Veterinary vaccine formulation, dosage form, processes for the production of inactivated g. parasuis antigens and of a vaccine formulation, combination of clinical strains and use thereof, and kit

Abstract

The present invention falls within the fields of molecular biology, medical sciences and immunology, and relates especially to preparations for medical purposes. In particular, the present invention relates to immunogenic compositions and / or formulations and / or polyvalent veterinary vaccines with a broad spectrum of protection against serovars of Glaesserella parasuis (G. parasuis), based on inactivated antigens, to a dosage form comprising said vaccine formulation, to the process for producing inactivated G. parasuis antigens, as well as to the process for producing said vaccine formulation. Additionally, the present invention relates to a combination of clinical strains, to the use of said combination of clinical strains in the manufacture of a polyvalent vaccine formulation for the prevention of infections caused by clinical strains of G. parasuis, regardless of their capsular type, which cause Glässers disease (GD), and to a kit comprising a combination of clinical strains and a preservative solution.

Description

Veterinary vaccine formulation, dosage form, processes for the production of inactivated G. antagonists. PARASUIS AND A VACCINE FORMULATION, COMBINATION OF CLINICAL STRAINS AND THEIR USE, AND KIT FIELD OF THE INVENTION

[0001] The present invention falls within the fields of Molecular Biology, Medical Sciences and Immunology, and in particular, relates to preparations for medical purposes.

[0002] Specifically, the present invention relates to immunogenic compositions and / or formulations and / or polyvalent veterinary vaccines, with a broad spectrum of protection, against serovars of Glaesserella parasuis (G. parasuis), from inactivated antigens, to a dosage form comprising said vaccine formulation, to the process for producing inactivated G. parasuis antigens, as well as to the process for producing said vaccine formulation. Additionally, the present invention relates to a combination of clinical strains, to the use of said combination of clinical strains in the manufacture of a polyvalent vaccine formulation for the prevention of infections caused by clinical strains of G. parasuis, regardless of their capsular type, that induce Glässer's disease (GD), and to a kit comprising a combination of clinical strains and a preservative solution. FUNDAMENTALS OF THE INVENTION

[0003] The following paragraphs are intended to introduce the reader to a more detailed description, but without intending to limit the subject matter claimed in this disclosure.

[0004] Vaccines capable of mediating an effective immune response are important in health strategies aimed at combating diseases caused by bacterial pathogens. The two basic strategies for inducing an effective immune response in the host involve administering to an animal (host) a "live" agent capable of replicating within the host, or administering inactivated microorganisms or antigens derived from them that are not capable of replicating in the host. Administering a live vaccine can pose a health risk to immunocompromised animals, as the rate of microorganism propagation may increase. If the host's immune system's ability to control the disease is exceeded, the disease will develop. Furthermore, another critical aspect of attenuated vaccines is the potential reversion of virulence of the attenuated microorganism once inoculated into the host; in this case, the vaccinated animal could develop the disease after vaccination. On the other hand, these risks are not associated with immunogenic compositions and / or formulations and / or vaccines based on inactivated microorganisms, such as those described in this patent application.

[0005] Glaesserella parasuis, a microorganism formerly classified as Haemophilus parasuis, is an early colonizer of the upper respiratory tract of swine. Specifically, G. parasuis is a Gram-negative bacterium belonging to the Pasteurellaceae family, with fifteen identified serovars (1 to 15). According to Rafael Frandoloso and Nubia Macedo, in the chapter "Glãsser Disease" (book "Swine Diseases"), G. parasuis causes a severe acute infection in piglets housed in the nursery phase. The different serovars of G. parasuis can be identified by various techniques such as polymerase chain reaction (PCR), indirect hemagglutination, and agar gel precipitation, and are classified into three groups according to their virulence. The serovars considered highly pathogenic are 1, 5, 7, 10, 12, and 14; with particular emphasis on serovars 1, 5 and 12, which are very common in Brazil and other pig-producing countries.Serovars 2, 4, 8, and 15 are considered moderately pathogenic, followed by serovars 3, 6, and 9, which are considered to have low pathogenicity or are non-pathogenic.

[0006] Despite the 15 serovars of G. parasuis already identified (Kielstein and Rapp-Gabrielson, 1992), the detection of a high percentage of non-typifiable (NT) strains demonstrates the existence of other serogroups not yet identified (Pires et al., 2019; Frandoloso and Dazzi 2023).

[0007] G. parasuis exclusively infects swine and is one of the first microorganisms to colonize the upper respiratory tract of piglets in the first few days of life, with the sow being the main source of infection (Kuchiishi et al., 2023). G. parasuis is considered an early colonizing agent that, under appropriate conditions, induces a severe systemic inflammatory pathology called Glässer's disease, which is considered one of the main emerging bacterial diseases in swine herds (Frandoloso, 2022). The disease can affect animals of any age. However, it is very common in piglets aged five to seven weeks. When present in the bloodstream, this agent leads to the depletion of TCR gamma / delta T lymphocytes and decreases the functional activity of monocytes, which facilitates the systemic diffusion of G. parasuis in the pig's body (Frandoloso et al., 2012; Dazzi et al., 2020).

[0008] Glässer's disease occupies a prominent position among the main infectious challenges of the nursery phase. Many factors can explain the increase in clinical cases of Glässer's disease, and among them, the following stand out: management practices (mixing of piglet batches with different microbiological and immunological backgrounds); the use of vaccines with limited or even absent cross-protection potential between serovars; and the circulation of highly virulent G. parasuis strains capable of triggering Glässer's disease in conventionally healthy animals (primary pathogen profile). Viral (porcine circovirus and Influenza A virus) and bacterial (Mycoplasma hyopneumoniae, Bordetella bronchisetica, Pasteurella multocida, and Actinobacillus pleuropneumoniae) co-infections generally facilitate the infection process of G. parasuis (Frandoloso; Schryvers, 2020; Ramos Prigol et al., 2022; Frandoloso and Dazzi, 2023). The lesions caused by G.Glässer's disease is characterized by polyserositis, polyarthritis, pericarditis, septicemia, and fibrinous meningitis. The economic losses associated with Glässer's disease are due to the high mortality of affected piglets, a high number of culls, and depreciation of carcasses at the slaughterhouse. The diagnosis of the disease is based on the clinical characteristics of the outbreak, macroscopic lesions, isolation, and molecular characterization of the bacteria (Frandoloso and Macedo, 2022). In this sense, it is noteworthy that the process of isolating the strains referenced in the present invention is not trivial; in fact, it is a complex and laborious process. This is remarkable since, until now, no veterinary pharmaceutical composition capable of providing broad-spectrum protection against the different existing serovars of G. parasuis was known.

[0009] Economically, uncontrolled infections caused by G. parasuis result in highly significant losses, which can exceed $80 million per year for the swine production chain (Holtkamp, ​​2007). This loss is the sum of several variables, such as: a) delayed growth; b) decreased feed conversion ratio; c) decreased daily weight gain; d) high costs resulting from the use of antibiotics; e) veterinary technical assistance; and f) high mortality rates that can reach 10% (Oliveira & Pijoan, 2004; Nedbalcova et al., 2016).

[0010] Controlling Glâsser's disease represents one of the main challenges for veterinary clinicians and the veterinary pharmaceutical industry, mainly due to the high antigenic diversity of the polysaccharide capsules of G. parasuis (Frandoloso and Schryvers, 2020; Frandoloso and Dazzi, 2023).

[0011] Vaccines are essential to control infections caused by G. parasuis, and consequently, Glasser's disease. Licensed vaccines available for G. parasuis are generally classic vaccines formulated with inactivated and / or attenuated whole bacterial bodies. In the experimental / scientific sphere, protein subunit vaccines based on structural antigens administered intramuscularly or orally have been evaluated in swine. In summary, vaccines against G. parasuis can be of the following types: i) inactivated bacterial vaccines; ii) live attenuated bacterial vaccines; and iii) subunit vaccines.

[0012] Inactivated bacterial vaccines are produced from strains of G. parasuis grown in the laboratory and subsequently inactivated so that they are unable to cause disease. Inactivation can be achieved by physical, chemical methods, or a combination of both. These vaccines typically contain a variety of bacterial antigens, including surface proteins, lipopolysaccharides (LPS), and a polysaccharide capsule. Administration is usually by intramuscular injection.

[0013] In turn, live attenuated bacterial vaccines are developed from virulent strains of G. parasuis that have undergone attenuation processes, in which their virulence is reduced without, however, completely losing their immunogenic properties. Live attenuated bacterial vaccines can be administered intramuscularly. On the other hand, subunit vaccines are those formulated with purified components of G. parasuis, such as specific proteins used by microorganisms during the pathogenesis of infection. These vaccines can be produced using genetic engineering techniques or extraction and purification of natural antigens; and their administration is carried out... usually administered intramuscularly.

[0014] Vaccination is an important preventive measure to reduce the transmission of G. parasuis among pigs and to prevent the development of Glässer disease. In line with the development of modern vaccines, the strategic use of transferrin-binding protein B (TbpB) as a vaccine antigen stands out as one of the main strategies to induce broad protection against all serovars of G. parasuis (Barasuol et al., 2017; Frandoloso et al., 2020; Frandoloso et al., 2015; Frandoloso; Schryvers, 2020; Guizzo et al., 2018; Ramos Prigol et al., 2022). TbpB PROTEIN

[0015] The TbpB protein is an immunogenic lipoprotein anchored to the outer membrane of G. parasuis, making it accessible to antibodies. Specifically, the function of the TbpB protein is to capture porcine transferrin and transport it to the surface of the TbpA protein, which internalizes an iron ion into the periplasmic space. This system is vital for G. parasuis and is the only iron uptake mechanism of this microorganism (Moraes, 2009; Noinaj et al., 2012a; Noinaj et al., 2012b; Frandoloso and Schryvers, 2020).

[0016] Molecularly, the TbpB protein can be classified into 3 molecular clusters (I, II and III) (Guizzo et al., 2018), with most G. parasuis strains, regardless of capsular type, expressing TbpBs belonging to cluster III.

[0017] Considering the need for an adequate vaccine formulation for the prevention of infections related to bacterial microorganisms belonging to the Pasteurellaceae family, and also considering that vaccines capable of mediating an effective and broad-spectrum immune response are essential to combat diseases caused by bacterial pathogens, the present invention proposes a method for the production of inactivated G. parasuis antigens, a single-dose polyvalent vaccine formulation with a broad spectrum of protection against G. parasuis, comprising the combination of G. parasuis serovars expressing TbpBs belonging to clusters I and III. Furthermore, this patent application discloses a method for preventing Glässer's disease in swine, in which the vaccine formulation is administered to the animal, preferably, in the first weeks of life.

[0018] Thus, the objective of the present invention is to describe an innovative and surprising strategy to overcome the capsular specificity of G. parasuis, in order to provide broad-spectrum protection to immunized animals. In this respect, it is noteworthy that the vaccine antigens employed in the present invention consist of whole, inactivated bacterial bodies with high expression of a structural lipoprotein antigen, which is responsible for conferring broad protection against any strain / serovar of G. parasuis. STATE OF THE ART

[0019] Some studies in the literature propose vaccines against G. parasuis to prevent Glässer's disease in swine. However, none of the vaccines currently available on the market are capable of inducing a broad-spectrum immune response like the vaccine formulation described in this patent application. Therefore, the objective of the present invention is to meet the existing need for an immunogenic, safe vaccine formulation capable of preventing pathologies caused by G. parasuis, in particular Glässer's disease, caused by a wide diversity of G. parasuis serovars.

[0020] US patent document 9132183 B2, owned by BOEHRINGER INGELHEIM ANIMAL HEALTH USA INC., published on September 6, 2012 (US 2012 / 0225091 A1), describes a live vaccine of a Haemophilus parasuis cell culture (former taxonomy of G. parasuis) exhibiting attenuated pathogenicity capable of triggering a protective immune response when administered to swine. According to the description in US 9132183 B2, the cell culture was modified from a pathogenic parental strain by MNNG mutagenesis (from the English “N-methyl-N'-nitro-N-nitrosoguanidine”) and was selected for complete streptomycin dependence for growth. Furthermore, according to the aforementioned document, several SNPs (single nucleotide polymorphisms) have been identified as being associated with specific proteins that are linked to virulence, as seen in the literature for H. parasuis or related bacterial species.Specifically, the aforementioned patent document describes a vaccine formulation obtained from the serovar 5 strain of H. parasuis, capable of providing heterologous protection against serovars 4 and 13 as well.

[0021] Thus, unlike the vaccine formulation described in the present invention, the vaccine formulation of US9132183 B2, although capable of providing heterologous protection against serovars 4 and 13, describes a composition comprising only 1 serovar, serovar 5, comprising attenuated and not inactivated bacteria. Furthermore, the aforementioned document does not describe the overexpression of TbpB in a non-recombinant form, a differentiating factor of the vaccine formulation of the present patent application.

[0022] In turn, document WO 2011 / 131789, owned by INTERVET INTERNATIONAL BV and published on October 27, 2011, refers to the use of the bacterium Haemophilus parasuis, serovar 5, which expresses a protein of approximately 60 kDa when cultured in liquid medium under iron restriction. The presence of this protein was demonstrated by Western blotting using serum from a convalescent animal that recovered from an infection by H. parasuis, serovar 4. Conversely, the aforementioned protein was not observed when H. parasuis, serovar 5, cultured under conditions of abundant iron, was incubated with serum from the same convalescent animal. This fact demonstrates that an iron-restricted environment is indispensable to stimulate the expression of the 60 kDa protein. Interestingly, and unexpectedly, no other protein bands were observed in Western blotting, suggesting that the 60 kDa protein is an immunodominant antigen among G. parasuis proteins.

