An immunogenic composition and uses thereof

Abstract

The present invention relates an immunogenic composition and the use thereof. The present invention also relates to an immunogenic diagnostic kit.

Description

[0001] AN IMMUNOGENIC COMPOSITION AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to an immunogenic composition. In particular, the present invention relates to an immunogenic composition comprising one or more recombinant E. coli- based antigens of each of a trimeric autotransporter fragment of protein 9 (VtaA9) of Glaesserella parasuis, a porcine circovirus type 2 genotype b (PCV2b) Open Reading Frame (ORF) 2 CAP protein and a porcine circovirus type 2 genotype a (PCV2a) Open Reading Frame (ORF) 1 REP protein, and the uses thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Glasser's disease caused by Glaesserella parasuis

[0006] Glaesserella parasuis, a Gram-negative member of the Pasteurellaceae family, poses a significant threat to pig populations worldwide, particularly affecting susceptible piglets aged 4-8 weeks. This pathogen swiftly colonizes the upper respiratory tract soon after birth, leading to Glasser's disease (GD), characterized by fibrinous polyserositis, arthritis, and meningitis. Despite primarily affecting piglets, sporadic cases can occur in adults, especially when introducing naive individuals into a healthy herd. The GD manifests with a sudden onset, short duration, and alarmingly high morbidity and mortality rates (up to 10% in conventional herds, and 75% in naive herds). Survivors may suffer from severe fibrosis in the abdominal and thoracic cavities, resulting in reduced growth rates and carcass condemnation at slaughter (Aragon, Segales et al. 2019).

[0007] The typical clinical signs of acute GD may include high fever (41.5 °C), severe coughing, abdominal breathing, swollen joints, CNS signs (e.g., lateral decubitus, paddling, and trembling). Typical lesions are shown in Figure 1. GD has become increasingly prevalent, especially with the emergence of porcine reproductive and respiratory syndrome. Isolating Gsuis in pure culture from infected animals poses a significant challenge due to its fastidious nature and the complications arising from antibiotic treatments. The mechanisms behind systemic invasion by Gsuis remain unclear, although virulent strains demonstrate the ability to evade phagocytosis, reduce antigen presentation to pulmonary alveolar macrophages, and delay antibody production. (Costa-Hurtado, Ballester et al. 2012, Olvera, Pina et al. 2012, Macedo, Gottschalk et al. 2021) The advent of high-health-status farms and early weaning practices has contributed to an increase in immunologically naive populations, amplifying disease-related morbidity and mortality rates. (Costa-Hurtado, Barba-Vidal et al. 2020) This trend carries significant economic and welfare implications for the pig farming industry. Given the growing concern surrounding antimicrobial administration, (EMA / CVMP 2021) vaccination emerges as the primary alternative for disease control. Thus, implementing effective vaccination strategies becomes imperative in mitigating the impact of this debilitating condition on swine populations and the agricultural sector at large.

[0008] Antibodies are essential in protecting against disease (Nedbalcova, Kucerova et al. 2011 , Costa-Hurtado, Ballester et al. 2012). VtaAs are outer membrane proteins that have a differential distribution between virulent and non-virulent strains of Gsuis. While group 3 VtaA are present in all Gsuis strains, group 1 and 2 VtaA are found only in virulent strains. (Bensaid, Pina-Pedrero et al. 2008, Olvera, Pina et al. 2010, Olvera, Pina et al. 2012, Olvera, Martinez- Moliner et al. 2013, Galofre-Mila, Correa-Fiz et al. 2017) The production of monoclonal antibodies against VtaA8 and 9 allowed the identification of one of them, 69C6, which recognized all VtaAs of group 1 (as mentioned above, associated with virulent strains). (Costa- Hurtado, Ballester et al. 2012) Table 1 shows the reaction of several monoclonal antibodies with VtaAs of the virulent Nagasaki strain (group 1 : VtaA1 to 9; group 2: VtaA10 and 11 ; group 3: VtaA 12 and 13).

[0009] Table 1. Reaction of monoclonal antibodies 72E10, 72F10, 80H8, 95F4 and 69C6 against the 13 VtaAs of the Nagasaki strain. The monoclonal antibody 69C6 was capable of opsonizing Gsuis and facilitated the recognition of the bacterium by alveolar macrophages, with subsequent phagocytosis (Costa-Hurtado, Ballester et al. 2012). This 69C6 antibody made it possible to identify the area of the protein exposed to the outside, which was also common to all VtaA1. Figure 2 schematically shows the fragments of VtaA9 that were expressed as recombinant proteins. The different fragments were expressed in pASK-IBA33plus as proteins fused to a tail of histidines. Its expression and purity were detected in a membrane by Ponceau staining (Figure 3) and the membrane was subsequently analyzed with anti-histidine antibody (anti-His) and with the monoclonal antibody 69C6 by Western blot (Figure 4 left). The reaction of the 69C6 antibody with the fragments was also measured by ELISA (Figure 4 right).

[0010] These results led to the selection of the F4 fragment as a possible immunogen, since it was exposed on the surface of the bacterium (recognized by 69C6), was common to all VtaA of group 1 and its size allowed effective purification. (Bensaid, Pina-Pedrero et al. 2008) As mentioned above said antigen is a recombinant E. co / / -based trimeric autotransporter protein described from highly virulent Gsuis designed to stop nostril-to-lung progression of pathogenic strains. VtaA9-F4 antigen is covered as a polypeptide and for use in a vaccine or immunogenic composition for treating an infection caused by Gsuis, which is the causal agent of GD, currently patented for our group (Bensaid, Pina-Pedrero et al. 2008).

[0011] Porcine circovirus associated diseases epidemiology caused by porcine circovirus type 2

[0012] Circoviruses (CV) include some of the smallest viruses known. They were named after their circularly arranged single-stranded DNA genome with a gene encoding a conserved replicase (Rep) protein on the sense strand. (Opriessnig, Karuppannan et al. 2020) Porcine circoviruses (PCVs) are a group of small, non-enveloped, single-stranded DNA viruses belonging to the Circoviridae family. PCVs are ubiquitous in global pig populations and uninfected herds are rarely found. Porcine circovirus 1 (PCV1), Porcine circovirus 2 (PCV2), Porcine circovirus 3 (PCV3) and most recently Porcine circovirus 4 (PCV4). It is generally accepted that PCV1 is non-pathogenic. In contrast, PCV2 is considered an important, economically challenging pathogen on a global scale with comprehensive vaccination schemes in place. The role of PCV3 is still controversial several years after its discovery. Propagation of PCV3 appears to be challenging and only one successful experimental infection model has been published to date. Similarly to PCV2, PCV3 is widespread and found in many pigs regardless of their health history, including high health herds. PCV4 has only recently been discovered and further information on this virus is required to understand its potential impact (Opriessnig, Karuppannan et al. 2020).

[0013] Among PCVs, PCV2 stands out as the primary causative agent of PCVAD, encompassing a spectrum of clinical conditions. These include post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, and subclinical infections (Segales 2012). PCVAD are pervasive viral infections affecting swine globally, leading to significant economic losses estimated at €1 ,240 million per year, and welfare concerns in affected herds. PCV2 exhibits a notable evolutionary rate, resulting in the emergence of various genetic variants. Currently, nine genotypes of PCV2 have been identified, with PCV2a, PCV2b, and PCV2d being the most prevalent. PCV2a was predominant in the 1990s, followed by a shift to PCV2b around 2000. PCV2 primarily affects pigs of all ages, with clinical signs typically appearing in post-weaning and growing pigs, although nursing piglets and adult animals can also be affected. The severity of PCVAD ranges from subclinical to severe systemic disease, with clinical manifestations including PMWS, PDNS, reproductive failure, and respiratory signs. These conditions contribute to high morbidity and mortality rates (Figure 5), significant economic losses, and welfare implications, with nearly 100% seroprevalence observed in domestic pig herds (Segales 2012). Among the different clinical presentations of PCVAD, PCV2-systemic disease (PCV2-SD) stands out as the most severe form, characterized by wasting, respiratory distress, enlarged lymph nodes, and systemic inflammation (Figure 6). It mainly affects young pigs after weaning and has a high mortality rate. PCV2-reproductive disease (PCV2-RD) affects pregnant sows, leading to reproductive failure such as abortions, stillbirths, and weak piglets, thereby impacting the health and immunity of offspring. PCV2-subclinical infection (PCV2-SI) may not cause overt clinical signs but affects pig growth, performance, and susceptibility to other infections (Segales 2012).

[0014] All four PCVs are similar in structure: they contain two main open reading frames (ORFs) oriented in opposite directions in the circular genome; the ORF1 or rep gene encodes for two pivotal proteins associated with replication, replication initiator protein (Rep) and Rep’, and the ORF2 or cap gene encodes for the capsid (Cap) protein. (Cheung 2012, Opriessnig, Karuppannan et al. 2020) Additionally, ORF1 encodes a group of RNAs and minor non- structural proteins (NS) associated RNAs (Rep3a, Rep3b, Rep3c, NS0, NS515, NS672), which are probably derived from the full Rep RNA through alternative splicing. ORF1 displays reduced entropy levels in its amino acid sequence compared to ORF2 in both PCV1 and PCV2, confirming a higher conservation degree in ORF1 relative to ORF2. The Rep protein is also highly conserved among PCV1 and PCV2 genotypes, exhibiting an 85% amino acid identity. Despite sequence variations, the Rep proteins from circoviruses share structural similarities in their endonuclease domains, suggesting a conserved overall fold and functional motifs. (Cheung 2003, Cheung 2004) Notably, Rep proteins of PCV1 and PCV2 are interchangeable in vivo, which demonstrates a significant level of conservation within the circovirus family. (Mankertz, Mueller et al. 2003) Pigs infected with PCV2 develop cell-mediated immunity against the Rep protein, assessed by detecting Rep-specific interferon-gamma secreting cells (IFN-y-SC) and IL-10 secreting cells (IL-10-SC) in peripheral blood mononuclear cells (PBMC) using ELISPOT immunoassays. The intensity of such response may be influenced by the level of PCV2 replication. Considering the substantial expression of Rep in cells supporting PCV2 replication, immune responses targeting this protein might be critical to constrain PCV2 replication and prevent the progression of PCV2 infection towards postweaning multisystemic wasting syndrome (PMWS). These findings postulate Rep as a promising and feasible candidate for the development of recombinant sub-unit vaccines aimed at providing broad protection against different PCV variants.

[0015] The ORF2 or cap gene encodes for the capsid (Cap) protein and it has been widely used for vaccine production with reasonable success and efficacy. (Guo, Hou et al. 2022) Different genotypes of PCV2 are encompassed in particular embodiments of the first aspect, such as PCV2a and PCV2b. Genotypes of PCV2 are determined by the number of nucleotide substitutions in ORF 2 (CAP) divided by the total nucleotides in ORF 2 (702 nt); this fraction is called p-distance. Recognizing the impossibility of defining a clear inter-cluster p-distance cutoff, a new phylogeny-grounded genotype definition based on three criteria has been proposed: maximum intra-genotype p-distance of 13% (calculated on the ORF2 gene), bootstrap support at the corresponding internal node higher than 70% and at least 15 available sequences. This scheme allowed defining 8 genotypes (PCV2a to PCV2h), which six of those had been previously proposed. (Franzo and Segales 2018) So far, all vaccines in the market have shown great efficacy in reducing clinical signs associated to diseases caused by PCV2, independently of the genotype present in the farm. Moreover, experimental data demonstrated the crossprotection of PCV2a vaccines against the most widespread genotypes (PCV2a, PCV2b, and PCV2d). Therefore, despite the significant number of genotypes described / proposed, it seems one single PCV2 serotype would exist so far.(Franzo and Segales 2020) The ORF2 of PCV2 encodes a protein of about 233 amino acids. Besides, and corroborating earlier observations in PCV1 , transcript mapping revealed that the rep gene of PCV2 encodes two products, the full-length protein Rep and the spliced version Rep' and that the simultaneous expression of Rep and Rep' proteins is essential for initiation of replication of PCV2. (Mankertz, Mueller et al. 2003) The rep gene products of PCV2 were not only able to bind the PCV2 origin but also the origin of PCV1 and vice versa, indicating that the cis- and trans-acting replication factors of the two viruses are functionally exchangeable. (Mankertz, Mueller et al. 2003, Opriessnig, Karuppannan et al. 2020) PCV2-infected pigs developed cell-mediated immunity to CAP and REP proteins and that, in the course of a sub-clinical infection, development and strength of such responses are possibly related to the levels of PCV2 replication. (Fort, Sibila et al. 2010) Therefore, joining PCV2b-related protective immune response coupled with highly conserved Rep-associated immune protective immune response seems to be a rational approach to confer a wider pan-PCV immunity. Any PCV2b ORF 2 (CAP) or PCV2a ORF1 (REP) are suitable to be used in the invention as functionally exchangeable PCV-related strong immunity developers in pigs.

[0016] In view of above, the present inventors have developed a recombinant E. co / / -based immunogenic composition comprising: (1) a fragment (F4) of trimeric autotransporter protein (VtaA9) described from highly virulent Glaesserella parasuis (Gsuis) (VTAA9-F4 Gsuis); (2) a porcine circovirus type 2 (PCV2b) ORF2 antigen coding for structural protein (CAP); and (3) a porcine circovirus type 1 (PCV2a) ORF 1 antigen coding for replicase protein (REP), suitable for the prevention and / or treatment of Glasser's disease (GD) and porcine circovirus- associated diseases (PCVAD) in animals, preferably livestock, more preferably porcine livestock.

