An adjuvant for the induction of potent, protective humoral responses to an H5N1 influenza virus vaccine with antigen-sparing effect in mice

Abstract

Intramuscular administration of inactivated influenza virus vaccine is the main vaccine platform used for the prevention of seasonal influenza virus infection. In clinical trials, inactivated H5N1 vaccines have been shown to be safe and capable of eliciting immune correlates of protection. However, the H5N1 vaccines are poorly immunogenic compared to seasonal influenza virus vaccines. Needle-free vaccination would be more efficient and economical in a pandemic, and the development of an effective and safe mucosal adjuvant will be an important milestone. A stabilized chemical analog of double-stranded RNA, PIKA, was previously reported to be a potent mucosal adjuvant in a murine model. While PIKA stimulates dendritic cells in vitro, little was known about its receptor and adjuvanting mechanism in vivo. In this study, we demonstrated that the immunostimulatory effect of PIKA resulted in an increased number of mature antigen-presenting cells, with the induction of proinflammatory cytokines at the inoculation site. In addition, coadministration of PIKA with a poorly immunogenic H5N1 subunit vaccine led to antigen sparing and quantitative and qualitative improvements of the immune responses over those achieved with an unadjuvanted vaccine in mice. The adjuvanted vaccine provided protection against lethal challenge with homologous and heterologous H5N1 wild-type viruses. Mice lacking functional TLR3 showed diminished cytokine production with PIKA stimulation, diminished antibody responses, and reduced protective efficacy against wild-type virus challenge following vaccination. These data suggest that TLR3 is important for the optimal performance of PIKA as an adjuvant. With its good safety profile and antigen-sparing effect, PIKA could be an attractive adjuvant for use in future pandemics.

FIG. 1.

Infiltration and maturation of antigen-presenting cells in the lungs of mice after intranasal administration of PIKA. Groups of three mice were given 100 μg of PIKA in a volume of 50 μl or 50 μl of PBS intranasally. One group continued to receive daily dosing of PIKA for two additional days. Lungs were harvested 24 h after the final dose. Single-cell suspensions were prepared, and different cell populations were identified by their differential surface expression of CD11b and CD11c. (A) Lung dendritic cells (lung DC [CD11b^int and CD11c^int]) expressed as a percentage of total cells in the lungs. The bars and error bars represent the means and standard errors for the group. d1, day 1. (B to D) Lung DC, alveolar macrophages (CD11b^lo and CD11c^hi), and interstitial macrophages (CD11b^hi and CD11⁻) were identified, and surface expression of CD86 was determined. Gray, isotype control; blue, PBS control; red, PIKA treated. The diagrams are representative of three individual animals from each group from a single experiment.

FIG. 2.

Intranasal administration of PIKA induced the production of proinflammatory cytokines. Groups of three BALB/c mice each were given either 100 μg of PIKA i.n. in PBS or 50 μl of PBS alone and were sacrificed 24 or 72 h later. The lungs were stored at −80°C until all samples were collected and homogenized in 1 ml of RPMI 1640 medium. To measure the concentrations of various cytokines, 50 μl of clarified samples was tested in duplicate using the Bio-plex protein array system. The concentration of each cytokine detected in the PIKA-treated group is expressed as the fold increase over the concentration detected in the PBS-treated group. The bars and error bars represent the means and standard errors for each group. Note that, due to the differences in the expression levels of the various cytokines, the scales of the y axes are different. These data are representative of two independent experiments. The absolute values of the cytokines in this experiment can be found in Table S1 in the supplemental material.

FIG. 3.

Coadministration of PIKA improved the immunogenicity of an H5N1 subunit vaccine, with an antigen-sparing effect. Groups of 5 mice were given two doses of the indicated amount of the H5N1 subunit vaccine, with or without PIKA, intranasally. (A and B) Influenza virus-specific serum antibody titers were determined on day 28 (A) and day 56 (B) by ELISA by using a BPL-inactivated H5N1 vaccine as the coating antigen. Each filled circle represents the antibody titer of an individual mouse, and each line represents the mean titer of the group of mice. The dashed lines represent the lower limit of detection. The asterisk indicates that the difference between the two groups was statistically significant (P < 0.05). (C) Serum neutralizing (MN) antibody titers on day 56 after vaccination against the wt A/Vietnam/1203/2004 virus. Each filled circle represents the MN titer of an individual mouse, and each line represents the geometric mean for the group of mice. (D) The IgG1 and IgG2a titers in day 56 sera from mice that received 1,500 ng of unadjuvanted or adjuvanted vaccine were determined by ELISA, and the ratios of IgG2a to IgG1 were calculated. (E) A group of 5 mice received 15 ng of adjuvanted vaccine i.n. and received a second dose on day 28. Serum samples were collected every 7 days after the initial vaccination to monitor the development of influenza virus-specific antibody responses by using an ELISA. The bars and error bars represent the means and standard errors for each group. The data were obtained from a single experiment.

