Novel method for production and purification of untagged pneumococcal surface protein A from clade 1.

Streptococcus pneumoniae can cause diseases with high mortality and morbidity. The licensed vaccines are based on capsular polysaccharides and induce antibodies with low cross reactivity, leading to restricted coverage of serotypes. For surpassing this limitation, new pneumococcal vaccines are needed for induction of broader protection. One important candidate is the pneumococcal surface protein A (PspA), which can be classified in 6 clades and 3 families. We have reported an efficient process for production and purification of untagged recombinant PspA from clade 4 (PspA4Pro). We now aim to obtain a highly pure recombinant PspA from clade 1 (PspA1) to be included, together with PspA4Pro, in a vaccine formulation to broaden response against pneumococci. The vector pET28a-pspA1 was constructed and used to transform Escherichia coli BL21(DE3) strain. One clone with high production of PspA1 was selected and adapted to high-density fermentation (HDF) medium. After biomass production in 6 L HDF using a bioreactor, the purification was defined after testing 3 protocols. During the batch bioreactor cultivation, plasmid stability remained above 90% and acetate formation was not detected. The final protein purification process included treatment with a cationic detergent after lysis, anion exchange chromatography, cryoprecipitation, cation exchange chromatography, and multimodal chromatography. The final purification process showed PspA1 purity of 93% with low endotoxin content and an overall recovery above 20%. The novel established process can be easily scaled-up and proved to be efficient to obtain a highly pure untagged PspA1 for inclusion in vaccine formulations. KEY POINTS: • Purification strategy for recombinant PspA1 from Streptococcus pneumoniae • Downstream processing for untagged protein antigens, the case of PspA1 • Purification strategy for PspA variants relies on buried amino acids in their sequences.

The metabolic slowdown caused by the deletion of pspA accelerates protein aggregation during stationary phase facilitating antibiotic persistence.

Entering a dormant state is a prevailing mechanism used by bacterial cells to transiently evade antibiotic attacks and become persisters. The dynamic progression of bacterial dormancy depths driven by protein aggregation has been found to be critical for antibiotic persistence in recent years. However, our current understanding of the endogenous genes that affects dormancy depth remains limited. Here, we discovered a novel role of phage shock protein A (pspA) gene in modulating bacterial dormancy depth. Deletion of pspA of Escherichia coli resulted in increased bacterial dormancy depths and prolonged lag times for resuscitation during the stationary phase. ∆pspA exhibited a higher persister ratio compared to the wild type when challenged with various antibiotics. Microscopic images revealed that ∆pspA showed accelerated formation of protein aggresomes, which were collections of endogenous protein aggregates. Time-lapse imaging established the positive correlation between protein aggregation and antibiotic persistence of ∆pspA at the single-cell level. To investigate the molecular mechanism underlying accelerated protein aggregation, we performed transcriptome profiling and found the increased abundance of chaperons and a general metabolic slowdown in the absence of pspA. Consistent with the transcriptomic results, the ∆pspA strain showed a decreased cellular ATP level, which could be rescued by glucose supplementation. Then, we verified that replenishment of cellular ATP levels by adding glucose could inhibit protein aggregation and reduce persister formation in ∆pspA. This study highlights the novel role of pspA in maintaining proteostasis, regulating dormancy depth, and affecting antibiotic persistence during stationary phase.

Novel method for production and purification of untagged pneumococcal surface protein A from clade 1

Streptococcus pneumoniae can cause diseases with high mortality and morbidity. The licensed vaccines are based on capsular polysaccharides and induce antibodies with low cross reactivity, leading to restricted coverage of serotypes. For surpassing this limitation, new pneumococcal vaccines are needed for induction of broader protection. One important candidate is the pneumococcal surface protein A (PspA), which can be classifed in 6 clades and 3 families. We have reported an efcient process for production and purifcation of untagged recombinant PspA from clade 4 (PspA4Pro). We now aim to obtain a highly pure recombinant PspA from clade 1 (PspA1) to be included, together with PspA4Pro, in a vaccine formulation to broaden response against pneumococci. The vector pET28a-pspA1 was constructed and used to transform Escherichia coli BL21(DE3) strain. One clone with high production of PspA1 was selected and adapted to high-density fermentation (HDF) medium. After biomass production in 6 L HDF using a bioreactor, the purifcation was defned after testing 3 protocols. During the batch bioreactor cultivation, plasmid stability remained above 90% and acetate formation was not detected. The fnal protein purifcation process included treatment with a cationic detergent after lysis, anion exchange chromatography, cryoprecipitation, cation exchange chromatography, and multimodal chromatography. The fnal purifcation process showed PspA1 purity of 93% with low endotoxin content and an overall recovery above 20%. The novel established process can be easily scaled-up and proved to be efcient to obtain a highly pure untagged PspA1 for inclusion in vaccine formulations.

