Molecule of recombinant nucleic acid, epitope of alpha toxin of recombinant c. novyi and use thereof, expression vector, vaccine composition, method for preventing or treating clostridiosis in an individual suffering from same and method for producing a vaccine composition against clostridiosis

Abstract

The present invention provides molecules of recombinant nucleic acid that encode epitopes of alpha toxin of recombinant C. novyi. Additionally, the invention provides vaccine compositions comprising said epitopes and use of same, and a method for preparing said compositions. Finally, the invention also provides a method for treating and preventing clostridiosis.

Description

“RECOMBINANT NUCLEIC ACID MOLECULE, RECOMBINANT C. NOVYI ALPHA TOXIN EPITOPE AND ITS USE, EXPRESSION VECTOR, VACCINE COMPOSITION, METHOD FOR PREVENTING OR TREATING CLOSTRIDIOSIS IN AN INDIVIDUAL SUFFERING THEREOF AND METHOD FOR PRODUCING A VACCINE COMPOSITION AGAINST CLOSTRIDIOSIS” FIELD OF THE INVENTION

[0001] The present invention relates to the field of veterinary medicine and biotechnology. Specifically, the present invention relates to the development and production of epitopes from C. novyi alpha toxin in the cultivation scale-up process in Escherichia coli BL21(DE3), and their use in vaccine formulations for clostridiosis in cattle. BACKGROUND OF THE INVENTION

[0002] Cattle farming is one of the most important economic activities in Brazil. The IBGE estimates that the country currently has more than 220 million head of cattle, with the Central-West region having the largest number of this type of livestock (IBGE, 2021).With the professionalization of cattle farming, disease control and the quality of products from this field have become increasingly strict (Ferraz and Felício, 2010; Carvalho and Zen, 2017).

[0003] There is a group of infections and poisonings that affect herds and are caused by anaerobic bacteria of the genus Clostridium, known as clostridiosis. Depending on the type of bacteria of the genus Clostridium, the following may occur: pulpy kidney disease, hemorrhagic enterotoxemia, infectious necrotizing hepatitis, and sudden death (Embrapa, 2001).

[0004] Clostridiosis is a typical disease that affects these herds, caused by bacteria of the genus Clostridium, which are characterized by being anaerobic, gram-positive, motile rods and causing significant losses in cattle farming (Ma et al., 2007; Lobato et al., 2013; Walker et al., 2016).

[0005] Strains of the genus cause diseases through the presence of toxins and / or tissue invasion, differing according to the presence and synthesis of genes (Uzal et al., 2010; Brandi et al, 2014; Brandi et al., 2016; Fouad et al., 2021).

[0006] C. novyi is a common agent of domestic animals and humans, and is divided based on toxin production into four types: A, B, C, and D, with type D recognized as C. haemolyticum. These potent exotoxins mediate clostridial pathogenesis (Uzal et al., 2010; Navarro et al., 2017).

[0007] C. novyi type B is an alpha toxin producer, the main cause of pathogenesis in this strain (Le Gratiet et al., 2021).This toxin is part of the group of large clostridial toxins, with a molecular mass of 250 kDa (Lima et al., 2011), with glycosyltransferase action, causing the inactivation of proteins present in the cytoskeleton and disorganization of actin filaments, resulting in cell rounding, loss of intercellular junctions and increased endothelial permeability (Gonçalves et al., 2013).

[0008] The alpha toxin of C. novyi is produced by types A and B, encoded by the chromosomal gene cpa or plc; the beta toxin is produced by types B and D, encoded by genes contained in plasmids. The alpha toxin is lethal; beta. toxin, classified as necrotizing and hemolytic, but is generally not produced in lethal amounts (Navarro et al., 2017).

[0009] Alpha toxin or PLC is part of the group of large clostridial toxins, with a molecular weight greater than 250 kDa. The action of this class causes cell rounding, an effect that alters the cytoskeleton of mammalian cells (Lima et al., 2020).

[0010] The alpha toxin produced is defined as a glycosyltransferase responsible for catalyzing the glycosylation of small GTPases, causing the inactivation of proteins present in the cytoskeleton and disorganization of actin filaments, resulting in cell rounding, loss of intercellular junctions, and increased endothelial permeability (Lobato et al., 2013, Lobato et al., 2021).

[0011] Studies of the three-dimensional structure of alpha toxin show two main domains: the N-terminal (amino-terminal) domain, presenting single-domain proteins, and the C-terminal (carboxy-terminal) domain, presenting proteins with two domains (Noshahri et al., 2016). The N-terminal domain of alpha toxin retains phospholipase activity and presents loss of hemolytic activity. The C-terminal domain influences the enzymatic activity of the N-terminal domain and recognizes membrane phospholipids, being extremely important for the hemolytic activity of alpha toxin (Nagahama et al., 2013).

[0012] That is, alpha toxin has a tripartite structure in which enzymatic activity is related to the N-terminal region, known as the catalytic domain; receptor binding is related to the C-terminal region. toxin; and the translocation of the toxin to the cytosol is related to the hydrophobic domain in the central region (Nagahama et al., 2013; Noshahri et al., 2016).

[0013] Hepatic necrosis is one of the diseases resulting from infection caused by C. novyi type B (Ma et al., 2007). This disease is associated with trematodes, such as Fasciola hepatica. However, there are indications of the disease in the absence of these parasites (Lobato et al., 2008; Lobato et al., 2013).

[0014] In its structure, the alpha toxin presents zinc metals strongly bound to an exchangeable divalent cation. Residues His-68, -126, and -136 bind to a divalent cation that is necessary for binding to membranes. His-148 and Glu-152 bind to a zinc ion that is essential for the active site of the toxin, and His-11 and Asp-130 tightly bind the other zinc ion, which is necessary for maintenance of the structure.

[0015] The amino acids Tyr-57 and -65 play roles in the penetration of the toxin into the membrane bilayer and also allow access to the sphingomyelin catalytic site (Nagahama et al., 2006; Nagahama et al., 2013; Oda et al., 2015).

[0016] The hemolytic, myotoxic and cytotoxic activity of alpha toxin may be a consequence of its interaction with host cell membranes and the subsequent hydrolysis of phospholipids (Alape-Girón et al., 2000). Furthermore, reports describe alpha toxin action with phospholipase C (PLC) activity and sphingomyelinase (SMase) activity.

[0017] Recent discoveries revealed that a membrane localization domain (MLD) was identified as one. conserved domain among numerous bacterial toxins, including the large family of clostridial glycosylating toxins (LCGT) that play key roles in plasma membrane localization (Liu et al., 2022).

[0018] As already described here, the alpha toxin or PLC, from C. novyi type B, belongs to the LCGT glycosylating group with phospholipase C and sphingomyelinase activity, with a C-terminal region directly related to binding to the membrane receptor that recognizes membrane phospholipids (Noshahri et al., 2016; (Liu et al., 2022).

[0019] Furthermore, in several strains of Clostridium, the plc gene is located in the same locus, close to the putative origin of replication and between two ribosomal RNA operons.It is worth noting that this is a highly relevant region, since this region of the chromosome is not only the first to be replicated, but also the most conserved segment of the genome, containing housekeeping genes (constitutive genes essential for the existence of a cell and expressed in all cells of an organism under normal and pathophysiological conditions).

[0020] Reports have already described that the nucleotide sequence of the plc gene is 2.2 kb, revealing an open reading region comprising amino acids 943 to 2137, corresponding to a protein with a molecular weight of 43.3 kDa. The following amino acid sequences encoded between the initiation codon and nucleotide 1027 form a signal sequence for the direct transport of the alpha toxin across the cell membrane (Tsutsui et al., 1995; Noshahri et al., 2016; Liu et al., 2022).

[0021] Another relevant aspect is that the alpha toxin can be displayed in two distinct conformations, one with. open form containing the accessible active site and a closed form with the covered active site, which encompasses residues 135 to 150. Predictions of the alpha toxin structure revealed that the N-terminal region contains 687 amino acid residues and is located in the first protein portion, being smaller than the C-terminal.

[0022] On the other hand, the C-terminal region of the protein, consisting of approximately 1000 amino acid residues, is more antigenic, given the greater presence of beta-sheet structures, with a greater immune response compared to the helical regions (Noshahri et al., 2016).

[0023] Furthermore, the binding of the C-terminal domain to the target membrane causes the active site to be positioned towards the lipid bilayer, causing the discovery of the active site, a fact that makes it important for the toxicity of the enzyme (Walker et al., 2000; Alape-Girón et al., 2000; Oda, et al., 2015).

[0024] Other studies have described that when immunizing animals using the C-terminal domain, the production of antibodies generated guarantees protection after challenge using a lethal dose of active toxin, revealing that it is an important, highly antigenic region with a high probability of humoral immune response.

[0025] On the other hand, regions of the N-terminal domain present a high potential for binding to the major histocompatibility complex II (MHCII) (Nagahama et al., 2013).

[0026] Infection occurs through latent spores that germinate in the liver, occurring in regions damaged by the migration of Fasciola hepatica. The spores pass into the vegetative form and trigger the production of large amounts of alpha toxin (Uzal, 2003; Lima et al., 2011).

[0027] Systemic effects cause animal death (Farias et al., 2014; Navarro et al., 2017). Multiplication of C. novyi type B in the liver can also cause sudden death (Lobato et al., 2008; Aquino et al., 2016; Radostits et al., 2002).

[0028] Some characteristics presented by the causative agent of clostridiosis make its eradication difficult, since these bacteria are capable of forming resistance spores and are ubiquitous in the gastrointestinal tract of animals and humans.

[0029] For this reason, control is based mainly on preventive measures and systematic vaccination of animals (Lima et al., 2011; Skarin; Segerman, 2014).

