consisting of three chromatography steps using Capto™ MMC, Q HyperD® F, and Sephacryl® S100 HR columns, corresponding to cationic, anionic, and molecular exclusion columns, respectively. Molecular exclusion chromatography is an expensive technique, difficult to scale up, due to the need to use large columns. The vaccine composition presented in this document is formed by the extemporaneous mixing of the PvCSP protein with the Poly-ICLC adjuvant (Hiltonol® / Oncovir). The chimeric protein of SEQ ID N º2 has repeating regions of amino acids, which are susceptible to hydrolysis when stored in solution and at temperatures between 2°C and 8°C. Therefore, unlike the formulation of the present invention, the formulation described in patent application BR 102022005915 2 does not exhibit adequate stability for commercial distribution of the product.

[0013] The present technology presents a process for producing the chimeric protein defined by the SEQ ID N º 2 comprising the three allelic variants [VK-210: (DRADGQPAG - SEQ ID N°3)2(DRAAGQPAG -SEQ ID N O 4)2 (DRADGQPAGD - SEQ ID N°5)2; P. vivax-like: (APGANQEGGAA - SEQ ID N°6); VK-247 (ANGAGNQPG - SEQ ID N°7)], which allows its production on an industrial scale and enables the obtaining of A large-scale vaccine composition with immunoprotective effects superior to those presented in the prior art. The process allows the purification of the chimeric SEQ ID N protein. º2 in just two steps, and obtaining a vaccine composition with greater efficacy compared to those presented in the state of the art. The formulation described in the present invention conferred 83.3% protection in a challenge study with Plasmodium berghei Pb / PvVK210, while the formulation described in patent application BR 10 2022 005915 2 conferred only 28.5% protection when evaluated in the same experimental model (MARQUES, RF; GIMENEZ, AM; CABALLERO, O.; SIMPSON, A.; SALAZAR, AM; AMINO, R.; GODIN, S.; GAZZINELLI, RT; SOARES, IS Non-clinical toxicity and immunogenicity evaluation of a Plasmodium vivax malaria vaccine using Poly-ICLC (Hiltonol®) as adjuvant. Vaccine, v. 42, n. 9, p. 2394-2406, 2024).

[0014] In addition to optimized yield for large-scale production of the chimeric SEQ ID N protein º2 and the improvement of immunological activity through the addition of the squalene-based adjuvant [CTVadl, CTVad2 (mixture of CTVadl and Hiltonol®) or CTVad3], the process presented here allows the composition to remain stable for at least 6 months at a temperature between 2 °C and 8 °C. Most formulations described in the prior art are composed of the extemporaneous mixing of the chimeric protein SEQ ID N º 2 in solution, with some adjuvant. As will be demonstrated in the examples presented in the present invention, under these conditions, in just 30 days of storage, the number of protein cleavage products increases significantly, reaching values ​​of 37.7%, and the purity of the protein, analyzed by capillary electrophoresis in a Bioanalyzer, decreases from 99.9% to 62.3%.

[0015] The development of formulations that can be stored and remain biologically active until the time of use is a A challenge to be overcome. It is estimated that half of the vaccines supplied become unusable due to degradation processes, which consequently limits the distribution of this type of product around the world (BRANDAU, DT et al. Thermal stability of vaccines. Journal of Pharmaceutical Sciences, v. 92, n. 2, p. 218-231, 2003. CLÉNET, D. Accurate prediction of vaccine stability under real storage conditions and during temperature excursions.

[0016] Although many factors contribute to vaccine instability, storage temperature and the pharmaceutical form of the product are the main ones. Aqueous liquid formulations, in particular, are more unstable, since degradative reactions occur more rapidly in an aqueous medium. Therefore, lyophilization has become one of the most important processes for the preservation of biological products. Since many proteins do not have adequate storage stability in solution, a solid pharmaceutical form can increase the product's shelf life. Lyophilized proteins exhibit greater long-term stability in the refrigerator and at room temperature, which offers advantages for storage, transport, and distribution (CARPENTER et al., 2010; CARPENTER et al., 2002). Lyophilized products are therefore more desirable, when applicable, since in the dry state there is less water mobility, which slows down the rate of degradative reactions.This process is very advantageous, especially for products intended for use in regions where maintaining a cold chain is difficult (BRANDAU, DT et al. Thermal stability of vaccines. Journal of Pharmaceutical Sciences, v. 92, no. 2, p. 218-231, 2003, CARPENTER, JF; IZUTSU, K; RANDOLPH, TW. Freezing- and Drying-Induced Perturbations of Protein Structure and Mechanisms of Protein Protection by Stabilizing Additives. In: Freeze Drying / Lyophilization of Pharmaceutical and Biological Products, 3 ed. Informa Healthcare, London, 2010; CHAN, MY; DUTILL, TS; KRAMER, RM Lyophilization of Adjuvanted Vaccines: Methods for Formulation of a Thermostable Freeze-Dried Product. In: Methods in molecular biology (Clifton, NJ). [sl: sn]. v. 1494p. 215-226).

[0017] In this regard, the present invention developed a new antimalarial vaccine composition comprising the chimeric protein SEQ ID N º2, containing sequences of the three allelic variants of P. vivax (VK-210: Seq ID n Q 3, P. vivax-like: Seq ID n Q 4, VK-247: Seq ID n Q5), as well as its lyophilized formulation, which ensures greater stability and allows for its distribution and commercial use. Furthermore, the new formulation developed here demonstrated superior immunological effects compared to those of the prior art, through the use of new squalene-based adjuvants (CTVad1, CTVad2, or CTVad3), with improved immunogenicity and protection in an experimental model. The present invention allowed for the reduction of both the API purification steps and the fermentation induction time, performed using a master cell bank. These changes yielded surprising results, such as scaling up the fermentation process (5x scale-up), scaling up the purification process (15x scale-up), and obtaining a chimeric protein with high purity and yield, without the need to use molecular exclusion chromatography, as used in patent application BR 10 2022 005915 2.Although this type of chromatography is used in industrial processes, it is a very expensive technique and difficult to scale up because it requires very large columns. It was expected that removing this step would compromise the purity of the protein obtained; however, surprisingly, it was possible to obtain a high-purity protein, allowing for a reduction in... Costs and production time are reduced, which is a major advantage at the industrial production level.

[0018] Therefore, it can be concluded that current technology presents the following advantages in relation to the documents cited in the prior art, namely: i. Definition of the best conditions for the fermentation stage in the production process of the chimeric protein defined by SEQ ID No. 2 (API of the malaria vaccine caused by Plasmodium vivax), reducing the time of this stage by half (36h) when compared to document BR 1020220059152 (72h); in addition, the improvements in the fermentation process allowed its scaling up to 50 L (5x increase in scale) (batch USP-01- USP-ENG1). ii. Defining the best conditions for purification in the API manufacturing process, reducing the number of chromatographic steps from 3 to 2; increasing purity (document BR 10 2022 005915 2: 98% by SDS-PAGE band densitometry; current process: 100% by RP-HPLC and 98.8% by SE-HPLC) and yield (document BR 10 2022 005915 2: 156 mg from 200 mL of supernatant; current process: 275.56 mg from 200 mL of supernatant and 5,082.5 mg from 3 L of supernatant). This change made it possible to obtain large quantities of protein with high purity through a faster, cheaper, and simpler process, representing another industrial advantage. iii. Scaling up the API manufacturing process. Previous process (document BR 1020220059152): 10 L of fermentation and 200 mL of purification; current process: 50 L of fermentation and 3 L of purification. iv. Development of a new vaccine formulation, composed of lyophilized active pharmaceutical ingredient (API) for reconstitution with the adjuvant at the time of use. The lyophilization process makes it possible to increase the stability and consequently the shelf life of the vaccine, which may represent a commercial advantage in the product distribution stages. (v. Vaccine composition containing a new squalene-based adjuvant) CTVad1, CTVad2, or CTVad3. The adjuvant used in document BR 10 2022 005915 2 is Poly-ICLC (Hiltonol / Oncovir), a synthetic complex of carboxymethylcellulose, polyinosinic-polycyticidylic acid, and poly-L-lysine double-stranded RNA, which is expensive and acquired through importation. Using CTVad1 or CTVad3, products manufactured in Brazil, makes it possible to reduce the cost of the final formulation and ensures autonomy in the manufacturing process. vi. The composition developed here proved superior in protection tests (83.3%) compared to formulations already described in the prior art (28.5%). vii. The production of the API of the present invention was carried out from a master cell bank. Previously filed patents do not utilize master cell banks. The use of this system allows minimizing the heterogeneity of the cell population and selecting stable and highly productive clones. Furthermore, the manufacture of this bank is a regulatory requirement for the industrial production of vaccines for human use.

[0019] Thus, the present invention relates to a process for producing a vaccine composition against malaria caused by Plasmodium vivax, developed in accordance with regulatory requirements. BRIEF DESCRIPTION OF THE FIGURES

[0020] Figure 1 presents the SDS-PAGE results, under denaturing conditions, for the expression test performed to produce a research cell bank. The SEQ ID No. 2 protein appears in the gel at a height of approximately 50 kDa.

[0021] Figure 2 presents the Western blot results for the expression test performed to produce the research cell bank. The SEQ ID No. 2 protein appears in the gel at a height of approximately 50 kDa.

[0022] Figure 3 refers to the quality control of the master cell bank: restriction analysis with endonucleases using the Southern blot technique on a stained agarose gel. 1. DNA marker II + DNA marker VII (23130, 9416, 6557, 4361, 2322, 2027, 1114, 900, 692, 501 bp) (3 μL + 3 μL). 2. DNA marker II + DNA marker VII (23130, 9416, 6557, 4361, 2322, 2027, 1114, 900, 692, 501 bp) (1.5 μL + 1.5 μL). 3. Empty. 4. Genomic DNA sample / EcoRI-HF + Eagl-HF (1 pg). 5. Control plasmid DNA / EcoRI-HF + Eagl-HF (350 pg). 6. Genomic DNA sample / BamHI-HF + Sacl-HF (1 pg). 7. Control plasmid DNA / BamHI-HF + Sacl-HF (350 pg). 8. Genomic DNA sample / Bgll (1 pg). 9. Control plasmid DNA / Bgll (350 pg). 10. Empty. 11. DNA marker II + DNA marker VII (23130, 9416, 6557, 4361, 2322, 2027, 1114, 900, 692, 501 bp) (1.5 μL + 1.5 μL). 12. DNA marker II + DNA marker VII (23130, 9416, 6557, 4361, 2322, 2027, 1114, 900, 692, 501 bp) (3 μL + 3 μL).

