Evaluation of the Efficacy of Infectious Bursal Disease Virus Multivalent Virus-Like-Particle

Antigens in Enzyme-Linked Immunosorbent Assays

Kimberly CarterMentor: Dr. Daral Jackwood

Food Animal Health Research Program

Infectious Bursal Disease Virus

• Highly contagious

• Targets immature B

lymphocytes

• Damages bursa of

Fabricius and other

lymphoid organs

• Immunosuppression

Infectious Bursal Disease Virus

Serotype 1

Classic Variant

Serotype 2

There are many different variant strains!

Antigenic Drift

Variations in P loops

New variant strains

VP2 Protein

Significance of Antigenic Drift

• Antibodies against classic strains cannot neutralize variant strains and vice versa

• Some antibodies against variant strains cannot be detected by an ELISA

Potential of multivalent virus-like particles

• Co-expression of pVP2

and VP3 produces

virus-like particles

(VLPs)

• Co-expression of

variant and classic

pVP2s produces

multivalent VLPs

Objective: Compare the efficacy of multivalent VLP antigens in ELISAs to the efficacy of antigens found in commercially-produced ELISAs in detecting classic and variant IBDV antibodies

Hypothesis: Multivalent VLP antigens in ELISAs will yield more positive results using known chicken anti-IBDV sera than commercial ELISA kits

Materials and Methods: Multivalent VLP Antigen Production

VP3 pVP2 Classic

pVP2 Variant

Sf9 insect cells

IBDV Multivalent VLP antigens

Materials and Methods: ELISA

96 well flat-bottomed plate

IBDV multivalent VLP antigens

96 well flat-bottomed plate

Various chicken anti-IBDV serum samples

96 well flat-bottomed plate

Peroxidase-labeled goat anti-chicken immunoglobulin G(conjugate)

ELISA (continued)

96 well flat-bottomed plate

ABTS substrate added and converted to a detectable product by the conjugate

96 well flat-bottomed plate

Stop solution added after 15 minutes to stop color development

Materials and Methods: Optical Density Measurement

• Plates were read on an ELISA reader at 405 nm

Results: FD181 VP3, pf33 pVP2, T1 pVP2, and pp34 pVP2 antigen

Vaxxitek 10 Dy14

Vaxxitek 5 Dg14

Control 1

0 Dy14

Control 9

Dy14

Control 8

Dg14

Control 6

Dg14

Control 5

Dy14

Control 4

Dy14

Control 3

Dy14

Control 2

Dg14

Control 3

Dy22

Control 4

Dy280.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

1.000

100200400800160032006400Control

Opti

cal D

ensi

ty (O

D40

5)

Vertical bars represent the mean of 12 serum samples and their dilutions from 1:100-1:6400.Horizontal bar represents 2 standard deviations above the negative control sera.

Results:FD181 VP3, Mo195 pVP2, T1 pVP2, and pp34 pVP2

Vaxxitek 10 Dy14

Vaxxitek 5 Dg14

Control 1

0 Dy14

Control 9

Dy14

Control 8

Dg14

Control 6

Dg14

Control 5

Dy14

Control 4

Dy14

Control 3

Dy14

Control 2

Dg14

Control 3

Dy22

Control 4

Dy280.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

1.000

1.100

100200400800160032006400Control

Opti

cal D

ensit

y (O

D405

)

Vertical bars represent the mean of 12 serum samples and their dilutions from 1:100-1:6400.Horizontal bar represents 2 standard deviations above the negative control sera.

Results:FD181 VP3, Mo195 pVP2, pf33 pVP2, T1 pVP2, and pp34 pVP2

Vaxxitek 10 Dy14

Vaxxitek 5 Dg14

Control 1

0 Dy14

Control 9

Dy14

Control 8

Dg14

Control 6

Dg14

Control 5

Dy14

Control 4

Dy14

Control 3

Dy14

Control 2

Dg14

Control 3

Dy22

Control 4

Dy280.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

1.000

100200400800160032006400Control

Opti

cal D

ensit

y (O

D405

)

Vertical bars represent the mean of 12 serum samples and their dilutions from 1:100-1:6400.Horizontal bar represents 2 standard deviations above the negative control sera.

Results:FD181 VP3, Mo195 pVP2, and pp34 pVP2

Vaxxitek 10 Dy14

Vaxxitek 5 Dg14

Control 1

0 Dy14

Control 9

Dy14

Control 8

Dg14

Control 6

Dg14

Control 5

Dy14

Control 4

Dy14

Control 3

Dy14

Control 2

Dg14

Control 3

Dy22

Control 4

Dy280.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

100200400800160032006400Control

Opti

cal D

ensit

y (O

D405

)

Vertical bars represent the mean of 12 serum samples and their dilutions from 1:100-1:6400.Horizontal bar represents 2 standard deviations above the negative control sera.

