Abstract:
The present continuation-in-part of the Application BR 7 10 2013 001893 refers to the obtainment of the viral capsid recombinant antigen of the Porcine circovirus 2 (PCV-2) and modifications thereof, upon expression in prokaryotic system, recovery of virus-like particles (VLPs) and its use in vaccine formulations. The antigens and vaccine formulations can be used in the immunization of animals in control programs of the diseases associated with PCV-2 in conventional swine production systems and represent alternatives to vaccines available on the market.

Description:
[0001]    The innovations and improvements herein refer to the removal of the viral capsid protein coding sequence of the porcine circovirus 2 (PCV-2) and transferred to a bacterial expression vector. This plasmid construct allows the expression of the recombinant protein without the 10-histidine tail. The expression of virus-like particles (VLP) by the recombinant protein was also verified. Assays were performed in murine and swine models, where the immunogenicity of the vaccine candidate of the patent object was confirmed. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0002]    The  FIG. 1  shows the analysis in agarose gel 1% of the cleavage reactions of the empty pET29a (channel 1), the cleavage of the recombinant plasmid pCap-rPCV2-29a with the XhoI and NdeI enzymes (channel 2), M: Ladder DNA marker 100 bp. 
           [0003]    The  FIG. 2  confirms the expression of rCAP-PCV-2 from the pCAP-rPCV2-29a. SDS-PAGE Gel 15% (left) and nitrocellulose membrane obtained by Western blotting (right). M: molecular weight marker; C−: Negative control (soluble fraction of the  E. coli  extract transformed with the empty bacterial expression plasmid and induced with IPTG); and 3: Sample (soluble fraction of the  E. coli  extract transformed with the pCAP-rPCV2-29a and induced with IPTG). The arrow indicates the band of approximately 27 kDa corresponding to rCap-PCV-2. 
           [0004]    The  FIG. 3  shows the fractions resulting from the precipitation process of the soluble fraction by saturation with ammonium sulfate. M: Molecular weight marker; 1: Soluble fraction—part not precipitated; 2: purified rCAP. The arrow indicates the RCAP-PCV2-29a. 
           [0005]    The  FIG. 4  show electron micrographs: formation of virus-like particles (VLPs) by rCap-PCV2-29a without histidine tail. The 200 mesh grids with formvar/carbon containing samples were analyzed by Transmission Electron Microscope using 85000× (left figure) and 140000× (middle and right drawings) magnifications. VLPs can be visualized by the arrows. 
           [0006]    The  FIG. 5  shows the specific humoral response induced by the rCap-PCV-2 in Balb/c mice before and after vaccination as measured by indirect ELISA. The animals were inoculated on days 0 and 21. The inoculated candidate containing the rCap-PCV-2 lacking histidine tail (G4) showed high levels of antibodies compared to controls (G1 and G2) used during the collections 2 (28 days) and 3 (42 days) of the experiment. 
           [0007]    The  FIG. 6  shows IFN-γ dosage by ELISA obtained through the lymphoproliferation assay of spleen cells from the various experimental groups stimulated with concanavalin A (ConA) as positive control, RPMI medium supplemented with bovine fetal serum as negative control and purified rCap-PCV-2 in concentrations of 0.05 μg/mL, 0.5 μg/mL and 5.0 μg/mL in the period of 72 hours. It was observed that the group inoculated with the proposed vaccine formulated (G4) when stimulated with 5.0 μg/mL of the purified PCV-2-rcap presented statistical difference compared to controls. It is noteworthy that the vaccine candidate induced the IFN-γ production, which is a cytokine of the T H 1 response profile and promoted a more efficient cellular immune response against the virus. 
           [0008]    The  FIG. 7  shows IL-10 dosage by ELISA obtained by the lymphoproliferation assay of splenic cell of the different experimental groups stimulated with concanavalin A (ConA) as positive control, RPMI medium supplemented with bovine fetal serum as negative control and purified rCap-PCV-2 in concentrations of 0.05 μg/mL, 0.5 μg/mL and 5.0 μg/mL in a period of 72 hours. It was observed that the group inoculated with the proposed vaccine formulated (G4) showed statistically significant IL-10 levels compared to controls, for both positive stimuli (ConA) and different concentrations of rCap-PCV-2. IL-10 is a characteristic cytokine of the T H 2 response profile (immunosuppressive) and inhibits the synthesis of pro-inflammatory cytokines such as IL-12, thus directing the response to humoral immunity. 
