Appendix 39

Appendix 39

Monoclonal antibody based detection of 3A containing non-structural proteins in Al (OH)3 adjuvanted Foot-and-Mouth disease vaccines

Abstract

Fuat Özyörük, Ünal Parlak, Gülhan Aynagöz, Hidayet Bozoğlu

Foot-and-Mouth Disease Institute, Ankara-Turkey

To detect 3A containing non-structural proteins [3A, 3AB and 3ABC (3A-NSPs)] in foot and mouth disease (FMD) vaccines, and to find out how they are eliminated are required. This study proposes a standard method based on enhanced detection of 3A-NSPs in Al(OH)3 adjuvanted FMD vaccines by an amplified Western blot assay (AWB). Limit of detection of AWB was found 11.5 ng/lane using anti-3A monoclonal antibody (MAb) 2C2 and recombinant 3ABC. Cellular debris (CD) of five baby hamster kidney cells (BHK-21) infected or non-infected with different type of FMDV, and elution fluids acquired from fifteen batches of FMD vaccines were analyzed for their reactivity with MAb 2C2 by AWB. All infected CD were reactive with Mab 2C2 at the molecular weight (MW) of 28 kDa identical to hypothetical MW of NSP-3AB, whereas none of the FMD vaccines tested had bands reactive with Mab 2C2. The results indicated that NSP-3AB was sedimented together with BHK-21CD, and FMD vaccines tested did not contain 3A-NSPs more than 12.3 ng/lane in the context of detection limit.

Introduction

Purity of the FMD vaccine is mainly associated with effective separation of virus in supernatant from CD (1, 4). It is important that vaccine be free from cellular proteins as well as non-structural viral proteins (NSP). Presence of NSPs in FMD vaccines would result in ambiguity whether or not vaccinated animals have FMD history, as NSP antibody response is the most reliable way to differentiate infected/convalescing from vaccinated animals in the field to facilitate prevention of new outbreaks. Many publication have revealed that anti-NSP 3ABC, 3AB, 3A, 2C responses in animals are specific indication of FMD history other than vaccination (2, 3, 8-11, 14). However these findings cannot justify that estabilshment of NSP detection procedures in FMD vaccine plants is unnecessary; because at what stage of the production, how, and how much of these NSPs are eliminated are not known except for 2C. In one study, absence of anti-2C antibodies from the sera of vaccinated animals, due to association of 2C with cellular debris that is separated from the virus harvest during vaccine production, have been reported (8). Present study proposed to establish a standard method based on enhanced detection of 3A-NSPs in FMD vaccines and CD of baby hamster kidney BHK-21 by an anti-3A monoclonal antibody. The results indicated that NSP-3AB was sedimented together with BHK-21 CD, and FMD vaccines examined did not contain 3ANSPs more than 12.3 ng/ml.

Material and methods

Cell cultures and viruses

A continuous cell line, BHK-21 Cl-13 cells, (provided from Animal Cell Culture Laboratory of FMD Institute, Ankara) were used for propagation of FMD vaccine strains currently used in Turkey; A Aydin 96, O1 Manisa and Asia-1. After development of cytopathic effect, cells were harvested by freezing and thawing three times. Alternatively, virus-cell suspensions in the vaccine production were homogenized with 2% percent chloroform at 4 ºC for 1 h followed by sedimentation over night. Clarification was improved by the treatment of diatoma earth and filters with 15, 5, 1.5 and 0.6 μm pore size. 1-2 ml of frozen-thawed cell virus suspension or chloroform treated cell sediment was centrifuged at 5000 rpm for 10 min.. Supernatant was discarded prior to pellet was washed with PBS by vortexing. The last two steps were repeated three times in sequence, and preparations were tested by AWB as described below.

MAb and recombinant protein

IgG1 MAb 2C2 recognizing a linear 3A epitope on FMDV was used to detect 3A-NSPs in vaccines and CDs (3). A recombinant protein, the 3ABC polypeptide of FMDV O1 Kaufbeuren, was used to figure out detection limit of AWB assay (3). Recombinant 3ABC concentration was determined by the combination of bicinchoninic acid assay (Micro BCATM; Pierce), sodium dodecyl sulphate polyacrylamide gel electrophoresis and Integrated Optical Density analysis (GelPro 3.1; Media Cybernetics).

Production of FMD vaccines adsorbed to aluminium hydroxide gel and preparation of elution fluids acquired from vaccines

Al(OH)3 adjuvanted FMD vaccines were produced according to manufacturer’s manual in the context of international standarts (7), and elution fluids containing viral proteins were acquired from aluminium hydroxide gel while concentrated 62 fold, as described previously (5). They were tested by liquid phase blocking ELISA in order to confirm the presence of viral proteins (6).