[0030] However, for some types of etiological agents of the clostridium genus, such as Clostridium novyi type B, there are still technical barriers to be overcome, such as the immunogenic properties of vaccine formulations (Nascimento et al. 2004). Vaccines used to control hepatic necrosis

[0031] The veterinary pharmaceutical market has been growing due to demand from the production animal segment, particularly livestock (Ferraz and Felício, 2010; Carvalho and Zen, 2017).

[0032] Immunization of these animals against clostridial diseases is carried out using concentrated toxoids, in which two doses of vaccine can confer immunity for up to one year. However, there is no official control to verify the vaccine efficacy of these immunogens (Lobato et al., 2008; Lima et al., 2011; Lobato et al., 2013).

[0033] This market is dominated by multinational companies whose clostridial vaccines contain inactivated toxins in their formulation, with multiple antigens and a wide variety of agents and toxins. The vast majority are composed of antigens for myonecrosis, enterotoxemia, bacillary hemoglobinuria, hepatic necrosis, botulism, and tetanus (Lobato et al., 2013).

[0034] In their study, Nascimento et al. (2004) analyzed vaccines against C. novyi type B and demonstrated low immunogenic power of the products sold in the national market.

[0035] Because it is a fastidious microorganism, obtaining the alpha toxin from C.novyi has been costly, leading to failures during industrial bioprocess stages (Zeng et al., 2011; Aquino et al., 2016; Navarro et al., 2017). Technological alternatives, such as recombinant protein expression, have been the target of in silico studies for the selection of vaccine epitope regions (Lobato et al., 2013; Lima et al., 2020; Le Gratiet et al., 2021). Research related to the development of vaccines formulated with recombinant protein, obtained from the fermentation of genetically modified microorganisms, reduces production costs and provides standardization of production. Analyses of protein hydrophobicity, accessibility, antigenicity, and flexibility can be performed using bioinformatics tools, enabling the selection of potential regions for protein construction (Santos et al., 2012). In this regard, a relevant aspect is the use of epitopes from the conserved gene region of C. novyi in vaccine formulations.Patent CN110041437-B uses tetanus toxin C fragments and three epitopes. from the N-terminal region and one from the C-terminal region of Clostridium novyi alpha toxin, however, they do not use genes from a conserved region in the C-terminal.

[0038] However, recombinant vaccines developed from antigens originating from conserved regions allow greater immunological stimulation of the host, since these are regions always present in bacterial infections, and which allows them to be described as primary targets in the development of recombinant vaccines. Furthermore, these regions have greater protection when compared to stimuli caused by bacterin-based vaccines, since the latter generates limited protection (Perez-Casal, J. et al., 2017; Baruah, N. et al., 2021; Cangussu, ASR et al., 2018).

[0039] Production of recombinant C.novyi alpha toxin has commonly been achieved by transforming Escherichia coli BL 21 (Zeng et al., 2011).Expression of recombinant proteins

[0040] In silico studies allow obtaining selectable epitopes that are used in the construction of expression vectors (Sobrinho et al., 2010; Santos et al., 2012; Santos et al., 2019; Brito et al., 2021), being validated after syntheses in cell cultures, as well as in the reactivity with sera from infected animals (Zeng et al., 2011; Silvestre et al., 2014; Moreira et al., 2016; Cangussu et al., 2018; Félix et al., 2019; Félix et al., 2022).

[0041] The production and expression of recombinant proteins makes use of different host cells in which these regions of interest are introduced. Through recombinant DNA technology, it is possible to obtain proteins. pure and functional proteins at low cost (Lee et al., 2003; Nosshahri et al., 2016; Kojima et al., 2022).

[0042] Several expression systems are used for this purpose, with the enterobacteria E. coli being the most used, with records of up to 60% in the use for the production of recombinant proteins (Duarte et al., 2021; Cardoso et al., 2022). Its choice is justified by the large volume of genetic, physiological, biochemical information and tools available for its genetic manipulation (Correa and Oppezzo, 2011; Zeng et al., 2011).

[0043] Several vectors are available for protein expression in E. coli, promoting high levels of expression, stability and versatility (Jonasson et al., 2002). In addition to these factors, the efficiency of clostridial vaccines is related to the adjuvants that compose them (Assis et al., 2002).Oily adjuvant and microemulsion

[0044] The adjuvant is one of the most important factors in vaccine composition and is directly linked to increased vaccine immunogenicity. Veterinary vaccines most often employ aluminum and mineral oil-based adjuvants (Park et al., 2014; Veenstra et al., 2017).

[0045] Aluminum-based adjuvants have several disadvantages, such as inducing short-lived antibody responses that lead to frequent revaccinations and side effects. In contrast, oil-based adjuvants have the advantage of inducing high titers and long-lasting antibody responses that lead to effective protection (Khorasani et al., 2016; Tehrani et al., 2016).

[0046] Emulsified oily adjuvants influence the antigen in time, location, and concentration through the "depot effect."The microemulsion forms a semi-solid mass at the injection site, slowly releasing the antigen into the tissue or blood, enhancing and prolonging the humoral response (Tehrani et al., 2016; Veenstra et al., 2017). These systems have the advantage of good injectability due to their low oil content and viscosity, allowing them to easily present the antigen (Leclercq et al., 2011; Sun et al., 2015). Antigens with emulsified adjuvants initiate the innate response that plays a role in activating the adaptive immune response by stimulating specific components of the humoral or cell-mediated immune response (Grun and Maurer, 1989; Veenstra et al., 2017).

[0049] In this context, given the relevance of these immunogens present in the formulations of vaccines against clostridial diseases, the present invention proposes the optimization and scaling of the obtaining of recombinant epitopes of C. novyi alpha toxin in recombinant E. coli BL21 pLysS (DE3) culture.

[0050] In this sense, three different epitope combinations (DE3 / Ep1, DE3 / Ep2 and DE3 / Ep1+DE3 / Ep2) of recombinant alpha toxin of C. novyi type B in W / O emulsion were proposed. Furthermore, the immunogenicity and protection parameters of immunized and challenged murine models were determined, aiming at new alternatives and advances for antigen adsorption applicable to the control of clostridiosis. In the epitope selection process, genetic engineering tools were used, in which the DE3 / Ep1 epitope comprises the region of the N-terminal domain (between the. amino acids 1 and 583, vaccine group G2), DE3 / Ep2 epitope comprising the region of the C-terminal domain (between amino acids 1599 and 2178, vaccine group G3), combined epitopes DE3 / Ep1+DE3 / Ep2 comprising both domains (vaccine group G4). Groups G2, G3 and G4 were compared with group G1 (MONTANIDE™ ISA 61 VG adjuvant, Seppic, Brazil), and showed better vaccine efficacy data.

[0051] Thus, a vaccine formulation was developed with two epitopes from the C.novyi alpha toxin sequence, GenBank CAA88565.1, located at amino acids 491° to 583° of the N-terminal region, and amino acids 1599° to 1779° of the C-terminal region. The formulation process was prepared in a water-oil emulsion in a 40 / 60 ratio, where tests were carried out in vivo and obtained results that indicate potential as an immunogenic composition in combating the group of diseases caused by the C.novyi type B agent.

[0052] The results indicate efficacy in its use as an immunogen in vaccine protection, regarding protection against damage caused by the active alpha toxin of C. novyi type B. The experimental group G3 (C-terminal) showed a higher vaccine efficacy rate (40%) when compared to group G2 (N-terminal), with 25%. When the domains were combined (G4), the immunization of mice caused a synergistic effect in protective potency, with 75% of animals surviving after a lethal challenge dose with active toxin.

[0053] Finally, a mechanically stirred tank biological reactor was used to promote the improvement of bioprocess conditions and enable proposals for scaling up the production of C. novyi alpha toxin epitopes in order to contribute to competitive gains for the veterinary vaccine producing industry. SUMMARY OF THE INVENTION

[0054] The present invention aims to provide epitopes of C. novyi alpha toxin for the production of a vaccine composition, as well as a method for producing the same. Preferably, the epitopes originate from the conserved region of the C.novyi gene.

[0055] In a first embodiment, the present invention provides recombinant nucleic acid molecules comprising the nucleic acid sequences defined by SEQ ID NO: 1 or SEQ ID NO: 3 and their degenerate sequences encoding the same polypeptide sequence.

[0056] In a second embodiment, the present invention provides recombinant C. novyi alpha toxin epitopes comprising the peptide sequences defined by SEQ ID NO: 2 or SEQ ID NO: 4.

[0057] In a third embodiment, the recombinant C. novyi alpha toxin epitopes further comprise a polyhistidine (6xHIS) tag at the amino-terminal end.

[0058] In a fourth embodiment, the present invention provides expression vectors comprising the nucleic acid molecules according to the present invention and their regulatory regions.

[0059] In a fifth embodiment, the expression vectors comprise the selected control regions of BamHI and EcoRI.

[0060] In a sixth embodiment, the present invention provides vaccine compositions comprising one or more epitopes according to the present invention and veterinarily acceptable excipients.

[0061] In a seventh embodiment, the vaccine compositions comprise the two epitopes according to the present invention and veterinarily acceptable excipients.

[0062] In an eighth embodiment, the vaccine compositions may be of the water / oil emulsion type and are for preventing or treating clostridiosis in a subject suffering therefrom.

[0063] In a ninth embodiment, the present invention provides the use of an epitope as disclosed in the present application in the preparation of a vaccine composition for preventing or treating clostridiosis in a subject suffering thereof.

[0064] In a tenth embodiment, the present invention provides a method for preventing or treating clostridiosis in a subject suffering thereof comprising administering the vaccine composition as disclosed by the present application.