           [0009]    The  FIG. 8  shows IL-12 dosage by ELISA obtained through the lymphoproliferation assay of spleen cells from the various experimental groups stimulated with concanavalin A (ConA) as positive control, RPMI medium supplemented with bovine fetal serum as negative control and purified rCap-PCV-2 in concentrations of 0.05 μg/mL, 0.5 μg/mL and 5.0 μg/mL in a period of 72 hours. Low quantification was observed for the IL-12 cytokine and there was no significant difference in the group inoculated with the vaccine candidate (G4) to the control groups. The low quantification of IL-12, a characteristic cytokine of the T H 1 response profile, is due to increased IL-10 levels ( FIG. 7 ) which is an immunosuppressive cytokine, and tends to inhibit the production of inflammatory cytokines, which are characteristics of T H 1 cells. It was observed that when quantifying the levels of different cytokines, the immune response mediated by the immunization with the vaccine candidate shown to be of the mixed type, i.e. there was a no predominance of T H 1 or T H 2 response. 
           [0010]    The  FIG. 9  shows the serological profile detected during the trial period in the farm I during the different collections. On the horizontal axis, the four collections are presented: Collection 1—Blood samples collected at 21 days old (day 0 post-vaccination); Collection 2—Blood samples collected at 53 days old (32 days post-vaccination); Collection 3—Blood samples collected at 102 days old (81 days post-vaccination); Collection 4—Blood samples collected at 166 days old (145 days post-vaccination). On the vertical axis are the levels of antibodies expressed in S/P. S/P=(test sample average−negative control average)/(positive control average−negative control average). The test group (G1) corresponds to the group that was inoculated with the formulation based on plasmid 29a, at a concentration of 100 μg. The positive control group (G2) corresponds to animals inoculated with the commercial vaccine. The negative control group (G3) are the animals that were inoculated with PBS. (*) Indicates statistical difference compared to the negative control (C − ). Therefore, the test formulations, G1, resulted in significantly higher levels than the positive and negative controls (G2 and G3), on the third and fourth sampling. 
           [0011]    The  FIG. 10  shows the serological profile observed during the trial period in the farm II during the different collections. The horizontal axis shows the four collections: Collection 1—Blood samples collected at 23 days old (day 0 post-vaccination); Collection 2—Blood samples collected at 65 days old (42 days post-vaccination); Collection 3—Blood samples collected at 116 days old (93 days post-vaccination); Collection 4—Blood samples collected at 158 days old (135 days post-vaccination). The vertical axis shows the levels of antibody expressed as S/P. S/P=(test sample average−negative control average)/(positive control average−negative control average). The test group G1 corresponds to the negative control group. The animals in this group were vaccinated with PBS. The test group G2 corresponds to the positive control group. The animals in this group were immunized with commercial vaccine. The test group G3 corresponds to the group of animals that received the research vaccine on the soluble fraction derived formulation. In addition, the G4 test group corresponds to the group of animals that received the research vaccine purified with ammonium sulfate. (*) Indicates statistical difference compared to the negative control (C − ). Therefore, the test formulations, soluble fraction, and ammonium sulfate purified vaccine generated significantly higher levels than the negative control since the second collection, keeping this behavior until the fourth and final collection. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Transferring of the Capsid Protein Coding Region of the Porcine Circovirus 2 to the Expression Vector in Bacterial System 
       [0012]    After verification of the correct sequence of the insert in the vector, the sample of the pCapPCV-2 plasmid DNA, SEQ ID NO: 01 (amplification vector) was subjected to an enzymatic assay where specific restriction sites were used to insert the gene ORF2 in another bacterial expression vector (pET-29a—Novagen). This expression vector is controlled by a T7 lac promoter, however the insert was directed so that the recombinant protein to be coded did not have the sequence encoding the 10-histidine tail in the N-terminal region, as in the vector earlier mentioned in BR 10 2013 001893 7, thereby targeting an increased production of virus-like particles (VLP&#39;s). After the cleavages, the products of the recurring digestions were liked using the enzyme T4 DNA ligase. The product of this liking reaction was then used to transform  E. coli  DH5α. Thus, the transformants clones were randomly selected from the colonies for the identification of the plasmids with the insert, and, to confirm the cloning, colonies PCRs were performed. The positive colonies were selected and each colony was subjected to PCR separately. This identification, in turn, is given by digestion reaction with the restriction enzymes XhoI and NdeI restriction. The same digestion reaction was also carried out with the pET29a without the ORF2 gene (empty). All the digestion reactions assays were carried out by electrophoresis in 1% agarose gel. Thus, the bands of the expected size were observed in 718 bp and 5371 bp, from the digestion with NdeI and XhoI, respectively, and a high molecular weight fragment corresponding to the plasmid remaining ( FIG. 1 ). The recombinant plasmids obtained were named PCAP-rPCV2-29 a , and the nucleotide and amino acid sequence confirmed as SEQ ID NO: 01 and SEQ ID NO: 02, respectively. Plasmids were stored in microcentrifuge tubes containing glycerol from 15 to 30% and kept at −80° C. 
