Avirulent Marek's disease virus type 1 strain 814 vectored vaccine expressing avian influenza (AI) virus H5 haemagglutinin induced better protection than turkey herpesvirus vectored AI vaccine

Abstract

Background: Herpesvirus of turkey (HVT) as a vector to express the haemagglutinin (HA) of avian influenza virus (AIV) H5 was developed and its protection against lethal Marek's disease virus (MDV) and highly pathogenic AIV (HPAIV) challenges was evaluated previously. It is well-known that avirulemt MDV type 1 vaccines are more effective than HVT in prevention of lethal MDV infection. To further increase protective efficacy against HPAIV and lethal MDV, a recombinant MDV type 1 strain 814 was developed to express HA gene of HPAIV H5N1.

Methodology/principal findings: A recombinant MDV-1 strain 814 expressing HA gene of HPAIV H5N1 virus A/goose/Guangdong/3/96 at the US2 site (rMDV-HA) was developed under the control of a human CMV immediate-early promoter. The HA expression in the rMDV-HA was tested by immunofluorescence and Western blot analyses, and in vitro and in vivo growth properties of rMDV-HA were also analyzed. Furthermore, we evaluated and compared the protective immunity of rMDV-HA and previously constructed rHVT-HA against HPAIV and lethal MDV. Vaccination of chickens with rMDV-HA induced 80% protection against HPAIV, which was better than the protection rate by rHVT-HA (66.7%). In the animal study with MDV challenge, chickens immunized with rMDV-HA were completely protected against virulent MDV strain J-1 whereas rHVT-HA only induced 80% protection with the same challenge dose.

Conclusions/significance: The rMDV-HA vaccine was more effective than rHVT-HA vaccine for protection against lethal MDV and HPAIV challenges. Therefore, avirulent MDV type 1 vaccine is a better vector than HVT for development of a recombinant live virus vaccine against virulent MDV and HPAIV in poultry.

Figure 1. Validation of rMDV-HA by PCR amplifications of MDV US2 gene region and HA open reading frame.

(M) DNA ladder; (R) the rMDV-HA infected CEF cells: the fragment of US2 region that covered the HA expression cassette and gpt selective marker, 4572 bp; the fragment of HA open reading frame, 1712bp. (WT) the MDV 814-infected CEF cells, the fragment of US2 region, 1180 bp; (NC) negative control.

Figure 2. Growth curves (one-step growth kinetics) of rMDV-HA, MDV 814 and rHVT-HA.

After inoculation of chickens with 100 PFU of each virus, virus titers were determined at different times and steadily increased from 48 to 96 h post-infection until maximal titers were reached. The data of the growth curve of rHVT-HA were cited from reference . Stars indicate that the differences were significant between the groups (P<0.05).

Figure 3. Immunofluorescence and Western blot analyses of recombinant HA protein expressed in rMDV-HA infected CEF cells.

AIV-specific chicken serum antibodies bound to the rMDV-HA infected CEF cells (A) by indirect immunofluorescence, but not to the CEF cells infected with MDV 814 (B) 72 h post-infection. CEF cells infected with rMDV-HA and non-infected cells probed with an AIV-specific chicken serum and IRDye™ 800-labeled polyclonal rabbit anti-chicken IgG (1∶4000). HA-specific bands corresponding to the cleaved HA1 and HA2 were detected in preparations of rMDV- HA-infected cells by Western blot, but not in MDV 814 infected cells (C).

Figure 4. Comparison of viremia levels between rMDV-HA, MDV 814 and rHVT-HA.

One-day old chicks were vaccinated with either rMDV-HA or MDV 814, and bled on 7, 14, 21 and 28 days post infection for determination of viremia. The data of the viremia level of rHVT-HA were cited from reference . Stars indicate that the differences were significant between the groups (P<0.05).

Figure 5. Protection of vaccinated chickens against virulent MDV challenge.

Cumulative survival of chickens from unvaccinated chickens or the chickens vaccinated with rMDV-HA, rHVT-HA or MDV 814 during the 60 day evaluation period after chickens were challenged with virulent MDV strain J-1.