Recombinant nucleic acid molecules, recombinant α-toxin epitopes from Clostridium noveni and their uses, expression vectors, vaccine compositions, methods for preventing or treating subjects with clostridial diseases, and methods for preparing vaccine compositions against clostridial diseases.

Abstract

This invention provides recombinant nucleic acid molecules encoding recombinant α-toxin epitopes of Clostridium novyi. Furthermore, this invention provides vaccine compositions comprising such epitopes and their uses, as well as methods for preparing such compositions. Finally, this invention also provides methods for treating and preventing clostridial diseases.

Description

Technical Field

[0001] This invention relates to the fields of veterinary medicine and biotechnology. Specifically, this invention relates to the use of *Escherichia coli* (E. coli) Escherichia coli During the culture and amplification of BL21(DE3), the development and production of products derived from Clostridium novie (… C. novyi The epitopes of the toxin, and its use in vaccine formulations for bovine clostridial disease. Background Technology

[0002] Cattle ranching is one of Brazil's most important economic activities. The Brazilian Institute of Geography and Statistics (IBGE) estimates that the country currently has over 220 million cattle, with the central and western regions having the largest herds (IBGE, 2021). As the cattle industry becomes more specialized, the requirements for disease control and product quality monitoring are becoming increasingly stringent (Ferraz and Felício, 2010; Carvalho and Zen, 2017).

[0003] There is a class of infectious and toxic diseases affecting cattle herds, caused by Clostridium ( ). Clostridium This condition is caused by anaerobic bacteria and is called "clostridiosis." Depending on the species of bacteria from the genus Clostridium, it can cause the following diseases: nephrotic syndrome, hemorrhagic enterotoxemia, necrotizing infectious hepatitis, and sudden death (Embrapa, 2001).

[0004] Clostridium infection is a typical disease affecting cattle, caused by bacteria of the genus Clostridium. These bacteria are anaerobic, Gram-positive, motile rod-shaped, and can cause huge losses to the cattle industry (Ma et al., 2007; Lobato et al., 2013; Walker et al., 2016).

[0005] Strains of this genus cause disease by producing toxins and / or invading tissues, which vary depending on the presence and synthesis of genes (Uzal et al., 2010; Brandi et al., 2014; Brandi et al., 2016; Fouad et al., 2021).

[0006] Clostridium novesiculum is a common pathogen in livestock and humans. Based on the type of toxin it produces, it can be classified into four types: A, B, C, and D, with type D being identified as hemolytic clostridium. C. haemolyticum These potent exotoxins mediate the pathogenic process of Clostridium (Uzal et al., 2010; Navarro et al., 2017).

[0007] Clostridium perfringens type B produces an alpha toxin, which is the primary cause of the strain's pathogenicity (Le Gratiet et al., 2021). This toxin belongs to the large clostridium toxin family and has a molecular weight of 250 kDa (Lima et al., 2011). It possesses glycosyltransferase activity, which leads to the inactivation of proteins in the cytoskeleton and the disruption of actin filament structure, resulting in cell rounding, loss of intercellular junctions, and increased endothelial permeability (Gonçalves et al., 2013).

[0008] Clostridium novesiculosus alpha toxins are produced by types A and B and encoded by chromosomal genes cpa or plc; while beta toxins are produced by types B and D and encoded by genes in plasmids. Alpha toxins are lethal; beta toxins, although classified as toxins with necrotizing and hemolytic effects, are not usually produced at lethal doses (Navarro et al., 2017).

[0009] Alpha toxins, or PLCs, belong to the large clostridium family and have a molecular weight exceeding 250 kDa. The mechanism of action of these toxins is to cause cells to become rounded, an effect that alters the cytoskeleton structure of mammalian cells (Lima et al., 2020).

[0010] The resulting alpha toxin is defined as a glycosyltransferase responsible for catalyzing the glycosylation reaction of small GTPases, leading to the inactivation of proteins in the cytoskeleton and the disorder of actin filament structure, which in turn causes cell rounding, loss of intercellular connections and increased endothelial permeability (Lobato et al., 2013; Lobato et al., 2021).

[0011] Studies of the three-dimensional structure of alpha toxin have revealed two domains: an N-terminal (amino-terminal) domain, characterized as a single-domain protein; and a C-terminal (carboxyl-terminal) domain, characterized as a two-domain protein (Noshahri et al., 2016). The N-terminal domain of alpha toxin retains phospholipase activity but loses hemolytic activity. The C-terminal domain influences the enzymatic activity of the N-terminal domain and recognizes membrane phospholipids, which is crucial for the hemolytic activity of alpha toxin (Nagahama et al., 2013).

[0012] That is, alpha toxin has a three-segment structure: its enzymatic activity is related to the N-terminal region (the so-called catalytic domain); its binding function to the receptor is related to the C-terminal region of the toxin; and the process of toxin transport into the cytosol is related to the hydrophobic domain of its middle region (Nagahama et al., 2013; Noshahri et al., 2016).

[0013] Liver necrosis is one of the diseases caused by infection with Clostridium novyi type B (Ma et al., 2007). This disease is associated with trematodes (such as Fasciola hepatica). However, cases of this disease have also occurred in the absence of such parasitic infections (Lobato et al., 2008; Lobato et al., 2013).

[0014] Structurally, alpha toxins are characterized by the presence of zinc ions that strongly bind to interchangeable divalent cations. His-68, His-126, and His-136 residues bind to the divalent cations, which are essential for the toxin's binding to the cell membrane. His-148 and Glu-152 bind to zinc ions, which are essential for the toxin's active site; His-11 and Asp-130 bind firmly to another zinc ion, which is essential for maintaining the structure.

[0015] Amino acids Tyr-57 and Tyr-65 play a role in the process of toxins penetrating the cell membrane bilayer and provide channels for toxins to contact sphingomyelin catalytic sites (Nagahama et al., 2006; Nagahama et al., 2013; Oda et al., 2015).

[0016] The hemolytic, myotoxic, and cytotoxic activities of alpha toxins can be attributed to their interaction with the host cell membrane and subsequent phospholipid hydrolysis (Alape-Girón et al., 2000). Furthermore, some reports describe alpha toxins possessing both phospholipase C (PLC) and sphingomyelinase (SMase) activities.

[0017] Recent research has revealed that among numerous bacterial toxins, including the large family of clostridial glycosylated toxins (LCGTs), the membrane localization domain (MLD) has been identified as a conserved domain that plays a crucial role in plasma membrane localization (Liu et al., 2022).

[0018] As described in this article, the α-toxin (i.e., PLC) of Clostridium novyi type B belongs to the LCGT glycosylated toxin family and has phospholipase C and sphingomyelinase activities; its C-terminal region is directly associated with the binding of a membrane receptor that can recognize membrane phospholipids (Noshahri et al., 2016; Liu et al., 2022).

[0019] Furthermore, in multiple Clostridium strains, the plc gene is located at the same site, adjacent to the presumed origin of replication and situated between two ribosomal RNA operons. Notably, this is a highly important region; because this region of the chromosome is not only the first part to be replicated, but also the most conserved segment of the genome containing "housekeeping genes" (housekeeping genes are constitutive genes essential for cell survival, expressed in all cells of an organism under normal and pathophysiological conditions).

[0020] Existing literature reports that the plc gene has a nucleotide sequence of 2.2 kb, containing an open reading frame encoding amino acid residues from position 943 to 2137, corresponding to a protein with a molecular weight of 43.3 kDa. The amino acid sequence encoded between the start codon and nucleotide 1027 constitutes a signal sequence for the direct transmembrane transport of α-toxin (Tsutsui et al., 1995; Noshahri et al., 2016; Liu et al., 2022).

[0021] Another aspect revealed is that alpha-toxins can exhibit two distinct conformations: an "open conformation" where the active site is accessible, and a "closed conformation" where the active site is masked, with the masked region encompassing amino acid residues from position 135 to 150. Predictions of the alpha-toxin structure indicate that its N-terminal region contains 687 amino acid residues, located in the first part of the protein, and is smaller than the C-terminal region.

[0022] On the other hand, the C-terminal region of this protein consists of about 1,000 amino acid residues. Due to the high content of β-sheet structure, it is more antigenic and can elicit a stronger immune response compared to the helical region (Noshahri et al., 2016).

[0023] Furthermore, the binding of the C-terminal domain to the target membrane causes the enzyme's active site to be positioned toward the lipid bilayer, thereby exposing it; this characteristic is crucial for the enzyme to exert its toxic effects (Walker et al., 2000; Alape-Girón et al., 2000; Oda et al., 2015).

[0024] Previous studies have shown that when animals are immunized with the C-terminal domain, the antibodies produced can ensure that the animals are protected after being challenged with a lethal dose of active toxin; this indicates that the region is an important and highly antigenic region that is very likely to trigger a humoral immune response.

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

[0026] Infection occurs via latent spores; these spores germinate within the liver and typically occur in areas damaged by liver fluke migration. The spores then develop into vegetative bodies and induce the production of large amounts of alpha toxin (Uzal, 2003; Lima et al., 2011).

[0027] The resulting systemic effects can lead to animal death (Farias et al., 2014; Navarro et al., 2017). In addition, the proliferation of Clostridium perfringens type B in the liver can also cause sudden death (Lobato et al., 2008; Aquino et al., 2016; Radostits et al., 2002).

[0028] The pathogens of clostridial diseases have certain characteristics that make their eradication very difficult; this is because these bacteria can form highly resistant spores and are widely present in the gastrointestinal tracts of animals and humans.

