Available avian influenza (AIV) serological diagnostic tests cannot distinguish vaccinated from naturally infected birds. Differentiation of vaccinated from infected animals (DIVA) is currently advocated as a means of achieving the full control of H5N1. In this study, for the first time, recombinant ectodomain of M2 protein (M2e) of avian influenza virus (H5N1 strain) was used for the DIVA serology test. M2e was cloned into pMAL-P4X vector and expressed in E. coli cells. We used Western blot to recognize the expressed M2e-MBP protein by chicken antisera produced against live H5N1 virus. Also, the specificity of M2e-MBP protein was compared to the M2e synthetic peptide via ELISA. In M2e-MBP ELISA, all sera raised against the live avian influenza viruses were positive for M2e antibodies, whereas sera from killed virus vaccination were negative. Furthermore, M2e-MBP ELISA of the field sera obtained from vaccinated and non-vaccinated chickens showed negative results, while challenged vaccinated chickens demonstrated strong positive reactions. H5N1-originated recombinant M2e protein induced broad-spectrum response and successfully reacted with antibodies against other AIV strains such as H5N2, H9N2, H7N7, and H11N6. The application of the recombinant protein instead of synthetic peptide has the advantages of continues access to an inexpensive reagent for performing a large scale screening. Moreover, recombinant proteins provide the possibility of testing the DIVA results with an additional technique such a Western blotting which is not possible in the case of synthetic proteins. All together, the results of the present investigation show that recombinant M2e-MBP can be used as a robust and inexpensive solution for DIVA test.

Although vaccination of poultry for control of highly pathogenic avian influenza virus (HPAIV) H5N1 has been practiced during the last decade in several countries, its effectiveness under field conditions remains largely unquantified. Effective HPAI vaccination is however essential in preventing incursions, silent infections and generation of new H5N1 antigenic variants. The objective of this study was to asses the level and duration of vaccine induced immunity in commercial layers in Indonesia. Titres of H5N1 haemagglutination inhibition (HI) antibodies were followed in individual birds from sixteen flocks, age 18–68 week old (wo). The study revealed that H5N1 vaccination had highly variable outcome, including vaccination failures, and was largely ineffective in providing long lasting protective immunity. Flocks were vaccinated with seven different vaccines, administer at various times that could be grouped into three regimes: In regime A, flocks (n = 8) were vaccinated two or three times before 19 wo; in regime B (n = 2), two times before and once after 19 wo; and in regime C (n = 6) three to four times before and two to three times after 19 wo. HI titres in regime C birds were significantly higher during the entire observation period in comparison to titres of regime A or B birds, which also differed significantly from each other. The HI titres of individual birds in each flock differed significantly from birds in other flocks, indicating that the effectiveness of field vaccination was highly variable and farm related. Protective HI titres of >4log2, were present in the majority of flocks at 18 wo, declined thereafter at variable rate and only two regime C flocks had protective HI titres at 68 wo. Laboratory challenge with HPAIV H5N1 of birds from regime A and C flocks confirmed that protective immunity differed significantly between flocks vaccinated by these two regimes. The study revealed that effectiveness of the currently applied H5N1 vaccination could be improved and measures to achieve this are discussed.

ABSTRAKSumarningsih, Tarigan S, Hemmatzadeh F, Ignjatovic J. 2019. Antigenik karakterisasi pada protein M2e menggunakan antibodi monoklonal anti-M2 dan antibodi poliklonal anti-M2e. JITV 24(3): 122-134.Protein Matrik 2 ektodomain (M2e) memiliki sifat lestari dan dianggap sebagai antigen potensial untuk mendeteksi infeksi virus influenza A pada unggas yang divaksinasi (DIVA test). Namun studi yang mempelajari antigenisitas M2 dan respon imun pada manusia atau hewan masih sangat terbatas. Pada studi ini sifat antigenik dari masing-masing tujuh belas M2e peptida dan enam belas protein rekombinan M2e (rM2e) yang memiliki variasi asam amino (aa) pada posisi 10, 11, 12, 13, 14, 16, 18 dan 20 dibandingkan dengan metode western blot (WB) dan enzyme-linked immunosorbent assay (ELISA) menggunakan antibodi monoklonal (mAb) 14C2 dari tikus, dan anti-M2e poliklonal antibody (pAb) yang berasal dari ayam dan kelinci. MAb 14C2 memiliki kekuatan pembeda terbaik dan aa posisi ke-11 merupakan imunodominan paling penting yang mempengaruhi ikatan mAb14C2 hingga tingkat yang terbesar. Perubahan pada posisi 14, 16 dan 18 juga mempengaruhi pengikatan mAb14C2, dan perubahan ini terdeteksi pada semua metode (WB atau ELISA) dan antigen yang digunakan (M2e peptida atau protein rM2e). Untuk anti-M2e pAb dari ayam dan kelinci, aa imunodominan ditemukan pada posisi 10 dan perubahan pada posisi 11 tidak mempengaruhi reaksi antibodi. Pengikatan pAb kelinci juga dipengaruhi oleh perubahan pada aa posisi 14 dan 16, hal ini mengkonfirmasi kontribusi posisi tersebut terhadap antigenisitas M2e. Posisi 10 adalah satu-satunya posisi yang penting untuk pengikatan pAb ayam terhadap M2e. Secara keseluruhan penelitian ini menunjukkan antigenik M2e terletak di antara residu 10 -18 dan perubahan aa pada posisi 10, 11, 12, 14, 16 dan 18, dapat mempengaruhi ikatan antibodi di dalam protein M2e. Kata kunci: Virus Influenza A, epitop M2e, antigenisitas ABSTRACT Sumarningsih, Tarigan S, Hemmatzadeh F, Ignjatovic J. 2019. Characterisation of M2e antigenicity using anti-M2 monoclonal antibody and anti-M2e polyclonal antibodies. JITV 24(3): 122-134.