BR102022005915A2 - PROCESS FOR PRODUCING AND PURIFYING A RECOMBINANT PROTEIN, RECOMBINANT PROTEIN, USE OF THE RECOMBINANT PROTEIN, IMMUNOBIOLOGICAL COMPOSITION, AND USE OF THE IMMUNOBIOLOGICAL COMPOSITION - Google Patents

Abstract

PROCESS FOR PRODUCTION AND PURIFICATION OF A RECOMBINANT PROTEIN, RECOMBINANT PROTEIN, USE OF THE RECOMBINANT PROTEIN, IMMUNOBIOLOGICAL COMPOSITION, AND USE OF THE IMMUNOBIOLOGICAL COMPOSITION The present invention relates to the development of recombinant proteins of allelic variants of the central domain of the circumsporozoite protein (CSP) of Plasmodium vivax used in the preparation of immunological compositions for the manufacture of vaccines capable of preventing malaria caused by P. vivax. Thus, the present invention relates to a process for the production and purification of recombinant protein, as well as the use of the previously obtained recombinant protein to produce an immunobiological composition that will be used to manufacture vaccines to prevent malaria caused by P. vivax .

Description

FIELD OF INVENTION

[001] The present invention falls within the field of Biotechnology, more precisely in the area of Molecular Biology, and refers to the development of recombinant proteins of allelic variants of the central domain of the Circumsporozoite Protein (CSP) of Plasmodium vivax used in the preparation of compositions immunological technologies for the manufacture of vaccines capable of preventing malaria caused by Plasmodium vivax.

BASICS OF THE INVENTION Malaria

[002] Malaria is caused by Plasmodium parasites, which are transmitted by the bite of the Anopheles mosquito in tropical regions. Recent data show that almost half of the world's population is exposed to the risk of this infection (World Health Organization, 2020).

[003] The development of alternative tools to control malaria is increasingly important due to the emergence of drug-resistant parasites and insecticide-resistant mosquitoes. According to the World Health Organization (WHO), in 2019, there were an estimated 229 million cases of malaria and 409,000 deaths caused by malaria worldwide, indicating that the disease continues to be a significant public health concern ( World Health Organization, 2020).

[004] In humans, malaria is caused by five species: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Among these species, most cases of the disease are attributed to P. falciparum and P. vivax. Globally, while P. falciparum is responsible for the most deaths, P. vivax is the most geographically widespread. In 2019, approximately 6.4 million cases of malaria were caused by P. vivax, which occurs mainly in the regions of Southeast Asia, the Western Pacific, the Eastern Mediterranean, and the Americas. Despite this, the development of vaccines against P. vivax malaria is lagging behind P. falciparum (World Health Organization, 2020).

[005] Mortality induced by P. vivax infection is considered very low when compared to P. falciparum infection. However, P. vivax-derived morbidity has increased significantly during recent years, especially due to the development of chloroquine resistance by the parasite. Furthermore, several countries have reported an increase in the number of cases of malaria caused by P. vivax with serious complications, including anemia, respiratory distress syndrome, cerebral malaria, malnutrition and kidney failure. For pregnant women, all forms of malaria, including P. vivax, pose an even greater threat, as malaria in pregnancy is associated with higher levels of parasitemia, increasing the risk of prematurity, miscarriage, low birth weight, death neonatal or maternal (Menkin-Smith and Winders, 2020).

Vaccines

[006] It is common knowledge in the art, the high complexity involved in the development of vaccines targeting parasites, such as those targeting Plasmodium protozoa, the causative agent of malaria.

[007] The most advanced malaria vaccine is MosquirixTM, also known as RTS,S, which consists of a recombinant vaccine produced in Saccharomyces cerevisiae and is based on the circumsporozoite protein (CSP) of P. falciparum. This vaccine was evaluated in a long Phase III trial that lasted five years and showed very promising efficacy results, until, in 2019, three countries in Sub-Saharan Africa - Ghana, Kenya and Malawi - began to lead the introduction of the vaccine in areas selected cases of moderate to high transmission of malaria as part of a large-scale pilot program coordinated by WHO. However, RTS,S is directed at P. falciparum and does not offer any protection against P. vivax, and there are few studies and clinical trials focused on P. vivax, especially in more advanced stages (World Health Organization, 2020; Garcon et al , 2003).

[008] Although studies on P. vivax malaria have been neglected in recent decades, there is a consensus that vaccine development efforts should also be directed to other species of Plasmodium in addition to P. falciparum.

[009] In this context, the development of a vaccine against P. vivax would represent a major advance in malaria control strategies. Thus, considering the promising results of RTS,S, the present inventors and other groups invested in CSP as a target for the development of vaccines targeting P. vivax.

Circumsporozoite protein (CSP) in vaccine development

[010] The first protein associated with effective protection against malaria, the circumsporozoite protein (CSP) is the most abundant antigen on the surface of Plasmodium sporozoites and is involved in the initial stages of hepatocyte invasion in mammalian hosts. Thus, CSP is an important target for antibodies and CD4+ and CD8+ T cells that can eliminate the pre-erythrocytic stages of the parasite (Nussenzweig et al, 1967; Cohen et al, 2010; Mueller et al, 2015; Birkett, 2016).

[011] The RTS,S vaccine comprises the conserved C-terminal domain and the central repeat domain of the P. falciparum CSP (PfCSP), in combination with the hepatitis B virus surface antigen (HBsAg). Expression occurs in the virus-like particle (VLP) assembly that is conjugated to the AS01 adjuvant. Immunological studies carried out 12 months after immunization with RTS,S demonstrated that protection against first or recurrent malaria events was related to the anti-CSP IgG antibodies generated, which targeted the repeat and C-terminal domains (Dobano et al , 2019).

[012] A disadvantage of formulations based on RTS,S is the excessive generation of antibodies directed to HBsAg, which may interfere with the specific efficacy against the circumsporozoite protein (CSP). To avoid this interference, a formulation called R21/Matrix-M was developed, which combines PfCSP with HbsAg monomers and forms VLP. R21/Matrix-M was recently tested in Phase IIb clinical trials, demonstrating high-level efficacy of 77% over 12 months of follow-up (Datoo et al, 2021).

[013] Antibodies against CSP confer protection against controlled human malaria infection (CHMI) caused by the P. falciparum parasite. However, although CSP is highly immunogenic, it does not induce long-lasting protection (Cawlfield et al, 2019).

[014] The P. vivax CSP (PvCSP) comprises a central repeat domain flanked by two conserved non-repetitive sequences, the N-(RI) and C-terminal domains. In contrast to PfCSP, three allelic variants of the central domain of PvCSP are described: VK210, VK247 and "P. vivax-like". These three variants have a worldwide distribution and can be identified by serological and molecular methods (Arnot et al, 1985; Rosenberg et al, 1989; Qari et al, 1993; De Souza-Neiras, 2007; Arruda et al, 2007; Raza et al , 2013; Pereira et al, 2018).

[015] Antibodies directed against the central repeat domain of PvCSP can neutralize the infectivity of sporozoites, allowing the acquisition of sterile immunity. However, strain diversity adds complexity to the development of a multi-allelic vaccine against P. vivax malaria.

[016] PvCSP-derived antigens have been combined in multivalent formulations or chimeric synthetic molecules in an attempt to obtain protective immunity against P. vivax. Recombinant PvCSP-derived proteins expressed in E. coli and yeast have been tested as vaccines with limited success (Collins et al, 1989).