[0029] Therefore, the prevention and control of this disease mainly relies on preventive measures and systematic vaccination of animals (Lima et al., 2011; Skarin; Segerman, 2014).

[0030] However, there are still some technical hurdles to be overcome for certain types of Clostridium pathogens (such as Clostridium botulinum type B), such as the immunogenicity of vaccine formulations (Nascimento et al., 2004).

[0031] Vaccines used to control liver necrosis

[0032] The veterinary drug market is showing growth due to demand from producing animals (especially livestock) (Ferraz and Felício, 2010; Carvalho and Zen, 2017).

[0033] The animals were immunized against clostridial disease using concentrated toxoids, with two doses of the vaccine providing immunity for up to one year; however, there is no official control to verify the efficacy of these immunogens (Lobato et al., 2008; Lima et al., 2011; Lobato et al., 2013).

[0034] This market is dominated by multinational corporations whose clostridial vaccines contain inactivated toxins in their formulations, and contain a variety of antigens and a wide range of pathogens and toxins. The vast majority consist of antigens targeting myonecrosis, enterotoxemia, hemoglobinuria, liver necrosis, botulism, and tetanus (Lobato et al., 2013).

[0035] In their 2004 study, Nascimento et al. analyzed vaccines against Clostridium bovis type B and demonstrated that products sold in the domestic market had low immunogenicity.

[0036] Because it is a difficult microorganism to cultivate, obtaining α-toxin from Clostridium novesiculosus has always been costly, leading to instances of failure in industrial bioprocessing stages (Zeng et al., 2011; Aquino et al., 2016; Navarro et al., 2017).

[0037] Technical alternatives, such as recombinant protein expression, have become the subject of computer simulation studies on the selection of vaccine epitope regions (Lobato et al., 2013; Lima et al., 2020; Le Gratiet et al., 2021).

[0038] Research related to vaccines developed using recombinant proteins (obtained from genetically modified microbial fermentation) has reduced production costs and provided standardization for manufacturing. Bioinformatics tools can be used to analyze hydrophobicity, accessibility, antigenicity, and protein flexibility, enabling the selection of potential regions for protein construction (Santos et al., 2012). Following these lines of thought, one relevant aspect is the use of epitopes from conserved gene regions of Clostridium novie in vaccine formulations. Patent CN110041437-B uses the C-fragment of tetanus toxin and three N-terminal epitopes and one C-terminal epitope of Clostridium novie alpha toxin; however, it does not use genes from conserved C-terminal regions.

[0039] However, recombinant vaccines developed from antigens derived from conserved regions allow for stronger immune stimulation of the host, as these regions are always present in bacterial infections, making them prime targets for recombinant vaccine development. Furthermore, these regions provide better protection compared to stimulation induced by inactivated bacterial vaccines, which offer limited protection (Perez-Casal, J. et al., 2017; Baruah, N. et al., 2021; Cangussu, ASR et al., 2018).

[0040] The production of recombinant Clostridium novie α-toxin is usually obtained by transforming Escherichia coli BL 21 (Zeng et al., 2011).

[0041] Expression of recombinant proteins

[0042] Computer simulation studies have enabled the acquisition of selectable epitopes used in the construction of expression vectors (Sobrinho et al., 2010; Santos et al., 2012; Santos et al., 2019; Brito et al., 2021), their synthesis in cell cultures, and their validation by sera reactivity with 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).

[0043] Recombinant protein production and expression utilize different host cells, introducing regions of interest into them. Recombinant DNA technology allows for the low-cost acquisition of pure and functional proteins (Lee et al., 2003; Nosshahri et al., 2016; Kojima et al., 2022).

[0044] Several expression systems were used for this purpose, with Enterobacter coli being the most commonly used, showing a recorded utilization rate of up to 60% for recombinant protein production (Duarte et al., 2021; Cardoso et al., 2022). These were chosen because of the abundance of genetic, physiological, and biochemical information available, as well as tools that can be used for their genetic manipulation (Correa and Oppezzo, 2011; Zeng et al., 2011).

[0045] Several vectors are available for protein expression in E. coli, promoting high expression levels, stability, and versatility (Jonasson et al., 2002). In addition to these factors, the efficacy of Clostridium vaccines is also related to the adjuvants that constitute them (Assis et al., 2002).

[0046] Oily adjuvants and microemulsions

[0047] Adjuvants are one of the most important factors in vaccine compositions and are directly related to increasing the immunogenicity of vaccines. Aluminum- and mineral oil-based adjuvants are most commonly used in veterinary vaccines (Park et al., 2014; Veenstra et al., 2017).

[0048] Aluminum-based adjuvants have several drawbacks, such as inducing short-lived antibody responses, leading to frequent revaccinations and side effects. In contrast, oil-based adjuvants have the advantage of inducing high antibody titers and long-lived antibody responses, resulting in effective protection (Khorasani et al., 2016; Tehrani et al., 2016).

[0049] Emulsified oil adjuvants influence the timing, location, and concentration of antigens through a "reservoir effect." Microemulsions form semi-solid clumps at the injection site, slowly releasing antigens into tissues or the bloodstream, increasing and prolonging humoral responses (Tehrani et al., 2016; Veenstra et al., 2017).

[0050] These systems have the advantage of good injection capability due to their low oil content and viscosity, and can easily present antigens (Leclercq et al., 2011; Sun et al., 2015).

[0051] Antigens with emulsifying adjuvants initiate innate immune responses and play a role in activating adaptive immune responses by stimulating specific components of humoral or cell-mediated immune responses (Grun and Maurer, 1989; Veenstra et al., 2017).

[0052] In view of this situation and considering the relevance of these immunogens in anti-clostridium vaccine formulations, the present invention proposes to optimize and scale up the acquisition of Clostridium novesiculum α-recombinant epitopes in recombinant Escherichia coli BL21 pLysS (DE3) cultures.

[0053] In this regard, three different epitope combinations (DE3 / Ep1, DE3 / Ep2, and DE3 / Ep1+DE3 / Ep2) of recombinant Clostridium novilis type B α-toxin were proposed in a W / O emulsion. Furthermore, immunogenicity and protective parameters in immunized and challenged mouse models were determined, aiming to explore new alternatives and advancements for antigen adsorption suitable for clostridial disease control. Genetic engineering tools were used in the epitope selection process, where the DE3 / Ep1 epitope contains an N-terminal domain region (between amino acids 1 and 583, vaccine group G2), the DE3 / Ep2 epitope contains a C-terminal domain region (between amino acids 1599 and 2178, vaccine group G3), and the combined epitope DE3 / Ep1+DE3 / Ep2 contains two domains (vaccine group G4). Groups G2, G3, and G4 were compared with group G1 (adjuvant MONTANIDE™ ISA 61 VG, Seppic, Brazil), showing better vaccine efficacy data.

[0054] Therefore, a vaccine formulation comprising two epitopes from the Clostridium novie α-toxin sequence (GenBank CAA88565.1), amino acids 491 to 583 in the N-terminal region and amino acids 1599 to 1779 in the C-terminal region, was developed. The formulation was prepared as an oil-in-water emulsion in a 40 / 60 ratio, and in vivo testing was conducted, yielding results indicating its potential as an immunogenic composition against diseases caused by Clostridium novie type B pathogen.

[0055] The results demonstrated its effectiveness as an immunogen in vaccine protection, as well as its protective effect against damage caused by the active alpha toxin of Clostridium novilis type B. The experimental group G3 (C-terminus) showed a higher vaccine efficacy (40% vs. 25%) than the G2 group (N-terminus). When the combined domain (G4) was used, mouse immunization exhibited a synergistic effect on protective efficacy, with 75% of animals surviving a lethal dose of the active toxin.

[0056] Finally, a mechanically stirred tank bioreactor was used to improve the conditions of the bioprocess and to develop epitopes for the scale-up production of Clostridium novesiculosus α-toxin, thereby contributing to a competitive advantage in the veterinary vaccine manufacturing industry. Summary of the Invention

[0057] The object of this invention is to provide an α-toxin epitope of Clostridium novitidis for use in the production of vaccine compositions, and a method for producing the epitope. Preferably, the epitope is derived from a conserved region of a Clostridium novitidis gene.

[0058] In a first embodiment, the present invention provides a recombinant nucleic acid molecule comprising a nucleic acid sequence as defined in SEQ ID NO: 1 or SEQ ID NO: 3 and a degenerate sequence encoding the same polypeptide sequence.

[0059] In a second embodiment, the present invention provides a recombinant α-toxin epitope of Clostridium novesiculum comprising a peptide sequence as defined in SEQ ID NO: 2 or SEQ ID NO: 4.

[0060] In the third embodiment, the recombinant α-toxin epitope of Clostridium novelisti also includes a polyhistidine tag (6×HIS) at the amino terminus.

[0061] In a fourth embodiment, the present invention provides an expression vector comprising a nucleic acid molecule and its regulatory region according to the present invention.

[0062] In the fifth embodiment, the expression vector contains a regulatory region selected from BamHI and EcoRI.

[0063] In a sixth embodiment, the present invention provides a vaccine composition comprising one or more epitopes and veterinary-acceptable excipients according to the invention.

[0064] In the seventh embodiment, the vaccine composition comprises two epitopes according to the invention and a veterinary-acceptable excipient.

[0065] In the eighth embodiment, the vaccine composition may be of the water / oil emulsion type and is used to prevent or treat clostridial disease in a subject.

