Patent Application
20160015799
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.
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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'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 (
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 (OD600) 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 OD600, 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.
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 (NaHCO350 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.
The samples (including negative controls) were analyzed in polyacrylamide gel 15% (Sambrook J., Russell D. W., Molecular Cloning: A laboratory manual, 3rd 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 (
Purification of the Capsid Recombinant Protein of the Porcine Circovirus 2 Expressed in pET29a Plasmid and Analysis on SDS-PAGE Gel
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; NaCO3H 50 mM; pH 7.3). The analysis of the results was made by polyacrylamide gel 15% (
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; NaCO3H 50 mM; pH 7.3) (
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
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).
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 PEG6000 solutions in previously standardized concentrations for the rCap-PCV-2-29a. The tubes containing the antigen to be recovered together with the PEG6000 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.
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.
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
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 1×106 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 CO2 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.
The supernatants collected in the lymphoproliferation concerned to the period of 72 hours were analyzed by ELISA for evaluation of the TH1 and TH2 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 TH2 profile: IL-4 and IL-10 (PeproTech Brazil—FUNPEC), according to the manufacturer's manual.
As can be seen in
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.
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.
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, Na2HPO4 10 mM, KH2PO4 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 (H2O2) 30%]. The reaction was incubated for 10 minutes and paralyzed with sulfuric acid solution 1.5 M (H2SO4). 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.
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 (
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.
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).
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.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102013001893-7 | Jan 2013 | BR | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14763121 | Jul 2015 | US |
| Child | 14875090 | US |