[017] An already known P. vivax malaria vaccine formulation is based on the recombinant protein VMP001, expressed in E. coli bacteria, which contains the repeated sequences of VK210 (nine repeats) and VK247 (one repeat) fused to the regions conserved N- and C-termini of PvCSP. The formulation called VMP001/AS01B, which combines the VMP001 protein with the AS01B adjuvant, was tested in Phase I/IIa clinical trials. The results of these trials showed that VMP001 is immunogenic, inducing humoral and cellular immune responses to the vaccine antigen in all volunteers. A significant delay in the development of parasitemia was also observed in 59% of vaccinated individuals compared to the control group. However, this vaccine did not induce sterile protection against P. vivax in any of the volunteers (Bennet et al, 2016).

[018] Another known P. vivax malaria vaccine formulation is called Rv21/Matrix-M, based on a protein comprising the central repeats of the chimeric VK210 and VK247 variants of PvCSP and the C-terminal sequence of PvCSP fused to the HBsAg antigen, with a four-amino acid sequence added at the C-terminus. The expression of this protein occurs in Pichia pastoris and the adjuvant used in the formulation is Matrix-M (Salman et al, 2017).

[019] However, these vaccines did not consider the three allelic variants of PvCSP (VK210, VK247 and P. vivax-like) that differ in terms of the amino acid sequence of the central protein region, which has B cell epitopes. the VMP001/AS01B and Rv21/Matrix-M vaccines show limited protection for only two of the three strains of P. vivax. Meanwhile, other studies have demonstrated the importance of the three alleles in developing a CSP-based vaccine that could confer worldwide protection against P. vivax malaria (Mueller et al, 2015).

[020] Previous studies were carried out on the expression of recombinant PvCSP proteins in bacterial (E. coli) and eukaryotic (P. pastoris) systems. In these studies, chimeric proteins were expressed by fusing the three central repeat regions of different PvCSP alleles (VK210, VK247 and P. vivax-like), and the adjuvants Poly (I:C) and Montanide were used (Teixeira et al, 2014 ; Gimenez et al, 2017; de Camargo et al, 2018).

[021] Furthermore, it appears that some documents present in the literature propose the production and use of recombinant proteins from P. vivax in the prevention of malaria, for example:

[022] The document in the name of Gimenez et al., entitled Vaccine containing the three allelic variants of the plasmodium vivax circumsporozoite induces antigens protection in mice after challenge with a transgenic rodent malaria parasite, reveals the use of recombinant bacterial and adenoviral proteins based on P. vivax circumsporozoite protein (CSP), demonstrating the possibility of strong immunological responses to each of the three forms of P. vivax. Although chimeric sequences containing the three PvCSP variants have been described in said document, in no case is the specific sequence of the recombinant protein proposed in the present invention described or inferred. Furthermore, it should be noted that the adjuvant used was Poly (I:C), while the adjuvant proposed in the present invention is Poly ICLC. Another difference refers to the purification systems for recombinant proteins. In that document, affinity chromatographies were used using His-trap columns (possibly due to the presence of histidine amino acids in the chimeric proteins described) and ion exchange; using Q-Hi-Trap speakers. However, in the present invention the chimeric proteins lack histidine amino acids at the N- or C-terminus. For purification, a new protocol was established that consisted of three steps of gel-filtration chromatography and exclusion; using Capto MMC, Q HyperD F and Sephacryl S100 HR columns. These combined differences generated more satisfactory results in purity, immunogenicity, stability and toxicity assays; therefore meaning an advantage over the formulations proposed in that document.

[023] The document in the name of Soares et al., entitled Recombinant Plasmodium vivax circumsporozoite surface protein allelic variants: antibody recognition by individuals from three communities in the Brazilian Amazon, refers to the evaluation of the immunogenicity of new recombinant proteins corresponding to each from the three allelic variants of P. vivax (VK210, VK247 and P. vivax-like) and the C-terminal region (shared by all PvCSP variants) to the use of this knowledge to prepare vaccines. Furthermore, recombinant proteins representing the repeat regions and the C-terminal domain of the PvCSP alleles were used to evaluate the seroprevalence of these alleles in the population. These recombinant proteins were generated containing various numbers and sequences of repeats of the PvCSP alleles. Consequently, the proteins generated are completely different from those described in the present invention. Furthermore, proteins were generated using distinct expression systems; the eukaryotic expression system in HEK-293T cells of said document, and Pichia pastoris being used in the present invention. Furthermore, the proteins mentioned in said document contain C-tag amino acids to facilitate purification through affinity chromatography, while in the present invention three steps of gel chromatography-filtration and exclusion were used; using Capto MMC, Q HyperD F and Sephacryl S100 HR columns.

[024] Document US 7150875 B2 discloses the state of the art for the production of a highly purified recombinant protein useful as a diagnostic reagent, for use in the production of antibodies and as a vaccine for P. vivax. The document reveals the variants of the aforementioned Plasmodium. Additionally, the technique describes the obtaining of a P. vivax protein unrelated to PvCSP (PvMSP-1 p42). For this reason, the mentioned Plasmodium variants do not correspond to the PvCSP allelic variants mentioned in the present invention. Furthermore, the expression system (E. coli), purification (Affinity Chromatography) and formulations are different from those proposed. Therefore, there is no direct or indirect relationship between both inventions, as it is a blood stage antigen of the parasite, while the present invention refers to a liver stage antigen.

[025] The document in the name of Teixeira et al., entitled Immunogenicity of a prime-boost vaccine containing the circumsporozoite proteins of plasmodium vivax in rodents, reveals the development of a P. vivax vaccine containing the three circulating allelic forms of P. vivax CSP. The document generates three recombinant bacterial proteins that represent the CSP alleles. Furthermore, mice were immunized with the mixture of recombinant proteins in a formulation containing the poly (I C) adjuvant. Additionally, in that document the antibodies recognized the three allelic forms of CSP, reacted to the repeated and non-repeated regions of CSP and recognized sporozoites expressing the VK210 and VK247 alleles. The recombinant proteins generated contain the N-terminal region of the PvCSP protein, absent in the sequences proposed in the present invention. Furthermore, the order of the repeated region sequences differs from the order proposed in the present invention; Since the proteins mentioned in the aforementioned document are expressed in bacteria, they need to be denatured to be used, and contain histidine amino acids to facilitate purification. All these modifications have a significant impact on the immune response that the proteins generate. Also, the purification system differs from Urea treatment and affinity/ion exchange chromatography in said document to Capto MMC, Q HyperD F and Sephacryl S100 HR columns in the present invention. As a result, the proteins and formulations generated are different from those described in the present invention.

[026] Document BR112018073454 A2 refers to vaccine compositions comprising fusion polypeptides or virus-like particles (VLPs) for use as vaccines for the prevention of malaria. Virus-like particles (VLPs) comprising fusion polypeptides feature selected repeat units derived from the repeat regions of Type I and Type II circumsporozoite proteins (CSP) of P. vivax (Pv), together with an amino acid sequence derived from C-terminal PvCSP sequence. It is also verified that proteins specifically containing surface antigen sequences derived from the Hepatitis B virus (HBV-S), require for the formation of VLPs, in addition to a minimum of 6 units (preferably 8-12) of PvCSP repeat. type I (VK210) and type II (VK247), plus the C-terminal region, in that specific order. In the present invention, 5 repeat units of PvCSP VK247 are used and, however, repeats of the P. vivax-like allele (absent in said document) are found between the VK210 and VK247 repeats. Therefore, the formulation proposed in the present invention has the advantage of considering the three PvCSP alleles, in addition to the differences mentioned.

[027] The document in the name of Atcheson & Reyes-Sandoval, entitled Protective efficacy of peptides from plasmodium vivax circumsporozoite protein, discloses a virus-like particle, Qβ, used as a platform to generate high levels of antibodies against PvCSP peptides. Minimal peptides from the VK210 and VK247 allelic variants of PvCSP are considered highly protective as vaccines. It can be seen that the peptides representing the repeated regions of the VK210 and VK247 variants are exposed separately on the surface of QB particles, used as a platform. Therefore, this document does not describe a chimeric protein containing both variants, nor the third allele contemplated in the present invention (P. vivax-like). Furthermore, proteins were generated using different expression systems; the eukaryotic expression system in HEK-293T cells being used in said document, and Pichia pastoris in the present invention.