[0066] In a ninth embodiment, the present invention provides the use of the epitope as disclosed herein in the preparation of a vaccine composition for the prevention or treatment of clostridial disease in a subject suffering from clostridial disease.

[0067] In a tenth embodiment, the present invention provides a method for preventing or treating clostridial disease in a subject, comprising administering a vaccine composition as disclosed in this application.

[0068] In the eleventh embodiment, the present invention provides a method for preparing a vaccine composition against clostridial disease, comprising the following steps:

[0069] i) Transform E. coli cells with an expression plasmid containing the molecules as defined above;

[0070] ii) The cells are cultured in a bioreactor using a fed-batch process;

[0071] iii) Purify the culture product from step ii);

[0072] iv) Prepare a vaccine composition containing the epitopes obtained after step iii). Attached Figure Description

[0073] Figure 1 : Figure 1 Computer simulation modeling and detection of vaccine epitopes. (A) 3D structure of α-toxin fragments from Clostridium novilis type B predicted by i-TASSER based on GenBank data: CAA88565.1

[48] . (B) and (C) are the 3D structures of DE3 / Ep1 and DE3 / Ep2, respectively. (D) Detection of DE3 / Ep1 (17 kDa) and DE3 / Ep2 (26 kDa) using 12% gel electrophoresis. (E) Western blotting of vaccine epitopes in the serum of Swiss mice infected with wild-type Clostridium novilis α-toxin.

[0074] Figure 2 : Figure 2 This relates to the α-toxin carriers ep1 and ep2 used in this invention.

[0075] Figure 3 : Figure 3 The surface response of DE3 culture in a rotary shaker, as well as the DCW ratio and inoculum ratio as a function of culture time (h), were analyzed using response equations and ANOVA software Origin Pro® 8.5. Data are presented as mean and standard deviation, taking p-values ​​into account (P < 0.05).

[0076] Figure 4 : Figure 4This study relates to optimizing the production of vaccine epitopes in a stirred tank bioreactor. (A) and (B) are kinetic profiles of DE3 in a simple batch reactor designed to synthesize epitopes DE3 / Ep1 and DE3 / Ep2. (C) and (D) are kinetic profiles of DE3 in a fed-batch reactor designed to synthesize DE3 / Ep1 and DE3 / Ep2. Data are derived from the culture of DE3 in SD medium containing ampicillin (100 µg / mL) in a benchtop bioreactor (Tecnal BIO-TEC®) at 37°C. Glucose (500 g / L) was added every 30 minutes until maximum DCW was reached. Induction was performed for 6 hours at 28°C using lactose (10 g / L) as an inducer. DCW – stem cell weight (g / L), O2 – dissolved oxygen (%), Hac – acetic acid concentration (g / L), and glucose (g / L). Induction begins, → Feeding begins. Data are presented as mean and standard deviation, with p-values ​​considered using Origin Pro® 8.5 software (p < 0.05).

[0077] Figure 5 : Figure 5 This study involves the scale-up production of vaccine epitopes in a non-stirred tank bioreactor. (A) and (B) are kinetic curves for the synthesis of epitopes DE3 / Ep1 and DE3 / Ep2. Data are derived from the culture of DE3 in SD medium containing ampicillin (100 µg / mL) in a Tecnal BIO-TEC® bioreactor using a fermenter modified from a Thermo Scientific Nalgene® round flask at 37°C. Glucose (500 g / L) was added every 30 minutes until maximum DCW was reached. Induction was performed for 6 hours at 28°C using a combination of lactose (10 g / L) and IPTG (0.2 mM) as inducers. DCW – stem cell weight (g / L), O2 – dissolved oxygen (%), Hac – acetic acid concentration (g / L), and glucose (g / L). Induction begins, → Feeding begins. Data are expressed as mean and standard deviation, using Origin Pro® 8.5 software, taking p-values ​​into account (p < 0.05).

[0078] Figure 6 : Figure 6 This study involves the purification of vaccine epitopes in a laboratory-scale stirred-tank bioreactor. (A) Chromatography of purified epitopes DE3 / Ep1 and DE3 / Ep2 obtained by simple batch processing. (B) Chromatography of purified epitopes DE3 / Ep1 and DE3 / Ep2 obtained by fed-batch processing. Data from Akta Purifier 10® chromatography system, GE Lifescience Healthcare®. Eluent was determined at 280 nm.

[0079] Figure 7 : Figure 7 This study relates to the purification of vaccine epitopes in a scaled-up, non-stirred tank bioreactor. (A) and (B) are spectra of purified epitopes DE3 / Ep1 and DE3 / Ep2 obtained via feed-batch chromatography. Data were obtained from an Akta Purifier 10® chromatography system (GE Lifescience Healthcare®). Eluent was determined at 280 nm.

[0080] Figure 8 : Figure 8 The study involved monitoring the body weight of mice in the experimental groups (A). Liver and spleen weight data (B and C). Control group (unimmunized 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 associated DE3 / Ep1 + DE3 / Ep2 ALFA-T / REC in W / O microemulsion); G5 (animals immunized with inactivated wild-type alpha toxin). P value p < 0.05 ( ).

[0081] Figure 9 : Figure 9 The vaccine efficacy testing involved ALFA-T / REC vaccine epitopes and a lethal dose of active alpha toxin 157 DL50g. -1 Mice were attacked. Control group (unimmunized animals). (G1) Animals inoculated with the adjuvant in the microemulsion. (A) (G2) Survival curves of mice immunized with ALFA-T / REC DE3 / Ep1 in the W / O microemulsion. (B) (G3) Survival curves of mice immunized with ALFA-T / REC DE3 / Ep2 in the W / O microemulsion. (C) (G4) Survival curves of mice immunized with the combined W / O microemulsion ALFA-T / REC DE3 / Ep1 + ALFA-T / REC DE3 / Ep2. (D) (G5) Survival curves of mice immunized with a lethal dose of active α-toxin 157DL50g. -1 Survival curves of mice inoculated with inactivated alpha toxin after attack. P-values ​​were determined using the Mantel-Cox test. p < 0.05 indicates a comparison between groups.

[0082] Figure 10 : Figure 10 This involves an enzyme-linked immunosorbent assay (ELISA). Data on humoral vaccine responses, including administration of a lethal dose of active alpha toxin (157 DL50 g), are relevant. -1The total IgG levels previously generated in unimmunized Swiss mice (control group), adjuvant group (G1), and immunized groups (G2, G3, and G4). A p-value of p < 0.05 indicates a comparison between the vaccine group and the control group.

[0083] Figure 11 : Figure 11 Immunization and challenge lethal doses of 157 DL50 g obtained from hematoxylin-eosin staining. -1 Histopathological data of the experimental groups. G1 (animals inoculated with adjuvants in W / O microemulsions). G2 and G3 - animals immunized with ALFA-T / REC DE3 / Ep1 and ALFA-T / REC DE3 / Ep2 in W / O microemulsions, respectively. G4 - animals immunized with associated ALFA-T / REC DE3 / Ep1 + ALFA-T / REC DE3 / Ep2 in W / O microemulsions. (A) Organ-related data: liver. (B) Organ-related data: spleen. Data: A1 - degeneration (40x), A2 - hemorrhage (40x), A3 - congestion (40x), B1 - degeneration (40x).

[0084] Figure 12 : Figure 12 A comparison of the conditions for inducing Ep1 protein is presented. (A) According to the initially proposed induction strategy, i.e., in *E. coli* Bl21(DE3) pLysS cells, at 28°C, for 6 h, lane 1: lactose inducer, 10 g / L; lane 2: IPTG inducer, 0.2 mM; lane 3: lactose inducer (10 g / L) + IPTG 0.2 mM. The initially proposed induction strategy is enclosed in rectangles. (B) According to the improved induction strategy, i.e., in *E. coli* Turner DE3 cells, at 37°C, lane 1: IPTG inducer, 1 mM, for 3 h; lane 2: IPTG inducer, 1 mM, for 16 h; lane 3: lactose inducer, 10 g / L, for 3 h; lane 4: lactose inducer, 10 g / L, for 16 h; lane 5: time zero, before induction. In lanes 1 and 3, the improved expression conditions are enclosed in rectangles.

[0085] Figure 13 : Figure 13A comparison of the conditions for inducing the protein Ep2. (A) According to the originally proposed induction strategy, i.e., in *E. coli* Bl21(DE3) pLysS cells, 28°C, 6 h induction, lane 1: lactose inducer, 10 g / L; lane 2: IPTG inducer, 0.2 mM; lane 3: lactose inducer (10 g / L) + IPTG 0.2 mM. In lane 3, the induction strategy described in the originally proposed patent is enclosed in a rectangle. (B) According to the improved induction strategy, i.e., in *E. coli* Bl21(DE3) pLysS cells, 37°C, lane 1: IPTG inducer, 1 mM, 16 h induction; lane 2: IPTG inducer, 0.1 mM, 16 h induction; lane 3: lactose inducer, 16 h induction. In lane 1, the improved expression conditions are enclosed in a rectangle. Detailed Implementation

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the scope of the teachings. Unless otherwise stated, all figures representing quantities, percentages, proportions, and other numerical values ​​used in the specification and claims should be understood to be modified in all cases by the term “about.” Therefore, unless stated to the contrary, the numerical parameters shown in the specification and claims are approximate values ​​that may vary depending on the desired properties.