[0017] This innovation supposes a real advantage in relation with bacterins-based vaccines against G. parasuis (aka. Haemophilus parasuis) used in association with commercial PCV2-based vaccines, postulating a double cross protection between both diseases and, certainly, supporting high efficacy against all highly virulent Gsuis strains. In addition, the present invention also provides an immunogenic diagnostic system to discriminate naturally infected from immunized animals comprising monoclonal and polyclonal antibodies of VtaA9-F4 Gsuis and monoclonal and polyclonal antibodies of PCV2a REP. In short, it provides an efficient and sustainable solution for the prevention and treatment of Glasser's disease (GD) and porcine circovirus-associated diseases (PCVAD) in preferably livestock, more preferably porcine livestock.

[0018] SUMMARY OF THE INVENTION

[0019] In a first aspect, the present invention relates to an immunogenic composition comprising one or more recombinant E. co / / -based antigens of each of a trimeric autotransporter fragment of protein 9 (VtaA9) of Glaesserella parasuis, a porcine circovirus type 2 genotype b (PCV2b) Open Reading Frame (ORF) 2 CAP protein, and a porcine circovirus type 2 genotype a (PCV2a) Open Reading Frame (ORF) 1 REP protein.

[0020] In a second aspect, the present invention relates to the uses of said immunogenic composition according to the first aspect of the invention.

[0021] The present invention refers to an immunogenic composition comprising carefully selected antigens that induce potent immune responses in preferably pigs compatible with protection against GD and PCVAD. VtaA9-F4 is a recombinant E. co / / -based trimeric autotransporter protein described from highly virulent Gsuis, designed to stop nostril-to-lung progression of pathogenic strains. VtaA9-F4 Gsuis fragment is capable to confer successful immunological protection against heterologous challenge of highly virulent Gsuis strains. Recombinant E. coli- based Open Reading Frame 2 (ORF-2) protein (CAP) from PCV2b is capable of selfassembling into immunogenic virus-like particles (VLPs). These VLPs are designed to induce cellular & humoral immune response. Recombinant E. co / / -based ORF1 protein (replicase, REP) from PCV2a is designed to reinforcing and ensure cross protection among PCV strains and genotypes, due to their exchangeable motifs and highly conserved sequence between PCV. Both recombinant PCV2 antigens are capable to confer bioequivalent or even superior immunological protective-related response comparing with market-leader commercial vaccines.

[0022] The present invention relates to any combination of antigens comprising recombinant fragment VtaA9-F4 Gsuis, ORF2 from PCV2b (CAP) plus ORF1 from PCV2a (REP) capable to produce a desired or intended immunological response in the host of a cellular or antibody-mediated type upon administration of the present invention, at least but not limited to have equivalent or even superior levels of the immune response considered as protective in bioequivalent commercial products, and / or even to confer successful immunological protection against heterologous challenge of highly virulent Gsuis strains.

[0023] In a third aspect, the present invention relates to an immunogenic diagnostic kit for discriminating naturally infected from immunized animals. In particular, said kit is useful for discriminating naturally infected from immunized animals based on monoclonal and polyclonal antibodies of VtaA9-F4 Gsuis & PCV2a REP (Strep tagged), which can be used in the diagnosis of GD and / or PCVAD by serum and / or blood sampling of livestock.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Embodiments of the present invention will now be further described in the following paragraphs of this specification, by means of illustrative example only, without intending to constitute any limitation of the scope of the present invention. The following description may be better understood when read in conjunction with the attached drawings, in which: Figure 1. Pathology caused by Glaesserella parasuis. (A) is the gross pathology illustrating meningitis that is prominent in the cerebellum in some cases. Severe pleuritis (B), fibrinosuppurative pericarditis (C), corneal opacity associated with congestion of the optic nerve (D) in endophthalmitis cases. Peritonitis with a dense yellowish fibrin content covered the abdominal cavity (E) and arthritis (F).

[0026] Figure 2. Schematic representation of the different fragments F1 to F6 that cover the complete protein VtaA9. The numbers represented in the complete vtaA9 gene (above) correspond to the positions of the nucleotides (nt). Below for each fragment, the first and last positions of the amino acids included in the fragments are indicated.

[0027] Figure 3. Expression and purification of fragments F1 to F6, which encompass the transient domain of VtaA9.

[0028] Figure 4. Detection of F1-F6 protein fragments by Western blotting (left) with anti-His antibody and 69C6 monoclonal antibody. Fragments F1 , F2, F3 and F5 were analyzed using 800 ng / lane, while F4 and F6 were analyzed at 2400 and 5600 ng / lane. The right panel shows the detection of the F1-F6 fragments (1 mg / well) by the 69C6 monoclonal antibody in ELISA.

[0029] Figure 5. Deceased piglet suffering from PCV2.

[0030] Figure 6. Healthy pig and pig with subclinical PCV2 infection.

[0031] Figure 7. Example of SDS-PAGE analysis of the VtaA9-F4 fragment purified from the soluble fractions through Immobilized Metal Affinity Chromatography (IMAC) using HisTrapTM FF Crude columns (Cytiva). SDS-PAGE analysis was performed in a 12% acrylamide gel run at 150 V for 65 minutes. Lane M: PageRuler Plus Prestained Protein Ladder (Thermo Fisher), 4 pL. The samples were diluted 1 :1 (v / v) in 2x Loading Buffer. The indicated volumes refer to the total sample volume loaded. TE (Total Extract), Sb (Soluble fraction), PI (Pellet, insoluble fraction), FT (Flowthrough), and F (Fractions) 7-20, 20 pL each. Induction conditions: 2 mg / L anhydrotetracycline, 25°C, 2 hours. VtaA9-F4, 19 kDa.

[0032] Figure 8. Validation of anti-VtaA9-F4 monoclonal antibodies purified through affinity chromatography using pre-packed columns with sepharose coupled to protein G (Cytiva). SDS-PAGE analysis was performed in a 12% acrylamide gel run at 150 V for 65 minutes. Lane M: PageRuler Plus Prestained Protein Ladder (Thermo Fisher), 4 pL. Samples were diluted 1 :1 in 2x loading buffer. VtaA9-F4 standard (2.5 mg / mL); 4 pL (125 ng), 6 pL (187.5 ng), and 10 pL (312.5 ng). TE (Total Extract) 1-3, 20 pL. Antibodies used for Western blot: anti-VtaA9- F4 monoclonal (1 :5000) (primary antibody, WorldPathol Global United S.A., ref. MR0134) and anti-Mouse IgG-HRP (1 :15000) (detection antibody, Sigma-Aldrich). VtaA9-F4, 19 kDa.

[0033] Figure 9. Validation of anti-VtaA9-F4 polyclonal antibodies purified from the collected serum using affinity chromatography with pre-packed columns with NHS-activated sepharose (Cytiva) coupled to VtaA9-F4 fragment. SDS-PAGE analysis was performed in a 12% acrylamide gel run at 150 V for 65 minutes. Lane M: PageRuler Plus Prestained Protein Ladder (Thermo Fisher), 4 pL. Samples were diluted 1 :1 in 2x loading buffer. VtaA9-F4 standard (2.5 mg / mL);

[0034] 4 pL (125 ng), 6 pL (187.5 ng), and 10 pL (312.5 ng). TE (Total Extract) 1-3, 20 pL. Antibodies used for Western blot: anti-VtaA9-F4 polyclonal (1 :5000) (primary antibody, WorldPathol Global United S.A., ref. MR0155) and anti-Rabbit IgG-HRP (1 :15000) (detection antibody, Sigma-Aldrich). VtaA9-F4, 19 kDa.

[0035] Figure 10. Representative graphic of VtaA9-F4 Gsuis immune response from 2 independent in vivo experiments with naturally infected Glaesserella parasuis piglets. Absorbance values at 450 nm. Sample bloods were collected before and after vaccination with 1 mL / piglet dose. The signal was compared with a placebo group vaccinated with PBS.

[0036] Figure 11. Antibodies measured by ELISA against F4 were measured at the time of first immunization (day 0), 2 weeks later (second immunization), and 2 weeks after the second immunization. F4 with Freund's adjuvant was used as a positive control and PBS in emulsion as a negative control.

[0037] Figure 12. Piglets’ immune responses vaccinated with VtaA9-F4 Gsuis and infected with different virulent G. parasuis strains. Absorbance values at 450 nm. A. Challenge made with Nagasaki strain. B. Challenge made with PV1-12 strain. Sample bloods were collected before and after each immunization, before and after challenge and before necropsy. The signal (black squares) was compared with a placebo group vaccinated with PBS (white circle).

[0038] Figure 13. Antibodies measured by ELISA against VtaA9-F4 Gsuis. Piglets vaccinated at 1 and 3 weeks of age with 100 ug (G3 and G6) had a higher antibody response than those vaccinated with a single dose of 200 ug of VtaA9-F4 Gsuis at 1 week of age (G2 and G5). This difference is clearly observed at 5 weeks of age.

[0039] Figure 14. Effects that were observed in piglets when their mothers (sows) were vaccinated with VtaA9-F4 Gsuis.

[0040] Figure 15. Antibodies against VtaA9-F4 Gsuis measured in ELISA in the sows' serum at the different times indicated. Vaccinated sows (mothers) had a high level of antibodies against VtaA9-F4 Gsuis after two doses of the immunogenic composition.

[0041] Figure 16. Antibodies to VtaA9-F4 Gsuis measured by ELISA in colostrum produced by sows (mothers). Vaccinated mothers had a high level of antibodies against VtaA9-F4 Gsuis.

[0042] Figure 17. ELISA-measured antibodies against VtaA9-F4 Gsuis in the serum of piglets born from vaccinated or unvaccinated sows.

[0043] Figure 18. Detection of TGFbeta in sows and their piglets at the indicated times.

[0044] Figure 19. Detection of antibodies against VtaA9-F4 Gsuis and surfactant protein D (SP-D) in bronchioalveolar lavage in piglets of vaccinated or not vaccinated sows with VtaA9-F4 Gsuis and challenged with a virulent strain of G. parasuis at 3 weeks of age.

[0045] Figure 20. Potency estimates of VtaA9-F4 samples by Enzyme-linked immunosorbent assay (ELISA) sandwich. Calibration curves corresponding to a set of three validation tests measured at 450 nm. Capture antibody mAb 1 (MR0134), 10 pg / mL. Detection antibody mAb 2-biotin (MR0137), 2 pg / mL. Streptavidin, 1 / 400 dilution. VtaA9-F4 concentration: 31.25, 62.5, 125, 250, 500, 1000, and 1500 ng / mL. TMB incubation time: 15 minutes. Validation test 1 is shown in blue, validation test 2 in orange, and validation test 3 in green.

[0046] Figure 21. Transmission electron microscopy (TEM) images obtained from the soluble fraction of PCV2b CAP Escherichia coli clarified extracts: A, PBS and B, TRIVAC formulation. Areas of negative contrast correspond to Virus-Like Particles (VLPs). Enlarged images for improved measurement of VLPs diameters for PBS. A1 TEM, 145, 000X magnification; A2 Histogram (19 nm particle diameter). Enlarged images for improved measurement of VLPs diameters for TRIVAC proof of concept (PoC). B1 TEM, 195, 000X magnification; B2 Histogram (10-11 nm particle diameter).

[0047] Figure 22. Example of SDS-PAGE and Western blot analysis of CAP protein isolated from the insoluble fraction. 12% Acrylamide SDS-PAGE gel. M, PeqLab peqGOLD Protein Marker V (Pre-Stained) (VWR), 5 pL. Samples were diluted 1 :1 (v / v) in 2x Loading Buffer. A total volume of 20 pL per sample was loaded. +, Positive Control (Porcine Circovirus Type 2 Capsid Protein, Sino Biological Europe GmbH, 23.8 kDa), 20 pL; TE (Total Extract), 20 pL; Sb (Soluble fraction), 20 pL; PI (Pellet, insoluble fraction), 20 pL. Induction conditions: 1 mM IPTG, 37 °C, 3 hours. A: Coomassie Blue staining; B Western blot with anti-His-HRP monoclonal antibodies (1 :5000) (primary and detection antibody, Sigma-Aldrich); C Western blot with anti-CAP monoclonal antibodies (1 :5000) (primary antibody, Life Technologies) and anti-Mouse IgG- HRP (1 :15000) (detection antibody, Sigma-Aldrich); D Western blot with anti-CAP polyclonal antibodies (1 :5000) (primary antibody, Bioss Antibodies) and anti-Rabbit IgG-HRP (1 :15000) (detection antibody, Sigma-Aldrich). PCV2b CAP, 28 kDa.

[0048] Figure 23. Comparative analysis of PCV2b CAP formulations versus Porcilis® PCV. Absorbance values at 450 nm were determined through the iNgezim PCV DAS ELISA assay. The results demonstrate the enhanced performance of the PCV2b CAP liquid paraffin formulation.

[0049] Figure 24. Validation of production robustness of PCV2b CAP liquid paraffin formulation. Absorbance values at 450 nm were determined through the iNgezim PCV DAS ELISA assay. PCV2b CAP liquid paraffin formulation demonstrates robust production, yielding twofold signal increase compared to Porcilis® PCV. These results suggest enhanced vaccine efficacy through improved antigen presentation.

