Appendix 41

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Appendix 41

Peparation of synthetic peptide FMD vaccine with newly developed antigen- polymere conjugates be used as immonogen and vaccine in veterinary medicine

S. I. Deliloglu Gürhan1, M. Mustafaev Akdeste2, Z. Mustafaeva Akdeste2, G. Aynagöz3, G. Unver 4 , N. Unal3, N. Celik3

1E.U. Bioengineering Dep., 35100 Bornova IZMIR- TURKEY, 2TUBITAK MAM GMBAE, Gebze KOCAELI- TURKEY, 3Foot and Mouth Disease Institute, PO Box 714, 06044 ANKARA- TURKEY, 4Veterinary Control and Research Institute, 35100 Bornova IZMIR- TURKEY

ABSTRACT

Preparation of newly developed immunogens and vaccine preparation by conjugation of synthetic polymers and peptide antigens of 40-60 and 135-160 amino acid sequences of immunogenic VP1 capsid protein of "A" type FMDV which causes epidemics in Turkey was the aim of this project. Thus, by the modification of the immunogenisity of the antigens, development of the new FMD vaccines, diagnostic reagents, pharmaceuticals and biotechnological preparations was considered.

The peptide chains were prepared by Fmoc-solid- phase synthesis chemistry. Electrostatic polycomplexes (EC) of bovine serum albumin- polypeptide conjugates (BSA.P) with polycations-copolymers of 4- vinyl- N- ethyl pyridine and 4- vinyl- N- cetylpyridine bromides and Cu2+ - induced ternary polycomplexes with anionic copolymers of acrylic acid with Nisopropyl- acryl amide and N- vinyl- pyrrolidone (PE) were used. A protein- peptide and synthetic polyelectrolyte (PE) - peptide conjugates consist of the samples of the different domains of foot-and-mouth disease virus (FMDV) VP1 which covalently bound to bovine serum albumin (BSA), polyacrilic acid (PAA), copolymers of acrylic acid (AA) with Nisopropylacrylamide (NIPAAm) (CP1) and N- vinylpyrolidone (CP2). For preparation of the conjugation systems Na- (2- ethylhexsyl) sulphosuccinate and octane were used as surface active material and regular organic phase respectively.

Primarily four different synthetic peptide vaccines prepared as described above were tested in mice for their antibody stimulating efficiency and in guinea-pigs for protection values against homolog challenge virus strain. The vaccine (VAC2) demonstrated minimum side effects in guinea-pigs and highest protection against the challenge virus was selected for vaccination of cattle. Peptide specific and intact virus specific antibody responses were demonstrated by enzyme- linked immunosorbant assay (ELISA) and neutralisation test (NT) with the 14 and 21 day post vaccination sera of the cattle vaccinated with (VAC2).

The results indicated that VAC2 is able to protect guinea-pigs against "A" type FMDV challenge but not induce sufficient specific antibody against neither the synthetic peptides nor to intact virus in cattle. However, some of the animals responded to the synthetic vaccine but the antibody level estimated with both LPB ELISA and NT was not in acceptable level for protection. Only one cattle performed VAC2 induced immune response just below the protection limit (NI=1.2) even 14 days post vaccination. It is clear that some quantitative and

qualitative modifications such as addition of T-cell epitope sequences or increasing the quantity of the peptide in the vaccine formula will aid to induce immune response in cattle.

In the author’s knowledge, this work is the first synthetic peptide vaccine trial in Turkey.

INTRODUCTION

The suggestion using synthetic peptides as foot-and-mouth disease (FMD) vaccine was made after the determination of the high neutralisation titre with one segment of VP1 about 20 years ago. In parallel with the developments in technology, at present, synthesis of some amino acid domains of VP1 can be realised rapidly and in a correct form [20]. Generally, the target is preparation of vaccine or diagnostic material for FMD.

The protection effect of synthesised biocojugates as a mixture in oil emulsions were detected in the immunisation of the animals [9]. In these studies the polypeptides synthesised as antigen were primarily coupled to a protein carrier then this conjugate was mixed with an oil adjuvant (e.g.: VP1-protein + IFA).

A number of researches are being carried out to develop synthetic peptide vaccines against viral pathogens including FMDV. Most of them are able to protect laboratory animals such as mice, rabbits and guinea pigs. However, there is no such vaccine which can be used in the field for the farm animals yet [6, 8, 17, 19, 25, 28].

The first synthetic peptide vaccine against an animal disease which fully protects the target animal is for the parvovirus infection of dogs [14]. Recently, edible synthetic peptide vaccines prepared by the expression of VP1 of FMDV to transgenic plants are developed [7, 23].