[0087] As used throughout this application, the term "amino acid" refers to a group of α-amino acids that can be encoded directly or in precursor form by nucleic acids. A single amino acid is encoded by a nucleic acid consisting of three nucleotides, called a codon or base triplet. 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 herein, refers to naturally occurring α-amino acids, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0088] The terms “peptide,” “polypeptide,” or “protein” are used interchangeably and refer to a polymer of amino acids linked by peptide bonds, regardless of the number of amino acid residues constituting the chain. As used herein, a polypeptide includes its “variants” or “derivatives,” which refer to polypeptides that include variations or modifications in their amino acid sequence relative to a reference polypeptide, such as substitutions, deletions, additions, or chemical modifications. Examples of chemical modifications are glycosylation, G-alkylation, PEGylation, phosphorylation, acetylation, amidation, etc. Polypeptides can be artificially produced from cloned nucleotide sequences using recombinant DNA technology or prepared via known chemical synthesis reactions.

[0089] More specifically, the term polypeptide in this invention can also be understood as an antigen, a multi-antigen, or a multi-epitope antigen, which consists of links of different epitopes that may or may not be linked by flexible or rigid linkers and are specific to a single pathogen or to different pathogens.

[0090] The term "epitope" refers to an antigenic determinant in a molecule, such as a portion of the molecule that is recognized by the immune system, for example, a portion recognized by an antibody. For example, an epitope is a discrete three-dimensional site on an antigen that is recognized by the immune system. Epitopes typically consist of chemically active surface clusters of a molecule, such as amino acid or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not lost. Epitopes of proteins preferably comprise continuous or discontinuous portions of the protein, and their length is preferably between 5 and 100 amino acids, more preferably between 5 and 50, more preferably between 8 and 30, and even more preferably between 10 and 25 amino acids. For example, epitope lengths can preferably be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0091] In a first embodiment, the present invention provides a Clostridium novie α-toxin vaccine peptide.

[0092] Using the IEDB-AR platform, gene regions of Clostridium novesiculosus α-toxin were predicted and selected from the GenBank database: CAA88565.1. The region corresponding to the vaccine epitope DE3 / Ep1 is contained within the N-terminal domain and includes a central hydrophobic domain that is partly responsible for toxin transport to the cytosol. The region corresponding to the DE3 / Ep2 epitope is located within the C-terminal domain and also includes a partial hydrophobic domain.

[0093] Epitopes were selected considering antigenicity, flexibility, and the ability to stimulate an immune response in computer simulation models. Expression in attenuated *E. coli* showed bands of 17 kDa and 26 kDa corresponding to DE3 / Ep1 and DE3 / Ep2, respectively. Figure 1 It should also be clarified that, after comparison using the Blast tool (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), no sequence identical to the sequence of this invention was found in the prior art.

[0094] To express the gene in *E. coli*, the epitope gene is inserted into an expression plasmid. In a preferred embodiment, the vector used is selected from pRSET, pColdl, pASK-IBA, pT7-7, pBAD-TOPO, pSC101, pACYC, and any vector used for expression in microbial cells (see [link to documentation]). Figure 2 ).

[0095] Preferably, the carrier used is pRSET.

[0096] The term "expression vector," or simply "vector," can be understood as a self-replicating DNA molecule used to transport exogenous DNA fragments. It is a delivery tool used for gene cloning. First, the DNA of interest is cloned into a suitable vector, and then, through transfection, the gene can be inserted into the host for expression.

[0097] Expression vectors are applicable to microbial host organisms as disclosed in this application and include, for example, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, including vectors for stable integration into the host chromosome and selection sequences or operable markers.

[0098] Furthermore, the expression vector may include one or more selectable marker genes and appropriate expression control sequences. Similarly, it may include selectable marker genes, for example, to provide resistance to antibiotics or toxins, compensate for auxotrophic deficiencies, or provide key nutrients absent in the culture medium.

[0099] Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and similar sequences well known in the art. When two or more exogenous coding nucleic acids are to be co-expressed, both nucleic acids may be inserted into, for example, a single expression vector or separate expression vectors.

[0100] For single-vector expression, the encoding nucleic acid can be operatively linked to a common expression control sequence or to different expression control sequences, such as inducible promoters and constitutive promoters.

[0101] The conversion of exogenous nucleic acid sequences involved in metabolic or synthetic pathways can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blotting or amplification of mRNA by polymerase chain reaction (PCR), or immunoblotting for gene product expression, or other analytical methods suitable for assessing the expression of an introduced nucleic acid sequence or its corresponding gene product.

[0102] Those skilled in the art will understand that exogenous nucleic acids are expressed in an amount sufficient to produce the desired product, and will also understand that expression levels can be optimized to achieve sufficient expression using methods well known in the art and disclosed herein.

[0103] In one instance, the Escherichia coli strain may be selected from the group consisting of, but not limited to: C41 (DE3), Rosetta (DE3), Origami (DE3), or Turner (DE3). Preferably, the Escherichia coli strain used in this invention is BL21 (DE3) pLysE.

[0104] Table 1:

[0105]

[0106] Table 2:

[0107]

[0108] The reactivity with serum from mice infected with wild-type Clostridium novesiculosus α toxin reveals the ability of these epitopes to recognize specific antibodies present in the infection. Figure 1 ).

[0109] The DE3 inoculum ratio was determined in rotary shakers and SD (semi-limited) media at ratios of 2.5%, 5%, and 10% (v / v). Response surface methodology data showed that a 10% (v / v) ratio achieved higher biomass yield and specific growth rate; subsequent studies all used this process condition. Figure 3 ).

[0110] To optimize the production of DE3 / Ep1 and DE3 / Ep2 epitopes, we evaluated the DE3 culture process in a 1L mechanically stirred tank bioreactor, which, as described herein, can be used in batch, fed-batch, or continuous modes, respectively.

[0111] Kinetic studies were conducted with a working volume of 700 mL, using three (3) 10 g / L lactose induction pulses, with the induction time maintained for 6 hours after reaching maximum cell density. Figure 4 ).

[0112] Kinetic curves of DE3 in single-batch cultures showed that DCW (stem cell weight) yields were 0.70 and 0.77 g / L, and no significant Hac (g / L) (acetic acid) production was observed under conditions of adequate O2 supply (%) and complete glucose depletion. Figure 3 AC). Regarding DE3 culture aimed at producing DE3 / Ep1 and DE3 / Ep2 epitopes, the specific growth rate (µ) and yield coefficient (Y) were compared. X / S and Y P / S ), and the volumetric productivity of products and cells (Q P and Q X There are no differences between them (Table 3).

[0113] Table 3:

[0114]

[0115] Under these same induction conditions, the kinetics of DE3 in fed-batch production showed a DCW yield of 1.03 g / L; with adequate oxygen supply (%), complete glucose depletion, and no significant HAC production (g / L) was observed. Figure 3 BD). Regarding DE3 culture aimed at producing DE3 / Ep1 and DE3 / Ep2 epitopes, the specific growth rate (µ) and yield coefficient (Y) were compared. X / S and Y P / S ), and the volumetric productivity of products and cells (Q P and Q X There were no differences between them (Table 3); however, all of these were superior to the results obtained in the simple batch culture mode (Table 3).

[0116] Epitope vaccine production scale-up was conducted in a fed-batch system, as this represented the highest yield achieved in previous studies. Therefore, we determined the scale-up of epitope production by setting the yield in a 20L unstirred tank bioreactor, with the aim of reducing operating costs.

[0117] Kinetics were performed in a 10 L working volume, with three (3) induction pulses associated with low concentrations of isopropyl-β-D-thiogalactopyranoside (IPTG) inducer (0.2 mM) and 10 g / L lactose solution, and the induction time was maintained for 6 hours after reaching maximum cell density. Figure 5 The kinetic profiles of DE3 in fed-batch production showed similar parameters between vaccine epitope (DE3 / Ep1 and DE3 / Ep2) production, achieving DCW yields of 1.07 and 1.20 g / L. O2 (%), glucose consumption (g / L), and Hac production (g / L) values ​​showed similar characteristics to those described in previous studies. Figure 5 AB).

[0118] In DE3 culture aimed at producing epitopes DE3 / Ep1 and DE3 / Ep2, specific growth rate (µ) and performance coefficient (µ) were measured. X / S and Y P / S ) and the volumetric productivity of products and cells (Q P and Q X There was no difference between them; however, all of these were superior to the results obtained in the mechanically stirred fed batch culture process (Table 4).

[0119] Using a 10 g / L lactose pulse induction strategy, the production dynamics of DE3 / Ep1 and DE3 / Ep2 were revealed in a 1 L mechanically stirred tank bioreactor under two processes (simple batch culture and fed-batch culture). The peak yields of DE3 / Ep1 and DE3 / Ep2 ranged from 300 mAU (simple batch culture) to 400 mAU (fed-batch culture), indicating that the fed-batch culture system yielded higher yields. Figure 6 ).

[0120] In a 10L scale-up culture, we used a combination of 10 g / L lactose and 0.2 mM IPTG, which resulted in increased yield. The peak yields of DE3 / Ep1 and DE3 / Ep2 reached approximately 1000 mAU. Figure 7 A) and 600mAU ( Figure 7 B).

[0121] This strategy of combining lactose and IPTG has shown promise; future scale-up studies can explore the induction of recombinant proteins in DE3 culture by evaluating different combinations. Purified 17kDa (DE3 / Ep1) and 26kDa (DE3 / Ep2) epitopes were obtained, corresponding to concentrations of 100 ng / µL and 110 ng / µL, respectively. Figure 1 (Table 4).

