Mucosal immunization with PspA (Pneumococcal surface protein A)-adsorbed nanoparticles targeting the lungs for protection against pneumococcal infection

Burden of pneumonia caused by Streptococcus pneumoniae remains high despite the availability of conjugate vaccines. Mucosal immunization targeting the lungs is an attractive alternative for the induction of local immune responses to improve protection against pneumonia. Our group had previously described the development of poly(glycerol adipate-co-ω-pentadecalactone) (PGA-co-PDL) polymeric nanoparticles (NPs) adsorbed with Pneumococcal surface protein A from clade 4 (PspA4Pro) within L-leucine microcarriers (nanocomposite microparticles-NCMPs) for mucosal delivery targeting the lungs (NP/NCMP PspA4Pro). NP/NCMP PspA4Pro was now used for immunization of mice. Inoculation of this formulation induced anti-PspA4Pro IgG antibodies in serum and lungs. Analysis of binding of serum IgG to intact bacteria showed efficient binding to bacteria expressing PspA from clades 3, 4 and 5 (family 2), but no binding to bacteria expressing PspA from clades 1 and 2 (family 1) was observed. Both mucosal immunization with NP/NCMP PspA4Pro and subcutaneous injection of the protein elicited partial protection against intranasal lethal pneumococcal challenge with a serotype 3 strain expressing PspA from clade 5 (PspA5). Although similar survival levels were observed for mucosal immunization with NP/NCMP PspA4Pro and subcutaneous immunization with purified protein, NP/NCMP PspA4Pro induced earlier control of the infection. Conversely, neither immunization with NP/NCMP PspA4Pro nor subcutaneous immunization with purified protein reduced bacterial burden in the lungs after challenge with a serotype 19F strain expressing PspA from clade 1 (PspA1). Mucosal immunization with NP/NCMP PspA4Pro targeting the lungs is thus able to induce local and systemic antibodies, conferring protection only against a strain expressing PspA from the homologous family 2

Pneumococcal load in BALF collected 24 hours after non-lethal challenge with EF3030.

<p>Mice were immunized with 2 doses of the indicated formulations and challenged with pneumococcal strain EF3030. Pneumococcal load in BALF was determined 24 hours after challenge. Symbols represent each individual. Means±standard errors are shown.</p

Binding of serum IgG to intact pneumococci.

<p>Sera from mice immunized with two doses of the indicated formulations were tested for the ability to bind to pneumococcal strains expressing PspA from clades 1 (A), 2 (B), 3 (C), 4 (D) and 5 (E). Results are shown as fluorescence intensity histograms and are representative of two experiments using sera from independent immunizations.</p

Immunophenotyping of cells recovered in BALF collected 12 hours after lethal challenge with ATCC6303.

<p>Mice were immunized with 2 doses of the indicated formulations and challenged with pneumococcal strain ATCC6303. BALF was collected 12 hours after challenge and recovered cells were immunophenotyped by flow cytometry. Percentages of alveolar macrophages (AM—F4/80+ CD11c+ CD11b-) (A), exudate macrophages (EM—F4/80+ CD11c- CD11b+) (B), B cells (F4/80- B220+) (C), CD4+ T cells (F4/80- CD4+) (D), CD8+ T cells (F4/80- CD8+) (E) and neutrophils (F4/80- Ly6G+) (F) are shown. Means and standard errors of each group are shown.</p

Induction of serum anti-PspA4Pro IgG antibodies by mucosal immunization targeting the lungs.

<p>The induction of anti-PspA4Pro IgG antibodies in sera from mice inoculated with the indicated formulations was determined by ELISA. Mice were inoculated with 1 (A) or 2 (B and C) doses of the formulations. Control mice were inoculated sc with saline or PspA4Pro. Log₁₀ anti-PspA4Pro IgG titers (A and B) and log₁₀ anti-PspA4Pro IgG1 titer/log₁₀ anti-PspA4Pro IgG2a titer ratios (C) are shown. * indicates statistically significant difference with saline and # indicates statistically significant difference with PspA4Pro sc (One-way ANOVA, Tukey’s Multicomparison Test for A and B; Unpaired t-test for C). Symbols represent each individual. Means±standard errors are shown. Representative of at least two independent experiments.</p

Induction of anti-PspA4Pro antibodies in BALF by mucosal immunization targeting the lungs.

<p>The induction of anti-PspA4Pro IgG (A) and IgA (B) antibodies in BALF from mice inoculated with the indicated formulations was determined by ELISA. Mice were inoculated with 2 doses of the formulations. Control mice were inoculated sc with saline or PspA4Pro. <i>A</i>_<i>405</i> nm of samples diluted 1:2 is shown. * indicates statistically significant difference with saline (One-way ANOVA, Tukey’s Multicomparison Test). Symbols represent each individual. Means±standard errors are shown.</p

Cytokine/chemokine levels in BALF collected 12 hours after lethal challenge with ATCC6303.

<p>Mice were immunized with 2 doses of the indicated formulations and challenged with pneumococcal strain ATCC6303. Levels of IL-6 (A), TNF-α (B), KC/CXCL1 (C) and MIP-2/CXCL2 (D) were determined in BALF collected 12 hours after challenge by Luminex. Naive mice were not immunized nor challenged. * indicates statistically significant difference (One-way ANOVA, Tukey’s Multicomparison Test). Symbols represent each individual. Means±standard errors are shown. Representative of two independent experiments.</p