Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge.

Avian influenza (AI) is a significant pub- lic health concern and serious economic threat to the commercial poultry industry worldwide. Previous re- search 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 at- tenuated Salmonella Enteritidis strains that express a protective M2e epitope in combination with a poten- tial immune-enhancing CD154 peptide sequence on the Salmonella outer membrane protein lamB. Commer- cial Leghorn chicks were orally immunized (immuniza- tion dose: 10 6 to 10 8 cfu/chick) with saline (negative control) or one of the recombinant Salmonella strains (ΔaroA M2e-CD154, ΔhtrA M2e-CD154, ΔaroA-ΔhtrA 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 ΔaroA M2e-CD154 exhibited signifi- cantly greater organ invasion in the liver and spleen at d 7 (P > 0.05); however, no marked differences in colo- nization of the cecal tonsils were observed. Vaccinated chickens exhibited significantly increased M2e-specific IgG responses, which were further enhanced by simul- taneous expression of CD154 (P < 0.05). Virus neutral- ization assays gave neutralizing indices of 6.6, 6.3, and 6.3 for ΔaroA M2e-CD154, ΔhtrA M2e-CD154, and ΔaroA-ΔhtrA M2e(4)-CD154 seven days post booster immunization, respectively, indicating effective neutral- ization of AI by serum IgG of vaccinated chickens. In a subsequent direct challenge study, specific-pathogen- free Leghorn chicks immunized with ΔaroA-ΔhtrA 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.