       Expression of the Capsid Recombinant Protein of the Porcine Circovirus 2 and Analysis on SDS-PAGE Gel 
       [0013]    The total expression of the recombinant proteins was done in medium scale in 1000 mL of TB (tryptone 12 g/L, yeast extract 24 g/L, glycerol 4 mL, monobasic potassium phosphate 2.31 g/L and dibasic potassium phosphate 12.54 g/U). For this, competent bacteria of the strain  E. coli  BL21-DE3-RIL codon plus were transformed with the pCap-rPCV2-29a construct analogous to that carried out with the amplification vector. Thus, approximately 20 nanograms of recombinant plasmid pCap-rPCV2-29a were added to 100 μL of competent cells and the mixture incubated on ice for 30 min. Then, the cells mixture and plasmid DNA were subjected to a thermal shock in a water bath at 42° C. for 1 minute, and again on ice for 2 minutes. Thereafter, 900 uL of LB medium (bacto-tryptone 10 g/L, yeast extract 5 g/L and sodium chloride 10 g/L) without antibiotic was added and cells incubated at 37° C. for 2 hours at 250 rpm. The cells were diluted hundred-fold (1:100) into LB medium containing kanamycin 50 μg/mL and incubated at 37° C. and 180 rpm for 12-16 hours (pre-inoculation). A negative control culture (same bacteria of strain  E. coli  BL21-DE3-RIL codon plus but not transformed) was also performed in liquid LB, pH 7.0, chloramphenicol 17 mg/mL. The cells were then diluted 1:100 in TB liquid medium, pH 7.0, kanamycin 50 μg/mL and the culture was grown at 30° C./180 rpm for approximately 4 hours until the optical density (OD 600 ) of from 0.6 to 0.8. It was performed the same way as for the negative control, using chloramphenicol 17 mg/mL. After reached the OD 600 , IPTG was added to final concentration of 0.25 mM for the expression of the recombinant protein of interest, the cultures were then left at a temperature of 30° C. for additional 4 hours, always under vigorous agitation and sufficient aeration. The same procedure was carried out for the negative control. After induction in optimal conditions, the samples from the inductions were centrifuged at 10,000 g for 20 min at 4° C. The supernatants were discarded and the precipitated cells were stored at −20° C. 
         [0014]    The precipitate resulting from a volume of 100 mL of the induced medium of the pCap-rPCV2-29a was thawed and resuspended in lysis buffer (NaHCO 3 50 mM, NaCl 60 mM, pH 7.3) to a final volume of approximately 5 mL. The process of cell lysis was performed with 6 cycles of 10 s sonication at 200-300 watts each, with intervals of 10 s and with the tubes on ice to prevent the sample warming. The cellular debris and the inclusion bodies were precipitated by centrifugation at 15,000×g for 30 min at 4° C. The supernatant (soluble fraction) was collected in a new tube and used for the purification of capsid recombinant protein of PCV-2, referred to herein as rCap-PCV2 (SEQ ID NO: 02) derived from the pCap-PCV2-29a induction. 
         [0015]    The samples (including negative controls) were analyzed in polyacrylamide gel 15% (Sambrook J., Russell D. W., Molecular Cloning: A laboratory manual, 3 rd  ed., Cold Spring Harbor Laboratory Press, New York, 2001). After the running, the gel was revealed by staining solution (Coomassie Brilliant Blue R-250 0.1%, acetic acid 9%, ethanol 45%). The electrophoresis analysis confirmed the presence of a band of approximately 27 kDa corresponding to the mass of the protein encoded by the ORF2 without the histidine tail (rCap-PCV2-29a). The confirmation of the expression was given by Western blotting technique ( FIG. 2 ). 
