Corynomicolic acids

Corynomicolic acids are part of the outer layer of the cell wall of the bacterium, which constitutes a protective and permeable barrier. The binding of these to trehalose molecules leads to the formation of curved structures, which blocks the access of molecules (antibiotics or lysozymes) to the peptidoglycan, conferring mechanical protection^30,31. Unlike the linear fatty acids of phospholipids, corynomicolic acids are 𝛽-hydroxy-branched fatty acids, which require carboxylation and condensation of two fatty acids for synthesis. The AccD2 and AccD3 enzymes are carboxylases widely conserved in the family Corynebacteriaceae, involved in the generation of intermediates for the synthesis of corynomicolic acids. Other enzymes involved in synthesis are AccD1, for the elongation of the mycolic acid chain, and FadD, an AMP ligase²⁹. Inoculation of corynomycolic acids in sheep showed that haptoglobin (Hp) increased its concentration three times, as well as twice the levels of serum amyloid A (SAA), proteins that indicate inflammation and acute infections⁴². These results indicate their virulent potential, since on their own, they are able to induce reactions of inflammation in the host, which contributes to the formation of granulomas.

Phospholipase D, PLD

The Phospholipase D exotoxin is considered the main virulence factor of C. pseudotuberculosis. The pld gene was identified and sequenced in 1990, is part of the pathogenicity island PiCp1 and encodes a 31.4KDa protein⁴³. Comparison of the sequence of the PLD protein of C. pseudotuberculosis revealed that it has greater similarity with Phospholipase A2; however, PLD does not belong to the family of phospholipases because it lacks the HKD motif conserved in this family⁴³. PLD is classified as a Sphingomyelinase D (SMasaD; EC 3.1.4.41), also known as sphingomyelin phosphodiesterases D or phospholipase D (PLD), which catalyzes the hydrolytic cleavage of sphingomyelin to produce choline and ceramide 1-phosphate or choline and lysophosphatidic acid (LPA)⁴⁴. Compounds derived from sphingomyelin degradation cause platelet aggregation, endothelial hyperpermeability, and pro-inflammatory responses⁴⁵. The enzymatic action of PLD hydrolyzes sphingomyelin, the main component of cell membranes, which leads to an alteration of the morphology of the target membrane. PLD contributes to the spread and persistence of the bacterium inside macrophages⁴⁶. The pld gene presents a widely conserved sequence in the strains of C. pseudotuberculosis, and when this is modified, the ability to produce the disease is hindered⁴¹.

Endoglycosidase, CP40

The CP40 protein is encoded by a gene with an open reading frame of 1,137 bp, which is located downstream of the pld gene in PiCp1. It was described as an enzyme with serine protease activity⁴⁷, but in another study, the analysis of the sequence allowed its grouping closer next to endoglycosidases and further away from serine protease sequences⁴⁸. In this work, it was proposed that its enzymatic activity is endoglycosidase, mediator of the hydrolysis of glycosidic bonds, proteins of the GH18 family, similar to the EndoE domain belonging to Enterococcus faecalis. GH18 enzymes contain a conserved consensus sequence motif (LIVMFY) - (DN) -G- (LIVMF) - (DN) - (LIVMF) - (DN) -X-E, where terminal glutamic acid is essential for enzymatic activity. By aligning the GH18 active site in CP40, its similarity to the EndoE and EndoS enzymes was established, only with changes in one or two amino acids respectively. The function as a virulence factor has been associated with the demonstrated ability in vitro to degrade the Fc region of IgG antibodies. CP40 endoglycosidase does not hydrolyze glycans in bovine and caprine IgG, while sheep IgG is partially hydrolyzed and equine IgG completely. The analysis was also performed with subclasses of human IgG, presenting activity in all and partially in IgG4. There was no detectable enzymatic activity in other glycoproteins, including some of the other immunoglobulin isotypes (IgA, IgD, and IgE)⁴⁸.

Secreted proteins PLD and CP40

According to various reports, proteins exported or secreted by bacteria are actively involved in the infection process⁴⁹. For this reason, the expression and secretion of the PLD and CP40 proteins have been highly studied. The development of an experimental infection showed by immunoblot that the production of antibodies was directed 88 % to the recognition of proteins of 30-31Kda (PLD) and in 75 to 88 % towards proteins of 38-41KDa (CP40), range in which are both proteins⁵⁰. The attenuated strain 1002, after several passes in a mouse model, was able to reverse the virulence. The analysis by mass spectrometry allowed the identification of the PLD and CP40 proteins only in the reactivated strain 1002, which indicates the participation of these proteins in the virulence⁵¹. On the other hand, through real-time PCR, the expression of several genes involved in virulence was identified in vitro and in vivo, including pld and cp40. This analysis made it possible to verify that, in the strains isolated from lymph nodes, the expression of these genes was higher compared to the strains obtained from in vitro cultures⁵².