[0023] Specifically, document WO 2011 / 131789 describes a vaccine preparation comprising inactivated H. parasuis serovar 5 cells capable of expressing an iron restriction-stimulated protein. However, unlike the vaccine formulation described in the present patent application, the vaccine formulation in WO 2011 / 131789 describes a composition comprising only one serovar, serovar 5, inactivated with formalin, and overexpressing a native 60 kDa protein belonging to a single antigenic cluster. Thus, the aforementioned document neither teaches nor even suggests a vaccine formulation like the one described in the present patent application comprising at least one serovar expressing TbpB from cluster I and at least one serovar expressing TbpB from cluster III, capable of promoting the prevention of Glässer's disease in a single dose.

[0024] The use of the inactivating agent formaldehyde, as detailed below, alters The antigenic characteristics of the TbpB protein, in immunological terms, eliminate the protective potential associated with this antigen. Thus, in order to overcome this technical problem, the present patent application describes a method for inactivating 4 clinical strains of G. parasuis with thimerosal, a molecule that acts on the genetic material of bacteria and, surprisingly, does not alter the structural and antigenic characteristics of the protein antigens, in particular, the TbpB protein.

[0025] Furthermore, another advantage of the present invention is related to the fact that the 4 strains of G. parasuis express TbpBs classified in genetic clusters I and III, which are found in 100% of the clinical strains of G. parasuis circulating in Brazil, the United States, Canada, Spain, and Asia (Curran et al., 2015; Guizzo et al., 2018; Fernandez et al., 2023). This fact demonstrates the importance of the present invention, since it reveals a safe and effective solution to a known technical problem that has not yet been overcome worldwide. In particular, the present invention proposes a pharmaceutical formulation characterized by providing an antigenic balance between the different serovars, which guarantees the development of antibodies against the structural antigens of the least immunogenic strain included in the present invention (for example, serovar 1).

[0026] With regard to non-patent references in the technical field of the present invention, the work of Frandoloso, R. et al., 2015, entitled “Nonbinding site-directed mutants of transferrin binding protein B exhibit enhanced immunogenicity and protective capabilities” is cited; Martinez-Martinez, S. et al., 2016, entitled “A vaccine based on a mutant transferrin-binding protein B from Haemophilus parasuis induces a strong T-helper 2 response and bacterial clearance after experimental infection”, - Barasuol, BM et al., 2017, entitled “New insights into functional and cross-linking properties of antibodies generated against recombinant TbpBs from Haemophilus parasuis”, - Guizzo, JA, et al., 2018, entitled “The amino acid selected to generate mutant TbpB antigens in transferrin binding may compromise in vivo protective capacity”, and de Prigol, SR et al., 2022, entitled “TbpB Y167A- Based vaccine can protect pigs against Glasser's disease triggered by G. parasuis SV7 expressing TbpB Cluster I” which define the state of the art in general, since (i) they do not describe any types of serovars of G. parasuis and, furthermore, (ii) do not use inactivated wild-type (non-recombinant) bacterial strains, as described in this invention. Moreover, the aforementioned documents describe the use of recombinant TbpB protein, unlike the present invention, and do not mention or even suggest an immunogenic composition comprising the combination of serovars 1 (SV1), 4 (SV4), 5 (SV5) and non-typifiable serovar (SVNT), for single-dose use for the prevention of Glässer disease.

[0027] Thus, unlike the solutions proposed in the prior art, the present invention, in addition to revealing a safe and effective method for inactivating 4 clinical strains of G. parasuis, reveals a polyvalent vaccine formulation with heterologous protection against any serovar of G. parasuis, regardless of its capsular type. It has the added advantage of being easy to administer and available in a single dose, produced from thimerosal-inactivated strains comprising a combination of serovars 1 (SV1), 4 (SV4), 5 (SV5) and a non-typifiable serovar (NT).Therefore, the vaccine formulation of the present invention facilitates the management of the immunization process by eliminating the need for a second vaccine dose, which is economically advantageous, generating savings on the extra vaccine dose, on the team and supplies for vaccination, and on animal losses due to the vaccination process, and for the animal it represents a gain in well-being by reducing stress and other risks associated with the restraint process to which animals are subjected during vaccination in the nursery phase. In addition to all this, the immunological effect of the vaccine formulation of the present invention is superior to compositions already available on the market, in the following characteristics: i) speed of the antibody-based immune response (onset of immunity); and ii) broad protection profile against any serovar of G. parasuis.

[0028] Therefore, the aforementioned prior art, even when combined with each other, and in any combination, would not result in the outcomes described in this patent application. SUMMARY OF THE INVENTION

[0029] The present invention aims to propose a safe and effective method for the production of inactivated antigens. In particular, the present invention aims to propose a method for the inactivation of 4 clinical strains. of G. parasuis, which correspond to serovars 1 (SV1), 4 (SV4), 5 (SV5) and non-typifiable serovar (NT), by means of the organometallic compound thimerosal, which has antiseptic and antifungal properties, whose structure is presented below according to Formula I. Formula 1.

[0030] Thimerosal is the chemical compound used to inactivate the four clinical strains of G. parasuis that make up the antigenic unit of the polyvalent vaccine formulation described in this patent application.

[0031] The clinical strains of G. parasuis used in the development of the technology described in this patent application were registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen), under registration number A95E38B.

[0032] Furthermore, the present invention also proposes a polyvalent vaccine formulation, in a single-dose presentation, with a broad spectrum of protection against G. parasuis. In particular, the present invention describes a broad-spectrum veterinary vaccine formulation for the prevention of infections caused by clinical strains of G. parasuis, regardless of their capsular type (serovars), which induce Glässer disease.

[0033] In the present invention, the ratio obtained between the antigen concentration (> 5x10 8 The number of CFU / dose) and the concentration of the adjuvant (> 5% adjuvant) in the aforementioned vaccine formulation surprisingly allows for the induction of seroconversion in just 14 days, without the need for a booster vaccine (2 â dose).

[0034] Specifically, the vaccine formulation disclosed in this patent application is based on the combination of 4 wild-type clinical strains of G. parasuis, comprising the nucleotide sequences SEQ ID Nos.: 1, 2, 3 and 4, which correspond to serovars 1 (SV1), 4 (SV4), 5 (SV5) and non-typifiable serovar (NT), respectively, which in turn correspond to polypeptide sequences with SEQ ID Nos.: 5, 6, 7 and 8. The G. parasuis serovars mentioned in this patent application are associated with 81% of Glãsser disease cases diagnosed in Brazil, considering the capsular cross-reactivity between SV5 and SV12 (Pires Espindola et al., 2019; Ramos Prigol et al., 2022). Therefore, taking into account the capsular antigens of the clinical strains under discussion, the vaccine formulation of the present invention could prevent more than 80% of cases of Glässer's disease occurring in Brazil, with the potential to achieve 100% coverage due to the strategic overexpression of the TbpB protein (cluster I and III proteins) on the surface of the 4 vaccine strains referenced above.

[0035] Furthermore, considering: i) the antigenic specificity of G. parasuis capsular antigens, i.e., the low or absent cross-reactivity between different serovars; and ii) the high diversity of G. parasuis serovars described in Brazil [9 classic serovars: SV1, SV2, SV4, SV5, SV7, SV12, SV13, SV14 and SV15 (Pires et al., 2019; Ramos Prigol et al., 2022) and 9 new serovars not yet serologically characterized (Pires Espindola et al., 2019)], the polyvalent and broad-spectrum vaccine formulation, as described in this patent application, presents itself as an important and necessary antigenic strategy to overcome the low or even absent heterologous protection between the different G. parasuis serovars.

[0036] With regard, in particular, to the antigenic strategy described in the present invention, a polyvalent formulation is proposed based on four clinical strains of G. parasuis, chemically modulated to overexpress the TbpB protein located in the outer membrane of these microorganisms.

[0037] The TbpB protein is found in some pathogenic bacteria. These bacteria use the TbpB protein to capture transferrin, a protein that transports iron in animal and human organisms. Transferrin represents the main source of iron for G. parasuis, as well as for many other invasive human bacteria. Specifically, the TbpB protein acts as a protein receptor located on the surface of G. parasuis and is responsible for initiating the iron uptake process. Iron is an essential chemical element for the survival and replication of G. parasuis in the environment. host.

[0038] As discussed above, the TbpB protein is a bacterial protein involved in the process of iron uptake from a host protein known as transferrin. TbpB binds specifically to transferrin, forming a protein complex that is transported to the surface of the TbpA protein, an integral membrane protein responsible for removing an iron ion from transferrin and sending it to the bacterial periplasmic space (Noinaj et al., 2012). Once inside the bacterium, the iron is used in various metabolic functions. Therefore, the presence of the TbpB protein in some invasive bacteria determines their survival within the host, as well as their ability to cause disease.Consequently, the overexpression of the TbpB protein, described in the present invention, is fundamental for the generation of specific antibodies (protective immune response) with a broad spectrum of protection (specific response against TbpB) in animals immunized with the vaccine formulation described herein.

[0039] The clinical strains of the vaccine formulation described in this patent application are representative strains of serovars SV1, SV4, SV5 and SVNT, and were deposited with the German Biological Material Deposit Authority Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on August 29, 2024, under numbers DSM 35165, DSM 35166, DSM 35167 and DSM 35168, respectively, and which, in turn, were strategically chosen taking into account the following factors: (i) immunogenic characteristics, (ii) belonging to clusters I and III, and (iii) high chemically inducible expression capacity of the TbpB protein.

[0040] With regard to the TbpB protein, the vaccine formulation described in this patent application includes its two main clusters expressed in G. parasuis, which represent 98% of the total antigenic diversity of this protein (Curran et al., 2015; Guizzo et al., 2018), and 100% of the diversity found in G. parasuis strains found in Brazil (Ramos et al., 2022). To achieve overexpression of the TbpB protein, an innovative chemical strategy is used during the production process of inactivated industrial G. parasuis antigens. This process also relates to one of the objects of protection of this patent application, which is related to mimicking the physiological conditions found in the host, i.e., the condition restrictive iron deficiency, and which stimulate the 4 strains related to the vaccine formulation of the present application to express high amounts of TbpB proteins. BRIEF DESCRIPTION OF THE FIGURES

[0041] To obtain a complete and thorough understanding of the objective of this invention, the following Figures are presented.

[0042] Figure 1 shows the serovars of G. parasuis that cause Glässer disease in Brazil.

[0043] Figure 2 presents the most prevalent G. parasuis serovars found in Brazil and the antigenic profile of the TbpB protein expressed by the clinical strains of these serovars. In this context, it is reiterated that (i) 72.3% of clinical cases of Glãsser's disease occurring in Brazil are caused by the SV1, SV4, SV5 and NT serovars of G. parasuis and (ii) all clinical strains of G. parasuis circulating in Brazil express the TbpB protein belonging to antigenic clusters I or III.

[0044] Figures 3A, 3B, 3C, and 3D illustrate the results obtained from the immunogenicity analysis of 40 clinical strains of G. parasuis tested in mice. For this analysis, the animals were immunized with two doses of monovalent vaccines based on SV1 (10 different strains), SV4 (10 different strains), SV5 (10 different strains), and NT (10 different strains) of G. parasuis. Fourteen days after revaccination, serum samples were collected and tested by indirect enzyme-linked immunosorbent assay (ELISA) to determine the systemic IgG titers against G. parasuis SV1, SV4, SV5, and NT.

[0045] The graphs in Figures 3A, 3B, 3C, and 3D mentioned above show the mean (bars) and standard deviation of the mean specific antibody titers in mice for each of the serovars tested. Additionally, the ability of the 40 G. parasuis strains to express the TbpB protein was tested by flow cytometry. TbpB expression was artificially induced by restricting the available iron in the culture medium. The G. parasuis strains SV1, SV4, SV5, and NT with the best combination of immunogenic profile and TbpB protein expression capacity are indicated in the graphs with an arrow, and represent the strains (SV1 - ID #02, SV4 - ID #11, SV5 - ID #29, and NT - ID #32) chosen for the development of the polyvalent vaccine formulation against G. parasuis.

[0046] Figure 4 presents a phylogenetic tree constructed with TbpB protein sequences from Brazilian clinical strains of G. parasuis and others collected from a public online repository. All Brazilian strains of G. parasuis are classified in antigenic clusters I and III of the TbpB protein, and predominantly, the Brazilian strains belong to cluster III.