         [0000]    Purification of the Capsid Recombinant Protein of the Porcine Circovirus 2 Expressed in pET29a Plasmid and Analysis on SDS-PAGE Gel 
         [0016]    For the recombinant protein (rCap-PCV2-29a) from the pCap-rPCV2-29a, the purification of the soluble fraction was performed by precipitation with ammonium sulphate 20%-40%. The soluble fraction was left under 180 rpm stirring at 0° C. for 2 hours. After this step, the sample was centrifuged at 10000×g at 4° C. for 30 minutes. Subsequently, the supernatant was separated and the precipitate resuspended in carbonate buffer (NaCl 60 mM; NaCO 3 H 50 mM; pH 7.3). The analysis of the results was made by polyacrylamide gel 15% ( FIG. 3 ). The amount of recombinant protein in the purified PCV-2 viral capsid was determined using the method described by Bradford (Bradford, M. M., 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dyebinding. Analytical biochemistry, V. 72, p. 248-254, 1976). The calculation was made by linear regression where the equation y=0.3267+0.0108x was obtained from the best fit to the optical density values for the tested BSA dilutions. 
       Preparation of the Vaccine Compound and Verification of the Virus-Like Particles (VLPs) Formation 
       [0017]    The purification of pCap-PCV2-29a in the soluble fraction from the pCap-rPCV2-29a was performed by precipitation with ammonium sulfate at 20% to 40%. The precipitate was resuspended in carbonate buffer (NaCl 60 mM; NaCO 3 H 50 mM; pH 7.3) ( FIG. 3 ). This sample was then used to prepare the vaccine candidate, where this preparation is given by the addition of an adjuvant in a previously quantized amount of sample. 
         [0018]    For VLPs verification, fractions from the CsCl gradient that showed a positive result on the Western-blotting were dialyzed separately against 500 mL of carbonate buffer (NaCl 300 mM, bicarbonate 50 mM, pH 7.0) two times for 4 hours each. Approximately 10 μL of each fraction were added to the 200 mesh grids covered with formvar/carbon and allowed to settle for 1 minute at room temperature. Then, the excess sample was removed with filter paper and a drop of uranyl acetate 2% was added in each grid and allowed to act for 1 minute. The excess of that contrast was removed with filter paper and the grids were left in a desiccator for 2 days. The analysis was performed in a transmission electron microscope and the images were photographed with 85000× and 140000× amplification. The results from this analysis can be seen in the three images of  FIG. 4 . A large production of VLPs was observed in the soluble fraction and for the vaccine formulated containing the pCap-PCV2-29a, which demonstrates that the histidine tag removal favored a larger production of virus-like particles in the samples. 
       Demonstration Experiments 
     Experiments in Mice 
       [0019]    25 female mice Balb/c of approximately 5 weeks old were used from the vivarium connected to the Life Sciences and Health Center (CCB) of the Federal University of Viçosa (UFV), which were equally divided into 5 groups. The procedures were performed according to the Animal Ethics and Experimentation Committee of the Federal University of Vigosa (UFV). 
         [0020]    The group 1 (G1) was vaccinated with PBS 1× (negative control), the group 2 (G2) was vaccinated with the commercial vaccine (positive control), the group 3 (G3) was vaccinated with the rCap protein purified in FPLC (rCap-PCV2), the group 4 was vaccinated with the vaccine formulated produced from pCap-rPCV2-29a plasmid. It should be noted that in this assay the recovery of the vaccines antigen for the vaccination of animals in the G4 group was performed by precipitation with polyethylene glycol (PEG6000) due to of its ability to purify viral particles. To the soluble fractions obtained after bacterial lysis process were added PEG 6000  solutions in previously standardized concentrations for the rCap-PCV-2-29a. The tubes containing the antigen to be recovered together with the PEG 6000  were cooled and left under stirring for the viral particles precipitation. After this step, the samples were centrifuged and the precipitates were suspended in lysis buffer. 
