Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys

Abstract

Highly pathogenic avian influenza virus (HPAIV) is a serious threat not only to domestic fowls but also to humans. Vaccines inducing long-lasting immunity against HPAIV are required. In the present study, we generated recombinant measles virus (MV) expressing the hemagglutinin protein of HPAIV without the multibasic site necessary for its pathogenicity in chickens using the backbone of an MV vaccine strain (rMV-Ed-H5HA) or a wild-type MV-derived mutant (rMV-HL-Vko-H5HA). We examined protective efficacy of the candidate vaccines in the monkey infection model by the challenge with a HPAIV (H5N1). Cynomolgus monkeys inoculated with the candidate vaccines produced both anti-H5 HA and anti-MV antibodies. They recovered earlier from influenza symptoms than unvaccinated monkeys after the challenge with the HPAIV strain. Chest radiography and histopathological analyses confirmed less severe pneumonia in the vaccinated monkeys. Vaccination tended to suppress viral shedding and reduced the interleukin-6 levels in the lungs. Furthermore, the vaccination with rMV-Ed-H5HA of monkeys with pre-existing anti-MV immunity induced the production of anti-H5 HA antibodies. These results suggest that both candidate vaccines effectively reduce disease severity in naïve hosts, and that rMV-Ed-H5HA is a particularly good candidate vaccine against HPAIV infection.

Figure 1

Generation of rMV-H5HA. (a) Structure of the full-genome cDNA of rMV-H5HA. (b) Comparison of growth curves for rMV-H5HA and the parental rMVs in the corresponding cell lines. Data are averages ± SEM (n = 3). Closed or open dots indicate rMV-H5HA or the parental virus, respectively. Expression of H5 HA protein in cells infected with rMV-H5HA was examined with immunofluorescent microscopy (c) and western blotting (d) Full-length blots of each protein are shown in Supplemental Figure S1) (c) H5 HA was detected with anti-H5 HA antibody (rabbit) and anti-rabbit IgG antibody conjugated with Alexa Fluor 568, and MV N was detected with an anti-CDV N monoclonal antibody (mouse) and anti-mouse IgG antibody conjugated with Alexa Fluor 488, respectively.

Figure 2

Clinical symptoms in monkeys after HPAIV challenge. (a) The vaccinated and unvaccinated monkeys were challenged with HPAIV (arrow). Fever onset was observed on the night of the day of challenge (black arrow heads). A second bout of fever was observed in the unvaccinated monkeys (gray arrow heads). Bodyweight (b) and respiratory rate (c) were monitored daily after the challenge. (d) Clinical symptoms were scored based on Supplemental Table 1. *p < 0.05, **p < 0.01, compared with the unvaccinated control on Dunnett’s test.

Figure 3

Inflammation of the lungs of HPAIV-challenged monkeys. (a) Chest X-rays taken at 4 and 8 dpi are shown. (b) Hematoxylin and eosin staining of the lower lobe of the right lung of the monkeys. Original maginifcation, 4× objective lens. (c) Histopathology of each lung lobe was scored as: 0, area of pulmonary alveoli was >60% of the observed section; 1, area of pulmonary alveoli was 40–60% of the observed section; 2, area of pulmonary alveoli was 30–40% of the observed section; 3, area of pulmonary alveoli was 20–30% of the observed section; 4, area of pulmonary alveoli was <20% of the observed section. The total score for each individual monkey was used in the analysis. *p < 0.05 compared with the unvaccinated control on Dunnett’s test.

Figure 4

Comparison of IL-6 levels in the lung. IL-6 levels in the lungs of H5N1-challenged monkeys were measured at 8 dpi. Each symbol shows the data from an individual monkey (n = 3 for each group). *p < 0.05 compared with the unvaccinated control on Dunnett’s test.

Figure 5

Comparison of IFN-γ levels in the serum or plasma. Serum or plasma samples were collected at 0 dpi (pre-challenge) and 1–7 dpi. IFN-γ levels for individual monkeys are indicated independently.