[0065] In an eleventh embodiment, the present invention provides a method for producing a vaccine composition against clostridiosis comprising the following steps: i) Transforming E. coli cells with an expression plasmid comprising the molecules as above; ii) Cultivating said cells in bioreactors through the fed-batch process; iii) Purifying the product of the cultivation step ii); iv) Preparing the vaccine composition comprising the epitopes obtained after step iii). BRIEF DESCRIPTION OF THE FIGURES

[0066] Figure 1: Figure 1 refers to insilicon modeling and detection of vaccine epitopes. (A) 3D structure of C. novyi type B alpha toxin fraction predicted by i-TASSER from GenBank data: CAA88565.1

[0048] . (B) and (C) 3D structures of DE3 / Ep1 and DE3 / Ep2, respectively. (D) Detection of DE3 / Ep1 (17kDa) and DE3 / Ep2 (26kDa) using 12% gel electrophoresis. (E) Western blotting of vaccine epitope reactivity with serum from Swiss mice infected with wild-type C. novyi alpha toxin.

[0067] Figure 2: Figure 2 refers to the alpha toxin vectors ep1 and ep2 used by the present invention

[0068] Figure 3: Figure 3 refers to the response surface of DE3 cultivation in a rotary shaker and DCW ratio, inoculum fraction as a function of cultivation time (h) using response equations and analysis of variations by Origin Pro® 8.5 software. Data are represented by mean and standard deviation, considering P-value (P<0.05).

[0069] Figure 4: Figure 4 refers to the optimization of vaccine epitope production in a stirred tank biological bioreactor. (A) and (B) Kinetic profiles of DE3 aiming at the synthesis of DE3 / Ep1 and DE3 / Ep2 epitopes in a single batch. (C) and (D) Kinetic profile of DE3 aiming at the synthesis of DE3 / Ep1 and DE3 / Ep2 in a fed-batch. Data obtained from DE3 cultures in SD medium containing ampicillin (100 µg / mL) carried out in a benchtop bioreactor (Tecnal BIO–TEC®), at 37 °C. Glucose supplementation (500 g / L) occurred at 30 min intervals up to maximum DCW. Induction carried out at 28 °C for 6 h using lactose (10 g / L) as an inducer. DCW – dry cell mass (g / L), O2 – dissolved oxygen (%), Hac – concentration of acetic acid (g / L) and glucose (g / L). (*) start of. induction, (→) start of feeding. Data are represented by mean and standard deviation, considering P-value (P<0.05) using Origin Pro® 8.5 software

[0070] Figure 5: Figure 5 refers to the Scaling of vaccine epitope production in a non-stirred tank biological bioreactor. (A) and (B) Kinetic profiles of DE3 aiming at the synthesis of DE3 / Ep1 and DE3 / Ep2 epitopes. Data obtained from DE3 cultures in SD medium containing ampicillin (100 µg / mL) carried out in a Tecnal BIO–TEC® bioreactor with an adapted tank using Thermo Scientific Nalgene® round carboys at 37 °C. Glucose supplementation (500 g / L) occurred at 30 min intervals up to maximum DCW. Induction conducted at 28 °C for 6 h using a combination of lactose (10 g / L) and IPTG (0.2 mM) as an inducer. DCW – dry cell mass (g / L), O2 – dissolved oxygen (%), Hac – concentration of acetic acid (g / L) and glucose (g / L). (*) start of induction, (→) start of feeding.Data are represented by mean and standard deviation, considering P-value (P<0.05) using Origin Pro® 8.5 software.

[0071] Figure 6: Figure 6 refers to the purification of vaccine epitopes in a lab-scale stirred tank biological bioreactor. (A) Profiles of purified DE3 / Ep1 and DE3 / Ep2 epitopes obtained by single batch. (B) Profiles of purified DE3 / Ep1 and DE3 / Ep2 epitopes obtained by fed-batch. Data obtained on an Akta Purifier 10® chromatography system, GE Lifescience Healthcare®). Elutes determined at 280 nm.

[0072] Figure 7: Figure 7 refers to the purification of vaccine epitopes in a scale-up non-stirred tank biological bioreactor. (A) and (B) Purified DE3 / Ep1 and DE3 / Ep2 epitope profiles obtained by fed-batch. Data. obtained in an Akta Purifier 10® chromatography system, GE Lifescience Healthcare®). Elutions determined at 280 nm.

[0073] Figure 8: Figure 8 refers to the monitoring of body weight of mice in experimental groups (A). Liver and spleen weight data (B and C). Control (non-immunized animals); G1 (animals inoculated with adjuvant in W / O microemulsion); G2 (animals immunized with DE3 / Ep1 ALFA-T / REC in W / O microemulsion); G3 (animals immunized with DE3 / Ep2 ALFA-T / REC in W / O microemulsion); G4 (animals immunized with DE3 / Ep1+DE3 / Ep2 ALFA-T / REC associated in W / O microemulsion); G5 (animals immunized with inactivated wild-type alpha toxin). P-value <0.05 (*).

[0074] Figure 9: Figure 9 refers to the vaccine efficiency test using ALFA-T / REC vaccine epitopes with mice challenged with a lethal dose of active alpha toxin 157 LD50.g -1. Control (non-immunized animals). (G1) Animals inoculated with adjuvant in microemulsion. (A) (G2) Survival curve of mice immunized with ALFA-T / REC DE3 / Ep1 in W / O microemulsion. (B) (G3) Survival curve of mice immunized with ALFA-T / REC DE3 / Ep2 in W / O microemulsion. (C) (G4) Survival curve of mice immunized with ALFA-T / REC DE3 / Ep1+ALFA-T / REC DE3 / Ep2 in combination and W / O microemulsion. (D) (G5) Survival curve of mice inoculated with inactivated alpha toxin, after challenged with a lethal dose of active alpha toxin 157 LD50.g -1 . P value determined by the log-rank test (Mantel-Cox). (*) p<0.05 represents comparative analysis between groups.

[0075] Figure 10: Figure 10 refers to the EnzymeLinked Immunosorbent Assay (ELISA). Stimulation of the humoral vaccine response, data referring to total IgG levels produced from the pre-application of the lethal dose of active alpha toxin (157 LD50.g -1) in non-immunized Swiss mice (Control), adjuvant group (G1) and immunized (G2, G3 and G4). (*) p-value <0.05 represents comparative analysis between the vaccine groups and the control group.

[0076] Figure 11: Figure 11 refers to the histopathological data of experimental groups after immunization and challenge with dose 157 LD50.g -1stained by the hematoxylin-eosin technique. G1 (animals inoculated with adjuvant in W / O microemulsion). G2 and G3 – animals immunized ALPHA-T / REC DE3 / Ep1 and ALPHA-T / REC DE3 / Ep2 in W / O microemulsion, respectively. G4 – animals immunized ALPHA-T / REC DE3 / Ep1+ALFA-T / REC DE3 / Ep2 associated in W / O microemulsion (A) Data related to liver organs. (B) Data related to spleen organs. Data: A1 – Degeneration (40x), A2 – Hemorrhage (40x), A3 – Hyperemia (40x), B1 – Degeneration (40x).

[0077] Figure 12: Figure 12 refers to the comparison of Ep1 protein induction conditions. (A) Expression of EP1 protein according to the initially proposed induction strategy, i.e., in E. coli Bl21 (DE3) pLysS cell, 28 °C, 6 h of induction, lane 1: lactose inducer, 10 g / L; lane 2: IPTG inducer, 0.2 mM; lane 3: lactose inducers (10 g / L)+IPTG 0.2 mM, initially proposed induction strategy highlighted with rectangular outline.(B) EP1 protein expression according to improved induction strategy, i.e., in E. coli Tuner DE3 cells, 37 °C, lane 1: IPTG inducer, 1 mM, 3 h induction; lane 2: IPTG inducer, 1 mM, 16 h induction; lane 3: Lactose inducer, 10 g / L, 3 h induction; lane 4: Lactose inducer, 10 g / L, 16 h induction; lane 5: time zero, before. induction. In channels 1 and 3, the expression conditions after improvement are highlighted with a rectangular outline.

[0078] Figure 13: Figure 13 refers to the comparison of the Ep2 protein induction conditions. (A) Expression of the EP2 protein according to the initially proposed induction strategy, that is, in E. coli Bl21 (DE3) pLysS cells, 28 °C, 6 h of induction, channel 1: lactose inducer, 10 g / L; channel 2: IPTG inducer, 0.2 mM; channel 3: lactose (10 g / L) + IPTG 0.2 mM inducers. In channel 3, the induction strategy described in the initially proposed patent is highlighted with a rectangular outline. (B) EP2 protein expression according to the improved induction strategy, i.e., in E. coli Bl21(DE3)pLysS cells, 37 °C, lane 1: 1 mM IPTG inducer, 16 h induction; lane 2: 0.1 mM IPTG inducer, 16 h induction; lane 3: lactose inducer, 16 h induction. In lane 1, the expression condition after improvement is highlighted with a rectangular outline.DETAILED DESCRIPTION OF THE INVENTION

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one skilled in the art to which the invention belongs. The terminology used in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the scope of the teachings. Unless otherwise indicated, all numbers expressing quantities, percentages and proportions, and other numerical values used in the specification and claims, should be understood as being modified, in all cases, by the term "about". Thus, unless otherwise indicated, the numerical parameters. The values shown in the specification and claims are approximations that may vary depending on the properties to be obtained.

[0080] As used throughout this application, the term "amino acid" denotes the group of α-amino acids that, directly or in the form of a precursor, can be encoded by a nucleic acid. Individual amino acids are encoded by nucleic acids consisting of three nucleotides, known as codons or base sets. Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is known as "degeneracy of the genetic code." The term “amino acid” as used in the present application denotes the naturally occurring α-amino acids comprising alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0081] The terms “peptide”, “polypeptide” or “protein” may be used interchangeably, and refer to a polymer of amino acids connected by peptide bonds, regardless of the number of amino acid residues constituting this chain. Polypeptides, as used herein, include “variants” or “derivatives” thereof, which refer to a polypeptide that includes variations or modifications, e.g., substitution, deletion, addition or chemical modifications in its amino acid sequence relative to the reference polypeptide. Examples of chemical modifications are glycosylation, G, PEG alkylation, alkylation, phosphorylation, acetylation, amidation, etc. The polypeptide can be produced artificially from. nucleotide sequences cloned through the recombinant DNA technique or can be prepared through a known chemical synthesis reaction.