[0023] Figure 4 represents the quality control of the master cell bank: restriction analysis with endonucleases using the hybridized Southern blot technique. 1. DNA marker II + DNA marker VII (23130, 9416, 6557, 4361, 2322, 2027, 1114, 900, 692, 501 bp) (3 μL + 3 μL). 2. DNA marker II + DNA marker VII (23130, 9416, 6557, 4361, 2322, 2027, 1114, 900, 692, 501 bp) (1.5 μL + 1.5 μL). 3. Empty. 4. Sample 1. Genomic DNA sample / EcoRI-HF + Eagl-HF (1 pg). 2. Control plasmid DNA I EcoRI-HF + Eagl-HF (350 pg). 3. Genomic DNA sample / BamHI-HF + Sacl-HF (1 pg). 4. Control plasmid DNA / BamHI-HF + Sacl-HF (350 pg). 5. Genomic DNA sample / Bgll (1 pg). 6. Control plasmid DNA / Bgll (350 pg). 7. Empty. 8. DNA marker II + DNA marker VII (23130, 9416, 6557, 4361, 2322, 2027, 1114, 900, 692, 501 bp) (1.5 μL + 1.5 μL). 12. DNA marker II + DNA marker VII (23130, 9416, 6557, 4361, 2322, 2027, 1114, 900, 692, 501 bp) (3 μL + 3 μL).

[0024] Figure 5 presents SDS-PAGE results, under denaturing conditions, relating to the development of the fermentation process for the production of the PvCSP-A / / epitope protein. The samples were diluted in a 1:100 ratio. (A) Lot USP-FDL-004. (B) Lot USP-FDL-005. (C) Lot USP-FDL-006. (D) Lot USP-FDL-007. (E) Lot USP-FDL-008. (F) USP-FDL-009. (G) Lots USP-FDL-010 and USP-FDL-011. (H) Lot USP-FDL-012.

[0025] Figure 6 shows the flowchart of the fermentation process on a bench scale (6 to 10 L).

[0026] Figure 7 shows the flowchart of the scaled-up fermentation process (50 L).

[0027] Figure 8 shows the flowchart of the purification process on a bench scale (200 mL of supernatant).

[0028] Figure 9 shows the flowchart of the step-by-step purification process (3L of supernatant).

[0029] Figure 10 shows SDS-PAGE results of the protein batch with SEQ ID No. 1. (A) USP-01-DEV1 and the batches relating to the present invention (B) 492-193; (C) USP-01-DSP-DEV2; and (D) USP-01-DSP-ENG1. The protein defined by SEQ ID No. 2 appears in the gel at a height of approximately 50 kDa.

[0030] Figure 11 shows Western blot results of the protein batch defined by SEQ ID No. 2. (A) USP-01-DEV1 and the batches relating to the present invention (B) 492-193; (C) USP-01-DSP-DEV2; and (D) USP-01-DSP-ENG1. The protein defined by SEQ ID No. 2 appears in the gel at a height of approximately 50 kDa.

[0031] Figure 12 represents the protein migration pattern defined by SEQ ID No. 2, in a 12% polyacrylamide gel, corresponding to the two main bands of approximately 50 kDa.

[0032] Figure 13 shows the amino acid sequence of bands 1 and 2 of the protein defined by SEQ ID No. 2. (A) The underlined sequence at the beginning of the protein indicates the difference at the N-terminal end for each band, which corresponds to a specific calculated mass for each of the SDS-PAGE gel bands. (B) Alignment of the primary sequences using the Clustal tool. Labeled 'protein' is the signal peptide (green), its cleavage site (red), and the beginning of the theoretical protein sequence (blue). The first 10 amino acids sequenced by Edman degradation from bands 1 and 2 are highlighted in brown and yellow, respectively.

[0033] Figure 14 represents the analysis of the secondary structure composition of the protein defined by SEQ ID No. 2 in the region between 197 and 260 nm. (A) Dichroic absorption spectrum of the protein defined by SEQ ID No. 2. (B) Secondary structure composition of the protein defined by SEQ ID No. 2 estimated from the spectra obtained by circular dichroism. The results were analyzed using Origin 8 software (OriginLab Corporation, Northampton, MA, USA).

[0034] Figure 15 represents the thermal denaturation analysis of the protein defined by SEQ ID No. 2 in the region between 197 and 260 nm. (A) Dichroic absorption spectrum defined by SEQ ID No. 2 at different temperatures. (B) Composition of secondary structures of the protein. (C) Denaturation curve using the signal at 202 nm during temperature increase. The results were analyzed using Origin 8 software (OriginLab Corporation, Northampton, MA, USA).

[0035] Figure 16 shows the three-dimensional model of the protein structure defined by SEQ ID No. 2, constructed using the trRosetta algorithm.

[0036] Figure 17 shows the fluorescence spectrum of the protein defined by SEQ ID No. 2.

[0037] Figure 18 represents the intact mass analysis of the protein defined by SEQ ID No. 2 by MALDI-TOF mass spectrometry. The masses corresponding to the two peaks of greatest intensity in the spectrum are highlighted in the table, which correspond to the two protein bands that appear on the SDS-PAGE gel.

[0038] Figure 19 shows the identification of the disulfide bridge between Cys239-Cys243 in the protein sequence defined by SEQ ID No. 2. (A) Chromatogram corresponding to the chromatographic run of the sample and highlighting in green the elution region of the target peptide. (B) Demonstration of the fragmentation pattern of the peptide containing the disulfide bridge. (C) Amino acid sequence defined by SEQ ID No. 2 where the peptide is observed (highlighted in yellow).

[0039] Figure 20 shows the biological activity of the protein defined by SEQ ID No. 2 calculated by the ELISA method. Rabbit serum was used as a negative control.

[0040] Figure 21 shows the assessment of the degree of protein aggregation defined by SEQ ID No. 2. (A) Chromatogram obtained by SE-FPLC analysis; (B) Histogram obtained by DLS analysis.

[0041] Figure 22 shows the stability assessment of the protein defined by SEQ ID No. 2 for 30 days, stored in a refrigerator (2 °C to 8 °C): SDS-PAGE and Western blot results.

[0042] Figure 23 shows the stability assessment of the protein defined by SEQ ID No. 2 for 30 days, stored in a refrigerator (2 °C to 8 °C): capillary electrophoresis results in a Bionalyzer.

[0043] Figure 24 shows the stability assessment of the protein defined by SEQ ID No. 2 for 15 days, stored in a refrigerator (2 °C to 8 °C): ELISA results.

[0044] Figure 25 shows the SDS-PAGE 12.5% ​​of that defined by SEQ ID No. 2 over 15 days at a temperature of 5°C. Bands 1 and 2 correspond to the previously described monomers of the protein defined by SEQ ID No. º 2. The bands analyzed are indicated by the numbers 3, 4, and 5. MM: Molecular mass in kDa. 0d, 3d, 6d, 9d, 12d, and 15d correspond to the days analyzed.

[0045] Figure 26 represents the identification of the amino acid sequence of bands 3, 4, and 5 by mass spectrometry. The sequence corresponding to the signal peptide is highlighted in gray. The peptides identified in each band by LC-MS / MS are underlined in yellow.

[0046] Figure 27 shows the magnitude of the IgG antibody response after immunization with the protein defined by SEQ ID No. 2 mixed with different adjuvants. (A) Antibodies against the PvCSP-VK210 variant. (B) Antibodies against the PvCSP-VK247 variant. (C) Antibodies against the PvCSP-P.vivax-like variant.

[0047] Figure 28 represents the humoral immune response in C57BL / 6 mice immunized with three doses of (i) 50 μL of CTVad1 (2.15 mg squalene); (ii) 50 μg of Hiltonol; (iii) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1 (2.15 mg squalene); (iv) 10 μg of defined by SEQ ID No. 2 + 50 μg of Hiltonol; (v) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1 (2.15 mg squalene) + 20 μg of Hiltonol; (vi) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1 (2.15 mg squalene). + 5 μg of Hiltonol. Results are expressed as total IgG antibody titers against Pv-CSP, P. vivax-like, VK210, and VK247. Comparison between groups was performed using the Mann-Whitney statistical test (ns = not significant, *p < 0.05).

[0048] Figure 29 shows the humoral immune response in C57BL / 6 mice immunized with three doses of ( / ) 50 pL of CTVadl (2.15 mg of squalene); ( / 7) 50 pg of Hiltonol; ( / 77) 10 pg of defined by SEQ ID Nº 2 + 50 μL of CTVad1 (2.15 mg of squalene); (iv) 10 μg of defined by SEQ ID No. 2 + 50 μg of Hiltonol; (v) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1 (2.15 mg of squalene) + 20 μg of Hiltonol; (vi) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1 (2.15 mg of squalene) + 5 μg of Hiltonol. Results are expressed as titers of IgG1 anti-Pv-CSP, anti-P.vivax-like, anti-VK210, and anti-VK247 antibodies. Comparison between groups was performed using the Mann-Whitney statistical test (ns = not significant, *p < 0.05).

[0049] Figure 30 shows the humoral immune response in C57BL / 6 mice immunized with three doses of ( / ) 50 pL of CTVadl (2.15 mg of squalene); ( / 7) 50 pg of Hiltonol; ( / 77) 10 pg of defined by SEQ ID N º2 + 50 μL of CTVad1 (2.15 mg of squalene); (iv) 10 μg of defined by SEQ ID No. 2 + 50 μg of Hiltonol; (v) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1 (2.15 mg of squalene) + 20 μg of Hiltonol; (vi) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1 (2.15 mg of squalene) + 5 μg of Hiltonol. Results are expressed as titers of IgG2c anti-Pv-CSP, anti-P.vivax-like, anti-VK210, and anti-VK247 antibodies. Comparison between groups was performed using the Mann-Whitney statistical test (ns = not significant, *p < 0.05).

[0050] Figure 31 shows the humoral immune response in C57BL / 6 mice immunized with three doses of (i) 50 μL of CTVad1 + 5 μg of Hiltonol; (ii) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1; (iii) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1 + 5 μg of Hiltonol. Results are expressed as total IgG antibody titers anti-Pv-CSP, anti-P. vivax-like, anti-VK210 and anti-VK247. Comparison between groups was performed using the Mann-Whitney statistical test (ns = not significant, *p < 0.05).

