The contribution of systemic and pulmonary immune effectors to vaccine-induced protection from H5N1 influenza virus infection

Abstract

Live attenuated influenza vaccines (LAIVs) are effective in providing protection against influenza challenge in animal models and in preventing disease in humans. We previously showed that LAIVs elicit a range of immune effectors and that successful induction of pulmonary cellular and humoral immunity in mice requires pulmonary replication of the vaccine virus. An upper respiratory tract immunization (URTI) model was developed in mice to mimic the human situation, in which the vaccine virus does not replicate in the lower respiratory tract, allowing us to assess the protective efficacy of an H5N1 LAIV against highly pathogenic H5N1 virus challenge in the absence of significant pulmonary immunity. Our results show that, after one dose of an H5N1 LAIV, pulmonary influenza-specific lymphocytes are the main contributors to clearance of challenge virus from the lungs and that contributions of influenza-specific enzyme-linked immunosorbent assay (ELISA) antibodies in serum and splenic CD8(+) T cells were negligible. Complete protection from H5N1 challenge was achieved after two doses of H5N1 LAIV and was associated with maturation of the antibody response. Although passive transfer of sera from mice that received two doses of vaccine prevented lethality in naive recipients following challenge, the mice showed significant weight loss, with high pulmonary titers of the H5N1 virus. These data highlight the importance of mucosal immunity in mediating optimal protection against H5N1 infection. Understanding the requirements for effective induction and establishment of these protective immune effectors in the respiratory tract paves the way for a more rational and effective vaccine approach in the future.

Fig 1

Restricted replication of VN04 (H5N1) LAIV by URTI is associated with diminished pulmonary humoral responses but does not affect serum antibody titers. (A and B) Groups of five mice were inoculated with the indicated doses of the VN04 H5N1 LAIV by URTI. Viral titers in the NT (A) and lungs (B) were determined 4 days pi and are expressed as log₁₀ TCID₅₀/g of tissues. The dotted lines represent the lower limit of detection. (C) BALB/c mice received 1 × 10⁶ or 1 × 10⁷ TCID₅₀ of VN04 H5N1 LAIV by URTI. The magnitudes of the serum antibody responses detected 28 days later by ELISA are presented as log₁₀ antibody titers. (D to F) Groups of 5 mice were vaccinated with the indicated doses of the VN04 H5N1 LAIV by URTI or TRTI. Serum samples (D) and homogenates of NT (E) and lung (F) were collected 28 days later. The titers of total influenza-specific antibodies in serum and influenza-specific IgA in the homogenates of NT and lungs were determined by ELISA. The bars and error bars represent the means and standard deviations of the group. *, P < 0.05.

Fig 2

Mice vaccinated by URTI showed diminished cellular responses in the lungs but not in the spleen. Groups of 5 mice were vaccinated as described in the legend to Fig. 1. Single-cell suspensions were obtained from lungs and spleens 28 days after vaccination. (A and B) The frequency of ASC in lungs (A) and spleen (B) was determined by B-cell enzyme-linked immunosorbent spot assay (ELISPOT). Due to the difference in frequencies, note that the scales of the y axes are different. (C and D) The frequency of NP₁₄₇-specific CD8⁺ T cells in lungs (C) and spleen (D) was determined by gamma interferon (IFN-γ) ELISPOT assay. The bars and error bars represent the means and standard deviations of the group. *, P < 0.05; N.D., not detected.