[0047] Figures 5A, 5B, 5C, 5D, and 5E present the results of an experiment evaluating adjuvants for the formulation of a broad-spectrum vaccine against G. parasuis. In this experiment, three monovalent vaccines based on the ID #02 strain of G. parasuis SV1 were potentiated with 3 different adjuvants. The formulations were named B276.001 (polymeric adjuvant based on sodium polyacrylate), B276. 002 (adjuvant emulsion of mineral oil, water and dimethyldioctadecyl ammonium bromide (DDA) and B276. 003 (aluminum hydroxide associated with mineral oil emulsion). The vaccines were applied to piglets free of specific pathogens at 21 and 35 days of age. Serum samples were collected throughout the study and tested by Indirect Enzyme-Linked Immunosorbent Assay (Indirect ELISA) to determine the titers of systemic anti-G IgGs.Statistical comparison was performed using the Kruskal-Wallis test (Figures 5A, 5B, 5C, and 5D) and two-way ANOVA (Figure 5E). Significant differences (p<0.05), when present, are indicated in the graphs. In this example, formulation B276.002, enhanced with the adjuvant emulsion of mineral oil, water, and dimethyldioctadecyl ammonium bromide (DDA) (Figure 5B), proved to be the most immunogenic formulation.

[0048] Figure 6 shows the functional capacity of antibodies from pigs immunized with formulations B276.001, B276.002, and B276.003 based on the ID #02 strain of G. parasuis SV1. Serum samples collected before primary vaccination (D21) and 14 days after revaccination (D49) were analyzed using the classical complement pathway activation assay. All vaccines induced the production of antibodies with a high capacity to activate the classical complement pathway.

[0049] Figures 7A, 7B, and 7C illustrate the biological effect of three chemical inactivators on the functional characteristics of the TbpB protein. In this In this experiment, the G. parasuis strains SV1 (ID #02), SV4 (ID #11), SV5 (ID #29), and NT (ID #32) were cultured under iron restriction and inactivated, under the same conditions, with formalin, formaldehyde, and thimerosal. The analysis of the biological effect of the chemical inactivators was performed using the Dot-Blot assay based on porcine transferrin (pTF) labeled with the peroxidase enzyme (HRP). In this test (Figure 7A), the production of a label (development of a black color) on the nitrocellulose membrane indicates the ability of the inactivated bacteria to capture transferrin via the TbpB protein. The absence of a signal indicates that the chemical inactivator altered the structure of the TbpB protein and removed its ability to bind to porcine transferrin. Figure 7B schematically illustrates the molecules involved in the positive reaction (ability of the inactivated bacteria to capture pTF-HRP). Figure 7C illustrates the interaction of recombinant TbpB protein with pTF-HRP (positive control of the test).The results of this experiment demonstrated that formalin is not suitable for the inactivation of G. parasuis because it alters the structure of the TbpB protein. Conversely, thimerosal and formalin can be used.

[0050] Figure 8 presents the antigenic characteristics of G. parasuis strains inactivated with formalin, formalin, and thimerosal. In this experiment, the G. parasuis strains SV1 (ID #02), SV4 (ID #11), SV5 (ID #29), and NT (ID #32) were cultured under iron restriction and inactivated, under the same conditions, with formalin, formalin, and thimerosal. The antigenicity analysis of the TbpB protein present on the surface of the inactivated bacteria was performed using flow cytometry with porcine antibodies (IgG) produced against recombinant TbpB protein. The results of this experiment demonstrate that formalin significantly alters the antigenic characteristics of the TbpB protein, exemplified by the inability of the anti-TbpB porcine antiserum to recognize the TbpB protein in the assay.Conversely, the anti-TbpB porcine antiserum was able to recognize the TbpB protein present on the surface of formalin- and thimerosal-inactivated bacteria, demonstrating the preservation of the original / native antigenic characteristics of the bacteria after inactivation.

[0051] Figure 9 demonstrates that the inactivating chemical molecule can compromise the clinical efficacy (protection) of a vaccine based on chemically inactivated microorganisms. In this experiment, two groups of SPF piglets were immunized; one with the polyvalent vaccine formulation based on... In two groups, piglets were tested: one group received a formalin-inactivated G. parasuis SV1, SV4, SV5, and NT strains, and another received a thimerosal-inactivated vaccine. A third group (control) received only one injection of PBS. Vaccines were administered at 21 days of age (single dose), and 3 weeks after vaccination, all animals were experimentally challenged with a lethal dose of G. parasuis SV5. One hundred percent (100%) of piglets immunized with the polyvalent vaccine formulation based on thimerosal-inactivated G. parasuis strains survived the experimental challenge. In contrast, no piglets (0%) immunized with the formalin-inactivated vaccine or inoculated with PBS survived the infection process. This example demonstrates that formalin cannot be used to inactivate G. parasuis strains with TbpB protein overexpression.

[0052] Figures 10A, 10B, 10C, and 10D demonstrate the effect of antigen concentration on the serological potency (immunogenicity) of the polyvalent vaccine formulation based on the SV1, SV4, SV5, and NT serovars of G. parasuis. In this experiment, SPF piglets were immunized with 3 formulations of the single-dose polyvalent vaccine formulation against G. parasuis containing 10 7 , 10 8 and 10 9G. parasuis SV1, SV4, SV5, and NT per vaccine dose. A fourth group was inoculated with PBS (negative control). Piglets were immunized (single dose) at 21 days of age, and serum samples were collected throughout the study and tested by Indirect ELISA to determine the systemic IgG titers against G. parasuis SV1, SV4, SV5, and NT. The graphs show the mean (bars) and standard deviation of the mean of specific antibody titers for each serovar tested. Statistical comparison was performed using the Kruskal-Wallis test. Significant differences (p<0.05), when present, are indicated in the graphs. In this example, the formulation B276.010.10 9 formulated with 10 9 bacteria / serovar / dose and enhanced with the adjuvant emulsion of mineral oil, water and dimethyldioctadecyl ammonium bromide (DDA) was consistently more immunogenic compared to the other two formulations prepared with lower antigen concentrations (10 7 and 108 ).

[0053] Figure 11 describes the steps in the industrial production process of the single-dose, broad-spectrum polyvalent vaccine formulation against G. parasuis. The first step in the process consists of activating the bacterial strains frozen at -80°C. Q C (Figure 11 A). The bacteria are seeded in chocolate agar plates (Figure 11 B) and incubated at 35 S C - 39 e C until colony formation is observed. Subsequently, the bacterial colonies are collected using a microbiological loop and inoculated into Erlenmeyer flasks (Figure 11C) containing supplemented Pleuropneumonia-Like Organism (PPLO) liquid culture medium. The liquid culture is incubated at 35°C. S C - 39 e C is stirred until the desired optical density is reached. At this point, the culture is transferred to a reactor containing supplemented PPLO liquid culture medium (Figure 11D). The culture is incubated at 35°C. S C - 39S C is agitated until the desired optical density is reached. At this point, the culture is transferred to a second reactor containing supplemented PPLO liquid culture medium (Figure E) and incubated under the same conditions as before for a period of 2 to 8 hours, until optical density values ​​between 0.5 and 2.0 are reached. Next, a solution containing deferoxamine is added to the culture to induce overexpression of the TbpB protein. Then, the bacteria are inactivated by adding a solution containing thimerosal to the reactor (Figure 11F). After bacterial inactivation, the antigens are concentrated and dialyzed with PBS by tangential filtration (Figure 11G), and stored in sterile, single-use plastic bags (Figure 11H) at a controlled temperature of 2 to 8°C. SC. Finally, after verifying the sterility of the produced antigens, the vaccine is formulated in a formulation tank (Figure 111). At this point, the 4 bacterial strains of G. parasuis (SV1, SV4, SV5 and NT), the adjuvant (emulsion of mineral oil, water and dimethyldioctadecyl ammonium bromide (DDA)), the diluent (PBS) and the preservative (thimerosal) are mixed, and the vaccine is packaged in vaccine vials (Figure 11J).

[0054] Figures 12A, 12B, 12C, and 12D illustrate the clinical protection capacity of the single-dose polyvalent vaccine formulation in pigs experimentally challenged with lethal doses of G. parasuis SV1, SV4, SV5, SV7, and NT. In this experiment, 5 groups of piglets were immunized with a single dose of the polyvalent vaccine formulation formulated with the thimerosal-inactivated G. parasuis serovars SV1, SV4, SV5, and NT, potentiated with an oil adjuvant. Additionally, another 5 groups of piglets were inoculated with PBS (control groups), and another 2 groups were immunized with the licensed vaccines Porcilis Glãsser (MSD Animal Health) and Hiprasuis Glãsser (Hipra Animal Health). The polyvalent vaccine (single dose) and the PBS (single dose) were The other vaccines were administered intramuscularly to SPF piglets at 21 days of age. The Porcilis Glãsser vaccine (MSD Animal Health) was administered intramuscularly to SPF piglets at 21 and 35 days of age (two doses), and the Hiprasuis Glãsser vaccine (Hipra Animal Health) was also administered intramuscularly to SPF piglets at 21 and 42 days of age (two doses). Twenty-two (22) days after the administration of the polyvalent vaccine, the piglets, according to the experimental groups (G1, G2, G3, G4, G5, G6, G7, G8, G9, G10), were experimentally challenged with lethal doses of G. parasuis SV1, SV4, SV5, SV7, and NT. Piglets from groups G11 and G12, immunized with the Porcilis Glãsser (MSD Animal Health) and Hiprasuis Glãsser (Hipra Animal Health) vaccines, respectively, were challenged 14 days after revaccination with serovars 5 and 1 of G. parasuis, respectively.The statistical comparison of survival rates between the groups was performed using the Logrank test. Significant differences (p0.05), when present, are indicated in the figures.

[0055] Figure 12A shows the survival rate (%) of piglets vaccinated with the polyvalent vaccine formulation (71.42%), with the Hiprasuis Glãsser - HIPRA vaccine (25%), and unvaccinated (0%) against G. parasuis SV1. Figure 12B shows the survival rate of piglets vaccinated (85.71%) and unvaccinated (0%) against G. parasuis SV4. Figure 12C shows the survival rate of piglets vaccinated with the polyvalent vaccine formulation (100%), with the Porcilis Glãsser - MSD vaccine (20%), and unvaccinated (0%) against G. parasuis SV5. Figure 12D shows the survival rate of piglets vaccinated (83.33%) and unvaccinated (0%) against G. parasuis NT. Figure 12E shows the survival rate of vaccinated (100%) and unvaccinated (14.28%) piglets against G. parasuis SV7. The results of this experiment demonstrate that the polyvalent vaccine formulation induces protection against G. parasuis serovars.parasites homologous to those included in its formulation (SV1, SV4, SV5 and NT) and heterologous (SV7), but not limited to these.

[0056] Figure 13 illustrates the ability of the single-dose polyvalent vaccine formulation against G. parasuis to stimulate the production of anti-TbpB antibodies in swine. In this experiment, sera from all of the Piglets included in the experiment described in Figure 12 were collected at the pre-experimental challenge time. Antibody (IgG) titration against recombinant TbpB protein was performed on the sera, as previously described by Guizzo et al., 2018. Results are expressed as the logarithm of the titers found in the indirect ELISA. Statistical comparison between groups was performed using the Kruskal-Wallis test. Significant differences (p<0.05), when present, are indicated in the graph. In this example, we demonstrate that only piglets immunized with the polyvalent vaccine developed antibodies against the TbpB protein. Surprisingly, neither of the two licensed vaccines (Porcilis Glãsser - MSD Animal Health and Hiprasuis Glãsser - Hipra Animal Health) stimulated the production of anti-TbpB IgGs 14 days after revaccination.

[0057] Figure 14 describes the protection spectrum of the single-dose polyvalent vaccine formulation against G. parasuis. This experiment was conducted with a total of 30 clinical strains of G. parasuis belonging to serovars SV1, SV2, SV4, SV5, SV6, SV7, SV11, SV13, SV14, SV15, and SVNT. The protection analysis, defined by the ability of the vaccine antiserum to recognize any strain of G. parasuis, regardless of its capsular type, was performed by flow cytometry. The results of this experiment demonstrate that the vaccine modulates the generation of antibodies (IgG) capable of recognizing all the tested strains of G. parasuis, demonstrating the broad protection spectrum of the single-dose polyvalent vaccine formulation against G. parasuis.

[0058] Figure 15 illustrates the evolution of rectal temperature before and after the application of the single-dose polyvalent vaccine formulation against G. parasuis. This experiment was conducted to evaluate the clinical safety of the vaccine. Statistical comparison was performed using the Kruskal-Wallis test. Post-vaccination time points were compared with values ​​obtained before vaccination (D0). Significant differences (p<0.05), when present, are indicated in the graph.

[0059] Figures 16A and 16B illustrate the evolution of rectal temperature before and after the application of the polyvalent vaccine formulation in pregnant sows. This experiment was conducted to evaluate the clinical safety of the vaccine in pregnant sows. The temperatures measured from the different sows... The results were compared between the experimental groups at each time point studied. Statistical comparison was performed using a two-way ANOVA test. Significant differences (p<0.05), when present, are indicated in the graphs.

[0060] Figure 17 shows the serological profile of the sows immunized during gestation. Sows in group G1 were immunized at 65 days of gestation. Sows in group G2 were immunized at 65 and 86 days of gestation. And sows in group G3 were inoculated with PBS at 65 and 86 days of gestation. Serum samples collected at 65, 86, 100, and 114 days of gestation were analyzed by indirect ELISA based on G. parasuis SV5. Statistical comparison between the different time points of each group was performed using the Kruskal-Wallis test. Significant differences (p<0.05), when present, are indicated in the graphs.

