Promising Multiple-Epitope Recombinant Vaccine against Foot-and-Mouth Disease Virus Type O in Swine Jung-Jun Shao, et al. ...
Background Foot-and-Mouth  Disease Virus (FMDV) <ul><li>Foot-and-Mouth Disease is a highly contagious viral disease of clo...
Background Foot-and-Mouth  Disease Virus (FMDV) Continues <ul><li>Recent outbreaks still occur mostly in the UK and Taiwan...
Current Treatments <ul><li>Can spread up to a 6km radius outside the infected heard.  This usually results in the mass cul...
Modified FMDV Type O Vaccine <ul><li>Previous studies have shown that recombinant proteins that contain one or more of the...
Methods <ul><li>46 Swine free of antibodies against structural proteins and 3ABC nonstructural proteins </li></ul><ul><li>...
Methods <ul><li>Design and Synthesis of a tandem-repeat multiple-epitope gene: </li></ul><ul><li>402 bp in length, contain...
Methods <ul><li>PCR amplification of the swine immunoglobin G heavy-chain constant region (scIgG) and RE gene: </li></ul><...
Methods <ul><li>Construction of a recombinant RE-scIG expression plasmid: </li></ul><ul><li>Amplified by overlapping PCR w...
Methods <ul><li>Expression and purification of recombinant protein: </li></ul><ul><li>Grown in  E. coli  in 100mL of LB me...
Materials & Methods <ul><li>Used to test for the immunoreactivity of the recombinant protein. </li></ul><ul><li>Steps:  </...
Methods <ul><li>Optimal Concentration of recombinant protein: 0.25 mg/dose </li></ul><ul><li>Recombinant protein then was ...
Methods <ul><li>13 Swine were broken up into 3 groups. </li></ul><ul><li>Group 1: inoculated intramuscularly (in the muscl...
Methods <ul><li>18 swine were split into 4 groups and used to determine the PD 50  of the vaccine in swine.  </li></ul><ul...
Methods <ul><li>Three sets of the multiple- epitope recombinant vaccines (A-C) were prepared. </li></ul><ul><li>15 swine w...
Methods <ul><li>LPB-ELISA </li></ul><ul><ul><li>Each test serum was placed in a well plate and viral antigen that was homo...
Methods <ul><li>The statistical analysis was done by statistical comparisons carried out and performed by SPSS software.  ...
Results Expression and characterization of recombinant RE-scIgG protein   Recombinant expression plasmid pET-22b-RE-scIgG ...
Results <ul><li>Potency of recombinant protein was evaluated according to titers of anti-FMDV specific antibodies in swine...
Results <ul><li>Potency of the multiple-epitope recombinant vaccine in swine </li></ul><ul><li>High titers of anti-FMDV an...
Results TABLE 2: Titers of antibodies against FMDV and swine challenge results
Results <ul><li>TABLE 3: Titers of antibodies against FMDV, protection ratios, and PD 50  in swine </li></ul><ul><li>PD 50...
Results <ul><li>Duration of immunity induced by the multiple-epitope recombinant vaccine in swine </li></ul><ul><li>All 3 ...
Results <ul><li>TABLE 4: Titers of antibodies to FMDV induced in swine by three batches of vaccine </li></ul><ul><li>Lymph...
Results <ul><li>Virus Isolation from Heparinized Blood and Nasal Swabs: </li></ul><ul><ul><li>Control Group: </li></ul></u...
Discussion <ul><li>Improved recombinant vaccine elicited high titers of anti-FMDV specific antibodies &  a high lymphocyte...
Discussion <ul><li>Advantages as alternative to traditional vaccines </li></ul><ul><li>Does not involve use of infectious ...
