Layton S L, Kapczynski D R, Higgins S, Higgins J, Wolfenden A D, Liljebjelke K A, Bottje W G, Swayne D, Berghman L R, Kwon Y M, Hargis B M, Cole K
Department of Poultry Science, University of Arkansas, Fayetteville 72701, USA.
Poult Sci. 2009 Nov;88(11):2244-52. doi: 10.3382/ps.2009-00251.
Avian influenza (AI) is a significant public health concern and serious economic threat to the commercial poultry industry worldwide. Previous research demonstrates that antibodies against M2e confer protection against influenza challenge. Using the Red recombinase system in combination with overlapping extension PCR, we recently developed several novel attenuated Salmonella Enteritidis strains that express a protective M2e epitope in combination with a potential immune-enhancing CD154 peptide sequence on the Salmonella outer membrane protein lamB. Commercial Leghorn chicks were orally immunized (immunization dose: 10(6) to 10(8) cfu/chick) with saline (negative control) or one of the recombinant Salmonella strains [DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, DeltaaroA-DeltahtrA M2e(4)-CD154] on day of hatch and 21 d posthatch. These candidate vaccine strains were evaluated for their ability to invade, colonize, and persist in tissues and elicit an M2e-specific antibody response. The vaccine candidate strain DeltaaroA M2e-CD154 exhibited significantly greater organ invasion in the liver and spleen at d 7 (P > 0.05); however, no marked differences in colonization of the cecal tonsils were observed. Vaccinated chickens exhibited significantly increased M2e-specific IgG responses, which were further enhanced by simultaneous expression of CD154 (P < 0.05). Virus neutralization assays gave neutralizing indices of 6.6, 6.3, and 6.3 for DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, and DeltaaroA-DeltahtrA M2e(4)-CD154 seven days post booster immunization, respectively, indicating effective neutralization of AI by serum IgG of vaccinated chickens. In a subsequent direct challenge study, specific-pathogen-free Leghorn chicks immunized with DeltaaroA-DeltahtrA M2e(4)-CD154 offered significant protection against direct challenge with low pathogenic AI H7N2, but not highly pathogenic H5N1 AI. Taken together, these data suggest that these Salmonella-vectored vaccines expressing M2e in association with CD154 are effective at protecting chickens against low pathogenic AI.
禽流感（AI）是一个重大的公共卫生问题，对全球商业家禽业构成严重的经济威胁。先前的研究表明，针对M2e的抗体可提供针对流感攻击的保护。我们最近使用Red重组酶系统结合重叠延伸PCR，开发了几种新型减毒肠炎沙门氏菌菌株，这些菌株在沙门氏菌外膜蛋白lamB上表达具有保护性的M2e表位，并结合潜在的免疫增强CD154肽序列。在孵化当天和孵化后21天，用盐水（阴性对照）或其中一种重组沙门氏菌菌株[ΔaroA M2e-CD154、ΔhtrA M2e-CD154、ΔaroA-ΔhtrA M2e(4)-CD154]对商品来航鸡进行口服免疫（免疫剂量：10(6)至10(8) cfu/只鸡）。评估了这些候选疫苗菌株在组织中侵袭、定植和持续存在的能力，以及引发M2e特异性抗体反应的能力。候选疫苗菌株ΔaroA M2e-CD154在第7天时在肝脏和脾脏中的器官侵袭显著更强（P>0.05）；然而，在盲肠扁桃体的定植方面未观察到明显差异。接种疫苗的鸡表现出显著增加的M2e特异性IgG反应，同时表达CD154可进一步增强这种反应（P<0.05）。在加强免疫7天后，针对ΔaroA M2e-CD154、ΔhtrA M2e-CD154和ΔaroA-ΔhtrA M2e(4)-CD154的病毒中和试验的中和指数分别为6.6、6.3和6.3，表明接种疫苗的鸡的血清IgG对禽流感有有效的中和作用。在随后的直接攻毒研究中，用ΔaroA-ΔhtrA M2e(4)-CD154免疫的无特定病原体来航鸡对低致病性AI H7N2的直接攻毒提供了显著的保护，但对高致病性H5N1 AI无效。综上所述，这些数据表明，这些表达与CD154相关的M2e的沙门氏菌载体疫苗在保护鸡免受低致病性禽流感方面是有效的。