AWB assay

Recombinant 3ABC, BHK-21 CD and elutes of FMD vaccines were evaluated for their reactivities with anti-3A MAb 2C2 by AWB. Samples were heated at 100ºC for 3 min in 0.03 M tris (pH 6.8) containing 2% SDS, 10% glycerol 0.01% bromphenol blue, and 100 mM dithiothreitol and loaded 30 μl/lane. Then they were subjected to electrophoresis in 4 to 20% SDS-PAGE. The separated proteins were transferred to nitrocellulose membrane (Pierce, Rockford, IL, USA) at 150 V for 1 h with a Mini TransBlot apparatus (Bio-Rad, Hercules, CA, USA). Membrane was blocked with blocking buffer (SuperBlock®; Pierce), reacted with anti-3A MAb 2C2 and secondary goat anti-mouse horseradish peroxidase conjugate(HRP) (Pierce). Binding of HRP conjugates were evaluated with a chemiluminescence reagent (SuperSignal® West Pico, Pierce) developed on X-ray film (X-Omat; Kodak, N.Y., USA).

Results and discussion

Detection limit of AWB assay

To establish a standard assay, recombinant 3ABC with known concentrations was tested in AWB using anti-3A MAb 2C2. As expected, MAb 2C2 was reactive with recombinant 3ABC and bands were visible up to 11.5 ng/lane (Fig. 1. lane 6). This detection limit (11.5 ng/lane)

was found extremely low compared to amount (0.003 ng/lane) stated by previous studies as well as manufacturer of the kit (12, 15). Low sensitivity in AWB remained to be determined.

78 kDa ► 1 2 3 4 5 6

FIG. 1. AWB of anti-3A MAb 2C2 with recombinant 3ABC. Lanes 1-6 were loaded with decreasing amount (ng/lane) of recombinant 3ABC; 390, 185, 92.5, 46.2, 23.1 and 11.5 respectively.

AWB of anti-3A MAb 2C2 with BHK-21 CDs

Previous studies (2, 3, 8-11, 14) revealing the association of NSP-2C with CD, and lack of antibody response against 3A, 3AB, and 3ABC in vaccinated herds let us to hypothesize that 3A-NSPs may as well be captured by CD during clarification. To test this hypothesis, FMD infected or non-infected BHK-21 CDs were analyzed whether they had 3A-NSPs. All examined CDs infected with FMDV had bands at 25-28 kDa reactive with anti-3A MAb 2C2 (Fig. 2. lane 1-4) while no band was observed with non-infected BHK-21 CD (Fig. 2. lane 5). Molecular weights of reactive bands were identical with the hypothetical location of native 3AB (Fig. 2. lane 6). Inferior bands were probably degradation products of 3AB but the possibility of 3A presence among those bands should not be disregarded. On the other hand absence of 3ABC band (corresponding to 48 kDa) reactive with MAb 2C2 could be explained by the fact that this polypeptide is neither translation nor cleavage product in picornaviridae (13).

1 2 3 4 5 6

60

39 28

18.8

FIG. 2. AWB of anti-3A MAb 2C2 with infected or non-infected BHK-21 CDs. All lanes were loaded with BHK-21 CDs either infected with an FMD vaccine strain (lanes1-4) [A Aydin 98, O1 Manisa, Asia-1, and Asia-1 (from production)] or non-infected (lane 5). Theoretical separation of 3ABC (48.1 kDa), 3AB (25.1 kDa) and 3A (17.3 kDa) according to their calculated molecular weights was shown in lane 6. Numbers to the left of each panel are in kilodaltons

AWB of anti-3A MAb 2C2 with elution fluids acquired from FMD vaccines

To determine if there was residual contamination of 3A-NSPs in the detection limit of AWB, elution fluids of FMD vaccines were analyzed in AWB using MAb 2C2. None of the tested FMD vaccines had bands corresponding to the hypothetical locations of 3A-NSPs (fig. 3. lane 1-15). These data indicated NSP 3AB were eliminated during clarification and not leaked through the FMD vaccines tested. If 3A-NSPs were exist but not determined due to out of detection limit, amounts of 3A-NSPs would not be more than 12.3 ng/ml in vaccines, taking to account of loading volume and detection limit. Even this amount seems very low to induce anti-3ABC

antibody response; it is not known how much 3ABC required for antibody response detectable with 3ABC-ELISA kits.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

FIG. 3. AWB of anti-3A MAb 2C2 with elutes acquired from FMDV vaccines. Type O vaccines (serial numbers: 14, 4, 5, 6, 7, 8 and 9) are in lanes 2, 4, 6, 7, 10, 13 and 14. Type A vaccines (serial numbers: 3, 11 and 10) are in lanes 5, 9 and 11. Type Asia-1 vaccines (serial numbers: 13, 15, 12, 2, and 1) are in lanes 1, 3, 8, 12 and 15. BHK-21 CD infected with type A virus as a positive control is in lane 16.

60

39 28

18.8

Acknowledgement

We thank Dr. E. Brocchi and Dr. F. De Simone for evaluating data, and providing MAb 2C2 and recombinant 3ABC protein.

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