Novel protein-based pneumococcal vaccines administered with the Th1-promoting adjuvant IC31 induce protective immunity against pneumococcal disease in neonatal mice

Abstract

Streptococcus pneumoniae is responsible for many vaccine-preventable deaths, annually causing around 1 million deaths in children younger than 5 years of age. A new generation of pneumococcal vaccines based on conserved proteins is being developed. We evaluated the immunogenicities and protective efficacies of four pneumococcal protein vaccine candidates, PcsB, StkP, PsaA, and PspA, in a neonatal mouse model. Mice were immunized three times and challenged intranasally with virulent pneumococci. All four proteins were immunogenic in neonatal mice, and antibody (Ab) responses were significantly enhanced by the novel adjuvant IC31, which consists of an antibacterial peptide (KLKL5KLK) and a synthetic oligodeoxynucleotide, ODN1a, that signals through Toll-like receptor 9 (TLR9). Two single proteins, StkP and PspA, combined with IC31 significantly reduced pneumococcal bacteremia but had no effects on lung infection. Three proteins, PcsB, StkP, and PsaA, were evaluated with alum or IC31. IC31 enhanced Ab responses and avidity to all three proteins, whereas alum enhanced Ab responses and avidity to StkP and PsaA only. Mice receiving the trivalent protein formulation with IC31 had significantly reduced bacteremia and lung infection compared to unvaccinated mice, but the level of protection was dependent on the dose of IC31. When PspA was added to the trivalent protein formulation, the dose of IC31 needed to obtain protective immunity could be reduced. These results demonstrate that a novel pneumococcal protein-based vaccine is immunogenic at an early age of mice and emphasize the benefits of using a combination of conserved proteins and an effective adjuvant to elicit potent protective immunity against invasive pneumococcal disease.

Fig 1

IgG antibody responses to PcsB, StkP, PspA, and PsaA are enhanced by IC31. Shown are data for PcsB (A)-, StkP (B)-, PsaA (C)-, and PspA (D)-specific Ab responses elicited by immunizations with each protein alone or in a quadrivalent protein formulation with or without IC31 (LD, 50 nmol KLK and 2 nmol ODN1a). IgG levels were measured by ELISA 2 weeks after one (week 2), two (week 4), and three (week 6) immunizations. The results for one experiment are shown (n = 8/group). Bars are shown as medians of each group with interquartile ranges. An asterisk indicates statistically higher Ab levels than those in the group receiving Tris buffer (P < 0.05). P values are shown for comparison between the groups receiving the 4 proteins with and without IC31, when statistically significant. The overall P values, calculated by using the Kruskal-Wallis test, were a P value of 0.0006 (week 2) and a P value of <0.0001 (weeks 4 and 6) for PcsB-specific Abs; a P value of <0.001 (weeks 2, 4, and 6) for StkP-specific Abs; a P value of 0.0381 (week 2), a P value of 0.0031 (week 4), and a P value of 0.0004 (week 6) for PsaA-specific Abs; and a P value of 0.0003 (week 2), a P value of <0.0001 (week 4), and a P value of 0.0002 (week 6) for PspA-specific Abs.

Fig 2

A quadrivalent protein formulation admixed with a lower dose of IC31 (LD) protects neonatally immunized mice from both bacteremia and lung infections, whereas StkP and PspA as single proteins admixed with the IC31 LD reduce only bacteremia. Pneumococcal CFU/ml in blood (A) and lungs (B) 24 h after challenge with S. pneumoniae serotype 1 is shown for each mouse, and the median for each group (n = 8/group) is indicated by a line. The IC31 LD consisted of 50 nmol KLK and 2 nmol ODN1a. The pneumococcal challenge dose was 1.18 × 10⁷ CFU/mouse. P values are shown for comparison between the immunized groups and the control group receiving Tris buffer when statistically significant. The results are shown for one experiment (n = 8/group). The overall P value for CFU in blood was 0.0009, and that for CFU in lungs was 0.0098. Further statistics are reported in Table S1 in the supplemental material.

