Molecular design and immunogenicity of a multiple-epitope foot-and-mouth disease virus antigen, adjuvants, and DNA vaccination / 生物工程学报

We designed and constructed a fuse expression gene OAAT and staphylococcal enterotoxin A (SEA) on the basis of the OAAT designed and constructed which consists of the structural protein VP1 genes from serotypes A and O FMDV, 5 major VP1 immunodominant epitopes from two genotypes of Asia1 serotype, and 3 Th2 epitopes originating from the non-structural protein, 3ABC gene and structural protein VP4 gene. The recombinant plasmids pEA was constructed using SEA as a genetic adjuvant. Expressions of target gene from the pEA in Hela cell were verified by IFA and Western blotting. The experiment of BALB/c mice immunized with the DNA vaccines showed that pA and pEA could induce simultaneously specific antibodies against serotypes A, Asia1, and O FMDV, and the highest antibody titres were found in the pEA and inactivated vaccine groups compared to pA vaccinating mice. Compared with the control, the levels of IL-2, IFN-gamma, IL-4, and IL-10 expression by splenic lymphocytes from mice immunized with pA and pEA were significantly increased. In addition, we found that the levels of IL-2, IFN-gamma and IL-4 from the mice immunized with pEA was higher than mice immunized with pA did. The results of viral challenge in guinea pigs showed the pA, pEA and inactivated vaccine provided full protection in 2/4, 2/4, 3/4, 3/4 and 4/4, 4/4 guinea pigs from challenge with FMDV O/NY00 and Asial/YNBS/58, respectively. The results demonstrated fuse protein OAAT and SEA may be potential immunoge against FMDV, furthermore, SEA may be an effective genetic adjuvant for DNA vaccine.

Immunogenicity of plasmids encoding P12A and 3C of FMDV and swine IL-18.

In this paper, two recombinant plasmids (pVIR-P12AIL18-3C and pVIR-P12A-3C) containing foot and mouth disease virus (FMDV) capsid polypeptide, 3C coding regions of O/NY00 and using/or not swine IL18 as a genetic adjuvant were constructed, and evaluated for their ability to induce humoral and cellular responses in mice and swine. In addition, the ability to protect swine against homologous virus challenge was examined. Mice and swine were given booster vaccination twice and once, respectively, and swine were challenged 10 days after the booster vaccination. Control groups were inoculated with pVAX1 or phosphate-buffered saline (PBS). All animals vaccinated with pVIR-P12AIL18-3C and pVIR-P12A-3C developed specific anti-FMDV ELISA antibody and neutralizing antibody and T lymphocyte proliferation and CTL cytotoxic activity was observed. In addition, we found that pVIR-P12AIL18-3C possessed stronger immunogenicity than pVIR-P12A-3C. The pVIR-P12AIL18-3C and pVIR-P12A-3C provided full protection in 3/4 and 2/4 swine from challenge with FMDV O/NY00, respectively. The results demonstrate the potential viability of a DNA vaccine in the control and prevention of FMDV infections.

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.