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Streptococcus pneumoniae is an infectious pathogen mainly infecting host bodies through the respiratory system. An effective pneumococcal vaccine would be targeted to the mucosa and provide not only protection against invasive infection but also against colonization in the respiratory system. In the present work, we applied bacterium-like particles (BLPs) as an adjuvant for the development of a PspA mucosal vaccine, in which the PspA protein was displayed on the surface of BLPs. Intranasal immunization with the PspA-BLP pneumococcal vaccine, comprised of PspA2 from pneumococcal family 1 and PspA4 from pneumococcal family 2, not only induced a high level of serum IgG antibodies but also a high level of mucosal SIgA antibodies. Analysis of binding of serum antibodies to intact bacteria showed a broad coverage of binding to pneumococcal strains expressing PspA from clade 1 to 5. Immunization with the PspA-BLP vaccine conferred protection against fatal intranasal challenge with both PspA family 1 and family 2 pneumococcal strains regardless of serotype. Therefore, the PspA-BLP pneumococcal vaccine was demonstrated to be a promising strategy for mucosal immunization to enhance both systemic and mucosal immune responses.

Streptococcus pneumoniae is a major pathogen that causes life-threatening diseases, such as pneumonia, otitis media, bacteremia, and meningitis, worldwide and especially in young children and the elderly. Pneumococcal surface protein A (PspA) is a widely studied candidate protein vaccine that represents a promising replacement for current polysaccharide and polysaccharide-conjugate vaccines. In this study, we describe a simple method to produce PspA of clade 4 from an Escherichia coli expression system using hydroxylapatite and ion-exchange chromatography. Using this method, we successfully expressed soluble PspA4 in 10 L of autoinducing culture medium, with a wet-cell yield of 19 g/L and a final PspA4 concentration of 22.8 mg/L. Additionally, we improved PspA4 purity from 17% to 70% in a single step through the use of hydroxylapatite, resulting in acquisition of recombinant PspA4 (>95% purity) at a final yield of 43% from the starting cell-lysis solution. We subsequently verified the secondary structure molecular weight of recombinant PspA4 by circular dichroism and mass spectrometry, respectively. These results demonstrated a highly efficient method for mass producing PspA4 protein and that can also be applied for purification of PspA proteins from other clades., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Streptococcus pneumoniae is a major cause of invasive pneumococcal disease, septicemia, and meningitis that can result in high morbidity rates in children under 5 years old. The current polysaccharide-based vaccines can provide type-specific immunity, but a broad-spectrum vaccine would provide greater coverage. Therefore, developing pneumococcal-protein-based vaccines that can extend to more serum types is highly important. In this study, we vaccinated mice via the subcutaneous (s.c.) route with a systemic vaccine that is a mixture of fusion protein PsaA-PspA23 and a single protein, PspA4, with aluminum hydroxide as an adjuvant. As a comparison, mice were immunized intranasally with a mucosal vaccine that is a mixture of PspA2-PA-BLP (where PA is protein anchor and BLP is bacterium-like particle) and PspA4-PA-BLP, via the intranasal (i.n.) route. The two immunization processes were followed by challenge with Streptococcus pneumoniae bacteria from two different PspA families. Specific IgG titers in the serum and specific IgA titers in the mucosa were determined following immunizations. Bacterial loads and survival rates after challenge were compared. Both the systemic vaccine and the mucosal vaccine induced a significant increase of IgG against PspAs. Only the mucosal vaccine also induced specific IgA in the mucosa. The two vaccines provided protection, but each vaccine showed an advantage. The systemic vaccine induced higher levels of serum antibodies, whereas the mucosal vaccine limited the bacterial load in the lung and blood. Therefore, coimmunizations with the two types of vaccines may be implemented in the future., (Copyright © 2018 American Society for Microbiology.)

