Sublingual Immunization with a Live Attenuated Influenza A Virus Lacking the Nonstructural Protein 1 Induces Broad Protective Immunity in Mice

The nonstructural protein 1 (NS1) of influenza A virus (IAV) enables the virus to disarm the host cell type 1 IFN defense system. Mutation or deletion of the NS1 gene leads to attenuation of the virus and enhances host antiviral response making such live-attenuated influenza viruses attractive vaccine candidates. Sublingual (SL) immunization with live influenza virus has been found to be safe and effective for inducing protective immune responses in mucosal and systemic compartments. Here we demonstrate that SL immunization with NS1 deleted IAV (DeltaNS1 H1N1 or DeltaNS1 H5N1) induced protection against challenge with homologous as well as heterosubtypic influenza viruses. Protection was comparable with that induced by intranasal (IN) immunization and was associated with high levels of virus-specific antibodies (Abs). SL immunization with DeltaNS1 virus induced broad Ab responses in mucosal and systemic compartments and stimulated immune cells in mucosa-associated and systemic lymphoid organs. Thus, SL immunization with DeltaNS1 offers a novel potential vaccination strategy for the control of influenza outbreaks including pandemics

Induction of HI Abs upon immunization with Delta H1N1.

<p>On the day before challenge as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039921#pone-0039921-g003" target="_blank">Figure 3</a>, the sera were collected and the titers of HI Abs were determined against A/PR8 virus. The values represent the mean + SEM antibody titers of sera from 5 mice per group.</p

SL immunization with DelNS1 vaccine activates mucosal and extramucosal CD4+ T cells.

<p>CFSE-labeled CD4⁺ T cells from HA-TCR transgenic mice were adoptively transferred into naïve mice. One day later, recipients were given SL 2×10⁷ pfu of Delta H1N1, 2×10⁵ pfu of mouse-adapted wt live or 40 µg of formalin-inactivated A/PR8. Three days after the immunization, proliferating (CFSE stained) HA-TCR CD4⁺ T cells were detected in cervical lymph nodes (CLN), lungs, mediastinal lymph nodes (MdLN) and spleens by FACS analysis. The data are representative of two experiments showing similar results.</p

Induction of homotypic and HSI upon immunization with Delta H1N1.

<p>BALB/c mice were immunized IN or SL with different doses of Delta H1N1 (▴H1N1), given once (1x) or twice (2x) at 2-week intervals. Four weeks later, animals were intranasally challenged with 5×LD₅₀ of homologous mouse-adapted A/PR8 (Fig. 3A) or heterosubtypic mouse-adapted A/Philippines (A/P) H3N2 (Fig. 3B) virus. Morbidity and mortality were monitored daily. Data are expressed as mean (body weight or % survival) determined on groups of 5–10 mice.</p

Levels of virus-specific IgA induced in mucosal and systemic compartments upon immunization with DeltaNS1 IAV.

<p>BALB/c mice were immunized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039921#pone-0039921-g006" target="_blank">Figure 6</a>. Four weeks later, titers of H1N1 (A/PR8) virus-specific IgA in sera and secretions were determined by ELISA. The values represent mean + SEM ELISA titers determined on groups of 5 mice.</p

Levels of virus-specific IgG induced in mucosal and systemic compartments upon immunization with DeltaNS1 IAV.

<p>BALB/c mice were immunized with different doses of Delta H1N1 (▴ H1N1) or wt live virus A/PR8/34 (PR8) H1N1 (WT) via the sublingual (SL) or intranasal (IN) route. Four weeks later, titers of H1N1 (A/PR8) virus-specific IgG in sera and secretions were determined by ELISA. The values represent mean + SEM ELISA titers determined on groups of 5 mice.</p

Induction of virus-specific IgG and IgA upon immunization with Delta H5N1.

<p>BALB/c mice were immunized with different doses of Delta H5N1 (▴ H5N1) or wt live virus A/PR8/34 (PR8) H1N1 via the sublingual (SL), nasal (N) or intranasal (IN) route. Four weeks later sera were collected and the levels of H5N1 virus-specific IgG and IgA were determined by Delta H5N1 virus-coated ELISA plates. The values represent the mean + SEM (vertical bars) end point ELISA antibody titers determined on 5 mice per group.</p

Induction of virus-specific Abs upon immunization with Delta H1N1.

<p>On the day before challenge as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039921#pone-0039921-g003" target="_blank">Figure 3</a>, the sera were collected and the titers of virus-specific Abs were determined by A/PR8 virus-coated ELISA plates. The values represent the mean + SEM antibody titers of sera from 5 mice per group.</p