Construction and immunogenicity of recombinant fowlpox vaccines coexpressing HA of AIV H5N1 and chicken IL18.
cDNAs of the HA genes of subtype H5N1 AIV were fused to form a single open reading frame, designated H5HA-H7HA. The H5HA-H7HA cDNA and chicken Interleukin-18 (IL18) were inserted into the fowlpox virus (FPV) expression vector pUTA-16-LacZ to produce pUTAL-H5-H7-IL18. cDNA of H5N1 AIV HA was inserted into the FPV expression vector pUTA2 to create the recombinant expression plasmid pUTA2-H5. Plasmids were then co-transected into CEF cells. The two recombinant fowlpox viruses (rFPV) were produced by three cycles with the BrdU and verified by RT-PCR, IFA and Western blotting. One-day-old specific pathogen free (SPF) chickens and 7-day-old commercial Leghorn egg-laying chickens were inoculated with 10(6) PFU recombinant or parental fowlpox vaccine viruses by wing-web puncture. Hemagglutination inhibition (HI) antibody titer and nonspecific cellular immunity level were assessed after 1-3 weeks post-immunization. We found that all rFPV-vaccinated groups produced HI-specific antibodies, and the level of cellular immunity induced by the rFPV-H5-H7-IL18 strain was significantly higher than that induced by rFPV-H5HA. At 3 weeks post-inoculation, immunized SPF and Leghorn chickens were challenged with H5N1 HP AIV. The rFPV-H5-H7-IL18 vaccine strains were able to induce complete (10/10) protection, while the rFPV-H5HA vaccine strain induced (9/10) protection. Cloacal swabbing samples were collected from immunized leghorn chickens during the first week post-challenge; no shedding was found in the rFPV-H5-H7-IL18 vaccinated group. The rFPV-H5-H7-IL18 vaccinated group displayed significantly increased weight gain relative to the rFPV-H5HA group. This study reports a significant step in the further development of new AIV vaccines.
Mingxiao
Ma
M
Genetic Engineering Laboratory, Academy of Military Medical Sciences, Changchun 130062, PR China.
Ningyi
Jin
J
Zhenguo
Wang
W
Ruilin
Wang
W
Dongliang
Fei
F
Min
Zheng
Z
Gefen
Yin
Y
Chang
Li
L
Leili
Jia
J
Kuoshi
Jin
J
Yingjiu
Zhang
Z
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D049109:Cell Proliferation; D002645:Chickens; D017931:DNA Primers; D005587:Fowlpox virus / Q000235:genetics / Q000276:immunology*; D006388:Hemagglutinins / Q000235:genetics*; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000276:immunology*; D020382:Interleukin-18 / Q000235:genetics*; D008214:Lymphocytes / Q000166:cytology; D010957:Plasmids; D013154:Spleen / Q000166:cytology; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology*
D017931:DNA Primers; D006388:Hemagglutinins; D020382:Interleukin-18; D014614:Vaccines, Synthetic