A new candidate epitope-based vaccine against PspA PhtD of Streptococcus pneumoniae: a computational experimental approach.

Introduction: Pneumococcus is an important respiratory pathogen that is associated with high rates of death in newborn children and the elderly. Given the disadvantages of current polysaccharide-based vaccines, the most promising alternative for developing improved vaccines may be to use protein antigens with different roles in pneumococcus virulence. PspA and PhtD, highly immunogenic surface proteins expressed by almost all pneumococcal strains, are capable of eliciting protective immunity against lethal infections. Methods: In this study using immunoinformatics approaches, we constructed one fusion construct (called PAD) by fusing the immunodominant regions of PspA from families 1 & 2 (PA) to the immunodominant regions of PhtD (PD). The objective of this project was to test the immunogenicity of the fusion protein PAD and to compare its protective activity against S. pneumoniae infection with PA or PD alone and a combination of PA and PD. The prediction of physicochemical properties, antigenicity, allergenicity, toxicity, and 3D-structure of the constructs, as well as molecular docking with HLA receptor and immune simulation were performed using computational tools. Finally, mice were immunized and the serum levels of antibodies/cytokines and functionality of antibodies in vitro were evaluated after immunization. The mice survival rates and decrease of bacterial loads in the blood/spleen were examined following the challenge. Results: The computational analyses indicated the proposed constructs could be antigenic, non-allergenic, non-toxic, soluble and able to elicit robust immune responses. The results of actual animal experiments revealed the candidate vaccines could induce the mice to produce high levels of antibodies and cytokines. The complement-mediated bactericidal activity of antibodies was confirmed and the antibodies provided favorable survival in immunized mice after bacterial challenge. In general, the experimental results verified the immunoinformatics studies. Conclusion: For the first time this report presents novel peptide-based vaccine candidates consisting of immunodominant regions of PspA and PhtD antigens. The obtained findings confirmed that the fusion formulation could be relatively more efficient than the individual and combination formulations. The results propose that the fusion protein alone could be used as a serotype-independent pneumococcal vaccine or as an effective partner protein for a conjugate polysaccharide vaccine.

PspA-mediated aggregation protects Streptococcus pneumoniae against desiccation on fomites.

IMPORTANCE: Spn is a dangerous human pathogen capable of causing pneumonia and invasive disease. The virulence factor PspA has been studied for nearly four decades with well-established roles in pneumococcal evasion of C-reactive protein and neutralization of lactoferricin. Herein, we show that mammalian (m)GAPDH in mucosal secretions promotes aggregation of pneumococci in a PspA-dependent fashion, whereas lactoferrin counters this effect. PspA-mediated GAPDH-dependent bacterial aggregation protected Spn in nasal lavage elutes and grown in vitro from desiccation on fomites. Furthermore, surviving pneumococci within these aggregates retained their ability to colonize naïve hosts after desiccation. We report that Spn binds to and forms protein complexes on its surface composed of PspA, mGAPDH, and lactoferrin. Changes in the levels of these proteins therefore most likely have critical implications on Spn colonization, survival on fomites, and transmission.

Negligible role for pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC) in the nasopharyngeal colonization of mice with a serotype 6B pneumococcal strain.