[0122] Table 4:

[0123]

[0124] In addition, to enhance the expression of recombinant proteins, novel induction strategies for obtaining Ep1 and Ep2 proteins were evaluated. Different temperatures (11, 22, and 37 °C), time (3 h and 16 h), competent cells (E. coli BL21(DE3) pLysS, E. coli Turner DE3, and E. coli Arctic Xpress DE3), and inducers (1 mM IPTG, 0.1 mM IPTG, and 10 g / L lactose) were evaluated.

[0125] Specifically, it was observed that when the conditions of 37°C, E. coli Tuner DE3, 1 mM IPTG, or 10 g / L lactose were used for 3 hours, the induction effect of Ep1 protein was significantly improved compared with the initially proposed induction strategy. Figure 12 B, lanes 1 and 3).

[0126] It is worth emphasizing that, compared to IPTG, lactose inducers are a non-toxic and low-cost reagent, making them well-suited for large-scale expression. The yield obtained in the improved induction strategy was approximately 10 mg / L, compared to approximately 0.05 mg / L in the initially proposed strategy.

[0127] Regarding the Ep2 protein, compared to the initially proposed strategy, an improvement in induction was also observed when induced for 16 hours at 37°C with E. coli BL21(DE3)pLysS and 1 mM IPTG. Figure 13 B, Lane 1).

[0128] After the same detection time, the lactose inducer (10 g / L) produced similar expression levels. Figure 13 (B, lane 2). It is worth emphasizing that, compared with IPTG, lactose inducer is a non-toxic and low-cost reagent, and therefore very suitable for large-scale expression. The yield obtained in the improved induction strategy was approximately 5 mg / L, compared with approximately 0.05 mg / L in the originally proposed strategy.

[0129] On the other hand, expression using the Escherichia coli Arctic Xpress DE3 strain did not show satisfactory results under any evaluation conditions.

[0130] Therefore, the improved induction strategy evaluated has proven promising for use with Escherichia coli Turner DE3 and Escherichia coli BL21(DE3)pLysS strains.

[0131] In a second embodiment, the present invention provides a vaccine composition comprising epitopes of Clostridium novie α-toxin.

[0132] In a preferred embodiment, the vaccine composition comprises the DE3 / Ep1 and DE3 / Ep2 epitopes disclosed herein; preferably, it is used as an antigen component at the concentrations described herein and mixed with an adjuvant.

[0133] Adjuvants also help enhance the immune response and / or improve the stability of vaccine formulations. Adjuvants are generally described as non-specific stimulants of the immune system, but they can also be used to target specific branches of the immune system. One or more compounds with such activities can be added to a vaccine.

[0134] Therefore, certain vaccines of the present invention also contain adjuvants. 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 mannoyl oleate derivatives in mineral oil solutions (e.g., MONTANIDE ISA 61), light mineral oils (e.g., DRAKEOL 6VR)), block polymers, ISCOM (immunostimulatory complex), vitamins and minerals (including vitamin E, vitamin A, selenium, and vitamin B12), and CARBOPOL®.

[0135] Preferably, the adjuvant is MONTANIDE™ ISA 61 VG, Seppic, Brazil.

[0136] Furthermore, the vaccine composition of the present invention may contain a diluent, an isotonic agent, a stabilizer, or an adjuvant. The diluent may include water, a saline solution, dextrose, ethanol, glycerol, etc. The isotonic agent may include sodium chloride, dextrose, mannitol, sorbitol, and lactose, etc. The stabilizer includes albumin and alkali metal salts of ethylenediaminetetraacetic acid, etc. Suitable adjuvants are known in the art.

[0137] Furthermore, according to the present invention, an “immune or immunological response” to a composition or vaccine is a cellular and / or antibody-mediated immune response in the host against 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 γδ T cells, specifically targeting one or more antigens contained in the composition or vaccine of interest. Preferably, the host will exhibit a therapeutic or protective immune response, resulting in increased resistance to new infections and / or reduced clinical severity of disease. This protective effect will be manifested through the reduction or disappearance of infection-related symptoms in the host.

[0138] In a third embodiment, the present invention provides the use of such vaccine epitopes in the preparation of vaccine compositions for the prevention and treatment of damage caused by active α-toxins of Clostridium botulinum type B.

[0139] The protein regions of the predicted and screened Clostridium novidis α-toxin showed potential for screening as epitopes: the region corresponding to DE3 / Ep1 was located in the N-terminal domain, while the region corresponding to DE3 / Ep2 was located in the C-terminal domain. Epitopes DE3 / Ep1 and DE3 / Ep2 showed bands of 17 kDa and 26 kDa, respectively, both in the in-silico model and after expression in the DE3 host. Figure 1 D).

[0140] Furthermore, the reactivity of these regions was confirmed by serum from mice infected with wild-type Clostridium novie α toxin, highlighting the importance of these selected regions in recognizing specific antibodies present in the infection. Figure 1 E).

[0141] Post-immunization follow-up revealed that the entire experimental group experienced weight gain, demonstrating the safety aspects of the formulation. Figure 8 A). Post-attack hematological data were used to assess changes in hematological parameters and liver function (Table 5).

[0142] Table 5 presents the results of the experimental group receiving 157 DL50g. -1 Hematological data after immunization and challenge. Group G1 (animals inoculated with the adjuvant in the W / O microemulsion); Groups G2 and G3: animals immunized with ALFA-T / REC DE3 / Ep1 and ALFA-T / REC DE3 / Ep2 in the W / O microemulsion, respectively; Group G4: animals immunized with the combination of ALFA-T / REC DE3 / Ep1 + ALFA-T / REC DE3 / Ep2 in the W / O microemulsion. Control group (-): unimmunized animals / unchallenged (healthy animals); Control group (+): unimmunized animals that received 157 DL50 g. -1 Animals subjected to dose-response. Ns (not significant): Comparison between serum from unimmunized / unchallenged mice and serum from mice inoculated with microemulsion adjuvant (G1 group). (significant): Hemoglobin (p<0.05); Hematocrit (p<0.05); Comparison between serum from immunized mice (G2, G3, and G4 groups) and positive control group.

[0143] Table 5:

[0144]

[0145] The positive control group showed clinical signs of polycythemia; in contrast, the data from immunized mice from groups (G2), (G3) and (G4) were consistent with the reference values ​​and relatively similar to those of healthy animals (Table 5).

[0146] Vaccine efficacy data showed no difference between the control group and the adjuvant group (G1), although a high mortality rate was observed after the challenge test. Figure 9 ).

[0147] Animals immunized with microemulsions of DE3 / Ep1 (G2) and DE3 / Ep2 (G3) showed protection rates of 25% and 40%, respectively. Microemulsions composed of the combined epitopes DE3 / Ep1 and DE3 / Ep2 (G4) exhibited an even higher protection rate of 75%.

[0148] Compared with data obtained using microemulsions containing inactivated wild-type alpha toxin epitopes (G5) (25% (Félix et al., 2019)), vaccines in groups G3 and G4 provided better efficacy data (P<0.005); while microemulsions containing DE3 / Ep1 epitopes (G2) provided comparable protective effects in animals to group G5.

[0149] Our data show that the obtained vaccine epitopes can serve as immunogens for vaccine protection against damage caused by active alpha toxins from Clostridium botulinum type B.

[0150] However, the hematological examination results (Table 5) not only yielded hematological data ( Figure 8 This is corroborated by data on vaccine effectiveness. Figure 9 ) and ELISA analysis data of mouse liver and spleen after challenge with Clostridium bovis type B active alpha toxin ( Figure 10 (This is consistent with)

[0151] This evidence suggests that computer simulation analysis of antigenic regions, followed by in vivo experiments, may be an effective strategy for developing suitable candidates for Clostridium noviperi type B vaccines. However, further studies optimizing epitope yield are needed to allow for adjustment of the concentration of each antigen in the vaccine dose. Histopathological analysis of liver and spleen tissue damage confirmed our findings, demonstrating the safety of the vaccine groups, with the G4 group showing greater efficacy. Similarly, ELISA immunogenicity assays used to validate antibody production indicated that the G4 vaccine group had a greater capacity to stimulate an immune response. Example

[0152] Example 1: Construction of Bioinformatics Tools and Vaccine Epitope Expression Vectors

[0153] Alpha toxin epitopes of Clostridium novyi were screened using a computer program (alpha toxin from Clostridium novyi, GenBank: CAA88565.1). Regions were predicted using the Immunoepitope Database Analysis Resource (IEDB-AR) (http: / / tools.immuneepitope.org / main), and two alpha toxin epitopes (DE3 / Ep1) located at amino acids 491–583 in the N-terminal region and (DE3 / Ep2) located at amino acids 1599–1779 in the C-terminal region were selected, both determined by considering the relationship between antigenicity, flexibility, and ability to stimulate adaptive immune responses.

[0154] The DE3 / Ep1 and DE3 / Ep2 genes, synthesized by Invitrogen-Thermo Fisher Scientific®, have codon preferences specific to E. coli and are inserted into the pRSET expression plasmid, which allows for the addition of polyhistidine tags (6×HIS) to the amino terminus of each protein to facilitate purification after cell lysis.

[0155] Following the manufacturer's instructions, use a Model 2510 electroporator (Eppendorf®) to transform the plasmid containing the synthetic gene into electrocompetent DE3 cells.