[0061] Figure 18 illustrates the kinetics of passive immunity in piglets born to vaccinated (G1 and G2) and unvaccinated (G3) sows during gestation. Serum samples from neonatal piglets were collected at 7, 14, and 21 days of age. Anti-G. parasuis SV5 IgG titers were determined by indirect ELISA. Statistical comparisons between different time points for each group were performed using the Kruskal-Wallis test. Significant differences (p<0.05), when present, are indicated in the graphs. The dashed line indicates the minimum IgG titer associated with experimental clinical protection. DETAILED DESCRIPTION OF THE INVENTION

[0062] Unless otherwise specified, the terms used throughout this descriptive report have their common meanings in the art, within the context of the disclosure, and in the specific context in which each term is used. Certain terms that are used to describe the disclosure are discussed below, or elsewhere in this descriptive report, to provide additional guidance to the technician regarding the description of the disclosure. Publications cited herein are specifically incorporated by reference in their entirety.

[0063] It will be appreciated that the same thing can be said in different ways. Consequently, alternative language and synonyms can be used for any one or more of the terms discussed here. None Special significance should be placed on whether a term is elaborated upon or discussed here. Synonyms for certain terms are provided in the text itself, but the exemplification of some synonyms does not preclude the potential use of others that may not be listed here.

[0064] The present invention relates, in a first embodiment, to a process for producing inactivated antigens of G. parasuis. The process for producing said antigens comprises the following steps: a) inoculating a seed (microorganism culture) of G. parasuis into a first culture medium, in a preferred embodiment, a chocolate agar plate is used, and then incubating the plate at a temperature capable of promoting seed propagation, in a preferred embodiment the temperature is 35 to 39 eC; b) inoculate the colonies from said first culture medium into a second culture medium, optionally a liquid culture medium (e.g., PPLO), and incubate the inoculum under uniform growth conditions; c) transfer said inoculum to a reactor containing optionally supplemented liquid culture medium and cultivate said inoculum until the optical density reaches a desired value; in one embodiment, the optical density OD is 0.5 to 2.0; d) transfer said inoculum to the cultivation reactors until the desired growth is achieved and add iron chelating solution; e) maintain the cultivation under the temperature, oxygen, and pH conditions for a predetermined period; in one embodiment, a temperature of 35 to 39°C may be used. SC, dissolved oxygen of 20 to 40%, pH of 7.2 to 7.8, for a period of 2 to 8 hours; f) inactivate the cultured microorganisms by different media available in the art, in a preferred embodiment the inactivation is carried out by adding an inactivating solution; g) if necessary, optionally process the antigens, after inactivation of the medium to obtain a concentrated solution by different techniques available in the art, such as using centrifugation or tangential filtration; h) collect the sterile processed final antigens from step “g”; i) optionally, take the sample for quality control analyses; and j) optionally, keep the antigens refrigerated at a temperature between 2 and 8 S C until the final formulation.

[0065] Therefore, to demonstrate the potential of the aforementioned process for producing inactivated G. parasuis antigens, the present invention will be described in detail regarding the steps performed and their respective parameters.

[0066] The production process for the inactivated antigens of the present invention employs an iron chelating solution. Said iron chelating solution is based on deferoxamine and / or 2,2'-bipyridine. In step “d” of said process for the production of inactivated G. parasuis antigens, the iron chelating solution based on deferoxamine is preferably used at a final concentration in the medium between approximately 50 and 150 pM.

[0067] With regard to the inactivation of G. parasuis, for example, in addition to the chemical inactivation strategy described in the present invention, it is also possible to achieve the inactivation of clinical strains of G. parasuis by physical methods (temperature) or even by a combination of physical and chemical methods.

[0068] Regarding the chemical methods of inactivating clinical strains of G. parasuis during the production process of inactivated antigens of the present invention, in one embodiment, a buffered formalin solution or a thimerosal solution is used as the inactivating solution.

[0069] In a preferred embodiment, in step “f” of the process for producing inactivated G. parasuis antigens, a thimerosal inactivating solution with a final concentration of 0.005% to 0.02% (w / v) or a buffered formalin solution with a concentration of 0.01% to 0.03% (v / v) is used.

[0070] In a second embodiment, the present invention describes a single-dose vaccine formulation capable of providing heterologous and broad-spectrum protection against G. parasuis. Specifically, the polyvalent vaccine formulation disclosed in this patent application is based on a combination of G. parasuis capable of expressing TbpB from Clusters I and III. In a preferred embodiment of the present invention, four clinical strains of G. parasuis are used, corresponding to serovars 1 (SV1), 4 (SV4), 5 (SV5) and a non-typifiable serovar (NT), which have been filed with the DSMZ under numbers DSM 35165, DSM 35166, DSM 35167 and DSM 35168, respectively, and, as such, presents itself as an efficient antigenic strategy due to the expression of TbpBs belonging to clusters I and III, which are present in more than 98% of clinical strains of G. parasuis.

[0071] More specifically, the present invention relates to a polyvalent vaccine formulation characterized by comprising a combination of representative serovars of clusters I and III of the TbpB protein of G. parasuis, an adjuvant, a preservative solution, and a pharmaceutically acceptable diluent. The serovars of the present invention refer to isolated, non-recombinant, inactivated serovars that overexpress the TbpB protein of two clusters (I and III) by adding an iron chelator, such as deferoxamine and / or 2,2'-bipyridine. In particular, the serovar capable of expressing the TbpB protein of cluster I of the present invention corresponds to serovar 5 (SV5), and the serovars capable of expressing the TbpB protein of cluster III correspond to serovars 1 (SV1), 4 (SV4) or NT.

[0072] Regarding the adjuvant used in the vaccine formulation disclosed in this patent application, a mineral oil, water, and dimethyldioctadecyl ammonium bromide (DDA) emulsion at a concentration of 5 to 30% w / v is preferably used. The adjuvants may also be based on aluminum hydroxide associated with a mineral oil and sodium polyacrylate emulsion in a phosphate saline solution, with 1 to 10% aluminum hydroxide, 5 to 30% mineral oil emulsion, and 5 to 30% sodium polyacrylate.

[0073] In a preferred embodiment, the vaccine formulation of the present invention uses a buffered thimerosal preservative solution, preferably prepared at a concentration between 0.5 and 10% w / v, wherein the thimerosal preservative solution is used to achieve a final concentration of the vaccine formulation between 0.005% and 0.02%.

[0074] Regarding the diluent used in the polyvalent vaccine formulation of the present invention, it is a buffered saline solution, preferably PBS at a concentration of 5 to 70% w / v.

[0075] A third embodiment of the present invention describes a dosage form of said vaccine formulation. Specifically, a vaccine formulation prepared for single-dose administration is described in this patent application. The dosage form of the present invention It comprises 2 ml of solution and the concentrated Glaesserella antigens SV1, SV4, SV5 and NT in the range of >1 x10 9 UFC, each.

[0076] In a fourth embodiment of the present invention, a combination of clinical strains of G. parasuis, deposited with the Depositary Authority DSMZ under numbers DSM 35165, DSM 35166, DSM 35167 and DSM 35168, corresponding to serovars of the SV1, SV4, SV5 and NT types, respectively, of strains of the species G. parasuis, is described.

[0077] In a fifth embodiment of the present invention, the use of said combination of clinical strains corresponding to serovars SV1, SV4, SV5 and NT of G. parasuis, adjuvant, preservative solution and pharmaceutically acceptable diluent in the manufacture of a vaccine formulation to prevent Glässer's disease in swine is described. In particular, the use of said combination of clinical strains corresponding to serovars SV1, SV4, SV5 and NT of G. parasuis, adjuvant, preservative solution and pharmaceutically acceptable diluent in the manufacture of a vaccine formulation to prevent Glässer's disease in swine is described, wherein said vaccine formulation is formulated to be administered to pregnant sows and piglets in the first weeks of life.

[0078] This patent application also refers to a sixth embodiment, a method for preventing Glässer's disease in swine, in which the vaccine formulation disclosed in the present invention is administered to the animal, preferably in the first weeks of life.

[0079] In a seventh embodiment, the present invention relates to a kit comprising a combination of any of the clinical strains corresponding to the serovars, of the SV1, SV4, SV5 and NT types of G. parasuis disclosed herein, adjuvant, preservative solution and pharmaceutically acceptable diluent in the manufacture of a vaccine formulation to prevent Glässer disease in swine. Particularly, the present invention describes a kit characterized by comprising a combination of clinical strains corresponding to the serovars, of the SV1, SV4, SV5 and NT types of G. parasuis and a preservative solution.

[0080] In an eighth embodiment of the present invention, a process for producing a vaccine formulation is described. During the development of the vaccine formulation, inactivation with formalin, in Concentrations between 0.2 and 0.5% v / v were successfully used to inactivate the bacteria. However, this process altered, as seen later, the antigenic characteristics of the vaccine antigens (as shown in line 3 of Figure 7). Therefore, vaccines formulated with these antigens did not achieve the desired efficacy (Figure 9).

[0081] Thus, other methods of bacterial inactivation were sought, ensuring adequate and safe inactivation without negatively impacting the antigenic characteristics of the TbpB protein overexpressed on the surface of the four bacterial vaccine strains. Two inactivation possibilities were found: thimerosal solution at a final concentration of 0.005% to 0.020% w / v and formalin solution of 0.01% to 0.03% w / v (both with positive dot-blot staining as shown in Figure 7).

[0082] As described in the present invention, the inactivation process is a critical factor for maintaining the antigenic characteristics of the TbpB protein of the present invention and, consequently, the immunogenic and antigenic potential of the vaccine formulation described in this application. In this regard, a possible preferred embodiment of the inactivation of the clinical strains of G. parasuis, which are used in the vaccine formulation of the present invention, will be described below. Inactivation of clinical strains

[0083] After the inactivation process of the G. parasuis bacteria with a thimerosal inactivating / preservative solution at a final concentration of 0.005% and 0.02%, or with a buffered formalin inactivating / preservative solution at a final concentration of 0.01 to 0.03% formalin, the samples must be removed from the reactor to confirm inactivation. In this patent application, the use of the inactivating solution ensures the inactivation of the culture without negatively impacting the antigenic characteristics of the TbpB protein, i.e., preserving the antigenic and functional characteristics of the TbpB protein. These characteristics are analyzed at this stage of the process using the Dot-blot technique. Dot-blot is an immunological technique that allows the analysis of the interaction between different molecules. In particular, in this invention, the ability of vaccine antigens immobilized on a nitrocellulose membrane to capture (bind) porcine transferrin conjugated with HRP was analyzed (Figure 7).

[0084] Upon achieving the desired growth of G. parasuis, which is Measured based on growth at OD600nm (0.5 - 1.0 absorbance units), an iron chelator, in this case a solution of deferoxamine and / or 2,2'-bipyridine, is added to the culture. Bacterial iron metabolism is a key point of the present invention and is intentionally used to increase the amount of TbpB protein on the surface of the 4 vaccine strains. Pigs immunized with the vaccine containing these antigens develop antibodies against the TbpB protein, which is present in 100% of G. parasuis strains. This is the theoretical basis that explains the broad spectrum of protection of this vaccine against any antigenic variant (serovars) of G. parasuis.We also emphasize that the protective capacity of these antigens may potentially not be restricted to the Glaesserella genus, since the antibodies generated against clusters I and III of TbpB can recognize the same proteins present on the surface of other bacteria such as Actinobacillus pleuropneumoniae and A. suis, which cause important pathologies in swine.

[0085] Iron chelators work by binding to iron and preventing it from being used by the bacteria during the propagation phase within the reactor. Consequently, after the bacteria deplete their cytoplasmic iron reserves, a survival mechanism is activated that consists of increasing the expression of proteins responsible for iron uptake from the host's transferrin; in this case, the need for overexpression of the TbpB protein stands out. This process mimics the restrictions encountered by the G. parasuis bacteria during the systemic infection phase in pigs.

[0086] The bacterial inactivation process described in the present invention surprisingly preserves the antigenic characteristics of the TbpB protein. Therefore, the vaccine formulation of the present invention is capable of inducing the production of antibodies that recognize native TbpB proteins, and consequently, confers broad protection against G. parasuis belonging to any serogroup of this bacterium.

[0087] In this regard, it is noteworthy that the heterologous protection provided by the vaccine formulation described in this patent application is not based on the polysaccharide capsule of G. parasuis. The heterologous protection provided by the vaccine formulation of this patent application is justified based on transferrin-binding protein B (TbpB). On the other hand, homologous protection based on the polysaccharide capsule refers to a classic immunization strategy in which polysaccharides derived from the capsules of the four strains included in the vaccine are used to induce a protective immune response against these four serovars (G. parasuis SV1, SV4, SV5 and NT).

[0088] TbpB plays a fundamental role in iron acquisition by many bacteria. Iron is an essential nutrient for bacterial survival and multiplication, and the ability to bind and capture iron from the host is crucial for the microorganism to establish infection and cause disease. The TbpB protein, along with the TbpA protein, forms the iron acquisition protein surface receptor of G. parasuis. Due to its biological function, the TbpB protein is considered an important virulence factor of this agent (Frandoloso & Schryvers, 2020). The polyvalent vaccine formulation of this patent application includes 4 virulent clinical strains of G. parasuis and, as previously mentioned, strategically expresses TbpBs belonging to clusters I and III, which are present in more than 98% of clinical strains of G. parasuis.