Streptococcus pneumoniae colonizes the human nasopharynx asymptomatically, but it can also cause several diseases, including otitis media, pneumonia, bacteremia, and meningitis. The colonization of the nasopharynx by the bacteria is an essential step for the pneumococcus to invade other sites and cause diseases. Pneumococcal surface protein A (PspA) and Pneumococcal surface Protein C (PspC) are important virulence factors and have been described to play roles in adhesion and immune evasion. In this study, we immunized mice subcutaneously with the recombinant α-helical region of PspA and/or PspC combined with different adjuvants to assess protection against colonization with the serotype 6B strain BHN418. Though high serum levels of specific IgG were detected, none of the formulations led to reduction in the colonization of the nasopharynx. The negative result may be due to the poor induction of IgG2c, which has been previously correlated with protection against pneumococcal colonization in mice. Furthermore, BHN418 pspA and pspC single and double knockouts were evaluated in colonization experiments and no differences in bacterial load were observed. In competition assays with the wild-type strain, borderline to no reduction was observed in the loads of the knockouts. Our results contrast with data from the literature using other pneumococcal strains, showing that the role of PspA and PspC in colonization can vary depending on the background of the knockout strain studied. BHN418 has been selected for its capacity to colonize humans in experimental challenge studies and may have redundant factors that compensate for the lack of PspA and PspC during nasopharyngeal colonization of mice.

Conserved structures of ESCRT-III superfamily members across domains of life.

Structural and evolutionary studies of cyanobacterial phage shock protein A (PspA) and inner membrane-associated protein of 30 kDa (IM30) have revealed that these proteins belong to the endosomal sorting complex required for transport-III (ESCRT-III) superfamily, which is conserved across all three domains of life. PspA and IM30 share secondary and tertiary structures with eukaryotic ESCRT-III proteins, whilst also oligomerizing via conserved interactions. Here, we examine the structures of bacterial ESCRT-III-like proteins and compare the monomeric and oligomerized forms with their eukaryotic counterparts. We discuss conserved interactions used for self-assembly and highlight key hinge regions that mediate oligomer ultrastructure versatility. Finally, we address the differences in nomenclature assigned to equivalent structural motifs in both the bacterial and eukaryotic fields and suggest a common nomenclature applicable across the ESCRT-III superfamily.

Development of a bivalent protein-based vaccine candidate against invasive pneumococcal diseases based on novel pneumococcal surface protein A in combination with pneumococcal histidine triad protein D.

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.

Establishment of a modified opsonophagocytic killing assay for anti-pneumococcal surface protein A antibody.

Streptococcus pneumoniae (pneumococcus) is a pathogenic gram-positive bacterium that causes pneumonia, meningitis, and sepsis. Pneumococcal surface protein A (PspA) induces antibodies that protect against lethal infections by pneumococci. PspA is a choline-binding protein present on the cell surface of almost all pneumococcal strains and is a non-capsular polysaccharide vaccine candidate. For research and development of PspA-based vaccines, an in-vitro test system to measure the activity of functional antibodies capable of killing pneumococci is essential. The opsonophagocytic killing (OPK) assay is used to evaluate the opsonic activity of functional antibodies induced by capsular polysaccharide (CPS)-based vaccines (standard OPK assay). Despite the potential of anti-PspA antibodies to protect against lethal infections in mice, the standard OPK assay fails to evaluate anti-PspA antibodies. Using a pneumococcal surface protein C-deficient strain and extending the incubation time of opsonized bacteria, complement, and HL-60 cells reportedly results in enhanced bactericidal activity (modified OPK assay). We aimed to measure the bactericidal activity of anti-PspA antibodies in intact pneumococcal strains. We optimized the pneumococcal culture method used in the OPK assay to increase the efficiency of anti-PspA antibody-mediated phagocytosis of HL-60 cells. As thick capsules hinder phagocytosis, we attempted to obtain pneumococci with thin capsules through an improved culture method. As pneumococci attached to cells exhibit thin capsules, pneumococci cultured in Todd Hewitt yeast extract (THY) broth were spread on blood agar plates and incubated for 4 h. cpsA mRNA transcript levels in pneumococci cultured on blood agar were lower than those in pneumococci cultured in THY broth. OPK activity against pneumococci expressing PspA of clades 1-5 was reasonably well detected using pneumococci cultured on blood agar in the modified OPK assay. The modified OPK assay for anti-PspA antibody using pneumococci cultured on blood agar represents a useful assay to determine the killing activity of functional anti-PspA antibodies against pneumococci.