[0050] Figure 25. Purification from the soluble fraction of the [PBS 1x NaCI 1 M & arginine 100 mM] - stabilized PCV2a Rep protein by IMAC affinity chromatography with nickel columns. 12% Acrylamide Gel SDS-PAGE. M, PageRuler Plus Prestained Protein Ladder Thermo Fisher, 4 pL. The samples were diluted 1 :1 (v / v) in 2x Loading Buffer. The volume indicated refers to the total volume of sample loaded. TE (Total Extract), Sb (Soluble fraction), PI (Pellet, insoluble fraction), FT (Flowthrough) and F (REP-positive rich Fractions) 12-15, 20 pL. Induction conditions: 1 mM IPTG, 37 °C, 4 hours. Antibodies used for Western Blotting: a-His-HRP (1 :5000), polyclonal anti-Rep (1 :5000) (primary) (ThermoFisher, ref. #PA5-112014) and Anti- IgG-HRP rabbit (1 :15000) (secondary) (Sigma-Aldrich, ref. RABHRP1). PCV2a Rep, 38 kDa. Figure 26. Purification from the insoluble fraction (inclusion bodies) of the [PBS 1x NaCI 1 M & arginine 100 mM] -stabilized PCV2a Rep protein by IMAC affinity chromatography with nickel columns. 12% Acrylamide Gel SDS-PAGE. M, PageRuler Plus Prestained Protein Ladder Thermo Fisher, 4 pL. The samples were diluted 1 :1 (v / v) in 2x Loading Buffer. The volume indicated refers to the total volume of sample loaded. PI (Pellet, insoluble fraction), FT (Flowthrough) and F (REP-positive rich Fractions) 6-12, 20 pL. Induction conditions: 1 mM IPTG, 37 °C, 4 hours. Antibodies used for Western Blotting: polyclonal Anti-Rep (1 :2000) (primary) (ThermoFisher, ref #PA5-112014) and rabbit Anti-IgG-HRP (1 :20000) (Sigma- Aldrich, ref. RABHRP1). PCV2a Rep, 38 kDa.

[0051] Figure 27. Direct ELISA for Glaesserella parasuis and Porcine circovirus type 2 ORF2 subunit antigen (genotype b). Absorbance of polyclonal anti-rabbit porcine IgG (horseradish peroxidase (HRP), Sigma-Aldrich, Spain) (1 / 400). Blocking: bovine serum albumin (BSA). Coating: VtaA9-F4 protein (1 / 400) & PCV2b Cap virus-like particles (VLPs) (1 / 400) (both, WorldPathol Manufacturing Ltd., Spain) in 0 (primary dose), 15 (booster dose, protocol B) and 30 days after immunization of commercial vaccine combination (Porcilis PCV2 Mhyo + HIPRA suis), TRIVAC 1 dose [E. coli-based PCVAD antigens (100 pg / mL dose) and Gsuis antigen (50 pg / mL dose)] (protocol A, PI38 / 24) and TRIVAC 2 doses (second shoot of TRIVAC) (protocol B, PI38 / 24).

[0052] Figure 28. Differentiating Infected from Vaccinated Animals (DIVA) immunochromatographic rapid test. A blood sample is dispensed in the sample window (S). The control line (C) serves as a procedural control to validate the correct performance of the reagents and the test. The absence of the control line invalidates the test. Interpretation of results: (1) A positive result for a vaccinated animal is indicated by the appearance of antibodies against VtaA9-F4 at the test line 1 (T1). (2a and 2b) Positive result for a naturally infected animal. (2a) Detection of antibodies against both, the VtaA9-F4 fragment (T1) and other epitopes distinct from F4 (VtaA9AF4, test line 2, T2). In this case, the presence of antibodies against VtaA9-F4 may be also attributed to vaccination. (2b) represents the scenario where the natural infected animal shows antibodies against VtaA9AF4 (T2) without reactivity towards the VtaA9-F4 fragment (T1).

[0053] DETAILED DESCRIPTION OF THE INVENTION Definitions

[0054] A skilled person in the art to which this invention belongs at the time of filing will understand all technical and scientific terms used herein the same way, unless otherwise specified. However, definitions contained herein prevail over any lexical or extrinsic definition in the event of ambiguity. Furthermore, singular terms shall include plurals, and plural terms shall include the singular, unless the context otherwise requires.

[0055] The term “antigen” refers to a molecule against which a subject can initiate a humoral or cellular immune response. There may be one or more antigens included depending on the intended function of the composition.

[0056] As used herein, the term "medicament" refers to a pharmaceutical or veterinary drug used to cure, treat or prevent disease in animals, including humans. The medicament derived of this invention can be classified as a biopharmaceutical, which include recombinant proteins, vaccines, blood products used therapeutically (such as IVIG), gene therapy, monoclonal antibodies, and cell therapy. A vaccine for veterinary use is preferred in the present invention.

[0057] An "active principle" or an "immunogenic composition" is referred to as a material accompanied by adequate excipients and / or carriers, that when administered to an animal, elicits, or is capable of eliciting, directly or indirectly, an immune response in the animal. Particularly, the immunogenic composition of the present invention elicits an immunological response in the host of a cellular or antibody-mediated type upon administration to a vertebrate, including humans, that has been shown to be equivalent or superior to those considered as protective in bioequivalent commercial products designed against target diseases.

[0058] The term “intramuscular” or “subcutaneous” refers to compositions administered through the dermis.

[0059] The term “combination” means that the immunogenic composition contains various antigens in a single preparation, protecting against two or more diseases or against one disease caused by two or more microorganisms. The immunogenic composition comprises molecules with antigenic properties, such as recombinant proteins and immunogenic polypeptides, among others. A molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. An antigenic polypeptide contains an epitope of at least about five, and particularly at least about 10, at least 15, at least 20 or at least 50 amino acids. The term “epitope” refers to an antigenic portion of a polypeptide. Epitope can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier polypeptide for immunization. The immunogenic composition relates according to this description, to the active molecule, composition comprising said molecule, or composition comprising more than one antigenic molecule to which a particular immune reaction is desired.

[0060] As for the expression "immunologically effective amount," or “immunologically effective dose” means the administration of that amount or dose of antigen, either in a single dose or as part of a series, that elicits, or is able to elicit, an immune response that reduces the incidence of or lessens the severity of infection or incident of disease in an animal for either the treatment or prevention of disease. The immunologically effective amount or effective dose is also able for inducing the production of antibody for either the treatment or prevention of disease, at least but not restricted to those considered as protective amounts of antibodies after administration of similar or bioequivalent antigens in market-validated regulators-approved medicaments. This amount shall vary depending upon a variety of factors, including the physical condition of the subject, and can be readily determined by someone of skill in the art.

[0061] The term “inactivated cells” refers to grown-cultured cells killed by physical means (such as heat) or chemical means (such as contact with formaldehyde, BEI, or any other well-known inactivation agent) as the main ways of inactivation methods, among others. The “inactivated cells” may be whole cells or partial cells, including cell lysates and supernatant recovered cells for example.

[0062] The term “livestock” relates to domesticated, or farm animals raised to produce commodities such us food. Particularly, it relates to food-producing animals such as cattle, sheep, goats, swine, poultry (including egg-producing poultry), and equine animal. More in particular, it relates to food-producing animals such as cattle, sheep, goats, swine, poultry and equine animals. Even more in particular, in the present invention livestock is porcine livestock.

[0063] The term "carrier" is to be understood as a pharmaceutically acceptable component other than the immunogenic component. The carrier can be organic, inorganic, or both. Suitable carriers well known to those of skill in the art and include, without limitation, large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) and inactive virus particles. The expression "pharmaceutically acceptable excipients and / or carriers" refers to pharmaceutically acceptable materials, compositions or vehicles. Pharmaceutically acceptable means being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit / risk ratio. In particular, it must be suitable for use in parenteral route.

[0064] As used herein, the term “host” or “subject” is intended for the target individuals in need thereof to whom the immunogenic composition of the invention is administered, among other humans, mammals, livestock, or any other animal species susceptible to be inoculated with the composition of the invention. Particularly, the mammal is from porcine species, more particularly is swine.

[0065] As used herein, the term “porcine”, “pig” or “swine” is intended for porcine species including, among others, pigs, boars, sows, gilts and piglets of any age or in any phase of their production cycle.

[0066] The term “preventing”, “to prevent” or “prevention”, include without limitation decreasing, reducing or ameliorating the risk of a symptom, clinical signs, disorder, condition, or disease, and protecting an animal from a symptom, clinical signs, disorder, condition, or disease. A prevention may be applied or administered prophylactically.

[0067] The term “treating”, “to treat” or “treatment”, include without limitation restraining, slowing, stopping, reducing, ameliorating, or reversing the progression or severity of an existing symptom, clinical sign, disorder, condition, or disease. A treatment may be applied or administered therapeutically.

[0068] The term “efficacy” as used herein, refers to the ability to produce a desired or intended immunological response in the host of a cellular or antibody-mediated type upon administration of the present invention, at least but not limited to have equivalent or even superior levels of the immune response considered as protective in bioequivalent commercial products or similar antigens.

[0069] The term “TRIVAC” in the present disclosure relates to the immunogenic composition according to the present invention. Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of”.

[0070] Description of the invention

[0071] In a first aspect the present invention relates to an immunogenic composition comprising one or more recombinant E. co / / -based antigens of each of a trimeric autotransporter fragment of protein 9 (VtaA9) of Glaesserella parasuis, a porcine circovirus type 2 genotype b (PCV2b) Open Reading Frame (ORF) 2 CAP protein, and a porcine circovirus type 2 genotype a (PCV2a) Open Reading Frame (ORF) 1 REP protein. CAP and REP are the names o the coded proteins (see https: / / www.ncbi.nlm.nih.gov / datasets / gene / 80510721 / ).

[0072] Herein sometimes the immunogenic composition is referred to as a trivalent immunogenic composition because of the main three components, but this does not exclude that other components such as carriers, adjuvants, etc. may be present.

[0073] In a preferred embodiment, the amount of the one or more recombinant E. co / / -based antigens of trimeric autotransporter fragment of protein 9 (VtaA9) of Glaesserella parasuis is from 0,5 pg to 200 pg of the whole composition.

[0074] In another preferred embodiment, the amount of the porcine circovirus type 2 genotype b (PCV2b) ORF2 CAP protein is from 0,1 pg to 200 pg of the whole composition.

[0075] In another preferred embodiment, the amount of the PCV2a ORF 1 REP protein is from 0.1 pg to 200 pg of the whole composition.

[0076] In another preferred embodiment, the immunogenic composition according to the present invention further comprises at least one pharmaceutically acceptable excipient and / or carrier.

[0077] In a second aspect, the present invention relates to an immunogenic composition according to the present invention for use as a medicament or as a combination and / or associated vaccine in livestock. In a more preferred embodiment, the immunogenic composition according to present invention is for use in the prevention and / or treatment of porcine enzootic pneumonia and / or Porcine Circovirus-Associated Diseases (PCVAD) and / or Glasser's disease in livestock. More preferably, said livestock is porcine livestock.

[0078] In another preferred embodiment, when the immunogenic composition according to present invention is used as a combination and / or associated vaccine, the vaccine further comprises one or more additional antigens from a microorganism selected from the group consisting of Actinobacillus sp., Brachyspira sp., Pasteurella multocida, Salmonella sp., Streptococcus sp., Isospora sp., Erysipelothrix rhusiopathiae, Leptospira sp., Staphylococcus sp., Bordetella bronchiseptica, Clostridium sp., Mycoplasma sp., Lawsonia intracellularis, Escherichia coli, porcine reproductive and respiratory syndrome virus (PRRS), swine influenza virus, contagious gastroenteritis virus, porcine parvovirus, encephalomyocarditis virus, coronavirus, rotavirus, porcine periweaning failure to thrive syndrome agent, classical swine fever virus, African swine fever virus, calicivirus, transmissible gastroenteritis coronavirus (TGEV), porcine epidemic diarrhea virus (PED) and torque teno virus (TTV) and combinations thereof. In this case, the immunogenic composition of the present invention can be used against another disease or pathological conditions affecting porcine livestock.

[0079] In another preferred embodiment, when the immunogenic composition according to present invention is used as a combination and / or associated vaccine, the vaccine further comprises an adjuvant. In a more preferred embodiment, said adjuvant is an adjuvant emulsion comprising mineral oil and an additive selected from a hydrophobic additive, an amphiphilic additive and combinations thereof.

[0080] In a third aspect, the present invention relates to an immunogenic diagnostic kit for discriminating naturally infected from immunized animals comprising:

[0081] (a) an immunogenic composition as defined in the present invention;

[0082] (b) monoclonal and polyclonal antibodies of VtaA9-F4 of Glaesserella parasuis, and

[0083] (c) optionally, monoclonal and polyclonal antibodies of PCV2a REP,

[0084] (d) optionally, instructions for its use.

[0085] In a more preferred embodiment, said animals are porcine livestock.

[0086] It is noted that the present invention is meant to encompass all VtaA9-F4 Gsuis variants, for example the ones with the GenBank accession numbers EU678341 (version EU678341.1), QGN52465 (version QGN52465.1), ATW45906 (version ATW45906.1), WP_021110501 (version WP_021110501.1), among others. It is noted that the present invention is meant to encompass all PCV2 ORF2 capsid protein variants, for example the ones with the GenBank accession numbers AAW79866 (version AAW79866.1), AAC35310 (version AAC35310.1), AAC35331 (version AAC35331.1), or ABX71779 (version ABX71779.1), among others.

[0087] It is noted that the present invention is meant to encompass all PCV ORF1 replicase protein variants, for example the ones with the GenBank accession numbers AAF35296 (version AAF35296.1), AAW78463 (version AAW78463.1 , type 2-B), AAD03061 (version AAD03061.1 , type 2-C), AAD12308 (version AAD12308.1 , type 2-D), AAD03071 (version AAD03071.1 , type 2-E), XCO51616 (version XCO51616.1), XCZ58777 (version XCZ58777.1), among others.

