Multigenerational stability and environmental safety of transgenic rice expressing a dimeric HN protein as a plant bioreactor for poultry Newcastle disease vaccine antigen production

Abstract

Newcastle disease (ND) remains among the most economically important viral diseases in poultry, and novel thermostable, low-cost and genotype VII-matched veterinary vaccine platforms are urgently needed to overcome the limitations of conventional vaccines, such as cold chain dependency, high application cost and insufficient cross-protection against prevailing genotype VII strains. Rice endosperm has emerged as an ideal plant bioreactor for veterinary vaccine antigen manufacturing owing to its low production cost, cold chain-independent antigen storage, high biomass yield and great potential for oral or feed-based poultry immunization. In this study, we systematically evaluated two independent transgenic rice lines (HN-1 and HN-2) expressing a dimeric hemagglutinin-neuraminidase (HN) antigen of Newcastle disease virus (NDV) across the T1–T3 generations under confined field conditions and aimed to screen elite plant bioreactor lines for large-scale production of an ND vaccine antigen. The exogenous HN gene exhibited stable genomic integration, with no sequence variations detected in its coding region, and maintained consistent transcriptional and protein expression levels over three successive generations. Agronomic assays demonstrated that the major agronomic and grain quality traits of the HN-1 line were highly comparable to those of wild-type TP309, whereas the grain chalkiness defects of HN-2 were clearly line specific. No detectable transgene flow to adjacent weeds was detected, and pollen viability, seed germination performance and nontarget arthropod diversity were not altered in the transgenic lines. Collectively, the HN-1 line possesses excellent genetic stability, agronomic adaptability and ecological safety and can serve as a promising elite plant bioreactor candidate for the industrial production of NDV HN vaccine antigens. This work provides genetic stability, agronomic performance, and preliminary biosafety data to support the further development and regulatory evaluation of rice-derived veterinary ND vaccines.

Keywords

Newcastle disease virus (NDV) / HN vaccine antigen / Transgenic rice / Poultry vaccine / Rice-based vaccine platform / Multigenerational stability / Environmental safety / Genotype VII

References

[1] Absalón AE, Cortés-Espinosa DV, Lucio E, Miller PJ, Afonso CL. Epidemiology, control, and prevention of Newcastle disease in endemic regions: Latin America. Tropical Animal Health and Production, 2019, 51510331048.

[2] Azegami T, Itoh H, Kiyono H, Yuki Y. Novel transgenic rice-based vaccines. Archives of Immunology and Therapy Experimentalis, 2015, 6328799.

[3] Bello MB, Yusoff K, Ideris A, Hair-Bejo M, Peeters BPH, Omar AR. Diagnostic and vaccination approaches for Newcastle disease virus in poultry: The current and emerging perspectives. BioMed Research International, 2018, 2018. 7278459

[4] Chu H, Zhang L, Hao Z, Hua Y, Hu W, Liu R, Lou Y, Gao W, Bian S, Li F, et al. Comprehensive evaluation of transgenic rice lines expressing CSFV E2 protein in genetic stability, environmental safety and field adaptation. Frontiers in Plant Science, 2025, 16: 1650765.

[5] Chu, H., Hao, Z., Zhang, L., Lou, Y., Hua, Y., Gao, W., Li, F., Han, L., Bian, S., Cheng, W., et al. 2025a. Evaluation of rice traits containing H9N2 subtype avian influenza HA protein before commercialization. Current Issues In Molecular Biology‌ 47(12). https://doi.org/10.3390/cimb47120986.

[6] Costa-Hurtado M, Afonso CL, Miller PJ, Shepherd E, Cha RM, Smith D, Spackman E, Kapczynski DR, Suarez DL, Swayne DE, et al. Previous infection with virulent strains of Newcastle disease virus reduces highly pathogenic avian influenza virus replication, disease, and mortality in chickens. Veterinary Research, 2015, 46197.

[7] Daniell H, Singh ND, Mason H, Streatfield SJ. Plant-made vaccine antigens and biopharmaceuticals. Trends in Plant Science, 2009, 1412669679.

[8] Dharmayanti NI, Nurjanah D, Nuradji H, Suyatno T, Indriani R. Newcastle disease virus: The past and current situation in Indonesia. Journal of Veterinary Science, 2024, 251e3

[9] Dimitrov KM, Afonso CL, Yu Q, Miller PJ. Newcastle disease vaccines-A solved problem or a continuous challenge?. Veterinary Microbiology, 2017, 206: 126-136.

[10] Eid, A. A. M., Hussein, A., Hassanin, O., Elbakrey, R. M., Daines, R., Sadeyen, J. R., Abdien, H. M. F., Chrzastek, K., Iqbal, M. 2022. Newcastle disease genotype VII prevalence in poultry and wild birds in egypt. Viruses 14(10). https://doi.org/10.3390/v14102244.

[11] Gomes CWC, Funkler G, Andretta I, Gonçalves MO, Santos HF, Cruz CEF. Newcastle disease vaccination in captive-bred wild birds. Tropical Animal Health and Production, 2018, 50613491353.

[12] Hein R, Koopman R, García M, Armour N, Dunn JR, Barbosa T, Martinez A. Review of poultry recombinant vector vaccines. Avian Diseases, 2021, 653438452.