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FMDV

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FMDV

  1. 1. Promising Multiple-Epitope Recombinant Vaccine against Foot-and-Mouth Disease Virus Type O in Swine Jung-Jun Shao, et al. Hong Kong University Of Science and Technology Clinical and Vaccine Immunology: January 2011
  2. 2. Background Foot-and-Mouth Disease Virus (FMDV) <ul><li>Foot-and-Mouth Disease is a highly contagious viral disease of cloven-hoofed ruminants (i.e.: cattle, swine, etc.) </li></ul><ul><li>The virus is a member of the genus Aphthovirus and of the Picornaviridae family </li></ul><ul><li>Characterized by fever and blister-like lesions followed by erosions on the tongue, mouth, teats and between the hooves </li></ul><ul><li>Most affected animals recover but not without severe losses of meat and milk (economically unfavorable) </li></ul><ul><li>Signs of virus can appear after and incubation period of 1-8 days and survives in lymph nodes and bone marrow at neutral pH </li></ul><ul><li>Can persist for up to a month and is very difficult to control </li></ul><ul><li>Spreads via animals, people, or materials that come into physical contact with susceptible animals </li></ul>
  3. 3. Background Foot-and-Mouth Disease Virus (FMDV) Continues <ul><li>Recent outbreaks still occur mostly in the UK and Taiwan </li></ul><ul><li>Cause extreme drop in meat consumption </li></ul><ul><li>The last of the nine outbreaks of Foot-and-Mouth Disease in the US was in 1929 </li></ul>
  4. 4. Current Treatments <ul><li>Can spread up to a 6km radius outside the infected heard. This usually results in the mass culling of livestock and the burning of the corpses to prevent spreading. </li></ul><ul><li>Vaccines exist but must be a perfect match to the specific type and subtype of the virus causing the outbreak and are chemically inactive whole-virus vaccine </li></ul><ul><li>Vaccines do not induce sterile immunity and therefore do not prevent carrier status and necessitate frequent revaccination </li></ul><ul><li>In production of these vaccines there is a risk of escape of live virus from biosafety facilities </li></ul>
  5. 5. Modified FMDV Type O Vaccine <ul><li>Previous studies have shown that recombinant proteins that contain one or more of the immunogenic epitopes (141 to 160 and 200 to 213 of the VPI protein) can confer full protection against a challenge is small animals </li></ul><ul><li>Immunogenicity (how well a vaccine elicits an immune response) of these vaccines was significantly lower than traditional vaccines </li></ul><ul><li>Could be due to rapid clearance of recombinant proteins and the lack of T-helper cell epitopes </li></ul><ul><li>In this study, to develop a completely safe replacement for traditional vaccines, a recombinant vaccine against FMDV type O was modified </li></ul><ul><li>Organism tested: Swine </li></ul><ul><li>Vaccine Manufacture Method: Escherichia coli </li></ul>
  6. 6. Methods <ul><li>46 Swine free of antibodies against structural proteins and 3ABC nonstructural proteins </li></ul><ul><li>Experiment One: measured the activity of the multiple epitope recombinant vaccine </li></ul><ul><li>Experiment Two: 50% pig protective dose was determined </li></ul><ul><li>Experiment Three: determined how long the vaccine lasted for </li></ul><ul><li>All experiments were done in areas of high-containment </li></ul><ul><li>All pig pens were separated with their own ventilation systems avoiding subject contamination </li></ul>
  7. 7. Methods <ul><li>Design and Synthesis of a tandem-repeat multiple-epitope gene: </li></ul><ul><li>402 bp in length, containing 3 copies of each sequence </li></ul><ul><li>Synthesized: 141 to 160, 200 to 213 aa, 141 to 160, 200 to 213 aa, 141 to 160, 200 to 213 aa </li></ul><ul><li>GGSSGG was used to separate the sequences </li></ul>
  8. 8. Methods <ul><li>PCR amplification of the swine immunoglobin G heavy-chain constant region (scIgG) and RE gene: </li></ul><ul><li>RE and scIgG genes were amplified separately through PCR with specific primers </li></ul><ul><li>Reaction mixture contained 5.0 µl of 10x buffer, 5.0 µl of deoxynucleoside triphosphates, 1.0 µl of ExTaq , 1.0 µl of primer, 0.5 µl of template DNA, and 36.5 µl of distilled water </li></ul><ul><li>PCR carried out by running 5 minutes at 94°C, 1 minute at 94°C (repeated 30 times), 30 seconds at 56°C, 30 seconds at 72°C, and finally 8 minutes at 72°C </li></ul><ul><li>Purified using an agarose DNA purification kit and stored at -20°C </li></ul>