[0088] Different genotypes of PCV2 are encompassed in particular embodiments of the first aspect, such as PCV2a and PCV2b. Genotypes of PCV2 are determined by the number of nucleotide substitutions in ORF 2 (CAP) divided by the total nucleotides in ORF 2 (702 nt); this fraction is called p-distance. Recognizing the impossibility of defining a clear inter-cluster p-distance cutoff, a new phylogeny-grounded genotype definition based on three criteria has been proposed: maximum intra-genotype p-distance of 13% (calculated on the ORF2 gene), bootstrap support at the corresponding internal node higher than 70% and at least 15 available sequences. This scheme allowed defining 8 genotypes (PCV2a to PCV2h), which six of those had been previously proposed. (Franzo and Segales 2018) So far, all vaccines in the market have shown great efficacy in reducing clinical signs associated to diseases caused by PCV2, independently of the genotype present in the farm. Moreover, experimental data demonstrated the crossprotection of PCV2a vaccines against the most widespread genotypes (PCV2a, PCV2b, and PCV2d). Therefore, despite the significant number of genotypes described / proposed, it seems one single PCV2 serotype would exist so far.(Franzo and Segales 2020) The ORF2 of PCV2 encodes a protein of about 233 amino acids. Besides, and corroborating earlier observations in PCV1 , transcript mapping revealed that the rep gene of PCV2 encodes two products, the full-length protein Rep and the spliced version Rep' and that the simultaneous expression of Rep and Rep' proteins is essential for initiation of replication of PCV2.(Mankertz, Mueller et al. 2003) The rep gene products of PCV2 were not only able to bind the PCV2 origin but also the origin of PCV1 and vice versa, indicating that the cis- and trans-acting replication factors of the two viruses are functionally exchangeable. (Mankertz, Mueller et al. 2003, Opriessnig, Karuppannan et al. 2020) As stated in prior art, PCV2-infected pigs developed cell-mediated immunity to Cap and Rep proteins and that, in the course of a sub-clinical infection, development and strength of such responses are possibly related to the levels of PCV2 replication. (Fort, Sibila et al. 2010) Therefore, joining PCV2b-related protective immune response coupled with highly conserved Rep-associated immune protective immune response seems to be a rational approach to confer a wider pan-PCV immunity. Any PCV2b ORF 2 (Cap) or PCV2a ORF1 (Rep) are suitable to be used in the invention as functionally exchangeable PCV-related strong immunity developers in pigs.

[0089] The one or more antigens of VtaA9-F4 Gsuis and the one or more antigens of PCV2 in the trivalent recombinant E. co / / -based immunogenic composition are in an immunologically effective amount. Accordingly, the present disclosure also includes a method of prevention and / or treatment of porcine enzootic pneumonia and / or Porcine Circovirus-Associated Diseases (PCVAD) and / or Glasser's disease in livestock, preferably porcine livestock, comprising the step of administering an immunologically effective amount of the immunogenic compositions according to the present invention according to any of the embodiments disclosed herein.

[0090] As shown in the examples below, commercial-bioequivalent efficient immunization of livestock was achieved with the immunogenic composition of the invention. The immunogenic composition was efficacious even in a single-shot protocol, not requiring additional booster doses, however the two-dose protocol was tested as immunologic strategy in PCVAD and GD acute outbreak situation.

[0091] Moreover, it supposes a real advantage in relation with bacterins-based vaccines against G. parasuis used in association with commercial PCV2-based vaccines, postulating a double cross protection between both diseases and, certainly, supporting high efficacy against all highly virulent Gsuis strains. To the best of the author's knowledge no prior immunogenic composition as the proposed has been described in peer-reviewed literature.

[0092] With this particular combination, and as illustrated in examples, effective immunization of swine was achieved against antigens of both highly virulent Glaesserella parasuis and of porcine circovirus.

[0093] In a particular embodiment of the first aspect, optionally in combination with any embodiment above or below, the one or more antigens of Glaesserella parasuis and of porcine circovirus are in a form selected from the group consisting of E.co / / -based VtaA9-F4 Gsuis, PCV2b-CAP VLPs & PCV2a-REP, isolated recombinant proteins, inactivated VtaA9-F4-positive, GAPpositive & REP-positive E. coli cells, inactivated VtaA9-F4-positive, CAP-positive & REP- positive E. coli cell lysates, and combinations thereof.

[0094] In a particular embodiment, optionally in combination with any embodiment above or below, the antigens in the parenteral immunogenic composition of the first aspect are provided as inactivated VtaA9-F4-positive, CAP-positive & REP-positive E. coli cell lysates, coming from E. coli mutant cells expressing one or more proteins of Glaesserella parasuis (VtaA9-F4) and porcine circovirus (VLPs of CAP and REP). These VtaA9-F4-positive, CAP-positive & REP- F4-positive Escherichia coli mutant strain cells (3 in total) comprise, in particular, one or more exogenous DNA sequence incorporated in the genome or in the cytosol thereof, said exogenous DNA sequence coding for at least one protein of VtaA9-F4 Gsuis, PCV2b (CAP) or PCV2a (REP).

[0095] In another particular embodiment of the first aspect, optionally in combination with any embodiment above or below, the VtaA9-F4-positive, CAP-positive & REP-positive Escherichia coli mutant strain cells (3 in total) of the immunogenic composition of the invention is from mutant strain of E. coli, deposited by Dr. Ivan Jose Galindo Cardiel (the applicant), and transferred to WorldPathol Global United, S.A. (another applicant), and WorldPathol Manufacturing S.L., (the manufacturer) in Leibniz-lnstitut Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and selected from a mutant strain of VtaA9-F4- positive E. coli (pending the deposit number); a mutant strain of CAP-positive E. coli with the accession number (pending the deposit number); a mutant strain of REP-positive E. coli with the accession number (pending the deposit number). All these mutant strains were deposited at DSMZ on September 27, 2024. They were declared viable. VtaA9-F4-positive E. coli mutant strain is disclosed in EP 1937712 (incorporated herewith by reference) (Bensaid, Pina-Pedrero et al. 2008).

[0096] In another particular embodiment, the dose of the immunogenic composition like the one of the invention, along with pharmaceutically acceptable excipients or carriers to become a combination vaccine or immunological product, is comprised from 0.5 to 2 mL.

[0097] The immunogenic composition can be administered in one or more dose. A multiple-dose immunization, as is well known in the art, consists of administering a first immunizing dose, followed by one or more additional doses which act as booster doses. In an embodiment of the invention, the immunogenic composition preferably is of single-dose administration, although the two-dose protocol was tested to be available as immunologic strategy in GD and PCVAD acute outbreak situation.

[0098] As detailed above, the present invention also provides an immunogenic diagnostic system to discriminate naturally infected from immunized animals comprising monoclonal and polyclonal antibodies of VtaA9-F4 Gsuis and monoclonal and polyclonal antibodies of PCV2b CAP. All particular embodiments of the immunogenic composition for use according to the first aspect of the invention also apply to the immunogenic diagnostic system to discriminate naturally infected from immunized animals, in particular livestock, more in particular porcine livestock.

[0099] In a particular embodiment, optionally in combination with any embodiment above or below, the monoclonal and polyclonal antibodies of any variant of VtaA9-F4 Gsuis and monoclonal and polyclonal antibodies of any variant of PCV2b CAP are used as substrate for lateral flow immunochromatography diagnostic kit.

[0100] In a particular embodiment, optionally in combination with any embodiment above or below, the immunogenic diagnostic system to discriminate naturally infected from immunized animals (DIVA) of the second aspect is for use as a diagnostic DIVA kit in animals, in particular livestock, more in particular porcine livestock.

[0101] More in particular, the immunogenic diagnostic system to discriminate naturally infected from immunized animals (DIVA) is for use in the diagnosis of GD and / or PCVAD by serum sampling of animals, in particular livestock, more in particular porcine livestock.

[0102] Other embodimentsincluded in the invention are as follows:

[0103] Clause 1. An immunogenic composition comprising:

[0104] - one or more antigens of Glaesserella parasuis,

[0105] - one or more antigens of porcine circovirus;

[0106] - pharmaceutically acceptable excipients and / or carriers.

[0107] Clause 2. The immunogenic composition according to clause 1 , wherein the one or more antigens of the composition comprises: (1) a fragment (F4) of trimeric autotransporter protein (VtaA9) described from highly virulent Glaesserella parasuis (Gsuis) (VTaA9-F4 Gsuis); (2) a porcine circovirus type 2 (PCV2b) ORF2 antigen coding for structural protein (CAP); and (3) a porcine circovirus type 1 (PCV2a) ORF 1 antigen coding for replicase protein (REP).

[0108] Clause 3. The immunogenic composition according to clauses 1-2, wherein the one or more antigens of the composition comprises: (1) a fragment (F4) of trimeric autotransporter protein (VtaA9) described from highly virulent Glaesserella parasuis (Gsuis) (VTaA9-F4 Gsuis); (2) a porcine circovirus type 2 (PCV2b) ORF2 antigen coding for structural protein (CAP); and (3) a porcine circovirus type 1 (PCV2a) ORF 1 antigen coding for replicase protein (REP), for the prevention and treatment of Glasser's disease (GD) and porcine circovirus-associated diseases (PCVAD) in animals, preferably in pigs.

[0109] Clause 4. The immunogenic composition according to any of clauses 1-3, wherein the one or more antigens of the composition consisting of E. co / / - based VtaA9-F4 Gsuis, PCV2b-CAP VLPs & PCV2a-REP, isolated recombinant proteins, inactivated VtaA9-F4-positive, CAP- positive & REP-positive E. coli cells, inactivated VtaA9-F4-positive, CAP-positive & REP- positive E. coli cell lysates, and combinations thereof.

[0110] Clause 5. The immunogenic composition according to any of clauses 1-4, wherein the one or more antigens based on fragment of trimeric autotransporter protein described from highly virulent Glaesserella parasuis (VtaA9-F4 Gsuis) are in the form of E. co / / -based VtaA9-F4- positive isolated recombinant proteins, inactivated VtaA9-F4-positive E. coli cells, inactivated VtaA9-F4-positive E. coli cell lysates, and combinations thereof.

[0111] Clause 6. The immunogenic composition according to any of clauses 1-4, wherein the one or more antigens of porcine circovirus type 2b (PCV2b) open reading framework (ORF) 2 antigen coding for structural protein (CAP) are in the form of E. co / / -based CAP-positive isolated recombinant proteins, inactivated CAP-positive E. coli cells, inactivated CAP-positive E. coli cell lysates, and combinations thereof.

[0112] Clause 7. The immunogenic composition according to any of clauses 1-4, wherein the one or more antigens of porcine circovirus type 2a (PCV2a) open reading framework (ORF) 1 antigen coding for replicase protein (REP) are in the form of E. co / / -based REP-positive isolated recombinant proteins, inactivated REP-positive E. coli cells, inactivated REP-positive E. coli cell lysates, and combinations thereof.

[0113] Clause 8. The immunogenic composition according to any of the previous clauses, wherein the VtaA9-F4-positive, CAP-positive & REP-positive Escherichia coli mutant strain cells (3 in total) of the immunogenic composition of the invention is from mutant strain of E. coli, deposited by Dr. Ivan Jose Galindo Cardiel (the applicant), and transferred to WorldPathol Global United, S.A. (another applicant), and WorldPathol Manufacturing S.L., (the manufacturer) in Leibniz- Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and selected from a mutant strain of VtaA9-F4-positive E. coli with the accession number (pending the deposit number); a mutant strain of CAP-positive E. coli with the accession number (pending the deposit number); a mutant strain of REP-positive E. coli with the accession number (pending the deposit number).

[0114] Clause 9. The immunogenic composition according to any of the previous clauses, further comprising one or more additional antigens wherein the additional antigen is selected from a group of microorganisms consisting of Actinobacillus, Bordetella, Borrelia, Brachyspira, Brucella, Campylobacter, Chlamydia and Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Erysipelothrix, Escherichia, Francisella, Haemophilus, Helicobacter, Isospora, Lawsonia, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio and Yersinia genus, and porcine reproductive and respiratory syndrome virus (PRRS), swine influenza virus, contagious gastroenteritis virus, porcine parvovirus, encephalomyocarditis virus, coronavirus, rotavirus, porcine periweaning failure to thrive syndrome agent, classical swine fever virus, African swine fever virus, calicivirus, torque teno virus (TTV), transmissible gastroenteritis coronavirus (TGEV), porcine epidemic diarrhea virus (PED) and combinations thereof.

[0115] Clause 10. An immunogenic composition as defined in any of clauses 1-9, for use as a medicament or combined vaccine in livestock.

[0116] Clause 11. The immunogenic composition for use according to clause 10, which is for use in the prevention and / or treatment of GD and PCVAD in animals, preferably in pigs, and optionally against another disease or pathological conditions affecting swine.

[0117] Clause 12. An immunogenic diagnostic kit to discriminate naturally infected from immunized animals comprising:

[0118] (a) an immunogenic composition as defined in any of clauses 1-10;

[0119] (b) monoclonal and polyclonal antibodies of VtaA9-F4 Gsuis; and

[0120] (c) monoclonal and polyclonal antibodies of PCV2a REP (Strep tagged)

[0121] (d) optionally, instructions for its use.

[0122] Clause 13. The immunogenic diagnostic kit to discriminate naturally infected from immunized animals as defined in clause 12 for use in the diagnosis of GD and PCVAD by serum and I or blood sampling of livestock. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, it is noted that any of the embodiments disclosed herein can be taken alone or combined with any other embodiment disclosed herein unless the context specifies otherwise. In other words, for example, a preferred option of a defined feature can be combined with a more or less preferred option of another feature.

[0123] EXAMPLES

[0124] Hereinafter, the invention described in the present application will be described in more detail by representing examples thereof. However, this application is not limited by the following examples.