Immune responses and protection against Streptococcus pneumoniae elicited by recombinant Bordetella pertussis adenylate cyclase (CyaA) carrying fragments of pneumococcal surface protein A, PspA.

Streptococcus pneumoniae is a common agent of important human diseases such as otitis media, pneumonia, meningitis and sepsis. Current available vaccines that target capsular polysaccharides induce protection against invasive disease and nasopharyngeal colonization in children, yet their efficacy is limited to the serotypes included in the formulations. The virulence factor Pneumococcal Surface Protein A (PspA) interacts with host immune system and helps the bacteria to evade phagocytosis. Due to its essential role in virulence, PspA is an important vaccine candidate. Here we have tested a delivery system based on the adenylate cyclase toxin of Bordetella pertussis (CyaA) to induce immune responses against PspA in mice. CyaA was engineered to express fragments of the N-terminal region of PspAs from clades 2 and 4 (A2 and A4) and the resulting proteins were used in immunization experiments in mice. The recombinant CyaA-A2 and CyaA-A4 proteins were able to induce high levels of anti-PspA antibodies that reacted with pneumococcal strains expressing either PspA2 or PspA4. Moreover, reactivity of the antibodies against pneumococcal strains that express PspAs from clades 3 and 5 (PspA3 and PspA5) was also observed. A formulation containing CyaA-A2 and CyaA-A4 was able to protect mice against invasive pneumococcal challenges with isolates that express PspA2, PspA4 or PspA5. Moreover, a CyaA-A2-A4 fusion protein induced antibodies at similar levels and with similar reactivity as the formulation containing both proteins, and protected mice against the invasive challenge. Our results indicate that CyaA-PspA proteins are good candidates to induce broad protection against pneumococcal isolates.

An immunoinformatics approach to epitope-based vaccine design against PspA in Streptococcus pneumoniae.

BACKGROUND: Streptococcus pneumoniae (SPN) is the agent responsible for causing respiratory diseases, including pneumonia, which causes severe health hazards and child deaths globally. Antibiotics are used to treat SPN as a first-line treatment, but nowadays, SPN is showing resistance to several antibiotics. A vaccine can overcome this global problem by preventing this deadly pathogen. The conventional methods of wet-laboratory vaccine design and development are an intense, lengthy, and costly procedure. In contrast, epitope-based in silico vaccine designing can save time, money, and energy. In this study, pneumococcal surface protein A (PspA), one of the major virulence factors of SPN, is used to design a multi-epitope vaccine. METHODS: For designing the vaccine, the sequence of PspA was retrieved, and then, phylogenetic analysis was performed. Several CTL epitopes, HTL epitopes, and LBL epitopes of PspA were all predicted by using several bioinformatics tools. After checking the antigenicity, allergenicity, and toxicity scores, the best epitopes were selected for the vaccine construction, and then, physicochemical and immunological properties were analyzed. Subsequently, vaccine 3D structure prediction, refinement, and validation were performed. Molecular docking, molecular dynamic simulation, and immune simulation were performed to ensure the binding between HLA and TLR4. Finally, codon adaptation and in silico cloning were performed to transfer into a suitable vector. RESULTS: The constructed multi-epitope vaccine showed a strong binding affinity with the receptor molecule TLR4. Analysis of molecular dynamic simulation, C-immune simulation, codon adaptation, and in silico cloning validated that our designed vaccine is a suitable candidate against SPN. CONCLUSION: The in silico analysis has proven the vaccine as an alternative medication to combat against S. pneumoniae. The designated vaccine can be further tested in the wet lab, and a novel vaccine can be developed.

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

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.

Peptide linker increased the stability of pneumococcal fusion protein vaccine candidate.