  9. 9. Methods <ul><li>Construction of a recombinant RE-scIG expression plasmid: </li></ul><ul><li>Amplified by overlapping PCR with specific primers </li></ul><ul><li>Reaction mixture contained 25.0 µl of 2x MightyAmp buffer, 1.0µl of each primer, 1.0µl of purified RE and scIgG, and 2.0µl of distilled water </li></ul><ul><li>PCR carried out for 2 minutes at 98°C, 10 seconds at 94°C (x30), 10 seconds at 60°C, 90 seconds at 68°C, and 8 minutes at 72°C </li></ul><ul><li>Purified and subcloned into the expression vector, pET-22b, which resulted in a recombinant expression plasmid, pET-22b-RE-scIgG </li></ul><ul><li>Plasmid was confirmed by BamHI/HindIII digestion </li></ul>
  10. 10. Methods <ul><li>Expression and purification of recombinant protein: </li></ul><ul><li>Grown in E. coli in 100mL of LB medium at 37°C overnight in a shaker </li></ul><ul><li>Growth was monitored by optical density and expression was induced with .8mM isopropyl-beta-D-thiogalacytopyranoside </li></ul><ul><li>RE-scIgG expressed in E. coli </li></ul><ul><li>Insoluble proteins were placed in 6M urea overnight </li></ul><ul><li>Concentration of each protein determined by the Bio-Rad Protein Assay </li></ul><ul><li>100µl of 2x staining reagent added to a 96 well micro-titer plate and standard bovine serum albumin was added to corresponding wells, then distilled water was added and plate was left at room temperature for 5 minutes </li></ul>
  11. 11. Materials & Methods <ul><li>Used to test for the immunoreactivity of the recombinant protein. </li></ul><ul><li>Steps: </li></ul><ul><ul><li>1. pure protein was subjected to 12% SDS- PAGE and transferred to a polyviynlidene diflouride membrane which was blocked by PBS (phosphate- buffered saline) with 10% Horse serum for 1 hour in a shaker. </li></ul></ul><ul><ul><li>2. The protein was then washed 3 times with PBS, that contained 0.05% Tween 20 (PBST). </li></ul></ul><ul><ul><li>3. The Membrane was incubated with a 1:500 dilution of positive serum from cattle infected with FML type O. </li></ul></ul><ul><ul><li>4. It was washed 3 times with PBST and this time incubated with 1:2,000 dilution of rabbit anti- cattle IgG antibody conjugated with horseradish peroxide for 1 hour. </li></ul></ul><ul><ul><li>5. After being washed 5 times with the PBST, the signals developed with 3,3’ 5,5’- tetramethylbenzidine. </li></ul></ul><ul><li>Western Blotting </li></ul>
  12. 12. Methods <ul><li>Optimal Concentration of recombinant protein: 0.25 mg/dose </li></ul><ul><li>Recombinant protein then was emulsified with Montanide ISA 206, this prepared the protein for the double emulsion formulation. </li></ul><ul><li>Protein was then diluted to a final concentration of 0.5 mg/ml and mixed with the same volume of oil adjuvant. </li></ul><ul><li>FINSIHED VACCINE FORMULATION: 0.25 mg of purified protein per ml and was stored at 4°C until use. </li></ul><ul><li>Preparation of Multiple- Epitope Recombinant Vaccine </li></ul>
  13. 13. Methods <ul><li>13 Swine were broken up into 3 groups. </li></ul><ul><li>Group 1: inoculated intramuscularly (in the muscle) behind the ear with 2ml of a commercially inactivated vaccine (type O), which was expected keep the swine immune for 6 months. </li></ul><ul><li>Group 2: vaccinated with full dose of the multiple- epitope recombinant vaccine. </li></ul><ul><li>Group 3: Consisted of 3 swine that only received 1 ml of PBS in oil adjuvant. </li></ul><ul><li>30 days after the vaccinations were administered, serum samples were collected and all animals were challenged intradermally (into the skin) in the bulb of the heel of the left hind foot with 10 3 ID 50 of the O/ China/ 99 Strain of FMDV. </li></ul><ul><li>The swine’s rectal temperatures and clinical signs were monitored for 10 days. </li></ul><ul><li>Comparison of Potency of the Multiple- Epitope Recombinant Vaccine with a Traditional Inactivated Vaccine in Swine </li></ul>
  14. 14. Methods <ul><li>18 swine were split into 4 groups and used to determine the PD 50 of the vaccine in swine. </li></ul><ul><li>Groups 1-3: vaccinated intramuscularly behind the ear with a full dose (1 ml), 1/3 of a dose (0.33 ml), and 1/9 (0.11 ml) of the multiple- epitope recombinant vaccine. </li></ul><ul><li>Group 4: was given 1 ml PBS in oil adjuvant. </li></ul><ul><li>30 days after being injected the swine were all bled and challenged. </li></ul><ul><li>In order to prevent the protected animals from getting an excess challenge from the infected animals, any of the animals that showed signs were quickly removed from the group. </li></ul><ul><li>PD 50 was estimated by Karber’s Method: Statistical analysis of dose-response curves. </li></ul><ul><li>Antibodies to FMDV were detected by liquid- phase blocking enzyme- linked immunosorbant assay (LPB-ELISA) and micro-neutralization assay. </li></ul><ul><li>Potency of the Multiple- Epitope Recombinant Vaccine in Swine </li></ul>
