Intranasal vaccination with a plant-derived H5 HA vaccine protects mice and ferrets against highly pathogenic avian influenza virus challenge

Abstract

Highly pathogenic avian influenza H5N1 infection remains a public health threat and vaccination is the best measure of limiting the impact of a potential pandemic. Mucosal vaccines have the advantage of eliciting immune responses at the site of viral entry, thereby preventing infection as well as further viral transmission. In this study, we assessed the protective efficacy of hemagglutinin (HA) from the A/Indonesia/05/05 (H5N1) strain of influenza virus that was produced by transient expression in plants. The plant-derived vaccine, in combination with the mucosal adjuvant (3',5')-cyclic dimeric guanylic acid (c-di-GMP) was used for intranasal immunization of mice and ferrets, before challenge with a lethal dose of the A/Indonesia/05/05 (H5N1) virus. Mice vaccinated with 15 μg or 5 μg of adjuvanted HA survived the viral challenge, while all control mice died within 10 d of challenge. Vaccinated animals elicited serum hemagglutination inhibition, IgG and IgA antibody titers. In the ferret challenge study, all animals vaccinated with the adjuvanted plant vaccine survived the lethal viral challenge, while 50% of the control animals died. In both the mouse and ferret models, the vaccinated animals were better protected from weight loss and body temperature changes associated with H5N1 infection compared with the non-vaccinated controls. Furthermore, the systemic spread of the virus was lower in the vaccinated animals compared with the controls. Results presented here suggest that the plant-produced HA-based influenza vaccine adjuvanted with c-di-GMP is a promising vaccine/adjuvant combination for the development of new mucosal influenza vaccines.

Keywords: (3′; 5′)-cyclic dimeric guanylic acid; adjuvant; c-di-GMP; ferret infection model; influenza H5N1; intranasal vaccination; mice, plant vaccine.

Figure 1.

HA-specific serum IgG (A) and IgA (B) responses in mice in the immunogenicity study. Groups of 6 mice were immunized twice (21 day interval) intranasally with plant derived H1N1 vaccine at 15, 5 or 1.5 μg HA in combination with 5 μg of c-di-GMP or with PBS plus 5 μg cdi-GMP (control). Blood samples were collected at days 0, 21, 28, 35 and 42 and serum IgG and IgA levels were analyzed by ELISA. * = significantly (P < 0.05) different from Day 0 titers.

Figure 2.

Serum HI antibody response in mice before challenge with the A/Indonesia/05/2005 (H5N1) virus. Groups of 16 mice were immunized twice (21 day interval) intranasally with plant derived vaccine at 15, 5 or 1.5 μg HA in combination with 5 μg of c-di-GMP or with PBS plus 5 μg cdi-GMP (control). Blood samples were collected pre-vaccination (Day −1) and at 20, 32, and 41 days post vaccination. The data show HI responses of each individual mouse and the horizontal lines represent geometric mean titers ± 95% confidence interval. The dotted line represents an HI titer of 40.

Figure 3.

Virus titers in the upper (nasal turbinates, A) and lower (lungs, B) respiratory tract after challenge with the A/Indonesia/05/2005 (H5N1) virus. Groups of 16 mice were immunized twice (21 day interval) intranasally with plant derived vaccine at 15, 5 or 1.5 μg HA in combination with 5 μg c-di-GMP or with 5 μg c-di-GMP/PBS (control). Mice were challenged by intranasal administration of highly pathogenic avian Influenza A/Indonesia/05/2005 (H5N1) on day 42. Groups of 3 mice were euthanized at days 3 (Day 45) and 5 (Day 47) post-challenge to examine for the presence of virus in the upper (nasal turbinates, A) and lower (lungs, B) respiratory tract. The limit of detection was 10^2.5 TCID₅₀/mL for nasal turbinates and 10^1.5 TCID₅₀/mL for lung tissue.

Figure 4.

Changes in body weight (A) and mortality (B) of vaccinated mice after the A/Indonesia/05/2005 (H5N1) virus challenge. Groups of 16 mice were immunized twice (21 day interval) intranasally with the adjuvanted vaccine at a HA content of 15, 5 or 1.5 μg or with 5 μg c-di-GMP/PBS (control). Mice were challenged by intranasal administration of highly pathogenic avian Influenza A/Indonesia/05/2005 (H5N1) on day 42. (A); Body weights were obtained daily for 2 weeks post-challenge (days 0-14). The weight of the animals as measured on the day of viral challenge (day 0) was used as the baseline weight to determine changes in body weight post viral challenge. (B); The survival rate (percentage) of mice was examined daily for 2 weeks post virus challenge.

Figure 5.

Virus recovery from the respiratory tract and systemic organs of vaccinated ferrets after challenge with A/Indonesia/05/2005 (H5N1) virus. Ferrets were vaccinated with 2 doses of the plant-derived vaccine (45 μg HA) adjuvanted with c-di-GMP (50 μg), non-adjuvanted vaccine or PBS (control) 14 days apart by the intranasal route. The ferrets were then challenged at 14 days after the second vaccine dose with the A/Indonesia/05/2005 (H5N1) live virus by the intranasal route. On day 3 post-challenge, 5 ferrets from each group were sacrificed and lung, nasal turbinate (NT), brain, olfactory bulb, spleen and nasal wash samples were collected. Tissue samples were homogenized and the clarified homogenates and nasal washes were assayed for the presence of A/Indonesia/05/2005 virus by titration on MDCK cells.

Figure 6.

Percentage weight loss (A) and changes in body temperature (B) in vaccinated and control ferrets following challenge with the A/Indonesia/05/2005 (H5N1) virus. Ferrets were vaccinated with 2 doses of 50 μg c-di-GMP adjuvanted vaccine (45 μg HA + c-di-GMP), vaccine alone (45 μg HA) and PBS (control) 14 days apart by the intranasal route. The ferrets were then challenged 14 days after the second vaccine dose with the A/Indonesia/05/2005 (H5N1) live virus by the intranasal route. Ferrets were monitored daily for 14 days following viral challenge for body weight and temperature.