The delivery site of a monovalent influenza vaccine within the respiratory tract impacts on the immune response

Abstract

Pulmonary vaccination is a promising immunization route. However, there still remains a crucial need to characterize the different parameters affecting the efficacy of inhaled vaccination. This study aimed at assessing the impact of antigen distribution within the respiratory tract on the immune response to a monovalent A/Panama/2007/99 H3N2 influenza split virus vaccine administered to BALB/c mice. Varying the administration technique allowed the targeting of the vaccine to different sites of the mouse respiratory tract, i.e. the nasal cavity, the upper or central airways, or the deep lung. This targeting was verified by using ovalbumin as a tracer compound. The immune responses generated following influenza vaccine administration to the different respiratory tract sites were compared to each other and to those elicited by intramuscular and peroral intragastric immunization. Delivery of the vaccine to the different respiratory regions generated systemic, local and cellular virus-specific immune responses, which increased with the depth of vaccine deposition, culminating in deep-lung vaccination. The latter induced virus-specific serum immunoglobulin G and neutralizing antibody titres as elevated as intramuscular vaccination, whereas the production of mucosal secretory immunoglobulin A was significantly superior in deep-lung-vaccinated animals. The analysis of cytokines secreted by mononuclear cells during an in vitro recall response indicated that deep-lung vaccination induced a local shift of the cellular immune response towards a T helper type 1 phenotype as compared to intramuscular vaccination. In conclusion, antigen distribution within the respiratory tract has a major effect on the immune response, with the deep lung as the best target for inhaled influenza vaccination.

Figure 1

Regional deposition in NMRI mice of instilled ovalbumin following delivery using the techniques described in Table 1. * Indicates higher regional deposition fraction versus the corresponding fraction of 10 µl i.t. (P < 0·05) and 25 µl i.t. (P < 0·01). ^# Indicates higher regional deposition fraction as compared to the corresponding fraction of 10 µl i.n. (P < 0·001), 50 µl i.n. (P < 0·01) and 10 µl i.t. (P < 0·05).

Figure 5

Impact of the delivery site of a monovalent A/Panama/2007/99 H3N2 influenza split virus vaccine on cellular immune responses. BALB/c mice were immunized as detailed in Table 2. (a) Proliferation of spleen and pulmonary lymph node (LN) mononuclear cells (MNCs) following in vitro split A/Panama/2007/99 virus stimulation. The results are expressed as stimulation indexes. § Indicates significant differences from the priming control group. One symbol indicates P < 0·05, two symbols indicate P < 0·01. The supernatants from the cultures of (b) pulmonary LN MNCs and (c) spleen MNCs were assayed for IFN-γ and IL-4. ^## Indicates highly sifgnificant difference from the pulmonary LN IL-4 production [Student's t-test versus LD(IL-4), P < 0·01]. ND: below limit of detection (LD); line A, LD for IFN-γ = 0·49; line B, LD for IL-4 = 0·25.

Figure 2

Impact of the delivery site of a monovalent A/Panama/2007/99 H3N2 influenza split virus vaccine on serum antibody responses. BALB/c mice were immunized as detailed in Table 2. (a) A/Panama/2007/99 virus neutralization titre. (b) Anti-A/Panama/2007/99 IgG titres. § Indicates significant difference from the priming control group. ^# Indicates significant difference from the deep-lung-immunized group. * Indicates significant difference from the i.m. immunized group. One symbol indicates P < 0·05, two symbols indicate P < 0·01.

Figure 3

Impact of the delivery site of a monovalent A/Panama/2007/99 H3N2 influenza split virus vaccine on the local respiratory antibody response. BALB/c mice were immunized as detailed in Table 2. (a) BAL and (b) nasal-wash anti-A/Panama/2007/99 IgA titres, (c) BAL anti-A/Panama/2007/99 IgG titres. § Indicates significant difference from the priming control group. ^# Indicates significant difference from the deep-lung-immunized group. * Indicates significant difference from the i.m. immunized group. One symbol indicates P < 0·05, two symbols indicate P < 0·01.

Figure 4

Impact of the delivery site of a monovalent A/Panama/2007/99 H3N2 influenza split virus vaccine on specific serum immunoglobulin G (IgG) subclasses response. BALB/c mice were immunized as detailed in Table 2. § Indicates significant difference from the priming control group. ^# Indicates significant difference from the deep-lung-immunized group. * Indicates significant difference from the i.m. immunized group. One symbol indicates P < 0·05, two symbols indicate P < 0·01.