Extensive efforts have been made toward improving effective strategies for pneumococcal vaccination, focusing on evaluating the potential of multivalent
protein-based
vaccines and overcoming the limitations of pneumococcal
polysaccharide-based
vaccines. In this study, we investigated the protective potential of mice co-immunization with the pneumococcal PhtD and novel rPspA
proteins against pneumococcal
sepsis infection. The formulations of each
antigen alone or in combination were administered intraperitoneally with
alum adjuvant into BALB/c mice three times at 14-day intervals. The production of
antigen-specific
IgG,
IgG1 and
IgG2a subclasses, and
IL-4 and IFN-γ
cytokines, were analyzed. Two in vitro
complement- and opsonophagocytic-mediated killing activities of raised
antibodies on day 42 were also assessed. Finally, the protection against an intraperitoneal challenge with 106 CFU/mouse of multi-drug resistance of Streptococcus pneumoniae ATCC49619 was investigated. Our findings showed a significant increase in the anti-PhtD and anti-rPspA sera
IgG levels in the immunized group with the PhtD+rPspA formulation compared to each alone. Moreover, the results demonstrated a synergistic effect with a 6.7- and 1.3- fold increase in anti-PhtD and anti-rPspA
IgG1, as well as a 5.59- and 1.08- fold increase in anti-PhtD and anti-rPspA
IgG2a, respectively. Co-administration of rPspA+PhtD elicited a mixture of Th-2 and Th-1 immune responses, more towards Th-2. In addition, the highest
complement-mediated killing activity was observed in the sera of the immunized group with PhtD+rPspA at 1/16 dilution, and the opsonophagocytic activity was increased from 74% to 86.3%. Finally, the survival rates showed that mice receiving the rPspA+PhtD formulation survived significantly longer (100%) than those receiving
protein alone or PBS and exhibited the strongest clearance with a 2 log10 decrease in bacterial load in the blood 24h after challenge compared to the control group. In conclusion, the rPspA+PhtD formulation can be considered a promising bivalent serotype-independent
vaccine candidate for protection against invasive
pneumococcal infection in the future.