Virulence factors involved in iron acquisition

In PiCp1 is the operon (fag ABCD)²⁹, composed of four genes, fagA, fagB, fagC, fagD, that are located downstream of the pld gene. These genes respectively encode an integral membrane protein, an iron-transporting enterobactin, an ATP-binding cytoplasmic membrane protein, and an iron-binding siderophore protein. FagA was identified as a membrane-associated protein with pathogenic potentialities, by mass spectrometry analysis of proteins expressed in a strain of C. pseudotuberculosis grown with animal serum⁵³. The culture of C. pseudotuberculosis in media with iron chelators (dipyridyl) caused the decrease in a logarithm order in the count of colony-forming units (CFUs), compared to bacteria grown in iron-enriched media. The evaluation of the transcriptional response of C. pseudotuberculosis, with iron restriction, allowed identifying the negative regulation of genes involved in the energy metabolism of the Krebs cycle (sdhC, sdhB, lpd), ATP production (atpF, atpH), pyruvate metabolism (lpd), oxidative phosphorylation (qcrC, qcrA, qcrB, ctaC, ctaF, ctaE, ctaD), ribosome processes, transport (rplJ, rplL, rplM, rpmA, rpsC, rpsI, rpsL, rpsM) and EF-G and EF-Ts elongation factors associated with the translation of mRNA (fusA, tsf). The gene analogous to dtxR of C. diphteriae was identified, with 79 % similarity in sequence, which encodes an iron-binding dependent protein, which acts as a regulatory factor for more than 40 genes⁵⁴. In PiCp3 and PiCp4, the genes belonging to the operon ciuABCDE involved in iron absorption, transport and biosynthesis of siderophores have been studied²⁹.

TetA Protein

In PiCp2, the tetA gene encodes a tetracycline efflux transporter protein that protects against the action of this antibiotic and confers resistance to the bacterium. The tetA gene is often carried by transmissible elements such as plasmids, transposons and integrons and has been identified in C. pseudotuberculosis²⁹.

Virulence factors for macrophage infection

On the pathogenicity island PiCp2 are the potG, sigK and dipZ genes, which respond to the mechanisms responsible for the intramacrophagic lifestyle of C. pseudotuberculosis. The potG gene encodes an ATP-binding membrane protein that provides energy for absorption of putrescine (polyamine) from the periplasmic space, it functions as a putrescine transporter system²⁹. Putrescines are polyamides produced by macrophages that induce decreased production of reactive nitrogen species and synthesis of pro-inflammatory cytokines. The dipZ gene has been identified in the phylum Actinobacteria, is regulated by sigK⁵⁵ and is activated during macrophage infection by Mycobacterium tuberculosis. In PiCp4, the presence of the gene that encodes the Sigma factor confers the ability to protect against oxidative stress, specifically against the action of nitrogen intermediate products, produced by macrophages⁵⁶. The FCR41 strain of C. pseudotuberculosis was used in the study of genes related to pili synthesis. The structure of the pili is composed of the major pili SpaA and SpaD; the minor pili SpaB and SpaE; and the pili type, SpaC, SpaF. A complete pili structure or even the minor pili can make initial contact with host cell receptors to facilitate the entry of microorganisms. In this strain, the spaC gene was identified, which encodes a protein responsible for anchoring the pili to the cell wall, which can allow initial contact with the cell receptors, to then facilitate intracellular invasion. It also presented the namH gene, which encodes an extracellular neuraminidase, which catalyzes the elimination of sialic acid groups present in a wide variety of glycoconjugates of the extracellular matrix of the host cell, which favors adherence to cells. In FCR41, the sodC gene was detected, which encodes a superoxide dismutase, an enzyme anchored to the membrane with extracellular domain, which eliminates oxygen free radicals, products of respiratory burst in macrophages⁵⁷.