[028] The document in the name of Tewari et al., entitled Poly(I:C) is an effective adjuvant for antibody and multifunctional CD4+ t cell responses to Plasmodium falciparum circumsporozoite protein (CSP) and αDEC-CSP in non-human primates, reveals that Poly(I:C) is an effective adjuvant for inducing potent antibodies and Th1 immunity with CSP-based vaccines that offers a potential alternative to existing protein-based vaccines. Furthermore, it reveals studies on the circumsporozoite protein of P. falciparum (Pf) (CSP). It is known that the theoretical action of Poly (I:C) as an adjuvant (increasing Th1 response) is known and in the public domain. However, experimental proof of the specific action of an adjuvant with a certain protein(s) is needed to prove or refute the sought effect. In the aforementioned document, it is considered that the information on a formulation containing PfCSP + Poly-ICLC cannot in any sense be extrapolated to a vaccine against P. vivax; due to the enormous differences between both parasites and the immune response they trigger, in addition to the sequences of the CS proteins being completely different.

[029] The document on behalf of Gimenez et al., entitled A universal vaccine candidate against Plasmodium vivax malaria confers protective immunity against the three PvCSP alleles, reveals the results and provides information on the development of a vaccine that targets multiple strains of P. vivax. After the fusion of two recombinant proteins representing three allelic variants of PvCSP (VK210, VK247 and P. vivax-like) to the mumps virus nucleocapsid protein, the protective efficacy in mice challenged with transgenic P. berghei parasites expressing allelic variants of PvCSP. Formulations containing Poly (I:C) as an adjuvant produced high and long-lasting IgG antibody titers specific to each PvCSP allelic variant. Although chimeric sequences containing the three PvCSP variants have been described in that document, the presence of the mumps virus nucleocapsid protein completely modifies the structure of the recombinant protein and, therefore, the type of antigen presentation and the antibodies that are generated are many different. Furthermore, the specific sequence of the recombinant protein proposed in the present invention is not described or inferred. It should also be noted that in the aforementioned document the adjuvant used was Poly (I:C), however the adjuvant proposed in the present invention is Poly-ICLC. Another difference concerns the purification systems for recombinant proteins. In that document, affinity chromatographies were used using His-trap columns (possibly due to the presence of histidine amino acids in the chimeric proteins described) and ion exchange; using Q-Hi-Trap speakers. However, in the present invention the chimeric proteins lack histidine amino acids at the N- or C-terminus. To proceed with the purification, three steps of gel-filtration and exclusion chromatography were used; using Capto MMC, Q HyperD F and Sephacryl S100 columns.

[030] Document WO2013142278 discloses vaccine compositions against P. vivax comprising immunogenic recombinant proteins, including a recombinant protein comprising the fusion of the three central repeat regions of the different PvCSP alleles: VK210, VK247 and P. vivax-like. Expression occurs in bacteria, preferably E. coli, and the preferred adjuvant is Poly (I:C). However, the production and purification processes of such proteins produced by bacteria were quite complex, requiring the recovery of insoluble fractions and treatment with urea to obtain the recombinants. Another important consideration is the need to reduce the potential toxicity of the final product, ensuring the total absence of endotoxins (LPS) and urea, in order to favor large-scale production (Teixeira et al, 2014). Furthermore, it appears that the proteins disclosed in the aforementioned document would require structural modifications to allow the effective production of soluble proteins and subsequent purification in the absence of histidine amino acids.

[031] In this sense, unlike the solutions found in the prior art, based on the simple absence of histidine amino acids at the C and N-terminal ends, the recombinant protein proposed in the present invention presents more satisfactory results in tests of purity, immunogenicity, stability and toxicity. Specifically, the total absence of histidine at the C-terminal end of the protein generates results superior to those presented in the prior art. Furthermore, the proposed immunological compositions presented the unexpected technical effect of generating a longer-lasting immune response when compared to the already known CSP-based recombinant proteins, with regard to the prevention of malaria caused by P. vivax, in addition to providing greater toxicity. low.

SUMMARY OF THE INVENTION

[032] The present invention aims to propose the development of recombinant proteins from allelic variants of the central domain of the circumsporozoite protein (CSP) of P. vivax used in the preparation of immunological compositions for the manufacture of vaccines capable of preventing malaria caused by P. vivax .

[033] Thus, the present invention primarily proposes a process for producing and purifying recombinant proteins that comprises the steps of: fusion of regions (i) a conserved region I (RI) of the circumsporozoite protein (CSP) of P. vivax; (ii) three central repeat regions from three different alleles (VK210, VK247 and P. vivax-like) of P. vivax CSP; (iii) the C-terminal domain region of PvCSP; and the purification of said proteins on a chromatographic column.

[034] Additionally, the present invention proposes recombinant proteins of allelic variants of the circumsporozoite protein (CSP) of P. vivax. Said recombinant proteins comprise three central repeat regions from different alleles (VK210, VK247 and P. vivax-like) of P. vivax CSP (PvCSP), are completely devoid of histidine at the C and/or N-terminal ends and, preferably expressed in Pichia pastoris yeast.

[035] Furthermore, the present invention proposes an immunobiological composition comprising said recombinant proteins and an adjuvant, preferably the Poly-ICLC adjuvant.

[036] Finally, the present invention proposes the use of recombinant proteins for the production of the immunobiological composition, as well as the use of the immunobiological composition for the manufacture of vaccines to prevent malaria caused by P. vivax.

DESCRIPTION OF FIGURES

[037] The following Figures are part of the present report and are included here in order to illustrate certain aspects of the invention.

[038] Figure 1 shows the time course of fermentation by SDS-PAGE. Culture supernatant samples were collected at the indicated times post-methanol induction and size-fractionated by 4%-12% PAGE, stained with Coomassie Blue, destained, and scanned. Comparable volumes of supernatant were loaded for each time point.

[039] Figure 2 shows the chromatogram and SDS-PAGE of the MMC column. Pooled product samples were collected and size fractionated by 4%-12% PAGE, stained with Coomassie Blue, destained and scanned. The amount of protein loaded into each channel is indicated.

[040] Figure 3 shows the chromatogram and SDS-PAGE of the Q HyperD F column. Pooled product samples were collected and size fractionated by 4%-12% PAGE, stained with Coomassie Blue, destained and scanned. The amount of protein loaded into each channel is indicated.

[041] Figure 4 shows an enlargement of the SDS-PAGE of the QHD column shown in Figure 3 to highlight the double band of the product.

[042] Figure 5 shows the Sephacryl S100HR chromatograms and SDS-PAGE. Pooled product samples were collected and size fractionated by 4%-12% PAGE, stained with Coomassie Blue, destained and scanned. The amount of protein loaded into each channel is indicated.

[043] Figure 6 shows the SDS-PAGE of the raw material. A sample of the raw material was freshly thawed or stored for 14 days and prepared for size fractionation by 4%-12% PAGE, stained with Coomassie Blue, destained and scanned. The amount of protein loaded into each channel is indicated.

[044] Figure 7 shows a Western Blot of the raw material. The raw material was fractionated by size by 4%-12% PAGE, transferred to PVDF membrane and incubated with mouse anti-CSP Ab 1°, washed with TBS, incubated with mouse anti-IgG Ab 2° conjugated with HRP, washed and developed using colorimetric TMB to visualize CSP. The amount of protein loaded into each channel is indicated.