[0125] EXAMPLE 1. Construction of recombinant F4 fragment of the VTaA9 protein from Glaesserella parasuis- positive Escherichia coli

[0126] 1.1. Plasmid construction of F4 fragment of the VtaA9 protein

[0127] The F4 protein fragment, consisting of amino acids 701 to 874 from the trimeric autotransporter VtaA9 from G. parasuis strain Nagasaki, is used as an immunogen. VtaA-F4 protein in silico analysis is performed and shown in Table 2. The F4 fragment of the VtaA9 gene from G. parasuis strain Nagasaki (GenBank: EU678341.1) is amplified by PCR using primers as follows: ATGGTAGGTCTCAAATGGCGGGTCCAACTGGAAATTCGG (SEQ ID No. 1 ; forward, with underlined Bsal site) and

[0128] ATGGTAGGTCTCAGCGCTACGACCAATATCTCTTGATAATTGAT (SEQ ID No. 2; reverse, with underlined Bsal site).(Bensaid, Pina-Pedrero et al. 2008) The PCR product is then cloned into the Bsal site of pASK-IBA33plus plasmid system (IBA-BioTAGnologies, Gottingen, Germany), in frame with an 6xHis tag at the C-terminus (pASK-IBA33plus_F4).

[0129] Table 2. In silico analysis data for recombinant protein F4

[0130] 1.2. Expression of F4 recombinant fragment of the VtaA9 protein from Glaesserella parasuis

[0131] The present invention concerns a successfully expressed and purified F4-6xHis recombinant protein in a BL21 RosettaTM (DE3) pLysS (Novagen) E. co / / -based pathogen-related GLP- validated vaccine manufacturing platform of manufacturer (297-50-065-AV, Zaragoza, Spain). Ampicillin resistant transformants are isolated on agar plates containing 100 pg / mL ampicillin and screened for the presence of F4 DNA inserts. Positive colonies are then selected and cultured in 1 L of Luria-Bertani (LB) medium containing 100 pg / mL of ampicillin. Culture is incubated at 37 °C with shaking at 250 rpm, until it reaches the exponential growth phase. Anhydrotetracycline (aTc) is added to the culture liquid to a final concentration of 0.2 mg / L. Thus, protein expression is induced for 2 hours, at 25 °C and 250 rpm. Upon completion of induction, E. coli cells are harvested by mild centrifugation (6000 rpm, 15 min and 4 °C) and washed once with phosphate-buffered saline (PBS) buffer.

[0132] Cell pellet is resuspended in lysis buffer and disrupted by multiple pulses of sonication. After ensuring the absence of unbroken cells in the lysate, insoluble material consisting of cell walls, inclusion bodies and membrane fragments is sedimented by centrifugation (6000 rpm for 15 min at 4 °C), yielding the insoluble fraction. The remaining supernatant represents the soluble fraction.

[0133] 1.3. Purification of recombinant F4 fragment from soluble and insoluble fractions The expressed recombinant F4 His-tagged fragment is purified by immobilized metal affinity chromatography (IMAC) using HisTrap™ FF Crude columns (Cytiva), according to the manufacturer’s instruction. To solubilize proteins accumulated in the inclusion bodies, 6M urea was added to the solubilization buffer. The amino acid sequence of the recombinant F4 His- tagged fragment is as follows: MAGPTGNSELKGITSIANGNDATKANGAKITLSAGSTDKTVNVNDAKITNVAAGTADTDAVN VSQLNTKAAASKTEVEAGKNVKVTSKTGANGQNIYNVSVSGDLSDITSISNGDTKVSLGKDK QGNPVVNMNGARITNVGDGSAEGDIVNVRQLNKVVSSVNTGFNQLSRDIGRSARGSHHHH HH (SEQ ID No. 3; with underlined 6xHis tag).(Bensaid, Pina-Pedrero et al. 2008)

[0134] 1.4. SDS-PAGE and Western blot analysis of F4 protein expression

[0135] Protein expression and purification of the recombinant VtaA9-F4 His-tagged fragment is confirmed by conducting a Western blot analysis in any of these raw materials; E.co / / -based VtaA9-F4 isolated recombinant proteins, inactivated VtaA9-F4-positive E. coli cells and inactivated VtaA9-F4-positive E.coli cell lysates. The cell extracts and fractions purified are diluted in 2x loading buffer. The samples are then separated by electrophoresis in a 12% polyacrylamide gel with sodium dodecylsuphate (SDS-PAGE) under reducing conditions. Separating gels are either stained with Coomassie brilliant blue or transferred by electroblotting onto a nitrocellulose membrane. Membranes are treated with blocking solution (PBS containing 5% skim milk) for 1 h at room temperature and incubated with anti-His monoclonal antibodies conjugated to horseradish peroxidase (1 :5000) for 2 h at room temperature, followed by three washes in PBST, 15 min each. The immunoreactive bands are visualized using an ECL kit for chemiluminescent detection. The Figure 7 illustrates a representative SDS-PAGE analysis used to demonstrate the effectiveness of the soluble recombinant VtaA9- F4 fragment purification process through IMAC.

[0136] 1.5. Production of monoclonal antibodies against F4 recombinant protein

[0137] Monoclonal antibodies can be produced using the hybridoma method, (Kohler and Milstein 1975), or through recombinant DNA methods. (Cabilly, Heyneker et al. 1989) Recombinant F4 fragment purified from the soluble fraction is administered intraperitoneally to three 3-month- old female BALB / c mice to elicit a targeted immune response. The immunization schedule includes an initial dose of 50 pg of F4 combined with Freund's complete adjuvant, followed by two booster doses of 25 pg of F4 with Freund's incomplete adjuvant, administered at 14-day intervals. To assess the immune response, blood is collected 7 days after the third immunization for antibody titer determination via indirect ELISA. Prior to cell fusion, a booster dose of 25 pg of F4 is administered intraperitoneally to the selected donor mouse 4-5 days before spleen extraction. Spleen cells are harvested 4-5 days after the booster dose and fused with SP2 / O- Ag-14 myeloma cells at a myeloma-to-splenocyte ratio of 1 :3, using polyethylene glycol (PEG 4000) as the fusing agent.(Goding 1996) Following cell fusion, the resulting hybridoma cells are seeded in 96-well plates containing selective Hypoxanthine-Aminopterin-Thymidine (HAT) medium at a density of 2x105cells per well. The medium supports the selective growth of hybridomas by inhibiting the proliferation of unfused myeloma cells.

[0138] Hybridoma cultures are screened for the production of monoclonal antibodies specific to the F4 antigen using indirect enzyme-linked immunoabsorbent assay (ELISA) and capture ELISA with biotin-conjugated F4 fragment, purified from the insoluble fraction through on-column refolding. Positive clones are then selected and subjected to limiting dilution cloning to ensure monoclonality. This process is repeated twice to confirm stable, antibody-producing clones. (Goding 1996)

[0139] The selected antibody-secreted hybridomas are cultured and expanded. Monoclonal antibodies are purified using pre-packed columns with sepharose coupled to protein G (Cytiva), to selectively bind and capture IgGs. The purified antibodies can be further characterized and utilized for potency tests, diagnostic or therapeutic applications.

[0140] 1.6. Production of polyclonal antibodies against F4 recombinant protein

[0141] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (s.c.) or intraperitoneal (i.p.) injections of the relevant antigen combined with an adjuvant. In this example, five female rabbits are immunized against the recombinant F4 fragment derived from the VtaA9 protein, designed to elicit a targeted immune response against G. parasuis. The immunization schedule consists of an initial injection of 250 pg of the recombinant F4, emulsified in Freund's complete adjuvant, followed by three booster injections of 125 pg of recombinant F4 emulsified in Freund's incomplete adjuvant. The booster injections are administered intraperitoneally at 3-week intervals. Eleven weeks after the initial immunization, a 0.5 mL serum sample is collected to determine the antibody titer using ELISA. A final bleed is performed seven days later (week 12), yielding 40-90 mL of serum per rabbit.

[0142] Antibody titers are assessed by indirect ELISA with the recombinant F4 protein, purified from the insoluble fraction through on-column refolding. Polyclonal antibodies are purified from the collected serum using affinity chromatography with pre-packed columns containing sepharose coupled with Protein A (Cytiva) for IgG purification and / or pre-packed columns with NHS- activated sepharose (Cytiva) coupled to F4, to capture specific immunoglobulins against the F4 fragment.

[0143] 1.7. SDS-PAGE and Western blot analysis for validation of monoclonal and polyclonal antibody production against VtaA9-F4 fragment

[0144] Western blot analyses are used to validate the specificity of anti-F4 monoclonal (see example 1 .5.) and polyclonal (see example 1 .6.) antibodies. The F4 protein is diluted in 2x loading buffer and loaded at different concentrations (125 ng, 187.5 ng, and 312.5 ng). The samples are then separated by electrophoresis in a 12% polyacrylamide gel with sodium dodecylsuphate (SDS- PAGE) under reducing conditions. Separating gels are either stained with Coomassie brilliant blue or transferred by electroblotting onto a nitrocellulose membrane. Membranes are treated with blocking solution (PBS containing 5% skim milk) for 1 h at room temperature and incubated with anti-F4 mouse monoclonal or rabbit polyclonal antibodies (1 :5000) for 2 h at room temperature, followed by three washes in PBS containing 0.05% Tween 20 (PBST), 15 min each. Membranes are incubated with a Horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit antibodies (1 :15000) for 1 h at room temperature, followed by three washes in PBST, 15 min each. The immunoreactive bands are visualized using an ECL kit for chemiluminescent detection. Figures 8 and 9 illustrate the validation of both monoclonal and polyclonal antibodies against the VtaA9-F4 fragment trough SDS-PAGE analyses complemented by Coomassie staining and Western blotting, to confirm antibodies specificity.

[0145] 1.8. ELISA-based potency assay for F4 recombinant protein

[0146] ELISA-based potency test assay were performed to ensure the immunization efficacy of the antigens of the immunogenic combination. The potency of the recombinant F4 fragment is determined though an endpoint ELISA utilizing monoclonal antibodies produced as described in example 1.5., in both the capture and detection phases. 96-well microtiter plates are coated with 10 pg / mL of monoclonal antibody 1 (mAb1) anti-F4 in carbonate-bicarbonate buffer (NaHCO3 / Na2CO3 0.1 M, pH 9.6) and incubated overnight at 4 °C. Following incubation, the plates are washed three times with PBS containing 0.05% Tween 20 (PBST) and blocked with blocking solution (PBS containing 5% skim milk) for 30 min at room temperature to prevent non-specific binding. Samples are diluted with PBS containing 1% BSA and 0.05% Tween, and 100 pL of the duplicate diluents are added into mAb1 -coated wells. After an incubation period of 2 hours at 37 °C, the plates are washed three times with PBST and incubated during 1 hour at 37 °C with the biotinylated monoclonal anti-F4 detection antibodies (mAb2) at a concentration of 2 pg / mL. After three washes with PBST, a streptavidin-HRP conjugate, diluted 1 / 400, is then added to bind to the biotinylated detection antibodies. After a 45 min incubation at room temperature, the plates are washed three times with PBST and developed at room temperature with tetramethyl-benzidine substrate (TMB) for 5-10 min in the dark. Then reactions are terminated by addition of 1 N HCI. Endpoint titers are considered valid if the absorbance is less than -2.000 for the test samples and does not exceed -0.150 for the negative controls, with the sample / negative control ratio required to be equal to or greater than 2-3. A measurement range from 31.25 ng / mL to 1500 ng / mL is defined.

[0147] 1.9. Proof of concept for VtaA9-F4 Gsuis -based immune response for Glasser's disease

[0148] Glaesserella parasuis is the causative agent of Glasser's disease (GD), which presents with non-specific clinical signs, such as fever, cough, swollen joints, prostration, nervous signs, among others, because of fibrinous polyserositis lesions and meningitis. G. parasuis presents strains with different degrees of virulence, from highly invasive to normal colonizers of the nasal microbiota. Antibodies are essential in protecting against disease (Nedbalcova, Kucerova et al. 2011 , Costa-Hurtado, Ballester et al. 2012). As stated in prior art, VtaA9-related previous results led to the selection of the F4 fragment as a possible immunogen, since it was exposed on the surface of the bacterium (recognized by 69C6), was common to all VtaA of group 1 and its size allowed effective purification. In the laboratory, when 50 mL was processed, 1.8 mg of F4 was obtained at a concentration of 2.000 ng / pL.

[0149] VtaA9-F4 Gsuis represents WPM’s preventive solution against GD. This subunit-based immunogenic composition is formulated with the F4 surface-exposed fragment derived from the group 1 and 2 of virulent-associated trimeric autotransporter proteins (VtaAs) of the Nagasaki strain (serovar 5), which is highly conserved in highly virulent Gsuis. This strategic composition not only addresses growing concerns related to antimicrobial use in disease management but also provides targeted broad-spectrum protection across a wide range of serovars. Based on the immunogenic potential of the VtaA9-F4 fragment, WPM has developed VtaA9-F4, an intramuscular subunit-based immunogenic composition, valid as vaccine candidate, poised as a safe and effective alternative to traditional bacterins and antibiotic therapies.

[0150] VtaA9-F4 immunogenic capacity has been validated through multiple in vivo assays conducted in piglets (Figure 10). In other immunization studies, the F4 fragment was found to be immunogenic when administered in emulsion, but not with aluminum salts (Figure 11). As a positive control, the F4 fragment with Freund's adjuvant was used. Previous studies ruled out propolis and inulin as adjuvants.