[0051] Figure 32 shows the humoral immune response in C57BL / 6 mice immunized with three doses of (i) 50 pL of CTVad1 + 5 pg of Hiltonol; (ii) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1; (iii) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1 + 5 μg of Hiltonol. Results are expressed as titers of IgG1 anti-Pv-CSP, anti-P. vivax-like, anti-VK210, and anti-VK247 antibodies. Comparison between groups was performed using the Mann-Whitney statistical test (ns = not significant, *p < 0.05).

[0052] Figure 33 shows the humoral immune response in C57BL / 6 mice immunized with three doses of (i) 50 μL of CTVad1 + 5 μg of Hiltonol; (ii) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1; (iii) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1 + 5 μg of Hiltonol. Results are expressed as titers of IgG2c anti-Pv-CSP, anti-P.vivax-like, anti-VK210, and anti-VK247 antibodies. Comparison between groups was performed using the Mann-Whitney statistical test (ns = not significant, *p < 0.05).

[0053] Figure 34 shows the cellular immune response in C57BL / 6 mice immunized with three doses of (i) 50 μL of CTVad1 + 5 μg of Hiltonol; (ii) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1; (iii) 10 μg of defined by SEQ ID No. 2 + 50 μL of CTVad1 + 5 μg of Hiltonol. Splenocytes from immunized animals were stimulated with PvCSP, RPMI, or the positive control ConA for 72 hours. IFN-γ levels in the supernatant were measured using the ELISA method. The comparison between the The analysis of the groups was performed using the Two-way ANOVA test and Tukey's multiple comparison test.

[0054] Figure 35 shows the percentage of uninfected animals after immunization with the proposed formulations followed by challenge with 5,000 PbANKA sporozoites.

[0055] Figure 36 shows the percentage of parasitemia after immunization with the proposed formulations followed by challenge with 5,000 PbANKA sporozoites. DETAILED DESCRIPTION OF THE INVENTION

[0056] The present invention relates to a process for the production of an antimalarial vaccine composition that has as its Active Pharmaceutical Ingredient (API) the recombinant chimeric protein defined by SEQ ID No. 2, containing core amino acid repeats of the different alleles (VK-210, P.vivax-like and VK-247) of the Plasmodium vivax circumsporozoite protein (PvCSP). The invention also relates to the vaccine composition produced by the process of the present technology. The vaccine composition is targeted against malaria caused by Plasmodium vivax, the major cause of the disease in Brazil, and exhibits high immunogenicity (in vitro biological activity = 29.10 x 10 4 U / mg; in vivo biological activity = total IgG titers: 10 6 ; IgG1: 10 7 ; IgG2c: 10 4) and protection (83.3% protection and total reduction of parasitemia, nine days after challenge with Plasmodium berghei Pb / PvVK210), with efficacy superior to that of the compositions presented in the prior art (28.5% protection and a tenfold reduction in parasitemia rate, five days after challenge with Plasmodium berghei Pb / PvVK210). The process disclosed in the present technology allows the production of a high-purity API (100% by RP-HPLC and 98.8% by SE-HPLC) and high yield (5,082.5 mg from 3L of purified supernatant).

[0057] The method for producing the antimalarial vaccine comprises the following steps: a. Clone the gene defined by SEQ ID No. 1 into circular plasmid DNA with the coding sequence of the α-factor secretion signal followed by the restriction enzyme sites SnaBI, EcoR, Avr II, Not I, promoter AOX1, and express the protein SEQ ID No. 2, in a master cell bank of methylotrophic P. pastoris yeast cells, under fermentation conditions of 6 to 50 L of culture, at a temperature of 26 °C to 30 °C, preferably 30 °C; with an induction rate of 0.015 g / ha 0.02 g / h of methanol, preferably 0.02 g / h; concentration of wet cells at the beginning of induction of 120 g / L to 300 g / L, preferably 250 g / L; induction time of 36 h to 72 h, preferably 36 h; use of antifoaming agent; b. Centrifuge the fermentation contents obtained in “a” at a force of 5,000 to 15,000 x g for 15 to 30 minutes, at a temperature of 4 °C to 15 °C, and collect the supernatant; c. Purify the protein present in the supernatant obtained in “b” through at least one pass through a cationic column (volume: 95 mL to 1.4 L; linear velocity: 400 to 600 cm / h; linear gradient with buffer composed of 100 mM to 25 mM succinate, 20 to 25 mM sodium phosphate and 25 mM to 100 mM CAPS, pH 4.0 to 7.0), followed by ultrafiltration for buffer exchange (20 to 25 mM sodium phosphate, pH 4.0 to 7.0), followed by at least one pass through an anionic column (volume: 40 mL to 700 mL; linear velocity: 150 cm / h; elution in fractions with 20 to 25 mM sodium phosphate buffer, pH 4.0 to 7.0) and followed by ultrafiltration for buffer exchange (phosphate-buffered saline); d. Solubilize the recombinant chimeric protein defined by SEQ ID No. 2 purified in “c” (10 to 100 pg / dose) in cryoprotective buffer. for lyophilization consisting of 50 to 500 pg of sucrose per dose, 1 to 10 mg of mannitol per dose, 9 to 90 pg of polysorbate 20 or 80 per dose, 9 mg of sodium chloride per dose, 0.144 mg of monobasic potassium phosphate per dose, 0.795 mg of dibasic sodium phosphate heptahydrate in 0.3 ml of water for injection per dose, with the ratio between sucrose and protein being 1:1, 3:1 or 5:1 and the mannitol and sucrose ratio preferably 20:1; e. Lyophilize the recombinant chimeric protein defined by SEQ ID No. 2 solubilized in the cryoprotective buffer described in “d”, performing freezing (-55 °C to -35 °C) for 1 to 3 h, followed by primary drying (-35 °C to -20 °C) for 1 to 10 h, followed by secondary drying (2 °C to 15 °C) for 1 to 10 h.

[0058] The process defined in the present invention allows the production of an antimalarial vaccine composition that features the recombinant protein defined by SEQ ID N. º2 lyophilized (10 to 100 pg / dose), for reconstitution at the time of use with the squalene-based adjuvant (CTVad1, CTVad2 or CTVad3; 1 mg to 25 mg of squalene / dose).

[0059] The adjuvant consists of 1 mg to 25 mg of squalene per dose; 0.004 pg to 0.1 pg of citric acid per dose; 0.02 ml to 0.5 ml of water for injection per dose; 0.07 pg to 1.4 pg of sodium citrate per dose; 0.01 pg to 3 pg of polysorbate 80 per dose; 0.01 pg to 3 pg of sorbitan trioleate per dose. It may or may not contain 5 to 50 pg of Hiltonol® per dose.

[0060] The components for lyophilization of SEQ ID No. 2 protein, as well as the pharmaceutical function of each excipient, are listed below (Table 1). Table 1. Description of components and pharmaceutical function. Concentration Function Component Pharmaceutical Technology (mg / mL) SEQ ID N º2 IFA 0.033 to 0.33 Sucrose Cryoprotectant 0.165 to 1.65 Polysorbate 20 or 80 Surfactant 0.03 to 0.30 Mannitol Diluent 3.3 to 33.00 Sodium chloride Buffering agent 9.00 Potassium phosphate Buffering agent 0.144 monobasic Dibasic sodium phosphate Buffering agent 0.795 heptahydrate Water for injection, vehicle q.s. 1.0

[0061] Sucrose is commonly used as a cryoprotectant and effective stabilizer in lyophilized formulations. A pyrogen-free form is available specifically for this use. To ensure good stability, a sugar-to-protein weight ratio of at least 1:1 is generally required, with ideal stability around 3:1 to 5:1. A further increase in sugar concentration can reach the stabilization limit or even destabilize a protein during lyophilization and storage (CARPENTER, JF; CHANG, BS; GARZON-RODRIGUEZ, W; RANDOLPHL, TW. Rational Design of Stable Lyophilized Protein Formulations: Theory and Practice). Carpenter and Manning. Kluwer Academic I Plenum Publishers, New York, 2002; SHESKEY, P.J.; COOK, WG; CABLE, CG. Handbook of Pharmaceutical Excipients 8 ed. London: Pharmaceutical Press 2017; THAKRAL, S; SONJE, J; MUNJAL, B; SURYANARAYANAN, R. Stabilizers and their interaction with formulation components in frozen and freezing- dried protein formulations. Advanced Drug Delivery Reviews, v. 173, p. 1–19, 2021).

[0062] Proteins are generally surface-active and vulnerable to aggregation due to surface-induced denaturation. In this context, the addition of surfactants to a formulation can stabilize proteins through two mechanisms: preferential binding of surfactant molecules at interfaces and / or the surfactant interacting with protein molecules forming a surfactant-protein complex, preventing the protein from interacting with other molecules that lead to its degradation (THAKRAL et al., 2021; CARPENTER et al., 2002). In this context, the use of non-ionic surfactants has been a successful strategy to prevent or inhibit surface-induced aggregation in liquid and lyophilized formulations. Polysorbates (20 and 80) are widely used surfactants in protein formulations, generally at concentrations <0.1% (w / v).The use of refined polysorbates and storage of the formulation under a nitrogen atmosphere or vacuum can prevent the occurrence of the known auto-oxidation of surfactants, which leads to the formation of peroxide, which can potentially lead to protein oxidation (CARPENTER, JF; CHANG, BS; GARZON-RODRIGUEZ, W; RANDOLPHL, TW. Rational Design of Stable Lyophilized Protein Formulations: Theory and Practice. Carpenter and Manning. Kluwer Academic I Plenum Publishers, New York, 2002; THAKRAL, S; SONJE, J; MUNJAL, B; SURYANARAYANAN, R. Stabilizers and their interaction with formulation components in frozen and freeze-dried protein formulations. Advanced Drug Delivery Reviews, v. 173, pp. 1–19, 2021).