[0089] During the infection process, G. parasuis significantly increases the expression of the TbpB protein on its surface, a situation that does not occur when the bacterium is cultivated in the laboratory, in vitro culture, using conventional culture media. This fact may explain, in part, why the classic licensed vaccines found in the state of the art, based on inactivated G. parasuis, do not induce antibodies against TbpB (Figure 13). In contrast, the G. parasuis strains included in the polyvalent vaccine formulation of this patent application are chemically stimulated during in vitro culture, so as to overexpress the TbpB proteins and, therefore, when applied to pigs, stimulate the development of antibodies against this structural antigen (Figure 13). Thus, the TbpB proteins are, in fact, responsible for ensuring heterologous and broad-spectrum protection against any strain of G. parasuis, as widely demonstrated in the literature.

[0090] The polyvalent vaccine formulation of the present patent application has, in addition, an extra advantage over existing vaccines. The vaccine utilizes a unique technology for maintaining the antigenic characteristics of the TbpB protein and other protein antigens, as known and disclosed in the state of the art. Therefore, the protein antigens expressed by the four G. parasuis strains are antigenically identical to those found on the surface of bacteria circulating in Brazilian farms that cause Glãsser's disease. This occurs due to the antigen expression modulation strategy developed during the chemical process of the industrial production stage of the vaccine strains (bacteria), which is capable, for example, of overexpressing the TbpB protein, mimicking the physiological conditions of iron restriction found in the host, and which stimulate the four vaccine strains to express high amounts of TbpB proteins. Consequently, the high expression of TbpB proteins aids in homologous and heterologous protection against G. parasuis serovars.parasites associated with Glässer's disease in Brazil, however, not being restricted solely to this country due to the universal characteristics of the polyvalent vaccine formulation of the present patent application. Production of antigens

[0091] The production process of the antigens that make up the vaccine formulation of the present invention is carried out separately for each of the 4 serovars [Serovars 1, 4, 5 and non-typifiable (NT), or SV1, SV4, SV5 and NT]. The strains used in this invention were isolated from clinical cases of Glässer disease. The serovars used in the vaccine express TbpBs from 2 clusters (I and III).

[0092] The aforementioned process for producing the antigens that make up the vaccine formulation of the present invention comprises the following steps: a) inoculating a seed (microorganism culture) of G. parasuis onto a chocolate agar plate and then incubating the plate at a temperature between 35 and 39 Qa) until seed propagation; b) inoculate the colonies from the chocolate agar plate into liquid culture medium (PPLO) and incubate the inoculum under uniform growth conditions, with controlled temperature between 35 and 39 °C and agitation between 120 and 180 rpm; c) transfer the inoculum to the reactor containing liquid culture medium supplemented with 5 to 15% inactivated bovine serum, 50 to 200 pg / mL NAD (nicotinamide adenine dinucleotide) and 0.025 to 5% dextrose and cultivate the inoculum until the optical density reaches a value between 0.5 and 2.0; d) transfer the inoculum to the culture reactors until the desired growth is achieved and add iron chelating solution at a final concentration of between approximately 50 and 150 pM; e) maintain the culture under temperature conditions between approximately 35 and 39 SC; dissolved oxygen between approximately 20 and 40%, pH between approximately 7.2 and 7.8, for a period of approximately 2 to 8 hours; f) inactivate the cultures by adding an inactivating solution at a final concentration of 0.005 to 0.02%; g) process the antigens, after inactivation, by concentrating the cultures using continuous flow centrifugation or tangential filtration; h) collect the final processed antigens in a sterile manner; i) optionally, take a sample for quality control analyses; e) optionally, keep the antigens refrigerated at a temperature between 2 and 8 e C up to the formulation.

[0093] The following steps are carried out to prepare the vaccine formulation: 1. Calculate the total quantity needed in colony-forming units (CFU), which must be greater than or equal to 1 x 10 9 CFU / dose per serovar, in order to meet the product formulation; 2. Calculate the volume of antigen needed to meet the formulation batches based on the total quantification required in CFU; 3. Transfer each inactivated G. parasuis serovar (serovars 1, 4, 5, and NT) to the reactor aseptically and homogenize; Vaccine formulation

[0094] For vaccine formulation, a prior step is required to homogenize mineral oil (5-30% w / v), water (94.5 to 65%) and dimethyldioctadecyl ammonium bromide (DDA) (0.5 to 5% w / v) aseptically, in order to obtain an emulsion for use as an adjuvant, with a concentration < 30% w / v in the formulation reactor.

[0095] Specifically, in one embodiment of the present invention, the steps related to the vaccine formulation process (as shown) The steps described in Figure 11 are as follows: a) homogenize the antigens; b) add the PBS diluent solution with thimerosal preservative solution, already solubilized and filtered with a sterilizing filter, to the formulation reactor; c) transfer the homogenized antigens to the formulation reactor; d) transfer the mineral oil, water, and DDA (adjuvant) emulsion to the formulation reactor; e) check the pH and, if necessary, adjust the desired pH (pH 7.0 - 7.5) with a sodium hydroxide or 1 N hydrochloric acid correction solution; f) homogenize the formulation; g) check the pH again and, if necessary, adjust the desired pH (pH 7.0 - 7.5) with a sodium hydroxide or 1 mol L⁻¹ hydrochloric acid correction solution. 1 h) complete the volume with PBS diluent solution and homogenize; i) maintain the product under agitation at 50 to 200 rpm in the formulation reactor until the start of filling; and j) keep the reactor cooled to a temperature between approximately 2 and 8 °C and pressurized.

[0096] Each dose of the single-dose vaccine formulation of the present patent application comprises 2 ml containing adjuvant, diluent, preservative, and concentrated Glaesserella NT antigen in the range of > 1 x 10 9 CFU, Glaesserella antigen concentrate SV1 in the range of > 1 x10 9 CFU, Glaesserella SV4 antigen concentrate in the range of > 1 x10 9 CFU and Glaesserella SV5 antigen concentration in the range of > 1 x10 9 UFC. Demonstration experiments Example 1 - In silico analysis of the antigenic diversity of clinical strains of G. parasuis associated with Glässer's disease in Brazil

[0097] Example 1 illustrates the diversity of G. parasuis serovars associated with clinical cases of Glässer disease diagnosed in Brazil. Based on serovar analysis, it is possible to establish a combination of clinical G. parasuis strains for the formulation of a broad-spectrum vaccine providing protection against the most prevalent serovars found in Brazil.

[0098] A total of 459 clinical strains of G. parasuis isolated from pigs. Animals housed on farms in Rio Grande do Sul (n=35), Santa Catarina (n=127), Paraná (n=56), São Paulo (n=36), Minas Gerais (n=154), Espírito Santo (n=2), Bahia (n=1), Mato Grosso (n=37), Mato Grosso do Sul (n=6) and Goiás (n=5), molecularly typified by Pires et al. (2019), were analyzed.

[0099] The serovar count and graphical representation of prevalence were performed using Prism software (GraphPad Prism Software, LLC, USA). The results are illustrated in Figure 1, and demonstrate that the G. parasuis serovars SV4 (26.6%), NT (17.6%), SV5 (15.0%), and SV1 (13.1%) are the most prevalent in Brazil. The sum of the prevalence of these serovars reaches 72.3%, and considering the cross-reactivity existing between some strains of SV5 and SV12 (Bak and Riising, 2002), a vaccine formulation containing the combination of clinical strains corresponding to the 4 G. parasuis serovars has the direct potential to prevent 80.8% of clinical cases of Glässer's disease occurring in Brazil.

[0100] Considering the distribution and prevalence of G. parasuis serovars observed worldwide (Macedo et al., 2021), a polyvalent vaccine formulation based on G. parasuis serovars 1, 4, 5 and NT would have the potential to prevent 52% of clinical cases of Glässer disease observed in the United States of America, 58.9% of those observed in Canada, 48.7% of those observed in Europe, 42% of those observed in China and 48.3% of those occurring in Vietnam.

[0101] As exemplified in Figure 2 and previously discussed, the TbpB protein is an essential virulence factor for G. parasuis. This protein can be antigenically classified into 3 clusters, named I, II, and III. Notably, 100% of G. parasuis strains found in Brazil express TbpBs belonging to clusters I and III (Guizzo et al., 2018; Prigol et al., 2022). The expression of the TbpB protein on the surface of G. parasuis can be artificially modulated during bacterial culture (Frandoloso et al., 2011). Therefore, selecting bacteria that express TbpBs belonging to clusters I and III and that simultaneously belong to serogroups SV1, SV4, SV5, and NT becomes a surprising and unique strategy for the development of a universal vaccine against G. parasuis, regardless of its capsular type. Example 2 - Immunogenicity analysis in C57BL / 6 mice and analysis of the expression of clinical strains of G. belonging to serogroups SV1, SV4, SV5 and NT

[0102] Example 2 highlights the existence of immunogenic variations among different clinical strains of G. parasuis classified within the same serogroup (Figure 3). Similarly, it demonstrates that the expression of the TbpB protein is strain-dependent. Therefore, this example presents the selection of the four clinical strains of G. parasuis belonging to SV1, SV4, SV5, and NT that possess the best combination of parameters (i) antibody production (immunogenicity) and (ii) the ability to express the TbpB protein artificially modulated through iron restriction.

[0103] Forty clinical strains of G. parasuis were used. Ten strains expressed polysaccharide capsules classified within serogroup SV1 (ID#01, ID#02, ID#03, ID#04, ID#05, ID#06, ID#07, ID#08, ID#09 and ID#10); 10 strains expressed polysaccharide capsules classified within serogroup SV4 (ID#11, ID#12, ID#13, ID#14, ID#15, ID#16, ID#17, ID#18, ID#19 and ID#20); Ten strains expressed polysaccharide capsules classified within serogroup SV5 (ID#21, ID#22, ID#23, ID#24, ID#25, ID#26, ID#27, ID#28, ID#29, and ID#30); and ten strains expressed non-typable (NT) polysaccharide capsules (ID#31, ID#32, ID#33, ID#34, ID#35, ID#36, ID#37, ID#38, ID#39, and ID#40).With the aforementioned strains, 40 potentiated vaccines were formulated with 50% oil adjuvant (complete and incomplete Freund's adjuvant, where the mineral oil and water emulsion containing killed mycobacteria (Mycobacterium spp) refers to the complete adjuvant; and the mineral oil and water emulsion refers to the incomplete adjuvant). Each vaccine contained, per ml, a concentration of 10. 9 bacteria. Two doses of each vaccine were administered intraperitoneally to C57BL / 6 mice 14 days apart. The first dose of the vaccines was potentiated with the complete Freund's adjuvant, and the second dose with the incomplete Freund's adjuvant. A volume of 0.2 ml of the vaccines was administered intraperitoneally in each immunization. Serum samples were collected 14 days after the second immunization.

[0104] The titer of anti-G. parasuis SV1, SV4, SV5 and NT antibodies (IgG) was determined by the indirect quantitative ELISA technique (Guizzo et al., 2018). The detection of the percentage of bacteria with TbpB overexpressed on the surface of G. parasuis was performed using flow cytometry with porcine transferrin (pTF) labeled with fluorescein isocyanate (FITC) (Barasuol et al., 2017).

[0105] As illustrated in Figure 3A, the immunogenicity of G. parasuis SV1 strains could be classified into three intensity levels: low (strains ID #04 and #06), medium (strains ID #01, 02, 05, 07, 08, and #10), and high (strains ID #02 and #09). The expression of the TbpB protein also varied according to the clinical strain of G. parasuis, with the lowest expression level detected in strain ID #07 (55.61%) and the highest in strain ID #06 (95.33%). Considering the combination of immunogenicity and TbpB protein expression parameters, strain ID #02 (indicated with an arrow, Figure 3A) was chosen as the representative microorganism of the SV1 serogroup of G. parasuis.

[0106] As illustrated in Figure 3B, the immunogenicity of G. parasuis SV4 strains varied markedly according to the clinical strain tested. Strains ID #11, #19, and #20 were the most immunogenic, stimulating the production of mean IgG titers greater than or equal to 1:5,600. TbpB protein expression also varied according to the clinical strain of G. parasuis, with the lowest expression level detected in strain ID #12 (35.49%) and the highest in strain ID #19 (84.55%). Considering the combination of immunogenicity and TbpB protein expression parameters, strain ID #11 (indicated with an arrow, Figure 3B) was chosen as the representative microorganism of the SV4 serogroup of G. parasuis.

[0107] As illustrated in Figure 3C, the immunogenicity of G. parasuis SV5 strains could be classified into three intensity levels: low (strains ID #22, #23, #27, #28, and #30), medium (strains ID #21, #24, and #35), and high (strains ID #26 and #29). The expression of the TbpB protein also varied according to the clinical strain of G. parasuis, with the lowest expression level detected in strain ID #22 (61.55%) and the highest in strain ID #29 (91.68%). Considering the combination of immunogenicity and TbpB protein expression parameters, strain ID #29 (indicated with an arrow, Figure 3A) was chosen as the representative microorganism of the SV5 serogroup of G. parasuis.

[0108] As illustrated in Figure 3D, the immunogenicity of G. parasuis NT strains could be classified into three levels of intensity: low (strains ID#31, #33, #35, #37, #38 and #40), medium (strains ID #34 and #39), and high (strains ID (#32 and #36). The expression of the TbpB protein also varied according to the clinical strain of G. parasuis, with the lowest level of expression detected in the strain ID #35 (62.66%) and the highest in strain ID #32 (93.44%). Considering the combination of immunogenicity parameters and TbpB protein expression, strain ID #32 (indicated with an arrow, Figure 3D) was chosen as the representative microorganism of the NT serogroup of G. parasuis. Example 3 - Sequencing and phylogenetic analysis of the TbpB protein from SV1 vaccination dates.

