Mucosal boosting increases protective efficacy of an influenza vaccine in mice

Abstract

Influenza virus infection is a significant cause of global mortality. However, the development of influenza vaccines that induce robust immune responses at the site of respiratory mucosal exposure has proven challenging. Here, we assessed immune responses and protective efficacy of a rhesus adenovirus serotype 52 (RhAd52) vectored influenza vaccine encoding the hemagglutinin (HA) glycoprotein from A/California/07/2009 administrated by systemic or mucosal routes of immunization. We observed robust and durable systemic and mucosal immunity, including IgA and tissue resident memory T cells in the respiratory mucosa, in mice that received the vaccine intranasally or intratracheally. In contrast, only systemic immune responses were observed in mice that received the vaccine intramuscularly. Moreover, a single intranasal or intratracheal dose of RhAd52-HA provided near complete protection against a lethal challenge with a mouse-adapted influenza virus strain, whereas intramuscular immunization with RhAd52-HA and mRNA-HA provided less robust protection. Our data demonstrate the importance of mucosal immunity for enhancing vaccine protection against influenza.

Keywords: Immunity; Virology.

Graphical abstract

Figure 1

Immunogenicity elicited by a single dose of RhAd52-HA and mRNA-HA in different delivery modes in mice (A–H) (A) Immunization plan. BALB/c mice were immunized with different vaccines in different delivery modes. At week 4, BALF (B) and nasal wash (C) were collected for the analysis of HA-specific IgA binding antibody titer. The HA-specific IgG binding antibody titer (D) and neutralization titer (E) in BALF were also analyzed. Mouse serum was collected for the analysis of HA-specific binding antibody titer (F) and neutralization titer (G). Cellular immunity in the lung, nasal turbinate (NT) and spleen were assessed with ICS assay (H). Black lines indicate the median value of each group, and dotted lines show the limit of detection (LOD) of the assay.

Figure 2

Immunogenicity elicited by a homologous prime/boost regimen (A–F) (A) Immunization plan. BALB/c mice were immunized with RhAd52-HA or mRNA HA at week 0 (prime) and boosted with same vaccine and same delivery route at week 10 (boost). Nasal wash was collected at the indicated time points and HA-specific IgA binding antibody titer was analyzed (B). Both HA-specific IgA (C) and IgG (D) binding antibody titers were analyzed in the BALF. The binding antibody titers in serum at the indicated time points were analyzed (E). The fold change of the median number of each cellular immunological parameter in the mice immunized with single dose and mice immunized with two doses in homologous “prime-boost” regimen was summarized (F).

Figure 3

Durable immune response elicited by a single dose of RhAd25-CA09 with mucosal delivery (A–D) (A) Immunization plan. BALB/c mice were immunized with a single dose of mRNA-HA IM or RhAd-CA09 HA IM or IN and terminated at month 7 to assess the durability of immune response. The HA-specific IgG antibody binding titers in serum and BALF and HA-specific IgA antibody binding titers in BALF and nasal wash were evaluated (B). The HA-specific IgG and IgA-secreting B cells in bone marrow (BM), lung and NT were evaluated (C). The number of IFN-γ⁺ T cells in lung, NT and spleen were assessed with ELISPOT assay (D).

Figure 4

Mucosal vaccinations of a single dose of RhAd52-HA completely protect mice from lethal challenge (A–D) (A) Immunization plan. BALB/c mice were immunized with different vaccines in different delivery modes and challenged with 22 LD₅₀ at week 4, then terminated at 3 dpi. Post challenge, the mice were monitored for clinical disease and body weight at the indicated time points (B). The viral load in the BALF and lung collected at 3 dpi were determined by TCID₅₀ assay (C). The RNA copies of influenza viruses in the BALF, nasal wash and lung collected at 3 dpi were determined (D). A parallel mouse study that BALB/c mice were immunized with different vaccines in different delivery modes and challenged with 22 LD₅₀ at week 4. Body weight post challenge were monitored for 16 days (error bar exhibits Mean ± SEM) (E).