Bakhsh Muhammad, Senevirathne Amal, Riaz Jamal, Kwon Jun, Aganja Ram Prasad, Cabarles Jaime C, Oh Sang-Ik, Lee John Hwa
College of Veterinary Medicine, Jeonbuk National University, Specialized Campus, Iksan 54596, Republic of Korea.
Central Department of Biotechnology, Tribhuvan University, Kirtipur 44618, Nepal.
Vaccines (Basel). 2025 Jul 25;13(8):790. doi: 10.3390/vaccines13080790.
Fowl typhoid (FT), a septicemic infection caused by Gallinarum (SG), and H9N2 avian influenza are two economically important diseases that significantly affect the global poultry industry. We exploited the live attenuated Gallinarum (SG) mutant JOL3062 (SG: ∆ ∆ ∆) as a delivery system for H9N2 antigens to induce an immunoprotective response against both H9N2 and FT. To enhance immune protection against H9N2, a prokaryotic and eukaryotic dual expression plasmid, pJHL270, was employed. The hemagglutinin (HA) consensus sequence from South Korean avian influenza A virus (AIV) was cloned under the Ptrc promoter for prokaryotic expression, and the B cell epitope of neuraminidase (NA) linked with matrix protein 2 (M2e) was placed for eukaryotic expression. In vitro and in vivo expressions of the H9N2 antigens were validated by qRT-PCR and Western blot, respectively. Oral immunization with JOL3121 induced a significant increase in SG and H9N2-specific serum IgY and cloacal swab IgA antibodies, confirming humoral and mucosal immune responses. Furthermore, FACS analysis showed increased CD4+ and CD8+ T cell populations. On day 28 post-immunization, there was a substantial rise in the hemagglutination inhibition titer in the immunized birds, demonstrating neutralization capabilities of immunization. Both IFN-γ and IL-4 demonstrated a significant increase, indicating a balance of Th1 and Th2 responses. Intranasal challenge with the H9N2 Y280 strain resulted in minimal to no clinical signs with significantly lower lung viral titer in the JOL3121 group. Upon SG wildtype challenge, the immunized birds in the JOL3121 group yielded 20% mortality, while 80% mortality was recorded in the PBS control group. Additionally, bacterial load in the spleen and liver was significantly lower in the immunized birds. The current vaccine model, designed with a host-specific pathogen, SG, delivers a robust immune boost that could enhance dual protection against FT and H9N2 infection, both being significant diseases in poultry, as well as ensure public health.
禽伤寒（FT）是由鸡沙门氏菌（SG）引起的一种败血性感染，H9N2禽流感是另外两种对全球家禽业有重大经济影响的疾病。我们利用减毒活鸡沙门氏菌（SG）突变株JOL3062（SG: ∆ ∆ ∆）作为H9N2抗原的递送系统，以诱导针对H9N2和FT的免疫保护反应。为增强对H9N2的免疫保护，使用了一种原核和真核双表达质粒pJHL270。将来自韩国甲型禽流感病毒（AIV）的血凝素（HA）共有序列克隆到Ptrc启动子下用于原核表达，并将与基质蛋白2（M2e）连接的神经氨酸酶（NA）的B细胞表位用于真核表达。分别通过qRT-PCR和蛋白质免疫印迹法验证了H9N2抗原的体外和体内表达。用JOL3121进行口服免疫可显著提高SG和H9N2特异性血清IgY和泄殖腔拭子IgA抗体水平，证实了体液免疫和黏膜免疫反应。此外，流式细胞术分析显示CD4+和CD8+ T细胞群体增加。免疫后第28天，免疫鸡的血凝抑制效价大幅上升，表明免疫具有中和能力。IFN-γ和IL-4均显著增加，表明Th1和Th2反应平衡。用H9N2 Y280毒株进行鼻内攻毒后，JOL3121组鸡的临床症状轻微或无临床症状，肺部病毒滴度显著降低。在用SG野生型攻毒后，JOL3121组的免疫鸡死亡率为20%，而PBS对照组的死亡率为80%。此外，免疫鸡脾脏和肝脏中的细菌载量显著降低。目前用宿主特异性病原体SG设计的疫苗模型可提供强大的免疫增强作用，既能增强对FT和H9N2感染（这两种都是家禽中的重要疾病）的双重保护，又能确保公共卫生。