[0063] Mannitol is traditionally used as a bulking agent due to its high propensity to crystallize in frozen solutions. In lyophilized preparations, mannitol has been used as a vehicle for... To produce a rigid and homogeneous cake, which improves the appearance of the lyophilized product in the vial. A pyrogen-free form is available specifically for this use. Commonly used concentrations range from 20 to 90% w / w (CARPENTER, JF; CHANG, BS; GARZON-RODRIGUEZ, W; RANDOLPHL, TW. Rational Design of Stable Lyophilized Protein Formulations: Theory and Practice. Carpenter and Manning. Kluwer Academic I Plenum Publishers, New York, 2002; SHESKEY, PJ; COOK, WG; CABLE, CG. Handbook of Pharmaceutical Excipients 8 ed. London: Pharmaceutical Press 2017; THAKRAL, S; SONJE, J; MUNJAL, B; SURYANARAYANAN, R. Stabilizers and their interaction with formulation components in frozen and freeze-dried protein formulations). Advanced Drug Delivery Reviews, vol. 173, p. 1–19, 2021).

[0064] Phosphate-buffered saline at pH 7.4 was selected due to the known protein solubility defined by SEQ ID N. º2 in this vehicle, in addition to being a buffer compatible with physiological pH. One point of attention is that the phosphate-saline buffer is an example where the crystallization of disodium phosphate dodecahydrate (the basic component of the buffer) during freezing reduces the pH of the concentrate, thus potentially altering the stability of the proteins. Therefore, it is desirable that the buffer components remain amorphous during freezing. In turn, sugars can inhibit salt crystallization in the buffer in frozen solutions, thus attenuating any pH change caused by the selective crystallization of the buffer components (THAKRAL, S; SONJE, J; MUNJAL, B; SURYANARAYANAN, R. Stabilizers and their interaction with formulation components in frozen and freeze-dried protein formulations. Advanced Drug Delivery Reviews, v. 173, pp. 1–19, 2021).

[0065] All the excipients chosen are safe, approved by the FDA and ANVISA for parenteral use, via intramuscular injection, and not... present incompatibilities with each other (FDA. Food and Drugs Administration. Inactive Ingredient Search for Approved Drug Products. Available at: https: / / www.accessdata.fda.gov / scripts / cder / iig / index.cfm Accessed on: April 14, 2024; SHESKEY, PJ; COOK, WG; CABLE, CG. Handbook of Pharmaceutical Excipients 8 ed. London: Pharmaceutical Press 2017).

[0066] Sodium chloride (NaCl), commonly used to adjust the osmolarity of parenteral solutions, is not used in lyophilized formulations due to its low eutectic temperature (-21 °C). In frozen solutions, NaCl crystallization is suppressed by amorphous solutes, such as sucrose, and by crystalline solutes, such as mannitol and glycine. This inability of NaCl to crystallize completely reduces the system's collapse temperature. Furthermore, NaCl also inhibits mannitol crystallization in frozen solutions (THAKRAL, S; SONJE, J; MUNJAL, B; SURYANARAYANAN, R. Stabilizers and their interaction with formulation components in frozen and freeze-dried protein formulations). Advanced Drug Delivery Reviews, vol. 173, p. 1–19, 2021).

[0067] The recombinant chimeric protein defined by SEQ ID N º2, the IFA of the Plasmodium vivax malaria vaccine, has a conserved region I (RI) linked to an immunodominant region, containing core amino acid repeats of the different alleles (VK-210, P.vivax-like and VK-247) of the circumsporozoite protein (PvCSP), which is predominant on the surface of sporozoites, the infective form of Plasmodium vivax, followed by the C-terminal domain region of the PvCSP protein.

[0068] The invention can be better understood from the following examples, which are not limiting. EXAMPLE 1 - EXPRESSION AND PURIFICATION OF THE RECOMBINANT PROTEIN DEFINED BY SEQ ID No. 2

[0069] Production of the Master Cell Bank. The recombinant chimeric protein defined by SEQ ID N º 2 was produced from a master cell bank. The production of this bank involved three steps, described below.

[0070] Construction of the pPIC9K-PvCSP vector containing the gene defined by SEQ ID Nº 1: the synthetic gene defined by SEQ ID N º 1 corresponding to the chimeric protein defined by SEQ ID N ºSequence 2 was synthesized by Genscript (USA), with a codon optimized for expression in the yeast Pichia pastoris. For sequence optimization, it was processed using the OptimumGene™ algorithm. The Codon Adaptation Index (CAI) value for the predicted sequence was determined to be 0.88 (CAI=1.0 being perfect for expression and CAI>0.8 optimal in terms of high levels of gene expression). The GC base pair content was optimized to prolong the mRNA half-life. Stem-loop structures were broken to facilitate and stabilize ribosomal binding to the mRNA. Potential N-glycosylation sites (NxS / T) were identified and altered (NxS / T to AxS / T) during the synthesis process. Next, the synthetic gene was cloned into the pPIC9K plasmid (Invitrogen™, catalog number V17520), using the EcoRI and Notl restriction sites, for expression under the control of the alcohol oxygenase 1 (AOX1) inducible methanol promoter.In particular, the recombinant protein comprises the fusion of: ( / ) a conserved I region (RI) of the circumsporozoite protein (CSP) of P. vivax, known to be the target of neutralizing antibodies; ( / 7) three central antigenic repeat regions of three different alleles (VK-210, P. vivax-like and VK-247) of P. vivax CSP; and (7 / 7) the C-terminal domain region of PvCSP, the recombinant protein being expressed in Pichia pastoris, devoid of histidine at the C and / or N-terminal ends. The strategy of joining the sequences into a single polypeptide optimizes, simplifies and reduces the time and costs of the process, when. Compared to the individual production of each variant separately, this process generates a single universal antigenic protein.

[0071] Expression test for the production of a research cell bank: the pPIC9K- vector containing the SEQ ID No. 1 gene was used to transform E. coli RR1 to obtain > 10 µg of purified plasmid DNA. An aliquot of approximately 5 pg of purified plasmid DNA linearized with Sail was electroporated into methylotrophic Pichia pastoris yeast. The transformed yeast were selected by growth in histidine-deficient medium, and the expression of the protein defined by SEQ ID No. 2 was verified by SDS-PAGE (Figure 1). Twenty-four clones were selected to identify the one with high protein production, after growth for approximately 48 h using glycerol as the sole carbon source and then growth for 72 h using methanol as the sole carbon source and inducer.The best clones were evaluated by Western blot (Figure 2), and clone C3 was selected for production of the research cell bank under Good Laboratory Practices conditions. For this purpose, a colony of clone C3, cultivated on an agar plate, was inoculated into a shaker flask containing 50 mL of BMGY medium, specific for the production of recombinant proteins in Pichia pastoris with methanol induction. The shaker flask was incubated at 28 °C, at 250 rpm, until the culture reached between the beginning and middle of the log phase (ODeoo approximately 5.0). Then, 25 mL of the culture were mixed with 25 mL of a 40% (v / v) glycerol solution for cryopreservation. 1 mL aliquots were placed in 40 cryotubes, labeled with batch number 388076FDL, and stored in liquid nitrogen.

[0072] Production of the master cell bank from the research cell bank: the master cell bank was produced under Good Manufacturing Practices conditions. For this, 150 mL of BMGY medium were used. Added to two shaker bottles. Aseptically, 0.25 ± 0.01 ml of the previously thawed research cell bank (lot 388076FDL) was transferred to each of the shaker bottles. The bottles were incubated at 30°C ± 2°C, at 250 rpm ± 10 rpm, until the target OD₂ was reached. OD₂ was monitored at 0, 17h, and every hour after 17h, until OD₂ was between 2.0 and 5.0. In bottle 1, the process was stopped after 19h of incubation (OD₂ = 2.19) and in bottle 2, the process was stopped after 20h of incubation (OD₂ = 2.64). Next, 120 ml of the culture from flask 2 were mixed with 120 ml of a 40% (v / v) glycerol solution for cryopreservation. 1 ml aliquots were placed in 200 cryotubes, labeled with batch number USP-01-MCB-01, and stored in liquid nitrogen.

[0073] Quality Control of the Master Cell Bank. The tests performed for quality control of the master cell bank are described in Table 1. Table 1. Quality control results for the master cell bank, batch USP-01-MCB-01. Test Specification Result Report the average of three samples before 6.3 x 10 6 UFC freeze Report the average of three Count of the total number of samples after 6.8 x 10 6 CFU viable cells freezing Samples before and after freezing Differences of less than 1 logarithm should be at most 1 log difference Identification by Gram-positive yeast Gram-positive yeast Gram staining Smooth colonies, smooth, circular colonies, circular in color. Identification by cream, translucent and cream, translucent and colony morphology with regular borders with regular borders (entire margin) (entire margin) Purity by means of Cultivation using agars. No contamination. No differential contamination. 100% equivalence Informative DNA sequence with the theoretical sequence Analysis of n º of copies Equivalent genome / 2 Informative Restriction analysis of gene copies Figure 3 Informative with endonucleases Figure 4

[0074] Development and scaling up of the fermentation process. Tests were carried out with 6 batches (USP-FDL-004 to USP-FDL-009) of 6L to 10L of fermentation to define the best conditions for the expression of the protein defined by SEQ ID No. 2. Temperatures of 26 °C or 30 °C were tested, with methanol induction rates of 0.015 g / h or 0.02 g / h and wet cell concentrations in the induction step of 120 g / L, 200 g / L, 250 g / L or 300 g / L (Table 2). Based on the tested conditions and the SDS-PAGE results (Figure 5), it was determined that the best condition for fermentation is a temperature of 30 °C, with an induction rate of 0.02 g / h of methanol, and a wet cell concentration in the induction step of 250 g / L (Table 2). This condition generates the highest concentration of wet cells at the end of fermentation (362.55 g / L) in the shortest processing time (36 h). Three more fermentation batches of 6 L to 7 L were produced to confirm the reproducibility of the process (USP-FDL-010 to USP-FDL-012) (Table 2). In production In these batches, the KFO 67 DyStar Polyolester antifoaming agent, specifically for use in the food industry and used in the prior art, was replaced by Polypropylene glycol 2000 (Sigma Aldrich), suitable for the pharmaceutical industry. Improvements in the fermentation process allowed its scaling up to 50 L (5x scale-up) (batch USP-01-USP-ENG1). The flowcharts of the fermentation processes at bench scale (6 to 10 L) and pilot scale (50 L) are described in Figures 6 and 7. Table 2. Conditions tested during the development of the fermentation process, batches USP-FDL-004 to USP-FDL-012. Lot / USP- USP- USP- USP- USP- USP- USP- USP- USP- Parameter FDL- FDL- FDL- FDL- FDL- FDL- FDL- FDL- FDL- o 004 005 006 007 008 009 010 011 012 Scale (L) 10 7 6 10 6 10 7 6 6 Temperature 30 30 30 30 30 26 30 30 26 ra (°C) Induction with 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.02 methanol 5 5 5 (g / h) Concentrate tion of mass cells 120 200 250 120 300 120 200 250 120 wet in beginning of induction (g / L) Concentrates 273, 343, 362, 302, 360, 265, 317, 322, 323, tion of 35 10 55 35 90 35 55 55 65 mass cells damp in end of fermentation ion (g / L) The production process conditions for batches USP-FDL-006 and USP-FDL-011 were defined as ideal because they generated a higher concentration of wet cell mass in a shorter processing time (36h).