  15. 15. Methods <ul><li>Three sets of the multiple- epitope recombinant vaccines (A-C) were prepared. </li></ul><ul><li>15 swine were split into 3 groups of 5 swine. All groups with vaccinated intramuscularly behind the ear with a full dose of vaccines A-C, respectively. </li></ul><ul><li>After they swine we vaccinated, serum samples were then collected monthly for 7 months. </li></ul><ul><li>Anti-FMDV antibodies were detected by LPB- ELISA. </li></ul><ul><li>There was a correlation determined between the number of dpv and the antibody level. </li></ul><ul><li>Titers of anti- FMDV anti-bodies from all vaccinated swine were then analyzed using the Student t test. </li></ul><ul><li>Duration of Immunity Induced by the Multiple- Epitope Recombinant Protein in Swine </li></ul>
  16. 16. Methods <ul><li>LPB-ELISA </li></ul><ul><ul><li>Each test serum was placed in a well plate and viral antigen that was homologous to the rabbit antisera that coats the plates </li></ul></ul><ul><ul><li>The serum was left overnight and was then transferred to an ELISA plate coated with rabbit anti-FMDV serum </li></ul></ul><ul><ul><li>The plate was washed and the rabbit anti-guinea pig IgG-HRP </li></ul></ul><ul><ul><ul><li>A horseradish peroxidase-conjugated antibody – allows for better detection of the target molecule </li></ul></ul></ul><ul><ul><li>The reaction was terminated by adding 2M H 2 SO 4 and the results were read by a spectrophotometer at 492nm </li></ul></ul>
  17. 17. Methods <ul><li>The statistical analysis was done by statistical comparisons carried out and performed by SPSS software. </li></ul><ul><li>Virus Neutralization Assay </li></ul><ul><ul><li>Follows a procedure outlined in a previous study and titers were calculated using the reciprocal of the last serum dilution </li></ul></ul><ul><li>Lymphocyte Proliferation Assay </li></ul><ul><ul><li>Flow cytometry over Ficoll-Hypaque selects for peripheral blood mononuclear cells </li></ul></ul><ul><li>Virus Isolation from Heparinized Blood and Nasal Swabs </li></ul><ul><ul><li>Follows a procedure outlined in a previous study to separate the virus from both blood samples and nasal swabs </li></ul></ul><ul><li>Detection of Antibodies to NSP of FMDV </li></ul><ul><ul><li>To determine if the swine had produced antibodies for NSP and FMDV, the researchers used a commercially available kit </li></ul></ul>
  18. 18. Results Expression and characterization of recombinant RE-scIgG protein Recombinant expression plasmid pET-22b-RE-scIgG constructed successfully Specific band of 52 kDa could be seen by 12% SDS-PAGE; no band found in lysates of E.coli /pET-22b cells In the Ni affinity column (Lane 5), SDS-PAGE showed recombinant protein purity of 95% FIG. 3: Lane 1: protein molecular size markers, Lane 2: plasmid pET-22b induced with IPTG after 4 hours, Lane 3: recombinant plasmid pET-22b-RE-scIgG induced with IPTG after 4 hours FIG. 4: Lane 1: protein molecular size markers, Lane 2: recombinant plasmid pET-22b-RE-scIgG preinduced with IPTG, Lane 3: recombinant plasmid pET-22b-RE-scIgG induced with IPTG after 6 hours, Lane 4: deposition of recombinant protein RE-scIgG after sonification, Lane 5: protein RE-scIgG purified with Ni-NTA agarose resin
  19. 19. Results <ul><li>Potency of recombinant protein was evaluated according to titers of anti-FMDV specific antibodies in swine </li></ul><ul><li>No drastic differences observed in antibody titers between recombinant vaccine and traditional vaccine </li></ul><ul><li>Recombinant vaccine provided full protection of FMDV O/China/99 strain in swine </li></ul><ul><li>FMDV signs: fever, depression, anorexia, lameness and formation of vesicles were present in all four feet and snout of each individual in control group </li></ul><ul><li>Comparison of the potency of the multiple-epitope recombinant vaccine with a commercial vaccine in swine </li></ul>FIG 5: Lane 1: protein molecular size markers, Lane 2: RE-scIgG reacted with serum negative for FMDV, Lane 3: RE-scIgG reacted with positive serum from cattle infected with FMDV FMDV (Type O) antibodies recognized RE-scIgG