[045] Figure 8 shows the intact mass spectroscopy of the raw material. The feedstock was desalted and stored for approximately four days at 4°C prior to electrospray ionization time-of-flight (ESI-TOF) mass spectroscopy analysis.

[046] Figure 9 shows the SDS-PAGE of the stability study. Raw material samples taken at the indicated time interval were prepared for size fractionation by 4%-12% PAGE, stained with Coomassie Blue, bleached and scanned. The amount of protein loaded is shown.

[047] Figure 10 shows the results of the immunogenicity assay described in Example 2, for days 42, 63 and 116. Comparison between the three graphs shows that there was a significant increase in antibody titers in each animal in Groups 2 and 3 after the third dose.

DETAILED DESCRIPTION OF THE INVENTION

[048] The present invention refers to the development of recombinant proteins from allelic variants of the central domain of the circumsporozoite protein (CSP) of Plasmodium vivax used in the preparation of immunological compositions for the manufacture of vaccines capable of preventing malaria caused by Plasmodium vivax.

[049] Thus, in a first embodiment, the present invention refers to a process for producing and purifying a recombinant protein comprising the steps: (a) fusing the following regions: (i) a conserved region I (RI) of circumsporozoite protein (CSP) of P. vivax; (ii) three central repeat regions from three different alleles (VK210, VK247 and P. vivax-like) of P. vivax CSP; and (iii) the C-terminal domain region of PvCSP; (b) purify the recombinant protein through the following steps in a chromatography column: (b.i) filter, dilute or adjust a solution comprising the recombinant protein; (b.ii) loading the solution obtained in step (b.i) into a first chromatography column; (b.iii) subject the product from step (b.ii) to ultrafiltration; (b.iv) loading the product resulting from step (b.iii) into a second chromatography column; (b.v) subjecting the product from step (b.iv) to ultrafiltration; (b.vi) loading the product resulting from step (b.v) into a third chromatography column; and (b.vii) subjecting the product from step (b.vi) to ultrafiltration.

[050] Wherein preferably the first chromatography column is Capto MMC; the second chromatography column is Q HyperD F; and the third chromatography column is Sephacryl S100 HR.

[051] Furthermore, it is noteworthy that the recombinant protein produced is expressed in Pichia pastoris and is partially or completely devoid of histidine at the C and/or N-terminal ends. Preferably, said recombinant protein is expressed in Pichia pastoris and is completely devoid of histidine at the C-terminal end.

[052] Thus, in a second embodiment, the present invention refers to the recombinant protein produced comprising the sequence SEQ ID No: 1.

[053] In a third embodiment, the present invention relates to an immunobiological composition comprising the recombinant protein as previously described and an adjuvant selected from the group consisting mainly of Poly-ICLC, Montanide ISA 720, Addavax, MF59, MPLA, Matrix- M, CPG-ODN and Aluminum Hydroxide, preferably Poly-ICLC.

[054] Poly-ICLC adjuvant (Hiltonol®), or polynosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose, is a synthetic double-stranded ribonucleic acid (dsRNA) viral mimic therapeutic “host-directed” and molecular pattern pathogen-associated (PAMP), with broad innate and adaptive immune boosting effects, vaccine adjuvant, antiviral and antiproliferative. They are mediated partially by their induction of a “natural mix” of interferons (IFNs), cytokines and chemokines, activation of natural killer cells, myeloid dendritic cells via TLR3 and MDA5, TCD4 and TCD8 cells, and activation of several cytoplasmic and nuclear enzyme systems (oligoadenylate synthetase, dsRNA-dependent protein kinase PKR, RIG-I Helicase and MDA5) that are involved in host antiviral and antitumor defenses. Said adjuvant also has other broad genetic regulatory actions.

[055] It is known that Poly-ICLC has been developed for the treatment of various disease indications in humans, mainly malignant gliomas, cancer, multiple sclerosis and HIV/AIDS, presenting satisfactory results regarding tolerance in most trials. For example, Poly-ICLC is being used as a PAMP adjuvant for several cancer vaccine platforms in nearly three dozen ongoing or recently completed Phase I/II clinical trials in patients with glioma, hepatoma, lymphoma, pancreas, prostate, ovary, breast, colon and cervical. More than 500 patients were safely treated in these studies.

[056] Furthermore, Poly-ICLC was used intranasally or mucosally in extensive studies of formal preclinical toxicology and viral challenge in mice and monkeys without significant adverse effects recorded at the clinical doses in use. Murine studies have revealed marked protection against otherwise lethal challenge with influenza viruses, pandemic influenza, SARS, Ebola viruses, inhalational anthrax, RSV, arboviruses, and others.

[057] Low-dose Poly-ICLC administered IM, IV, IN, IT or SC has been well tolerated in several clinical trials of patients with various types of cancer and in normal volunteers. Several additional studies have used it in combination with various cancer or HIV vaccines. The most common adverse events experienced in these studies were temporary discomfort at the injection site and temporary feverish malaise or flu-like symptoms. Occasionally, patients on low dose Poly-ICLC have developed a flu-like syndrome with high fever, chills, and malaise. This syndrome usually resolves within 12 to 24 hours, responds to acetaminophen, and typically does not recur on subsequent dosing. Subcutaneous dosing, alone or with vaccine, may also result in transient erythema at the injection site.

[058] Finally, in a fourth and fifth embodiments, the present invention refers to the use of the recombinant protein for the production of the immunobiological composition, as well as the use of the immunobiological composition for the manufacture of vaccines for the prevention of malaria caused by P. vivax .

[059] To allow a better understanding of the present invention and clearly demonstrate the technical advances obtained, the results of the different pre-clinical trials carried out are presented below, as examples.

EXAMPLES OF THE INVENTION

[060] The following examples are intended only to illustrate, without limiting the scope of the present invention. Any variations capable of being carried out by a person skilled in the art must be considered to be within the scope, scope and scope of the present invention.

Example 1: Expression in Pichia pastoris Fermentation of yeast in a bioreactor

[061] Growth in culture flasks was conducted in two steps to bring the culture to the desired cell density for inoculation in the fermentor. The 250 mL initial culture flask contained 50 mL of BMGY medium (buffered media glycerol yeast extract) and was inoculated with a colony from an agar plate. The culture was incubated for 24 hours at 28°C and 250 rpm. When it reached OD600 > 2, 1 mL of culture was transferred from the 50 mL flask to 600 mL of BMGY in a 2 L flask with a 2 L crown. The culture was then incubated for 24 hours at 28°C and 250 rpm. Once the culture reached OD600 > 5, the entire contents were transferred to a sterile 1 L glass flask equipped with silicone tubing. The entire contents of the glass flask were used to inoculate a fermenter containing 10 L of basal saline medium supplemented with 4.35 mL/L of PTM1 saline and 5% of the antifoaming agent KFO673. Before inoculation, the basal salt medium (BSM) was sterilized at 122°C for 30 minutes and 200 rpm by steam-in-place (SIP).

[062] Fermentations were conducted at 30°C with the air flow set at 10 L/min and the pressure set at 0.20 bar. The pH during the production phase was maintained at 5.0 using 28-30% ammonium hydroxide and the dissolved oxygen was maintained at 40% with agitation set at a cascade of 400 - 1200 rpm and oxygen set at a cascade of 1 - 30 L/min. When a peak in OD was observed, glycerol feeding was started with a 63% (w/v) glycerol solution at a rate of 20 g/L/h. After one hour, the glycerol feeding rate was reduced from 20 g/L/h to 0 g/L/h over three hours and induction was started with the amount of 1.5 mL/L methanol.