Streptococcus pneumoniae is a bacterial pathogen exclusive to humans, responsible for respiratory and systemic diseases. Pneumococcal protein vaccines have been proposed as serotype-independent alternatives to currently used conjugated polysaccharide vaccines, which have presented limitations regarding their coverage. Previously in our group, pneumococcal surface protein A (PspA) and detoxified pneumolysin (PdT) were genetically fused and the hybrid protein protected mice against pneumococcal challenge, offered higher cross-protection against different strains and showed greater opsonophagocytosis rate than co-administered proteins. As juxtaposed fusion was unstable to upscale production of the protein, flexible (PspA-FL-PdT) and rigid (PspA-RL-PdT) molecular linkers were inserted between the antigens to increase stability. This work aimed to produce recombinant fusion proteins, evaluate their stability after linker insertion, both in silico and experimentally, and enable the production of two antigens in a single process. The two constructs with linkers were cloned into Escherichia coli and hybrid proteins were purified using chromatography; purity was evaluated by SDS-PAGE and stability by Western blot and high performance size exclusion chromatography. PspA-FL-PdT showed higher stability at -20°C and 4°C, without additional preservatives. In silico analyses also showed differences regarding stability of the fusion proteins, with molecule without linker presenting disallowed amino acid positions in Ramachandran plot and PspA-FL-PdT showing the best scores, in agreement with experimental results. Mice were immunized with three doses and different amounts of each protein. Both fusion proteins protected all groups of mice against intranasal lethal challenge. The results show the importance of hybrid protein structure on the stability of the products, which is essential for a successful bioprocess development.

Immunoinformatics-aided design of a new multi-epitope vaccine adjuvanted with domain 4 of pneumolysin against Streptococcus pneumoniae strains.

BACKGROUND: Streptococcus pneumoniae (Pneumococcus) has remained a leading cause of fatal infections such as pneumonia, meningitis, and sepsis. Moreover, this pathogen plays a major role in bacterial co-infection in patients with life-threatening respiratory virus diseases such as influenza and COVID-19. High morbidity and mortality in over one million cases, especially in very young children and the elderly, are the main motivations for pneumococcal vaccine development. Due to the limitations of the currently marketed polysaccharide-based vaccines, non-serotype-specific protein-based vaccines have received wide research interest in recent years. One step further is to identify high antigenic regions within multiple highly-conserved proteins in order to develop peptide vaccines that can affect various stages of pneumococcal infection, providing broader serotype coverage and more effective protection. In this study, immunoinformatics tools were used to design an effective multi-epitope vaccine in order to elicit neutralizing antibodies against multiple strains of pneumococcus. RESULTS: The B- and T-cell epitopes from highly protective antigens PspA (clades 1-5) and PhtD were predicted and immunodominant peptides were linked to each other with proper linkers. The domain 4 of Ply, as a potential TLR4 agonist adjuvant candidate, was attached to the end of the construct to enhance the immunogenicity of the epitope vaccine. The evaluation of the physicochemical and immunological properties showed that the final construct was stable, soluble, antigenic, and non-allergenic. Furthermore, the protein was found to be acidic and hydrophilic in nature. The protein 3D-structure was built and refined, and the Ramachandran plot, ProSA-web, ERRAT, and Verify3D validated the quality of the final model. Molecular docking analysis showed that the designed construct via Ply domain 4 had a strong interaction with TLR4. The structural stability of the docked complex was confirmed by molecular dynamics. Finally, codon optimization was performed for gene expression in E. coli, followed by in silico cloning in the pET28a(+) vector. CONCLUSION: The computational analysis of the construct showed acceptable results, however, the suggested vaccine needs to be experimentally verified in laboratory to ensure its safety and immunogenicity.

In-silico design and evaluation of an epitope-based serotype-independent promising vaccine candidate for highly cross-reactive regions of pneumococcal surface protein A.