[0151] Following the initial vaccination, a primary antibody response against VtaA9 was detected, showing a gradual increase in antibody concentration that peaked approximately two weeks post-vaccination. Subsequent administration of a second dose further augmented the antibody concentration, ensuring a sustained protection in piglets. The efficacy of the immunogenic composition against both homologous (serovar 5, Nagasaki) and heterologous (serovar 1 , PV1-12) Gsuis strains was confirmed in an in vivo assay. Seronegative piglets against Gsuis were vaccinated with two doses of VtaA9-F4 with a 15-day interval between each dose. The placebo control group was treated with an equivalent dosage of PBS. Two weeks after the second immunization, the piglets were intratracheal ly challenged with a dose of 107'8CFU / piglet of either serovar 5 or serovar 1 of Gsuis, based on their assigned groups. Following infection, clinical signs were recorded daily for two weeks. Surviving animals were euthanized at the end of the trial. Notably, no clinical signs or pathological findings were observed in all vaccinated pigs.

[0152] After infection, the immunized group exhibited a substantial increase in protective antibody activity compared to the non-immunized group, underscoring the successful outcome of the trial and the effectiveness of the immunogenic composition in providing protection against Gsuis (Figure 12). Furthermore, no mortality was recorded in the challenged immunized group, and lesion severity was notably reduced. Moreover, VtaA9-F4 Gsuis demonstrated crossimmunity and protection against Gsuis regardless of serovar, proving highly effective in controlling disease outbreaks by restoring pulmonary alveolar macrophages (PAMs) function in heavily infected virulent Gsuis animals. Additionally, it holds the potential to prevent GD in Gsuis-affected herds worldwide. The recommended vaccination of all 3-week-old piglets aligns with the objective of reducing antibiotic use in animal production, as advocated by the Committee for Medicinal Products for Veterinary Use (CVMP) of the EMA.

[0153] When different regimens of immunization were tested, we observed that piglets vaccinated at 1 and 3 weeks of age with 100 pg had a greater antibody response against F4 than those vaccinated with a single dose of 200 pg of F4 at 1 week of age. This difference was observed at 5 weeks of age (Figure 13). Piglets were inoculated with 2 virulent strains of G. parasuis at 5 weeks of age, but protection could not be clearly observed because Glasser's disease did not reproduce with inoculation. Although the disease did not develop after the challenge, a reduction in the levels of antibodies against F4 was observed, which seems to indicate that the antibodies induced by the vaccine are opsonizing and after the challenge are "absorbed" on the surface of the bacteria and used for their elimination.

[0154] Accurate adjuvanted formulations help to induce a stronger and more persistent immune reaction, provide immunity with a smaller amount of the active ingredient, reduce the dose needed, and sometimes help to stimulate a more effective immune response in populations with weaker immune systems, like neonatal or immunocompromised pigs. This formulation results in improved vaccine efficacy and long-lasting protection against infectious diseases.

[0155] The VtaA9-F4 vaccine prototype, composed of recombinant E. co / / -based trimeric autotransporter protein VtaA9-F4 (100 pg / mL dose), is formulated on an emulsion with squalene (60%) plus a-tocopherol (40%) and elicited strong protective immune response both in piglets and sows, vaccinated 5 and 2 weeks before delivery (Lopez-Serrano, Neila-lbanez et al. 2021). This galenic formulation has shown a robust 12-months stability. The effects of maternal vaccination are summarized in Figure 14. Vaccination of the dams induced a high level of antibodies against F4 in serum (Figure 15) and colostrum (Figure 16), which was transmitted to piglets (Figure 17). No adverse effects were observed in dams with the F4 vaccine.

[0156] In parallel with the increase in antibodies, the F4 vaccine also resulted in an increase in the serum level of TGF-p in both vaccinated sows and their piglets in the first days of life (Figure

[0157] 18). Following the challenge to piglets with a virulent strain, lower body temperature and higher weight were observed in the piglet group from vaccinated sows. In addition, as in previous studies, a reduction in antibodies was observed after virulent challenge that would indicate its opsonizing capacity. Necropsies performed 2 weeks later detected anti-F4 antibodies and a higher level of SP-D (surfactant protein) in the BALF of piglets from vaccinated mothers (Figure

[0158] 19).

[0159] Vaccinating the sows also produced changes in the nasal microbiota of the piglets, including the reduction of G. parasuis strains. (Blanco-Fuertes, Correa-Fiz et al. 2022)

[0160] Recent work performed by WPM has additionally shown that sow immunization with F4 emulsified with Carbopol Polymer adjuvant not only induced anti-F4 specific immune responses but also modulated the immunity traits in their offspring with an increase of circulating TGF-p. Neonatal pig vaccination is essential for early disease protection, reducing mortality rate and antibiotic reliance, and ensuring swine industry sustainability. Accordingly, WPM has explored the vaccine potential of the VtaA9-F4 fragment in neonate pigs, testing the efficacy of two novel adjuvants: the Cationic Adjuvant Formulation 01 (CAF®01) and the bis-(3,5)-cyclic dimeric adenosine monophosphate (CDA), specifically designed to stimulate different immune pathways. While additional research is required for a comprehensive understanding of the effects, both adjuvants demonstrated a capacity to trigger immune responses in the piglets, notwithstanding some variability and potential influence from factors such as maternal antibodies and natural colonization by virulent Gsuis (Lopez-Serrano, Mahmmod et al. 2023). The piglets were vaccinated with two doses of vaccine, at 14 days of age and 21 days later. The immune response induced against the F4 fragment varied depending on the adjuvant used. Piglets vaccinated with the F4+CDA vaccine developed specific anti-F4 IgG, with a biased response towards inducing lgG1 responses, while the F4 vaccine with CAF01 did not induce antibodies against F4. Piglets immunized with both formulations showed balanced memory T cell responses, evidenced after in vitro restimulation of peripheral blood mononuclear cells with F4. Interestingly, pigs immunized with F4+CAF01 more efficiently controlled the natural nasal colonization that arose naturally during the experimental procedure.

[0161] CAF®01 exhibited a significantly reduced nasal colonization by Gsuis, suggesting a robust potential for mucosal immunity, despite not eliciting a strong systemic humoral response. Conversely, piglets vaccinated with the VtaA9-F4 + CDA combination showed efficient induction of anti-F4 lgG1 antibodies by day 38, indicative of a Th2 skewed response. Moreover, two piglets did not become colonized at all, suggesting some level of protective immunity.

[0162] A potency assay is a regulatory requirement for the release of every immunological active ingredient batch. Potency is a critical quality attribute intended to ensure the stability, integrity and immunocompetence of an antigen in its final formulation. As the principles of reduction, refinement, and replacement (3 Rs) are extensively promoted and increasingly recognized, the World Health Organization (WHO), regulatory authorities, and vaccine manufacturers remain committed to substituting in vivo methods for vaccine assessment. Based on the ICH (international conference on harmonization) Q14 and Q2 (R2) requirements, WPM has developed an in vitro double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) with proprietary monoclonal antibodies raised against VtaA9-F4 to determine the antigen content in the VtaA9-F4 prototype (Figure 20).

[0163] EXAMPLE 2. Construction of PCV2b ORF2 (CAP) positive recombinant Escherichia coli

[0164] 2.1. Construction of CAP plasmid The PCV2b CAP protein encoded by the ORF2 is used as an immunogen. PCV2b ORF 2(CAP) protein in silico analysis is performed and shown in Table 3. The ORF2 gene sequence (GenBank: AAW79866.1) is synthesized using a custom DNA synthesis service (DNA 2.0) to ensure accurate sequence replication. The ORF2 gene sequence is codon-optimized for E. coli expression and cloned into the pJ411 vector (DNA 2.0), in frame with a 6xHis tag at the C- terminus (pJ411_Cap).

[0165] Table 3. In silico analysis data for recombinant protein PCV2b ORF2 (CAP).

[0166] 2.2. Expression of PCV2b ORF2 (CAP) recombinant protein and self-assembly of VLPs in vitro

[0167] The present invention concerns a successfully expressed and purified full-length ORF2 CAP- 6xHis recombinant protein in a BL21-CodonPlus (DE3)-RIL E. co / / -based pathogen-related GLP-validated vaccine manufacturing platform of manufacturer (297-50-065-AV, Zaragoza, Spain). Kanamycin resistant transformants are isolated on agar plates containing 40 pg / mL kanamycin and screened for the presence of CAP DNA inserts. Positive clones are further confirmed through restriction digestion analysis and Sanger sequencing. Verified colonies are then cultured in 1 L of LB medium containing 40 pg / mL of kanamycin and incubated at 37 °C with shaking at 250 rpm, until reaching the exponential growth phase. Protein expression is induced by the addition of isopropyl p-D-1 -thiogalactopyranoside (IPTG) to a final concentration of 1 mM, followed by an incubation period of 4 hours, at 37 °C with continuous shaking at 250 rpm. Upon completion of induction, E. coli cells are harvested by mild centrifugation (6.000 rpm, 15 min and 4 °C) and washed once with phosphate-buffered saline (PBS) buffer. Cell pellet is suspended in lysis buffer and disrupted by multiple pulses of sonication. The homogenate is cleared by centrifugation (6.000 rpm for 15 min at 4 °C) and insoluble material consisting of cell walls, inclusion bodies and membrane fragments is sedimented, yielding an insoluble fraction enriched in CAP.

[0168] The CAP protein is suspended in PBS and allowed to assemble into virus-like particles (VLPs). This protein self-assembles into immunogenic Virus-Like Particles (VLPs) with a normal Cap appearance, closely mimicking the PCV2b capsid. Transmission electron microscopy analysis (TEM) revealed that CAP PCV2b VLPs expressed in the E. coli insoluble fraction displayed spherical particles, with diameters of approximately 19 nm in PBS solution (Figure 21 A1 y A2) and 10-11 nm in TRIVAC formulation (Figure 21 B1 y B2), consistent with prior publications. (Guo, Lu et al. 2011 , Bian, Yu et al. 2021 , Jiao, Yang et al. 2023)

[0169] The amino acid sequence of the recombinant CAP His-tagged protein is as follows: MTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTFGYTIKRTT VKTPSWAVDMMRFNINDFLPPGGGSNPRSVPFEYYRIRKVKVEFWPCSPITQGDRGVGSS AVILDDNFVTKATALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQL WLRLQTAGNVDHVGLGTAFENSIYDQEYNIRVTMYVQFREFNLKDPPLKPASHHHHHH (SEQ ID No. 4; with underlined 6xHis).

[0170] 2.3. SDS-PAGE and Western blot analysis of PCV2b ORF2 (CAP) protein expression

[0171] Protein expression and purification of the recombinant Strep-CAP-6xHis protein is confirmed by conducting a Western blot analysis in any of these raw materials; E.co / / -based PCV2b CAP VLPs isolated recombinant proteins, inactivated CAP-positive E. coli cells and inactivated CAP-positive E.coli cell lysates. Membranes are incubated for 2 h at room temperature with either anti-His monoclonal antibodies conjugated to horseradish peroxidase (1 :5000) as the primary and detection antibody, or anti-CAP antibody (Bioss Antibodies, 1 :5000) as the primary antibody followed by a 1 h incubation at room temperature with anti-IgG-HRP (Sigma-Aldrich, 1 :15000) as the detection antibody. The immunoreactive bands are visualized using an ECL kit for chemiluminescent detection. Figure 22 illustrates a representative SDS-PAGE stained with Coomassie Blue alongside a Western blot analysis, demonstrating the presence of the CAP protein in the insoluble fraction suspended in PBS and allowed to assemble into VLPs.

[0172] 2.4. ELISA-based potency assay for PCV2b ORF2 (CAP)

[0173] ELISA-based potency tests are conducted to ensure the immunogenic efficacy of PCV2b ORF2 (CAP). These tests utilize commercially available ELISA kits for detecting circovirus Viral Protein 2 (VP2) antigen (INgezim PC DAS, Ingenasa) or, alternatively, proprietary or commercially sourced antibodies specific to CAP.

[0174] 2.5. Proof of concept for PCV2b-CAP-based immune response for PCVAD

[0175] In previous research, WPM developed two PCV2b ORF2 (CAP) VLPs formulations based on a carbomer-squalene emulsion and a liquid paraffin emulsion, respectively, and compared them against the leader-of-market reference vaccine (Porcilis® PCV and derivatives, Intervet Inc). The experimental design encompassed two test groups, one without dilution and the other with a 1 / 400 dilution. As a reference control, the soluble fraction of an E. coli lysate expressing PCV2b ORF2 (CAP) VLPs was included. Preliminary results showed that the liquid paraffin emulsion stood out as the most stable and robust formulation, with no evidence of antigen masking (Figure 23).

[0176] Following the selection of the PCV2b CAP VLP vaccine prototype formulation, the production robustness was verified through the manufacture of three independent batches. For a real comparative analysis, PCV2b CAP VLP doses were adjusted to contain the same viral protein titration as the leader-of-market reference vaccine. As depicted in Figure 24, the anti-CAP- positive commercial ELISA signal of the E. coli lysate and the PCV2b CAP VLP emulsion in liquid paraffin exhibited a twofold increase compared to that obtained with leader-of-market reference vaccine. It is worthy to mention that the immune response after administration of leader-of-market reference vaccine was monitored and confirmed by determining anti-CAP antibody titers in the vaccinated, as stated EMA-approved technical files from manufacturer (EMEA 2009, Medicines 2017).

[0177] This enhancement was consistent across all three replicas and tested dilutions. Additionally, the signal obtained with PCV2b CAP VLP liquid paraffin closely resembled that obtained in the E. coli lysate, suggesting that the PCV2b CAP-6xHis protein present in the emulsion is recognized as VLPs. These findings reinforce the idea that the PCV2b CAP VLP formulation have the potential to enhance vaccine efficacy by improving antigen presentation, outperforming existing commercial vaccines.