[0082] More specifically, the term polypeptide of the present invention can also be understood as antigen, polyantigen or multi-epitope antigen, which consist of a junction of different epitopes that may or may not be interconnected by flexible or rigid linkers, specific for a single pathogen or for different pathogens.

[0083] The term "epitope" refers to an antigenic determinant on a molecule, for example, to the part of a molecule that is recognized by the immune system, for example, that is recognized by an antibody. For example, epitopes are the discrete three-dimensional sites on an antigen that are recognized by the immune system.Epitopes typically consist of chemically active surface clusters of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the fact that binding to the former, but not the latter, is lost in the presence of denaturing solvents. A protein epitope preferably comprises a continuous or discontinuous portion of said protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, most preferably between 10 and 25 amino acids in length; for example, the epitope may preferably be 8, 9, 10, or 12 amino acids long. 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.

[0084] In a first embodiment, the invention provides C. novyi alpha toxin vaccine peptides.

[0085] The C. novyi alpha toxin gene regions were predicted and selected using the IEDB-AR platform, from the GenBank database: CAA88565.1. The regions corresponding to the DE3 / Ep1 vaccine epitope are comprised in the N-terminal domain, with part of the hydrophobic domain in the central region for toxin translocation to the cytosol. The regions corresponding to the DE3 / Ep2 epitope are comprised in the C-terminal domain, also with part of the hydrophobic domain.

[0086] The epitopes were selected considering aspects of antigenicity, flexibility and stimulation of the immune response in the in silico model, expressed in attenuated E. coli revealing bands of 17 kDa and 26 kDa corresponding to DE3 / Ep1 and DE3 / Ep2, respectively (Figure 1).It should also be clarified that, after performing alignments using the Blast tool (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), no sequences identical to those of the present invention were found in the prior art.

[0087] For expression in E. coli, the epitope genes were inserted into expression plasmids. In a preferred embodiment, the vector used is selected from the group pRSET, pColdl pASK-IBA, pT7-7, pBAD-TOPO, pSC101, pACYC, and any other vector for expression in microbial cells (see figure 2).

[0088] Preferably, the vector used is pRSET.

[0089] The term “expression vector” or simply “vector” can be understood as DNA molecules that replicate autonomously and that can be used to transport exogenous DNA fragments. It is a vehicle used in gene cloning. The DNA of interest is first cloned into an appropriate vector and then, by transfection, the gene can be inserted into the host for expression.

[0090] Expression vectors are applicable for use in the microbial host organisms as disclosed in the present application and include, for example, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome.

[0091] Furthermore, expression vectors may include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes may also be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture medium.

[0092] Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like that are well known in the art.When two or more exogenous coding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or into separate expression vectors.

[0093] For single-vector expression, the coding nucleic acids can be operably linked to a common expression control sequence or linked to different expression control sequences, such as an inducible promoter and a constitutive promoter.

[0094] Transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art.Such methods include, for example, nucleic acid analysis, such as Northern blot or mRNA amplification by polymerase chain reaction (PCR), or immunoblotting for expression of gene products or other suitable analytical methods for testing the expression of an introduced nucleic acid sequence or its corresponding gene product.

[0095] It is understood by those skilled in the art that the exogenous nucleic acid is expressed in sufficient quantity to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

[0096] In one example, the E. coli strain can be selected from the group comprising, but not limited to: C41 (DE3), Rosetta (DE3), Origami (DE3), or Turner (DE3). Preferably, the E. coli strain used in the present invention is BL21 (DE3) pLysE. Table 1: Table 2:

[0097] Reactivity with serum from mice infected with wild-type C. novyi alpha toxin revealed the ability of these epitopes to recognize specific antibodies present in the infection (Figure 1).

[0098] The determination of the DE3 inoculum fraction was carried out in orbital shakers and SD medium (semi-defined medium) in the proportions of 2.5, 5 and 10% (v / v). Data from response surface modeling revealed that the proportion of 10% (v / v) allowed for greater biomass and specific growth rate, being conditions adopted in subsequent studies (Figure 3).

[0099] Aiming to optimize the production of the DE3 / Ep1 and DE3 / Ep2 epitopes, we evaluated DE3 cultivations in a 1 L mechanically stirred tank biological reactor, which can be performed in batch, fed-batch or continuous manner, as disclosed in this document.

[0100] The kinetics were conducted with a working volume of 700 mL, with three (3) 10 g / L lactose induction pulses, with the induction time maintained for 6 h after reaching maximum cell density (Figure 4).

[0101] The kinetic profile of DE3 in a simple batch revealed a DCW (Dry Cell Weight) yield of 0.70 and 0.77 g / L, with adequate O2 supply (%) and total glucose depletion, with no significant Hac (g / L) (acetic acid) production being evidenced (Fig. 3AC). Specific growth rate (µ), yield coefficient (Y. X / S and Y P / S ) and volumetric productivity of products and cells (Q P and Q X ), do not differ from each other regarding DE3 cultivations aiming at the production of the DE3 / Ep1 and DE3 / Ep2 epitopes (Table 3). Q X ) h • L 9 9 4 / m 4, 8 , 5 3 2 7, 1 1, 9 2 1 d n / S e P m / r l m 6 2 5 9 7 Y o / l , 0 3, 0 2, 0 3, e d mo 0 m e t m ( m n e ) i L ci m m g ( ) L P m / l o 6 m 5 6 1 1 ,0 5, 0 6, 0 6, 0m ( µ 1- ) h 4 8 5 ( 2, 0 2 9 ,0 2, 0 2, 0o ã ç e s e s e s e s u d t oc t oc t o t o n I a l a l c a l c a l r t o e o a d e a i r e o l ó t d o a a i r e o l ó t d a a i r e o ló t d i r a al ó t r i a c s r o a b c s r o a b c s r o a b c a s r o B E a l E a l E a l E b a l l a l u 3 3 3 3 é E D E E E C D D D a a - a a d a - a a m e d t a s l i e d a a t l t e n e d a l e d a d a l t e n e S a t b a B m i t l a a tb a B m i l a e : d A A r m e ot g a s e a n s e r T E T o l p S E S V c x R R a e i p p e d c a n a n o p n i ê í T u et í q o e r t o o e s P r ) P d e e n a fl a n 1 ) p a f a n 2 p m o e g A i x / E l A ix / E N t o 3 e d ( t o 3 e d (

[0102] The kinetic profile of DE3 in fed-batch, under these same induction conditions, revealed a DCW yield of 1.03 g / L, with adequate O2 supply (%) and total glucose depletion, with no significant Hac production (g / L) being evidenced (Fig. 3BD). Specific growth rate (µ), yield coefficient (Y X / S and Y P / S ) and volumetric productivity of products and cells (Q P and Q X), do not differ from each other regarding DE3 cultures aiming at the production of the DE3 / Ep1 and DE3 / Ep2 epitopes; however, all were superior to those obtained in the simple batch (Table 3).

[0103] The scale-up of vaccine epitope production was carried out in a fed-batch system as it represents the highest yields obtained in previous studies. Thus, we established the scale-up of epitope production by determining the yield in a 20 L non-stirred tank biological reactor, with the expectation of reducing operating costs.

[0104] The kinetics was carried out with a working volume of 10 L, with three (3) induction pulses associating a low concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) inducer (0.2 mM) with 10 g / L lactose solution, with the induction time being maintained for 6 h, after reaching maximum cell density (Figure 5).The kinetic profile of DE3 in fed-batch revealed similar parameters between the production of vaccine epitopes (DE3 / Ep1 and DE3 / Ep2), achieving DCW yields of 1.07 and 1.20 g / L. Values of O2 (%), glucose consumption (g / L) and Hac production (g / L) presented profiles similar to those described in previous studies (Figure 5AB).

[0105] Specific growth rate (µ), yield coefficient (Y. X / S and Y P / S ) and volumetric productivity of products and cells (Q P and Q X) do not differ from each other regarding DE3 cultivations aiming at the production of the DE3 / Ep1 and DE3 / Ep2 epitopes; however, all were superior to those obtained in the mechanically stirred fed-batch (Table 4).

[0106] The induction strategy using 10 g / L lactose pulses revealed DE3 / Ep1 and DE3 / Ep2 production profiles in both processes (single and fed-batch) conducted in the 1 L mechanically stirred tank biological reactor. DE3 / Ep1 and DE3 / Ep2 production peaks are revealed in the ranges from 300 mAU (single batch) to 400 mAU (fed-batch), revealing higher production in the fed-batch system (Figure 6).

[0107] In the 10 L scale-up, we used the association of 10 g / L lactose and 0.2 mM IPTG, which revealed increases. Production peaks of DE3 / Ep1 and DE3 / Ep2 are revealed at 1000 mAU (Figure 7A) and 600 mAU (Figure 7B) bands, respectively.