[0156] Next, cells were plated on Luria Bertani (LB) medium (Kasvi®) containing ampicillin (100 µg / mL) (Multilab®) and chloramphenicol (11.4 µg / mL) (Kasvi®) and cultured overnight at 37°C. Epitope expression in DE3 was then performed using isopropyl-β-D-thiogalactopyranoside (IPTG) (Invitrogen®) in LB medium containing ampicillin and chloramphenicol.

[0157] Cultures were incubated at 37°C until an OD600 of 0.6 was achieved, followed by 1 mM IPTG induction and then temperature adjustment to 28°C. DE3 culture samples were treated with denaturing buffer (20% (v / v) glycerol (Synth®); 4% (w / v) sodium dodecyl sulfate (SDS) (Synth®); 100 mM Tris pH 6.8 (Synth®); 0.2% (w / v) bromophenol blue (Synth®); 200 mM β-mercaptoethanol (BioRad®)) and then boiled for 10 minutes.

[0158] Polyacrylamide gel electrophoresis (SDS-PAGE 12%) was performed to confirm the expression of the corresponding epitopes of the DE3 / Ep1 and DE3 / Ep2 genes, followed by Western blotting.

[0159] The design and presentation of DE3 / Ep1 and DE3 / Ep2 in DE3 were carried out in collaboration with the Laboratory for Diagnostic and Controllable Infectious Diseases in the Amazon (LDCDIA), which is affiliated with the Leônidas & Maria Deane Institute (ILMD / FiocruzAmazônia).

[0160] Example 2: DE3 culture in a rotary shaker

[0161] DE3 colonies were inoculated into semi-limited (SD) medium supplemented with 100 μg / mL ampicillin. The composition of the SD medium was: glucose 5.0 g / L (Synth®), yeast extract 5.0 g / L (Synth®), dipotassium hydrogen phosphate (K2HPO4) 9.4 g / L (Synth®), potassium dihydrogen 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®).

[0162] This study was conducted in triplicate using an SL-223 / F rotary shaker (Solab Cientifica®), maintained at 37°C and 200 rpm for 16 hours. Samples were collected, optical density (OD600) was read, and specific growth rate (µh) was determined. -1 (and DCW, assessing vaccination rates corresponding to 2.5%, 5%, and 10%).

[0163] Example 3: Cultivation of DE3 in SD medium in a stirred tank bioreactor

[0164] Batch culture and fed-batch culture were carried out in a TecnalBIO-TEC® 1L stirred tank bioreactor. The reactor consists of a jacketed glass vessel and a digital control unit; pH is controlled at 7 at 37°C using 3 M sodium hydroxide (NaOH) (Synth®); and a dissolved oxygen (DO) replenishment strategy with cascaded stirring control from 100-1000 rpm and a fixed specific air feed rate of 3 vvm.

[0165] Inoculum proliferation was first carried out by activating each DE3 stock culture (1.5 mL) in 8.5 mL SD medium, stirring at 200 rpm for 12 hours at 37°C, and maintaining an inoculum ratio of 10% in the bioreactor.

[0166] During the fed-batch process, 7 mL of a 500 g / L glucose solution containing 100 μg / mL ampicillin was added every 30 minutes until the steady-state growth phase was reached. Samples were then collected every hour to determine growth kinetics, acetate and glucose consumption, and the synthesis of DE3 / Ep1 and DE3 / Ep2.

[0167] Example 4: Scale-up of DE3 culture in SD medium in a non-stirred tank bioreactor

[0168] Feed batch culture was carried out in a 20 L unstirred tank bioreactor, which was a modified round bottle (Thermo Scientific Nalgene®) from a round narrow-mouth bottle with a low-density polyethylene (LDPE) handle for easy transport and dispensing, and a white polypropylene (PP) screw cap (83B).

[0169] Cultures were performed at 37°C with pH maintained at 7 using 3 M NaOH and a dissolved oxygen (DO) supplementation strategy with a fixed 3 vvm inlet, without mechanical agitation. Inoculum proliferation was carried out using a stock culture (1.5 mL) of each DE3 in 8.5 mL SD medium, stirred at 200 rpm for 12 h at 37°C, maintaining a 10% inoculum ratio for cultivation in a 10 L working volume bioreactor.

[0170] During the fed-batch process, 100 mL of a 500 g / L glucose solution containing 100 μg / mL ampicillin was added every 30 minutes until the steady-state growth phase was reached. Samples were then collected every hour to determine growth kinetics, acetate and glucose consumption, and the synthesis of DE3 / Ep1 and DE3 / Ep2.

[0171] Example 5: Induction and purification system for recombinant DE3 / Ep1 and DE3 / Ep2 epitopes

[0172] In the first induction phase, in our culture conducted in a stirred tank bioreactor, three 35 mL pulses of 10 g / L lactose solution (Synth®) were applied immediately after reaching maximum cell growth, followed by a second application two hours later. During scale-up using a non-stirred tank bioreactor, we used three 500 mL pulses of 10 g / L lactose solution with 0.2 mMIPTG at the same time intervals as in our laboratory-scale experiments.

[0173] All induction studies were performed at 28°C for 6 hours. A chromatography system (Akta Purifier 10®, GE Lifescience Healthcare®) was used in the purification steps of DE3 / Ep1 and DE3 / Ep2. The collected fractions were lyophilized and stored at -20°C for analysis. Reversed-phase chromatography was performed according to the method described by Field et al. (2021).

[0174] DE3 / Ep1 and DE3 / Ep2 fractions obtained from affinity chromatography were lyophilized, dissolved 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®) pre-equilibrated with solution A and subjected to 0 to 70% solution B (acetonitrile 99.9% (Merck®)) over 5 column volumes. ® The solution was eluted with a gradient of 0.1% TFA and 0.1% TFA at a flow rate of 1 mL / min.

[0175] The eluent was monitored at 280 nm. DE3 / Ep1 and DE3 / Ep2 spectra were obtained to confirm the achieved degree of purification and product concentration. The collected fractions were again visualized on 12% SDS-PAGE to check purity, and antigenicity was confirmed by Western blotting.

[0176] Example 6: Western blotting

[0177] The DE3 / Ep1 and DE3 / Ep2 bands in a 12% SDS-PAGE gel were transferred to a nitrocellulose membrane following the BioRad® protocol. Primary antibodies were derived from serum of Swiss mice infected with wild-type Clostridium novilis α-toxin. Negative controls were derived from serum of healthy Swiss mice.

[0178] The secondary antibody used was anti-mouse IgG-rabbit A9044 (Sigma®) (conjugated to peroxidase at a ratio of 1:2000, and 0.05% (v / v) of substrate 3,3′-diaminobenzidine (DAB)). Its reactivity with purified DE3 / Ep1 and DE3 / Ep2 was verified by comparison with inactive wild-type alpha toxin.

[0179] Example 7: Fermentation Parameters

[0180] Example 7.1: Determination of glucose consumption

[0181] The determination of glucose consumption was performed using the liquid enzymatic glucose system (Labtest®) of formula (1), which is based on the principle described by Bergmeyer et al. (1986), using two reactions involving glucose oxidase (GOD) and peroxidase (POD).

[0182] Equation (1)

[0183]

[0184] The resulting product, 4-antipyrine quinone imine, is red, and its intensity is directly proportional to the glucose concentration. Prepare the assay solution in a 2 mL shaking tube, incubate at 37°C for 5 minutes, and read the value at 510 nm. The glucose concentration is calculated as follows:

[0185] Equation (2)

[0186] Example 7.2: Determination of Hac and DCW yields

[0187] Hac was determined using a Shimadzu® chromatograph (SPD-M2OA) equipped with a high-precision LC-6AD pump (CBM-20). a A UV detector was used for Hac detection, measuring the absorbance range from 190 to 700 nm. An ion-size exclusion column, a Shim-pack® C-18 column (250 × 4.6 mm, Shimadzu®), was used. Analysis was performed at 30 °C for 12 min at a mobile phase flow rate of 0.6 mL / min. The mobile phase consisted of 1% phosphoric acid (H3PO4) solution, with an injection volume of 20 µL. Samples were filtered using a Chromabono® C-18 membrane (3 mL / 500 mg) and read at 204 nm. Hac quantification was performed using a standard curve and linear regression.

[0188] The DCW value is obtained by the difference between mass A (mA) and mass B (mB), where mA represents the mass of the crucible without sample, mB represents the mass of the crucible with culture sample, and Al represents the volume of the sample. The DCW value is obtained by correlating the dry weight of the culture sample with OD600, where 1 OD600 unit corresponds to 0.61 g / L of cell dry weight. The dry weight correlation is determined as described in equation (3).

[0189] Equation (3)

[0190] Example 7.3: Performance Coefficient

[0191] The specific growth rate (μ) is determined during the exponential phase of bacterial growth, during which the specific growth rate is constant and reaches its maximum value (µx=µm). The μ value is obtained using equation (4), where dX represents the difference in OD600 values ​​between a specific time point and subsequent time points, dt represents the change over time, and X corresponds to the cell concentration at a specific time point.

[0192] Equation (4)

[0193] Regarding biomass and products performance coefficient Analysis was performed, as shown in equations (5) and (6). The biomass-product conversion coefficient was calculated. and substrate consumption Cell growth factors were considered; the relationship between the two was established by equation (5). When calculating product yield, the amount of product generated was taken into account. and substrate consumption The relationship between the two is established by equation (6). Equations (7) and (8) define the products. and biomass Volumetric productivity, and its relationship with time A connection has been established.

