Adjuvanted influenza vaccine administered intradermally elicits robust long-term immune responses that confer protection from lethal challenge

Abstract

Background: The respiratory illnesses caused by influenza virus can be dramatically reduced by vaccination. The current trivalent inactivated influenza vaccine is effective in eliciting systemic virus-specific antibodies sufficient to control viral replication. However, influenza protection generated after parenteral immunization could be improved by the induction of mucosal immune responses.

Methodology/principal findings: Transcutaneous immunization, a non-invasive vaccine delivery method, was used to investigate the quality, duration and effectiveness of the immune responses induced in the presence of inactivated influenza virus co-administered with retinoic acid or oleic acid. We observed an increased migration of dendritic cells to the draining lymph nodes after dermal vaccination. Here we demonstrate that this route of vaccine delivery in combination with certain immunomodulators can induce potent immune responses that result in long-term protective immunity. Additionally, mice vaccinated with inactivated virus in combination with retinoic acid show an enhanced sIgA antibody response, increased number of antibody secreting cells in the mucosal tissues, and protection from a higher influenza lethal dose.

Conclusions/significance: The present study demonstrates that transdermal administration of inactivated virus in combination with immunomodulators stimulates dendritic cell migration, results in long-lived systemic and mucosal responses that confer effective protective immunity.

Figure 1. Neutralizing antibody titers after vaccination.

Virus-neutralizing antibody activities in sera collected 2 weeks after immunization. Dilutions of sera were incubated with approximately 100 plaque forming units of PR8 virus for 1 hr at room temperature, applied to monolayers of confluent MDCK cells, and a standard plaque reduction assay was performed. Representative data are shown from at least three independent experiments. Data shown are average and standard error of mean (SEM) from 6 mice per group. Groups are as described in legend of Table 1.

Figure 2. IL-4 and IFN-γ secreting cells in response to inactivated influenza antigen.

The cellular immune responses were analyzed in splenocytes by ELISPOT for IL-4 and IFN-γ production 2 weeks after vaccination by re-stimulation with nucleoprotein (NP) and hemagglutinin (HA) class I (A,B) and class II (C,D) restricted peptides. Groups are as described in the legend of Table 1. Data are pooled from three independent experiments and are the average ± SEM for six mice per group. Statistical significance of the differences between groups in comparison was calculated by Student's test (p<0.05). Statistics; a: p<0.05 comparing all groups to the PR8 alone group, b: <0.05 comparing adjuvanted groups to PR8-CT group (PR8-CT-RA, PR8-CT-OA, PR8-OA-RA).

Figure 3. Protection of mice from lethal influenza virus challenge.

Survival rates of immunized mice were monitored for 10 days after i.n. infection with mouse adapted PR8 virus. (A) Kaplan-Meier curve showing the percent of mice that survived a 5x LD₅₀ PR8 virus challenge. (B) Percent of mice that survived a 20x LD₅₀ PR8 virus challenge. (C) Percentage of body weight after i.n. challenge with 20x LD₅₀ PR8 virus. PBS, PR8, PR8-OA-RA, PR8-CT, PR8-CT-OA, and PR8-CT-RA groups are as described in the legend of Table 1. Data represent the average of six mice per group for each challenge study.

Figure 4. Antibody titers of post-challenged mice 4 and 8 days after intranasal infection with live A/PR/8/34 influenza virus.

Total IgG anti-influenza titers determined by quantitative ELISA from immunized mice. Serum IgG1 and IgG2a isotype titers at day 4 (B,D) and day 8 (C,D) post-challenge. Ratios for IgG1/IgG2a were derived from the results in panels B and C. Mucosal IgA (E) and IgG (F) antibody titers by ELISA in bronchoalveolar lavage fluid (BALF) at day 4 and 8 after challenge. Data shown are average and standard error of mean (SEM) from 6 mice per group. Groups are as described in the legend of Table 1. Statistics; a: p<0.05 comparing all groups to the PR8 alone group.

Figure 5. Influenza specific antibody secreting cells (ASC) in spleen and lungs of immunized mice.

(A) Splenocytes and (B) single cell lung suspensions of vaccinated and control mice were assessed by ELISPOT 4 days post-challenge. Data shown are average and standard error of mean (SEM) from 5 mice per group. Groups are as described in the legend of Table 1. N: naïve (unimmunized) mice; PBS: mock immunized with PBS, challenged mice. Statistics; *: p<0.05 comparing all groups to the naive group;**: p<0.05 comparing all groups to the PBS alone group; a: p<0.05 comparing all groups to the PR8 alone group; b: <0.05 comparing adjuvanted groups to PR8-CT group (PR8-CT-RA, PR8-CT-OA, PR8-OA-RA).

Figure 6. Dendritic cell mobilization to the lymph nodes upon TCI immunization.

Dendritic cells were gated as triple positive populations (CD11c+/CD11b+/CD205+ cells). The total number of triple positive population/mouse lymph node was calculated and the data of each group of mice were averaged. Groups are as described in the legend of Table 1.