Protective immunity to lethal challenge of the 1918 pandemic influenza virus by vaccination

Abstract

The remarkable infectivity and virulence of the 1918 influenza virus resulted in an unprecedented pandemic, raising the question of whether it is possible to develop protective immunity to this virus and whether immune evasion may have contributed to its spread. Here, we report that the highly lethal 1918 virus is susceptible to immune protection by a preventive vaccine, and we define its mechanism of action. Immunization with plasmid expression vectors encoding hemagglutinin (HA) elicited potent CD4 and CD8 cellular responses as well as neutralizing antibodies. Antibody specificity and titer were defined by a microneutralization and a pseudotype assay that could assess antibody specificity without the need for high-level biocontainment. This pseudotype inhibition assay can define evolving serotypes of influenza viruses and facilitate the development of immune sera and neutralizing monoclonal antibodies that may help contain pandemic influenza. Notably, mice vaccinated with 1918 HA plasmid DNAs showed complete protection to lethal challenge. T cell depletion had no effect on immunity, but passive transfer of purified IgG from anti-H1(1918) immunized mice provided protective immunity for naïve mice challenged with infectious 1918 virus. Thus, humoral immunity directed at the viral HA can protect against the 1918 pandemic virus.

Fig. 1.

Expression of viral HAs. (A) The structure of the vectors, together with the indicated specific mutations in the cleavage site, for immunogens and lentiviral vector pseudotypes. (B) Expression of the indicated viral HAs was determined by Western blot analysis with antisera reactive to the 1918 influenza HA. Expression was evaluated after transfection of the indicated plasmids in 293T cells. Arrows indicate the relevant viral HA bands.

Fig. 2.

Humoral and cellular immune responses to 1918 influenza HA after DNA vaccination. (A) Antibody responses induced by DNA vaccination against the 1918 influenza HA measured by ELISA (Left) or microneutralization (Right). The microneutralization assay using dilutions of heat-inactivated sera and titers of virus neutralizing antibody were determined as the reciprocal of the highest dilution of serum that neutralized 100 plaque-forming units of virus in Madin-Darby canine kidney (MDCK) cell cultures on a 96-well plate (Right). ELISA for viral nucleocapsid protein (NP) was performed for determining the presence of the virus as the read-out. Data are presented as the mean for each group. (B) Intracellular cytokine staining was performed to analyze the T cell response to viral HA peptides. The percentage of activated T cells that produced either IFN-γ and/or TNF-α in response to stimulation with overlapping peptides in CD4 (Left) or CD8 (Right) is shown. Lymphocytes from mice (n = 5 per group) immunized with empty plasmid vector (control) or mice (n = 10 per group) immunized with the indicated plasmid at 0, 3, 6, and 12 weeks were assessed, and immune responses were measured 11 days after the final boost. Nonstimulated cells gave responses similar to controls at background levels. Symbols indicate the response of individual animals, and the median value is shown with a horizontal bar.

Fig. 3.

Immune protection conferred against lethal challenge of 1918 influenza and lack of T cell dependence. (A) Immunization with H1(1918), H1(1918)ΔCS, or negative control plasmid expression vectors was performed in mice (n = 10 per group) as described (11), and survival (Upper) and weight loss (Lower) were evaluated. The statistical significance between these groups are P = 1.08 × 10⁻⁵ and P = 1.08 × 10⁻⁵ with respect to controls by Fisher's exact test. (B) Monoclonal rat anti-mouse anti-CD4, CD8, and CD90 (T cell depletion) were used to deplete T cells in H1(1918)ΔCS immunized mice, in comparison with a control group of vaccinated animals injected with nonimmune IgG (Control IgG). Vector-immunized animals that received no depletion served as additional controls (Vector). Mice were administered IgG at −3, +3, +9, and +15 days after viral challenge. Mice (n = 10 per group) were then evaluated for survival (Upper) and weight loss (Lower). No decrease in immune protection was observed in T cell-depleted animals.

Fig. 4.

Immune mechanism of protection, showing dependence on Ig. (A) Activity of control, nonimmune, IgG (control), or anti-HA immune IgG [anti-H1(1918)], purified as described (12), was confirmed by ELISA before passive transfer into naïve recipients (n = 10 per group). (B) Passive transfer. To assess the protective effects of immune IgG, mice received immune or control IgG 24 h before infection with 100 LD₅₀ of 1918 virus. Mice were then monitored for survival and weight loss throughout a 21-day observation period. The difference between the immune [α−H1(1918) IgG] and control IgG (IgG) groups was significant (P = 0.0007).

Fig. 5.

Development of HA-pseudotyped lentiviral vectors. (A) Gene transfer mediated by lentiviral vectors pseudotyped with H1(1918), H5(Kan-1), or other HAs containing the H5 protease cleavage site was measured with a luciferase reporter assay. (B) Neutralization by antisera from mice immunized with the indicated HA plasmid expression vectors or no insert (control) plasmid DNA vectors was measured with the luciferase assay with the HA-pseudotyped lentiviral vectors. Reduction of gene transfer in the presence of immune sera was observed in a dose-dependent fashion (data not shown).