Protection Elicited by Nasal Immunization with Recombinant Pneumococcal Surface Protein A (rPspA) Adjuvanted with Whole-Cell Pertussis Vaccine (wP) against Co-Colonization of Mice with <i>Streptococcus pneumoniae</i>

<div><p>A promising alternative vaccine candidate to reduce the burden of pneumococcal diseases is the protein antigen PspA (Pneumococcal surface protein A). Since concomitant colonization with two or more pneumococcal strains is very common in children, we aimed to determine if immunization with PspA would be able to control co-colonization. We evaluated nasal immunization with recombinant PspA (rPspA) in a model of co-colonization with two strains expressing different PspAs. Mice were immunized intranasally with rPspAs from clades 1 to 4 (rPspA1, rPspA2, rPspA3 or rPspA4) using whole-cell pertussis vaccine (wP) as adjuvant. Mice were then challenged with a mixture of two serotype 6B isolates St491/00 (PspA1) and St472/96 (PspA4). Immunization with rPspA1+wP and rPspA4+wP reduced colonization with both strains and the mixture of rPspA1+rPspA4+wP induced greater reduction than a single antigen. Immunization rPspA1+rPspA4+wP also reduced colonization when challenge experiments were performed with a mixture of isolates of serotypes 6B (PspA3) and 23F (PspA2). Furthermore, none of the tested formulations led to a pronounced increase in colonization of one isolate over the other, showing that the vaccine strategy would not favor replacement. Interestingly, the adjuvant wP by itself already led to some reduction in pneumococcal colonization, indicating the induction of non-specific immune responses. Anti-rPspA IgG was observed in serum, nasal wash (NW) and bronchoalveolar lavage fluid (BALF) samples, whereas animals inoculated with formulations containing the adjuvant wP (with or without rPspA) showed higher levels of IL-6 and KC in NW and increase in tissue macrophages, B cells and CD4⁺T cells in BALF.</p></div

Recovery of pneumococci from the nasopharynx of mice challenged with a mixture of serotype 23F and serotype 6B strains–rPspA1 and rPspA4 mixture.

<p>Mice were immunized intranasally with two doses of the indicated formulations and challenged with a mixture of serotype 23F and serotype 6B strains expressing different PspAs. The adjuvant wP was inoculated only in the first dose. Recovery of strains 23F OPKA (PspA2) (A) and 6B OPKA (PspA3) (B) in nasal washes performed 5 days after challenge is shown.</p

Recovery of pneumococci from the nasopharynx of mice challenged with a mixture of two 6B strains–rPspA1 and rPspA4 mixture.

<p>Mice were immunized intranasally with two doses of the indicated formulations and challenged with a mixture of two 6B strains expressing different PspAs. Recovery of strains St491/00 (PspA1) (A) and St472/96 (PspA4) (B) in nasal washes performed 5 days after challenge is shown. * indicates statistical difference with saline (One-way ANOVA, Tukey’s Multicomparison Test—** P≤0.01).</p

Immunophenotyping of BALF cells.

<p>BALF samples were collected from mice immunized with the indicated formulations and infiltrated cells were immunophenotyped by flow cytometry. BALF was collected before (D0), at day 1 (D1) and at day 5 (D5) after intranasal challenge with a mixture of strains 23F OPKA (PspA2) and 6B OPKA (PspA3). Percentages of F4/80⁺ CD11b⁺ (A), F4/80⁺ CD11c⁺ (B), F4/80^- B220⁺ (C), F4/80^- CD4⁺ (D), F4/80^- CD8⁺ (E) and F4/80^- Ly6G⁺ (F) are shown. Differences with saline at the same day and differences within the same group at different days are shown (One-way ANOVA, Tukey’s Multicomparison Test—* P≤0.05; **P≤0.01, ***P≤0.001). Results are representative of two independent experiments.</p

Induction of anti-rPspA IgG serum antibodies.

