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

Indoleamine 2,3-dioxygenase controls fungal loads and immunity in Paracoccidioidomicosis but is more important to susceptible than resistant hosts.

Paracoccidioidomycosis, a primary fungal infection restricted to Latin America, is acquired by inhalation of fungal particles. The immunoregulatory mechanisms that control the severe and mild forms of paracoccidioidomycosis are still unclear. Indoleamine 2,3-dioxygenase (IDO), an IFN-γ induced enzyme that catalyzes tryptophan metabolism, can control host-pathogen interaction by inhibiting pathogen growth, T cell immunity and tissue inflammation.In this study, we investigated the role of IDO in pulmonary paracoccidioidomycosis of susceptible and resistant mice. IDO was blocked by 1-methyl-dl-tryptophan (1MT), and fungal infection studied in vitro and in vivo. Paracoccidioides brasiliensis infection was more severe in 1MT treated than untreated macrophages of resistant and susceptible mice, concurrently with decreased production of kynurenines and IDO mRNA. Similar results were observed in the pulmonary infection. Independent of the host genetic pattern, IDO inhibition reduced fungal clearance but enhanced T cell immunity. The early IDO inhibition resulted in increased differentiation of dendritic and Th17 cells, accompanied by reduced responses of Th1 and Treg cells. Despite these equivalent biological effects, only in susceptible mice the temporary IDO blockade caused sustained fungal growth, increased tissue pathology and mortality rates. In contrast, resistant mice were able to recover the transitory IDO blockade by the late control of fungal burdens without enhanced tissue pathology.Our studies demonstrate for the first time that in pulmonary paracoccidioidomycosis, IDO is an important immunoregulatory enzyme that promotes fungal clearance and inhibits T cell immunity and inflammation, with prominent importance to susceptible hosts. In fact, only in the susceptible background IDO inhibition resulted in uncontrolled tissue pathology and mortality rates. Our findings open new perspectives to understand the immunopathology of paracoccidioidomycosis, and suggest that an insufficient IDO activity could be associated with the severe cases of human PCM characterized by inefficient fungal clearance and excessive inflammation

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

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

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

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

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

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

Correction: Evaluation of inactivated Bordetella pertussis as a delivery system for the immunization of mice with Pneumococcal Surface Antigen A

International audienceAbstract[This corrects the article DOI: 10.1371/journal.pone.0228055.].Erratum forEvaluation of inactivated Bordetella pertussis as a delivery system for the immunization of mice with Pneumococcal Surface Antigen A.Castro JT, Oliveira GS, Nishigasako MA, Debrie AS, Miyaji EN, Soares-Schanoski A, Akamatsu MA, Locht C, Ho PL, Mielcarek N, Oliveira MLS.PLoS One. 2020 Jan 16;15(1):e0228055. doi: 10.1371/journal.pone.0228055. eCollection 2020