PppA, a Surface-Exposed Protein ofStreptococcus pneumoniae, Elicits Cross-Reactive Antibodies That Reduce Colonization in a Murine Intranasal Immunization and Challenge Model

Abstract

The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the present conjugate vaccine may still cause pneumococcal disease. To address these serotypes, and the remaining otitis media due to Streptococcus pneumoniae, efforts have been devoted to identifying protective protein antigens. Immunity to conserved surface proteins important for adhesion, nutrient acquisition, or other functions could result in a reduction of colonization and a lower disease potential. We have been searching for conserved surface-exposed proteins from S. pneumoniae that may be involved in pathogenesis to test as vaccine candidates. Here, an ϳ20-kDa protein that has significant homology to a nonheme iron-containing ferritin protein from Listeria innocua and other bactoferritins was identified as pneumococcal protective protein A (PppA). We expressed and purified recombinant PppA (rPppA) and evaluated its potential as a vaccine candidate. The antibodies elicited by purified rPppA were cross-reactive with PppA from multiple strains of S. pneumoniae and were directed against surface-exposed epitopes. Intranasal immunization of BALB/c mice with PppA protein and either a synthetic monophosphoryl lipid A analog, RC529AF, or a cholera toxin mutant, CT-E29H, used as an adjuvant reduced nasopharyngeal colonization in mice following intranasal challenge with a heterologous pneumococcal strain. PppA-specific systemic and local immunoglobulin G (IgG) and IgA antibody responses were induced. The antisera reacted with whole cells of a heterologous S. pneumoniae type 3 strain. These observations indicate that PppA may be a promising candidate for inclusion in a vaccine against pneumococcal otitis media.Infections with Streptococcus pneumoniae are a major cause of human diseases, such as otitis media, bacteremia, meningitis, and fatal pneumonia, worldwide (9). The rapid emergence of multidrug-resistant pneumococcal strains throughout the world has led to an increased emphasis on prevention of pneumococcal infections by vaccination (18). The presently available 23-valent pneumococcal capsular polysaccharide vaccine is not effective in children less than 2 years of age or immunocompromised patients, two of the major populations at risk for pneumococcal infection (14). A seven-valent pneumococcal polysaccharide-protein conjugate vaccine, recently licensed in the United States, was shown to be highly effective in infants and children against systemic pneumococcal disease caused by the vaccine serotypes and against cross-reactive capsular serotypes (36). However, parenteral immunization with the sevenvalent vaccine was only 60% effective against serotype-specific otitis media (17), demonstrating the need for additional immunization strategies (e.g., intranasal [i.n.] immunization), additional noncapsular antigens, or both. Therefore, there is an immediate need for a cost-effective vaccine to cover most or all of the disease-causin...

Nasal Immunization with Lactococcus lactis Expressing the Pneumococcal Protective Protein A Induces Protective Immunity in Mice

“…A 534-bp DNA fragment containing the pppA gene of S. pneumoniae T14 was amplified with the primers EC1 and EC2 (Table 2), as described by Green et al (15). The purified PCR product was cloned into the NcoI and SalI sites of plasmid pET28b to generate pET-PppA, which was recovered by transformation into E. coli DH10B.…”

“…The PppA is a small S. pneumoniae protein that can be recovered in large quantities from the wash fluids of intact pneumococcal cells with PBS (15). PppA is antigenically conserved and exhibits a high level of sequence conservation in serotypes 3, 5, 9, 14, 19, and 23 (15).…”

“…Restriction endonuclease and ligation reactions were carried out according to the recommendations of the suppliers. The pppA gene was amplified from the chromosomal DNA of S. pneumoniae T14 (Table 1) with the primers EC1 and EC2 (Table 2), following the conditions described by Green et al (15). PCR amplifications were performed with a DNA thermocycler (MyCycler 580BR; Bio-Rad) by using 28 cycles consisting of a denaturation step at 94°C for 60 s, a primer annealing step at 52°C for 60 s, and a primer extension step at 72°C for 60 s. The PCR product was purified from agarose gels, using homemade silica beads, and treated with the restriction enzymes NcoI and SalI.…”

“…Serum and BAL fluid antibodies against PppA protein were determined by a modification of the enzyme-linked immunosorbent assay (ELISA) protocol described by Green et al (15). Briefly, plates were coated with rPppA (100 l of a 5-g/ml stock in sodium carbonate-bicarbonate buffer [pH 9.6] per well).…”

“…Thus, worldwide research on this subject is focused on the search for possible additional candidates that are antigenically conserved and that elicit antibodies that reduce colonization or protect against systemic disease or both. A novel pneumococcal surface protein, identified as pneumococcal protective protein A (PppA), has been described (15). This protein is antigenically conserved among different serotype strains of S. pneumoniae, and it has been reported that nasal immunization of adult mice with recombinant PppA administered with mucosal adjuvants elicits antibodies that are effective in reducing pneumococcal nasal colonization.…”