[063] Methanol feeding was started as soon as the initial amount of methanol was consumed. Methanol feeding consisted of methanol supplementation in 12 mL/L of PTM1 salts. The methanol feeding rate was increased exponentially using a growth rate of 0.02 h-1 and a consumption rate of 0.014 gmethanol/h*gPichia. Induction was conducted for 72 hours. The optical density of the fermented culture was determined using the Carry 50 spectrophotometer at a wavelength of 600 nm (OD600) in a cuvette with an optical path length of 10 mm. The optical densities of the samples over the fermentation time were obtained by subtracting the OD600 of the medium from the OD600 of the culture using water as a control. SDS-PAGE of samples over time was presented in Figure 1.

Purification and characterization of the protein expressed in Pichia pastoris

[064] The objective of purification was to produce at least 30 mg of purified P. vivax circumsporozoite protein antigen expressed in Pichia pastoris and at least 95% purity by SDS-PAGE. For this, a purification process based on three columns was used, the steps of which are described below.

[065] Dilution of the supernatant: the process began by removing 200.4 mL of the frozen supernatant (Lot USP-FDL-001). The supernatant was thawed over a period of two hours at room temperature. This was then adjusted by adding 1400 mL of water for injection (WFI; 7mL/mL supernatant), 2000 mL of 100 mM succinate, pH 4.0 (10mL/mL), and 400 mL of 2M sodium chloride (2mL/mL ) and left to mix for approximately five minutes. The resulting solution was not filtered again, as it was clear and had been previously filtered during collection.

[066] Capto MMC: the adjusted supernatant was loaded onto a 103 mL Capto MMC column (GE Healthcare, 17-5317) (2.6 x 18 cm) that was previously cleaned with 1N sodium hydroxide and equilibrated with 3 volumes of column (CV) of 100 mM sodium succinate, 25 mM sodium phosphate, 25 mM CAPS, at pH 4.0 at 600 cm/h (53 mL/min). All steps for the Capto MMC column were performed at 600 cm/h.

[067] All chromatography and ultrafiltration steps were carried out at room temperature (approximately 19 °C), unless otherwise indicated.

[068] After loading, the column was washed with 5 CV of 100 mM sodium succinate, 25 mM sodium phosphate, 25 mM CAPS, at pH 4.0. The bound product was eluted by a 20 CV linear gradient going from 100 mM sodium succinate, 25 mM sodium phosphate, 25 mM CAPS, at pH 4.0 to 25 mM sodium succinate, 25 mM sodium phosphate, CAPS 100 mM, 2M sodium chloride, at pH 10.0. Once UV280 began to increase, four fractions were collected until UV280 returned to baseline.

[069] The fractions were analyzed via BCA, SDS-PAGE and RP-HPLC. Based on BCA and SDS-PAGE, fractions 1 and 2 were pooled and resulted in an MMC pool volume of 1000 mL. The MMC pool was stored overnight at 4°C.

[070] UFDF#1: The MMC pool was concentrated to approximately 1 mg/mL and diafiltered with five volumes of 20 mM sodium phosphate, 2 mM EDTA, at pH 7.0. Concentration was performed using 2 x 0.1 m2 5 kDa Biomax membranes (Millipore, P2B005A01) with inlet/outlet pressure settings of 42 and 30 psi, respectively. Before use, the system was cleaned with 0.5 N sodium hydroxide and then equilibrated with 20 mM sodium phosphate at pH 7.0. The resulting 300 mL of retentate had a pH of 6.9 and a conductivity of 3.2 mS/cm. No precipitation was observed during this stage of the process. The retentate was stored overnight at 4°C.

[071] Q HyperD F: The UFDF#1 retentate was loaded onto a 42 mL Q HyperD F column (Pall, 20066-023) (2.6 x 8 cm) that was previously cleaned with 1N sodium hydroxide/sodium chloride 1M sodium and equilibrated with 1 CV of 20 mM sodium phosphate, 1M sodium chloride, at pH 7.0 followed by 3 CV of 20 mM sodium phosphate, pH 7.0 at 150 cm/h (13 mL/min) . All steps for the Q HyperD F column were performed at 150 cm/h. CSP does not bond with this resin, so all material flowing through the resin was collected as product. After loading, the column was washed with 5 CV of 20 mM sodium phosphate at pH 7.0. Material was collected until UV280 returned to baseline. The flow-through product (296 mL) was stored overnight at 4 °C. The resin was regenerated by eluting binding contaminants with 5 CV of 20 mM sodium phosphate, 1 M sodium chloride, at pH 7.0.

[072] UFDF#2: The flow product (296 mL) through the Q HyperD F column was concentrated using a 5 kDa Pellicon XL membrane (Millipore, PXB005A50) to 60 mL. Before use, this system was cleaned with 0.5N sodium hydroxide and then equilibrated with 20 mM sodium phosphate, 150 mM sodium chloride, at pH 7.4. No precipitation was observed.

[073] Sephacryl S100 HR: Before use, a 1374 mL Sephacryl S100HR column (GE Healthcare, 17-0612) (50 x 70 cm) was cleaned with 0.2N sodium hydroxide and equilibrated with 2 CV of sodium phosphate. 20 mM sodium, 150 mM sodium chloride, at pH 7.4 run at 40 cm/h (13 mL/min). All processing with this column was performed at this flow rate. A 148 mL aliquot of the flow through Q HyperD F (<4% CV) was loaded directly onto the column. As UV280 began to increase, three fractions were collected. The product eluted as two peaks, fraction 1 and fraction 2. A second cycle was performed and the corresponding fractions from each cycle were pooled and stored frozen at -80 °C.

[074] UFDF#3: a pool of the two product peaks was made, which were analyzed as a single product. Fractions were thawed and pooled to a total volume of 475 mL. This material was then concentrated to approximately 1 mg/mL using the same 2 x 0.1 m2 5 kDa membrane system used for UFDF#1 after cleaning with 0.5 N sodium hydroxide and then equilibration with sodium phosphate. 20 mM, 150 mM sodium chloride, at pH 7.4.

[075] Bulking: All open manipulations of the product were performed within a Class 100 biosafety cabinet. The retentate was 0.2 μm filtered with an Acropak 20 filter (Pall, 12201) using a sterile 60 mL syringe into sterile containers of 15 mL. The filtered material was divided into aliquots of approximately 5 mL in 39 polypropylene tubes (15 mL, Falcon, 352097) and stored at -80 °C. A mass balance of the raw material shows a total fill of 195 mL. Raw material testing included endotoxin (LAL), bioburden, host cell protein, SDS-PAGE, BCA and intact mass spectroscopy.

[076] Figure 1 shows a time course of fermentation showing that the product was present in the cells.

[077] The expression of CSP in Pichia pastoris was sufficient to generate the amount of material required. From 200 mL of supernatant, 153 mg of purified protein was produced (0.765 mg CSP/mL of supernatant). Processing through the first three columns took four days with overnight holding steps stored at 4°C. Yields were better than expected as the process was not optimized.

[078] The MMC column was able to remove most of the contaminating host cell proteins as shown in Figure 2. This column uses ionic interaction and hydrophobic interaction to separate proteins. Before performing the size exclusion chromatography step, the pooled product was concentrated to approximately 5 mg/mL to minimize loading volume.

[079] Figure 3 presents the results of the Q hyperD F column. This step is performed to remove the remaining higher molecular weight impurities. A significant amount of CSP is lost during this step and would be a focus for future optimization. It is evident at this point in the purification that two protein bands are present in the pooled product. It also appears that the smaller protein of the two may bind to the Q column more than the larger protein, see Figure 4.

[080] After passing through the first three columns, the product eluted in size exclusion HPLC as two peaks. Two cycles were performed for the size exclusion column and both chromatograms look very similar (Figure 5). The two fractions were analyzed using intact mass spectroscopy to determine whether they were suitable for use. Both were considered acceptable and were placed in a pool.