Resistance and adaptation virulence factors

Chaperone proteins (HSPs) are highly conserved and are expressed under heat stress, as well as nutrient reduction, hypotaxia, breakdown of metabolism and other cellular processes. In C. pseudotuberculosis, Hsp10 (groES) and Hsp60 (groEL)⁵⁸ have been studied. In strain 1002, resistance to various types of abiotic stress, such as acidity, high temperatures and osmotic stress, associated with the presence of these proteins, was evaluated⁵⁹. The hspR gene encodes the regulatory factor of the expression (repressor protein) of the operon with the genes dnaK, grpE, dnaJ and hspR, which encode heat shock proteins. In the absence of stress, the HspR protein attaches to an inverted repeat sequence that represses the promoters responsible for controlling the expression of the Hsp operon. In another study, the separation of proteins by two-dimensional electrophoresis allowed the identification of 11 new extracellular proteins, 3 with unknown functions and 8 related to the elongation factor Tu, GroEL (HSP60), enolase, glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase (SodA), which depend on a non-classical secretion method via SecA. Both SecA genes (SecA1 and SecA2) were identified in the strains studied, possibly involved in the secretion system of C. pseudotuberculosis⁶⁰. The study of the virulence factors of C. pseudotuberculosis as candidate molecules for the development of more powerful and effective vaccines is still being continued.

Inactivated vaccines and toxoids

Inactivated vaccines are composed of non-viable whole cultures of the bacterium or inactivated toxins, either by chemical or physical methods. In these formulations, the microorganism is dead, so they do not confer danger of development of the disease; however, the response is mainly humoral, less intense, requires high concentrations of the microorganism and several doses. They are not subjected to any purification procedure, so they contain all the biochemical components of the bacterium, which are more reactogenous and can produce adverse effects. The protein precipitate of a strain of C. pseudotuberculosis isolated from alpaca in Peru was evaluated in a group of 20 BALB/c mice, where it induced protection from challenge with 10⁴ CFUs of a virulent strain of C. pseudotuberculosis. The vaccine reduced the toxic effects caused by the bacterium, which was observed with the decrease in the number and size of abscesses in the animals of the vaccinated group (40 % affected) compared to the multiple abscesses of greater size at the subcutaneous level and in kidney and liver in the animals of the control group (95 % affected)⁷⁶.

Vaccine formulations based on 250 - 500 mg/ml of cell wall and 133-265 mg/ml of PLD toxin, all supplemented with 20 mg/ml of muramyl dipeptide as an adjuvant, were evaluated in alpacas, subjected to the challenge with 10⁶ CFUs of a virulent strain of C. pseudotuberculosis. The animals vaccinated with the highest dose of PLD did not show abscesses, unlike the vaccinated group with the lowest concentration, where the formation of abscesses at the inoculation site and in renal lymph nodes was observed. Formulations that included cell wall showed a lower degree of protection, with the formation of superficial and internal abscesses. The results suggest that the concentration of PLD toxin may influence the protective capacity, being dose dependent on the immunization of alpacas⁷⁷.

The degree of protection conferred by vaccination with PLD toxin adsorbed in aluminum hydroxide gel (Group 1) or its combination with toxins of Clostridium perfringens D, Clostridium novyi B, Clostridium tetani, Clostridium septicum and Clostridium chauvoei (Group 2), as well as toxins of Clostridium spp., PLD and 1.2 mg/ml of sodium selenate (Group 3); it was evaluated in an ovine model, challenged with 1.3 x 10⁸ CFU of a virulent strain of C. pseudotuberculosis. The percentage of affected animals in group 1 was 10.5 % (4 of 38 animals showed a superficial lesion), for group 2 7.9 % (3 of 38 animals showed a superficial lesion) and 8.3 % in group 3 (2 of 24 animals, each with a superficial lesion and at the lung level), in contrast to the results obtained in the control group of unvaccinated animals with an impact of 51.5 % (17/33), with 60 lesions at the lung level and 16 in carcasses, with 4.5 lesions per animal. The results indicated that there was no reduction of protective potency as a result of the combination of PLD with Clostridium antigens⁷⁸.

The efficacy of four non-commercial vaccines based on PLD as an antigen was evaluated in sheep challenged with a virulent strain. The levels of superoxide ions were determined as a non-specific immune response, these being elevated in the group vaccinated with PLD + inactivated bacterium, followed by the group vaccinated with PLD Toxoid. Lysozyme activity was higher in the group vaccinated with PLD + inactivated bacterium, followed by PLD Toxoid, PLD + commercial Covexin8 vaccine and a local experimental vaccine. The group vaccinated only with PLD showed a marked positive response of lymphocyte proliferation compared to the rest of the groups. The results indicated that PLD stimulated the specific and non-specific cellular immune response⁷⁹.