[0075] Development and scaling up of the purification process. The purification of the recombinant protein defined by SEQ ID No. 2 was performed using two sequential chromatographic steps: cation-exchange chromatography using a Capto™ MMC column and anion-exchange chromatography using a Q Ceramic HyperD® F column. The flowcharts of the bench-scale process (purification of 200 mL of supernatant) and pilot-scale process (purification of 3 L of supernatant) are detailed in Figures 8 and 9, respectively. The scale-up achieved was 15x compared to what is described in the prior art.

[0076] With the new purification process, it was possible to obtain a high yield (275.56 mg from 200 ml of supernatant and 5,082.5 mg from 3 L of supernatant) and a protein with high purity (100% by RP-HPLC and 98.8% by SE-HPLC), without needing the molecular exclusion chromatography step, presented in document BR 10 2022 005915 2 (purity: 98% by SDS-PAGE band densitometry and yield: 156 mg from 200 ml of supernatant). Molecular exclusion chromatography is used in industrial processes; however, it is a very expensive technique and difficult to scale up because it requires very large columns. It was expected that by removing this step from the purification, the purity of the protein obtained would be... compromised. However, surprisingly, it was possible to obtain a high-purity protein using one less step in the purification process, reducing costs and manufacturing time. This represents another industrial advantage. Table 3 describes the quality control results of the protein defined by SEQ ID No. 2, purified by the process described in document BR 1020220059152 (batch USP-01-DEV1), and by the process described in the present invention (development batch: 492-193; reproducibility batch: USP-01-DSP-DEV2; and scaling-up batch: USP-01-DSP-ENG1). The protein is carried in phosphate-buffered saline (8 mM monobasic sodium phosphate, 2.3 mM dibasic sodium phosphate, 130 mM sodium chloride, pH 7.4). Table 3. Quality control and yield results of the Defined Protein batch by SEQ ID No. 2 produced by the method described in document BR 10 2022 005915 2 (USP-01 -DEV1) and of the batches produced by the method described in the present invention (492-193; USP-01 -DSP-DEV2; USP-01 -DSP-ENG1). USP-01- USP-01- USP-01 - Lot / Parameter 492-193 DEV1 DSP-DEV2 DSP-ENG1 Scale purification 200 ml_ 200 mL 200 ml_ 3 L (volume of supernatant) Identification Figure 10 Figure 10 Figure 10 Figure 10 by SDS-PAGE Identification by Western- Figure 11 Figure 11 Figure 11 Figure 11 blot Concentration 0.8 mg / mL 0.84 mg / mL 0.83 mg / mL 1.00 mg / mL determined by the method of acid bicinchoninine (BCA) No Purity 98%* determined 89.6%*** 98.8%*** Protein residual 45.4 ng / mg 24.63 ng / mg 25.7 ng / mg 19.9 ng / mg cell host protein (ng / mg 0.1 EU / pg No 0.005 EU / pg 0.007 EU / pg Endotoxins specific protein protein No Bioburden 0 CFU / mL 0 CFU / mL 0 CFU / mL determined Quantity of protein: 195 mL, 268 mL, 332 mL, 5082.5 mL 156 mg 225.12 mg 275.56 mg 5,082.5 mg produced *Purity determined by band densitometry of the SDS-PAGE gel. **Purity determined by RP-HPLC. “* Purity determined by SE-HPLC. EXAMPLE 2 - CHARACTERIZATION OF THE PROTEIN DEFINED BY THE SEQ ID NO2

[0077] Primary structure. The following is the amino acid sequence that makes up the primary structure of the protein defined by SEQ ID No. 2: PRENKLKQPGPGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAG QPAGDRADGQPAGDRADGQPAGDRADAPGANQEGGAAAPGANQE GGAAAPGANQEGGAAAAPGANQEGGAAAPGANQEGGAAAPGANQ EGGAAAANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGAN GAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKV RATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT

[0078] To confirm the listed amino acid sequence, advanced mass spectrometry analyses and N-terminal sequencing, using the Edman degradation method (EDMAN, 1950), were performed on samples from the two protein bands that appear near 50 kDa on the SDS-PAGE gel (Figure 12). The alignment of the theoretical primary amino acid sequence of the protein, together with the experimentally obtained sequence for each of the two bands, was performed to identify the difference between them (Figure 13).

[0079] Secondary Structure. The determination of the secondary structure of the protein defined by SEQ ID No. 2 was performed by circular dichroism using a JASCO-J815 spectropolarimeter (Jasco Inc., Easton, USA) equipped with a Peltier temperature control system (Analytical Instruments, Japan). Sample preparation consisted of diluting the protein dispersed in phosphate-buffered saline (PBS) and the control buffer with ultrapure water to achieve a working concentration that maintained the detector voltage below 700 V in the 190–260 nm region. As a result, the sample was diluted to a final protein concentration of 3.82 pM (0.1 mg / mL), which allowed recording the dichroic signal in the spectral range of 195–260 nm and 197–260 nm. Due to sample dilution, the ionic composition was approximately 1.3 mM Na2HPO4, 0.23 mM KH2PO4, 0.35 mM KCl, and 17.8 mM NaCl.Dichroic spectra were obtained in a quartz cuvette with an optical path length of 0.1 cm at 25 °C. Ten dichroic spectra were accumulated for each sample at wavelengths 195 to 260 and 197 to 260 nm, with intervals of 0.1 nm, a scanning rate of 100 nm / min, and a time of 0.1 nm. with a response time of 1 second and a bandwidth of 1.7 nm. The average of the readings was corrected by subtracting the contributions of the buffers. The ellipticities obtained for each wavelength (0À) were converted to molar ellipticity by residuals (0)À using Equation 1: (6)À = QÁ / 10 cn L (Equation 1)

[0080] Where: 0À is the ellipticity (mdegrees°); n, the number of amino acid residues; c, the protein concentration (mol / L) and L, the optical path length of the quartz cuvette (cm). The value of (0)À is expressed in mdegrees.cm 2 / dmol. The protein's secondary structure content was estimated using the Bestsel internet server (http: / / bestsel.elte.hu / index.dhp).

[0081] The data obtained (Figure 14) indicated reproducibility in the results and that the protein has few helical structures, with a majority presence of antiparallel beta-sheets (mean of 31.0 ± 4.9%), followed by beta-curves (mean of 22.4 ± 1.7%) and 46.4 ± 3.4% of other disordered structures (Table 4). Table 4. Composition of secondary protein structures defined by SEQ ID No. 2 estimated from spectra obtained by circular dichroism. Structures % 195 to 260 nm % 197 to 260 nm Average α-helices 0.2 0.1 0.15 + 0.07 p-sheets 34.5 27.5 31.0 + 4.90 antiparallel p sheets 0.0 0.0 0.0 + 0.0 parallel Beta curves 21.2 23.6 22.4 + 1.7 Other structures 44.0 48.8 46.4 ± 3.4 disordered

[0082] Thermal stability: The structural stability of the protein defined by SEQ ID No. 2 was evaluated by monitoring changes in ellipticity at 202 nm, over a temperature range of 25 °C to 100 °C, with a heating rate of 1 °C / min and a reading interval of 0.2 °C. To determine the transition temperatures (Tm) between the native and denatured states, denaturation curves were fitted using the Boltzmann equation with sigmoidal fit in the OriginPro 2023 program (OriginLab Corporation, Northampton, MA, USA). Simultaneously, at 10 °C intervals, dichroic spectra were collected in the range of 197 to 260 nm (far UV region), using a data step of 0.1 nm. Dichroic spectra were obtained using a Jasco J-815 spectropolarimeter equipped with a Peltier temperature control system (Jasco Analytical Instruments, Tokyo, Japan). Measurements were performed at constant temperature using quartz cuvettes with a path length of 0.1 cm.Standard sensitivity, a bandwidth of 1.71 nm, a digital integration time of 0.5 s, and a scan rate of 100 nm / min in continuous scan mode were employed. To ensure the accuracy of the analyses, five consecutive readings were accumulated, and the average of the spectra was recorded. Furthermore, the signal contribution from the buffers was subtracted from each reading and protein spectrum collected. The results shown in Figure 15, letter A, demonstrate that as the temperature increases, the protein spectra exhibit surprising stability, suggesting that the protein structure resisted thermal denaturation throughout the entire analysis range.

[0083] In Figure 15, letter B, it can be observed that the composition of secondary structures remained unchanged during the process of Denaturation. The signal at 202 nm remained constant across the temperature range (as shown in Figure 15, letter C), except in the 210-220 nm region, where a slight increase in the negative signal was detected. This phenomenon suggests a slight tendency towards protein structuring or aggregation (responsible for some light scattering) in this specific region. Interestingly, the protein in question has only three cysteine ​​(Cys) residues, which, at first glance, would not be sufficient to confer significant stability, especially considering the possibility of multiple disulfide bridge formation. However, the results shown in Figure 15, letter A, challenge this expectation by revealing a remarkable resistance to thermal variations.

[0084] Prediction of the three-dimensional structure: the structural model of the protein defined by SEQ ID No. 2 (Figure 16) was designed using the trRosetta algorithm, a protein structure prediction tool accessible through the portal (https: / / vanqlab.nankai.edu.cn / trRosetta). In this process, five models were built and refined through meticulous energy optimization. The degree of confidence obtained during modeling was low, forcing the construction of the models based on de novo folding, guided by deep learning constraints. The model suggested the presence of a disulfide bridge between cysteines 239 and 273.