         [0021]    With the exception of the group inoculated with commercial vaccines, the vaccines were administered with aluminum hydroxide adjuvant at a concentration of 1 mg/mL. The animals were vaccinated twice (two doses) subcutaneously at an interval of 21 days between doses. The amount of rCap-PCV2 vaccinated was 50 μg for the first dose and 25 μg for the second dose. Blood samples were collected via ocular sinus puncture before each inoculation, corresponding to the days 0, 28, and 42 for the serological analysis. The evaluation of the humoral immune response was made by indirect ELISA technique. The optimum working concentrations of the antigen rCap-PCV-2 and the best dilution of serum (primary antibody) were evaluated by Checker board titrating (Crowther J. R. ELISA. Theory and Practice. Methods in Molecular Biology. V. 42, p. 1-223, 1995). It was determined as the optimal antigen concentration (rCap-PCV-2), 28 μg/well and 1:100 as the ideal serum dilution (primary antibody). The analyses were performed using a positive sample of mouse serum for PCV-2. 
         [0022]    The mice vaccinated with the vaccine candidate containing the rCap-PCV2-29a (without histidine tail) had antibody levels higher than those obtained for mice vaccinated with commercial vaccines in periods corresponding to collections 2 (28 days) and 3 (42 days), as shown in  FIG. 5 . The presence of VLPs in the proposed vaccine candidate ( FIG. 4 ) possibly contributed to a better immune response in this group of animals. 
         [0023]    The mice from all groups were euthanased and the spleen were removed aseptically and divulsed in EDTA 200 mM to obtain total spleen cells. These cells were washed with RPMI 1640, supplemented with streptomycin 1.00 g/L and penicillin 0.75 g/L, centrifuged at 1.000×g for 10 min at 4° C. for 4 min and incubated with lysis buffer (9 parts of ammonium chloride 0.16 M and 1 part of Tris-HCl 0.17 M) at room temperature. The cells were again washed and suspended to a concentration of 1×10 6  cells/mL in RPMI medium supplemented with fetal bovine serum 5% (FBS). Subsequently, the cells were added to 24-well plates (in duplicate for each experimental group) (1000 μL/well) and incubated at 37° C. under atmosphere of CO 2  5% 72 hours using the treatments with RPMI medium supplemented with FBS 5% (negative control), with concanavalin A stimulation (ConA 2 μg/mL) used as positive control, and with the recombinant capsid protein of PCV2 purified on affinity column at concentrations of 0.05 μg/ml, 0.5 μg/mL and 5.0 μg/mL. After the incubation period, the plates were centrifuged at 1000×g for 5 min for cells sedimentation and the supernatants collected for evaluation of the cytokines profile by ELISA. 
         [0024]    The supernatants collected in the lymphoproliferation concerned to the period of 72 hours were analyzed by ELISA for evaluation of the T H 1 and T H 2 profile. The Murine IFN-gamma ELISA kit, Murine IL-12 ELISA kit, Murine IL-10 ELISA Kit and Murine IL-4 ELISA kit commercial kits were used for the Tel profile evaluation: IL-12 and IFN-γ and T H 2 profile: IL-4 and IL-10 (PeproTech Brazil—FUNPEC), according to the manufacturer&#39;s manual. 
         [0025]    As can be seen in  FIGS. 6 to 8  the dosage of the INF-γ, IL-10 and IL-12 cytokines in ng/mL showed a mixed response profile, i.e. not being predominantly T H 1 or T H 2. High levels of INF-γ favor a T H 1 profile response in which the cellular immunity, essential for combating intracellular pathogens (virus), is preferred. However, the largest quantifications for IL-10 indicate a direction of the immune response to T H 2 profile, where the antibody production is favored. This result confirms what was observed in  FIG. 5 , indicating a higher serological level for mice vaccinated with the proposed vaccine candidate (G4—rCap-PCV-2-29a) in relation to the controls (G1 and G2) used in this experiment. 
       Trial in Swine 
     Farm I 
       [0026]    The Trial I was conducted in a commercial farm of complete cycle, naturally infected, in the region of Zona da Mata—MG respecting the management adopted by the farm. This study was conducted in accordance to the ethical standards of animal testing and requirements of the Ethics Committee on Animal Use (CEUA) of the UFV. 105 swine were used, males and females, Choice Genetics commercial line, approximately 21 day old, divided into 3 groups of 35 animals. 
         [0027]    At 21 days of age, the piglets were identified with an ear tag proper for swine and subsequently weighed for the of group division. This division was done by random experimental lineation, so that all groups had similar mean weight. The immunization was performed by intramuscular route with doses of 1 mL in the piglets with an average age of 21 days, so that the group 1 received the formula from the pCap-PCV2-29a protein at a concentration of 100 μg, the group 2 was the positive control and received the commercial vaccine. Group 3 was the negative control and, therefore, received PBS. Blood samples were collected on days 0, 32, 81 and 145 post-vaccination to obtain the serum. 