[0108] The lactose and IPTG association strategy proved promising and can be explored by evaluating different combinations in future scaling studies for recombinant protein induction in DE3 cultures. The purified epitopes of 17 kDa (DE3 / Ep1) and 26 kDa (DE3 / Ep2) were obtained with a concentration corresponding to 100 ng / µl and 110 ng / µl, respectively (Figure 1, Table 4). h X • L 7 7 Q m / ) 9 , 5 , g ( 2 1 4 1) h • P L Q m / l 4 o0 4 , 00 , o 0 t name m ( m i ( 1 ) d n • e S L - ) r / P m / L and l d Y o m / 6 l 3 8 the , 30, 0 emt n m ( m ) and i in L f and S / X m • l 6 5 o o 7 , 8 , C Y m 5 6 1 m / 7 g ( ) L P m / l o 6 5m , 1 06 , 0m ( ) µ 1 -h 0 (3 , 1 03 , / 0º ã ç e / s e u o d t n c G T s o t c G T I a P I a P I r L L o t a o t a o e rn e l a t a c n l a m s e c e m s o i u e u e B A d A e d a l u l é 3 E 3 E C D D a a a m e d d a a d a a t d a t t s l i e n a e l e n e S t a t B m i l a a B m i l : a e d m e a s A A : r 4 o g s t a e T T e n r pE S E S a V o l c x e R R l p p e e a b i c a a a d n o n ê í e n í e T p i T u q t o t e s r o P r P o ) ) d e e n a f l a n 1 p 2 E a f p l a n m o e g i A x / 3 i x E / 3 N o t e A d ( o t e d (

[0109] Additionally, seeking to improve the expression of recombinant proteins, new induction strategies to obtain Ep1 and Ep2 proteins were evaluated. Different temperatures (11, 22 and 37 °C), times (3 h and 16 h), competent cells (E. coli Bl21 (DE3) pLysS, E. coli Tuner DE3, E. coli Arctic Xpress DE3) and inducers (1 mM IPTG, 0.1 mM IPTG and 10 g / L Lactose) were tested.

[0110] In particular, it was observed that when the conditions 37 °C, E. coli Tuner DE3, 1 mM IPTG or 10 g / L lactose, for 3 hours were applied, the Ep1 protein showed an improvement in induction compared to the induction strategy initially proposed, as can be seen in Figure 12B, lanes 1 and 3.

[0111] It is important to emphasize that the lactose inducer is a non-toxic and low-cost reagent when compared to IPTG, therefore being ideal for expression in large volumes.The approximate yield obtained in the improved induction strategy was 10 mg / L, while in the initially proposed strategy it was approximately 0.05 mg / L.

[0112] Regarding the Ep2 protein, an improvement in induction was also observed, compared to the initially proposed strategy, when the conditions 37 °C, E. coli Bl21 (DE3) pLysS, 1 mM IPTG, for 16 hours of induction were employed. As can be seen in Figure 13B, lane 1.

[0113] The inducer lactose (10 g / L) produced similar expression levels after the same time tested (Figure 13B, lane 2). It is important to emphasize that the lactose inducer is a non-toxic and low-cost reagent when compared to IPTG, therefore being ideal for expression in large volumes. The approximate yield obtained in the induction strategy. improved was 5 mg / L, while in the initially proposed strategy it was approximately 0.05 mg / L.

[0114] On the other hand, the expressions using the E. coli Arctic Xpress DE3 strain did not show satisfactory results in any condition tested.

[0115] Thus, the tested improved induction strategies showed promise when employed in E. coli Tuner DE3 and E. coli Bl21 (DE3) pLysS.

[0116] In a second embodiment, the invention provides a vaccine composition comprising the alpha toxin epitopes of C. novyi.

[0117] In a preferred embodiment, the vaccine composition comprises the DE3 / Ep1 and DE3 / Ep2 epitopes as disclosed herein, preferably at the concentrations described in the present application as an antigenic component, which is mixed with an adjuvant.

[0118] Adjuvants are also useful for improving the immune response and / or increasing the stability of vaccine preparations.Adjuvants are typically described as non-specific stimulators of the immune system, but they can also be useful for targeting specific arms of the immune system. One or more compounds possessing this activity can be added to the vaccine.

[0119] Therefore, particular vaccines of the present invention further comprise an adjuvant. Examples of chemical compounds that can be used as adjuvants include, but are not limited to, aluminum compounds (e.g., aluminum hydroxide), metabolizable and non-metabolizable oils, mineral oils, including mande oleate derivatives in mineral oil solution (e.g., MONTANIDE ISA 61), and light mineral oils such as DRAKEOL 6VR. block polymers, ISCOM (immunostimulating complexes), vitamins and minerals (including, but not limited to: vitamin E, vitamin A, selenium and vitamin B12) and CARBOPOL ®;

[0120] Preferably, the adjuvant is MONTANIDE™ ISA 61 VG, Seppic, Brazil.

[0121] Furthermore, the vaccine compositions of the present invention may include diluents, isotonic agents, stabilizers, or adjuvants. Diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkaline salts of ethylenediaminetetraacetic acid, among others. Suitable adjuvants are known in the art.

[0122] Furthermore, according to the invention, an “immunological or immune response” to a composition or vaccine is the development in the host of a cellular and / or antibody-mediated immune response to the composition or vaccine of interest.Typically, an “immune response” includes, but is not limited to, one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells, and / or gamma-delta T cells, specifically directed to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will exhibit a therapeutic or protective immune response, such that resistance to further infection is increased and / or the clinical severity of the disease is reduced. Such protection will be demonstrated by a reduction or absence of symptoms associated with the host’s infections.

[0123] In a third embodiment, the invention provides the use of said vaccine epitopes in the preparation of a vaccine composition for the prevention and treatment of damage caused by the active alpha toxin of C. novyi type B.

[0124] The protein regions of the alpha toxin of C.novyi predicted and selected regions revealed potential for epitope selection, with the region corresponding to DE3 / Ep1 located in the N-terminal domain and the DE3 / Ep2 region belonging to the C-terminal domain. Both in the in silico model and after expression in DE3, the DE3 / Ep1 and DE3 / Ep2 epitopes revealed bands of 17 kDa and 26 kDa, respectively (Figure 1D).

[0125] Furthermore, their reactivity with serum from mice infected with wild-type C. novyi alpha toxin was demonstrated, highlighting the importance of these selected regions in recognizing specific antibodies present in the infection (Figure 1E).

[0126] Post-immunization follow-up revealed that the entire experimental group gained body weight, evidencing aspects of the formulation's harmlessness (Figure 8A). Post-challenge hematological data served to evaluate changes in hematological parameters and liver function (Table 5).

[0127] Table 5 shows the hematological data of experimental groups after immunization and challenge with dose 157 LD50.g. -1 . G1 (animals inoculated with adjuvant in W / O microemulsion). G2 and G3 – animals immunized with ALFA-T / REC DE3 / Ep1 and ALFA-T / REC DE3 / Ep2 in W / O microemulsion, respectively. G4 – animals immunized with ALFA-T / REC DE3 / Ep1+ALFA-T / REC DE3 / Ep2 in W / O microemulsion. Control (-) non-immunized / non-challenged animals (animals healthy); Control (+) non-immunized animals challenged doses 157 LD50.g -1 . 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[0128] The positive control group presented clinical pictures of polycythemia, in contrast, immunized mice from groups (G2), (G3) and (G4) showed data in accordance with reference values, and comparatively similar to data from healthy animals (Table 5).

[0129] Vaccine efficacy data revealed that animals from the control and Adjuvant groups (G1) did not differ from each other, with a high lethality rate being observed after the challenge test (Figure 9).

[0130] Animals immunized with microemulsion using DE3 / Ep1 (G2) and DE3 / Ep2 (G3) revealed a protection rate of 25% and 40%, respectively. The microemulsion composed of associated epitopes DE3 / Ep1 and DE3 / Ep2 (G4) provided a higher protection rate, with 75%.

[0131] Vaccines from groups G3 and G4 provided superior efficiency data to those using microemulsion containing the wild-type inactivated alpha toxin epitope (G5), with 25% (Félix et al. 2019) (P<0.005), while the microemulsion containing the DE3 / Ep1 epitope (G2) provided the same protection as G5 animals.

[0132] Our data showed that the obtained vaccine epitopes can be used as immunogens in vaccine protection against damage caused by the active alpha toxin of C. novyi type B.

[0133] However, the hematological findings (Table 5) are confirmed by the hematological data (Figure 8) and also correlate with the vaccine efficacy data (Figure 9), as well as in ELISA analysis data of liver and spleen of mice challenged with active alpha toxin of C. novyi type B (Figure 10).

[0134] This evidence indicates that in silico analysis of antigenic regions, followed by in vivo experiments, may be an effective strategy for considering a suitable candidate for vaccine development against C. novyi type B. However, advances in studies to optimize epitope yields are needed to enable adjustments in the concentration of each antigen in the vaccine dose.Histopathological analysis of liver and spleen tissue damage corroborated our findings, indicating the harmlessness of the vaccine groups with greater effectiveness in group G4, as well as the ELISA immunogenicity test to verify antibody production, indicating a greater capacity to stimulate the immune response of the vaccine group G4. Examples Example 1 – Bioinformatics tools and construction of the vaccine epitope expression vector

[0135] Computer programs were used to select epitopes of C. novyi alpha toxin (Clostridium novyi alpha-toxin – GenBank: CAA88565.1). The regions were predicted using Immune Epitope Database Analysis Resource (IEDB-AR) (http: / / tools.immuneepitope.org / main), selecting two alpha toxin epitopes located in amino acid 491° to 583° of the N-terminal region (DE3 / Ep1), and amino acids 1599° to 1779° of the C-terminal region (DE3 / Ep2), both obtained considering the relationship of antigenicity, flexibility and capacity to stimulate the adaptive immune response.

[0136] The DE3 / Ep1 and DE3 / Ep2 genes were synthesized by Invitrogen – Thermo Fisher Scientific. ® , with codon bias for E. coli inserted into the pRSET expression plasmid, which allows the addition of a tail polyhistidine (6xHIS) at the amino-terminal end of each protein in order to facilitate purification after cell lysis.

[0137] The plasmid containing the synthetic gene was transformed into electrocompetent DE3 using the 2510 electroporator (Eppendorf ® ), following the manufacturer's manual.