[0178] Based on these results, WPM conducted an in vivo assay to evaluate the efficacy of the vaccine prototype. Piglets were assigned to four experimental groups and immunized intramuscularly with PCV2b CAP VLP, in different vaccination schedules and dosages, and with a commercial reference vaccine (Porcilis® PCV, single dose). Serum samples collected at DO (first dose), D14 (second dose), and D28 were analyzed for PCV2-specific viral protein 2 using the iNgezim PCV DAS ELISA assay. Despite the high level of maternal antibodies against PCV2 detected at the beginning of the assay (DO, 3 weeks of life), the analysis revealed similar PCV2-specific IgG levels in all immunized groups, correlating with bioequivalent immune response against PCV2b VLP of leader-of-market reference vaccine. These promising results validate the effectiveness and successful proof of concept of PCV2b CAP VLP industrial design, underscoring its potential as a PCV2 sub-unit vaccine alone, or in combinations thereof.

[0179] EXAMPLE 3. Construction of PCV2a ORF1 (REP) positive recombinant Escherichia coli

[0180] 3.1. Construction of REP plasmid

[0181] PCV2a REP protein encoded by the ORF1 is used as an immunogen. PCV2a ORF 1 (REP) protein in silico analysis is performed and shown in Table 4. Similar to EXAMPLE 2, the ORF1 gene sequence (GenBank: AAF35296.1) is synthesized using a custom DNA synthesis service (DNA 2.0) to ensure accurate sequence replication. The ORF1 gene sequence is codon- optimized for E. coli expression and cloned into the pJ401 vector (DNA 2.0), in frame with a Strep tag at the N-terminus and a 6xHis tag at the C-terminus (pJ401_Rep).

[0182] Table 4. In silico analysis data for recombinant protein PCV2a ORF1 (REP).

[0183] 3.2. Expression of PCV2a ORF1 (REP) recombinant protein

[0184] The present invention concerns a successfully expressed and purified full-length ORF1 Strep- REP-6xHis recombinant protein in a BL21 (C2530H, New England Biolabs) E. co / / -based pathogen-related GLP-validated vaccine manufacturing platform of manufacturer (297-50-065- AV, Zaragoza, Spain). Kanamycin resistant transformants are isolated on agar plates containing 30 pg / mL kanamycin and screened for the presence of REP DNA inserts. Positive clones are further confirmed through restriction digestion analysis and Sanger sequencing. Verified colonies are then cultured in 1 L of LB medium containing 30 pg / mL of kanamycin and incubated at 37 °C with shaking at 250 rpm, until reaching the exponential growth phase. Protein expression is induced by the addition of isopropyl p-D-1 -thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM, followed by an incubation period of 4 hours, at 37 °C with continuous shaking at 250 rpm. Upon completion of induction, E. coli cells are harvested by mild centrifugation (6.000 rpm, 15 min and 4 °C) and washed once with phosphate-buffered saline (PBS) buffer.

[0185] 3.3. Purification of PCV2a ORF1 (REP) recombinant protein

[0186] The expressed recombinant Strep-REP-6xHis protein is purified by immobilized metal affinity chromatography (IMAC) using HisTrap™ FF Crude columns (Cytiva) and / or StrepTrap™ HP (Cytiva), according to the manufacturer’s instruction.

[0187] The amino acid sequence of the recombinant Rep Strep-His-tagged protein is as follows: WSHPQFEKSAMPSKKNGRSGPQPHKRVWFTLNNPSEDERKKIRELPISLFDYFIVGEEGNE EGRTPHLQGFANFVKKQTFNKVKWYLGARCHIEKAKGTDQQNKEYCSKEGNLLMECGAPR SQGQRSDLSTAVSTLLESGSLVTVAEQHPVTFVRNFRGLAELLKVSGKMQKRDWKTNVHVI VGPPGCGKSKWAANFADPETTYWKPPRNKWWDGYHGEEVWIDDFYGWLPWDDLLRLC DRYPLTVETKGGTVPFLARSILITSNQTPLEWYSSAAVPAVEALYRRITSLVFWKNATEQSTE EGGQFVTLSPPCPEFPYEINYASHHHHHH (SEQ ID No. 5; with underlined Strep (N- terminus) and 6xHis (C-terminus) tags, and the linker region in bold).

[0188] 3.4. SDS-PAGE and Western blot analysis of PCV2a ORF1 (REP) protein expression

[0189] Protein expression and purification of the recombinant Strep-REP-6xHis protein is confirmed by conducting a Western blot analysis in any of these raw materials; E.co / / -based PCV2a REP isolated recombinant proteins, inactivated REP-positive E. coli cells and inactivated REP- positive E. coli cell lysates. Membranes are incubated for 2 h at room temperature with either anti-His monoclonal antibodies conjugated to horseradish peroxidase (1 :5000) as the primary and detection antibody, or anti-REP antibody (Thermo Fisher Scientific, 1 :5000) as the primary antibody followed by a 1 h incubation at room temperature with anti-IgG-HRP (Sigma-Aldrich, 1 :15000) as the detection antibody. The immunoreactive bands are visualized using an ECL kit for chemiluminescent detection.

[0190] The yield of the recombinant PCV2a Rep protein purified through IMAC from either soluble fraction (Figure 25) or inclusion bodies (Figure 25) was 2,10 mg PCV2a REP per 8,26 g cultured REP-positive E.coli. To improve this yield, optimization of the protein expression conditions, purification methods, and protein solubility enhancement techniques will be refined with the integration of the E. co / / -based GLP-validated platform (EIC TRIVAC Project©).

[0191] 3.5. ELISA-based potency assay for PCV2a ORF1 (REP)

[0192] Proprietary PCV2a ORF1 (REP)-specific ELISA-based potency tests are being conducted to ensure the immunization efficacy of in any of these raw materials; E.co / / -based PCV2a REP isolated recombinant proteins, inactivated REP-positive E. coli cells and inactivated REP- positive E. coli cell lysates.

[0193] 3.6. Production of monoclonal antibodies against PCV2a ORF1 (REP) recombinant protein

[0194] Monoclonal antibodies can be produced using the hybridoma method, (Kohler and Milstein 1975), or through recombinant DNA methods. (Cabilly, Heyneker et al. 1989) Recombinant PCV2a ORF1 (REP) protein purified from the soluble fraction will be administered intraperitoneally to three 3-month-old female BALB / c mice to elicit a targeted immune response. The immunization schedule includes an initial dose of 50 pg of PCV2a ORF1 (REP) combined with Freund's complete adjuvant, followed by two booster doses of 25 pg of PCV2a ORF1 (REP) with Freund's incomplete adjuvant, administered at 14-day intervals (new GMP- manufactured PCV2a REP protein).

[0195] To assess the immune response, blood will be collected 7 days after the third immunization for antibody titer determination via indirect ELISA. Prior to cell fusion, a booster dose of 25 pg of PCV2a ORF1 (REP) will be administered intraperitoneally to the selected donor mouse 4-5 days before spleen extraction. Spleen cells will be harvested 4-5 days after the booster dose and fused with SP2 / O-Ag-14 myeloma cells at a myeloma-to-splenocyte ratio of 1 :3, using polyethylene glycol (PEG 4000) as the fusing agent.(Goding 1996) Following cell fusion, the resulting hybridoma cells will be seeded in 96-well plates containing selective Hypoxanthine- Aminopterin-Thymidine (HAT) medium at a density of 2x105cells per well. The medium supports the selective growth of hybridomas by inhibiting the proliferation of unfused myeloma cells.

[0196] Hybridoma cultures will be screened for the production of monoclonal antibodies specific to the PCV2a ORF1 (REP) antigen using indirect enzyme-linked immunoabsorbent assay (ELISA) and capture ELISA with biotin-conjugated PCV2a ORF1 (REP), purified from the insoluble fraction through on-column refolding. Positive clones will be then selected and subjected to limiting dilution cloning to ensure monoclonality. This process will be repeated twice to confirm stable, antibody-producing clones. (Goding 1996) The selected antibody- secreted hybridomas will be cultured and expanded, and their supernatants will be collected for antibody purification. Monoclonal antibodies will be purified using pre-packed columns with sepharose coupled to protein G (Cytiva), to selectively bind and capture IgGs.

[0197] Alternatively, polyclonal antibodies specific to REP will be purified from mouse serum via affinity chromatography using a HiTrap™ protein A column. This approach selectively enriches the sample with mouse immunoglobulins, ensuring a higher concentration and purity of the target antibodies. The purified antibodies can be further characterized and utilized for potency tests, diagnostic or therapeutic applications.

[0198] 3.7. Production of polyclonal antibodies against F4 recombinant protein

[0199] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (s.c.) or intraperitoneal (i.p.) injections of the relevant antigen combined with an adjuvant. In this example, five female rabbits will be immunized against the PCV2a REP, designed to elicit a targeted immune response against Rep (new GMP-manufactured PCV2a REP protein). The immunization schedule consists of an initial injection of 250 pg of the PCV2a REP, emulsified in Freund's complete adjuvant, followed by three booster injections of 125 pg of PCV2a REP emulsified in Freund's incomplete adjuvant. The booster injections are administered intraperitoneally at 3-week intervals. Eleven weeks after the initial immunization, a 0.5 mL serum sample is collected to determine the antibody titer using ELISA. A final bleed is performed seven days later (week 12), yielding 40-90 mL of serum per rabbit.

[0200] Antibody titers will be assessed by indirect ELISA with the PCV2a REP, purified from the insoluble fraction through on-column refolding. Polyclonal antibodies will be purified from the collected serum using affinity chromatography with pre-packed columns containing sepharose coupled with Protein A for IgG purification and / or pre-packed columns with NHS-activated sepharose coupled to PCV2a REP, to capture specific immunoglobulins against the Rep protein.

[0201] 3.8. Proof of concept PCV2a-REP-based immune response for PCVAD

[0202] ORF1 PCV2a-REP recombinant soluble & refolding protein from REP-positive E. coli have been proven to be stable from -4 to -80 °C (manufacturer's internal report, data not shown). Currently, recombinant ORF1 PCV2a-REP soluble & refolded proteins are being studied to assess their galenic compatibility with the same immunogenic composition and its pharmaceutically acceptable excipients or carriers. Previous data indicates that the immunologically effective dose of recombinant ORF1 PCV2a-REP soluble & refolded proteins may be comparable to those required for a protective immune response against PCV2b CAP. Ongoing studies in pig models are focused on determining antibody production, protective dynamics and the overall immunological response to the REP antigen.

[0203] EXAMPLE 4. Proof of concept for TRIVAC immunological protective-related response comparing with market-leader commercial vaccines in piglets

[0204] Efficacy is defined for this invention as the ability to produce a desired or intended immunological response in the host of a cellular or antibody-mediated type upon administration of the present invention, at least but not limited to have equivalent or even superior levels of the immune response considered as protective in bioequivalent commercial products or similar antigens. Efficacy as bioequivalent immunogenic response has been considered the most appropriate strategy to test author's hypothesis & invention avoiding unnecessary suffering to animals, keeping challenge test, based on intended pathogenic infections to deliberately cause disease, when had enough direct evidence to justify pathogen iatrogenic exposition of pigs. (Smith, Glutton et al. 2017) Several previous studies with the active principles comprising the invention have been settled to determine immunologically effective dose of each immunogenic composition. However, no previous data exist about specific, total or partial, combinations of the active principles comprising the invention.

[0205] 4.1. TRIVAC® partial (non-galenized) Proof of Concept (PoC): immune response dynamics

[0206] Two dosing experiments to demonstrate TRIVAC-associated immune dynamics compared to protective-related commercial immune response have been performed in piglets (ref. PI38 / 24). A single dose protocol (A) and a one-dose (primary dose) coupled to a 15-days-separated two- dose (booster dose) immunization protocol (B), both composed of recombinant E. co / / -based PCVAD-related antigens (100 pg / mL dose) and Gsuis antigen (50 pg / mL dose) (GLP-batch, WG2408350, TRIVAC®), formulated with light liquid paraffin base, were compared to conventional immunization protocol of the Spanish market leader- vaccines combination for PCVAD & Mhyo [Porcilis® PCV (Porcine circovirus type 2 ORF2 subunit antigen > 2828 All*) M Hyo > 2,69 UPR (Intervet)] & Glasser's disease [HIPRASUIS® GLASSER (inactivated Glaesserella parasuis (aka Haemophilus parasuis, serotype 1 > 1 / 16 MAT*) (Hipra)]. Commercial vaccine combination was chosen after direct interviews with Heads of veterinary clinical practitioners of four European companies which manage 17 M pigs / year in total. Interviews were done between June 2022- September 2023.

[0207] Fifteen PCVAD & Gsuis- seronegative 8-days-old minimal disease (MD) piglets were housed at Centre of Biomedical Investigations of Aragon (Zaragoza, Spain) in biosafety level 2 conditions. Three experimental groups were defined as follows: (1) Commercial, composed by a vaccine combination of single-shoot treatment of Porcilis PCV2 Mhyo (2 mL / dose) + HIPRA suis (2 mL / dose), the most recommended treatment for clinical practitioners in moderate to sub-clinical outbreak of GD and / or PCVAD; (2) Protocol A, composed by a single-shoot treatment of TRIVAC PoC [E. co / / -based PCVAD-related antigens (100 pg / mL dose) and Gsuis antigen (50 pg / mL dose)] (2 mL / dose) (as bioequivalent PoC); and (3) Protocol B, composed by a two-shoot (primary + booster) treatment of TRIVAC PoC (as previously defined) (modelling most followed prescriptions of clinical practitioners when faces a severe or acute outbreak of GD and / or PCVAD). Serum samples were taken in 0 (primary dose, all groups), 15 (booster dose, protocol B), 30 and 45 days after immunization. Direct ELISA determination for Glaesserella parasuis and Porcine circovirus type 2 ORF2 subunit antigen (genotype b) was performed. Absorbance of polyclonal anti-rabbit porcine IgG (horseradish peroxidase (HRP), Sigma-Aldrich, Spain) (1 / 400) was measured. Blocking was done with bovine serum albumin (BSA). Coating of the plates were performed with VtaA9-F4 (1 / 400) (autotransporter protein fragment of group 1 < Pan- Glaesserella>) & PCV2b Cap virus-like particles (VLPs) (1 / 400) (same active principle of commercial vaccine) (both manufactured by WorldPathol Manufacturing Ltd., Spain).