In fact, synthetic peptides are not strong antigens. They need to be conjugated to a suitable adjuvant. Most of the vaccine development studies are focussed on the preparation of a good effective adjuvant [18]. The adjuvant is very important as well as inoculation route, dose and formulation of the antigen. If a suitable adjuvant was selected, controlled release of the antigen and, as a result of it, long lasting immunity with single inoculation can be achieved [16].

The 200-213 and 140-160 amino acid residues of VP1 protein conjugated with key hole limpet haemocyanine could stimulate neutralising antibody response and protect the guinea pigs against FMDV challenge [5, 10]. Higher peptide mass (5mg) and boosting the cattle could stimulate antibody response in cattle. However, complete protection against intact virus was not reported [24].

Although the hopeful results were reported with modified vaccines, biosynthetic vaccines and synthetic peptide vaccines none of them can compete with the conventional inactivated vaccines yet. Additionally, researches on the modified vaccines lost their importance after the prohibition of EC countries the importation of the meat from the countries where modified intact virus vaccines are applied [13].

Recently, a synthetic peptide vaccine against O type FMDV effective in swine was reported [30]. Some others are also carrying out the field trials [12].

In addition to the receptor sites for protein antibodies, the unbound peptides can interact with helper T cell receptors and Ia antigens. Starting from this fact it was hypothesized that the protection problem of cattle could be solved by the addition of an extra T cell epitope into the vaccine formulation [11]. Another encouraging technique is utilisation of the non immunogenic synthetic polyelectrolyte (PE) as antigen carriers [21].

In the present study, a novel approach to a totally synthetic vaccine, which consists of FMDV VP1 peptides (40-60 and 135-160 residues), prepared by chemical synthesis and nonimmunogenic membrane active polyelectrolyte (PE) is reported.

MATERIAL AND METHODS

Peptide Synthesis:

40- 60 and 135- 160 amino acid residues of "A" Aydin 98 (A Iran 96) serotype of FMDV were synthesized by Merrifield solid-phase technique with an automatic synthesizer (Millipore’ s Automated Peptide Synthesizer, USA) [23, 26]. Those peptides were purified and characterized with chromatographic, spectroscopic and fluorimetric analysis. The experiments were repeated with the same sequences synthesized by SIGMA GENO§YS (USA).

P1 40-60 (21 mere) Val-Lys-Ile-Asn-Asn-Thr-Ser-Pro-Thr-His-Val-Ile-Asp-Leu-Met-Gln-Thr-His-Gln-His-Gly

P3 135-160(26 mere) Lys-Tyr-Ser-Ala-Thr-Gly-Glu-Arg-Thr-Arg-Gly-Asp-Leu-Gly-Ala-Leu-Ala-Ala-Arg-Val-Ala-Thr-Gln-LeuPro-Ala Table 1. : Synthesised amino acid sequences of A Aydın98 FMDV strain [1].

Polyelectrolyte Sythesis: Cationic polyelectrolytes (PE) are the copolymers of 4-vinyl-Nethylpyridine (PEVP) and 4-vinyl-N-cethylpyridine (PECVP). PE was obtained by quaternization of narrow fractions of poly-4-vinylpyridine (Pn = 103) with ethyl and cethylbromides by the method previously desribed [15]. The anionic PEs are polyacrylic acid (PAA), copolymers of acrylic acid (AA) with N-isopropylacrylamide (NIPAAm) (CP1) and N-vinylpyrolidone (VP) (CP2). CP1 was prepared by the radical copolymerization of AA with NIPAAm in the presence of 2-oxoglutaric acid as a UV initiator by irradiation of UV light (365 nm) under nitrogen at room temperature. CP2 was obtained by the radical copolymerization of AA with VP in a methanol solution in presence of a radical initiatorazodinitrile-bis-isobutiric acid at 60 0C. The copolymers obtained were purified by three precipitations from methanol into ether.

Polyelectrolyte-peptide and protein-peptide conjugate synthesis: To carry out PE-peptide (PE.Pep) and BSA-peptide (BSA.Pep) conjugation reactions, surfactant stabilized micro emulsion of water in organic solvents, i.e. hydrated reversed micelle (HRM) systems (BSA –Bovine Serum Albumin Mw = 70000, pI = 4.9, from Sigma Chemical Co., St.Louis, MO) was used. Using the HRM technique, a series of protein-peptide and PE-peptide conjugates were prepared including FMDV VP1 epitopes of different regions which were covalently bounded to BSA, CP1 and CP2.