FIG. 4.

The enhanced immunogenicity of the adjuvanted H5N1 subunit vaccine was associated with better protection against lethal challenge with wt H5N1 viruses. (A to C) Groups of 5 mice were primed and boosted with the indicated vaccine doses as described for Fig. 3. On day 56, the mice were challenged intranasally with 10⁵ TCID₅₀ of wt VN04 (H5N1) virus. Viral titers on day 4 p.i. in the lung (A), nasal turbinates (B), and brain (C) are expressed as log₁₀ TCID₅₀/g of tissue. Each filled circle represents the titer of an individual mouse, and each line represents the mean for the group of mice. An asterisk indicates that the difference between two groups was statistically significant (P < 0.05). An asterisk shown above a single column of data indicates that the difference between this group and the PBS group was statistically significant (P < 0.05). (D and E) Groups of 5 mice were primed and boosted with 100 ng of vaccine as described for Fig. 3 and were challenged with 10⁵ TCID₅₀ of wt A/Indonesia/05/2005 (H5N1) virus (clade 2.1) on day 56. The viral titers on day 4 p.i. in nasal turbinates (D) and the lung (E) are expressed as log₁₀ TCID₅₀/g of tissue. The data are representative of two independent experiments.

FIG. 5.

The adjuvanting effect of PIKA can be achieved by multiple routes of administration. (A and B) Groups of 5 mice were vaccinated with two doses of 100 ng of the unadjuvanted or adjuvanted vaccine by the indicated routes. Influenza virus-specific serum antibody titers were determined on day 28 (A) and day 56 (B) by ELISA using a BPL-inactivated H5N1 vaccine as the coating antigen. (C) Groups of 5 mice were vaccinated with two doses of 500 ng of PIKA-adjuvanted vaccines by the indicated routes, and pulmonary IgA titers were determined by ELISA on day 56. (D) The IgG1 and IgG2a titers in day 56 sera from mice that received 100 ng of unadjuvanted or adjuvanted vaccine were determined by ELISA, and the ratios of IgG2a to IgG1 were calculated. The IgG2a/IgG1 ratio for the group that received the unadjuvanted vaccine through the i.n. route could not be determined, because the ELISA antibody titer was below the lower detection limit of the assay, and is denoted as N.A. (E and F) Groups of 5 mice were vaccinated as described for panel B and were challenged with 10⁵ TCID₅₀ of wt VN04 (H5N1) virus. Viral titers on day 4 p.i. in nasal turbinates (E) and the lung (F) are expressed as log₁₀ TCID₅₀/g of tissue. Each filled circle represents the titer of an individual mouse, and each line represents the mean for the group of mice. The asterisk indicates that the difference between two groups was statistically significant (P < 0.05). The data were obtained from a single experiment. A separate experiment showed that 100% of the mice that received 500 ng of the adjuvanted vaccine survived lethal challenge.

FIG. 6.

TLR3 is important for the adjuvanting property of PIKA in mice. (A) Groups of three TLR3^−/− or TLR3^+/+ mice each were given either 100 μg of PIKA i.n. in PBS or 50 μl of PBS alone and were sacrificed 24 h later. The lungs were homogenized in 1 ml of RPMI-1640 medium and were stored at −80°C. To measure the concentrations of various cytokines, 50 μl of clarified samples was tested in duplicate using the Bio-plex protein array system. The concentration of each cytokine detected in the PIKA-treated group was expressed as the fold increase over the concentration in the PBS control sample. The bars and error bars represent the means and standard errors for each group. Note that, due to the differences in the expression levels of the various cytokines, the scales of the y axes are different. (B) Groups of 5 mice were vaccinated with two 100-ng doses of the unadjuvanted or adjuvanted vaccine i.n. Influenza virus-specific serum antibody titers were determined on day 56 by ELISA using a BPL-inactivated H5N1 vaccine as the coating antigen. Each filled circle represents the titer for an individual mouse, and each line represents the mean for the group of mice. (C and D) The mice were challenged with 1 × 10⁵ TCID₅₀ of wt VN04 virus on day 56. The viral titers on day 4 p.i. in nasal turbinates (C) and the lung (D) are expressed as log₁₀ TCID₅₀/g of tissue. An asterisk indicates that the difference between two groups was statistically significant (P < 0.05). The data were obtained from a single experiment.