[0208] Commercial group shows a minimal increase of immune response in day 15 following for a continuous decrease of signals for Cap antigen from day 30 onwards. A slight trend but no significant increase of signal for Gsuis antigen was observed in commercial group. Protocol A group (single-shoot PoC TRIVAC) was enough to obtain a 2-fold increase of immune response in both antigens, PCV2b-Cap (as marker for TRIVAC®, main active principle of this partial TRIVAC PoC) & VtaA9-F4 (pan-G / aessere / / a), by day 15, comparing to commercial treatment. Protocol B group (two-shoot PoC TRIVAC) obtained a 3-fold increase of immune response in both antigens by day 30, comparing to commercial treatment. Both dosing treatments have indicated a robust superior immune response in conditions of bioequivalent protective-related positive-immunizing commercial dosing (Figure 26).

[0209] As we stated in description of prior art, homologous neutralizing antibodies are essential in protecting against GD (Nedbalcova, Kucerova et al. 2011 , Costa-Hurtado, Ballester et al. 2012). VtaAs are outer membrane proteins that have a differential distribution between virulent and non-virulent strains of Gsuis. While group 3 VtaA are present in all Gsuis strains, group 1 and 2 VtaA are found only in virulent strains. (Bensaid, Pina-Pedrero et al. 2008, Olvera, Pina et al. 2010, Olvera, Pina et al. 2012, Olvera, Martinez-Molineret al. 2013, Galofre-Mila, Correa- Fiz et al. 2017). VtaA-F4 fragment is a protective immunogen, since it is exposed on the surface of the bacterium and is common to all VtaA of group 1 (virulent strains). (Bensaid, Pina- Pedrero et al. 2008) Highly virulent Gsuis strains bases GD pathology on the nostril-to-lung progression, driven by autotransporter proteins of group 1. (Aragon, Segales et al. 2019) Therefore, protection against these antigens has to be achieved to protect pigs against such nostril-to-lung progression. High-pathogenic whole-bacteria-based commercial treatment achieved a slight increase signal after immunization of Gsuis antigen (less than 0.5-fold), antigen marking this corresponding special protection against autotransporter proteins (Figure 27). However, dose-dependent immune response of partial TRIVAC PoC confirms robustness and protection-related virulent-homologous significant higher antibody production.

[0210] As we stated in description of prior art, PCV2 stands out as the primary causative agent of PCVAD, encompassing a spectrum of clinical conditions. These include post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, and subclinical infections (Segales 2012). All four PCVs are similar in structure: they contain two main open reading frames (ORFs) oriented in opposite directions in the circular genome; the ORF1 or rep gene encodes for two pivotal proteins associated with replication, replication initiator protein (Rep) and Rep’, and the ORF2 or cap gene encodes for the capsid (Cap) protein. (Cheung 2012, Opriessnig, Karuppannan et al. 2020) Experimental data demonstrated the cross-protection of PCV2a vaccines against the most widespread genotypes (PCV2a, PCV2b, and PCV2d). Therefore, despite the significant number of genotypes described / proposed, it seems one single PCV2 serotype would exist so far.(Franzo and Segales 2020) PCV2-infected pigs developed cell-mediated immunity to CAP and REP proteins and that, in the course of a sub-clinical infection, development and strength of such responses are possibly related to the levels of PCV2 replication. (Fort, Sibila et al. 2010) Any PCV2b ORF 2 (CAP) or PCV2a ORF1 (REP) are suitable to be used in the invention as functionally exchangeable PCV-related strong immunity developers in pigs. In the beginning of PCV2a-related PCVAD epidemics (Early’s 2000) two-shoot treatments were administered to achieve immunological protection. (Segales 2015) Afterwards, conventional expected immune response for PCV2-based Cap protein were practically standardized by several products, leading to different situations and vaccination practices related with clinical or sub- clinical status. (Segales 2015) However, reported field failures of PCV2-related vaccination protocols and new PCV strains emergence postulates the need of a better single or with booster pan-PCV treatment. The PCVAD-related most recommended preventive treatment for clinical practitioners tested in this experiment elicits a slight increase of PCV2-related neutralizing antibodies. It is worthy to mention that ELISA used in this experiment wants to determine a measurement of all antibodies linking to the antigen, both mono and polyclonal. Partial TRIVAC PoC treatments elicited a 2 (single-shoot) to 3-fold higher (two-shoots) increase of immunological effective amounts of antibodies.

[0211] The results might indicate potentially two non-exclusive situations; (1) TRIVAC® proof-of- concept (PoC) has been overdosed, therefore, we will obtain more doses per manufactured mL after adjustment; and / or (2) TRIVAC® PoC has 2 (one dose) to 3-fold (two doses) higher immune response of market leader counterpart, leading towards a more efficient & efficacy immunological composition. Authors hypothesize that these TRIVAC-related higher signals will ensure wider longer pan-PCV pan-G / aessere / / a protection even in a single-shoot, comparing with an, otherwise, standard well-known clinically efficient products. Such robust immune response should prevent vaccination failures even in a single-shoot vaccination program. Special formulation of the immunogenic composition comprising carefully selected antigens, such as VtaA9-F4 Gsuis fragment and PCV2b-CAP and PCV2a-REP proteins, might induce potent immune responses in pigs compatible with heterologous cross-protection against pathogenic strains of GD and PCVAD.

[0212] 4.2. Challenge test to assess clinical efficacy beyond immunological response

[0213] Protection against a virulent form of the pathogen is necessary to define the level of immunity in order to assess clinical efficacy. (Segales 2015) Protection against challenge has been used by competitor products to assess and define such clinical efficacy and immunity. (EM EA 2009, AEMPS 2014, Medicines 2017) Although efficacy is defined for this invention as the ability to produce a desired or intended immunological response in the host of a cellular or antibody- mediated type upon administration of the present invention, at least but not limited to have equivalent or even superior levels of the immune response considered as protective in bioequivalent commercial products or similar antigens. Once this target has been demonstrated, a challenge-based approach is being used for further validation of superior immunological protective-related observed response. (Smith, Glutton et al. 2017)

[0214] Challenge protocol for PCVAD and GD can be particularly difficult given the abundance of different serotypes. Virulent PCV2 viruses shall be obtained from most common field isolates at the moment of experiment or from European culture collections, and grown in vitro using a porcine kidney cell substrate, and traditional virological methods. High virulent G. parasuis shall be similarly obtained and grown in culture medium. Virus and bacterial stocks shall be titrated, and graded doses of each pathogen shall be administered to different groups of minimum age pigs (10 pigs per group). The pigs will be observed for overt clinical signs (primary indicators), as well as gross pathological lesions (e.g. lung lesions, lymph node pathology (secondary indicators) and virus / bacterial isolation and histopathology (tertiary indicators). The doses that give clinical signs typical of those seen in the field, with consistent secondary and tertiary indicators of infection will be chosen for challenge trials. If 5-10 groups of pigs are used for each pathogen and 2 doses are tested (“High” and “low”), the total number of pigs will be between 20 & 40 piglets. These piglets must be PCV, and G. parasuis free prior to infection, and have not been vaccinated. The order in which trials shall be done will be (a) Gsuis & (b) PCV2b.

[0215] EXAMPLE 5. Differentiating Infected from Vaccinated Animals (DIVA)-technology based diagnostic kit related to the immunogenic composition

[0216] 5.1. Design of TRIVAC® DIVA IC Test The invention presents a novel and efficient DIVA (Differentiating Infected from Vaccinated Animals) strategy utilizing a rapid immunochromatographic test. This innovative method allows for the distinction between vaccinated and naturally infected populations in under 15 minutes, directly at the pig farm, offering significant advantages over currently available products. Upon vaccination with the TRIVAC formulation, pigs produce antibodies against the F4 antigen of G. su / s, without eliciting an immune response to other VtaA9 epitopes typically associated with natural infections. Additionally, the incorporation of a Strep-tag at the C-terminal end of the REP protein further refines the differentiation capability. This specific modification enables the rapid immunochromatographic test to precisely recognize antibodies targeting the Strep- tagged REP protein, which are exclusively generated in vaccinated animals. Since these antibodies are absent in naturally infected animals, the test can unambiguously distinguish between vaccination-induced immune responses and those resulting from natural infection, ensuring a higher level of accuracy in identifying the immunological status of the animals.

[0217] TRIVAC device technology is based on lateral flow test (LFT) assay (aka, lateral flow device (LFD), lateral flow immunochromatographic assay, or rapid test) (Figure 28).(Koczula and Gallotta 2016) LFTs operate on the same principles of affinity chromatography as the enzyme- linked immunosorbent assays (ELISA). In essence, these tests run the liquid sample along the surface of a pad with reactive molecules that show a visual positive or negative result. The pads are based on a series of capillary beds, such as pieces comprising at least but not limited to porous paper, micro structured polymer, or sintered polymer. Each of these pads has the capacity to transport fluid (e.g., urine, blood, saliva) spontaneously. On the basis of this invention, specific VtaA9-F4 & PCV2a-REP antibodies in blood is the preferred fluid to measure. The sample pad acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid flows to the second conjugate pad in which the manufacturer has stored freeze dried bio-active particles called conjugates in a salt-sugar matrix. The conjugate pad contains all the reagents required for an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., antibody) that has been immobilized on the particle's surface. This marks target particles as they pass through the pad and continue across to the test and control lines. The test line shows a signal, often a color as in pregnancy tests. The control line contains affinity ligands which show whether the sample has flowed through and the bio-molecules in the conjugate pad are active. After passing these reaction zones, the fluid enters the final porous material, the wick, that simply acts as a waste container. LFTs can operate as either competitive or sandwich assays.

[0218] This rapid immunochromatographic test, based on blood samples obtained through puncture, enables the detection of antibodies derived from vaccination versus those from natural infection in less than 15 minutes. This capability provides an enhanced tool for surveillance and control in herd management, allowing for effective outbreak tracking, verification of vaccination success, and the implementation of targeted strategies for controlling G. suis & PCV infections.

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Claims

CLAIMS1. An immunogenic composition comprising one or more recombinant E. co / / -based antigens of each of:- a trimeric autotransporter fragment of protein 9 (VtaA9) of Glaesserella parasuis,- a porcine circovirus type 2 genotype b (PCV2b) Open Reading Frame (ORF) 2 CAP protein and,- a porcine circovirus type 2 genotype a (PCV2a) Open Reading Frame (ORF) 1 REP protein.

2. The immunogenic composition according to claim 1 , wherein the amount of the one or more recombinant E. co / / -based antigens of trimeric autotransporter fragment of protein 9 (VtaA9) of Glaesserella parasuis is from 0,5 pg to 200 pg of the whole composition.

3. The immunogenic composition according to any of claims 1 or 2, wherein the amount of the porcine circovirus type 2 genotype b (PCV2b) ORF2 CAP protein is from 0,1 pg to 200 pg of the whole composition.

4. The immunogenic composition according to any of claims 1 to 3, wherein the amount of the PCV2a ORF 1 REP protein is from 0.1 pg to 200 pg of the whole composition.

5. The immunogenic composition according to any of claims 1 to 4, further comprising at least one pharmaceutically acceptable excipient and / or carrier.

6. The immunogenic composition according to any of claims 1 to 5, for use as a medicament or as a combination and / or associated vaccine in livestock.

7. The immunogenic composition according to claim 6, for use in the prevention and / or treatment of porcine enzootic pneumonia and / or Porcine Circovirus-Associated Diseases (PCVAD) and / or Glasser's disease in livestock.

8. The immunogenic composition for use according to claim 6 or 7, wherein the livestock is porcine.

9. The immunogenic composition for use according to any of claims 6 to 8, wherein when used as a combination and / or associated vaccine, the vaccine further comprises one or more additional antigens from a microorganism selected from the group consisting of Actinobacillus sp., Brachyspira sp., Pasteurella multocida, Salmonella sp., Streptococcus sp., Isospora sp.,Erysipelothrix rhusiopathiae, Leptospira sp., Staphylococcus sp., Bordetella bronchiseptica, Clostridium sp., Mycoplasma sp., Lawsonia intracellularis, Escherichia coli, porcine reproductive and respiratory syndrome virus (PRRS), swine influenza virus, contagious gastroenteritis virus, porcine parvovirus, encephalomyocarditis virus, coronavirus, rotavirus, porcine periweaning failure to thrive syndrome agent, classical swine fever virus, African swine fever virus, calicivirus, transmissible gastroenteritis coronavirus (TGEV), porcine epidemic diarrhea virus (PED) and torque teno virus (TTV) and combinations thereof.

10. The immunogenic composition for use according to any of claims 6 to 9, wherein when used as a combination and / or associated vaccine, the vaccine further comprises an adjuvant.

11. The immunogenic composition for use according to claim 10, wherein the adjuvant is an adjuvant emulsion comprising mineral oil and an additive selected from a hydrophobic additive, an amphiphilic additive and combinations thereof.

12. An immunogenic diagnostic kit for discriminating naturally infected from immunized animals comprising:(a) an immunogenic composition as defined in any of claims 1 to 5;(b) monoclonal and polyclonal antibodies of VtaA9-F4 of Glaesserella parasuis, and(c) optionally, monoclonal and polyclonal antibodies of PCV2a REP,(d) optionally, instructions for its use.

13. The immunogenic diagnostic kit according to claim 12, wherein said animals are porcine livestock.

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