Comparison of the effectiveness of antibody and cell-mediated immunity against inhaled and instilled influenza virus challenge

Abstract

Background: To evaluate immunity against influenza, mouse challenge studies are typically performed by intranasal instillation of a virus suspension to anesthetized animals. This results in an unnatural environment in the lower respiratory tract during infection, and therefore there is some concern that immune mechanisms identified in this model may not reflect those that protect against infectious virus particles delivered directly to the lower respiratory tract as an aerosol.

Method: To evaluate differences in protection against instilled and inhaled virus, mice were immunized with influenza antigens known to induce antibody or cell-mediated responses and then challenged with 100 LD50 A/PR/8/34 (PR8) in the form of aerosol (inhaled) or liquid suspension (instilled).

Results: Mice immunized with recombinant adenovirus (Ad) expressing hemagglutinin were protected against weight loss and death in both challenge models, however immunization with Ad expressing nucleoprotein of influenza A (NPA) or M2 resulted in greater protection against inhaled aerosolized virus than virus instilled in liquid suspension. Ad-M2, but not Ad-NPA-immunized mice were protected against a lower instillation challenge dose.

Conclusions: These results demonstrate differences in protection that are dependent on challenge method, and suggest that cell-mediated immunity may be more accurately demonstrated in mouse inhalation studies. Furthermore, the data suggest immune mechanisms generally characterized as incomplete or weak in mouse models using liquid intranasal challenge may offer greater immunity against influenza infection than previously thought.

Figure 1

Comparison of virus titers in the lungs of vaccinated mice after instillation and inhalation PR8 challenge. Geometric mean virus titers (TCID₅₀/ml) are shown for mice challenged by instillation (grey bars) and inhalation (white bars). Virus titers were determined for lungs (5 mice per group) collected on day 4 post-challenge, and homogenized in 1 ml of serum-free medium. Error bars indicate standard deviation and the dashed line is the limit of quantification for this assay.

Figure 2

Mice immunized with rAd-M2 and rAd-NP are protected against weight loss when virus is inhaled, but not when virus is instilled. The average percent of starting weight for each group is shown for mice that were challenged by (A) instillation and (B) inhalation. Mice in each group (n = 5) were weighed individually on each day after challenge. Each graph shows the percent group mean change in weight relative to the baseline body weights, with error bars indicating the standard deviation for the group.

Figure 3

Mice immunized with rAd-M2 and rAd-NPA survive inhaled, but not instilled, PR8 challenge. Survival of immunized mice is shown for (A) instilled, and (B) inhaled virus challenge. Mice in all groups (n = 10) were challenged on the same day; to more easily visualize differences, naïve, PR8 and X-31-exposed mice are shown in graphs on the left hand side, and groups vaccinated with adenovirus recombinants expressing HA, M2, NP_A and NP_B are shown graphically on the right hand side. Since 5 mice were sacrificed in each group on day 4, survival of the remaining 5 was monitored to the end of the study (12 days post challenge). A key indicating the color code for each group is provided on the figure. Survival against both instilled or inhaled virus challenge was significantly greater for mice previously infected with either PR8 or X31 than naïve mice (Mantel-Cox test, p < 0.001); similarly survival of mice immunized with Ad-HA was greater than Ad-NP_B-immunized mice (control group) when challenged with instilled or inhaled virus challenge (p < 0.001). The difference in survival between Ad-NP_B-immunized and Ad-M2 or Ad-NP_A-immunized mice was not statistically different when challenged with instilled virus, but trended towards significance (p = 0.054) for mice challenged by inhalation.

Figure 4

(A) Virus titers, (B) weight loss and (C) mortality of 10LD₅₀ instilled PR8. Groups of mice (n = 10) were immunized as previously and challenged by instillation of PR8. Four days after challenge, 5 mice in each group were euthanized to allow titration of virus in the lungs, the weight and survival of the remaining mice was monitored until day 12. (A) shows geometric mean virus titers, together with the standard deviation; the dashed line is the limit of quantification for this assay. (B) shows the percent reduction in weight relative to the baseline body weights, with error bars showing standard deviation. A key for the groups is shown to the right of this graph. (C) shows percent survival of mice in each group. A key for these groups is shown to the right of the graph. Survival of mice vaccinated with Ad-HA and Ad-M2 was significantly greater (Mantel-Cox test, p = 0.0016) than mice immunized with Ad-NP_A or Ad-NP_B. The latter 2 groups were not different from one another or naïve mice.