<p>Mice were immunized intranasally with two doses of the indicated formulations and anti-rPspA1(A), anti-rPspA2 (B), anti-rPspA3 (C) and anti-rPspA4 (D) IgG in serum samples were detected by ELISA. * indicates statistical difference with saline (One-way ANOVA, Tukey’s Multicomparison Test—*** P≤0.001). Results are representative of two independent experiments.</p

IL-6 and KC levels in nasal wash samples.

<p>NW samples were collected from mice immunized with the indicated formulations before (D0), at day 1 (D1) and at day 5 (D5) after intranasal challenge with a mixture of strains 23F OPKA (PspA2) and 6B OPKA (PspA3). IL-6 (A-C) and KC (D-F) were detected using a Luminex-based assay. * indicates statistical difference with saline (One-way ANOVA, Tukey’s Multicomparison Test—* P≤0.05; **P≤0.01, ***P≤0001). Results are representative of two independent experiments.</p

Recovery of pneumococci from the nasopharynx of mice challenged with a mixture of serotype 23F and serotype 6B strains.

<p>Mice were immunized intranasally with two doses of the indicated formulations and challenged with a mixture of serotype 23F and serotype 6B strains expressing different PspAs. Recovery of strains 23F OPKA (PspA2) (A) and 6B OPKA (PspA3) (B) in nasal washes performed 5 days after challenge is shown. * indicates statistical difference with saline (One-way ANOVA, Tukey’s Multicomparison Test- * P≤0.05; **P≤0.01).</p

Recovery of pneumococci from the nasopharynx of mice challenged with a mixture of two 6B strains.

<p>Mice were immunized intranasally with two doses of the indicated formulations and challenged with a mixture of two 6B strains expressing different PspAs. Recovery of strains St491/00 (PspA1) (A) and St472/96 (PspA4) (B) in nasal washes performed 5 days after challenge is shown. * and ^# indicate statistical difference with saline and wP, respectively (One-way ANOVA, Tukey’s Multicomparison Test—* or ^# P≤0.05; ** P≤0.01; *** P≤0.001). Results are from two independent experiments.</p

In pulmonary paracoccidioidomycosis, 1MT treatment increases fungal loads and pulmonary NO but reduces kynurenines levels and IDO mRNA production.

<p>(<b>A, B</b>) Determination of fungal loads by CFU assays in the lungs of A/J and B10.A mice (n = 5–6), treated or not with 1MT, at weeks 2 and 8 after infection with 1×10⁶ yeasts. (<b>C, D</b>) Groups (n = 6–8) of B10.A and A/J mice received subcutaneous implants of 1MT releasing or control pellets (5 mg/ml/day) and disease severity assessed by CFU assays at weeks 4 and 8 after infection with 1×10⁶ yeast cells of <i>P. brasiliensis</i>. The bars depict means ± SEM of log10 CFU. The results are representative of 2 experiments with equivalent results (**<i>p</i><0.01, and ***<i>p</i><0.001). Levels of (<b>E, F</b>) nitric oxide and (<b>G, H</b>) kynurenines were measured in lung homogenates. NO production was measured by Griess reagent, and kynurenines were evaluated using Ehrlich's reagent. (<b>I, J</b>) IDO mRNA was measured using TaqMan real-time PCR assay. Data are means ± SEM of three independent experiments at weeks 2 and 8 after infection with similar results (*<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001).</p

1MT treatment reduces IL-12 and increases IL-6 and TGF-β production by A/J and B10.A macrophages.

<p>(<b>A, B</b>) IFN-γ-primed and unprimed macrophages were treated with 1MT or left untreated and then cultivated for 48 h. Some cultures were infected with viable <i>P. brasiliensis</i> yeasts (1∶25, fungus∶macrophages ratio) for 48 h. Supernatants were removed and used for cytokine measurements by means of ELISA. Data are means ± SEM of triplicate samples from three independent determinations (*<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001).</p