Inactivated Eyedrop Influenza Vaccine Adjuvanted with Poly(I:C) Is Safe and Effective for Inducing Protective Systemic and Mucosal Immunity

Abstract

The eye route has been evaluated as an efficient vaccine delivery routes. However, in order to induce sufficient antibody production with inactivated vaccine, testing of the safety and efficacy of the use of inactivated antigen plus adjuvant is needed. Here, we assessed various types of adjuvants in eyedrop as an anti-influenza serum and mucosal Ab production-enhancer in BALB/c mice. Among the adjuvants, poly (I:C) showed as much enhancement in antigen-specific serum IgG and mucosal IgA antibody production as cholera toxin (CT) after vaccinations with trivalent hemagglutinin-subunits or split H1N1 vaccine antigen in mice. Vaccination with split H1N1 eyedrop vaccine antigen plus poly(I:C) showed a similar or slightly lower efficacy in inducing antibody production than intranasal vaccination; the eyedrop vaccine-induced immunity was enough to protect mice from lethal homologous influenza A/California/04/09 (H1N1) virus challenge. Additionally, ocular inoculation with poly(I:C) plus vaccine antigen generated no signs of inflammation within 24 hours: no increases in the mRNA expression levels of inflammatory cytokines nor in the infiltration of mononuclear cells to administration sites. In contrast, CT administration induced increased expression of IL-6 cytokine mRNA and mononuclear cell infiltration in the conjunctiva within 24 hours of vaccination. Moreover, inoculated visualizing materials by eyedrop did not contaminate the surface of the olfactory bulb in mice; meanwhile, intranasally administered materials defiled the surface of the brain. On the basis of these findings, we propose that the use of eyedrop inactivated influenza vaccine plus poly(I:C) is a safe and effective mucosal vaccine strategy for inducing protective anti-influenza immunity.

Fig 1. CT and Poly(I:C) enhances systemic and mucosal Ab production by eyedrop vaccination of OVA or HA.

Groups of female BALB/c mice received OVA (100 μg) (A) or HA (1 μg) (B) plus CT (2 μg), poly(I:C) (10 μg), MPLA (10 μg), Imject Alum (320 μg) resolved in 5 μl of PBS, or 5 μl of PBS alone by drops on both eyes every week (three times). Ag-specific Ab levels were measured in serum and in various mucosal fluids 1 wk after final vaccination by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001 versus Ag alone; §§ p < 0.01, §§§ p < 0.001 versus poly(I:C) group; ‘n.s.’, non-significant. Results are representative of three independent experiments, with three mice in each group.

Fig 2. Comparison of antibody production by the amount of hemagglutinin (HA) or poly(I:C).

(A). Dose-dependent HA vaccine antigens were administered with 10 μg poly(I:C) resolved in 5 μl of PBS by drops on both eyes two times at a 2-week interval in female BALB/c mice. HA-specific Ab levels were measured in serum and in various mucosal fluids 2 weeks after final vaccination by ELISA. (B) 1 μg of HA vaccine antigen plus dose-dependent poly(I:C) was vaccinated by drops on both eyes two times at a 2-week interval in female BALB/c mice. HA-specific Ab levels were measured in serum and in various mucosal secretions 2 weeks after final vaccination by ELISA. * p < 0.05; ** p < 0.01 versus Ag alone group; ‘n.s.’, non-significant. Results are representative of three independent experiments, with three mice in each group.

Fig 3. Comparison of Ab production between intranasal and eyedrop administration routes.

(A) Dose-dependent HA vaccine antigens were administrated with 10 μg poly(I:C) resolved in 5 μl PBS by drops on both eyes two times at a 2-week interval in female BALB/c mice. HA-specific Ab levels were measured in serum and in various mucosal secretions 2 weeks after final vaccination by ELISA. (B) 1 μg H1N1 split vaccine antigen plus dose-dependent poly(I:C) resolved in 5 μl PBS were vaccinated by drops on both eyes two times at a 2-week interval in female BALB/c mice. Ag-specific Ab levels were measured in serum and in various mucosal secretions 2 weeks after final vaccination by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001 versus Ag alone; § p < 0.05, §§ p < 0.01, §§§ p < 0.001 versus poly(I:C) group. Results are representative of three independent experiments, with four mice in each group.