Conclusions and Future Directions

• Effective in yielding positive results at a dilution of 1:100–Not nearly as effective at higher dilutions

• FD181 VP3, Mo195 pVP2, and pp34 pVP2 antigen appeared to be most effective–Unexpected

• Commercially-produced ELISAs need to be conducted– Are ELISAs containing multivalent VLP

antigens more effective than commercial ELISA kits?

Multivalent vs. Monovalent VLPs• IBDV monovalent VLP antigens yielded

more positive results at higher dilutions– Positive results at a 1:200 dilution in a majority

of serum samples– Multiple positive results at a dilution of 1:400

• Suggests monovalent VLP antigens are more effective than multivalent VLP antigens

ReferencesBenton, W. J., M.S. Cover, J. K. Rosenberger, and R. S. Lake. Physicochemical properties of the infectious bursal agent (IBA). Avian Dis. 11:438-

445. 1967.

Briggs, D. J., C. E. Whitfill, J. K. Skeeles, J. D. Story, and K. D. Reed. Application of the positive/negative ratio method of analysis to quantitate

antibody responses to infectious bursal disease virus using a commercially available ELISA. Avian Dis 30(1):216-218.1986.

Cheville, N. F. Studies on the pathogenesis of Gumboro disease in the bursa of Fabricius, spleen, and the thymus of the chicken. Am. J. Path.

51:527-551.1967.

Coulibaly, F., C. Chevalier, I. Gutsche, J. Pous, J. Navaza, S. Bressanelli, B. Delmas, and F. A. Rey. The birnavirus crystal structure reveals

structural relationships among icosahedral viruses. Cell. 120:761-772. 2005.

Faragher, J. T., W. H. Allan, and P. J. Wyeth. Immunosuppressive effect of infectious bursal agent on vaccination against Newcastle disease. Vet.

Rec. 95:385-388. 1974.

Ismail, N. M. and Y. M. Saif. Differentiation between antibodies to serotypes 1 and 2 infectious bursal disease viruses in chicken sera. Avian Dis.

1002-1004. 1990.

Ismail, N. M., Y. M. Saif, W. L. Wigle, G. B. Havenstein, and C. Jackson. Infectious bursal disease virus variant from commercial leghorn pullets.

Avian Dis. 34:141-145. 1990.

Jackwood, D. J. Multivalent virus-like particle vaccine protects against classic and variant infectious bursal disease viruses. Avian Dis. 57:41-50.

2013.

Jackwood, D. J., Y. M. Saif, and P. D. Moorhead. Immunogenicity and antigenicity of infectious bursal disease virus serotypes I and II in chickens.

Avian Dis. 29(4):1184-1194. 1985.

Jackwood, D. H. and Y. M. Saif. Antigenic diversity of infectious bursal disease viruses. Avian Dis. 31:766-770. 1987.

Kibenge, F. S. B., A. S. Dhillon, and R. G. Russell. Biochemistry and immunology of infectious bursal disease virus. J. Gen. Virol. 69:1757-1775.

1988.

Letzel, T., F. Coulibaly, F. A. Rey, B. Delmas, E. Jagt, A. A. M. W. van Loon, and E. Mundt. Molecular and structural bases for the antigenicity of

VP2 of infectious bursal disease virus. J. Virol. 81(23):12827-12835. 2007.

Müller, H. Replication of infectious bursal disease virus in lymphoid cells. Arch. Virol. 87:191-203. 1986.

Oña, A., D. Luque, F. Abaitua, A. Maraver, J. R. Castón, and J. F. Rodríguez. The C-terminal domain of the pVP2 precursor is essential for the

interaction between VP2 and VP3, the capsid polypeptides of infectious bursal disease virus. Virology. 322:135- 142. 2004.

Thayer, S. G., P. Villegas, and O. J. Fletcher. Comparison of two commercial enzyme-linked immunosorbent assays and conventional methods for

avian serology. Avian Dis. 31(1):120-124. 1987.

van den Berg, T. P., N. Eterradossi, D. Toquin, G. Meulemans. Infectious bursal disease (Gumboro disease). Rev. Sci. Tech. Off. Int. Epiz.

19(2):527-543. 2000.

Acknowledgements

• Dr. Daral Jackwood• Fellow colleagues in

Dr. Jackwood’s lab• All personnel in the

Food Animal Health Research Program

• All who make ORIP possible

Questions?