[0109] Example 3 presents the classification of TbpB proteins expressed by the 4 clinical strains of G. parasuis included in the vaccine. The variability of the amino acid sequence of the TbpB protein expressed by SV1, SV4, SV5, and NT was analyzed by constructing a phylogenetic tree encompassing the 4 sequences of the TbpBs included in the vaccine (expressed by the vaccine strains), and other sequences available in a public repository.

[0110] The TbpB gene was obtained from 46 nucleotide sequences deposited in the public repository GenBank (accessed August 2, 2022). The sequences obtained were from G. parasuis strains isolated in Brazil (n=27), the United States of America (n=3), Canada (n=5), Spain (n=6), and China (n=5). In addition to these 46 strains, the 4 tbpB gene sequences from vaccine strains were included in the analysis, totaling 50 strains. All strains were initially isolated from pigs with Glässer disease. [0011 1] The TbpB protein sequences were aligned using MUSCLE software. The phylogeny of the sequences was generated using PhyML software, and the phylogenetic tree rendering was performed using Fig Tree software (Figure 4).

[0112] With regard to Figure 4, the TbpB protein variants represented in the phylogenetic tree can be classified into three clusters (cluster I, cluster II, and cluster III). The location of the 4 vaccine strains is indicated in the figure, with SV5 classified within cluster I and SVs 1, 4, and NT within cluster III. Example 4 - Immunogenicity analysis of candidate vaccine clinical strains of G. parasuis in combination with different adjuvants in Liyres piglets.

[0113] Example 4 presents the effect of different adjuvants on the serological potency of a monovalent vaccine based on strain #02 of G. parasuis SV1. Strain #02 is the least immunogenic of the 4 vaccine strains, as shown in Figure 3A (murine model). The formulations were evaluated in piglets free of specific pathogens and deprived of maternal colostrum (target species).

[0114] In this respect, three different adjuvants were evaluated: a polymer based on sodium polyacrylate, an emulsion of mineral oil, water and oily dimethyldioctadecyl ammonium bromide (DDA), and aluminum hydroxide associated with a mineral oil emulsion. A total of 4 experimental groups were formed. Groups G1 (n=10), G2 (n=10), and G3 (n=10) were immunized at 21 and 35 days of age with 2 ml doses of formulations B276.001 (polymer based on sodium polyacrylate), B276.002 (emulsion of mineral oil, water, and dimethyldioctadecyl ammonium bromide (DDA)), and B276.003 (aluminum hydroxide associated with mineral oil emulsion), respectively. Group G4 (n=6) received two 2 ml inoculations of PBS pH 7.2 at 21 and 35 days of age.

[0115] All animals immunized with the formulations containing adjuvants B276.001, B276.002, and B276.003 produced high titers of anti-G. parasuis SV1 IgG after completion of the vaccination protocol (Figure 5). Surprisingly, animals in group G2, immunized with the formulation enhanced with the adjuvant emulsion of mineral oil, water, and dimethyldioctadecyl ammonium bromide (DDA), developed high IgG titers as early as 7 days after vaccine administration (Figure 5B). At 14 days post-vaccination, the mean circulating IgG titer was 1:2,960, and increased after revaccination (Figure 5B). As illustrated in Figure 5E, the IgG titers found at 7 and 14 days after primary vaccination were significantly higher (p<0.05) in animals immunized with the formulation comprising adjuvant B276.002 compared to the titers observed in animals vaccinated with the formulation comprising adjuvants B276.001 and B276.003 (in this case, statistical differences were observed only at 14 days). This result demonstrated that the adjuvant emulsion of mineral oil, water and dimethyldioctadecyl ammonium bromide (DDA) could be explored in a single-dose vaccine formulation for the prevention of Glâsser's disease, since animals immunized with only one dose of the formulation comprising the adjuvant B276.002 were able to significantly increase antibody titers over the first 14 days of vaccination, and therefore, achieving protective antibody titers at the beginning of the critical period for the development of Glâsser's disease (5 weeks of age). Thus, it was observed that the speed of the immune response modulated by the aforementioned adjuvant was surprising, and far superior when compared to the speed of immune responses modulated by currently commercially available vaccines (Porcilis Glâsser - MSD, and Hiprasuis Glâsser - Hipra).

[0116] As illustrated in Figure 6, the 3 adjuvants modulated the production of functional anti-G. parasuis antibodies, and therefore, capable of activating the classical pathway of the complement system. Serum samples collected from piglets 14 days after revaccination (D49) significantly activated (p<0.0001) the complement system more compared to samples collected at the pre-vaccination time (D21). Example 5 - In vitro analysis of the impact of chemical inactivation on the functional and antigenic characteristics of the TbpB protein.

[0117] Example 5 demonstrates that the type of chemical inactivator can alter the antigenic characteristics of the TbpB protein and, consequently, compromise the efficacy of a polyvalent vaccine formulation based on G. parasuis strains with TbpB protein overexpression.

[0118] As previously described, the four strains of G. parasuis SV1, SV4, SV5, and NT of the present invention, deposited with the Depositary Authority DSMZ under numbers DSM 35165, DSM 35166, DSM 35167, and DSM 35168, respectively, were cultured in liquid Pleuropenumonia-Like Organism medium supplemented with Nicotinamide Adenine Dinucleotide (NAD, 50-200 ng / mL) and dextrose (0.025-5%), and under iron-restrictive conditions (deferoxamine, 50-150 pM). After bacterial propagation, the cultures were inactivated with: i) thimerosal (0.005%-0.02%), ii) formalin solution (0.01% - 0.03%) and iii) formaldehyde (0.25% - 0.5%).

[0119] Inactivated bacteria were immobilized on a nitrocellulose membrane (dot-blot test), and after a blocking process with skim milk (5%), were incubated with porcine transferrin (pTF) conjugated with the peroxidase enzyme (HRP). Subsequently, the membrane was revealed with a chromogenic solution, which, when in contact with HRP, confers a chemiluminescent signal, indicating the presence of the TbpB protein on the bacterial surface. In the test, recombinant TbpB protein was used as a positive control to demonstrate the biological uptake activity of pTF-HRP.

[0120] As illustrated in Figure 7, bacteria inactivated with thimerosal and with formalin solution < 0.025% (final concentration in culture) did not undergo antigenic changes. Conversely, bacteria inactivated with formalin at a concentration > 0.25% (final concentration in culture) underwent significant antigenic and functional changes, observed by the inability of the TbpB protein to capture pTF-HRP (absence of chemiluminescent signal).

[0121] Additionally, the same inactivated bacteria were subjected to antigenicity analysis using flow cytometry, as described by Barasuol et al. (2017). Briefly, a total of 10 6Inactivated and non-inactivated bacteria were incubated with a porcine antiserum specific for the TbpB protein. Subsequently, a phycoerythrin-conjugated secondary antibody specific for porcine IgG was added to the reaction, and the bacteria were then acquired and analyzed using a FACVerse flow cytometer (Becton Dickinson, USA).

[0122] As illustrated in Figure 8, porcine anti-TbpB recombinant antibodies (IgG) were able to bind to bacteria inactivated with thimerosal and formalin. Conversely, few formalin-inactivated bacteria showed anti-TbpB antibodies associated with their surface, indicating that formalin altered the antigenic characteristics of the TbpB protein. The percentage of formalin-inactivated bacteria with associated anti-TbpB IgGs was less than 3%, while, conversely, more than 88% of thimerosal- and formalin-inactivated bacteria showed IgGs associated with the TbpB protein. Statistical differences (p<0.0001) were found between the antigenic characteristics of formalin-treated bacteria compared to those treated with formalin. with those inactivated with thimerosal and / or formalin. This result demonstrates that formalin, in addition to altering the biological activity of the TbpB protein (Figure 7), also alters its antigenic structure (Figure 8). Therefore, based on the experiments performed, it can be concluded that formalin is not a suitable inactivating agent for microorganisms with overexpression of the superficial TbpB protein. Example 6 - In vivo analysis of the protective capacity of antigens inactivated with thimerosal and formalin in piglets free of specific pathogens.

[0123] Example 6 demonstrates that vaccines based on G. parasuis strains with overexpressed TbpB protein and inactivated with formalin at a concentration > 0.25% do not induce protective responses against Glãsser disease caused by G. parasuis serovar 5.

[0124] In this experiment, a total of 24 specific pathogen-free piglets were used. The piglets were distributed into three experimental groups, namely: Group 1 (G1, n = 8) immunized with the polyvalent formulation containing formalin-inactivated G. parasuis SV1, SV4, SV5, and NT. Group 2 (G2, n = 8) immunized with the polyvalent formulation containing thimerosal-inactivated G. parasuis strains. Group 3 (G3, n = 8) inoculated with PBS, pH = 7.2. The vaccines were potentiated with an oil-type adjuvant. The animals were immunized at 21 days of age via intramuscular injection, and three weeks later were experimentally challenged with 10 6 G. parasuis instilled intranasally. After the challenge, the animals were clinically monitored for 14 days.

[0125] As shown in Figure 9, all unvaccinated animals challenged with G. parasuis SV5 died between days 2 and 4 post-infection. Surprisingly, none (0%) of the animals immunized with the formalin-inactivated G. parasuis strain-based vaccine survived the infection. Deaths were observed between days 4 and 6 post-infection. Conversely, 100% of the animals immunized with thimerosal-inactivated bacteria survived the experimental challenge.

[0126] This result, demonstrated through an in vivo study in the target species, showed that the negative effects of formalin on the biological function and antigenic characteristics of TbpB result in an antigen incapable of stimulating a protective antibody response in the target species. Therefore Conversely, inactivating bacteria with TbpB overexpression using thimerosal, which does not alter the functional and antigenic characteristics of the proteins of the inactivated microorganisms, results in a protective antigen. Example 7 - Immunogenicity assessment of the single-dose polyvalent vaccine formulation enhanced with an oil adjuvant.

[0127] Example 7 illustrates the serological potency (immunogenicity) and clinical protection capacity (survival rate) of the polyvalent vaccine formulation based on the SV1, SV4, SV5 and NT serovars of G. parasuis in antigen and adjuvant concentrations of mineral oil, water and dimethyldioctadecyl ammonium bromide (DDA) emulsion type optimized for single dose application.

[0128] In this example, three vaccine formulations were evaluated, differing only in antigen concentration. All formulations included G. parasuis SV1, SV4, SV5, and thimerosal-inactivated NT. Formulation B276.010.10 7 , was formulated with 10 7 G. parasuis SV1, SV4, SV5 and NT enhanced with 20% adjuvant emulsion of mineral oil, water and dimethyldioctadecyl ammonium bromide (DDA). Formulation B276.010.10 8 , was formulated with 10 8 G. parasuis SV1, SV4, SV5 and NT enhanced with 20% oily adjuvant. Formulation B276.010.10 9 , was formulated with 2x10 9 G. parasuis SV1, SV4, SV5 and NT potentiated with 20% adjuvant. The vaccines were packaged in 100 mL vials and evaluated for sterility.

[0129] To evaluate the serological potency of the vaccines, four experimental groups composed of colostrum-deprived piglets were formed. Group 1 (G1, n = 6) was immunized with the B276.010.10 formulation. 7 Group 2 (G2, n = 6) was immunized with the B276.010.10 formulation. 8 Group 3 (G3, n = 6) was immunized with the B276.010.10 formulation. 9 Group 4 (G4, n = 6) was inoculated with PBS, pH 7.2. A single 2 mL dose of the vaccines and / or PBS pH 7.2 was administered via deep intramuscular injection in the side of the neck to piglets at 21 days of age.

[0130] Blood (serum) samples were collected throughout the vaccination study at the following times: D0 (before vaccination), D+14, D+21 (animals at 42 days of age, a critical age for the development of Glässer's disease). Serum samples were used to determine systemic IgG titers against the SV1, SV4, SV5, and NT serotypes of G. parasuis by means of... Quantitative Indirect ELISA, based on vaccine strains. Statistical comparison between experimental groups at each time point of the study was performed using a two-way ANOVA test. Statistical differences (p<0.05) are indicated in the graphs.

[0131] As illustrated in Figure 10, the serological potency (systemic IgG titers) of formulation B276.010.10 9 was significantly superior (p<0.05) compared to formulation B276.010.10 7 for all serovars of G. parasuis. The same profile of serological superiority was observed in relation to formulation B276.010.10 8 However, in this case, statistical differences were only found 14 days after vaccination and with respect to serovars SV4 and NT. Low titers (1:100) of polyreactive antibodies were found in some animals against SV1, SV4, SV5, and NT at time D0 (in all groups) and throughout the study in animals in group G4.

[0132] This example demonstrates that the formulation B276.010.10 9 It has the surprising ability to stimulate a potent antibody (IgG) response 14 days after a single dose of the vaccine. The serological potency was dependent on the combination of antigenic load and the type of adjuvant used.

