Enhancement of the immunogenicity and protective efficacy of a mucosal influenza subunit vaccine by the saponin adjuvant GPI-0100

Abstract

Identification of safe and effective adjuvants remains an urgent need for the development of inactivated influenza vaccines for mucosal administration. Here, we used a murine challenge model to evaluate the adjuvant activity of GPI-0100, a saponin-derived adjuvant, on influenza subunit vaccine administered via the intranasal or the intrapulmonary route. Balb/c mice were immunized with 1 µg A/PR/8 (H1N1) subunit antigen alone or in combination with varying doses of GPI-0100. The addition of GPI-0100 was required for induction of mucosal and systemic antibody responses to intranasally administered influenza vaccine and significantly enhanced the immunogenicity of vaccine administered via the intrapulmonary route. Remarkably, GPI-0100-adjuvanted influenza vaccine given at a low dose of 2×1 µg either in the nares or directly into the lungs provided complete protection against homologous influenza virus infection.

Figure 1. Effects of unadjuvanted and GPI-0100-adjuvanted mucosal influenza vaccine on lung virus titers.

Mice were immunized on day 0 and on day 20 with 1 µg A/PR/8 subunit vaccine alone or adjuvanted with the indicated doses of GPI-0100. The control mice received HBS buffer. The immunizations were given via intramuscular (i.m.), intranasal (i.n.) or intrapulmonary (pul.) route. 2 weeks after the second immunization, mice were infected with live A/PR/8 virus. Lung samples were collected 3 days later upon termination. Virus titer is expressed as the 10log virus titer per gram of lung tissue for individual mice. The black line represents the geometric mean virus titer per group. One mouse from the HBS control group was sacrificed before the challenge due to abnormal tissue growth with unknown reason. Levels of significance are depicted as follows: *p<0.05, **p<0.01 and ***p<0.005.

Figure 2. Influenza-specific IgG and hemagluttination inhibition (HAI) titers elicited by unadjuvanted and GPI-0100-adjuvanted mucosal influenza vaccine.

Serum samples from the mice described in the legend to Fig. 1 were collected on day 20 and day 34. (A) Total IgG responses after a single immunization. Average 10log IgG titers ± standard error of the mean (S.E.M.), n  = 6 mice per group. The detection limit of the assay is represented by the dotted line. (B) Total IgG responses after two immunizations. (C) HAI titers after two immunizations. The results are expressed as 2log HAI titers of individual mice. The black line represents the geometric mean virus titer per group. Due to technical reasons only 5 and 4 samples from mice receiving 30 µg GPI-0100 adjuvanted intranasal immunization and unadjuvanted intrapulmonary immunization were available for the HAI assay.

Figure 3. Phenotype of the influenza-specific antibody responses.

Serum samples from the mice described in the legend to Fig. 1 were collected on day 37. (A) Average quantities (µg/ml) of influenza-specific IgG1± S.E.M., n  = 6 mice per group. (B) Average quantities (µg/ml) of influenza-specific IgG2a ± S.E.M.

Figure 4. Effect of GPI-0100 on influenza-specific IL-4-producing T cells.

Mice were immunized on day 0 and on day 20 with 1 µg A/PR/8 subunit vaccine alone or adjuvanted with the indicated doses of GPI-0100. The immunizations were given via intramuscular (i.m.), intranasal (i.n.) or intrapulmonary (pul.) route. Spleen samples were collected one week later upon termination. Splenocytes were isolated and stimulated overnight with PR8 subunit. The result is expressed as cytokine producing splenocytes per 5×10⁵ cells of individual mice.

Figure 5. Influenza-specific mucosal IgA and IgG responses elicited by unadjuvanted and GPI-0100-adjuvanted mucosal influenza vaccine.

Nose and lung wash samples from the mice described in the legend to Fig. 4 were collected on day 27 upon termination. (A) Nasal IgA responses after two immunizations. Average OD492 at each dilution ± S.E.M., n  = 6 mice per group. The starting and ending dilution is 2 and 4096 respectively. (B) Lung IgA responses after two immunizations. (C) Nasal IgG responses after two immunizations. Average IgG titers ± S.E.M. Due to technical reasons only 5 samples from mice receiving unadjuvanted intrapulmonary immunization were available. (D) Lung IgG responses after two immunizations. Due to technical reasons only 5 samples from mice receiving HBS and intrapulmonary immunization were available.

Figure 6. Effect of GPI-0100 on lung histology.

On day 0 and on day 20 mice received either HBS buffer (A, C, E, G) or GPI-0100 at the indicated doses (B, D, F, H) via the intranasal (A, B, E, F) or the intrapulmonary (C, D, G, H) route. Lung samples were collected 3 days after the second treatment upon termination. Lung sections were prepared and stained for Ly-6G and Ly-6C (A-D) or CD68 (E-H) to identify neutrophils and macrophages, respectively. Representative pictures of histological analyses of each treatment group are shown. The brown colored cells indicated by an arrow is a neutrophil (N) or a macrophage (M).

Figure 7. Effect of GPI-0100 on recruitment of inflammatory cells to the lung.

Quantification of (A) neutrophils and (B) macrophages on micrographs as described in the legend to Figure 6. For each staining section, regions of interest (ROI) were selected using HistoQuest software. The total number of cells within the regions were defined by the nucleus staining. Neutrophils or macrophages are defined as nucleated brown cells. The results are presented as % neutrophils or % macrophages per stained sample from individual mouse.