+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 TechnologyClinical and Vaccine Immunology:January 2011

+Background Foot-and-Mouth Disease Virus (FMDV)

Foot-and-Mouth Disease is a highly contagious viral disease of cloven-hoofed ruminants (i.e.: cattle, swine, etc.)

The virus is a member of the genus Aphthovirus and of the Picornaviridae family

Characterized by fever and blister-like lesions followed by erosions on the tongue, mouth, teats and between the hooves

Most affected animals recover but not without severe losses of meat and milk (economically unfavorable)

Signs of virus can appear after and incubation period of 1-8 days and survives in lymph nodes and bone marrow at neutral pH

Can persist for up to a month and is very difficult to control

Spreads via animals, people, or materials that come into physical contact with susceptible animals

+Background Foot-and-Mouth Disease Virus (FMDV) Continues

Recent outbreaks still occur mostly in the UK and Taiwan

Cause extreme drop in meat consumption

The last of the nine outbreaks of Foot-and-Mouth Disease in the US was in 1929

+Current Treatments

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.

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

Vaccines do not induce sterile immunity and therefore do not prevent carrier status and necessitate frequent revaccination

In production of these vaccines there is a risk of escape of live virus from biosafety facilities

+Modified FMDV Type O Vaccine

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

Immunogenicity (how well a vaccine elicits an immune response) of these vaccines was significantly lower than traditional vaccines

Could be due to rapid clearance of recombinant proteins and the lack of T-helper cell epitopes

In this study, to develop a completely safe replacement for traditional vaccines, a recombinant vaccine against FMDV type O was modified

Organism tested: Swine

Vaccine Manufacture Method: Escherichia coli

+Methods

- 46 Swine free of antibodies against structural proteins and 3ABC nonstructural proteins

- Experiment One: measured the activity of the multiple epitope recombinant vaccine

- Experiment Two: 50% pig protective dose was determined

- Experiment Three: determined how long the vaccine lasted for

- All experiments were done in areas of high-containment

- All pig pens were separated with their own ventilation systems avoiding subject contamination

+Methods

Design and Synthesis of a tandem-repeat multiple-epitope gene:

-402 bp in length, containing 3 copies of each sequence-Synthesized: 141 to 160, 200 to 213 aa, 141 to 160, 200 to 213 aa, 141 to 160, 200 to 213 aa-GGSSGG was used to separate the sequences

+Methods

PCR amplification of the swine immunoglobin G heavy-chain constant region (scIgG) and RE gene:

- RE and scIgG genes were amplified separately through PCR with specific primers

- 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

- 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

- Purified using an agarose DNA purification kit and stored at -20°C

+

Methods

Construction of a recombinant RE-scIG expression plasmid:

- Amplified by overlapping PCR with specific primers- 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- 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- Purified and subcloned into the expression vector, pET-22b, which

resulted in a recombinant expression plasmid, pET-22b-RE-scIgG- Plasmid was confirmed by BamHI/HindIII digestion

+Methods

Expression and purification of recombinant protein:

- Grown in E. coli in 100mL of LB medium at 37°C overnight in a shaker

- Growth was monitored by optical density and expression was induced with .8mM isopropyl-beta-D-thiogalacytopyranoside

- RE-scIgG expressed in E. coli

- Insoluble proteins were placed in 6M urea overnight

- Concentration of each protein determined by the Bio-Rad Protein Assay

- 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

+Materials & Methods

Used to test for the immunoreactivity of the recombinant protein.

Steps: 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.

2. The protein was then washed 3 times with PBS, that contained 0.05% Tween 20 (PBST).

3. The Membrane was incubated with a 1:500 dilution of positive serum from cattle infected with FML type O.

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.

5. After being washed 5 times with the PBST, the signals developed with 3,3’ 5,5’- tetramethylbenzidine.

Western Blotting

+Methods

Optimal Concentration of recombinant protein: 0.25 mg/dose

Recombinant protein then was emulsified with Montanide ISA 206, this prepared the protein for the double emulsion formulation.

Protein was then diluted to a final concentration of 0.5 mg/ml and mixed with the same volume of oil adjuvant.

FINSIHED VACCINE FORMULATION: 0.25 mg of purified protein per ml and was stored at 4°C until use.

Preparation of Multiple- Epitope Recombinant Vaccine

+Methods

13 Swine were broken up into 3 groups.

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.

Group 2: vaccinated with full dose of the multiple- epitope recombinant vaccine.

Group 3: Consisted of 3 swine that only received 1 ml of PBS in oil adjuvant.

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 103 ID50 of the O/ China/ 99 Strain of FMDV.

The swine’s rectal temperatures and clinical signs were monitored for 10 days.

Comparison of Potency of the Multiple- Epitope Recombinant Vaccine with a Traditional Inactivated Vaccine in Swine

+Methods

18 swine were split into 4 groups and used to determine the PD50 of the vaccine in swine.

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.

Group 4: was given 1 ml PBS in oil adjuvant.

30 days after being injected the swine were all bled and challenged.

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.

PD50 was estimated by Karber’s Method: Statistical analysis of dose-response curves.

