Multiple-clade H5N1 influenza split vaccine elicits broad cross protection against lethal influenza virus challenge in mice by intranasal vaccination

Abstract

Background: The increase in recent outbreaks and unpredictable changes of highly pathogenic avian influenza (HPAI) H5N1 in birds and humans highlights the urgent need to develop a cross-protective H5N1 vaccine. We here report our development of a multiple-clade H5N1 influenza vaccine tested for immunogenicity and efficacy to confer cross-protection in an animal model.

Methodology/principal findings: Mice received two doses of influenza split vaccine with oil-in-water emulsion adjuvant SP01 by intranasal administration separated by two weeks. Single vaccines (3 µg HA per dose) included rg-A/Vietnam/1203/2004(Clade 1), rg-A/Indonesia/05/2005(Clade 2.1), and rg-A/Anhui/1/2005(Clade 2.3.4). The trivalent vaccine contained 1 µg HA per dose of each single vaccine. Importantly, complete cross-protection was observed in mice immunized using trivalent vaccine with oil-in-water emulsion adjuvant SP01 that was subsequently challenged with the lethal A/OT/SZ/097/03 influenza strain (Clade 0), whereas only the survival rate was up to 60% in single A/Anhui/1/2005 vaccine group.

Conclusion/significance: Our findings demonstrated that the multiple-clade H5N1 influenza vaccine was able to elicit a cross-protective immune response to heterologous HPAI H5N1 virus, thus giving rise to a broadly cross-reactive vaccine to potential prevention use ahead of the strain-specific pandemic influenza vaccine in the event of an HPAI H5N1 influenza outbreak. Also, the multiple-clade adjuvanted vaccine could be useful in allowing timely initiation of vaccination against unknown pandemic virus.

Figure 1. HAI titers of mice immunized with single or trivalent H5N1 influenza split vaccines.

Mice were immunized i.n. with single H5N1 influenza split vaccines (3 µg HA per dose) and a trivalent vaccine that contained 1 µg HA per dose of each single vaccine in combination with adjuvant on day 0 and 14, and bled on day 28. Four HA units of VN/1203 (Fig. 1A), ID/05 (Fig. 1B), AH/01 (Fig. 1C), and China097 (Fig. 1D) viral antigen were used. Results are the geometric mean titers of positive sera (HI titer >10). The values are means ± SEM from six mice. * p<0.05 and ** p<0.01. The dashed horizontal line indicates the lower limit of detection.

Figure 2. Mucosal antibody response in BALB/c.

Secretion of anti-HA IgA antibodies against VN/1203, ID/05, and AH/01 H5N1 inactivated antigen in nasal and lung lavage (dilution 1∶5) from mice immunized i.n. with single H5N1 influenza split vaccines (3 µg HA per dose) and a trivalent vaccine containing 1 µg HA per dose of each single vaccine in combination with adjuvant. The values are means ± SEM from six mice. * p<0.05 and ** p<0.01. The dashed horizontal line indicates the lower limit of detection.

Figure 3. sIgA-ASC, INF-γ, and IL-4 after vaccination as examined by ELISPOT assay.

Mice were immunized twice at a 2-week interval with single H5N1 influenza split vaccines (3 µg HA per dose) and a trivalent vaccine that contained 1 µg HA per dose of each single vaccine in combination with adjuvant. On day 14 after the final immunization, pools of three mice for each group were killed and single-cell suspensions were prepared from spleen for the ELISPOT assay. (A) The figure shows antibody-secreting cells (ASCs) per 500,000 lymphocytes. The bars indicate the mean number of specific ASCs per 500,000 lymphocytes ± SD. (B) The INF-γ and IL-4 secreted by spleen lymphocytes were detected using ELISPOT after culture for 40–44 h. The values are means ± SEM from two experiments. * p<0.05 and ** p<0.01.

Figure 4. Protective effect of multiple-clade influenza vaccine in mice challenged with A/OT/SZ/097/03.

Groups of mice (n = 10/group) were immunized i.n. with single H5N1 influenza split vaccines (3 µg HA per dose) and a trivalent vaccine that contained 1 µg HA per dose of each single vaccine in combination with adjuvant and challenged i.n. with 50LD₅₀ OT/SZ/097/03 virus suspension. Mice were monitored for weight change (A) and survival rates (B) throughout a 14-day observation period.

Figure 5. Histopathological changes in lungs and virus titers of infected mice.

Mice (n = 6/group) immunized twice i.n. with single H5N1 influenza split vaccines (3 µg HA per dose) and a trivalent vaccine that contained 1 µg HA per dose of each single vaccine in combination with adjuvant were infected with 50LD₅₀ OT/SZ/097/03 virus 2 weeks after the second immunization. (A) Histopathological changes in the lungs on 4 day post challenge. The figure indicates the representative images of histopathological damage from H & E-stained lungs of 5 mice in PBS group and trivalent vaccine group. (magnification, 100×). (B) On day 4 after virus inoculation, mice were killed and the nasal turbinates, lungs, spleen, kidney, and brains were collected. Virus titers in tissue homogenates were determined by TCID₅₀ assay using MDCK cells. The values are means ± SEM from six mice. * p<0.05 and ** p<0.01. The dashed horizontal line indicates the lower limit of detection.