Four different antigenic extracts obtained from the attenuated strain T1 were evaluated in goats of Canindé breed, for the study of the humoral and cellular immune response. Animals in group 1 (immunized with 0.5ml of the culture supernatant of the T1 strain in 1:1 ratio with Freund’s incomplete adjuvant, FIA) and group 3 (immunized with 100 mg of extracellular concentrate, 250 mg of CpG oligodeoxynucleotides and 0.5 ml of adjuvant FIA) showed the highest levels of antibodies and IFN-ɣ after immunization, as well as post-challenge with 10⁵ CFUs of the virulent strain VD57, compared to groups 2 (1ml of suspension of 2 x 10⁶ CFU/ml strain T1), group 4 (formulation of group 3 without FIA) and control group. Only 25 % of the animals in group 1, 33.3 % in group 2 and 22.2 % in group 3 did not develop lesions, in group 4 and 5, 100 % of the animals developed some type of lesion⁸⁰. These results show that the production of specific anti-PLD antibodies only decreases the spread of the bacterium and the appearance of lesions in tissues other than the inoculation site, not controlling the infection. Bacterin or toxoid vaccines contribute to reducing the clinical manifestations of the disease, being more efficient in sheep than goats, although in neither species is it possible to control the infection, leaving a percentage of affected animals that can spread the disease.

Attenuated vaccines

Attenuated vaccines have live immunizing agents that can replicate in the body without causing disease, since they lack certain structures or molecules that decrease their virulence. In principle, they confer a very intense and long-lasting immune response, since they give rise to an infection similar to the natural one, but they constitute a risk, since in some cases the virulence can be reversed. The first experimental attenuated vaccines for CLA used a strain called Toxminus, whose pld gene was modified by site-specific mutation. The necropsy of the animals vaccinated with 10⁵ to 10⁷ CFUs of the attenuated strain Toxminus allowed observing that no abscesses formed in the animals challenged with 10⁶ CFUs of a virulent strain, compared to the control group where abscesses of 2.5 cm developed in popliteal ganglia. However, the vaccine produced an undesirable abscess at the inoculation site, the antibody titer in the groups vaccinated with 10⁵ to 10⁷ CFUs was similar, so the response was non-dose dependent and the virulent challenge strain induced a superior antibody response at weeks 5 and 9. There was also a reduction in the ability of the Toxminus strain to remain in the host, due to the absence of PLD, an antigen that favors persistence, in addition to highly activating the humoral immune response⁸¹.

In another study, the Toxminus strain was transformed with a plasmid containing the pld gene modified to obtain the exotoxin with a change of histidine for a tryptophan at position 20, which eliminates enzymatic activity. Immunization with the orally administered Toxminus strain induced a predominant humoral response of IgG1 type, while IgG2 isotype levels were higher in subcutaneously vaccinated sheep. Th1 cells are responsible for cell-mediated cellular immunity, they produce IFN-ɣ, IL-2, and tumor necrosis factor beta (TNF-β), cytokines that activate macrophages and complement the activation of B lymphocytes to produce antibodies of the IgG2 isotype. On the other hand, Th2 clones secrete IL-4 and preferably induce the production of IgG1, IgA and IgE in B cells. Consequently, with the results obtained, the orally vaccinated animals did not present significant levels of protection, because the increase in IgG1 is indicative of the absence of a Th1 response, which is essential for the activation of cytotoxic T cells, essential for the elimination of intracellular pathogens, such as C. pseudotuberculosis. The incidence and degree of abscess formation were very low (abscesses of 0.2 cm and 1 cm), occurring only in two animals at the site of inoculation of the virulent strain for the challenge. In the group of animals vaccinated with the non-modified Toxminus strain, 50 % of the sheep developed abscesses, as well as 66 % of animals in the unvaccinated control group. The Toxminus strain did not allow for elevated PLD expression and evidence of the excretion of the live attenuated bacterium through feces was found⁸².

The CZ171053 strain mutated in the ciuA gene, by means of the transposon-TnFuZ system, presented a reduced ability to survive in vitro within the macrophages of the J774 cell line. The immunization of BALB/c mice with this attenuated strain allowed the survival of 80 % of the animals challenged with 10⁶ CFUs of the virulent strain MIC-6. These results suggest that the CZ171053 strain could be evaluated as a live attenuated vaccine in the target hosts of the disease.

