Abstract
During the past decades, with the implementation of pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccines (PCVs), a dramatic reduction in vaccine type diseases and transmissions has occurred. However, it is necessary to develop a less expensive, serotype-independent pneumococcal vaccine due to the emergence of nonvaccine-type pneumococcal diseases and the limited effect of vaccines on colonization. As next-generation vaccines, conserved proteins, such as neuraminidase A (NanA), elongation factor Tu (Tuf), and pneumolysin (Ply), are promising targets against pneumococcal infections. Here, we designed and constructed a novel fusion protein, NanAT1-TufT1-PlyD4, using the structural and functional domains of full-length NanA, Tuf and Ply proteins with suitable linkers based on bioinformatics analysis and molecular cloning technology. Then, we tested whether the protein protected against focal and lethal pneumococcal infections and examined its potential protective mechanisms. The fusion protein NanAT1-TufT1-PlyD4 consists of 627 amino acids, which exhibits a relatively high level of thermostability, high stability, solubility and a high antigenic index without allergenicity. The purified fusion protein was used to subcutaneously immunize C57BL/6 mice, and NanAT1-TufT1-PlyD4 induced a strong and significant humoral immune response. The anti-NanAT1-TufT1-PlyD4 specific IgG antibody assays increased after the first immunization and reached the highest value at the 35th day. The results from in vitro experiments showed that anti-NanAT1-TufT1-PlyD4 antisera could inhibit the adhesion of Streptococcus pneumoniae (S. pneumoniae) to A549 cells. In addition, immunization with NanAT1-TufT1-PlyD4 significantly reduced S. pneumoniae colonization in the lung and decreased the damage to the lung tissues induced by S. pneumoniae infection. After challenge with a lethal dose of serotype 3 (NC_WCSUH32403), a better protection effect was observed with NanAT1-TufT1-PlyD4-immunized mice than with the separate full-length proteins and the adjuvant control; the survival rate was 50%, which met the standard of the marketed vaccine. Moreover, we showed that the humoral immune response and the Th1, Th2 and Th17-cellular immune pathways are involved in the immune protection of NanAT1-TufT1-PlyD4 to the host. Collectively, our results support that the novel fusion protein NanAT1-TufT1-PlyD4 exhibits extensive immune stimulation and is effective against pneumococcal challenges, and these properties are partially attributed to humoral and cellular-mediated immune responses.