Combination of pneumococcal surface protein A (PspA) with whole cell pertussis vaccine increases protection against pneumococcal challenge in mice

Abstract

Streptococcus pneumoniae is the leading cause of respiratory acute infections around the world. In Latin America, approximately 20,000 children under 5 years of age die of pneumococcal diseases annually. Pneumococcal surface protein A (PspA) is among the best-characterized pneumococcal antigens that confer protection in animal models of pneumococcal infections and, as such, is a good alternative for the currently available conjugated vaccines. Efficient immune responses directed to PspA in animal models have already been described. Nevertheless, few low cost adjuvants for a subunit pneumococcal vaccine have been proposed to date. Here, we have tested the adjuvant properties of the whole cell Bordetella pertussis vaccine (wP) that is currently part of the DTP (diphtheria-tetanus-pertussis) vaccine administrated to children in several countries, as an adjuvant to PspA. Nasal immunization of BALB/c mice with a combination of PspA5 and wP or wP(low)--a new generation vaccine that contains low levels of B. pertussis LPS--conferred protection against a respiratory lethal challenge with S. pneumoniae. Both PspA5-wP and PspA5-wP(low) vaccines induced high levels of systemic and mucosal antibodies against PspA5, with similar profile, indicating no essential requirement for B. pertussis LPS in the adjuvant properties of wP. Accordingly, nasal immunization of C3H/HeJ mice with PspA5-wP conferred protection against the pneumococcal challenge, thus ruling out a role for TLR4 responses in the adjuvant activity and the protection mechanisms triggered by the vaccines. The high levels of anti-PspA5 antibodies correlated with increased cross-reactivity against PspAs from different clades and also reflected in cross-protection. In addition, passive immunization experiments indicated that antibodies played an important role in protection in this model. Finally, subcutaneous immunization with a combination of PspA5 with DTP(low) protected mice against challenge with two different pneumococcal strains, opening the possibility for the development of a combined infant vaccine composed of DTP and PspA.

Figure 1. Pneumococcal loads in lungs and sera.

Lungs (A, C and E) and sera (B, D and F) from non-immunized BALB/c mice (Non) or mice immunized with wP or PspA5-wP were collected at different time points after intranasal challenge with ATCC6303 pneumococcal strain (PspA5). CFU were determined in four mice per group, after plating the samples in blood-agar. Circles represent individual mice and lines represent the mean for each group. (d = days). In conditions where no bacteria were detected, 1 CFU was considered.

Figure 2. Evaluation of anti-PspA5 antibodies in mice immunized with the different vaccine formulations.

Three weeks after the last immunization anti-PspA5 IgG (A), IgG1 and IgG2a (B) in the sera and IgG (C) and IgA (D) in BALF were detected through ELISA. Concentration of antibodies (mean of 6 animals for A and B and 4 animals for C and D) are shown. IgG1∶IgG2a ratios are indicated above the bars (B). Results are representative of two experiments (A and B). Asterisks represent significant differences from control groups or from group immunized with PspA5, when indicated (* P<0.05; ** P<0.005, Mann-Whitney U test).

Figure 3. Induction of anti-PspA5 antibodies in C3H/HePas and C3H/HeJ mice.

C3H/HePas (A) and C3H/HeJ (B and C) were immunized with the different vaccine formulations through the nasal route. Three weeks after the last immunization anti-PspA5 IgG in the sera were detected through ELISA. Concentration of antibodies (mean of 6 animals) is shown. Results are representative of two experiments. Asterisks represent significant differences from control groups or from group immunized with PspA5, when indicated (* P<0.05; ** P<0.005, Mann-Whitney U test).

Figure 4. Binding to pneumococcal surface, complement deposition and passive protection elicited by anti-PspA5 antibodies.

Sera from BALB/c mice immunized with PspA5 (dotted black lines), PspA5-wP (solid black lines) or PspA5-wP_low (solid dark gray lines) were tested for the ability to bind to the pneumococcal surface (A) and to mediate C3 deposition (B). S. pneumoniae ATCC 6303(PspA5) was incubated with 2% (A) or 10% of each group serum (B). Sera from non-immunized animals (gray areas) and immunized with wP (solid light gray lines) or wP_low (dotted light gray lines) were used as control. The median fluorescence of bacteria is shown for each sample. Data are representative of two independent experiments. Naïve mice were inoculated trough the intraperitoneal route with a 1∶100 dilution of each serum 2 h before pneumococcal challenge. Survival was monitored until 10 days after challenge (C). ***P<0.001 when compared with control groups and P = 0.004 when compared with mice vaccinated with PspA5 by Fisher exact test. Data were composed with two independent experiments.

Figure 5. Cross-reactivity and cross protection induced by immunization of mice with PspA5-wP.

Western-blot analyses were carried out using total protein lysates from different pneumococcal strains and a 1∶500 dilution of sera from BALB/c mice immunized with PspA5 (A) or PspA5-wP (B). Mice were immunized through the nasal route with the different indicated vaccines. Three weeks after the last immunization mice were challenged with the 0603 pneumococcal strain (PspA1) through the nasal route and CFU were determined in nasal washes, 5 days after challenge. Circles represent individual pneumococcal loads and lines represent the mean for each mice group. Asterisks indicate significant differences when compared with indicated groups (*P = 0.01; **P = 0.003; ***P = 0.001, Mann-Whitney U test). Results were composed with two independent experiments.

Figure 6. Cross-reactivity induced by immunization of mice with PspA3-wP.

Sera from mice immunized with PspA3 (dotted black lines) or PspA3-wP (solid heavy black lines) were tested for the ability to bind to the pneumococcal surface (A) and to mediate C3 deposition (B). S. pneumoniae ATCC 6303 (PspA5) was incubated with 2% (A) or 10% of each group serum (B). Sera from non-immunized animals (gray areas) and immunized with wP (solid light gray lines) were used as control. The median fluorescence of bacteria is shown for each sample. Data are representative of two independent experiments.

Figure 7. Evaluation of anti-PspA5 antibodies in mice immunized with the different vaccine formulations.

Three weeks after the last immunization anti-PspA5 IgG (A), IgG1 and IgG2a (B) in the sera were detected through ELISA. Concentration of antibodies (mean of 6 animals) is shown. IgG1∶IgG2a ratios are indicated above the bars (B). Results are representative of two experiments. Asterisks represent significant differences from indicated groups (**P<0.005, when compared with non-immunized mice or with the respective control groups and *P<0.01, when compared with mice immunized with PspA5, Mann-Whitney U test).