A live attenuated cold-adapted influenza A H7N3 virus vaccine provides protection against homologous and heterologous H7 viruses in mice and ferrets

Abstract

The appearance of human infections caused by avian influenza A H7 subtype viruses underscores their pandemic potential and the need to develop vaccines to protect humans from viruses of this subtype. A live attenuated H7N3 virus vaccine was generated by reverse genetics using the HA and NA genes of a low pathogenicity A/chicken/BC/CN-6/04 (H7N3) virus and the six internal protein genes of the cold-adapted A/Ann Arbor/6/60 ca (H2N2) virus. The reassortant H7N3 BC 04 ca vaccine virus was temperature sensitive and showed attenuation in mice and ferrets. Intranasal immunization with one dose of the vaccine protected mice and ferrets when challenged with homologous and heterologous H7 viruses. The reassortant H7N3 BC 04 ca vaccine virus showed comparable levels of attenuation, immunogenicity and efficacy in mice and ferret models. The safety, immunogenicity, and efficacy of this vaccine in mice and ferrets support the evaluation of this vaccine in clinical trials.

Figures

Figure. 1

Percent change in body weight following i.n. inoculation of H7N3 viruses. Groups of 5 mice received 106 TCID50/mouse of BC 04 ca (□), H7N3 BC 04 wt LP (■), H7N3 BC 04 wt HP (▲) viruses. Mock infected mice (●) received L-15.

Figure. 2

Replication of the H7N3 BC 04 ca virus in mice. Level of replication of ca and wt viruses in mice following i.n. inoculation with 106 TCID50/mouse of H7N3 BC 04 wt HP (hatched bars), H7N3 BC 04 wt LP (solid bars), H7N3 BC 04 ca (gray bars), AA wt (open bars) and AA ca (horizontal hatch bars) viruses. Virus titers in the (A) nasal turbinates, (B) lungs, (C) spleen, and (D) brain of 4 mice per group sacrificed on 2, 3, and 4 dpi, respectively are expressed as mean ± SE log10TCID50/gm of tissue. The dashed horizontal line indicates the lower limit of detection. * indicates statistically significant (p<0.05) reduction in virus titers compared to the corresponding wt virus.

Figure. 3

Replication of the H7N3 BC 04 ca virus in ferrets. Level of replication of ca and wt viruses in ferrets following i.n. inoculation with 107 TCID50/ferret of H7N3 BC 04 wt HP (hatched bars), H7N3 BC 04 wt LP (solid bars), H7N3 BC 04 ca (gray bars), AA wt (open bars), and AA ca (horizontal hatch bars) viruses on 3 dpi. Virus titers are expressed as mean ± SE log10EID50/gm of tissue. The dashed horizontal line indicates the lower limit of detection. * indicates statistically significant (p<0.05) reduction in virus titer compared to the H7N3 BC 04 wt viruses.

Figure. 4

Percent change in body weight of mice i.n. immunized with a single dose of the H7N3 BC 04 ca vaccine virus following lethal challenge with HP H7 viruses. Groups of 8 mice that were immunized with 106 TCID50 of H7N3 BC 04 ca vaccine virus (□) or were mock-immunized (L-15) (▲) were challenged 4 weeks later with 50 LD50 of (A) H7N3 BC 04 wt HP, (B) NL/03 (H7N7), and (C) EG/63 (H7N3) viruses. All immunized mice survived and mock-immunized mice succumbed to death between 6 and 8 dpi.

Figure. 5

Replication of H7 wt challenge viruses in the respiratory tract of mice immunized with the H7N3 BC 04 ca vaccine virus. Groups of mice were immunized with 1 (open bars) or 2 (gray bars) doses of 106 TCID50 of the H7N3 BC 04 ca vaccine virus or were mock-immunized (L-15) (solid bars) and 4 mice per group were challenged with 105 TCID50 of each indicated virus (A) 4 weeks or (B) 8 weeks post-immunization. H7N3 BC 04 wt LP and H7N3 BC 04 wt HP are homologous and UT/95 and VA/02 are heterologous LP viruses from the North American lineage. EG/63 (H7N3) and NL/03 (H7N7) are antigenically heterologous HP viruses from the Eurasian lineage. Virus titers are expressed as mean ± SE log10TCID50/gm of tissue from groups of 4 mice. The dashed horizontal line indicates the lower limit of detection. The reduction in virus titer in the respiratory tract of the immunized mice compared to mock-immunized group was statistically significant (p<0.05).

Figure. 6

Replication of H7 wt challenge viruses in ferrets immunized with H7N3 BC 04 ca vaccine virus. Groups of ferrets immunized with 1 (open bars) or 2 (gray bars) doses of 107 TCID50 of H7N3 BC 04 ca vaccine virus and mock-immunized (L-15) (solid bars) ferrets were challenged 4 weeks post-dose 1 and 2 with 106 TCID50 of the indicated viruses. The H7N3 BC 04 wt LP and H7N3 BC 04 wt HP viruses are homologous viruses from the North American lineage and NL/03 (H7N7) is a heterologous HP virus from the Eurasian lineage. Virus titers in the nasal turbinates, olfactory bulbs, and brain are expressed as mean ± SE log10TCID50/gm of tissue. Virus titers in the lungs are expressed as mean ± SE log10EID50/gm of tissue. * indicates statistically significant (p<0.05) reduction in the virus titers. The dashed horizontal line indicates the lower limit of detection.