Fig 3

Mice vaccinated by URTI succumbed to lethal challenge with H5N1 wt virus. (A) Groups of 5 mice were vaccinated as described in Fig. 1 and were challenged with 1 × 10⁵ TCID₅₀ of VN04 (H5N1, clade 1) wt virus i.n. 28 days later. Survival was monitored for 3 weeks after challenge. The symbol for each group is as follows: ●, 10⁶ TCID₅₀ of VN04 H5N1 LAIV by TRTI; x, 10⁶ TCID₅₀ by URTI; ♦, 10⁷ TCID₅₀ by URTI; ▼, mock-vaccinated group. As the mortality rates for the 10⁶ TCID₅₀ by URTI and mock-vaccinated groups were identical, the symbols for these groups are superimposed. (B and C) Mice were vaccinated and challenged as described in panel A. Viral titers in NT (B) and lungs (C) at the indicated time points were determined on MDCK monolayers. The dotted lines represent the lower limit of detection. *, P < 0.05; #, there was a significant difference between this group and the mock-vaccinated group.

Fig 4

CD4⁺ and CD8⁺ lymphocytes are important in mediating accelerated viral clearance from the lungs. Groups of 5 mice were vaccinated with 1 × 10⁶ TCID₅₀ of VN04 (H5N1) LAIV by TRTI and were challenged with wt VN04 (H5N1) 28 days later. Specific monoclonal antibodies were injected i.p. 2 days before and after challenge to deplete specific subsets of lymphocytes. Lungs were harvested on day 4 pi. (A) Single-cell suspensions were prepared to verify the depletion of the indicated cell subsets in the lungs. (B to E) Vaccinated mice were euthanized 2 (B, C) or 4 (D, E) days postchallenge. Viral titers in NT (B, D) and lungs (C, E) were determined on MDCK monolayers. *, P < 0.05; #, there was a significant difference between this group and the mock-vaccinated group. N.A. indicates that all mice succumbed to the infection prior to sample collection. From an independent study (25), the average viral titers in NT and lungs of mock-infected mice on d4 postinfection were 10⁶ and 10^7.2 TCID₅₀/g of tissues, respectively.

Fig 5

The effect of boosting on the immunogenicity of the VN04 (H5N1) LAIV given by URTI. Groups of 5 mice were given the VN04 (H5N1) LAIV by URTI or TRTI as indicated on days 0 and 28. (A to C) Serum samples (A), lung homogenates (B), and NT homogenates (C) were collected on day 56. The titers of total influenza-specific antibodies in serum and influenza-specific IgA in the homogenates of NT and lungs were determined by ELISA. (D) Serum-neutralizing antibody titers against 100 TCID₅₀ of VN04 H5N1 wt virus were determined in an MN assay. (E and F) Mice vaccinated as indicated by URTI were challenged with 1 × 10⁵ TCID₅₀ of VN04 H5N1 wt virus. Survival (E) and weight loss (F) were monitored for 15 days. (G and H) Groups of 5 mice were vaccinated as indicated and challenged with 1 × 10⁵ TCID₅₀ of VN04 H5N1 wt virus on day 56. Viral titers in NT (G) and lungs (H) on day 4 postchallenge were determined on MDCK monolayers. *, P < 0.05.

Fig 6

Passive transfer of postvaccination sera (PVS) confers variable protection against lethal challenge but does not prevent pulmonary viral replication of the VN04 (H5N1) wt challenge virus. (A to C) Two groups of mice were given 1 × 10⁶ TCID₅₀ of VN04 H5N1 LAIV by TRTI. One group was euthanized for serum (d28-1 dose PVS) on d28 and the other group received a boost, and sera were collected on d56 (d56-2 dose PVS). Groups of 5 naïve mice received 0.5 ml of the indicated PVS i.p. 1 day before wt virus challenge. For active immunization, a group of mice was vaccinated with two doses of 500 ng of an adjuvanted subunit VN04 H5N1 vaccine s.c. 28 days apart. All mice were challenged with 1 × 10⁵ TCID₅₀ of VN04 H5N1 wt virus on day 56. Survival (A) and weight loss (B) were monitored for 15 days. (C and D) The viral titers in NT (C) and lungs (D) were determined on day 4 postchallenge. *, there was a significant difference between this group and the L15 mock-vaccinated group. The dotted line represents the lower limit of detection.