A Cross-Reactive Protein Vaccine Combined with PCV-13 Prevents Streptococcus pneumoniae- and Haemophilus influenzae-Mediated Acute Otitis Media

Abstract

Acute otitis media is one of the most common childhood infections worldwide. Currently licensed vaccines against the common otopathogen Streptococcus pneumoniae target the bacterial capsular polysaccharide and confer no protection against nonencapsulated strains or capsular types outside vaccine coverage. Mucosal infections such as acute otitis media remain prevalent, even those caused by vaccine-covered serotypes. Here, we report that a protein-based vaccine, a fusion construct of epitopes of CbpA to pneumolysin toxoid, confers effective protection against pneumococcal acute otitis media for non-PCV-13 serotypes and enhances protection for PCV-13 serotypes when coadministered with PCV-13. Having cross-reactive epitopes, the fusion protein also induces potent antibody responses against nontypeable Haemophilus influenzae and S. pneumoniae, engendering protection against acute otitis media caused by emerging unencapsulated otopathogens. These data suggest that augmenting capsule-based vaccination with conserved, cross-reactive protein-based vaccines broadens and enhances protection against acute otitis media.

Keywords: Haemophilus influenzae, Streptococcus pneumoniae; otitis media; vaccines.

FIG 1

Protein-based vaccines enhance IgG antibody responses against S. pneumoniae. ELISA-based measurements of cross-reactive IgG from vaccinated mice receiving alum (vehicle control), PCV-13, YLN, or PCV-13 with YLN. Sera from vaccinated animals were tested against whole-cell lysates of serotype 4 (a), 19F (b), 7F (c), or 2 (d) pneumococcal strains. Each data point represents titers from an individual animal. Groups were different by ANOVA, with P values of <0.0001 for serotypes 4, 19F, and 2 and a P value of 0.0002 for serotype 7F. Indicated pairwise comparisons were made by Mann-Whitney testing. P values are indicated, with a P value of <0.05 being considered significant. Horizontal bars indicate the medians for each group.

FIG 2

Impact of vaccine interventions on serotype 19F-mediated acute otitis media. (a) Vaccination with either PCV-13 or PCV-13 with YLN significantly reduced bacterial burden of serotype 19F S. pneumoniae in the middle ear 24 h postchallenge. Each data point represents titers from an individual animal. Groups were significantly different by ANOVA at a P value of 0.0011. Indicated pairwise comparisons were made by Mann-Whitney testing, with a P value of <0.05 being considered significant. Horizontal bars indicate the medians for each group. (b and c) Percentages of animals with bacteria recovered from the ears (b) or the percentage of bacterium-positive ears (c) indicates that PCV-13 supplemented with YLN significantly reduced the overall burden of AOM. Comparison was made by chi-square test. *, P < 0.05 compared to alum-vaccinated mice. Representative Xenogen images of alum (d)-, PCV-13 (e)-, YLN (f)-, and PCV-13 with YLN (g)-vaccinated animals.

FIG 3

Impact of vaccine interventions on serotype 7F-mediated acute otitis media. (a) Twenty-four hours postchallenge, 7F S. pneumoniae-inoculated animals that were vaccinated with either PCV-13 or PCV-13 with YLN had a significantly lower bacterial burden in the middle ear than did those that received other vaccinations. Each data point represents titers from an individual animal/ear. Groups were not significantly different by ANOVA, with a P value of 0.2226. Indicated pairwise comparisons were made by Mann-Whitney testing, with a P value of <0.05 being considered significant. Horizontal bars indicate the median for each group. (b) The percentage of animals with bacteria recovered from the ears or the percentage of bacterium-positive ears indicate that PCV-13 supplemented with YLN significantly reduced the overall burden of AOM. Comparison was made by chi-square test. *, P < 0.05 compared to alum-vaccinated mice. (c and d) Representative Xenogen images of animals vaccinated with alum (c), PCV-13 (d), or PCV-13 with YLN (e).

FIG 4

YLN vaccination induces potent antibody responses and protection against nonencapsulated S. pneumoniae AOM. Shown are ELISA-based measurements of cross-reactive IgG from vaccinated mice receiving alum (vehicle control), PCV-13, YLN, or PCV-13 with YLN. (a) Sera from vaccinated animals were tested against whole-cell lysates or nonencapsulated strain MNZ67. Each data point represents titers from an individual animal/ear. Groups were significant by ANOVA at P values less than 0.0001. Indicated pairwise comparisons were done by Mann-Whitney test. P values are indicated, and a P value of <0.05 was considered significant. (b) Twenty-four hours postchallenge, nonencapsulated S. pneumoniae-inoculated animals that were vaccinated with either PCV-13 or PCV-13 with YLN had a significantly lower bacterial burden in the middle ear than did those that received other vaccinations. Groups were not significantly different by ANOVA, with a P value of 0.5669. Indicated pairwise comparisons were made by Mann-Whitney testing, with a P value of <0.05 being considered significant. Horizontal bars indicate the medians for each group.

FIG 5

YDN vaccination antibody responses and protective capacity against S. pneumoniae AOM. Shown are ELISA-based measurements of cross-reactive IgG from vaccinated mice receiving alum (vehicle control), PCV-13, YDN, or PCV-13 and YDN simultaneously. (a and b) Sera from vaccinated animals were tested against whole-cell lysates from serotype 4 (a) and 19F (b) strains. Groups were significant by ANOVA at P values less than 0.0001. Indicated pairwise comparisons were done by Mann-Whitney testing. P values are indicated, and a P value of <0.05 was considered significant. Each data point represents titers from an individual animal. (c) Vaccination with either PCV-13 or PCV-13 with YDN significantly reduced bacterial burden of S. pneumoniae in the middle ear 24 h postchallenge. Groups were significantly different by ANOVA at a P value of 0.0475. Indicated pairwise comparisons were made by Mann-Whitney testing, with a P value of <0.05 being considered significant. Horizontal bars indicate the medians for each group. (d) Percentage of animals with bacteria recovered from the ears or the percentage of bacterium-positive ears indicates that PCV-13 supplemented with YDN significantly reduced the overall burden of AOM. Comparison was made by chi-square test. *, P < 0.05 compared to alum-vaccinated mice.

FIG 6

Efficacy of YLN and YDN against NTHi AOM. (a) ELISA-based measurements of cross-reactive IgG from vaccinated mice receiving alum (vehicle control), PCV-13, YDN, YLN, or PCV-13 with YDN/YLN. Sera from vaccinated animals were tested against whole-cell lysates from NTHi, with each point being the serum from an individual animal. Groups were not significantly different by ANOVA, with a P value of 0.0766. Indicated pairwise comparisons were done by Mann-Whitney testing. P values are indicated, and a P value of <0.05 was considered significant. (b) Ear tissues were harvested 72 h postinfection with NTHi, and bacterial burdens were enumerated. Each data point represents titers from an individual ear. Horizontal bars indicate the medians for each group. Groups were not significantly different by ANOVA, with a P value of 0.3125. Indicated pairwise comparisons were made by Mann-Whitney testing, with a P value of <0.05 being considered significant.