Polyelectrolyte complex synthesis: To prepare a PE-BSA.Pep electrostatic complexes, various concentrations of the BSA.Pep conjugate solutions were added to PEVP (or PECVP), dissolved in phosphate buffer (PBS), pH 7.2. To produce the PE-Cu2+ complex, the

CuSO4.5H2O (pH 4) solution was added to PE, dissolved in PBS. The desired pH values were adjusted with 1 M NaOH. The ternary PE- Cu2+ -protein complexes were, in turn, prepared by adding BSA.Pep conjugate solution to the PE- Cu2+ solution. Both of the amino acid sequences (P1 and P3) coupled to 4 different polymeric complexes (CP1, CP2, PECVP and CP-Cu2+ -BSA). Thus, 4 different vaccine compositions were prepared (VAC1, VAC2, VAC3 and VAC4).

VAC1 VAC2 VAC3 VAC4

(CP1-P1) + (CP1-P3) (CP2-P1) + (CP2-P3) (PECVP-BSA.P1) + (PECVP-BSA.P3) (CP-Cu2+ -BSA.P1) + (CP-Cu2+ - BSA.P3)

Table 2: Composition of the synthetic peptide vaccines.

Side Effects in Guinea pigs: 4 animals for each vaccine were inoculated s.c. (2ml/ animal). The local and general reactions were detected and recorded during the 1 month of inspection period.

Side Effects in Cattle: VAC2 which passed both guinea pig tests was inoculated to 13 cattle. The local and systemic adverse reactions were inspected clinically for 7 days.

Immunisation Studies:

Vaccine Doses: Synthetic peptide quantities of vaccines which used in immunisation and potency trials are summarized in Table 3.

Animal

Mouse

P1 (µg/dose)

50 Guinea-pig 500 Cattle 1500

P3 (µg/dose)

50 500 1500

P1 + P3 (µg/dose)

100 1000 3000

Vaccine dose (ml)

0.2 2 2

Table 3: Final synthetic peptide concentration of one vaccine dose for animals.

Mouse: 8 week old Balb/c mice were immunized intra venous (i.v.) with 4 vaccine candidates. Synthetic peptide combination (P1+P3) was used as negative control. Animals were bled at weekly intervals and antibody titres were estimated with indirect ELISA.

Cattle: 10 cattle (in a fattening farm near Ankara) were inoculated with VAC2 s.c. Animals were bled before vaccination (on the day of vaccination), 14th and 21st days pv.. Antibody response against both synthetic peptides and intact virus were tested with indirect ELISA and LPB ELISA respectively. Development of neutralizing antibodies against whole virus was also detected with NT with BHK cells.

Protection Test: Guinea-pig protection test was applied as described by Barnett, CARABİN 2002 AND Wotzler et al 2002. [3, 31]. Groups of four animals were immunized with twofold dilutions of the 4 vaccine candidates and an aluminium hydroxide-saponine adjuvant vaccine prepared with the inactivated A98 virus as control. Immunization was done subcutaneously 2ml vaccine/ guinea-pig where the final synthetic peptide concentration in each dilution was 1 mg, 0.5mg, 0.25mg and 0.125mg. 21 days post vaccination animals were challenged with 400 guinea-pig ID50 "A" 98 virus/ animal. A group of four unvaccinated guinea-pigs was also infected with the challenge virus as control.

RESULTS AND DISCUSSION

Vaccine Site Reactions in Guinea-pigs: Four vaccine formulations were tested in guineapigs. There was abnormal reaction in the animals vaccinated with VAC1 and VAC3. A local hyperaemia in the inoculation zone was detected for 1-2 days in the animals after vaccination with VAC2. Nevertheless, severe local and systemic reactions appeared just a few minutes after inoculation of VAC4. In coordinated pace, loss of appetite for 1 day, apses with large hyperaemia and large swelling zone in the inoculation area. This severe side effects with VAC4 were attributed its Cu+2 content of the polymer.

Vaccine Site Reactions in Cattle: All of the cattle were inspected for 1 week post vaccination. Mild reactions were detected such as increase in body temperature of two animals (39.1-40.1) for 1 or 2 days and a small lump with 30-40 mm Ø insensitive to pressure lasting for 7 days maximum.

Immunogenicity:

Mice: The mice immunised intravenously with adjuvant-free polypeptide were not responded to the antigen. Whereas, primary peptide specific immune response increased in the first 7 days and the titres were steady up to 23rd day p.v. in the animals vaccinated intravenously with PE-polypeptide conjugates (Table 4). Since peptides are known as poor immunogens, unresponsiveness to the peptides without adjuvant was expected. The highest antibody level was detected in the sera of the mice inoculated with VAC2 14 days post vaccination and starting from the 21. day pv peptide specific antibody titre decreased gradually.