  20. 20. Results <ul><li>Potency of the multiple-epitope recombinant vaccine in swine </li></ul><ul><li>High titers of anti-FMDV antibodies found in swine  titers lower when recombinant vaccine dosage reduced </li></ul><ul><li>One pig given 1/3 dose, three pigs given 1/9 dose </li></ul><ul><ul><li>Clinical signs of FMDV </li></ul></ul><ul><ul><li>Disease onset delayed and disease severity reduced </li></ul></ul><ul><li>Control swine developed clinical signs and vesicles in feet and snout </li></ul>
  21. 21. Results TABLE 2: Titers of antibodies against FMDV and swine challenge results
  22. 22. Results <ul><li>TABLE 3: Titers of antibodies against FMDV, protection ratios, and PD 50 in swine </li></ul><ul><li>PD 50 of 6.47 higher than recommended by OIE </li></ul><ul><li>Virus Neutralization Antibodies: </li></ul><ul><ul><li>According to OIE, it is recommended that swine produce titer values of ≥1.65 log10 when given the virus neutralization test </li></ul></ul><ul><ul><li>Table: </li></ul></ul><ul><ul><ul><li>When given a full dose of multiple-epitope recombinant vaccine all swine met or exceeded the recommended value. </li></ul></ul></ul><ul><ul><ul><li>When given 1/3 of the dose, one swine did not meet the requirement. When given 1/9 of the dose, 3 swine did not meet the requirement. </li></ul></ul></ul>
  23. 23. Results <ul><li>Duration of immunity induced by the multiple-epitope recombinant vaccine in swine </li></ul><ul><li>All 3 sets of recombinant vaccine provided strong immune responses in swine </li></ul><ul><li>High antibody titers lasted more than 120 days, anti-FMDV antibody titers in 30% swine significantly decreased </li></ul><ul><li>No drastic differences between antibody titers obtained from the recombinant vaccines A, B, C in swine were seen </li></ul>
  24. 24. Results <ul><li>TABLE 4: Titers of antibodies to FMDV induced in swine by three batches of vaccine </li></ul><ul><li>Lymphocyte Proliferation Assay: </li></ul><ul><ul><li>A higher percentage of lymphocyte proliferation was obtained with RE-scIgG (recombinant DNA) </li></ul></ul><ul><ul><ul><li>Although the recombinant group differed significantly from the control group, there was no significant difference between the traditional inactivated vaccine and the multiple epitope recombinant vaccine. </li></ul></ul></ul>
  25. 25. Results <ul><li>Virus Isolation from Heparinized Blood and Nasal Swabs: </li></ul><ul><ul><li>Control Group: </li></ul></ul><ul><ul><ul><li>All were positive for virus isolation from plasma and nasal swabs from day 2 to day 9 after the challenge </li></ul></ul></ul><ul><ul><li>Experimental Group: </li></ul></ul><ul><ul><ul><li>4 of 5 that were vaccinated with a full dose were negative for virus isolation from plasma and nasal swabs </li></ul></ul></ul><ul><li>Detection of Antibodies to NSP (nonstructural proteins) of FMDV: </li></ul><ul><ul><li>Control Group: </li></ul></ul><ul><ul><ul><li>In 2 of the 3 pigs – antibodies to NSP of FMDV were detected for the first time at either 7 to 9 days after the infection </li></ul></ul></ul><ul><ul><li>Experimental Group: </li></ul></ul><ul><ul><ul><li>Had been vaccinated with multiple-epitope recombinant vaccine – no detectable antibodies to NSP of FMDV </li></ul></ul></ul>
  26. 26. Discussion <ul><li>Improved recombinant vaccine elicited high titers of anti-FMDV specific antibodies & a high lymphocyte proliferation response </li></ul><ul><li>Single recombinant vaccine achieved long-lasting immunity, for up to 6 months </li></ul><ul><li>Vaccine elicited host immune response through cytokines, specifically interferons </li></ul><ul><li>By increasing the number of antigenic epitopes, the immunogenicity increased significantly </li></ul><ul><li>IgG may prolong half-life of the antigenic epitopes & help deliver peptides to MHC molecules on lymphocytes </li></ul>
  27. 27. Discussion <ul><li>Advantages as alternative to traditional vaccines </li></ul><ul><li>Does not involve use of infectious viral particles </li></ul><ul><li>Recombinant protein can be stored easily </li></ul><ul><li>Recombinant protein is more than 50% of total cellular protein and purity of the target protein is >95% = lower production costs </li></ul><ul><li>Large quantity of protein can be obtained from E.coli in vivo </li></ul><ul><li>Development of recombinant protein is easier than genetic engineering and bioinformatics </li></ul>

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