The behavior of the humoral and cellular immune response was evaluated in BALB/c mice inoculated with 10⁷ CFUs of the attenuated strain T1. An increase in the titer of of IgG1 and IgG2 was observed, no lesions characteristic of the disease were shown and the culture of spleen cells, stimulated in vitro with antigens secreted by T1, presented a greater proliferation compared to cells stimulated with intracellular antigens⁸³.

DNA vaccines

Advances in molecular biology techniques have allowed the development of new generation vaccines, among which are naked DNA vaccines. These vaccines are only DNA (plasmids with the genes of interest), they do not have envelopes or protein structures, so the route of administration, the dose, and re-immunization are very important, since they are factors that influence the potency and type of immune response. The disadvantage of this type of vaccine lies in the ability to express the antigen of interest, since in most cases, the particles have a low adsorption and the quantity of plasmids that are introduced into the cells is limited.

The design of a plasmid carrying the gene encoding the extracellular domain of bovine CTLA-4 fused to the inactivated pld gene (boCTLA-4-HIg-ΔPLD) was evaluated as a naked DNA vaccine in sheep. CTLA-4 binds with high affinity to B7 membrane antigen in antigen-presenting cells (APCs), improving the humoral immune response. Although the antibody titer increased significantly, the protection of immunized sheep was partial against the experimental challenge with a virulent strain⁸⁴. Different immunization routes were evaluated: intramuscular, subcutaneous and gene gun bombardment. The maximum levels of total IgG antibodies were 12x10³ in the group vaccinated intramuscularly, while in the groups with the other routes of administration, values of 3-4 x 10³ were reached. The protection conferred by the intramuscular vaccine was 45 % (9 of 20 animals), compared to the rest of the groups (subcutaneously and with gene gun) that only protected 10 % of the animals⁸⁵.

The potential of a DNA vaccine formulated based on the pTARGET plasmid transformed with the protein esterase cp09720⁸⁶ was compared with a subunit vaccine with recombinant CP09720 adjuvated with aluminum hydroxide. Both were evaluated in BALB/c mice, with the recombinant protein vaccine being the one that induced the highest titer of IgG1 and IgG2 antibodies. The two vaccines were able to increase IFN-γ expression, although the subunit vaccine presented the highest levels of IFN-γ mRNA. Protection levels against the challenge were 58.3 % in animals vaccinated with recombinant esterase, while the pTARGET DNA/protein esterase cp09720 vaccine only protected 16.6 %.

Recombinant protein subunit vaccines

Subunit vaccines combine antigens such as lipopolysaccharides, recombinant proteins, or synthetic peptides. These vaccines are very safe, but not very immunogenic, so adjuvant substances that enhance the response of the immune system are used. The PLD protein obtained by recombinant route has been one of the most used for the development of subunit vaccines. A group of researchers from the United Kingdom determined the potentialities of a vaccine from 50 μg of PLD obtained recombinantly (PLDr) in E. coli and its combination with 1.25×10¹⁰ cells/ml of whole cultures of C. pseudotuberculosis inactivated with formalin. In this work, the control group was vaccinated with the commercial Glanvac 3 vaccine (Commonwealth Serum Laboratories (CSL) Ltd., Victoria, Australia). The highest levels of antibodies were detected in the groups immunized with the PLDr vaccine and the vaccine of PLDr + inactivated whole cells, compared to the control groups⁷³.

The PLDr protein together with whole cultures of C. pseudotuberculosis biovar ovis and equi, inactivated with formalin were used for the immunization of sheep. The detection of anti-PLD antibody levels by ELISA allowed detecting that the vaccinated animals presented an increase in IgG after the second booster dose, but after the challenge, there was a decrease in the OD from 0.65 to 0.55, although the levels remained above the cut-off value for 20 weeks. No lesions were observed in external and internal lymph nodes, compared to the unvaccinated control group where 80 % of the animals presented lesions and manifestations of the disease. Both vaccines were able to protect the animals from the challenge with a virulent strain. In this work for the first time, sheep are immunized with a biovar equi strain in combination with PLDr⁸⁷.

Different recombinant proteins rCP09720 (esterase), rCP01850 (L14 protein binding to the 50S rRNA subunit) and PLD (rPLD) have also been evaluated in the immunization of BALB/c mice. In this study, survival rates after challenge with a virulent strain were 30 % (rPLD), 40 % (rPLD + rCP09720) and 50 % (rPLD + rCP01850). The vaccine rPLD + rCP01850 was able to induce a cellular immune response, significantly increasing levels of IFN-γ and TNF-α, while IL4 and IL12 production was not detected⁸⁸.