         [0028]    To determine the humoral immune response of the immunized pigs, commercial 96-well microplates were used. The microplates were coated with the solution of the rCap-PCV-2 (1.125 μg/mL) diluted in carbonate buffer 0.05 M overnight at 4° C. After this period, the plates were washed with PBS containing Tween 20 (PBS/T—NaCl 137 mM, KCl 2.7 mM, Na 2 HPO 4  10 mM, KH 2 PO 4  2 mM, Tween 20 0.05%; pH 7.2), blocked with blocking solution (PBS/T containing BSA) under stirring of 500 rpm and at 37° C. in incubator for commercial microplate. Then the plates were incubated with sera samples diluted (1:800) in dilution solution (PBS/T containing BSA 0.5%), with each sample being tested in triplicate. After washing again, the plates were incubated with secondary antibody (swine anti-IgG conjugated with peroxidase produced in rabbit, Sigma), diluted in dilution solution. Subsequently, the plates were washed again and for the colorimetric reaction a solution containing the chromogenic substrate was added [10 mL of citrate buffer 0.1 M; pH 5; o-Phenylenediamine 4 mg (OPD); 5 μL of hydrogen peroxide (H 2 O 2 ) 30%]. The reaction was incubated for 10 minutes and paralyzed with sulfuric acid solution 1.5 M (H 2 SO 4 ). The presence of antibody was determined by reading the absorbance at 492 nm (D0492 nm) in commercial microplate reader. The results were analyzed by the Prisma statistical program and by the Newman-Keuls multiple comparison test. 
         [0029]    In the first blood sample (day 0), which was before applying the vaccine, the three groups showed homogeneous results with respect to the antibody levels. In the second collection (32 days post-vaccination), there was no statistically significant difference between the vaccine group and control group. In the third (81 days post vaccination) and fourth collections (145 post-vaccination), the results were similar, wherein the vaccine candidate generated a higher antibody response compared to the positive and negative control groups ( FIG. 9 ). 
       Farm II 
       [0030]    The trial II was conducted in a commercial farm of complete cycle naturally infected with PCV2. The study was conducted in the region of Zona da Mata—MG, respecting the management adopted by the farm and in accordance with the ethical standards of animal testing and requirements of the Ethics Committee on Animal Use (CEUA) of the UFV. It was used 120 swine, males and females, with commercial genetic of approximately 23 days old, divided into 4 groups of 30 animals. At 23 days of age, the piglets were identified with an ear tag proper for swine, and thereafter weighed for the groups division. This division was made by random experimental lineation, so that all groups had similar average weight. 
         [0031]    The vaccination was performed in a single 1 mL dose intramuscularly at weaning (animals at approximately 3 weeks of age). The group 1 was the negative control group and the animals were vaccinated with PBS 1 mL (saline solution). The group 2 was considered a positive control group in which animals received 1 mL of a commercial vaccine. The group 3 was the group that received 1 mL of vaccine prepared from the partially purified soluble fraction (150 μg of antigen). The group 4 was the group that received 1 mL of vaccine test purified with ammonium sulfate (150 μg of antigen). 
         [0032]    Blood samples were collected through the jugular vein using sterile needles in blood collection tubes of 8 mL with clot activator, being stored in the first hours at room temperature to clot retraction and obtaining serum. The sample collections were performed at 0, 42, 93, and 135 days after vaccination. Antibody profiles were performed by indirect ELISA as described in the experiment of Farm I. The results were analyzed by the statistical software Prisma and the Newman-Keuls multiple comparison test. 
         [0033]    The first blood collection was performed at the time of weaning at 23 days old (0 days of vaccination), prior to vaccination, and at that time, the piglets showed results homogenous regarding the antibody levels. In the second collection, which occurred at 42 days post-vaccination with the animals averaging 65 days of age, there was a statistical difference in antibody levels between treatments. Groups 2, 3, and 4 were statistically different from group 1. In addition, groups 3 and 4 were statistically different from Group 2. In the third collection, 93 days after vaccination, with the animals at an average of 116 days of age, Groups 2, 3, and 4 were statistically different from group 1. The groups 3 and 4 differ significantly from group 2. In the fourth and last collecting, at 135 days post vaccination and animals at 158 days of age, groups 3 and 4, prepared from the vaccine candidate were statistically different from group 1, whereas there was no statistical difference at this stage between the positive and negative controls.