[0085] The analysis of the model's structural composition was conducted using the DSSP-web server (http: / / bioinformatica.isa.cnr.it / SUSAN / DSSP-web / ). Notably, the most stable model showed a predominance of π-curve structures (25.9%), in addition to exhibiting unstructured areas (47.1%). These findings are similar to the experimentally obtained results (Table 4). However, it is evident that the proportions of helical and π-sheet structures differ significantly from the results obtained experimentally. Experimental data showed a 9.5% presence of helical structures, in contrast to the 0.15% observed experimentally. Similarly, the occurrence of antiparallel p-sheets was estimated at 4.4%, contrasting with the 31.0% observed in the experimental data. It is also noted that the model suggests a proportion of 13.1% of parallel p-sheets, which were not identified experimentally. Therefore, the results of the three-dimensional modeling should be used with caution.

[0086] Tertiary structure. The protein Defined by SEQ ID No. 2 has aromatic amino acids in its primary sequence, with band 1 having 2 tyrosine residues, 1 tryptophan residue, and 1 phenylalanine residue; and band 2 having 1 tyrosine residue and 1 tryptophan residue. The fluorescence spectra related to the tertiary structure of the protein Defined by SEQ ID No. 2 are shown in Figure 17. For this analysis, 75 pL of the protein (in triplicate) were pipetted onto NUNC 384 black plates, as well as the blank solution (PBS). The samples were excited at 280 nm with emission between 300 and 390 nm, and subsequently the samples were excited at 295 nm with emission between 313 and 403 nm. Readings were performed on a Varioskan LUX instrument. The maximum fluorescence intensity obtained was observed at 327 nm, demonstrating that the protein is folded with tryptophan residues exposed in the hydrophilic region.

[0087] Relative molecular mass. The theoretical molecular mass of the protein defined by SEQ ID No. 2 is equal to 25.0 kDa; however, in SDS-PAGE analyses (Figure 12), this protein shows a migration profile in the gel, corresponding to two main bands of approximately 50 kDa.

[0088] To confirm and understand this profile, intact mass analyses were performed using MALDI-TOF mass spectrometry. (Figure 18). It was detected that the protein is composed of two monomers, with experimental masses of 25,639.622 Da and 24,861.682 Da, respectively, corroborating the result obtained by sequencing for each of the bands of the SDS-PAGE gel (Figure 13). The migration of the bands, recorded by electrophoresis, corresponds to the formation of dimers equal to 51,277.88 Da (band 1) and 49,694.08 Da (band 2). It is believed that this phenomenon occurs due to the large quantity of hydrophobic amino acids in the central repeat region of the protein, which, being in solution, would be attracted into dimers by repulsion action by the hydrophilic medium, phosphate-buffered saline, in which the protein is immersed.

[0089] Characterization of the resulting post-translational modification forms. In the protein sequence, the theoretical presence of a disulfide bridge between cysteine ​​239 and cysteine ​​243 is verified in the ATVGTEWTPCSVTCGVGVR peptide, identified by nano LC-MS / MS mass spectrometry after digestion of the protein with the enzyme trypsin, which performs cleavage after arginine residues. To confirm the theoretical prediction, the sample was digested with the same enzyme, but without prior reduction / alkylation of the disulfide bridges. The generated data were evaluated in the BiopharmaFinder 5.1 program using the Disulfide Bond Default method (Figure 19).It is possible to observe the impossibility of identifying only one fragment of the sequence, as demonstrated by the identification of an additional fraction in the obtained chromatogram (Figure 19, letter A), and its corresponding theoretical sequence (Figure 19, letter C), showing that the amino acids, labeled γ11, γ12, and γ13 ions, present between the cysteines corresponding to γ10 and γ14 ions, could not be cleaved, ionized, and subsequently sequenced (Figure 19, letter B). This result highlights the presence of a disulfide bridge between them.

[0090] Comparison of the physicochemical and structural characteristics between the recombinant chimeric protein defined by SEQ ID No. 2 and the original proteins. The physicochemical and structural characteristics of the protein defined by SEQ ID No. 2 and the original PvCSP proteins are listed in Table 5. Table 5. Comparative physicochemical and structural characteristics of the recombinant protein defined by SEQ ID No. 2 in relation to the variants PvCSP-VK21 0, PvCSP-P. vivax like and PvCSP-VK247. Defined PvCSP- PvCSP-P. PvCSP- Characteristic by SEQ ID VK210 2 vivax like 3 VK247 4 No. 2 1 Number of 266 336 387 342 amino acids Mass molecular 25, 0 5 33.1 37.0 32.2 (kDa) Point 4.61 5.11 5.97 5.33 isoelectric aliphatic index 40.08 49.14 55.97 45.94 Modifications Bridges Bridges Bridges Bridges post-disulfide: disulfide: disulfide: disulfide: translations 277 ↔ 281 325 ↔ 329 283 ↔ 287 231 ↔ 235 Predicted 311 ↔ 316 359 ↔ 364 317 ↔ 322 at: https: / / web.expasy.org / cgi-bin / protparam / . Accessed on: April 15, 2024. From: https: / / www.uniprot.org / uniprotkb / A0A4D6G1Y5 / entry. Accessed on: April 15, 2024. From: https: / / www.uniprot.org / uniprotkb / D3JYZ9 / entry. Accessed on: April 15, 2024. From: https: / / www.uniprot.org / uniprotkb / A0A4D6G202 / entry. Accessed on: April 15, 2024. 5 The theoretical molecular mass of the protein defined by SEQ ID No. 2 is equal to 25.0 kDa; however, in SDS-PAGE analyses, this protein shows a migration profile in the gel, corresponding to two main bands of approximately 50 kDa.

[0091] In vitro biological activity. The in vitro biological activity of the recombinant protein defined by SEQ ID No. 2 was determined from the EC50 parameter, defined as the protein concentration required to generate 50% of the maximum response observed in an ELISA titration curve (Figure 20). For this analysis, serial dilutions of the protein defined by SEQ ID No. 2 were performed, starting from 400 ng / well with an 8-point experimental design, using a dilution factor of 4 times (4 pg / mL to 0.004 pg / mL and 0 pg / mL) in sensitization buffer with overnight incubation at 5 °C, followed by plate blocking for 2 hours at room temperature. After discarding the volume, rabbit-produced polyclonal antibody (anti-PvCSP) was added to the plates according to the standardization, with incubation in an incubator at 37 °C.Next, the plates were washed and the secondary antibody anti-rabbit IgG conjugated to horseradish peroxidase (anti-IgG-HRP) was applied, according to the standard, with incubation in an oven at 37 °C. Then, the plates were washed again and incubated with tetramethylbenzidine (TMB) substrate, according to the manufacturer's instructions. The reaction was stopped by adding sulfuric acid and absorbance readings were taken at 450 nm. The data obtained in the ELISA were normalized and subjected to standard curve analysis based on the function "[Agonist] vs. Normalized response", using the GraphPad Prism program (GraphPad Software Inc., San Diego, CA, USA) to obtain the EC50. The specific activity in units / mg from the EC50 (ng / mL) was calculated using Equation 2:. Specific activity (units / mg) = 10 6 units / mg (Equation 2) EC50 (ng / mL)

[0092] The specific biological activity result obtained was equal to 29.10 x 10 4 U / mg.

[0093] Data on aggregates. In order to obtain data on aggregates, the chimeric protein Defined by SEQ ID No. 2 was subjected to size exclusion fast protein liquid chromatography (SE-FPLC) and dynamic light scattering (DLS) assays. The chromatogram in Figure 21, letter A, shows two peaks, both corresponding to the chimeric protein Defined by SEQ ID No. 2. The first population is less frequent in the sample and has 266.43 kDa (approximately 10.5 monomers). The second identified population has 136.07 kDa (approximately 5.4 monomers) and is present in greater quantity in the sample. There is no indication of aggregate formation or protein degradation. The histogram presented in Figure 21, letter B, represents the size distribution of the protein Defined by SEQ ID No. 2 evaluated by DLS.The values ​​for average protein diameter, polydispersity index, and percentage of aggregates obtained were: 7.59 ± 0.09 d.nm; 0.27 ± 0.01 and 0.02 ± 0.01%, respectively.

[0094] Stability. A preliminary stability study was conducted with the protein defined by SEQ ID No. 2 in solution, stored for 30 days in a refrigerator (2°C to 8°C). The results of SDS-PAGE and Western blot (Figure 22), capillary electrophoresis in Bioanalyzer (Figure 23), and in vitro biological activity by ELISA (Figure 24) demonstrated that cleavage products increase as a function of storage time and are recognized by rabbit polyclonal antibody. Furthermore, biological activity and protein purity decrease significantly (Table 6). Table 6. Results of preliminary stability analysis of the protein defined by SEQ ID No. 2 stored for 30 days in solution in a refrigerator (2 °C to 8 °C). Time Criteria Test Method 12 days 15 days 30 days initial acceptance Purity Greater than 99.0 ± 95.3 ± 80.3 ± 62.3 ± (%) 80% 0.9 3.2 2.5 9.5 _ Electrophoresis _ Products capillary in from Less than 1.0 ± 4.7 ± 19.7 ± 37.7 ± Bioanalyzer cleavage 20% 0.9 3.2 2.5 9.5 (%) Activity 29.1 x 1.6 x Not if biological ELISA Informative 10 4 10 4 applies applies in vitro U / mg U / mg