Background: Streptococcus pneumoniae is a major pathogen accounting for a large number of pneumococcal disease in worldwide. Due to the mucosal immune pathway induces both systemic and mucosal immune responses, the potential strategy to prevent pneumococcal disease may be to develop a mucosal vaccine., Method: In this study, we developed an intranasal pneumococcal protein vaccine based on a bacterium-like particle (BLP) delivery system. PspA is expressed and exposed on the surface of all pneumococcal strains, which confers the potential to induce immune responses to protect against pneumococcal infection. We fused one of the pneumococcal surface proteins (PspA, family2 clade4) with the protein anchor (PA) protein in order to display PspA on the surface of BLPs., Result: The current results showed that intranasal immunization with BLPs/PspA-PA efficiently induced both PspA-specific IgG in the serum and PspA-specific IgA in mucosal washes. And intranasal immunization of BLPs/PspA-PA could provide complete protection in a mouse challenge model with pneumococci of different two clades of both homologous and heterologous PspA families., Discussion and Conclusion: Thus, targeted delivery of multiple bacterial antigens via BLPs may prevent pneumococcal disease by inducing both systemic and mucosal immune responses.

Streptococcus pneumoniae is an important pathogen accounting for a large number of deaths worldwide. Due to drawbacks of the current polysaccharide-based vaccine, the most promising way to generate an improved vaccine may be to utilize protection-eliciting pneumococcal proteins. Pneumococcal surface adhesin A (PsaA) and pneumococcal surface protein A (PspA) are two vaccine candidates which have been evaluated against S. pneumoniae infection in animal models or human clinical trials with encouraging results. In this study, the efficacy of the fusion protein PsaA-PspA, which includes PsaA part and PspA part, in inducing immunoprotective effects against fatal pneumococcal challenge was evaluated in an animal model. PspA part of PsaA-PspA fusion protein contains both family1 N-terminal region and family 2 N-terminal clade-defining region of PspA. Immunization with the PsaA-PspA fusion protein induced high levels of antibodies against both PsaA and PspA, which could bind to intact S. pneumoniae strains bearing different PspAs. Ex vivo stimulation of splenocytes from mice immunized with PsaA-PspA induced IL-17A secretion. Mice immunized with PsaA-PspA showed reduced S. pneumoniae levels in the blood and lungs compared with the PBS group after intranasal infection. Finally, mice immunized with PsaA-PspA fusion proteins were protected against fatal challenge with pneumococcal strains expressing different PspAs regardless of the challenge route. These results support the PsaA-PspA fusion protein as a promising vaccine strategy, as demonstrated by its ability to enhance the immune response and stimulate production of high titer antibodies against S. pneumoniae strains bearing heterologous PspAs, as well as confer protection against fatal challenge with PspA family 1 and family 2 strains.

Streptococcus pneumoniae is a major cause of infectious disease and complications worldwide, such as pneumonia, otitis media, bacteremia and meningitis. New generation protein-based pneumococcal vaccines are recognized as alternative vaccine candidates. Pneumolysin (Ply) is a cholesterol-dependent cytolysin produced by all clinical isolates of S. pneumoniae. Our research group previously developed a highly detoxified Ply mutant designated Plym2 by replacement of two animo acids (C428G and W433F). Exhibiting undetectable levels of cytotoxicity, Plym2 could still elicit high titer neutralizing antibodies against the native toxin. However, evaluation of the active immunoprotective effects of Plym2 by subcutaneous immunization and lethal challenge with S. pneumoniae in mice did not yield favorable results. In the present work, we confirmed the previous observations by using passive immunization and systemic challenge. Results of the passive immunization were consistent with those of active immunization. Further experiments were conducted to explain the inability of high titer neutralizing antibodies against Ply to protect mice from S. pneumoniae challenge. Pneumococcal Ply is known to be the major factor responsible for the induction of inflammation that benefits the host. Proinflammatory cytokines facilitate the clearance of invaders by the recruitment and activation of leukocytes at the early infection stage. We demonstrated that Plym2 could induce proinflammatory cytokines similarly to wild-type Ply. A systemic infection model was used to clarify that Plym2 lacking cytolytic activity could protect mice from intraperitoneal challenge directly, while antibodies to the mutant had no effect. Therefore, the protective function of Plym2 may be due to its induction of proinflammatory cytokines. When used in the systemic infection model, Plym2 antibodies may block the induction of proinflammatory cytokines by Ply. These findings demonstrate that a Ply-based vaccine would not be an effective primary vaccine component, but it may be beneficial as an adjuvant to stimulate cytokine production.