Examination of the H5N1 avian influenza virus epitopes for differentiation between infected and vaccinated animals

Abstract

© 2015 Dr. Maria Theresia Khrisdiana PutriAvian Influenza Virus H5N1 has been a significant pathogen in veterinary and public health since it first appearance in 1996, particularly in countries such as Indonesia where the virus has become endemic. The disease eradication by vaccination has reduced the risk of clinical disease and mortality in the poultry flocks, however full protection has not been achieved. Furthermore, monitoring new infection by serological method has been challenging due to difficulties to discriminate between vaccinated and infected animals (DIVA). Viral proteins M2e and HA2 have been shown to have potential as a DIVA antigen, and are likely to be useful in AIV control in countries where AIV is endemic. The potential of M2e protein for DIVA has been extensively studied previously. In chapter 2 of this study, a library of rabbit antibody against chicken M2e was constructed in the phagemid pSEX81, but several attempts to recover a clone containing the target fragment were unsuccessful. In chapter 3 to 5 of this study, the potency of HA subunit 2 (HA2) protein for DIVA in chicken was examined. This was initially done by epitope mapping the entire HA2 of consensus sequence of Indonesian avian H5N1 strains, using several overlapping recombinant proteins and sera obtained from infected and vaccinated chickens. From this study, a protein spanning aa positions 380-461 (H5 numbering) or 49-130 (HA2 numbering) was found antigenic but reacted strongly with both infected and vaccinated chicken sera. Another protein spanning aa positions 483-565 (H5 numbering) or 152-234 (HA2 numbering) was found to react strongly with infected chicken sera but less strongly with vaccinated chicken sera, indicating the existence of DIVA epitope. This finding was consistent with a report by Khurana, et al., (2011) who identified that a peptide (refer as E29) encompassing aa positions 488-516 of H5 was suitable as a DIVA antigen in human. In order to characterise the potential of the E29 epitope as a DIVA in chickens, several regions of the E29 peptide including aa 492-505 (refer as E15) predicted to be highly immunogenic were produced and examined. The results suggested that aa positions 492Y, 501E, 502E, 503I, 504S and 505G were important for binding of mouse anti-E15 monoclonal antibody while aa positions 495A to 505G were important for binding of monospecific anti-E15 chicken antibodies. The capacity of E29 and E15 as DIVA antigens in chicken was further examined using synthetic (both modified and unmodified) peptides and a limited number of reference sera obtained from experimentally infected or vaccinated chicken sera in ELISA. After optimisation of ELISA conditions, the E29 was found to exhibit the greatest discrimination power compared to other antigen forms between vaccinated and infected chicken sera. The newly developed E29 ELISA was then evaluated against a large number of field sera collected from infected or vaccinated sera and shown to have potential as a DIVA. The finding described in this thesis form the basis for future investigations on application of HA2 epitopes as DIVA reagent for better control of AI in Indonesia and other countries where AIV is endemic, and are ultimately expected to reduce socioeconomical losses incurred due to AI globally