The magnitude of local immunity in the lungs of mice induced by live attenuated influenza vaccines is determined by local viral replication and induction of cytokines

Abstract

While live attenuated influenza vaccines (LAIVs) have been shown to be efficacious and have been licensed for human use, the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) have to be updated for optimal protective efficacy. Little is known about the effect of different HA and NA proteins on the immunogenicity of LAIVs developed using the same backbone. A panel of LAIVs that share the internal protein genes, with unique HA and NA gene segments from different influenza subtypes, was rescued by reverse genetics, and a comparative study of immune responses induced by these vaccines was conducted in mice. The results suggest that the magnitude of lung immunity, including pulmonary IgA antibody and memory CD8(+) T lymphocytes, induced by the vaccines depends on the replication efficiency of the LAIVs, as well as the induction of cytokines/chemokines in the lungs. However, these factors are not important in determining systemic immunity such as serum antibody titers and memory CD8(+) T cells in the spleen. A qualitative analysis of immune responses induced by a single dose of an H5N1 LAIV revealed that the vaccine induced robust systemic and mucosal immunity in mice. In addition, antibodies and memory lymphocytes established in the lungs following vaccination were required for protection against lethal challenge with homologous and heterologous H5N1 viruses. Our results highlight the different requirements for inducing systemic and lung immunity that can be explored for the development of pulmonary immunity for protection against respiratory pathogens.

FIG. 1.

Different LAIVs differ in their replication in mice. Groups of five mice each were inoculated with 1 × 10⁶ TCID₅₀ of various LAIVs i.n. Lungs and NT were harvested on the indicated days p.v. Virus titers in the NT (A) and lungs (B) were determined on MDCK monolayers and are expressed as log₁₀ TCID₅₀/g of tissue. The lower limit of detection is represented by the dotted line. The bars and error bars represent the means and standard deviations, respectively, of the group. d, day.

FIG. 2.

The induction of systemic and mucosal humoral immune responses by various LAIVs in mice. (A) Groups of three mice were vaccinated i.n. with various LAIVs as previously described, and serum samples were collected 28 days later. Influenza virus-specific serum antibody titers were determined by ELISA, using homologous BPL-inactivated whole virion preparations as the coating antigen. (B) The isotype distribution of influenza virus-specific antibodies in serum. The lower limit of detection is represented by the dotted line. (C) The serum neutralizing antibody titer on day 28 p.v. against the homologous LAIV virus. Plates were scored for cytopathic effect after 6 days of incubation at 33°C. The bars and error bars represent the means and standard deviations, respectively, for the groups. (D and E) Influenza virus-specific antibodies in lungs (D) and NT (E) determined by ELISA. PR8 wt virus was used as a positive control (n = 5).

FIG. 3.

The induction of antibody-secreting cells in lungs of mice. (A to C) Groups of three LAIV-vaccinated mice were killed on day 28 p.v., and the numbers of influenza virus-specific antibody-secreting cells in lungs (A), spleen (B), and bone marrow (C) were determined by B-cell ELISPOT assay. (D to F) The isotype distribution of ASCs in lungs: IgA-specific (D), IgG_2a-specific (E), and IgG1-specific (F). The bars and error bars represent the means and standard deviations, respectively, for the groups. (G and H) Groups of five mice were vaccinated with the VN04 (H5N1) ca vaccine i.n., and the isotype distribution of influenza virus-specific antibodies in the nasal wash (G) and lung homogenates (H) was determined by ELISA. Samples were diluted appropriately and added in three sets of wells that were coated with BPL-inactivated VN04 (H5N1) ca. Different isotype-specific antibodies were added in indicated sets and developed as mentioned in Materials and Methods. The optical density at 405 nm was measured. (I) IgA antibody titers in the lungs of mice 28 days after receiving various LAIV. *, the difference between the groups was statistically significant (P < 0.05). PR8 wt virus was used as a positive control.

FIG. 4.