[0095] On the other hand, when the protein is lyophilized, using the vaccine composition described in the present invention, it remains stable for at least 6 months (Table 7). Table 7. Results of preliminary stability analysis of the protein defined by SEQ ID No. 2 stored for 6 months, lyophilized, in a refrigerator (2 °C to 8 °C). Specifications Test interval Time Criteria 2 3 6 Test Method Initial 7 days 15 days 1 month acceptance months months months 31 / 01 / 23 Mass Aspect of Compliance Compliance Compliance Compliance Compliance Compliance Compliance Compliance Local Development white freeze-dried powder the test the test the test the test the test the test the test homogeneous Liquid Aspect after Compliance Compliance Compliance Compliance Compliance Compliance Compliance Compliance Clear local development and reconstitution the test the test the test the test the test the test the colorless test Pharmacopoeia Between 6.0 and 6.48 ± 6.48 ± 6.43 ± 6.44 ± 6.50 ± 6.45 ± 6.48 ± PH Brazilian, 6th edition, 8.0 0.049 0.034 0.066 0.02 0.032 0.015 0.002 2019. General method 5.2.19 Pharmacopoeia Size of Americana, USP43- 23.94± 26.38± 40.75± 25.57± 21.64± 26.7± 34.04± Between 5 and 50 particle F38, 2020. Method 2.56 7.38 8.75 5.00 1.43 10.2 4.34 1430.3. Pharmacopoeia Americana index, USP43- 0.333± 0.361 ± 0.361 ± 0.326± 0.275± 0.295± ​​0.426± Less than 0.7 Polydispersity F38, 2020. Method 0.039 0.027 0.027 0.028 0.006 0.005 0.043 1430.3. Pharmacopoeia American, USP43 - Lower than 16.39± 21.09± 12.35± 9.52± 17.79± 19.08± 3.232± % aggregates F38, 2020. Method 25% 1.54 5.2 3.09 3.69 1.13 4.00 3.5 1430.3. Pharmacopoeia Brazilian Osmolality, 6 a edition, Between 400 and 477, 33± 460± 449± Not applicable Not applicable Not applicable Not applicable (mosmol / kg) 2019. General method 700 5.13 2.65 13.00 applies applies applies applies 5.2.28 Evaluation of products cleavage by Local development Lower than Not applicable Not applicable Not applicable Electrophoresis 0 0 0 Agilent Protein 230 kit 25% applies applies applies applies in Bioanalyzer (%) Identification Identification of two Competes Competes Competes Competes Competes Competes Competes by DSS - Local Development bands of the test the test the test the test the test the test the test EGPA 50kDa Identification Identification by Western - of two Complies Complies Complies Complies Complies Complies Complies Blot in Local Development bands of the test the test the test the test the test the test condition 50kDa denaturant Activity 4.68 x 1.65 x 3.82 x 5.90 x 3.93 x 3.12 x Biological by Local Development Informative 4.7 x 10 5 10 5 10 5 10 5 10 4 10 4 10 4 ELISA Waste Tryptophan structure It is not fulfilled. It is not fulfilled. It is fulfilled. It is fulfilled. It is fulfilled. Tertiary development by local development exposed in the test applies applies the test the test the test the test fluorescence region hydrophilic EXAMPLE 3 - DEVELOPMENT AND PRODUCTION OF THE VACCINE COMPOSITION

[0096] A preliminary study was conducted to evaluate the stability of the protein defined by SEQ ID No. 2 dispersed in phosphate-buffered saline over 15 days, stored in a refrigerator (5 °C ± 3 °C). SDS-PAGE results (Figure 25) showed the formation of three degradation bands. These bands were extracted from the electrophoretic gel mesh for identity assessment by LC MS / MS mass spectrometry (Figure 26). The results of this analysis allowed us to conclude that all peptides identified in the three bands cut from the SDS-PAGE gel (bands 3, 4, and 5) correspond to fractions of the protein defined by SEQ ID No. 2 (bands 1 and 2), and are therefore products of protein cleavage and not contaminated by other possible compounds.Based on this protein stability profile defined by SEQ ID No. 2 in solution, it was determined that the presentation of the malaria vaccine caused by Plasmodium vivax would consist of the lyophilized active pharmaceutical ingredient (API) for reconstitution with the adjuvant at the time of use.

[0097] Production of lyophilized API: For lyophilization of 1 mL of API, the protein defined by SEQ ID No. 2 (0.033 to 0.33 mg / mL) is mixed with a solution containing sucrose (1.65 mg / mL), polysorbate 20 or 80 (0.30 mg / mL), mannitol (33.0 mg / mL), sodium chloride (9.0 mg / mL), monobasic potassium phosphate (0.144 mg / mL), dibasic sodium phosphate heptahydrate (0.795 mg / mL), and water for injection (q.s.p. 1.0 mL). The concentration of sucrose and mannitol in the formulation should be calculated so that the sucrose:protein ratio is 5:1 and the mannitol:sucrose ratio is 20:1. This formulation is packaged in cryoresistant vials and subjected to the following 21-hour freeze-drying cycle: ( / ) freezing stage: -52 °C, cooling rate of 1.8 °C / min for 40 minutes; ( / 7) primary drying stage: first condition -35 °C, 0.1 mbar, 1 h; second condition -35 °C, 0.1 mbar, 8 h; (iii) first condition 15 °C, 0.1 mbar, 2 h; second condition 15 °C, 0.1 mbar, 6 h; third condition: 2 °C, 0.01 mbar, 30 min; fourth condition: 2 °C, 0.01 mbar, 2 h. The results presented in Table 8 show that after lyophilization the protein maintains its physicochemical and structural characteristics and that the lyophilization process was effective in reducing cleavage products, indicating that possibly the cleavage products are generated in the protein in solution due to a hydrolysis process. Table 8. Characterization results of the protein defined by SEQ ID No. 2 in solution and after lyophilization process. Protein Result Technique Protein test in lyophilized milk after analytical analysis. Solution reconstitution with water. Clear liquid. Appearance: Visual inspection. colorless colorless pH Potentiometry 7.44 ± 0.02 7.21 ± 0.03 Zeta potential ELS -4.03 ± 0.53 mV -6.77 ± 1.43 mV Decrease of 321.30 + 2.90 415.67 + 4.93 Osmolality point mOsmol / Kg mOsmol / Kg freezing Average diameter DLS 7.59 + 0.09 d. nm 8.99 + 0.53 d. particle nm index DLS 0.27 + 0.01 0.74 + 0.14 polydispersity Percentage of aggregates DLS 0.02 + 0.01% 1.05 + 0.36% Electrophoresis products 1.00 + 0.95% 0.28 + 0.63% capillary cleavage in Bioanalyzer SDS-PAGE Identification and two bands of two bands of Western-blot in Identification of conditions close to being ... approximately approximately denaturing 50 kDa 50 kDa Activity ELISA 2.91 x 10 5 U / mg 1.65 x 10 5 U / mg biological in vitro Spectroscopy Maximum absorption Maximum absorption Tertiary structure fluorescence at 327 nm at 327 nm

[0098] Adjuvant production: The nanoemulsion with adjuvant properties (CTVadl) is prepared through an initial homogenization of the aqueous phase, composed of sodium citrate and citric acid (10 mM citrate buffer, pH 6.5) and polysorbate 80, and the oil phase, containing squalene and sorbitan trioleate 85. After obtaining a conventional emulsion, the emulsion is homogenized under high pressure (using pressure between 800 and 1500 bar, 3 to 10 cycles) to obtain the nanoemulsion. CTVadl is prepared from the dilution of CTVadl in 10 mM citrate buffer, pH 6.5. CTVadl is used in the vaccine composition consisting of the extemporaneous mixing of the API in solution with the adjuvant. CTVad3 is used in the vaccine composition consisting of lyophilized active pharmaceutical ingredient (API) for reconstitution at the time of use. This is necessary to allow intramuscular administration of the adjuvant at doses between 1 mg and 25 mg of squalene.Table 9 describes the characterization results for CTVadl and CTVad3. Table 9. Characterization results of the CTVadl and CTVad3 adjuvants. Test CTVadl batch 1471 / 23 B CTVad3 batch 1472 / 23 B Appearance White emulsion White opalescent and homogeneous emulsion pH 6.36 + 0.01 6.38 + 0.15 Particle size 174.70 + 1.01 d. nm 174.4 + 1.25 d.nm Polydispersity index 0.074 + 0.016 0.055 + 0.011 Zeta potential -28.03 + 0.28 mV -26.56 + 0.77 mV Viscosity 1.19 + 0.02 Cp 1.17 + 0.01 Cp Osmolality 37.67 + 0.58 mosmol / kg 36.67 + 0.58 mosmol / kg Identification of the main peak retention time of the main peak retention time in the HPLC chromatogram of the sample solution corresponds to that of the standard preparation, both obtained in assay. Assay of 101.15 ± 1.97% 96.88 + 0.49% squalene by RP - 43.50 ± 0.85 mg / mL a 34.68 ±0.17 mg / mL HPLC EXAMPLE 4 - IMMUNOGENICITY ASSAYS USING VACCINE COMPOSITION IN AN EXPERIMENTAL MODEL

[0099] Humoral response to different adjuvants: the state of the art describes a vaccine composition composed of the amino acid sequence defined by SEQ ID No. 2 and Poly-ICLC (document BR 10 2022 005915 2). Knowing that the adjuvant plays a critical role in To induce protective immunity against malaria, we tested other adjuvants besides Poly-ICLC for comparative purposes. These tests are relevant because the efficacy and final cost of the vaccine can vary depending on the adjuvant used. In this sense, C57BL / 6 mice (n=6 / group) were immunized subcutaneously (100 pL / dose) with 10 pg of the protein defined by SEQ ID No. 2, using as adjuvant 50 pg of Poly-ICLC (Hiltonol / Oncovir) or 50 pL of AddaVax (InvivoGen) or 50 pL of IB160 (kindly provided by Dr. Paulo Lee Ho, Butantan Institute) or 70 pL of Montanide ISA 720 (Seppic) or 50 pg of Poly-IC (InvivoGen). This study was conducted at the Animal Facility of the Chemistry Complex of the University of São Paulo (CEUA FCF 74.2016-P531).The results revealed that after 3 doses, with a 14-day interval between doses, all adjuvants tested were effective in inducing high titers of specific PvCSP antibodies at a level consistent with protective immunity (Figure 27).

[0100] Comparison between Poly-ICLC and CTVadl adjuvants: In order to compare the immune response induced by Poly-ICLC (Hiltonol / Oncovir) and CTVadl adjuvants and their combination, an experiment in C57BL / 6 mice (n=6 / group) was performed. The animals were immunized subcutaneously (100 pL / dose) with 3 doses, with a 30-day interval between doses of: ( / ) 50 pL of CTVadl (2.15 mg of squalene); ( / 7) 50 pg of Hiltonol; ( / 77) 10 pg of Defined by SEQ ID No. 2 + 50 pL of CTVadl (2.15 mg of squalene); ( / ) 10 pg of Defined by SEQ ID No. 2 + 50 pg of Hiltonol; (v) 10 pg of Defined by SEQ ID No. 2 + 50 pL of CTVadl (2.15 mg squalene) + 20 pg of Hiltonol; vi) 10 pg of Defined by SEQ ID No. 2 + 50 pL of CTVadl (2.15 mg squalene) + 5 pg of Hiltonol. As represented in Figure 28, animals immunized with the different formulations produce high titers of total anti-PvCSP IgG after the primer, and these levels increase significantly after the Administration of the second boost. The same occurs with the production of total IgG in relation to the reactivity of the different alleles, with P. vivax-like being the variant with the highest reactivity when compared to VK-210 and VK-247, but with lower titers than the PvCSP chimera. Another important point to be observed is the antibody production in the test groups of the CTVadl + Hiltonol adjuvant combination at concentrations of 20 pg and 5 pg, showing that when analyzed with the comparative group Hiltonol at a concentration of 50 pg, both test groups showed equivalent or even better antibody production, proving the same response efficacy with up to 10x less Hiltonol in the formulation.

