University of Tehran Iranian Journal of Veterinary Medicine 2251-8894 19 1 2025 01 01 Serological Evaluation of H9-RBD-Pichia, a Novel Recombinant Influenza Vaccine, in BALB/c Mice 23 30 93996 10.32598/ijvm.19.1.1005421 EN Amir Asghari Baghkheirati Department of Avian Health and Diseases, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. 0000-0002-2109-4574 Mohammad Hadi Sekhavati Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran. 0000-0001-8091-3798 Seyed Mostafa Peighambari Department of Avian Health and Diseases, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. 0000-0001-9166-1303 Kiarash Ghazvini Department of Microbiology and Virology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. 0000-0002-8538-1425 Jamshid Razmyar Department of Avian Health and Diseases, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. 0000-0002-1247-4591 Journal Article 2023 07 20 <strong>Background</strong>: The H9N2 influenza virus is one of the most important subtypes of influenza that has caused irreparable damage to the poultry industry, and many efforts are underway to develop its recombinant vaccines.<br /><strong>Objectives</strong>: This study aims to evaluate the effectiveness of H9-RBD-Pichia, a novel recombinant subunit vaccine, in stimulating the immune system and producing hemagglutinin-inhibiting antibodies in mice.<br /><strong>Methods</strong>: A total of 24 female BALB/c mice were purchased and randomly divided into six groups: Group 1 received oral administration of lysed yeast twice, group 2 received one injection of lysed yeast, group 3 received two injections of lysed yeast, group 4 received one injection of pure recombinant protein, group 5 received two injections of pure recombinant protein and group 6 was negative control. Prime and booster immunizations were performed on days 14 and 28, respectively. Sera samples were collected on day 42. Hemagglutination (HA) and hemagglutination inhibition (HI) assays were performed on antigen and sera samples, respectively.<br /><strong>Results</strong>: HA titers of 1:4, 1:4, and 1:32 were seen in the positive control, intact, and bead-treated yeasts, respectively, showing the functionality of the expressed H9-RBD recombinant protein. HI assay showed this vaccine could stimulate the immune system and produce anti-hemagglutinin antibodies in mice. The highest antibody titer (1:512) was observed when yeast lysate was injected twice on days 14 and 28.<br /><strong>Conclusion</strong>: The H9-RBD-Pichia vaccine has been produced based on genetic engineering technology and could produce high titers of hemagglutinin-inhibiting antibodies in mice.  https://ijvm.ut.ac.ir/article_93996_ba8a3feda934b7345cb696839ba982ee.pdf