Maternal immunization with pneumococcal surface protein A provides the immune memories of offspring against pneumococcal infection

Abstract

Introduction: Streptococcus pneumoniae (S. pneumoniae) is one of the most widespread pathogens in the world and one of the largest infectious causes of infant mortality. Although current vaccines have various benefits, antibiotic resistance and the inability to vaccinate infants less than one year old demands the development of new protective strategies. One strategy, 'maternal immunization', is to protect infants by passive immunity from an immunized mother, although its mechanism is still not fully understood.

Materials and methods: The current study aimed to acquire immunity against S. pneumoniae in infants by maternal immunization with pneumococcal common antigen, pneumococcal surface protein A (PspA). Four-week-old female mice were immunized with recombinant PspA intranasally twice a week for three weeks. Females were mated with age-matched males after immunization, and delivered offspring.

Results: The week-old offspring derived from and fostered by immunized mothers had more anti-PspA-specific antibody producing cells in the spleen than those derived from sham-immunized mothers. The offspring were raised up to four weeks old and were subcutaneously stimulated with recombinant PspA. The levels of anti-PspA IgG in sera after stimulation were significantly higher in the offspring derived from the immunized mothers and the induced specific antibody to PspA showed protective efficacy against systemic pneumococcal infection.

Discussion: Maternal immunization is suggested to be able to provide a sustained immune memory to offspring. The current study would be a milestone in the field of maternal immunization toward a universal pneumococcal vaccine.

Keywords: PspA; Streptococcus pneumoniae; immunological memory; invasive infection; maternal immunization.

Figure 1

SDS-PAGE of rPspA and schematic of the experiment. (A) SDS-PAGE of rPspA after purification. Quantified rPspA was purified by nickel affinity chromatography. The appropriate size of the product and a single band were confirmed by SDS-PAGE. (B) Schedule from immunization of adult mice until experiments. Four-week-old female mice were immunized intranasally twice a week with rPspA 1 μg and CTB for the first two weeks and only rPspA for the last week. Control mice were given CTB only in the first two weeks and only saline during the last week. After immunization, the female mice were mated with male mice. Three weeks after mating, the offspring were obtained. Splenocytes of week-old offspring were analyzed for the ability to produce antibodies and cytokines. Subcutaneous stimulation of rPspA was performed at four weeks of age. The serum levels of anti-PspA antibodies were analyzed 3, 7, 14 and 21 days after the stimulation. At two weeks after the stimulation with rPspA (at six-week-old), offspring were intraperitoneally challenged with S. pneumoniae.

Figure 2

Number of anti-PspA specific IgG producing cells in the spleens of 7-day old offspring. The splenocytes were incubated with 5 µg of rPspA overnight, then incubated in polyvinylidene difluoride membrane 96 wells coated with rPspA overnight. After the cells were removed, the immobilized antibodies on the membrane were incubated with biotinylated detection antibody to mouse IgG, then incubated with alkaline phosphatase conjugates with streptavidin. The spots were visualized by BCIP/NBT. Circle and blue bar; Group A (n=8), triangle and green bar; Group B (n=8), diamond and yellow bar; Group C (n=8), square and red bar; Group D (n=8). Each dot represents individual data of the mouse. Each data is shown as mean with standard error of the mean. Kruskal-Wallis test with Dunn’s multiple comparison was performed for a comparison between the groups. *; P<0.05.

Figure 3

Change of levels of anti-PspA antibody in offspring’s sera with time after stimulation with rPspA. Four-week-old offspring were subcutaneously stimulated with a single dose of 10 µg of rPspA without any adjuvants. The levels of serum antibodies against PspA were evaluated by ELISA at day 3, day 7, day 14, and day 21 after the stimulation with rPspA. Data is shown as mean with standard error. Blue bar, green bar, yellow bar, and red bar represent Group A (n=9), Group B (n=8), Group C (n=9), and Group D (n=8) respectively. (A) serum IgM, (B) serum IgG1, (C) serum IgG2a, (D) serum IgG2b. Kruskal-Wallis test with Dunn’s multiple comparison was performed for comparison between the groups in each day of the blood sampling. *; P<0.05, **; P<0.01.

Figure 4

Cytokine production from maternal splenocytes by PspA stimulation. At the age of 8-week-old, the splenocytes from female mice were incubated with rPspA (1 or 5 µg) or concanavalin A (con A) for positive control. Cytokines in the supernatant were quantified by ELISA. (A) IL-4, (B) IFN-γ, (C) IL-17A. Data is shown as mean with standard error. Cyan bar and orange bar indicate immunized female (n=12) and non-immunized female (n=12) respectively. Mann-Whitney’s U test was performed for comparison between the groups. ***; P<0.001, ****; P<0.0001.

Figure 5

Cytokine production from splenocytes of offspring by PspA stimulation. At the age of week-old, the splenocytes from offspring were incubated with rPspA (1 or 5 µg) or concanavalin A for positive control. Cytokines in the supernatant were quantified by ELISA. (A) IL-4, (B) IFN-γ, (C) IL-17A. Data is shown as mean with standard error. Blue bar, green bar, yellow bar, and red bar represent Group A (n=6), Group B (n=6), Group C (n=6), and Group D (n=6) respectively. Kruskal-Wallis test with Dunn’s multiple comparison was performed for comparison was performed for comparison between the groups. *; P<0.05, ***; P<0.001.

Figure 6

Survival after pneumococcal systemic infection in matured offspring. After two weeks of the subcutaneous stimulation with rPspA (at 6 weeks old), the offspring were intraperitoneally infected with S. pneumoniae and monitored every 8 hours until they appeared severely sick. Blue circle with solid line represents offspring derived from and fostered by immunized mother (Group A) (n=10). Red square with dashed line represents offspring derived from and fostered by non-immunized mother (Group D) (n=9). Log rank test was performed for comparison between the two groups. **; P<0.01.