Fig 3

IC31 induces higher and more rapid Ab responses to all three proteins in the trivalent formulation, whereas Alum enhances only StkP- and PsaA-specific Abs. Shown are the median IgG Ab levels specific for PcsB (A), StkP (B), and PsaA (C) 2, 4, and 6 weeks after the first neonatal immunization at 7 days of age. The IC31 LD consisted of 50 nmol KLK and 2 nmol ODN1a, and the IC31 HD consisted of 90 nmol KLK and 3.6 nmol ODN1a. The results for one of two comparable experiments are shown (n = 8 mice/group). The overall P values were a P value of 0.0676 (week 2) and a P value of <0.0001 (weeks 4 and 6) for PcsB-specific Abs, a P value of <0.0001 (weeks 2, 4, and 6) for StkP-specific Abs, and a P value of <0.0001 (weeks 2, 4, and 6) for PsaA-specific Abs.

Fig 4

IC31 enhances the levels of both IgG1 and IgG2a Abs to the three proteins in the trivalent vaccine more than alum. Shown are levels of IgG1 and IgG2a specific for PcsB (A), StkP (B), and PsaA (C) 2 weeks after the third immunization of neonatal mice with the trivalent protein formulation with high- or low-dose IC31, alum, or no adjuvant. The IC31 LD consisted of 50 nmol KLK and 2 nmol ODN1a, and the IC31 HD consisted of 90 nmol KLK and 3.6 nmol ODN1a. Bars represent the medians of each group with interquartile ranges. *, P < 0.05; **, P < 0.001 (compared to the group receiving alum/IC31). The results for one of two comparable experiments are shown. The overall P values were a P value of <0.0001 (IgG1) and a P value of 0.0134 (IgG2a) for PcsB-specific Abs, a P value of <0.0001 (IgG1) and a P value of 0.0024 (IgG2a) for StkP-specific Abs, and a P value of <0.0001 (IgG1 and IgG2a) for PsaA-specific Abs.

Fig 5

Affinity maturation is enhanced by IC31 more than by alum. Shown are avidity indexes of IgG Abs specific for PcsB, StkP, and PsaA following three immunizations of neonatal mice with the trivalent protein formulation admixed with alum, a low or a high dose of IC31, or no adjuvant. The IC31 LD consists of 50 nmol KLK and 2 nmol ODN1a, and the IC31 HD consists of 90 nmol KLK and 3.6 nmol ODN1a. Results for one of two comparable experiments are shown. *, P < 0.05; **, P < 0.001 (compared to the group receiving the three proteins without an adjuvant). The overall P values were a P value of 0.0002 for PcsB-specific Abs, a P value of <0.0001 M for StkP-specific Abs, and a P value of <0.0001 for PsaA-specific Abs.

Fig 6

Neonatal mice immunized with the trivalent protein formulation admixed with high-dose IC31 are protected against both bacteremia and lung infection. Pneumococcal CFU/ml in blood (A) and lungs (B) 24 h after challenge with S. pneumoniae serotype 1 is shown for each mouse. The median for each group (n = 8/group) is indicated by a line. The IC31 LD consisted of 50 nmol KLK and 2 nmol ODN1a, and the IC31 HD consisted of 90 nmol KLK and 3.6 nmol ODN1a. The pneumococcal challenge dose was 2.80 × 10⁸ CFU/mouse. The results for one of two comparable experiments are shown. The level of bacteremia (CFU/ml) did not differ significantly between the groups, as analyzed by the Mann-Whitney test. The Fisher exact test was performed to compare the frequencies of protected mice between the groups by using CFU/ml lower than the mean CFU minus 2× the standard deviation in the control group (3.48 CFU/ml in blood and 5.13 CFU/ml in lung) as a measure of protection. P values that were statistically significant by the Fisher exact test are shown. The overall P values were 0.8467 (blood infection) and 0.1698 (lung infection). Further statistical analysis is presented in Table S2 in the supplemental material.