[13] Hu Z, He X, Deng J, Hu J, Liu X. Current situation and future direction of Newcastle disease vaccines. Veterinary Research, 2022, 53199

[14] Huma, I., Azeem, S., Naz, S., Yaqub, T., Mushtaq, M. H., Azam, M. 2024. A Newcastle disease live virus vaccine is safe and efficacious at various storage conditions. Veterinaria Italiana 59(3). https://doi.org/10.12834/VetIt.2980.19377.1.

[15] LeBlanc, Z., Waterhouse, P., Bally, J. 2020. Plant-based vaccines: The way ahead? Viruses 13(1). https://doi.org/10.3390/v13010005.

[16] Li Q, Wang L, Sun Y, Liu J, Ma F, Yang J, Zhao D, Zhang Y, Luo J, Guo J, et al. Evaluation of an immunochromatographic strip for detection of avian avulavirus 1 (Newcastle disease virus). Journal of Veterinary Diagnostic Investigation, 2019, 313475480.

[17] Li J, Ling M, Sun Y, Di H, Cong Y, Yu H, Cong Y. Characterization of Newcastle disease virus obtained from toco toucan. Journal of Veterinary Science, 2020, 212e19

[18] Liu Y, Sun C, Chi M, Wen H, Zhao L, Song Y, Liu N, Wang Z. Genetic characterization and phylogenetic analysis of Newcastle disease virus from China. Infection, Genetics and Evolution, 2019, 75. 103958

[19] Liu M, Shen X, Li J, Yu Y, Fan J, Jia X, Dai Y. Efficacy of Newcastle disease LaSota vaccine-induced hemagglutination inhibition antibodies against challenges with heterologous virulent strains of genotypes VII and IX. Veterinary Immunology and Immunopathology, 2023, 259. 110591

[20] Loza-Rubio, E., and E. Rojas-Anaya. 2010. Vaccine production in plant systems – an aid to the control of viral diseases in domestic animals: a review. Acta Veterinaria Hungarica 58 (4): 511–522. https://doi.org/10.1556/avet.58.2010.4.11.

[21] Ma F, Xu Q, Wang A, Yang D, Li Q, Guo J, Zhang L, Ou J, Li R, Yin H, et al. A universal design of restructured dimer antigens: Development of a superior vaccine against the paramyxovirus in transgenic rice. Proceedings of the National Academy of Sciences of the United States of America, 2024, 1214e2305745121

[22] Ma, F., Zhang, E., Li, Q., Xu, Q., Ou, J., Yin, H., Li, K., Wang, L., Zhao, X., Niu, X., et al. 2020. A plant-produced recombinant fusion protein-based Newcastle disease subunit vaccine and rapid differential diagnosis platform. Vaccines (Basel) 8(1). https://doi.org/10.3390/vaccines8010122.

[23] Mayers J, Mansfield KL, Brown IH. The role of vaccination in risk mitigation and control of Newcastle disease in poultry. Vaccine, 2017, 354459745980.

[24] Ruan B, Liu Q, Chen Y, Niu X, Wang X, Zhang C, Guo M, Zhang X, Cao Y, Wu Y. Generation and evaluation of a vaccine candidate of attenuated and heat-resistant genotype VIII Newcastle disease virus. Poultry Science, 2020, 99734373444.

[25] Shahar E, Haddas R, Goldenberg D, Lublin A, Bloch I, Bachner Hinenzon N, Pitcovski J. Newcastle disease virus: Is an updated attenuated vaccine needed?. Avian Pathology, 2018, 475467478.

[26] Shahid, N., T. R. Rehman, S. Samiullah, A. Shakoor, A. Latif, S. Yasmeen, A. A. Azam, T. Shahid, A. Q. Husnain, and M. Rao. 2020. Early stage development of a Newcastle disease vaccine candidate in corn. Frontiers in Veterinary Science 7: 00499. https://doi.org/10.3389/fvets.2020.00499.

[27] Stander J, Mbewana S, Meyers AE. Plant-derived human vaccines: Recent developments. BioDrugs, 2022, 365573589.

[28] Sultan HA, Talaat S, Elfeil WK, Selim K, Kutkat MA, Amer SA, Choi KS. Protective efficacy of the Newcastle disease virus genotype VII-matched vaccine in commercial layers. Poultry Science, 2020, 99312751286.

[29] Ul-Rahman A, Ishaq HM, Raza MA, Shabbir MZ. Zoonotic potential of Newcastle disease virus: Old and novel perspectives related to public health. Reviews in Medical Virology, 2022, 321e2246

[30] Webster DE, Thomas MC, Huang Z, Wesselingh SL. The development of a plant-based vaccine for measles. Vaccine, 2005, 231518591865.

[31] Yuki Y, Kiyono H. MucoRice: Development of rice-based oral vaccine. Nihon Rinsho Men'eki Gakkai Kaishi, 2008, 315369374.

[32] Zhang, D., Ding, Z., Xu, X. 2023. Pathologic mechanisms of the Newcastle disease virus. Viruses 15(4). https://doi.org/10.3390/v15040864.

Funding

the Key Scientific and Technological Projects of Henan Province(222102110210)

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