[081] Table 1 below brings together the purification results for each stage of the process, while Table 2 shows raw material analysis results. Table 1 *Purity based on densitometry with load > 6μg per channel, Coomassie dye. Table 2

[082] A total of 153 mg of purified CSP (98% purity) was obtained from 200 mL of Pichia pastoris supernatant. Analytical testing of the purified protein showed that the product had low endotoxin contamination (Table 2). During raw material testing, a sample that was left at 4°C for approximately 1 week exhibited degradation (Figure 6). A stability study at 4 °C was performed to confirm this observation and evaluate degradation over time (Figure 9). The product showed signs of degradation after one day and slow but consistent degradation continued resulting in two fragments of approximately 25 kDa and 10 kDa. Additional process development is needed to improve overall yield and to remove the putative protease that causes degradation. It should be noted that mass spectroscopy was performed after 4 days of storage at 4 °C before carrying out the stability study. Little or no degradation was observed when the product was stored at -80 °C.

Example 2: Immunogenicity after subcutaneous injection of the Plasmodium vivax CSP vaccine in male and female C57BL/6 mice

[083] The objective of the assay was to evaluate the antibody response after the administration of three doses of the immunobiological composition described in the present invention by subcutaneous injection in C57BL/6 mice. The adjuvant selected in this trial was Poly-ICLC. C57BL/6 mice were selected because they represent a suitable and well-established model to evaluate the immune response to a specific vaccine and to provide a prediction of the response in humans.

[084] Animals were initially accepted into the randomization pool based on pre-study body weights and physical examinations. Animals were assigned to study groups using computer-generated random numbers so that the mean body weight of each group was not statistically different (p < 0.05) from the control mean. Males and females were randomized separately and each study animal was assigned a unique number. Sixty C57BL/6 mice (30/sex) were randomly assigned to 3 groups (10 animals/sex/group). At the first dose, all mice were between 7 and 8 weeks old; males weighed between 21.1 and 26.9 g and females weighed between 17.4 and 20.8 g.

[085] Table 3 below shows information on the tested protein, adjuvant and diluent used in the composition. The P. vivax CSP protein and adjuvant were considered 100% pure for formulation purposes. Table 3

[086] The adjuvant was supplied at a concentration of 2 mg/mL. Adjuvant dilutions were made with sterile PBS using aseptic procedures to provide concentrations of 0.5 mg/mL for Group 1 and 1 mg/mL for Groups 2 and 3 on each dosing day. The compositions were kept on wet ice during dosing.

[087] The compositions were prepared on each dosing day. Stock P. vivax CSP at 0.8 mg/mL was thawed at room temperature and diluted with PBS to give concentrations of 100 μg/mL and 200 μg/mL for Groups 2 and 3, respectively. Each of these was mixed 1:1 (v:v) with the adjuvant at a concentration of 1 mg/mL to prepare final dosage solutions for administration to Groups 2 and 3, respectively. After preparation, the compositions were placed on wet ice and used within 4 hours.

[088] The animals were administered with adjuvant (Poly-ICLC) at 50 μg/dose or CSP vaccine P. vivax/Poly-ICLC at 5/50, or 10/50 μg/dose by subcutaneous injection in the right flank on days 1 and 43 and on the left flank on the 22nd, totaling 3 doses. Table 4 shows the designation of the groups and respective doses applied. Table 4

[089] Blood samples were collected on days 42 and 63, on which the animals were not fasting for collection. Final blood was collected on day 116 under 70% CO2/30% O2 inhalational anesthesia. Immediately after final blood collection, while under anesthesia, animals were sacrificed by cervical dislocation and discarded without necropsy.

[090] To assess immunogenicity, serum IgG responses to CSP antigen were determined in male (M) and female (F) C57BL/6 mice immunized by subcutaneous injection with purified proteins in combination with Poly-ICLC adjuvant compared to groups who received only the adjuvant. From blood collected on days 42, 63 and 116, serum was prepared and stored frozen at -75 ± 15 °C until sent to the analysis site, where it was stored at -20 °C until analysis. The antibody titer to the homologous protein was determined by ELISA, and the results were expressed as mean IgG titers (log10 ± SEM). The results show that there was an increase in titers in each animal in Groups 2 and 3 after the third dose (Figure 10), with the exception of two females in Group 2. The high antibody titers lasted for a period of 116 days after the first immunization . No statistically significant difference (P > 0.05) was observed between the groups receiving the antigen in combination with the adjuvant on day 42, 63, or 116, indicating that mice vaccinated with 5 or 10 μg/dose responded similarly. Likewise, there was no significant difference between the sexes in response.

[091] During the trial, electronic data collection, including randomization, dose formulations and dispensing, dosing, animal husbandry, environmental enrichment, physical examinations, cageside observations, body weights, and body weight changes was performed using ProvantisTM Version 8 (Instem LSS, Limited; Stone, UK). Environmental monitoring of the animals was also performed throughout the trial using the Rees Environmental Monitoring System (Rees Scientific Corporation; Trenton, NJ).

[092] The data collected also shows that administration of the P. vivax CSP vaccine at doses up to 10/50 μg/dose was well tolerated and had no effect on mortality, physical examinations, cage observations, body weights or weight changes body.

Example 3: Immunogenicity after subcutaneous injection of Plasmodium vivax CSP vaccine in male and female New Zealand white rabbits

[093] The objective of the trial was to evaluate the antibody response after the administration of four doses of the immunobiological composition described in the present invention by subcutaneous injection in New Zealand rabbits. The adjuvant selected in this trial was Poly-ICLC.

[094] The vaccine was administered four times by subcutaneous injection into male and female New Zealand white rabbits, followed by a recovery period of 21 days. A complete interim necropsy of the first five animals/sex in each group was performed on day 31. At the conclusion of the treatment period, animals assigned to the main phase (five animals/sex/group) were necropsied on day 45, two days after last injection. Animals assigned to the recovery phase were maintained for a 21-day interval without treatment to assess the persistence or reversibility of any observations. The animals received the adjuvant (Poly-ICLC, 50 μg/dose) or the CSP P. vivax/Poly-ICLC vaccine (10/50 μg/dose) by subcutaneous injection with a needle and syringe into the skin of the right and left flanks in the days 1, 15, 29 and 43. Animals underwent a full necropsy on day 31 (provisional), 45 (terminal) or 63 (recovery).

[095] Sixty (30/sex) New Zealand White rabbits were randomly assigned to two groups. Tables 5 and 6 show the designation of the groups and respective doses applied. Table 5 M = males; F= females. Table 6 M = males; F= females. *The animal was moved to the provisional necropsy group as per the veterinarian's recommendation due to a skin abrasion on the right hip.

[096] Serum IgG responses to the P. vivax CSP vaccine were determined in male and female New Zealand white rabbits immunized by subcutaneous injection with the vaccine in combination with the adjuvant and in animals that received the adjuvant alone. Blood was collected before the first dose and on days 14, 28, 31, 42, 45, and 63. Blood was processed into serum and serum was stored frozen at -75 ± 15°C. Samples were sent to the analysis site on dry ice and stored at -20 °C until analyzed. The antibody titer to the homologous protein was determined by ELISA. The results are expressed as IgG titers in table 7.

[097] After the third dose, high titers of IgG antibodies against the P. vivax CSP vaccine were detected in 13 males (out of 15) and 14 females (out of 15) in animals immunized with the vaccine plus the adjuvant . As expected, no IgG antibodies against the vaccine were detected in the groups that received only adjuvant. Table 7 n/a - does not apply.