Antibodies to FMDV were detected by liquid- phase blocking enzyme- linked immunosorbant assay (LPB-ELISA) and micro-neutralization assay.

Potency of the Multiple- Epitope Recombinant Vaccine in Swine

+Methods

Three sets of the multiple- epitope recombinant vaccines (A-C) were prepared.

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.

After they swine we vaccinated, serum samples were then collected monthly for 7 months.

Anti-FMDV antibodies were detected by LPB- ELISA.

There was a correlation determined between the number of dpv and the antibody level.

Titers of anti- FMDV anti-bodies from all vaccinated swine were then analyzed using the Student t test.

Duration of Immunity Induced by the Multiple- Epitope Recombinant Protein in Swine

+Methods

LPB-ELISA Each test serum was placed in a well plate and viral antigen

that was homologous to the rabbit antisera that coats the plates

The serum was left overnight and was then transferred to an ELISA plate coated with rabbit anti-FMDV serum

The plate was washed and the rabbit anti-guinea pig IgG-HRP A horseradish peroxidase-conjugated antibody – allows

for better detection of the target molecule The reaction was terminated by adding 2M H2SO4 and the

results were read by a spectrophotometer at 492nm

+Methods

The statistical analysis was done by statistical comparisons carried out and performed by SPSS software.

Virus Neutralization Assay Follows a procedure outlined in a previous study and titers were

calculated using the reciprocal of the last serum dilution

Lymphocyte Proliferation Assay Flow cytometry over Ficoll-Hypaque selects for peripheral blood

mononuclear cells

Virus Isolation from Heparinized Blood and Nasal Swabs Follows a procedure outlined in a previous study to separate the

virus from both blood samples and nasal swabs

Detection of Antibodies to NSP of FMDV To determine if the swine had produced antibodies for NSP and

FMDV, the researchers used a commercially available kit

+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

+Results

• Potency of recombinant protein was evaluated according to titers of anti-FMDV specific antibodies in swine

• No drastic differences observed in antibody titers between recombinant vaccine and traditional vaccine

• Recombinant vaccine provided full protection of FMDV O/China/99 strain in swine

• FMDV signs: fever, depression, anorexia, lameness and formation of vesicles were present in all four feet and snout of each individual in control group

Comparison of the potency of the multiple-epitope recombinant vaccine with a commercial vaccine in swine

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

+Results

Potency of the multiple-epitope recombinant vaccine in swine

High titers of anti-FMDV antibodies found in swine titers lower when recombinant vaccine dosage reduced

One pig given 1/3 dose, three pigs given 1/9 dose Clinical signs of FMDV Disease onset delayed and disease severity reduced

Control swine developed clinical signs and vesicles in feet and snout

+Results

TABLE 2: Titers of antibodies against FMDV and swine challenge results

+ ResultsTABLE 3: Titers of antibodies against FMDV, protection ratios, and PD50 in swine

PD50 of 6.47 higher than recommended by OIE

Virus Neutralization Antibodies:

–According to OIE, it is recommended that swine produce titer values of ≥1.65 log10 when given the virus neutralization test

–Table:

•When given a full dose of multiple-epitope recombinant vaccine all swine met or exceeded the recommended value.

•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.

+Results

Duration of immunity induced by the multiple-epitope recombinant vaccine in swine

All 3 sets of recombinant vaccine provided strong immune responses in swine

High antibody titers lasted more than 120 days, anti-FMDV antibody titers in 30% swine significantly decreased

No drastic differences between antibody titers obtained from the recombinant vaccines A, B, C in swine were seen

+ Results

TABLE 4: Titers of antibodies to FMDV induced in swine by three batches of vaccine

Lymphocyte Proliferation Assay:

A higher percentage of lymphocyte proliferation was obtained with RE-scIgG (recombinant DNA)

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.

+Results

Virus Isolation from Heparinized Blood and Nasal Swabs: Control Group:

All were positive for virus isolation from plasma and nasal swabs from day 2 to day 9 after the challenge

Experimental Group: 4 of 5 that were vaccinated with a full dose were negative for

virus isolation from plasma and nasal swabs

Detection of Antibodies to NSP (nonstructural proteins) of FMDV:

Control Group: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

Experimental Group:Had been vaccinated with multiple-epitope recombinant vaccine – no detectable antibodies to NSP of FMDV

+Discussion

Improved recombinant vaccine elicited high titers of anti-FMDV specific antibodies & a high lymphocyte proliferation response

Single recombinant vaccine achieved long-lasting immunity, for up to 6 months

Vaccine elicited host immune response through cytokines, specifically interferons

By increasing the number of antigenic epitopes, the immunogenicity increased significantly

IgG may prolong half-life of the antigenic epitopes & help deliver peptides to MHC molecules on lymphocytes

+

Discussion

Advantages as alternative to traditional vaccines

Does not involve use of infectious viral particles

Recombinant protein can be stored easily

Recombinant protein is more than 50% of total cellular protein and purity of the target protein is >95% = lower production costs

Large quantity of protein can be obtained from E.coli in vivo

Development of recombinant protein is easier than genetic engineering and bioinformatics