A live attenuated strain of Mycobacterium bovis BCG (Bacillus-Calmette-Guerin) was also used for the expression of recombinant PLD in the pUS2000 plasmid. The system was not efficient for elevated expression of the PLD protein but was effective for vaccination and protection in a mouse model. Immunization of BALB/c mice with 10⁶ CFUs of M. bovis pUS2000/PLD for PLD expression, as well as with M. bovis pUS2000/PLD + 50μg of purified PLDr and the unmodified M. bovis strain, induced elevated antibody production compared to the negative control (100 μl of NaCl 0.9 %), but without significant differences between the vaccinated groups. This is because the M. bovis strain alone is able to induce an elevated humoral and cellular immune response. However, faced with the challenge with 2 x 10⁴ CFUs of the virulent strain MIC-6, the group vaccinated with M. bovis pUS2000/PLD experienced a significant increase in IgG levels compared to the rest of the groups. The cellular immune response was evaluated by measuring the production levels of IFN-γ and IL-10 in the spleen cell culture supernatants of the vaccinated animals, after being stimulated with 8 μg/ml of PLDr. The levels of IFN-γ and IL-10 were higher in the cell culture of the group that received a reactivation of the vaccination with 50 μg of PLDr. The level of protection conferred by these formulations was 88 % in animals vaccinated with M bovis pUS2000/PLD+ 50 μg of PLDr, 77 % for group M. bovis pUS2000/PLD and 66 % for the non-modified M. bovis group. The protective immune response generated by this whole cell vaccine of M. bovis BCG modified to express PLD could cause the activation of several populations of T cells due to the variety of antigens (lipids, proteins and carbohydrates) of the formulation. Then re-immunization with 50 μg of PLD obtained recombinantly stimulates increased proliferation of T cells specific to this particular antigen⁸⁹.

Subunit vaccines have also been developed using the recombinant CP40 protein. The preparation of PLD vaccines from C. pseudotuberculosis culture supernatants usually contain other antigens which could be contributing to the protective immune response. The CP40 protein was identified in inactivated vaccine preparations through immunoblot assays, where it was observed that sera from animals vaccinated with Glanvac 6 could intermittently recognize this protein, suggesting that it was present in some vaccine batches⁸². In an experimental study in sheep, immunization with 100 μg of recombinant CP40 protected 82 % of the animals, with a decrease in lung lesions by 98 %. No relationship was found between decreased development of lung lesions and antibody titer, so it was assumed that cellular response, as antibody-dependent cellular cytotoxicity, could be responsible for protection⁴³.

Subsequently, the comparative evaluation of four vaccine formulations was carried out, which used as immunogens the recombinant CP40 protein and the CP09 strain attenuated by induced mutagenesis. The live attenuated strain CP09 of C. pseudotuberculosis was not able to induce a humoral immune response in the vaccinated mice, nor challenged with a virulent strain. Animals vaccinated with formulations that included CP40r had a significant increase in the titer of IgG1 antibodies. However, these groups, after the challenge, experienced a significant increase in IgG2 levels, the maximum being reached by animals immunized with CP40r. The formulation based on CP40r protected 90 % of the animals from the challenge with the virulent strain, followed by the vaccinated group with the attenuated strain CP09 + CP40r with 70 %, while vaccination with CP40r followed by re-immunization with CP09 only protected 60 %⁹⁰.

Another group of researchers performed the evaluation in BALB/c mice of a CP40r subunit vaccine with different adjuvants, saponin or Freud’s complete adjuvant (FCA). Animals immunized with CP40r/saponin showed elevated values in complete levels of antibodies and IgG2a, IgG2b and IgG3, with statistically significant differences with respect to the control group. The group vaccinated with CP40r/ACF showed significant differences in complete levels of IgG, IgG2a and IgG2b. Both vaccine formulations protected 100 % of the animals challenged with 10⁴ CFUs of the virulent strain C57 of C. pseudotuberculosis, with a tendency towards a Th1 response. Reactivity and production of specific isotypes IgG2a, IgG2b and IgG3 are associated with the action of pro-inflammatory cytokines such as IFN-γ and CD8+ T cells, which activate B cells by modifying the immunoglobulin heavy chain. The use of different adjuvants did not influence the antibody response, so the use of saponin is proposed to replace Freud’s adjuvant, which is toxic in sheep⁹¹.