Example 4: Systemic and local toxicity following subcutaneous injections of Plasmodium vivax CSP vaccine in male and female C57BL/6 mice, followed by a 21-day recovery period

[098] The objective of the trial was to evaluate the potential local and systemic toxicity of the vaccine when administered four times by subcutaneous injection in male and female C57BL/6 mice, followed by a recovery period of 21 days. The adjuvant selected in this trial was Poly-ICLC.

[099] High titers of IgG antibodies against the P. vivax CSP vaccine (test article) were detected after the third dose in all groups immunized with the vaccine plus adjuvant.

[100] Significant increases in body temperatures occurred in Group 2 males 6 hours after dosing on days 1 and 15 and in Group 2 females 24 hours after the final dose. However, these effects were not considered adverse due to the magnitude of the increase and rapid resolution.

[101] At terminal sacrifice on day 45, males and females administered the test article/adjuvant and adjuvant alone exhibited minimal to moderate mixed infiltration at the injection sites. Test article/adjuvant administration was associated with an increase in the incidence and severity of mixed infiltrates at injection sites compared to animals receiving adjuvant alone. Minimal to mild mononuclear infiltration was also identified at the injection sites of males receiving the test article/adjuvant and adjuvant only on day 45, at similar levels of incidence and severity.

[102] Females receiving the test article/adjuvant had a decrease in the number of red blood cells, percentage hematocrit, and hemoglobin concentration with an increase in the breadth of red blood cell distribution; a decrease in platelet count and an increase in mean platelet volume; and an increase in monocyte count. Monocyte counts were also increased for males receiving the test article/adjuvant. The increase in monocyte count microscopically correlated with and was attributed to an increase in the incidence and severity of mixed infiltration at injection sites on day 45. Decreased platelet counts in females receiving the test article/adjuvant may also be related to the increased incidence and severity of mixed infiltration at injection sites, due to the role of platelets in the inflammatory response. The increase in mean platelet volume indicates a regenerative response to the decrease in platelet count. Changes in red cell parameters were attributed to mild suppression of erythropoiesis secondary to increased monocyte production in the bone marrow.

[103] Spleen weights were increased for males and females given the test article/adjuvant on day 45, without a microscopic correlate. Increased spleen weight may be a response to stimulation of the immune response by vaccination. Thymus weights were decreased for both males and females given the test/adjuvant article on day 45, without a microscopic correlate. Decreased thymus weights may be a response to stress associated with mixed infiltrates at injection sites. All results at day 45 were consistent with the expected response to vaccination and none were considered adverse.

[104] At recovery sacrifice on day 63, minimal to mild mononuclear infiltrates were identified at the injection sites of males and females given the test article/adjuvant and adjuvant only at similar levels of incidence and severity. Mononuclear cell infiltrates were considered the chronic/resolving phase of the response to injections. A slight decrease in the number of red blood cells, hematocrit percentage and hemoglobin concentration persisted for females. However, changes compared to animals receiving adjuvant alone were smaller on day 63 than on day 45, indicating reversal of the suppression of erythropoiesis. Complete resolution of reductions in circulating red blood cell mass is expected to occur within a short period of time.

[105] In conclusion, under the conditions of this study, repeated subcutaneous administration of the P. vivax CSP/Poly-ICLC vaccine at 10/50 μg/dose to C57BL/6 mice was well tolerated. All observations were considered non-adverse and consistent with the expected inflammatory response and immune stimulation following vaccine administration.

Example 5: Systemic and local toxicity following subcutaneous injections of Plasmodium vivax CSP vaccine in male and female New Zealand white rabbits followed by a 21-day recovery period

[106] The aim of the trial was to evaluate the potential local and systemic toxicity of the vaccine when administered four times by subcutaneous injection in male and female New Zealand white rabbits, followed by a 21-day recovery period. The adjuvant selected in this trial was Poly-ICLC.

[107] After the third dose, high titers of IgG antibodies against the P. vivax CSP vaccine were detected in 13 males (15 total) and 14 females (15 total) in animals immunized with the vaccine plus adjuvant.

[108] Administration of the P. vivax CSP/Poly-ICLC vaccine resulted in minimal to moderate edema and erythema in both groups 2-4 days after each dose and severe erythema in one Group 2 male three days after the first dose on day 19. The incidence and severity were higher in animals that received the vaccine/adjuvant, but were not adverse due to rapid resolution after dosing. Administration of the P. vivax CSP/Poly-ICLC vaccine also resulted in an increase in body temperature six hours after each dose, but this was not considered adverse because the increases were minimal and resolved within 24 hours after dose administration.

[109] Microscopic observations at the injection sites of animals administered the test article/adjuvant and adjuvant only consisted of mixed cellular infiltration, lymphocytic infiltration, focal muscle degeneration, and hemorrhage.

[110] Mixed cellular infiltration at the injection sites was characterized by the presence of monocytes/macrophages, heterophils, lymphocytes, and occasional plasma cells, which were mainly located in adipose and connective tissue deep to the panniculus muscle. In some animals with moderate mixed infiltration, the infiltrates extend more deeply to involve the fascia associated with the underlying skeletal muscle, or more superficially, into the dermis and periadnexal connective tissue within the dermis.

[111] Lymphocytic infiltration at injection sites was characterized by the presence of small clusters of lymphocytes in adipose and connective tissue deep and superficial to the panniculus muscle.

[112] Focal muscle degeneration at the injection sites was characterized by individual degenerated myofibers or, more commonly, small groups of degenerated myofibers extending through the entire thickness of the subdermal panniculus muscle. These were considered an expected response to needle insertion through the muscle during the subcutaneous injection procedure. Hemorrhage at the injection sites was also considered to be a result of the injection procedure.

[113] In conclusion, under the conditions of this study, repeated subcutaneous administration of the P. vivax CSP/Poly-ICLC vaccine at 10/50 μg/dose four times over 43 days to New Zealand White rabbits was well tolerated. All observations were considered non-adverse and consistent with the expected inflammatory response and immune stimulation following vaccine administration.

BIBLIOGRAPHIC REFERENCES

[114] World Health Organization. World malaria report 2020: 20 years of global progress and challenges. Geneva: World Health Organization; 2020. License: CC BY-NC-SA 3.0 IGO.

[115] Menkin-Smith L, Winders WT. Plasmodium Vivax Malaria. [Updated 2020 Aug 11]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; Jan. 2021. Available at: https://www.ncbi.nlm.nih.gov/books/NBK538333/.

[116] World Health Organization. Malaria: The malaria vaccine implementation program (MVIP), Mar. 2020. Available at: https://www.who.int/news-room/q-a-detail/malaria-vaccine-implementation-programme.

[117] Dobano, C.; Sanz, H.; Sorgho, H.; Dosoo, D.; Mpina, M.; Ubillos, I.; Aguilar, R.; Ford, T.; Diez-Padrisa, N.; Williams, N.A.; et al. Concentration and avidity of antibodies to different circumsporozoite epitopes correlate with RTS,S/AS01E malaria vaccine efficacy. Nat. Commun. 2019, 10, 2174. Available at: https://www.nature.com/articles/s41467-019-10195-z.

[118] Datoo, Mehreen S. et al, High Efficacy of a Low Dose Candidate Malaria Vaccine, R21 in 1 Adjuvant Matrix-M™, with Seasonal Administration to Children in Burkina Faso (April 20, 2021). Available at: http://dx.doi.org/10.2139/ssrn.3830681.

[119] 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 7, 46482 (2017). Available at: https://doi.org/10.1038/srep46482.

[120] Garcon, N., Heppner; D. G. & Cohen, J. Development of RTS,S/AS02: a purified subunit-based malaria vaccine candidate formulated with a novel adjuvant. Expert Rev Vaccines 2, 231-238 (2003).