[0133] The speed of serological modulation induced by formulation B276.010.10 9 This is surprising and essential for the prevention of Glässer's disease, which, in most farms, appears from 35 days of age in piglets (Frandoloso and Macedo, 2022). Therefore, this formulation has the ability to provide immunological protection when maternal immunity ceases to be protective, guaranteeing piglets immunological coverage during the periods of greatest susceptibility to the development of Glässer's disease caused by any serovar of G. parasuis. Example 8 - Industrial production of vaccine antigens and formulation of the polyvalent vaccine formulation based on serotypes SV1, SV4, SV5 and NT of

[0134] Example 8 illustrates the steps in the production process of the polyvalent vaccine formulation based on the SV1, SV4, SV5 and NT serovars of G. parasuis with overexpression of the TbpB proteins belonging to clusters I and III.

[0135] The summary of the vaccine development process is... illustrated in Figure 11. The process described is specific to the bacterial strain included in the vaccine. The first step in the vaccine production process consists of activating the bacterial strains frozen at -80°C. S C (Figure 11 A). The bacteria are seeded onto chocolate agar plates (Figure 11 B) and incubated for 35 to 39 eC until observation of colony formation. Subsequently, bacterial colonies were collected using a microbiological loop and inoculated into Erlenmeyer flasks (Figure 11C) containing 1000 mL of Pleuropneumonia-Like Organism (PPLO) liquid culture medium supplemented with Dextrose (0.025 to 5% w / v) and Nicotinamide Dinucleotide (50-200 pg / mL w / v). The liquid culture was incubated for 35 to 39 e C is stirred (100-200 rpm) until an optical density value of 0.5-1.0 absorbance units is reached at a wavelength of 600 nm. At this point, the culture is transferred to a reactor containing 90-100 liters of supplemented PPLO liquid culture medium (Figure 11 D). The culture is incubated for 35 to 39 QC is stirred (50-200 rpm) with dissolved oxygen injected at a ratio of 20% to 40%, with the pH adjusted between 7.2 and 7.8, until an optical density between 0.5 and 1.0 is achieved. At this point, the culture is transferred to a reactor containing 450-500 liters of supplemented PPLO liquid culture medium (Figure E) and incubated under the same conditions for a period of 2 to 8 hours, obtaining optical density values ​​between 0.5 and 2.0. Subsequently, a solution containing deferoxamine (50 to 150 pM, final concentration) is added to the culture to induce overexpression of the TbpB protein. Therefore, the bacteria are inactivated by adding a solution containing thimerosal (0.005% and 0.02%) to the reactor (Figure 11F). After bacterial inactivation, the antigens are concentrated and dialyzed with PBS pH 7.2 by tangential filtration (Figure 11G), and stored in sterile, single-use plastic bags (Figure 11H) at a controlled temperature of 2 to 8°C. QC. Finally, after verifying the sterility of the produced antigens, the vaccine is formulated in a formulation tank (Figure 111). At this point, the 4 bacterial strains of G. parasuis (SV1, SV4, SV5 and NT), the adjuvant (emulsion of mineral oil, water and dimethyldioctadecyl ammonium bromide (DDA)), the diluent (PBS pH 7.2-7.6) and the preservative (thimerosal 0.025-0.05%) are mixed, and the vaccine is packaged in 50, 100 and 150 mL vials (Figure 11J). Example 9 - Protective capacity of the single-dose polyvalent vaccine formulation in piglets immunized and experimentally challenged with G. parasuis SV1, SV4, SV5, SV7 and NT.

[0136] Example 9 demonstrates the clinical efficacy of the single-dose vaccine formulated with G. parasuis serovars SV1, SV4, SV5, and NT against controlled experimental challenge with lethal doses of clinical strains of G. parasuis belonging to serogroups homologous to the vaccine antigens SV1, SV4, SV5, and NT, and heterologous, represented by serovar SV7.

[0137] A total of 85 specific pathogen-free piglets were included in this study. The animals were divided into 10 experimental groups. Groups G1, G2, G3, G5, G6, and G10 were formed with 7 piglets each; group G4 with 6 piglets; groups G7, G8, G9, and G12 with 8 piglets each; and group G11 with 5 piglets.

[0138] Piglets were immunized at 21 days of age with a single 2 ml dose of a polyvalent vaccine formulation based on the SV1, SV4, SV5, and NT serovars of G. parasuis with overexpression of the TbpB protein belonging to clusters I and III, also called a polyvalent vaccine formulation. The vaccine was administered intramuscularly in the side of the neck near the ear. In addition to this vaccine, this study also evaluated the licensed vaccines Porcilis Glãsser (MSD Animal Health) and Hiprasuis Glãsser (Hipra Animal Health), and a PBS solution pH 7.2. The Porcilis Glãsser vaccine vaccination protocol consisted of the intramuscular administration of two 2 ml doses, the first administered at 21 days of age and the second at 35 days of age. In the case of the Hiprasuis Glässer vaccine, the first dose of 2 ml was administered at 21 days of age, and the second at 42 days of age. PBS was inoculated (2 ml) once at 21 days of age.

[0139] Groups G1 to G5 were immunized with the polyvalent vaccine formulation of the present invention. Groups G6 to G10 were inoculated with PBS, and groups G11 and G12 were vaccinated with the Porcilis Glãsser (MSD Animal Health) and Hiprasuis Glãsser vaccines, respectively. At 43 days of age, the animals vaccinated with the polyvalent vaccine (described in this patent application) were challenged with different serovars of G. parasuis. The animals in the group immunized with the Porcilis Glãsser and Hiprasuis Glãsser vaccines were challenged 14 days after revaccination. Groups G1, G6, and G12 were Groups G1 and G2 were challenged with serovar SV1. Groups G2 and G7 were challenged with serovar SV4. Groups G3, G8, and G11 were challenged with serovar SV5. Groups G4 and G9 were challenged with the untyped serovar (NT). Groups G5 and G10 were challenged with serovar SV7. After the experimental challenge, performed intranasally by inoculating 10 6The animals were clinically monitored for a period of 14 days. All animals that died as a consequence of the infection or that survived until the end of the study were necropsied for recording of macroscopic lesions. At that time, samples from systemic sites were collected for bacterial isolation.

[0140] As illustrated in Figure 12, all animals inoculated with PBS and experimentally challenged with G. parasuis SV1 (Figure 12A), SV4 (Figure 12B), SV5 (Figure 12C), and NT (Figure 12D) died as a consequence of the experimental infection. Approximately eighty-five percent (85.72%) of the unvaccinated animals challenged with serovar 7 died after the experimental challenge (Figure 12E). In contrast, the polyvalent vaccine formulation conferred 100% protection against serovar SV5 (Figure 12C), 85.71% against serovar SV4 (Figure 12B), 83.33% against serovar NT, and 71.42% against serovar SV1 (Figure 12A). Surprisingly, the Hiprasuis Glãsser (Hipra Animal Health) and Porcilis Glãsser (MSD Animal Health) vaccines conferred only 25% and 20% protection against experimental challenges based on serovars SV1 and SV5, respectively (Figures 12A and 12C). Therefore, a low level of protection against capsular types homologous to those included in the formulation of these vaccines.Furthermore, the polyvalent vaccine formulation conferred 100% protection against serovar SV7 (Figure 12E), a heterologous serovar in relation to the composition of this vaccine. This demonstrates that the surprising protective potential of the polyvalent vaccine formulation of the present invention goes beyond the serovars included in its composition (heterologous protection).

[0141] During necropsy, animals vaccinated with the polyvalent vaccine formulation of the present invention showed a reduced frequency of macroscopic lesions compared to the groups inoculated with PBS (G6 to G10) by: 90.5% when challenged with G. parasuis SV7; 86.4% when challenged with G. parasuis NT; 77.1% when challenged with G. parasuis SV5; 65.2% when Challenged with G. parasuis SV4, the number of lesions decreased by 51%, and when challenged with G. parasuis SV1, the number of lesions decreased by 51%. In contrast, the Hiprasuis (Hipra Animal Health) and Porcilis Glãsser (MSD Animal Health) vaccines reduced the number of lesions by only 22% and 19.2%, respectively, compared to the PBS inoculated groups (G6 and G8).

[0142] Clinically, the polyvalent vaccine formulation of the present invention reduced the observation of respiratory signs (cough, dyspnea, and sneezing) compared to control groups by up to: 100% when animals were challenged with serovar SV7; 86% when animals were challenged with serovars SV1 and SV5; and 84% and 80% when animals were challenged with serovars SV4 and NT, respectively. Regarding the locomotor system, the polyvalent vaccine formulation of the present invention reduced the observation of lameness by up to: 86% when animals were challenged with serovars SV5 and SV7; 84% when animals were challenged with serovar NT; and 72% when animals were challenged with serovar SV1.With regard to neurological signs, the polyvalent vaccine formulation of the present invention reduced the observation of pedaling / lateral recumbency movement with muscle tremors by up to: 100% when animals were challenged with serovars SV4, SV5 and SV7; 84% when animals were challenged with serovar NT; and 72% when animals were challenged with serovar SV1.

[0143] As illustrated in Figure 13, at the pre-experimental challenge stage, all animals in groups G1, G2, G3, G4, and G5, immunized with the polyvalent vaccine of the present invention, showed antibodies against the TbpB protein. Conversely, no animals in groups G6, G7, G8, G9, G10, G11, and G12 showed anti-TbpB antibodies. Example 10 - Prediction of heterologous in vitro protection based on antigenicity assay by flow cytometry

[0144] Example 10 illustrates the heterologous protection profile of the polyvalent vaccine formulation of the present invention against all serovars of G. parasuis isolated from pigs with Glässer disease in Brazil.

[0145] For this study, a total of 30 clinical strains of G. parasuis representative of serovars SV1 (n=3), SV2 (n=3), SV4 (n=3), SV5 (n=3), SV7 (n=3), SV12 (n=3), SV13 (n=3), SV14 (n=3), SV15 (n=3), and NT (n=3) were used. All strains except two (174, 84-22113, and 84-15995) were isolated from Clinical cases of Glasser's disease occurring in Brazil in 2021 and 2022 were analyzed. Antigenicity assessment was performed by flow cytometry using the protocol described by Barasuol et al. (2017). Briefly, G. parasuis strains were incubated with sera from piglets immunized with the polyvalent vaccine formulation of the present patent application (primary antibody). After the incubation period of the primary antibody and removal of molecules not associated with the structural antigens of the bacteria, a phycoerythrin-labeled porcine anti-IgG goat serum was added to the reaction. After incubation and washing steps, the bacteria were acquired in a FACSVerse flow cytometer (Becton Dickinson, USA) and analyzed for the presence of porcine IgGs associated with their surface. The percentage of bacteria with associated IgG represents the number of bacteria (percentage referring to 10 6bacteria) from a clonal population recognized by antibodies stimulated by the polyvalent vaccine formulation of the present invention.

[0146] As demonstrated in Figure 14, the polyvalent vaccine formulation of the present invention stimulated the development of IgG in immunized piglets capable of recognizing the 30 evaluated G. parasuis strains. The percentage of recognition was dependent on the serovar and the clinical strain within the specific serogroup studied. The minimum in vitro recognition percentage that predicts in vivo clinical protection in the target species was defined as 36%. In this case, and in particular, considering the average reactivity value of the sera of animals immunized with the polyvalent vaccine formulation of the present invention (example 9) against the SV5 ID #21 vaccine strain (Figure 14), which was 36%, and which conferred 100% protection against G. parasuis SV5 (ID #21 strain) (Figure 12) in the immunization and experimental challenge study using specific pathogen-free piglets (example 9).

[0147] Using the same prediction strategy, however, in this heterologous case, the average recognition of the vaccine serum against the G. parasuis SV7 174 strain was 38% (Figure 14), this reactivity percentage being above the established cutoff point of 36%. In this case, the in vivo efficiency of the vaccine against strain 174 was, again, 100% (example 9, Figure 12). Therefore, cytometric analysis demonstrated that the polyvalent vaccine formulation of the present invention is surprisingly capable of inducing the production of antibodies (IgG) capable of recognizing all serovars of G. parasuis associated with the development of Glâsser's disease in Brazil, which do not differ from those found in all other countries with industrial pig production. Example 11 - Clinical safety study of a single-dose inert vaccine formulation based on G. β serotypes SV1, SV4, SV5 and NT.

[0148] Example 1 describes that the polyvalent vaccine formulation of the present invention is safe when applied to young piglets 21 days old. For the purpose of the clinical safety study, 46 conventional piglets, 21 days old, were used. Of this total, 48 animals were vaccinated intramuscularly (lateral neck region) with 2 ml of the polyvalent vaccine formulation of the present invention, and the remaining 8 animals were inoculated with 2 ml of PBS pH 7.2 via the same route of administration. Before vaccine application (DO), the vaccination site / general clinical aspects of all animals were evaluated. After vaccine application, and at times D0+4 hours (period between vaccination and 4 hours after), D+24 hours, D+48 hours, D+72 hours and D+96 hours, the same parameters were evaluated.

[0149] Regarding clinical aspects, piglets in the control group did not show any behavioral changes after PBS application. Conversely, animals immunized with the polyvalent vaccine formulation of the present invention showed mild to moderate prostration during the first 4 hours after product application. This behavior disappeared completely 24 hours after vaccination. Isolated cases of vomiting (2 / 38) and tachypnea (1 / 38) were observed during the first 90 minutes after vaccination. No deaths (0 / 38) as a consequence of product injection were observed during the study.