[0101] The same profile is observed in Figure 29. There was a significant increase in IgG1 antibody production as immunizations were administered, reaching a plateau after the third dose. The PvCSP chimera again showed high titers in all formulation tests, with no significant difference between the groups evaluated. Regarding the variants, there was again a significant increase in antibody production after the three immunization doses, where P. vivax-like showed greater reactivity when compared to VK-21 O and VK-247.

[0102] Figure 30 shows that between the prime and boost doses there was a significant increase in IgG2c antibody production for both PvCSP and variants; however, no significant differences were observed between the first and second boost doses for all conditions tested. Among the analyzed groups, it was concluded that there was no difference in antibody production between the test groups (PvCSP + CTVadl + Hiltonol 20 pg or 5 pg) under all conditions tested, again showing that the group containing 10 times less Hiltonol presented the same efficacy profile when compared. to the group immunized with 50 pg of Hiltonol. Unlike the other immunoglobulins tested, the response of the CTVadl + PvCSP group showed significantly lower titers of IgG2c antibody production when compared to the other vaccinated groups.

[0103] Following this initial study with different adjuvants, it was observed that even when the concentration of Hiltonol was reduced by up to 10 times (from 50 pg to 5 pg), the humoral response efficacy remained without any significant difference between the groups. Based on these data, we conducted a new immunogenicity study to evaluate the reproducibility of the humoral response and now also the cellular response.

[0104] In the new experiment, C57BL / 6 mice (n=6 / group) were immunized intramuscularly (100 pL / dose) with 3 doses, with a 30-day interval between doses: ( / ) 50 pL of CTVadl + 5 pg of Hiltonol; ( / 7) 10 pg of Defined by SEQ ID No. 2 + 50 pL of CTVadl; ( / 77) 10 pg of Defined by SEQ ID No. 2 + 50 pL of CTVadl + 5 pg of Hiltonol. The results revealed that animals immunized with the different formulations produced high titers of total anti-PvCSP IgG after the prime, and these levels increased significantly after administration of the second boost (Figure 31). The same occurs with the production of total anti-variant IgG, with P. vivax-like being the variant with the highest reactivity when compared to VK-210 and VK-247, but with lower titers than those obtained for the chimera defined by SEQ ID No. 2 (Figure 31).

[0105] The same profile is observed in Figure 32. There was a significant increase in IgG 1 antibody production as immunizations were administered, reaching a plateau after the third dose. The PvCSP chimera again showed high titers, with no significant difference between the groups evaluated.

[0106] In Figure 33, the same pattern from the first experiment was observed. The group immunized with the CTVadl + Hiltonol 5 formulation |jg + PvCSP showed a significant increase in the production of immunoglobulin lgG2c, when compared to the group immunized only with CTVadl + PvCSP. This can be justified by the type of Toll receptor 1 response that is triggered by the Hiltonol adjuvant, being mainly involved in the Th1 cellular response.

[0107] The cellular immune response was evaluated by detecting IFN-γ in the culture supernatant of splenocytes from immunized mice stimulated in vitro with the PvCSP chimera. As a negative control, cells were stimulated with RPMI medium, while as a positive control, cells were stimulated with Concanavalin A. As observed in Figure 34, cells from animals immunized with the PvCSP + CTVadl chimera and animals immunized with the combination of adjuvants PvCSP + CTVadl + Hiltonol 5 pg are stimulated to produce IFN-γ when stimulated with the chimera, at two different concentrations of 10 pg and 20 pg, which is not observed with the CTVadl + Hiltonol 5 pg control group. There was no significant difference in IFN-γ production between the groups evaluated with the different adjuvants.

[0108] These studies were conducted at the Animal Facility of the René Rachou Institute, Fiocruz, MG (CEUA LW-31 / 23). EXAMPLE 5 - PROTECTION TEST USING VACCINE COMPOSITION IN AN EXPERIMENTAL MODEL

[0109] This study was conducted in C57BL / 6 mice at the Pasteur Institute (Paris, France) under the approval of the Ethics Committee (CEUA APAFIS#32422-2021071317049057 v2 and APAFIS #32989-2021091516594748 v1). The animals (n=6 / group) were immunized intramuscularly (100 pL / dose) with 3 doses, with a 30-day interval between doses: ( / ) 50 pL of CTVadl; ( / 7) 50 pg of Hiltonol; ( / 77) 50 pg of CTVadl + 5 pg of Hiltonol; ( / ) 10 pg of PvCSP + 50 pL of CTVadl; (v) 10 µg of PvCSP + 50 µg of Hiltonol; (vi) 10 µg PvCSP + 50 µL CTVad1 + 5 µg Hiltonol.

[0110] In order to evaluate the vaccine efficacy of the chimera defined by SEQ ID No. 2, thirty days after the administration of the third dose, the animals were challenged with 5,000 sporozoites of Plasmodium berghei ANKA. The inoculation protocol was via the plantar pad, and from time zero of the challenge, blood collection was performed from the fourth to the tenth day after inoculation to evaluate the percentage of parasitemia in the animals using flow cytometry.

[0111] For parasitemia analysis, a drop of blood from the tail of each animal was collected and diluted in 5,000 pL of 1X PBS. In the cytometer (Cytoflex Daily QC / Fluorospheres Beckman Coulter), a total of 250,000 to 500,000 erythrocytes were examined per sample.

[0112] Based on the results obtained, it was observed that among all the proposed vaccine formulations containing the sequence defined by SEQ ID No. 2, the group of animals immunized with the CTVadl + PvCSP All-epitopes formulation showed 83.3% of uninfected animals after the challenge, that is, significant protection in the detection of circulating sporozoites (Figure 35). The CTVadl + Hiltonol + PvCSP All-epitopes group showed the second best protection, around 66.6% of uninfected animals after the challenge, followed by the Hiltonol + PvCSP All-epitopes group with 33.3% protection. The parasitemia rate evaluated in the animals after the challenge is shown in Figure 36.

Claims

CLAIMS 1. Method for producing an antimalarial vaccine composition characterized by comprising the following steps: a. Clone the gene defined by SEQ ID No. 1 into circular plasmid DNA with the coding sequence of the α-factor secretion signal followed by the restriction enzyme sites SnaB I, EcoR, Avr II, Not I, promoter AOX1, and express the protein SEQ ID No. 2, in a master cell bank of methylotrophic P. pastoris yeast cells, under fermentation conditions of 6 to 50 L of culture; at a temperature of 26 °C to 30 °C, preferably 30 °C; with an induction rate of 0.015 g / ha 0.02 g / h of methanol, preferably 0.02 g / h; concentration of wet cells at the beginning of induction of 120 g / L to 300 g / L, preferably 250 g / L; induction time of 36 h to 72 h, preferably 36 h; use of antifoaming agent; b. Centrifuge the fermentation contents obtained in “a” at a force of 5,000 to 15,000 x g for 15 to 30 minutes, at a temperature of 4 °C to 15 °C, and collect the supernatant; c. Purify the protein present in the supernatant obtained in “b” through at least one pass through a cationic column (volume: 95 ml to 1.4 L; linear velocity: 400 to 600 cm / h; linear gradient with buffer composed of 100 mM to 25 mM succinate, 20 to 25 mM sodium phosphate and 25 mM to 100 mM CAPS, pH 4.0 to 7.0), followed by ultrafiltration for buffer exchange (20 to 25 mM sodium phosphate, pH 4.0 to 7.0), followed by at least one pass through an anionic column (volume: 40 ml to 700 ml; linear velocity: 150 cm / h; elution in fractions with 20 to 25 mM sodium phosphate buffer, pH 4.0 to 7.0) and followed by ultrafiltration for buffer exchange (saline phosphate buffer); d. Solubilize the recombinant chimeric protein defined by SEQ ID No. 2 purified in “c” (10 to 100 pg / dose) (10 to 100 pg / dose) in cryoprotective buffer for lyophilization composed of 50 to 500 pg of sucrose per dose, 1 to 10 mg of mannitol per dose, 9 to 90 pg of polysorbate 20 or 80 per dose, 9 mg of sodium chloride per dose, 0.144 mg of monobasic potassium phosphate per dose, 0.795 mg of dibasic sodium phosphate heptahydrate in 0.3 ml of water for injection per dose, with the sucrose to protein ratio being 1:1, 3:1 or 5:1 and the mannitol to sucrose ratio preferably 20:1; e. Lyophilize the recombinant chimeric protein defined by SEQ ID No. 2 solubilized in the cryoprotective buffer described in “d”, performing freezing (-55 °C to -35 °C) for 1 to 3 h, followed by primary drying (-35 °C to -20 °C) for 1 to 10 h, followed by secondary drying (2 °C to 15 °C) for 1 to 10 h.

2. Antimalarial vaccine composition obtained by the process defined in claim 1, characterized by comprising the recombinant protein defined by SEQ ID N º 2 (10 to 100 pg / dose) lyophilized, reconstituted with the squalene-based adjuvant at the time of use, consisting of 1 mg to 25 mg of squalene per dose; 0.004 pg to 0.1 pg of citric acid per dose; 0.02 ml to 0.5 ml of water for injection per dose; 0.07 pg to 1.4 pg of sodium citrate per dose; 0.01 pg to 3 pg of polysorbate 80 per dose; 0.01 pg to 3 pg of sorbitan trioleate per dose.

3. Antimalarial vaccine composition, according to claim 2, characterized by comprising the composition defined in claim 2 plus 5 to 50 pg of Hiltonol® / dose.

4. Use of the vaccine composition defined in either of claims 2 and 3, characterized by being for the production of an antimalarial vaccine.

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