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, 2 (1), 63-73

Mucosal Immunization of Mice With Recombinant OMP P2 Induces Antibodies That Bind to Surface Epitopes of Multiple Strains of Nontypeable Haemophilus Influenzae

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Mucosal Immunization of Mice With Recombinant OMP P2 Induces Antibodies That Bind to Surface Epitopes of Multiple Strains of Nontypeable Haemophilus Influenzae

K L Ostberg et al. Mucosal Immunol.

Abstract

Nontypeable Haemophilus influenzae (NTHI) is a significant cause of otitis media in children and exacerbations in patients with chronic obstructive pulmonary disease. Vaccine research for NTHI has focused on the outer membrane proteins (OMPs) of NTHI. The goal of this study was to evaluate mucosal and systemic immune responses to recombinant OMP P2 (rP2) of NTHI. Enzyme-linked immunosorbent assay (ELISA) demonstrated that both mucosal and systemic routes of immunization resulted in antibodies to rP2. Whole-cell ELISA and flow cytometry indicated that mucosal immunization induced antibodies to epitopes that are on the bacterial surface of the homologous strain as well as several heterologous strains. In contrast, systemic immunization induced antibodies to non-surface exposed epitopes. These data show for the first time that mucosal immunization of mice with rP2 induces antibodies that recognize surface exposed epitopes on multiple strains, indicating that P2 is a candidate for development of a mucosal vaccine for NTHI.

Conflict of interest statement

DISCLOSURE

The authors declared no conflict of interest.

Figures

Figure 1
Figure 1
SDS-polyacrylamide gel (PAGE) of recombinant P2. Proteins were resolved on a 12% acrylamide gel and stained with Coomassie Blue. Molecular mass markers are listed in kDa on the left.
Figure 2
Figure 2
Serum antibodies induced by SQ and IN immunization with recombinant P2 (rP2) assayed by whole-cell enzyme-linked immunosorbent assay (ELISA) using the homologous strain of nontypeable Haemophilus influenzae (NTHI). All assays were performed with a serum dilution of 1:500. Assays were performed in triplicate and results are expressed as the mean±s.e.m. P-values were calculated using Student’s t-test. OD, optical density; SQ, subcutaneous; IFA, incomplete Freund’s adjuvant; IN, intranasal; CT, cholera toxin.
Figure 3
Figure 3
Serum antibodies induced by SQ and IN immunization with recombinant P2 (rP2) assayed by flow cytometry with antisera raised to rP2. Flow cytometry was performed with the homologous strain in triplicate and median fluorescence intensity was determined. IN sera were tested at a 1:10 dilution. SQ sera were tested at a 1:100 dilution. Results are expressed as the mean±s.e.m. Student’s t-test was used to test for significance. SQ, subcutaneous; NMS, normal mouse serum; IN, intranasal.
Figure 4
Figure 4
P2 expression in outer membrane preparations of wild-type and P2-deficient nontypeable Haemophilus influenzae (NTHI). (a) Proteins were resolved on a 12% acrylamide gel and stained with Coomassie Blue. Molecular mass standards are noted in kDa on the left. Arrows indicate presence of the P2 protein in the wild-type strain; (1) wild-type and (2) P2-deficient mutant. (b) Immunoblot assay of wild-type and P2-deficient mutants. Whole-cell lysate was resolved on a 12% acrylamide gel and transferred to nitrocellulose. Blots were probed with antiserum to its homologous P2. Bound antibodies were detected with anti-mouse IgG-horseradish peroxidase (HRP). Molecular mass markers are noted in kDa on the left; (1) wild type and (2) P2-deficient mutant.
Figure 5
Figure 5
Assessment of serum antibodies induced by intranasal (IN) immunization by flow cytometry with wild-type and P2-deficient strains. Strains are noted along the X-axis. Results are expressed as the mean±s.e.m. Significance was determined with Student’s t-test; WT: wild-type and ΔP2: P2-deficient mutant.
Figure 6
Figure 6
Assessment of serum antibodies induced by intranasal (IN) immunization by whole-cell enzyme-linked immunosorbent assays (ELISAs) with heterologous strains of nontypeable Haemophilus influenzae (NTHI). Assays were performed in triplicate and results are expressed as the mean±s.e.m. Significance of antibody binding in comparison to normal mouse serum was determined using Student’s t-test. IN, intranasal; OD, optical density; NMS, normal mouse serum. *P-value < 0.05.
Figure 7
Figure 7
Assessment of serum antibodies induced by intranasal (IN) immunization by flow cytometry with heterologous strains of nontypeable Haemophilus influenzae (NTHI). Assays were performed in triplicate and results are expressed as the mean±s.e.m. Note that serum from mice immunized with rP254 was limited and was therefore only tested with 10 strains. Significance of antibody binding in comparison to normal mouse serum was determined using Student’s t-test. IN, intranasal; NMS, normal mouse serum. *P-value < 0.05.
Figure 8
Figure 8
Avidity of anti-recombinant P2 (rP2) antibodies induced by subcutaneous (SQ) vs. intranasal (IN) immunization. Avidity was tested by enzyme-linked immunosorbent assay (ELISA), using sodium thiocyanate (NaSCN) to disrupt antigen–antibody binding. Experiments were performed in triplicate. Results are expressed as the mean±s.e.m.

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