[0138] Then, the cells were plated in Luria Bertani (LB) culture medium (Kasvi ® ) containing ampicillin (100 µg / mL) (Multilab® ) and chloramphenicol (11.4 µg / mL) (Kasvi ® ), and incubated overnight at 37 °C. Expression of epitopes in DE3 was performed with isopropyl-β-D-thiogalactopyranoside (IPTG) induction (Invitrogen ® ), in LB medium with ampicillin and chloramphenicol.

[0139] The cultures were carried out in the first hours until growth of OD600 (0.6) at 37 °C, and after, induction with 1 mM IPTG, followed by temperature adjustment to 28 °C. Samples of DE3 cultures were treated with denaturing buffer (20% (v / v) glycerol (Synth ® ); sodium dodecyl sulfate (SDS) 4% (w / v) (Synth ® ); Tris pH 6.8 (100 mM) (Synth ® ); bromophenol blue 0.2% (w / v) (Synth ® ); beta-mercaptoethanol (200 mM) (BioRad ®)), and subsequent boiling treatment for 10 min.

[0140] Polyacrylamide gel electrophoresis (SDS-PAGE 12%) was performed to certify the expression of the corresponding epitopes of DE3 / Ep1 and DE3 / Ep2 genes, followed by Western blotting.

[0141] The design and expression of DE3 / Ep1 and DE3 / Ep2 in DE3 were performed in partnership with the Laboratory of Diagnosis and Control of Infectious Diseases of the Amazon (DCDIA), from the Leônidas & Maria Deane Institute (ILMD / Fiocruz Amazônia). Example 2 – Cultivation of DE3 on a rotary shaker

[0142] Isolated colonies of DE3 were inoculated in semi-defined culture medium (SD) supplemented with 100 μg / mL ampicillin. The SD medium was composed of 5.0 g / L glucose (Synth ® ); yeast extract 5.0 g / L (Synth ® ); potassium phosphate bibasic (K2HPO4) 9.4 g / L (Synth ® ); monobasic potassium phosphate (KH2PO4) 5.0 g / L; sodium chloride (NaCl) 5.0 g / L (Synth ® ), zinc chelate (EDTA-Zn) 1.0 g / L (Comnagro® ) and manganese chelate (EDTA-Mn) 0.2 g / L (Comnagro ® ).

[0143] The studies were conducted in triplicate on a rotary shaker SL-223 / F (Solab Cientifica ® ) controlled at 37°C, 200 rpm for 16 h. Inoculum fractions corresponding to 2.5%, 5% and 10% were evaluated, collecting samples, readings of optical densities (OD600), determining the specific growth rate (µh -1 ) and DCW. Example 3 – Cultivation of DE3 in SD medium in a stirred-tank biological reactor

[0144] Batch and fed-batch cultivations were conducted in a Tecnal BIO–TEC stirred-tank biological reactor ® with a capacity of 1 L, consisting of a jacketed glass container; digital control unit; and pH 7 control, using 3 M sodium hydroxide (NaOH) (Synth ®), at 37 °C; and dissolved oxygen (DO) supplementation strategy, with agitation cascade control of 100-1000 rpm, specific air feed flow input fixed at 3 vvm.

[0145] Inoculum propagation was initially performed from the stock culture of each DE3 (1.5 mL) activated in 8.5 mL of SD medium, under agitation at 200 rpm, 12 h at 37 °C, maintaining an inoculum fraction of 10% in the bioreactor.

[0146] During the fed-batch process, 7 mL of a 500 g / L glucose solution containing ampicillin at a concentration of 100 μg / mL were added at every 30 min intervals, until reaching the stationary growth phase. Samples were collected at 1-h intervals to determine growth kinetics, acetate and glucose consumption, as well as DE3 / Ep1 and DE3 / Ep2 synthesis.Example 4 – Scale-up of DE3 cultivation in SD medium in a non-stirred tank biological reactor

[0147] Fed-batch cultivations were conducted in a 20 L non-stirred tank biological reactor, consisting of a vat adapted from a round carboy (Thermo Scientific Nalgene. ®) with a wide mouth, with low-density polyethylene (LDPE) handles, which facilitate transport and dispensing, containing a screw cap (83B) in white polypropylene (PP).

[0148] The cultures were conducted with pH 7 control using 3 M NaOH, at 37 °C; and a dissolved oxygen (DO) supplementation strategy with a fixed air input at 3 vvm, without mechanical agitation. Inoculum propagation was performed using a stock culture of each DE3 (1.5 mL) activated in 8.5 mL of SD medium, under agitation at 200 rpm, 12 h at 37 °C, maintaining an inoculum fraction of 10% for cultivation in the bioreactor with a working volume of 10 L.

[0149] During the fed-batch process, 100 mL of a 500 g / L glucose solution plus 100 μg / mL ampicillin were added, at 30 min intervals until reaching the stationary growth phase. Samples were collected at 1 h intervals to determine growth kinetics, acetate and glucose consumption, as well as for DE3 / Ep1 and DE3 / Ep2 synthesis. Example 5 – Induction and purification system of recombinant DE3 / Ep1 and DE3 / Ep2 epitopes

[0150] In the first induction step, we used 3 pulses of 35 mL of 10 g / L lactose solution (Synth ® ), in cultures performed in the stirred tank biological reactor, immediately after maximum cell growth was achieved, followed by another addition after two hours of subsequent cultivation. In the scale-up using a non-stirred tank biological reactor, we used three pulses of 500 mL of the combination of 10 g / L lactose with 0.2 mM IPTG, at the same time intervals applied in the lab-scale experiments.

[0151] All induction studies were conducted for 6 h at 28 °C. A chromatography system (Akta Purifier 10 ® , GE Lifescience Healthcare® ) was used in the purification step of DE3 / Ep1 and DE3 / Ep2. The collected fractions were lyophilized and stored at -20 °C for analysis. Reverse-phase chromatography was performed according to that proposed by Field et al. (2021).

[0152] The DE3 / Ep1 and DE3 / Ep2 fractions, from affinity chromatography, after lyophilization, were solubilized in 0.1% trifluoroacetic acid (TFA) (solution A) (Merck ® ) and subjected to high-performance liquid chromatography (HPLC) on a C-18 column (25 mm x 4.6 mm, Supelco ® ), previously equilibrated with solution A and eluted under a gradient of 0 to 70% with solution B (acetonitrile 99.9% (Merck ®) and TFA 0.1%) in 5 column volumes, under a flow rate of 1 mL / min.

[0153] Elutions were monitored at 280 nm. Profiles for DE3 / Ep1 and DE3 / Ep2 were obtained, confirming the degree of purification and product concentration. The collected fractions were visualized again in 12% SDS-PAGE to check purity and confirm antigenicity by western blotting. Example 6 – Western blotting

[0154] Bands for DE3 / Ep1 and DE3 / Ep2, present in 12% SDS-PAGE gel, were transferred to nitrocellulose membranes following the BioRad protocol ® . A primary antibody derived from blood serum of Swiss mice infected with wild-type alpha toxin of C. novyi type B was used. The negative control used blood serum from healthy Swiss mice.

[0155] The secondary antibody anti-mouse IgG-rabbitA9044 (Sigma ®) (conjugated with peroxidase in the proportion 1:2,000 and 0.05% (v / v) of the substrate 3,3′-Diaminobenzidine (DAB)) was used to certify the reactivity with purified DE3 / Ep1 and DE3 / Ep2, compared with inactivated wild-type alpha toxin. Example 7 – Fermentation parameters Example 7.1 – Determination of glucose consumption

[0156] The determination of glucose consumption was performed by the liquid enzymatic glucose system (Labtest®) Eq. (1), where the principle consists of two reactions using the enzymes glucose oxidase (GOD) and peroxidase (POD) according to Bergmeyer et al. (1986). Eq. (1) G licose + O2 + H2O GOD Gluconic Acid + H2O22 H2O2 + 4 ― Aminoantipyrine + phenolPOD Antipyrliquinonimine + 4 H2

[0157] The product formed, 4-antipyrliquinonimine, has a reddish coloration, its intensity being directly proportional to the glucose concentration. The assays were prepared in shaken 2 mL tubes, incubated for 5 min at 37 °C, and the reading was performed at 510 nm. The glucose concentration was calculated as described in Eq. (2). Eq. (2) Example 7.2 – Determination of Hac and DCW production

[0158] The determination of Hac was carried out using the Shimadzu chromatograph ® (SPD – M2OA), equipped with a high-precision LC-6AD (CBM-20ª) pump and ultraviolet detectors that detect in the absorbance range of 190 to 700 nm, for the detection of Hac. The ion exclusion column used was the C-18 Shim-pack column ® (250 x 4.6mm, Shimadzu ®). The analysis was carried out for 12 min at 30 °C with a mobile phase flow rate of 0.6 mL / min. The mobile phase consisted of a 1% phosphoric acid (H3PO4) solution and an injection volume of 20 µL. The samples were filtered using a C-18 Chromabon membrane. ® (3mL / 500mg), previously and readings were taken at 204 nm. The Hac determination was performed using a standard curve and linear regression.