[121] Mueller I, Shakri AR, Chitnis CE. Development of vaccines for Plasmodium vivax malaria. Vaccine (2015) 33:7489-95. https://doi:10.1016/j.vaccine.2015.09.060.

[122] Birkett AJ. Status of vaccine research and development of vaccines for malaria. Vaccine (2016) 34:291520. https://doi:10.1016/j.vaccine.2015.12.074.

[123] Nussenzweig RS, Vanderberg J, Most H, Orton C. Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. Nature (1967) 216:160-2. https://doi:10.1038/216160a0.

[124] Clyde DF. Immunization of man against falciparum and vivax malaria by use of attenuated sporozoites. Am J Trop Med Hyg (1975) 24:397-401. https://doi:10.4269/ajtmh.1975.24.397.

[125] Cohen J, Nussenzweig V, Nussenzweig R, Vekemans J, Leach A. From the circumsporozoite protein to the RTS, S/AS candidate vaccine. Hum Vaccin (2010) 6:90-6. https://doi:10.4161/hv.6.1.9677.

[126] Arnot, D. E.; Barnwell, J. W.; Tam, J.P.; Nussenzweig, V.; Nussenzweig, R.S.; Enea, V. Circumsporozoite protein of Plasmodium vivax: Gene cloning and characterization of the immunodominant epitope. Science 1985, 230, 815-818.

[127] Rosenberg, R.; Wirtz, R. A.; Lanar, D.E.; Sattabongkot, J.; Hall, T.; Waters, A.P.; Prasittisuk, C. Circumsporozoite protein heterogeneity in the human malaria parasite Plasmodium vivax. Science 1989, 245, 973-976.

[128] Qari, S. H.; Shi, Y.P.; Goldman, I.F.; Udhayakumar, V.; Alpers, M.P.; Collins, W. E.; Lal, A. A. Identification of Plasmodium vivax-like human malaria parasite. Lancet 1993, 341, 780-783.

[129] De Souza-Neiras, W.C.; de Melo, L.M.; Machado, R.L. The genetic diversity of Plasmodium vivax—A review. Mem Inst. Oswaldo Cruz 2007, 102, 245-254.

[130] Raza, A.; Ghanchi, N.K.; Thaver, A. M.; Jafri, S.; Beg, M.A. Genetic diversity of Plasmodium vivax clinical isolates from southern Pakistan using pvcsp and pvmsp1 genetic markers. Malaria J. 2013, 12, 16.

[131] Pereira, V.A.; Sanchez-Arcila, J.C.; Vasconcelos, M.P.A.; Ferreira, A.R.; de Souza Videira, L.; Teva, A.; Perce-da-Silva, D.; Marques, M.T.Q.; de Carvalho, L.H.; Banic, D. M.; et al. Evaluating seroprevalence to circumsporozoite protein to estimate exposure to three species of Plasmodium in the Brazilian Amazon. Infect. Dis. Poverty 2018, 7, 46.

[132] Arruda, M.E.; Zimmerman, R. H.; Souza, R.M.; Oliveira-Ferreira, J. Prevalence and level of antibodies to the circumsporozoite protein of human malaria parasites in five states of the Amazon region of Brazil. Mem. Inst. Oswaldo Cruz 2007, 102, 367-371.

[133] Collins WE, Nussenzweig RS, Ballou WR, Ruebush TK II, Nardin EH, Chulay JD, et al. Immunization of Saimiri sciureus boliviensis with recombinant vaccines based on the circumsporozoite protein of Plasmodium vivax. Am J Trop Med Hyg (1989) 40:455-64. https://doi:10.4269/ajtmh.1989.40.455.

[134] 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 Trop Dis (2016) 10:e0004423. https://doi:10.1371/journal.pntd.0004423.

[135] de Camargo, T. M., de Freitas, E. O., Gimenez, A. M. et al. Prime-boost vaccination with recombinant protein and adenovirus-vector expressing Plasmodium vivax circumsporozoite protein (CSP) partially protects mice against Pb/Pv sporozoite challenge. Sci Rep 8, 1118 (2018). Available at: https://doi.org/10.1038/s41598-017-19063-6.

[136] 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. 2014 Feb;82(2):793-807. https://doi:10.1128/IAI.01410-13. Epub Dec, 2013.

[137] Lima LC, Marques RF, Gimenez AM, et al. A Multistage Formulation Based on Full-Length CSP and AMA-1 Ectodomain of Plasmodium vivax Induces High Antibody Titers and T-cells and Partially Protects Mice Challenged with a Transgenic Plasmodium berghei Parasite. Microorganisms. 2020;8(6):916. 17 Jun. 2020. https://doi:10.3390/microorganisms8060916.

[138] Gimenez AM, Lima LC, Françoso KS, Denapoli PMA, Panatieri R, Bargieri DY, Thiberge JM, Andolina C, Nosten F, Renia L, Nussenzweig RS, Nussenzweig V, Amino R, Rodrigues MM, Soares IS. Vaccine Containing the Three Allelic Variants of the Plasmodium vivax Circumsporozoite Antigen Induces Protection in Mice after Challenge with a Transgenic Rodent Malaria Parasite. Front Immunol. Oct. 2017. 11;8:1275. https://doi:10.3389/fimmu.2017.01275. eCollection 2017.

Claims (10)

1. Process for producing and purifying a recombinant protein, characterized by the fact that it comprises the following steps: (a) fusing the following regions: (i) a conserved region I (RI) of the circumsporozoite protein (CSP) of P. vivax ; (ii) three central repeat regions from three different alleles (VK210, VK247 and P. vivax-like) of P. vivax CSP; and (iii) the C-terminal domain region of PvCSP; and (b) purifying the recombinant protein through the following steps in a chromatography column: (b.i) filtering, diluting or adjusting a solution comprising the recombinant protein; (b.ii) loading the solution obtained in step (b.i) into a first chromatography column; (b.iii) subject the product from step (b.ii) to ultrafiltration; (b.iv) loading the product resulting from step (b.iii) into a second chromatography column; (b.v) subjecting the product from step (b.iv) to ultrafiltration; (b.vi) loading the product resulting from step (b.v) into a third chromatography column; and (b.vii) subjecting the product from step (b.vi) to ultrafiltration. 2. Process, according to claim 1, characterized by the fact that preferably the first chromatography column is Capto MMC; the second chromatography column is Q HyperD F and the third chromatography column is Sephacryl S100 HR. 3. Process, according to claim 1, characterized by the fact that the recombinant protein produced is expressed in Pichia pastoris and is partially or totally devoid of histidine at the C and/or N-terminal ends, preferably totally devoid of histidine at the C-terminal. 4. Recombinant protein produced and purified according to the process defined in any one of claims 1 to 3, characterized by the fact that it comprises the amino acid sequence of SEQ ID No: 1. 5. Recombinant protein, according to claim 4, characterized by the fact that it is at least 95%, preferably 98% pure. 6. Use of the recombinant protein, as defined in claim 4 or 5, characterized by the fact that it is for the production of an immunobiological composition. 7. Immunobiological composition, characterized by the fact that it comprises the recombinant protein as defined in claim 4 or 5, and an adjuvant selected from the group consisting mainly of Poly-ICLC, Montanide ISA 720, Addavax, MF59, MPLA, Matrix-M, CPG -ODN and Aluminum Hydroxide, preferably Poly-ICLC. 8. Use of the immunobiological composition, as defined in claim 7, characterized by the fact that it is in the preparation of vaccines to prevent malaria caused by P. vivax. 9. Use of the immunobiological composition, according to claim 8, characterized by the fact that the vaccine is tolerated at a concentration of 10/50 μg/dose of CSP P. vivax/Poly-ICLC respectively. 10. Use of the immunobiological composition according to claim 8 or 9, characterized by the fact that the vaccine is administered by subcutaneous injection.