[0194] Equation (5)

[0195] Equation (6)

[0196] Equation (7)

[0197] Equation (8)

[0198] Example 8: Vaccine Formulation

[0199] The purified DE3 / Ep1 and DE3 / Ep2 were emulsified in adjuvant MONTANIDE™ ISA 61 VG (Seppic, Brazil). The aqueous phase (W) of the vaccine consisted of 40% recombinant antigen, and the oil phase (O) consisted of 60% adjuvant. Microemulsification was performed using 10 mL sterile syringes interconnected by silicone tubing. The aqueous and oil phase components were repeatedly and forcefully injected between the two syringes over 10 minutes.

[0200] Example 9: Experimental Design

[0201] Using 4-6 week old Swiss mice weighing between 17-23 grams, both male and female, this study has been approved by the Committee on Animal Use Ethics (CEUA) of the Federal University of Tocantins (Approval No.: 23.101.006.832 / 2017-53).

[0202] The experimental group was divided into 5 (five) groups, with 10 (ten) animals in each group, as follows: PBS (unimmunized); adjuvant group in W / O microemulsion (G1); DE3 / Ep1 vaccine group in W / O microemulsion (G2); DE3 / Ep2 vaccine group in W / O microemulsion (G3); and DE3 / Ep1 + DE3 / Ep2 combined vaccine group in W / O microemulsion (G4). Comparisons were also made with the group immunized with wild-type inactivated alpha toxin (G5) (Félix et al., 2019).

[0203] All groups were administered the medication via intramuscular injection, in three doses of 100 µL each, with a dosing interval of 15 days. The weight of each animal was monitored during the experiment.

[0204] Animals in each group received a challenge inoculation (challenge dose was 157DL 50 g). -1 The lethal dose of active alpha toxin was determined, and Kaplan-Meier survival curves were established. Blood samples were collected for specific antibody, hematological, and biochemical analyses. Liver and spleen were also collected from the animals for histopathological analysis.

[0205] For surviving animals, they were first anesthetized, then euthanized with ketamine (300 mg / kg) (Vetbrands, Brazil) and xylazine (22.5 mg / kg) (Syntec®, Brazil), and their level of consciousness was confirmed by heartbeat and respiratory movements.

[0206] Example 10: Immunogenicity, hematological and biochemical data

[0207] Immunogenicity was determined by analyzing serum from immunized Swiss mice using an ELISA immunoassay. Blood samples were collected from mice on day 45 after the last immunization to assess stimulation of total IgG production. ELISA was performed using purified DE3 / Ep1 and DE2 / Ep2 proteins for normalization and antigen fixation on 96-well microplates (Nunc MaxiSorp) at 4 °C.

[0208] After blocking with 2% casein-PBS buffer (blocking buffer) for 1 hour at room temperature, the plate was washed four times with 0.05% Tween-20-PBS buffer. The serum was diluted to 1:50 with blocking buffer and incubated at 4°C for 16 hours.

[0209] After washing, anti-mouse IgG peroxidase conjugate (Sigma, USA) was used at a ratio of 1:2000 in blocking buffer. Following washing, 3,3',5,5'-tetramethylbenzidine (BD Biosciences, USA) was added and reacted for 15 minutes, then the reaction was terminated with 2.5M H₂SO₄ (Dinâmica, Brazil). Density was measured at 450 nm; values ​​above the critical value were considered positive.

[0210] The cutoff value was determined based on the mean optical density of pre-immunization serum. Cutoff values ​​were calculated using serum from unimmunized animals. Furthermore, hematological and biochemical data were obtained by centrifuging blood in microtubes at 3500 rpm for 15 minutes, and parameters of white blood cell differential counts (total white blood cells, neutrophils, and monocytes) and red blood cell differential counts (erythrocytes, hemoglobin, hematocrit, VCM, HCM, CHCM, and platelets) were assessed.

[0211] Example 11.1: Histopathology

[0212] We used enzyme-linked immunosorbent assay (indirect ELISA) technology, following the protocol established by the Amazon Infectious Disease Diagnosis and Control Laboratory (LDCDIA), to assess the total IgG levels produced in mice challenged with active alpha toxin from Clostridium novilis type B.

[0213] To capture IgG, we sensitized plates with antigen (inactivated Clostridium novie type B α toxin, 4 µg / ml, diluted in carbonate / bicarbonate buffer, pH 9.6) and incubated overnight at 4°C. After incubation, the plates were blocked with 1×PBS / 3% BSA for 2 hours, washed with 0.05% PBS Tween, and then serum (1:1) from mice challenged with active α toxin was added. After adding the primary antibody, the plates were incubated in a humidified chamber at 37°C for 1 hour, followed by washing. Subsequently, a colorimetric step was performed using a horseradish peroxidase-conjugated anti-mouse IgG polyclonal antibody from Rhea Biotech®. Finally, 100 µL of the chromogenic substrate tetramethylbenzidine (TMB-Sigma®) was added to each well, allowed to react for 10 minutes, and the reaction was terminated with 2M sulfuric acid (H2SO4).

[0214] Optical density (OD) was determined using an ELISA reader (GLOMAX®) with a 450 nm filter. To determine antibody levels, antibody titration profiles were constructed using mouse IgG from Rhea Biotech® at different concentrations (1000 ng, 500 ng, 250 ng, 125 ng, 62.5 ng, and 31.25 ng).

[0215] 11.2 Histopathological Analysis

[0216] Liver and spleen were removed from each group of Swiss mice via necropsy and fixed in 10% buffered formalin for histopathological analysis. The tissues were processed and embedded in paraffin, then stained with H&E.

[0217] Assess major liver lesions such as hepatocyte hydrops, inflammation, perivasculitis, congestion, and necrosis. In the spleen, assess periarteriolar and follicular hyperplasia in the white and red pulp, as well as inflammatory infiltrates.

[0218] Based on the severity of the lesions, each animal was analyzed according to the following scoring system: 0) No lesions; 1) Mild lesions, occupying less than 25% of the field of view; 2) Moderate lesions, occupying more than 25% but less than 50% of the field of view; 3) Severe lesions, occupying more than 50% of the field of view.

[0219] Post-immunization humoral response analysis showed that the G4 group (combined epitopes DE3 / Ep1 and DE3 / Ep2) had a greater increase in IgG levels, approaching 1000 ng / mL, significantly higher than the IgG levels obtained in the control group (450 ng / mL), the G1 group (adjuvant, 700 ng / mL), the G2 group (DE3 / Ep1, 820 ng / mL), and the G3 group (DE3 / Ep2, 915 ng / mL) (p < 0.05), demonstrating a stronger ability to stimulate an immune response against Clostridium botulinum. Figure 11 ).

[0220] A sepsis infection model challenged with active alpha toxin from Clostridium novidis type B showed that immunization with 157 LD50·g -1 Following dose challenge, tissue damage occurred in the liver and spleen of mice. Histopathological results showed greater severity of degeneration, hemorrhage, and congestion in the G1 (adjuvant), G2 (DE3 / Ep1), and G3 (DE3 / Ep2) groups. In contrast, minor changes were observed in the G4 group (combined DE3 / Ep1 and DE3 / Ep2 epitopes), similar to those observed in the control group. Figure 11 ).

[0221] Example 12: Statistical Analysis

[0222] The experiment employed a completely randomized design and used analysis of variance (ANOVA). Kinetic parameters obtained in stirred and non-stirred systems were compared and analyzed.

[0223] The PBS group (non-immunized group) and the vaccine group were used for comparison. The mean of the data was analyzed using the Student t-test, with significance considered for p-values ​​(P < 0.05) using Origin Pro® 8.5 software. Kaplan-Meier survival curves were used to determine the probability of vaccine efficacy.

[0224] Example 13: Transformation and expression of EP1 and EP2 proteins in Escherichia coli BL21(DE3)pLysS

[0225] The procedure for transforming E. coli BL21(DE3)pLysS™ using the pRSET plasmid containing the EP1 and EP2 genes is as follows:

[0226] First, 0.5 μL (equivalent to 25 ng) of plasmid was added to a total of 50 μL of competent Escherichia coli BL21(DE3)pLysS cells. Subsequently, an electrical pulse of 1.9 kV was applied to the mixture using an Eppendorf 2510 electroporator.

[0227] Next, the cells were resuspended in 450 μL of liquid LB medium, and the contents were transferred to a 2.0 mL microtube. The microtube was incubated at 37°C with constant stirring at 150 rpm for 1 hour. Then, 100 µL of the transformant was aliquoted onto culture plates containing LB agar medium, and 100 μg / mL ampicillin and 25 μg / mL chloramphenicol were added. These plates were incubated at 37°C for a total of 16 hours.

[0228] Subsequently, colonies randomly selected from the plate were pre-inoculated into test tubes, each containing 3 mL of liquid LB medium containing ampicillin (100 µg / mL) and chloramphenicol (25 µg / mL) as screening antibiotics. After incubation at 37°C with shaking for 16 hours, the cultures were transferred to 100 mL of liquid LB medium containing the screening antibiotics. The temperature was maintained at 37°C on a shaker until the optical density (OD) reached 0.6 (measured at 600 nm using a spectrophotometer).

[0229] At this point, 15 mL of bacterial culture was dispensed into six conical tubes, and IPTG (isopropyl-β-D-thiogalactopyranoside) was added to each tube to a final concentration of 0.1 mM and 1 mM, and lactose was added to a final concentration of 10 g / L.

