Response of BALB/c mice to a monovalent influenza A (H1N1) 2009 split vaccine

Abstract

The novel influenza A (H1N1) 2009 virus has emerged to cause the first pandemic of the twenty-first century. Disease outbreaks caused by the influenza A (H1N1) virus have prompted concerns about the potential for a pandemic and have driven the development of vaccines against this subtype of influenza A. In this study, we developed a monovalent influenza A (H1N1) split vaccine and evaluated its effects in BALB/c mice. Mice were immunized subcutaneously with 2 doses of the vaccine containing hemagglutinin (HA) alone or HA plus an aluminum hydroxide (Al(OH)(3)) adjuvant. Immunization with varying doses of HA (3.75, 7.5, 15, 30, 45 or 60 microg) was performed to induce the production of neutralizing antibodies. The vaccine elicited strong hemagglutination inhibition (HI) and microneutralization, and addition of the adjuvant augmented the antibody response. A preliminary safety evaluation showed that the vaccine was not toxic at large doses (0.5 ml containing 60 microg HA+600 microg Al(OH)(3) or 60 microg HA). Moreover, the vaccine was found to be safe at a dose of 120 microg HA+1200 microg Al(OH)(3) or 120 microg HA in 1.0 ml in rats. In conclusion, the present study provides support for the clinical evaluation of influenza A (H1N1) vaccination as a public health intervention to mitigate a possible pandemic. Additionally, our findings support the further evaluation of the vaccine used in this study in primates or humans.

Figure 1

Adjuvant and non-adjuvant influenza A (H1N1) vaccine-enhanced HI antibody responses. Eight groups of BALB/c mice were immunized at weeks 0 and 2 with influenza A (H1N1) split vaccine alone or combined with 1.2 mg/ml Al(OH)₃. The data are presented as mean±SD from three experiments performed in duplicate. Serum HI titers against influenza A (H1N1) at 2 weeks after the first and second doses of vaccine alone (a) or with adjuvant (b) are shown. HI, hemagglutination inhibition; PBS, phosphate-buffered saline.

Figure 2

Addition of Al(OH)₃ to the influenza A (H1N1) split vaccine further enhances the HI-antibody response. BALB/c mice were immunized at weeks 0 and 2 with 3.75, 7.5, 15, 30, 45 and 60 µg of influenza A (H1N1) split vaccine alone or containing 1.2 mg/ml Al (OH)₃. Shown are the geometric mean±SD of the serum HI titer against influenza A (H1N1) at 2 weeks after the first and second doses of vaccine alone (a) or with adjuvant (b), respectively. The vaccine doses are shown on the x axis. Each point represents the arithmetic mean±SD (n=5). The data are representative of three similar experiments. HI, hemagglutination inhibition.

Figure 3

Influenza A (H1N1) split vaccines with Al(OH)₃ as an adjuvant further enhance the virus-neutralizing antibody response. BALB/c mice (five mice per group) were immunized at weeks 0 and 2 with 3.75, 7.5, 15, 30, 45 and 60 µg of influenza A (H1N1) split vaccine alone or containing 1.2 mg/ml Al(OH)₃. The data represent mean±SD of three experiments performed in duplicate. Serum neutralization antibody titers against influenza A (H1N1) at 2 weeks after the first (a) and second (b) immunization with vaccine alone or containing adjuvant are shown. PBS, phosphate-buffered saline.

Figure 4

Serum IgG-antibody responses after vaccination. Mice (five mice per group) were immunized subcutaneously twice at a 2-week interval with HA from the influenza A (H1N1) vaccine. On days 14, 21 and 28 after the first immunization, influenza-specific serum IgG responses were measured by ELISA. The sampling days after vaccination are shown on the x axis. The data are mean±SD from three experiments performed in duplicate. ASC, antibody-secreting cell; HA, hemagglutinin; IgG, immunoglobulin G.

Figure 5

Examination of the IgG antibody response after vaccination by ELISPOT assay. Mice were immunized subcutaneously twice at a 2-week interval with HA from the influenza A (H1N1) vaccine. On day 14 after the final immunization, pools of three mice per group were killed and single-cell suspensions were prepared from the spleens for ELISPOT assay. The number of ASCs is shown per 500 000 lymphocytes. The bars show the mean number of specific ASCs per 500 000 lymphocytes±SD. The data are mean±SD from two experiments. ASC, antibody-secreting cell; ELISPOT, enzyme-linked immunospot; HA, hemagglutinin; IgG, immunoglobulin G.

Figure 6

Analysis of INF-γ and IL-4 by ELISPOT. Mice were immunized subcutaneously twice at a 2-week interval with HA from the influenza A (H1N1) vaccine. On day 14 after the final immunization, pools of three mice for each group were killed and single-cell suspensions were prepared from the spleens. The cells were cultured at 1×10⁵ cells in 200 µl with 5 µg/ml influenza A (H1N1) vaccine virus HA. INF-γ (a) and IL-4 (b) secretion by the splenic lymphocytes was detected by ELISPOT after culture for 40–44 h. The data are mean±SD from two experiments. ELISPOT, enzyme-linked immunospot; HA, hemagglutinin; INF-γ, interferon-γ.