In fact, the characteristics of antibody development and the level of immune response was not dependant with the preparation method of the biopolymer systems and the structure of the polymer carriers.

Potency in Guinea-pigs: Guinea-pig potency test is still an acceptable and reliable method in determination of the potency of the FMD vaccines [4]. Starting from that point, 4 PEpolypeptide conjugates (VAC1, VAC2, VAC3, and VAC4) which developed the immunogenic activity in the mice were selected to be used in guinea-pig potency test. The conventional vaccine, with Al (OH)3 adjuvant, inactivated virus which contains 8.9µg 146S antigen was protected all of the animals. Guinea-pigs vaccinated with VAC2 which contains 1mg synthetic peptide/dose was also protected the entire animal. The protection ratio in the animals vaccinated with the same dose (1mg) of peptide conjugated with different polymers (VAC3 and VAC4) was ¾. However, VAC1 developed weak protection. The importance of the adjuvant in the potency of the inactivated or subunit vaccines is a well known reality [11, 20]. For that reason, recently most of the vaccine development studies are targeted to find out more effective adjuvant with minimum side effects. Also in the present study, guinea-pigs vaccinated with the same quantity of synthetic peptides conjugated with different PE’s were protected against the same quantity of the virus in different levels (Table 4).

VAC2 was selected as primary candidate for the further experiments because of its higher protective capacity and lower toxicity in lab animals.

Vaccine Trial with Cattle: 15-18 months old 10 cattle were vaccinated subcutaneously with VAC2 which contain 3mg synthetic peptide / doses. Animals were bled 14 and 21 days post vaccination. Antibody response to homolog intact virus was evaluated with both LPB-ELISA

and NT in BHK21 cell line. Although there was a mild increase in the titres of 8 animals out of 10, none of them passed over the acceptable levels. Since, NI 0.9-1.3 considered uncertain. Only one cattle (ear tag No.99) could reach up to that level with NI 1.2 in 14th day p.v. However, to be honest, this animal was probably primed before vaccination (NI 0.3 at day 0).

In the present study vaccination of the guinea-pigs with 1mg of PE- conjugated synthetic peptides (40-60 and 135-160 amino acid. residues of VP1) developed complete protection. Antibody response in mice with 100µg of the same peptide vaccine conferred the effectiveness of preparation. Nevertheless, 2 ml vaccine with 2 mg synthetic peptide was not sufficient to develop antibody response against homolog virus.

Antoni et al (1988) showed that the cattle with high antibody titre at 21st day p.v. against synthetic peptide vaccine developed generalised lesions after challenge with 10.000 ID50 of the homolog virus [2]. Contrary, in some experiments some of the cattle with insufficient antibody titre could be protected after challenge [27]. It is clear that there is some other factors play important role in the protection mechanism of the animals. This can be cellular immunity or other type immunological responses [3].

For this study increasing the quantity of the synthetic peptide per dose could also be a solution to the problem. Tam et al (1989) showed that sufficient protection in cattle could be achieved with 5mg synthetic peptide [24]. Another alternative is preparation of new vaccine combinations with some additional amino acid residues. Volpina el al (1999) declared that besides mice, guinea-pigs and rabbits also sheep and cattle responded to the vaccination with synthetic peptide vaccines contain 170-188 amino acid residues of VP1 protein of A type FMDV [29]. The last solution but not the least is to make some modifications in synthetic polymer composition and coupling mechanisms.

Figure 1: Peptide specific antibody formation dynamics in the mice immunised with biopolymer systems combined by two polypeptides (40-60 and 135-160 sequences) containing PE-peptide conjugates, electrostatic and ion coordination bonds (Cu2+). (1■) VAC1, (2●) VAC2, (3▲) VAC3, (4▼) VAC4, (5♦) P1+P3.

TESTS

Mice

Peptide specific Ab response (indirect ELISA)

Guinea-pigs

Side effects

Potency

Side effects

Cattle

Virus specific Ab response (NT&ELISA) Potency *number of protected guinea-pigs /number of challenged ND- not done

RESULTS

VAC1 –VAC2 + VAC3 + VAC4 ND VAC1 no VAC2 negligible (1-2 days) VAC3 no VAC4 sever V AC1 – (0/4)* ≤2ml (≤1mg) VAC2 + (4/4)* 2ml (1mg) VAC3 + (3/4)* 2ml (1mg) VAC4 + (3/4)* 2ml (1mg)

VAC2 no ± ND

Table 4: Summary evaluation of the control tests applied to the synthetic peptide vaccines.

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