Fig 4. Eyedrop inactivated influenza vaccine plus poly(I:C) administration protects mice from lethal influenza virus challenge.

Female BALB/c mice were given PBS (△) or H1N1 split vaccine Ag alone (eyedrop, ○; IN, □) or Ag plus 10 μg poly(I:C) (eyedrop, ●; IN, ■) by eyedrop or IN two times at a 2-week interval. Eyedrop groups were vaccinated with 5 μl of vaccine on both eyes and IN groups were received with 20 μl of vaccine. At 2 weeks after the last administration, mice were challenged IN with 10X LD₅₀ of homologous mouse-adapted H1N1 influenza virus. Body weights (A) and survival rates (B) were monitored daily. (C) Nine mice in each group were vaccinated by eyedrop or intranasal immunization with 1 μg of H1N1 split vaccine plus 10 μg of poly(I:C). At two weeks after the final vaccination, mice were anesthetized and challenged with 50 μl of mouse-adapted live influenza A/California/04/09 (H1N1) virus suspension (10 X LD50; 0.75 TCID50) via the IN route. To measure the viral titers in the lung organs, three mice per group were sacrificed on day +1, +4 and +7. After the lungs were removed, they were homogenized in 1ml PBS using a small motor and upper respiratory tract was rinsed with 1ml PBS. Samples were centrifuged at 12,000×g and the supernatant fluids were removed, and then the supernatants were stored at −70°C until assayed for viral titers. Viral titers were assayed by plaque assay with MDCK cells. * p < 0.05; *** p < 0.001 between Ag plus 10 μg poly(I:C)_eye and PBS; § p < 0.05, §§ p < 0.01, §§§ p < 0.001 between Ag plus poly(I:C) treated eye and IN groups. Results are representative of three independent experiments, with five mice in each group.

Fig 5. No inflammation in the eyes after administration of HA antigen plus poly(I:C) in mice.

(A-D) Female BALB/c mice were administered 1 μg of HA vaccine alone, 1 μg HA plus 10 μg poly(I:C), or plus 2 μg CT resolved in 5 μl of PBS on the eyes for various time periods. Total RNA was extracted from homogenized conjunctival tissues with TRI reagents for reverse transcription and real-time PCR analysis. Gene expression levels were calculated as a relative ratio to the average value of house-keeping genes, β-actin. ** p < 0.01 versus 0 h. Data represent means ± S.D. of 3 independent experiments.

Fig 6. No induction of cellular infiltration in conjunctival tissues after administration of HA antigen plus poly(I:C).

(A) Female BALB/c mice were administered PBS, 1 μg / 10 μl of HA vaccine antigens plus 10 μg /10 μl poly(I:C), or 2 μg/10μl CT on the eyes, and eye tissues were prepared and stained with H&E. Every representative figure of the same position of the rectangle (Aa) are shown (Aa-b, PBS; Ac, poly(I:C); Ad, CT). (B) Mononuclear cells were counted in sub-epithelial regions of tarsal conjunctival areas and plotted as a graph (cells per 1000 μm²). * p < 0.05; ** p < 0.01 versus Ag alone group. Results are representative of three independent experiments, with three mice in each group.

Fig 7. No detection of contrast medium on the olfactory bulbs from mice administered eyedrops.

Female BALB/c mice were administered 10 μl or 50 μl of contrast medium by eyedrop or IN, respectively. After 30 min of contrast medium treatment, CT pictures of the brains of each mouse were taken. Frontal view (A), top view (B), or dimmed top view (C). Inner dashed circle, olfactory bulbs; blue arrows and red dots, spots of the contrast medium.