[0150] Regarding the vaccine application site, edema was observed at the vaccination site in some animals at 4 hours (9 / 38), 24 hours (7 / 38), 48 hours (2 / 38), and 72 hours (1 / 38) after vaccination. Erythema was observed in 1 animal 4 hours after vaccination and in 5 animals 24 hours after injection. No nodules (0 / 38), abscesses (0 / 38), or wounds (0 / 38) were observed at the vaccine application site. Example 12 - Clinical safety study of a single-dose polyvalent vaccine formulation based on G. β serotypes SV1, SV4, SV5 and NT.

[0151] Example 12 demonstrates that the polyvalent vaccine formulation of the present invention is safe when applied to pregnant sows in a single dose at 65 days of gestation, or in two doses at 65 and 86 days of gestation. For the clinical safety assessment, 36 pregnant sows were included in the study. Of this total, 12 sows (G1) were vaccinated intramuscularly (lateral neck region) with 2 ml of the polyvalent vaccine formulation at 65 days of gestation. Another 12 sows (G2) were immunized with the same formulation at 65 and 86 days of gestation. Finally, another 12 sows (G3) were inoculated with 2 ml of PBS pH 7.2 at 65 and 86 days of gestation. Before vaccine application (DO), the vaccination site, rectal temperature, and general clinical aspects of all animals were evaluated.After vaccine administration, and at times D65 + 2 hours, D65 + 4 hours (the period between vaccination and 4 hours after), D66, and daily until 14 days after primary vaccination, the same parameters were evaluated. The same procedure was repeated before and after revaccination of group G2 and application of PBS in group G3.

[0152] Regarding clinical aspects, the sows in group G3 did not show any behavioral changes after PBS inoculation. The same trend was observed in the sows of groups G1 and G2, immunized with one or two doses of the polyvalent vaccine. As illustrated in Figure 16A, a significant increase (p=0.0248) in rectal temperature was observed 4 hours after vaccination in the sows of group G1 compared to group G3 (PBS). The average rectal temperature observed in group G1 at that time was 38.6 Q C (low-grade fever), while in group G3 it was 38.1 Q C (increase of only 0.5 eC) After revaccination (Figure 16B), the same profile of rectal temperature elevation was observed 24 hours after the application of the second vaccine dose in group G2 compared to group G3 (PBS). In this case, although the temperature differences between these two groups (G2: 37.95 e C versus G3: 37.4 e C) Although the difference was significant (p=0.0464), the values ​​found in both groups were within the physiological range for this animal category (< 38.0 s C); therefore, it is not considered a fever. Example 13 - Analysis of serological potency in pregnant sows and immunity kinetics in newborn piglets.

[0153] In this example, we demonstrate that the polyvalent vaccine is immunogenic in pregnant sows and can be used to increase the colostral antibody titers necessary to prevent the occurrence of Glâsser's disease in newborn piglets. Piglets receive specific maternal immunity exclusively through colostrum (antibodies and effector cells), and therefore, colostrum modulation through vaccines is essential for the prevention of diseases that occur during the first weeks of life.

[0154] In this study, a total of 12 sows (G1) were vaccinated intramuscularly (lateral neck region) with 2 ml of the polyvalent vaccine formulation at 65 days of gestation. The same number of sows (n=12, G2) were immunized with the same formulation at 65 and 86 days of gestation. Finally, another 12 sows (G3) were inoculated with 2 ml of PBS pH 7.2 at 65 and 86 days of gestation. Blood samples were collected from all sows at the following gestation times: D65, D86, D100 and D114.

[0155] As illustrated in Figure 17, both the single-dose protocol applied at 65 days of gestation and the two-dose protocol (D65 and D86) induced an increase in anti-G. parasuis antibodies in the sows. At the time of primary vaccination, pre-existing antibodies in the sows of group G1 were 1.7 times higher than those observed in the sows of group G2. In group G1, we observed a maintenance of already high antibody levels throughout gestation, but without significant differences between the analyzed time points. Conversely, we observed a significant increase (p < 0.001) in antibodies in sows immunized with two doses of the polyvalent vaccine. Both protocols stimulated antibody production at significantly higher titers (p < 0.001) than those observed in the sows of the control group (G3) at 100 days of gestation.

[0156] From the total number of sows analyzed, 40 neonatal piglets per sow group were randomly selected and identified with ear tags. Blood samples were collected from these animals at 7, 14, and 21 days of age. The titration of maternal anti-G. parasuis SV5 antibodies acquired from maternal colostrum was performed by quantitative indirect ELISA. As illustrated in Figure 18, piglets born from sows in group G1, immunized with a single dose of the polyvalent vaccine administered at 65 days of gestation, had protective IgG anti-G. parasuis SV5 titers up to the The same was observed with piglets born from sows in group G2, immunized at 65 and 86 days of gestation. Conversely, piglets born from vaccinated sows showed low titers of anti-G. parasuis SV5 IgG during the first two weeks of life (D7 and D14). In this group, IgG titers increased between D14 and D21, indicating that the piglets were undergoing an active infection process and did not have protective levels of passive antibodies. Therefore, this study demonstrated that the duration of maternal immunity observed in piglets born from non-immunized sows (G3) was significantly (p < 0.001) shorter than the immunity observed in piglets that ingested colostrum from vaccinated sows. The highest titers of specific IgGs were observed in piglets born from sows vaccinated with two doses of the polyvalent vaccine at 65 and 86 days of gestation.The duration of protective passive immunity stimulated by the polyvalent vaccine was at least 3 weeks, regardless of the vaccination protocol used. Piglets born to sows immunized with two doses of the polyvalent vaccine had 2.47 times more antibodies at weaning (D21) compared to piglets born to sows immunized only once during gestation.

[0157] The present invention is defined herein in terms of its preferred embodiment. Nevertheless, a person skilled in the art is perfectly capable of observing that modifications may be made to the information described herein, such modifications still being covered by the same scope of the subject matter described and claimed.

Claims

MODIFIED CLAIMS Received by the International Secretariat on December 10, 2025 (10.12.2025) 1. Veterinary vaccine formulation, characterized by comprising a combination of clinical strains of G. parasuis corresponding to serovars capable of expressing the TbpB protein of clusters I and III, and one or more components selected from the group comprising: an adjuvant, a preservative solution and a pharmaceutically acceptable diluent.

2. Veterinary vaccine formulation, according to claim 1, characterized in that the serovars capable of expressing the TbpB protein of cluster I correspond to serovar 5 (SV5), and the serovars capable of expressing the TbpB protein of cluster III correspond to serovars 1 (SV1), 4 (SV4) or NT (SVNT).

3. Veterinary vaccine formulation, according to claim 1 or 2, characterized in that said formulation comprises inactivated G. parasuis serovars that overexpress TbpB in a non-recombinant manner.

4. Veterinary vaccine formulation, according to any one of claims 1 to 3, characterized in that the overexpression of TbpB occurs by the addition of iron chelators selected from deferoxamine or 2,2'-bipyridine at a concentration of 50 to 150 pM.

5. Veterinary vaccine formulation, according to any one of claims 1 to 4, characterized in that the G. parasuis strains correspond to any one of the strains deposited with the German Depositary Authority DSMZ under numbers DSM 35165, DSM 35166, DSM 35167 and DSM 35168.

6. Veterinary vaccine formulation, according to any one of claims 1 to 5, characterized in that it comprises a combination of wild clinical strains of G. parasuis, comprising nucleotide sequences SEQ ID Nos.: 1, 2, 3 and 4, which correspond to serovars SV1, SV4, SV5 and SVNT, respectively, which in turn correspond to polypeptide sequences SEQ ID Nos.: 5, 6, 7 and 8.

7. Veterinary vaccine formulation, according to any one of claims 1 to 6, characterized in that the preservative solution comprises thimerosal in buffered solution, preferably prepared in MODIFIED SHEET (ARTICLE 19) a concentration between 0.5 and 10% w / v, in which the thimerosal preservative solution is used in the final concentration of the vaccine formulation between 0.005% and 0.02%.

8. Veterinary vaccine formulation, according to any one of claims 1 to 7, characterized in that the diluent used in said vaccine formulation is a buffered saline solution, preferably PBS, at a concentration between 5 and 70% w / v.

9. Veterinary vaccine formulation, according to any one of claims 1 to 8, characterized in that the adjuvant used in said vaccine formulation is preferably an emulsion of mineral oil, water and dimethyldioctadecyl ammonium bromide (DDA) at a concentration of 5 to 30% w / v.

10. Veterinary vaccine formulation, according to any one of claims 1 to 9, characterized in that, alternatively, the adjuvant used in said vaccine formulation is aluminum hydroxide associated with an emulsion of mineral oil or sodium polyacrylate in phosphate saline solution, being 1 to 10% aluminum hydroxide, 5 to 30% mineral oil emulsion and 5 to 30% sodium polyacrylate.

11. Veterinary vaccine formulation, according to any one of claims 1 to 10, characterized in that the antigen concentration in said vaccine formulation is > 5x10 8 The CFU / dose and the adjuvant concentration in the vaccine formulation must be > 5%.

12. Veterinary vaccine formulation, according to any one of claims 1 to 11, characterized by providing heterologous and broad-spectrum protection against Glaesserella parasuis.

13. Veterinary vaccine formulation, according to any one of claims 1 to 12, characterized in that it is prepared to be administered in a single dose.

14. Dosage form, characterized in that it comprises the vaccine formulation, as defined in any one of claims 1 to 13, prepared in a single dose.

15. Dosage form, according to claim 14, characterized in that it comprises 2 ml of solution and concentrated Glaesserella NT antigen in the range of >1 x10 9 UFC, concentrated Glaesserella antigen MODIFIED SHEET (ARTICLE 19) SV1 in the range >1 x10 9 CFU, Glaesserella SV4 antigen concentrate in the range of >1 x10 9 CFU and Glaesserella SV5 antigen concentration in the range of >1 x 10 9 UFC.

16. Process for the production of inactivated G. parasuis antigens, characterized by comprising the following steps: a) inoculating G. parasuis microorganism seed into a first culture medium and incubating at a temperature capable of promoting seed propagation; b) inoculating colonies from said first culture medium into a second culture medium and incubating this inoculum under uniform growth conditions; c) transferring the inoculum to a reactor containing optionally supplemented liquid culture medium and cultivating the inoculum until the optical density reaches a predetermined value; d) transferring the inoculum to the culture reactors until the desired growth is achieved and adding iron chelating solution; e) maintaining the culture under predetermined conditions of temperature, dissolved oxygen, and pH for a predetermined period of time; f) inactivating the cultures, optionally by adding an inactivating solution.

17. Process, according to claim 16, characterized in that in step b) the uniform inoculum growth conditions refer to incubation under controlled temperature conditions between 35 and 39 °C and agitation between 120 and 180 rpm.

18. Process, according to claim 1 6, characterized in that in step c) the optical density achieved is between 0.5 and 2.

0.

19. Process according to claim 1 6, characterized in that the iron chelating solution in step d) is a deferoxamine or 2,2'-bipyridine solution.

20. Process, according to claim 1 6, characterized in that the inactivating solution in step “f)” is a thimerosal solution or a buffered formalin solution.

21. Process according to claim 16, characterized in that the inactivating solution in step f) is a thimerosal solution at a concentration MODIFIED SHEET (ARTICLE 19) final of 0.01%.

22. Process for the production of a vaccine formulation, as defined in any one of claims 1 to 13, comprising a combination of inactivated G. parasuis antigens, as produced by the process defined in any one of claims 16 to 21, characterized in that it comprises the following steps: a) homogenizing the antigens; b) adding to the formulation reactor the PBS diluent solution with thimerosal preservative solution already solubilized through sterilizing filtration; c) transferring the homogenized antigens to the formulation reactor; d) transferring the mineral oil, water and DDA emulsion to the formulation reactor; e) checking the pH and, if necessary, adjusting with sodium hydroxide or 1 N hydrochloric acid corrector solution to pH 7.0 - 7.5; f) homogenizing the formulation for at least 4 hours; f) Check the pH again, and, if necessary, adjust the pH to the range of 7.00 - 7.5 with a corrective solution of sodium hydroxide or 1 N hydrochloric acid;g) complete the volume with PBS diluent solution and homogenize; h) keep the product under agitation at 50-200 rpm in the formulation reactor until the start of filling; ei) keep the reactor cooled to a temperature between approximately 2 and 8 °C and pressurized.

23. Combination of clinical strains, characterized by being strains of the species G. parasuis corresponding to serovars of clusters I and III.

24. Combination according to claim 23, characterized in that the serovars of <3. parasuis are of type SV1, SV4, SV5 and NT.

25. Combination according to claim 23 or 24, characterized in that the clinical strains are those deposited with the DSMZ and correspond to DSM 35165, DSM 35166, DSM 35167 and DSM 35168 registrations, respectively.

26. Use of a combination of clinical strains, as defined in any one of claims 23 to 25, characterized in that it is used in the manufacture of a polyvalent veterinary vaccine formulation to prevent Glässer's disease in an animal. MODIFIED SHEET (ARTICLE 19) 27. Use according to claim 26, characterized in that the animal is a pig.

28. Use, according to claims 26 or 27, characterized in that the vaccine formulation is prepared to be administered to pregnant sows and piglets in their first weeks of life.

29. Kit, characterized in that it comprises: i. a combination of clinical strains, as defined in any one of claims 23 to 25; and ii. a preservative solution. MODIFIED SHEET (ARTICLE 19)

Images