[0159] The DCW determination was obtained by the difference between mass A (mA) and mass B (mB), where mA represents the mass of the crucibles without sample, and mB the mass of the crucibles containing culture samples, and Al the volume of the sample. DCW values were generated by correlating the weights dry and OD600 of culture samples, where one (1) unit of OD600 corresponds to 0.61 g / L of dry weight of cells. The dry mass correlation was determined as described in Eq. (3). Eq. (3) ^^^^^^ = (^^^^ ― ^^^^)^^^^ . 1000 (g / L)Example 7.3 – Yield coefficients

[0160] The specific growth rate (μ) was determined during the exponential phase of bacterial growth, in which the specific growth rate is constant and maximum (µ x = µ m ). Values of μ were obtained using Eq. (4), where dX corresponds to the difference in DO 600 at a specific time point and a subsequent time point, dt corresponds to the time change and X corresponds to the cell concentration at that specific time point. Eq. (4) ^ ^ = ( ^^^^ ^ ^^^ ) / ^^ (h-1)

[0161] The yield coefficients ( ^^) were analyzed for biomass ( ^^^^) and product ( ^^^^) ^^ ^^ ,visualized in Eqs. (5) and (6). To calculate the biomass-to-product conversion factor, cell growth ( ^^) and substrate consumption ( ^^) were considered, which were related by Eq. (5). To calculate the product yield, the amount of product formed ( ^^) and substrate consumption ( ^^) were considered, which were related by Eq. (6). Eq. (7) and (8) define the volumetric productivity of the product (^^ ^^ ) and biomass (^^ ^^ ), in relation to time (^^). Eq. (5) ^ ^ ― ^^ ^^ ^^ = ^^ ^^ ^^ ― ^ (g / mmol.mL) ^^ ^^ ^^^ Eq. (6) Eq. (7) ^ ^ ― ^^^^ = ^^ ^^^^^^(mmol / mL.h)Eq. (8) Example 8 – Vaccine Formulation

[0162] Purified DE3 / Ep1 and DE3 / Ep2 were emulsified in MONTANIDE™ ISA 61 VG adjuvant, Seppic, Brazil. The aqueous phase of the vaccine (W) consisted of 40% recombinant antigen and the oil phase (O) of 60% adjuvant. The microemulsion was performed using sterilized 10 mL syringes connected to each other by silicone hoses. Volumes of the aqueous and oil fractions were forcibly transferred between the syringes over 10 minutes. Example 9 – Experimental Design

[0163] Swiss mice of both sexes, 4-6 weeks old and weighing between 17-23 g were used after approval by the Animal Use Ethics Committee (CEUA) of the Federal University of Tocantins (UFT), no. 23.101.006.832 / 2017-53.

[0164] The experimental groups were separated into 5 (five) groups, with 10 (ten) animals per group, being the groups: PBS (Non-immunized); Adjuvant in W / O microemulsion (G1); DE3 / Ep1 vaccine W / O microemulsion (G2); DE3 / Ep2 vaccine in W / O microemulsion (G3); DE3 / Ep1+DE3 / Ep2 vaccine in association and W / O microemulsion (G4). The groups were also compared with groups immunized with wild-type inactivated alpha toxin (G5) (Félix et al., 2019).

[0165] Each constituent was administered intramuscularly, in 3 (three) doses of 100 µL, with intervals of 15 (fifteen) days between each application and the weight of each animal was monitored during the experimental time.

[0166] The groups were challenged with a challenge dose (157 LD50.g. -1– lethal dose of active alpha toxin) and the Kaplan-Meier survival curve was determined. Blood samples were collected for specific antibody, hematological, and biochemical analyses. Livers and spleens of animals were also collected for histopathological analyses.

[0167] Surviving animals were previously anesthetized and euthanized with ketamine (300 mg / kg) (Vetbrands, Brazil) and xylazine (22.5 mg / kg) (Syntec ® , Brazil), and consciousness was certified by heartbeat and respiratory movements. Example 10 – Immunogenicity, hematological and biochemical data

[0168] Immunogenicity was determined by analyzing sera from immunized Swiss mice using an ELISA immunoassay. Blood samples from mice after the last immunization (45D) were evaluated for stimulation of total IgG production. The ELISA was applied using purified DE3 / Ep1 and DE2 / Ep2 proteins for standardization and antigen fixation in 96-well microplates (Nunc MaxiSorp durante) at 4°C.

[0169] After blocking for 1 hour with 2% casein-PBS buffer (blocking buffer) at room temperature, the plates were washed four times with 0.05% Tween-20-PBS buffer. The serum was diluted 1:50 with blocking buffer and incubated for 16 h at 4°C.

[0170] After washing, anti-mouse IgG peroxidase conjugate (Sigma, USA) was used at a ratio of 1:2000 in the blocking buffer. After washing, 3,3',5,5'-tetramethylbenzidine (BD Biosciences, USA) was added for 15 min and blocked with 2.5 M H2SO4 (Dinâmica, Brazil). Optical density was measured at 450 nm, and values above the cutoff were considered positive.

[0171] The mean optical density of the serum determined the cutoff point before immunization. Sera from non-immunized animals were used to calculate the cutoff.Furthermore, hematological and biochemical data were obtained by centrifuging the blood in microtubes at 3500 rpm for 15 minutes, with leukogram (total leukocytes, neutrophils and monocytes) and erythrocyte (erythrocytes, hemoglobin, hematocrit, MCV, MCH, MCHC and platelets) parameters being evaluated. Example 11.1 – Histopathology

[0172] We evaluated the levels of total IgG produced by mice challenged with active alpha toxin of C. novyi type B using the Enzyme Linked Immunosorbent Assay (indirect ELISA) technique, according to the protocol pre-established by the Laboratory for Diagnosis and Control of Infectious Diseases of the Amazon (LDCDIA).

[0173] To capture IgG, we sensitized the plate with antigen (inactivated alpha toxin of C. novyi type B, at a concentration of 4µg / ml, diluted in carbonate / bicarbonate buffer pH 9.6 and left overnight at 4°C.After incubation, we blocked the plate with PBS 1x / BSA 3% for 2 hours, followed by a washing process with PBS Tween 0.05% to add sera (1:1) from mice challenged with active alpha toxin. After adding the primer, the plate was incubated in a humid chamber in an oven at 37°C for 1 h, followed by the washing process. Subsequently, a polyclonal anti-mouse IgG antibody conjugated with peroxidase from Rhea Biotech® was applied for the development step. Finally, 100 µL of the chromogenic substrate Tetramethylbenzidine (TMB – Sigma®) was added to each well and left to react for 10 minutes, stopping the reaction with 2M sulfuric acid (H2SO4).

[0174] The optical density (OD) was determined by an ELISA reader (GLOMAX®) using a 450nm filter. To determine antibody levels, an antibody titration curve was performed using Rhea Biotech® mouse IgG at different concentrations (1000ng, 500ng, 250ng, 125ng, 62.5ng, and 31.25ng). 11.2 HISTOPATHOLOGICAL ANALYSIS

[0175] Liver and spleen of Swiss mice from each experimental group were removed by necropsy and fixed in 10% buffered formalin for histopathological analysis. The tissues were processed and embedded in paraffin and stained by H&E staining.

[0176] The main lesions in the livers, such as hydropic degeneration of hepatocytes, inflammation, perivasculitis, hyperemia and necrosis were evaluated. In the spleen. periarteriolar and follicular hyperplasia in white and red pulps, and inflammatory infiltrates were evaluated.

[0177] Each animal was analyzed according to the degree of lesions following the scoring system: 0) absent lesions; 1) discrete lesions up to 25% of the observation field; 2) moderate lesions greater than 25% and less than 50% of the observation field; 3) strong lesions greater than 50% of the observation field.

[0178] Analysis of the post-immunization humoral response revealed greater stimulation in IgG levels for group G4 (associated epitopes DE3 / Ep1 and DE3 / Ep2), with values close to 1000ng / mL, levels significantly higher than the IgG levels obtained in the Control (450ng / mL), G1 (Adjuvant) (700ng / mL), G2 (DE3 / Ep1) (820ng / mL) and G3 (DE3 / Ep2) (915ng / mL) groups (p<0.05), evidencing greater capacity to stimulate the immune response against C. novyi type B (Figure 11).

[0179] The sepsis model of infection with active alpha toxin of C.novyi type B showed tissue damage in the livers and spleens of mice after immunization and challenge with a dose of 157 LD50.g. -1 . Histopathological findings revealed greater severity of degeneration, hemorrhage, and hyperemia in groups G1 (Adjuvant), G2 (DE3 / Ep1), and G3 (DE3 / Ep2). In contrast, discrete changes were observed in group G4 (associated epitopes DE3 / Ep1 and DE3 / Ep2), similar to the data observed in the Control group (Figure 11). Example 12 – Statistical Analysis

[0180] The experiments were conducted following a completely randomized design using analysis of variance (ANOVA). Kinetic parameters obtained in agitated and non-agitated systems were analyzed comparatively.

[0181] The PBS vaccine group (non-immunized) was used to compare the immunized groups. Data means were analyzed by Student's t-test, considering p-value (P<0.05) as significant using Origin Pro software. ®8.5. Kaplan-Meier survival curve defined the probability of vaccine efficacy. Example 13 – Transformation and Expression of EP1 and EP2 proteins in E. coli BL21 (DE3) pLysS

[0182] The transformation of Escherichia coli BL21 (DE3) pLysS™ bacteria with pRSET plasmids containing the EP1 and EP2 genes was performed following the following procedure:

[0183] First, 0.5 μL (corresponding to 25 ng) of the plasmid was added to a total of 50 μL of the competent E. coli BL21 (DE3) pLysS cell. Subsequently, this mixture was exposed to an electric pulse with an intensity of 1.9 kV using a 2510 electroporator (Eppendorf).

[0184] Immediately afterwards, the cells were resuspended with 450 mL of liquid LB medium and the contents were then placed in a 2.0 mL microtube. This microtube was kept at 37 ºC, with constant agitation at 150 rpm, for a period of 1 hour.Then, 100 µL of the transformed strain was distributed on a culture plate containing LB agar medium, to which 100 μg / mL of ampicillin and 25 μg / mL of chloramphenicol were added. These plates were incubated at 37 ºC for a total of 16 hours.

[0185] Then, randomly selected colonies from the plates were pre-inoculated separately into test tubes, each containing 3 mL of LB liquid medium, and containing ampicillin (100 µg / mL) and chloramphenicol (25. μg / mL) as antibiotics for selection. After a shaking incubation period at 37 °C for 16 hours, the culture was transferred to 100 mL of liquid LB medium containing the selection antibiotics. The temperature was maintained at 37 °C on the shaker until the optical density (OD) reached 0.6, measured in a spectrophotometer reading at 600 nm.

