A new generation of modified live-attenuated avian influenza viruses using a two-strategy combination as potential vaccine candidates

Abstract

In light of the recurrent outbreaks of low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI), there is a pressing need for the development of vaccines that allow rapid mass vaccination. In this study, we introduced by reverse genetics temperature-sensitive mutations in the PB1 and PB2 genes of an avian influenza virus, A/Guinea Fowl/Hong Kong/WF10/99 (H9N2) (WF10). Further genetic modifications were introduced into the PB1 gene to enhance the attenuated (att) phenotype of the virus in vivo. Using the att WF10 as a backbone, we substituted neuraminidase (NA) for hemagglutinin (HA) for vaccine purposes. In chickens, a vaccination scheme consisting of a single dose of an att H7N2 vaccine virus at 2 weeks of age and subsequent challenge with the wild-type H7N2 LPAI virus resulted in complete protection. We further extended our vaccination strategy against the HPAI H5N1. In this case, we reconstituted an att H5N1 vaccine virus, whose HA and NA genes were derived from an Asian H5N1 virus. A single-dose immunization in ovo with the att H5N1 vaccine virus in 18-day-old chicken embryos resulted in more than 60% protection for 4-week-old chickens and 100% protection for 9- to 12-week-old chickens. Boosting at 2 weeks posthatching provided 100% protection against challenge with the HPAI H5N1 virus for chickens as young as 4 weeks old, with undetectable virus shedding postchallenge. Our results highlight the potential of live att avian influenza vaccines for mass vaccination in poultry.

FIG. 1.

Schematic representation of avian influenza virus PB1 and PB2 constructs for the generation of ts and HA-tagged mutant viruses. (A) Site-directed mutagenesis was used to introduce three ts mutations (K391E, E581G, and A661T) into the PB1 gene and one (N265S) into the PB2 gene of the WF10 (H9N2) virus. The PB1 gene was further modified by incorporating an HA tag sequence in frame with the C terminus of the PB1 protein. The HAtagR primer is unique for the HA tag sequences, whereas the PB1-2147F and PB1-2341R primers anneal to sequences in the PB1 gene. (B) Viral RNA was isolated from the recombinant viruses grown in embryonated chicken eggs. The RNA was RT-PCR amplified using two sets of primers as indicated. The RT-PCR products were analyzed on a 2% agarose gel in the presence of ethidium bromide. A 100-bp ladder was used as a molecular weight marker. WT, tag, ts, and att correspond to four different virus strains: 7WF10:1malH7, 7tagWF10:1malH7, 7tsWF10:1malH7, and 7attWF10:1malH7.

FIG. 2.

Plaque morphologies of mutant avian influenza viruses at various temperatures. Confluent MDCK cells (A) or CEK cells (B) seeded in six-well plates were infected with recombinant viruses. The numbers 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴, and 10⁻³ on the plaque pictures indicate the virus dilution used to infect cells at the indicated temperature. At 3 days postinfection for cells incubated at 37°C, 39°C, or 41°C and 4 days postinfection for those incubated at 32°C, cell monolayers were fixed and the viral antigen was visualized by immunostaining as described in Materials and Methods. The plaques were counted, and virus titers were represented as the log₁₀ PFU/ml, as indicated below the individual plaque picture. A titer of <3.0 log₁₀ PFU/ml) indicates that no virus was detected at 10⁻³ dilution. The nonpermissive temperature was defined as the lowest temperature that had a titer reduction of 100-fold or greater compared to 37°C. Titers that define the shutoff temperature are shown in bold. Arrows in panel A indicate pinpoint plaques. Note: Adobe Photoshop version 7.0 was used for panel A to enhance the contrast of the plaque immunostaining in MDCK cells.

FIG. 3.

Immunoblot analysis of HA epitope expression in virus-infected cells. MDCK cells were infected with different recombinant viruses or A/Memphis/98 (H3N2) (positive control for HA tag expression) at an MOI of 1.0 at 37°C and harvested at 16 h postinfection. After lysis, cellular proteins were separated by 10% SDS-PAGE, blotted onto nitrocellulose, and incubated with monoclonal rat anti-HA (upper panel) or mouse anti-WF10 polyclonal antibody (lower panel). The membrane was subsequently incubated with goat anti-mouse or goat anti-rat IgG conjugated to horseradish peroxidase and developed by enhanced chemiluminescence. A ∼90-kDa band corresponding to the PB1-HA fusion protein is observed in extracts of cells infected with the HA tag-expressing viruses. HA0 and HA1 of the H3 virus are shown. The expression of NP is shown as loading control. The size of protein markers (in kDa) is shown on the left. WT, tag, ts, and att correspond to four different virus strains: 7WF10:1malH7, 7tagWF10:1malH7, 7tsWF10:1malH7, and 7attWF10:1malH7.

FIG. 4.

Viral protein accumulation at various temperatures in infected cells. MDCK and CEK cells were infected with recombinant viruses at an MOI of 10 at 37°C, 39°C, and 41°C, and harvested at 6 h postinfection. Cell lysates were prepared and run on a 10% SDS-PAGE. As described in the legend of Fig. 3 and in Materials and Methods, the expression of viral proteins NP and M1 was detected with a mouse anti-WF10 polyclonal antibody and mouse anti-M1 monoclonal antibody, respectively. As loading control, the expression of actin was monitored by using a mouse antiactin monoclonal antibody. WT, tag, ts, and att correspond to four different virus strains: 7WF10:1malH7, 7tagWF10:1malH7, 7tsWF10:1malH7, and 7attWF10:1malH7.

FIG. 5.

Kinetics of HI antibody production in chickens after single-dose (10⁶ EID₅₀) in ovo vaccination with H5N1 live att vaccine. Eighteen-day-old embryos were vaccinated with 10⁶ EID₅₀ 6attWF10:2H5ΔN1 virus in ovo. Sera were collected randomly from eight chickens on a weekly basis and tested for neutralizing antibodies against the A/VN/1203/04 (H5N1) virus by the HI test. GMT, geometric mean reciprocal end-point tier. Horizontal lines indicate the mean HI titer.