[0230] Induction with IPTG and lactose was performed under the following conditions: 3 h and 16 h; at 37 °C, 22 °C, and 11 °C. Immediately afterwards, the bacterial pellet was centrifuged at 3500 × g for 15 min at 4 °C. The bacterial pellet was stored in 50 mL conical tubes and kept at -20 °C pending the subsequent sonication step. This step involved resuspending the bacterial pellet in MCAC-0 buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 10% glycerol, 1 mM PMSF) and then sonicating using a standardized procedure developed by our team. The sonicated material was centrifuged at 10,000 × g for 15 min at 4 °C. Following the sonication procedure, the material was analyzed by SDS-PAGE with sodium dodecyl sulfate and Western blot analysis.

[0231] Example 14: Transformation and expression of EP1 protein in Escherichia coli Arctic Xpress DE3

[0232] The procedure for transforming E. coli Arctic Xpress DE3 with the pRSET plasmid containing the EP1 gene is as follows:

[0233] First, 1 μL (equivalent to 50 ng) of plasmid was added to a total of 50 μL of competent Escherichia coli Arctic XpressDE3 cells. Then, an electrical pulse of 1.9 kV was applied to the mixture using an Eppendorf 2510 electroporator.

[0234] Next, the cells were resuspended in 450 μL of liquid LB medium, and the contents were transferred to a 2.0 mL microtube. The microtube was incubated at 37°C with constant stirring at 150 rpm for 1 hour. Then, 100 µL of the transformant was aliquoted onto culture plates containing LB agar medium, and 100 μg / mL ampicillin was added. These plates were incubated at 37°C for a total of 16 hours.

[0235] Subsequently, colonies randomly selected from the plate were pre-inoculated into test tubes, each containing 3 mL of liquid LB medium and ampicillin as a screening antibiotic. After incubation at 37°C with shaking for 16 hours, the cultures were transferred to 50 mL of liquid LB medium containing the screening antibiotic. The temperature was maintained at 37°C on a shaker until the optical density (OD) reached 0.6 (measured at 600 nm using a spectrophotometer).

[0236] At this point, the bacterial culture was aliquoted into six conical tubes, and IPTG (isopropyl-β-D-thiogalactopyranoside) was added to each tube to a final concentration of 0.1 mM and 1 mM, and lactose was added to a final concentration of 10 g / L.

[0237] Induction with IPTG and lactose was performed at 11°C for 3 and 16 hours. Immediately afterwards, the bacterial pellet was centrifuged at 4000 rpm for 15 minutes at 4°C. The bacterial pellet was stored in 50 mL conical tubes and kept at -20°C pending the subsequent sonication step. This step involved resuspending the bacterial pellet in MCAC-O buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 10% glycerol, 1 mM PMSF) and then sonicating using a standardized procedure developed by our team. The sonicated material was centrifuged at 14,000 rpm for 15 minutes at 4°C. Following the sonication procedure, the material was analyzed by SDS-PAGE with sodium dodecyl sulfate and Western blot analysis.

[0238] Example 15: Transformation and expression of EP1 protein in Escherichia coli Tuner DE3

[0239] The procedure for transforming E. coli Turner DE3 using the pRSET plasmid containing the EP1 gene is as follows:

[0240] First, 1 μL (equivalent to 50 ng) of plasmid was added to a total of 50 μL of competent E. coli Turner DE3 cell suspension. Then, an electrical pulse of 1.9 kV was applied to the mixture using an Eppendorf 2510 electroporator.

[0241] Next, the cells were resuspended in 450 μL of liquid LB medium, and the contents were transferred to a 2.0 mL microtube. The microtube was incubated at 37°C with constant stirring at 150 rpm for 1 hour. Then, 100 µL of the transformant was aliquoted onto culture plates containing LB agar medium, and 100 μg / mL ampicillin was added. These plates were incubated at 37°C for a total of 16 hours.

[0242] Subsequently, colonies randomly selected from the plate were pre-inoculated into test tubes, each containing 3 mL of liquid LB medium and ampicillin as a screening antibiotic. After incubation at 37°C with shaking for 16 hours, the cultures were transferred to 50 mL of liquid LB medium containing the screening antibiotic. The temperature was maintained at 37°C on a shaker until the optical density (OD) reached 0.6 (measured at 600 nm using a spectrophotometer).

[0243] At this point, the bacterial culture was aliquoted into two conical tubes, and IPTG (isopropyl-β-D-thiogalactopyranoside) was added to each tube to a final concentration of 0.1 mM and 1 mM, respectively, and lactose was added to a final concentration of 10 g / L.

[0244] Induction with IPTG and lactose was performed under the following conditions: 3 hours and 16 hours; at 37°C and 22°C. Immediately afterwards, the bacterial pellet was centrifuged at 4000 rpm for 15 minutes at 4°C. The bacterial pellet was stored in 50 mL conical tubes and kept at -20°C pending the subsequent sonication step. This step involved resuspending the bacterial pellet in MCAC-O buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 10% glycerol, 1 mM PMSF) and then sonicating using a standardized procedure developed by our team. The sonicated material was centrifuged at 14,000 rpm for 15 minutes at 4°C. Following the sonication procedure, the material was analyzed by SDS-PAGE with sodium dodecyl sulfate and Western blot analysis.

[0245] Example 17: Purification of recombinant EP1 and EP2 proteins obtained in Examples 13 to 16

[0246] Proteins were purified using immobilized metal affinity chromatography (IMAC). For this purpose, a Ni-containing... 2+ Ion purification was performed using a QIAGEN column according to the manufacturer's instructions. Protein elution was performed using an imidazole concentration profile ranging from 100 mM to 500 mM. Subsequently, samples were quantified using Bradford reagent (Bio-rad) according to the manufacturer's instructions. Yield was determined based on the total protein concentration (in milligrams) obtained in each elution of the isolated protein, combined with the initial bacterial growth volume used. Therefore, if 10 mg and 5 mg of protein were obtained in 1 L of induction, the estimated yields were 10 mg / L and 5 mg / L, respectively.

[0247] Example 18: Analysis of recombinant proteins obtained in Examples 13 to 16 by polyacrylamide gel electrophoresis and Western blotting

[0248] Recombinant protein expression was assessed using polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis was performed in Tris-glycine buffer at 150 V for 1 hour and 30 minutes. The molecular weight marker "Blueye Prestained Protein Marker" (Sigma Aldrich) was used as a reference. Samples were resuspended in resuspension buffer (200 mM Tris-HCl pH 6.8, 0.1% bromophenol blue, 4% SDS, 4% β-mercaptoethanol, 20% glycerol). Subsequently, the samples were denatured at 95 °C for 10 minutes and then loaded onto the gel. Gel staining was performed using commercial buffer from Scienco to visualize protein bands.

[0249] For Western blot assays, membranes were prepared using an unstained SDS-PAGE gel containing the sample and a semi-dry transfer system (Bio-rad) in tris-glycine-SDS buffer. The transfer system was run at a constant voltage of 10 V for 1 hour and 10 minutes. After removal from the chamber, the membranes were stored in 1×PBS at 4°C until color development. Next, color development was performed by first blocking the membrane with 1×PBS containing 3% BSA for 30 minutes, then washing the membrane with MilliQ water for 5 minutes, followed by adding anti-6×hisG monoclonal antibody (Invitrogen) diluted to a concentration of 1:3000 for 1 hour. The membranes were then washed three times with 1×PBS supplemented with 0.05% Tween 20 for 5 minutes each time. Anti-mouse IgG secondary antibody diluted 1:10000 + alkaline phosphatase (Seracare) was added, and incubation was performed for 1 hour. The membranes were then washed again with PBS Tween and water as described above. Then, develop the color reaction with BCIP / NBT buffer (Invitrogen) for up to 10 minutes, and then take a picture to record the result.

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Claims

1. A recombinant nucleic acid molecule, characterized in that, The recombinant nucleic acid molecule comprises a nucleic acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3, and its degenerate sequence encoding the same polypeptide sequence.

2. An epitope of a recombinant α-toxin derived from Clostridium novie, characterized in that, The epitope comprises a peptide sequence as shown in SEQ ID NO: 2 or SEQ ID NO:

4.

3. The epitope according to claim 2, characterized in that, The amino terminus of the epitope also includes a multihistidine tail (6×HIS).

4. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule and its regulatory region as described in claim 1.

5. The expression vector according to claim 4, characterized in that, The control regions are selected from BamHI and EcoRI.

6. A vaccine composition, characterized in that, The vaccine composition comprises one or more epitopes as described in claim 2 and veterinary-acceptable excipients.

7. A vaccine composition, characterized in that, The vaccine composition comprises the epitope as described in claim 2 and a veterinary-acceptable excipient.

8. The vaccine composition according to claim 6, characterized in that, The vaccine composition is an oil / water emulsion.

9. The vaccine composition according to claim 6, characterized in that, The vaccine composition is used to prevent or treat clostridium infection in a subject.

10. The use of the epitope according to claim 2 or more, characterized in that, The epitope is used to prepare a vaccine composition or to prevent or treat clostridial disease in a subject.

11. A method for preventing or treating clostridium infection in a subject, characterized in that, The method includes administering the vaccine composition of claim 6.

12. A method for preparing a vaccine composition against clostridial disease, characterized in that, The method includes the following steps: i) Transform E. coli cells with an expression plasmid containing the molecule of claim 1; ii) The cells are cultured in a bioreactor using a fed-batch process; iii) Purify the culture product from step ii); iv) Prepare a vaccine composition containing the epitopes obtained after step iii).

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