Cellular immunity elicited by LAIV in mice. (A) Groups of three mice were vaccinated with 10⁶ TCID₅₀ of VN04 (H5N1) ca vaccine i.n. and sacrificed at the indicated time (x axis), and the frequency of epitope-specific CD8⁺ CTLs in lungs was determined by intracellular cytokine staining (y axis). The solid line indicates NP₁₄₇-specific and the dotted line indicates HA₅₁₈ specific data. The error bars are the standard deviation of the group. (B) Groups of three mice were vaccinated with the indicated dose of the H5N1 vaccine i.n. and were sacrificed 8 days later. The frequency of NP₁₄₇-specific CD8⁺ T cells in the lungs was determined by ICS. The bars and error bars are the means and standard deviations, respectively, of the groups. *, the difference between the groups was statistically significant (P < 0.05). (C and D) Upregulation of surface expression of CD107a/b on epitope-specific CD8⁺ CTL. Pulmonary lymphocytes were stimulated with NP₁₄₇ epitope in vitro for 5 h, and epitope-specific CD8⁺ CTLs were identified by IFN-γ production (area B). Monoclonal antibodies against CD107a/b were used to detect exocytosis of lytic granules, and the surface expression of CD107a/b on epitope-specific CD8⁺ CTLs is shown in panel D. (E) Groups of three vaccinated or mock-vaccinated mice received 5 × 10⁶ NP₁₄₇-pulsed (hatched bar) or HA₅₁₈-pulsed cells (white bar) on day 8 p.v. by intravenous injection. An equal number of unpulsed targets was coinjected to measure nonspecific lysis. The degree of specific lysis was determined 16 h later by calculating the ratio between the epitope-pulsed and unpulsed targets in the spleen. The bars and error bars are the means and standard deviations, respectively, of the groups. ND, not detected. (F and G) The frequency of epitope-specific CD8⁺ CTLs induced by various LAIVs. Groups of three mice were vaccinated with 1 × 10⁶ TCID₅₀ of different LAIVs i.n., and the frequency of NP₁₄₇-specific (F) and HA₅₁₈-specific (G) CD8⁺ T cells in lungs on day 8 p.v. was detected by ICS. The asterisk indicates that the LAIV strain has an altered HA₅₁₈ epitope. PR8 wt virus was used as a positive control.

FIG. 5.

The induction of cellular immune memory by LAIV. (A and B) Groups of three vaccinated mice were sacrificed on day 28 to examine the frequency of NP₁₄₇-specific CD8⁺ T cells in the lungs (A) and spleen (B). The bars and error bars are the means and standard deviations, respectively, of the groups. (C) The level of IFN-γ produced by splenic NP₁₄₇-specific CD8⁺ T cells based on the mean fluorescence (MFL) intensity in the detecting channel. (D) The degree of in vivo lytic activity in vaccinated mice at indicated time points (x axis) was determined by an in vivo cytotoxicity assay. Open symbols represent lytic activity detected in vaccinated mice. Filled symbols represent mock-vaccinated mice. The error bars represent the standard deviations of the means. In panels A and B, PR8 wt virus was used as a positive control.

FIG. 6.

The immunity induced by Panama99 (H3N2) ca vaccine provides significant protection in mice. Groups of five vaccinated or mock-vaccinated mice were challenged i.n. with 1 × 10⁶ TCID₅₀ of Panama99 (H3N2) wt virus on day 28 p.v. Viral titers in NT (A) and lungs (B) at indicated time points were determined on MDCK monolayers. The short solid and dotted lines represent the means of the L15 and Panama99 (H3N2) ca groups, respectively. The lower limit of detection is denoted by a long dotted line. *, difference between the vaccinated and the mock-vaccinated group was statistically significant (P < 0.05).

FIG. 7.

The immunity induced by the VN04 (H5N1) ca vaccine provides significant protection in the NT and accelerated viral clearance from the lungs following challenge with homologous and heterologous wt viruses. (A to C) Groups of 10 vaccinated or mock-vaccinated mice were challenged i.n. with 1 × 10⁵ TCID₅₀ of VN04 (H5N1) wt virus on day 28 p.v. Viral titers in NT (A) and lungs (B) at indicated time points were determined on MDCK monolayers. (C) Viral titers in brain 4 days after the infection. (D to F) Vaccinated mice were challenged with 1 × 10⁵ TCID₅₀ of Indo 05 (H5N1) wt virus. Viral titers in NT (D) and lungs (E) at indicated time points were determined on MDCK monolayers. (F) Viral titers in brain 4 days after the infection. The lower limit of detection is denoted by a long dotted line. The short solid and dotted lines represent the means of the L15 and VN04 (H5N1) ca groups, respectively. *, the difference between the two groups was statistically significant (P < 0.05). The results are the representative of two individual experiments.