[0186] At this point, 15 mL of the bacterial culture were distributed into six conical tubes, and a final concentration of 0.1 mM and 1 mM IPTG (isopropyl-β-D-thiogalactopyranose) and a final concentration of 10 g / L lactose, respectively, were added to each tube.

[0187] Induction with IPTG and lactose was performed under the following conditions: 3 hours and 16 hours; at 37 °C, 22 °C, and 11 °C. Immediately after, centrifugation was performed at 3500 xg for 15 minutes at 4 °C. The bacterial pellets were stored in 50 mL conical tubes and stored at -20 °C, awaiting the subsequent sonication step.This procedure consisted of resuspending the bacterial pellets in MCAC-0 buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 10% glycerol, and 1 mM PMSF), followed by sonication using a procedure standardized by our team. The sonicated material was centrifuged at 10,000 xg, 4 °C, for 15 min. After sonication, analysis was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Example 14 – Transformation and Expression of the EP1 protein in E. coli Arctic Xpress DE3

[0188] The transformation of the Escherichia coli Arctic Xpress DE3 bacteria with the pRSET plasmids containing the EP1 gene was performed following the following procedure:

[0189] First, 1 μL (corresponding to 50 ng) of the plasmid was added to a total of 50 μL of the competent E. coli Arctic Xpress DE3 cell.Subsequently, this mixture was exposed to an electric pulse with an intensity of 1.9 kV using a 2510 electroporator (Eppendorf).

[0190] Immediately thereafter, the cells were resuspended with 450 mL of liquid LB medium and the contents were then placed in a 2.0 mL microtube. This microtube was kept at 37 ºC, with constant shaking at 150 rpm, for a period of 1 hour. Then, 100 µL of the transformed material was distributed on culture plates containing LB agar medium, to which 100 μg / mL of ampicillin was added. These plates were incubated at 37 ºC for a total of 16 hours.

[0191] Then, randomly selected colonies from the plates were pre-inoculated separately into test tubes, each containing 3 mL of liquid LB medium, containing ampicillin as antibiotics for selection. After a period of incubation with shaking at 37°C for 16 hours, the culture was transferred to 50 mL of liquid LB medium containing the selection antibiotic.The temperature was maintained at 37 °C on the shaker until the optical density (OD) reached 0.6, measured in a spectrophotometer reading at 600 nm.

[0192] At this point, the bacterial culture was distributed into six conical tubes, and a final concentration of 0.1 mM and 1 mM of IPTG (isopropyl-β-D-thiogalactopyranose) and lactose (10 g / L, final concentration), respectively, was added to each tube.

[0193] Induction with IPTG and lactose was performed under the following conditions: 3 hours and 16 hours; at 11 °C. Immediately after, centrifugation was performed at 4000 rpm for 15 minutes. at 4°C. The bacterial pellets were stored in 50 mL conical tubes at -20°C, awaiting the subsequent sonication step. This procedure consisted of resuspending the bacterial pellets in MCAC-O buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 10% glycerol, and 1 mM PMSF), followed by sonication using a procedure already standardized by our team. The sonicated material was centrifuged at 14,000 rpm, 4°C, for 15 minutes. After the sonication procedure, analysis was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Example 15 – Transformation and Expression of EP1 Protein in E.coli Tuner DE3

[0194] Transformation of Escherichia coli Tuner DE3 bacteria with pRSET plasmids containing the EP1 gene was performed following the procedure:

[0195] First, 1 μL (corresponding to 50 ng) of the plasmid was added to a total of 50 μL of E. coli Tuner DE3 competent cells. Subsequently, this mixture was exposed to an electric pulse with an intensity of 1.9 kV using a 2510 electroporator (Eppendorf).

[0196] Immediately afterwards, the cells were resuspended with 450 mL of liquid LB medium and the contents were then placed in a 2.0 mL microtube. This microtube was kept at 37ºC, with constant shaking at 150 rpm, for a period of 1 hour. Then, 100 µl of the transformed material was distributed on culture plates containing LB agar medium, to which 100 μg / mL of ampicillin was added. These plates were incubated at 37ºC for a total of 16 hours.

[0197] Then, randomly selected colonies from the plates were pre-inoculated separately.in test tubes, each containing 3 mL of liquid LB medium, containing ampicillin as antibiotics for selection. After a period of incubation with shaking at 37°C for 16 hours, the culture was transferred to 50 mL of liquid LB medium containing the selection antibiotic. The temperature was maintained at 37°C on the shaker until the optical density (OD) reached 0.6 measured in a spectrophotometer reading at 600 nm.

[0198] At this point, the bacterial culture was distributed into two conical tubes, in which IPTG (isopropyl-β-D-thiogalactopyranose) was added at a final concentration of 0.1 mM and 1.0 mM, and lactose (10 g / L, final concentration), respectively, in each tube.

[0199] Induction with IPTG and lactose was performed under the following conditions: 3 hours and 16 hours; at 37°C and 22°C. Immediately after, centrifugation was performed at 4000 rpm for 15 minutes at 4°C. The bacterial pellets were stored in 50 mL conical tubes and stored at -20°C, awaiting the subsequent sonication step.This procedure consisted of resuspending the bacterial pellets in MCAC-O buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 10% glycerol, and 1 mM PMSF), followed by sonication using a procedure standardized by our team. The sonicated material was centrifuged at 14,000 rpm, 4 °C, for 15 minutes. After the sonication procedure, analysis was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Example 17 – Purification of recombinant proteins EP1 and EP2 obtained in Examples 13 to 16

[0200] The proteins were purified by immobilized metal affinity chromatography (IMAC). For this purpose, a purification column containing Ni2+ ions (QIAGEN) was used, following the manufacturer's instructions. Protein elution was performed using an imidazole concentration curve from 100 mM to 500 mM. Samples were then quantified using Bradford reagent (Bio-rad), following the manufacturer's instructions. Yield was determined by summing the protein concentrations in milligrams obtained in each elution of the isolated proteins in parallel with the volume of bacterial growth initially used. Therefore, if 10 mg and 5 mg of proteins were obtained in 1 L of induction, the estimated yields are 10 mg / L and 5 mg / L, respectively. Example 18 – Analysis by polyacrylamide gel electrophoresis and Western Blot of the recombinant proteins obtained in Examples 13 to 16

[0201] The evaluation of the expression of the recombinant proteins was performed in polyacrylamide gel (SDS-PAGE).Electrophoresis was performed in Tris-glycine buffer for 1h30min at 150V. The molecular mass marker "Blueye Prestained Protein Marker" (Sigma Aldrich) was used as a reference. The samples were resuspended in resuspension buffer (200 mM Tris-HCl pH 6.8, 0.1% bromophenol blue, 4% SDS, 4% β-mercaptoethanol, and 20% glycerol). Subsequently, the samples were denatured at 95°C for 10 min and then applied to the gels. The gels were stained to visualize the protein bands using a commercial buffer from Scienco.

[0202] To perform Western blot assays, the membranes were prepared using an SDS-PAGE gel with the samples (unstained) and a Semi-dry system. (Bio-rad), in tris-glycine-SDS buffer. The system was subjected to a constant voltage of 10 V for 1 hour and 10 minutes. After being removed from the tank, the membrane was stored in 1X PBS at 4 °C until development. Then, development was performed by first blocking the membrane for 30 minutes using a 1X PBS solution with 3% BSA. After that, the membrane was washed with MiliQ water for 5 minutes and then the anti-6xhisG monoclonal antibody (Invitrogen), diluted at a concentration of 1:3000, was added for 1 hour. The membrane was then washed 3 times, for 5 minutes each, with 1X PBS solution added with 0.05% Tween 20. The secondary antibody anti-mouse IgG + alkaline phosphatase (Seracare), diluted 1:10000, was added and incubated for 1 hour. 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Claims

CLAIMS 1. Recombinant nucleic acid molecule characterized by the fact that it comprises the nucleic acid sequences defined by SEQ ID NO: 1 or SEQ ID NO: 3 and their degenerate sequences that encode the same polypeptide sequence.

2. Recombinant C. novyi alpha toxin epitope characterized by the fact that it comprises the peptide sequences defined by SEQ ID NO: 2 or SEQ ID NO:

4.

3. Epitope, according to claim 2, characterized by the fact that it further comprises a polyhistidine tag (6xHIS) at the amino-terminal end.

4. Expression vector characterized by the fact that it comprises the nucleic acid molecules as defined in claim 1 and their regulatory regions.

5. Expression vector, according to claim 4, characterized by the fact that the controlling regions are selected from BamHI and EcoRI. 6.Vaccine composition characterized by the fact that it comprises one or more epitopes as defined in claim 2 and veterinarily acceptable excipients.

7. Vaccine composition characterized by the fact that it comprises the two epitopes as defined in claim 2 and veterinarily acceptable excipients.

8. Vaccine composition, according to claim 6, characterized by the fact that it is a water / oil emulsion.

9. Vaccine composition, according to claim 6, characterized by the fact that it is for the prevention or treatment of clostridiosis in an individual suffering from it.

10. Use of one or more epitopes as defined in. claim 2, characterized in that it is in the preparation of a vaccine composition to prevent or treat clostridiosis in an individual suffering from the same.

11. Method for preventing or treating clostridiosis in an individual suffering from the same, characterized in that it comprises administering the vaccine composition as defined in claim 6.

12. Method for producing a vaccine composition against clostridiosis, characterized in that it comprises the following steps: i) Transforming E. coli cells with an expression plasmid comprising the molecules as defined in claim 1; ii) Cultivating said cells in bioreactors through the fed-batch process; iii) Purifying the product from cultivation step ii); iv) Preparing the vaccine composition comprising the epitopes obtained after step iii).

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