BACKGROUND: The pathogenicity of pneumococcus with high morbidity, mortality, and multi-drug resistance patterns has been increasing. The limited coverage of the licensed polysaccharide-based vaccines and the replacement of the non-vaccine serotypes are the main reasons for producing a successful serotype-independent vaccine. Pneumococcal surface protein A (PspA) is an extremely important virulence factor and an interesting candidate for conserved protein-based pneumococcal vaccine classified into two prominent families containing five clades. PspA family-elicited immunity is clade-dependent, and the level of the PspA cross-reactivity is restricted to the same family. METHODS: To cover and overcome the clade-dependent immunity of the PspAs in this study, we designed and tested a PspA1-5c+p vaccine candidate composed of the highest immunodominant coverage of B- and T-cell epitope truncated domain of each clade focusing on two cross-reactive B and C regions of the PspAs. The antigenicity, toxicity, physicochemical properties, 3D structure prediction, stability and flexibility of the designed protein using molecular dynamic (MD) simulation, molecular docking of the construct withHLADRB1*(01:01) and human lactoferrin N-lop, and immune simulation were assessed using immunoinformatics tools. In the experimental section, after intraperitoneal immunization of the mice with Alum adjuvanted recombinant PspA1-5c+p, we evaluated the immune response, cross-reactivity, and functionality of the Anti-PspA1-5c+p antibody using ELISA, Opsonophagocytic killing activity, and serum bactericidal assay. RESULTS: For the first time, this work suggested a novel PspA-based vaccine candidate using immunoinformatics tools. The designed PspA1-5c+p protein is predicted to be highly antigenic, non-toxic, soluble, stable with low flexibility in MD simulation, and able to stimulate both humoral and cellular immune responses. The designed protein also could interact strongly with HLADRB1*(01:01) and human lactoferrin N-lop in the docking study. Our immunoinformatics predictions were validated using experimental data. Results showed that the anti-PspA1-5c+p IgG not only had a high titer with strong and same cross-reactivity coverage against all pneumococcal serotypes used but also had high and effective bioactivity for pneumococcal clearance using complement system and phagocytic cells. CONCLUSION: Our findings elucidated the potential application of the PspA1-5c+p vaccine candidate as a serotype-independent pneumococcal vaccine with a strong cross-reactivity feature. Further in-vitro and in-vivo investigations against other PspA clades should be performed to confirm the full protection of the PspA1-5c+p vaccine candidate.

Negligible role for pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC) in the nasopharyngeal colonization of mice with a serotype 6B pneumococcal strain

Streptococcus pneumoniae colonizes the human nasopharynx asymptomatically, but it can also cause several diseases, including otitis media, pneumonia, bacteremia, and meningitis. The colonization of the nasopharynx by the bacteria is an essential step for the pneumococcus to invade other sites and cause diseases. Pneumococcal surface protein A (PspA) and Pneumococcal surface Protein C (PspC) are important virulence factors and have been described to play roles in adhesion and immune evasion. In this study, we immunized mice subcutaneously with the recombinant α-helical region of PspA and/or PspC combined with different adjuvants to assess protection against colonization with the serotype 6B strain BHN418. Though high serum levels of specific IgG were detected, none of the formulations led to reduction in the colonization of the nasopharynx. The negative result may be due to the poor induction of IgG2c, which has been previously correlated with protection against pneumococcal colonization in mice. Furthermore, BHN418 pspA and pspC single and double knockouts were evaluated in colonization experiments and no differences in bacterial load were observed. In competition assays with the wild-type strain, borderline to no reduction was observed in the loads of the knockouts. Our results contrast with data from the literature using other pneumococcal strains, showing that the role of PspA and PspC in colonization can vary depending on the background of the knockout strain studied. BHN418 has been selected for its capacity to colonize humans in experimental challenge studies and may have redundant factors that compensate for the lack of PspA and PspC during nasopharyngeal colonization of mice.

Negligible role for pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC) in the nasopharyngeal colonization of mice with a serotype 6B pneumococcal strain

Streptococcus pneumoniae colonizes the human nasopharynx asymptomatically, but it can also cause several diseases, including otitis media, pneumonia, bacteremia, and meningitis. The colonization of the nasopharynx by the bacteria is an essential step for the pneumococcus to invade other sites and cause diseases. Pneumococcal surface protein A (PspA) and Pneumococcal surface Protein C (PspC) are important virulence factors and have been described to play roles in adhesion and immune evasion. In this study, we immunized mice subcutaneously with the recombinant α-helical region of PspA and/or PspC combined with different adjuvants to assess protection against colonization with the serotype 6B strain BHN418. Though high serum levels of specific IgG were detected, none of the formulations led to reduction in the colonization of the nasopharynx. The negative result may be due to the poor induction of IgG2c, which has been previously correlated with protection against pneumococcal colonization in mice. Furthermore, BHN418 pspA and pspC single and double knockouts were evaluated in colonization experiments and no differences in bacterial load were observed. In competition assays with the wild-type strain, borderline to no reduction was observed in the loads of the knockouts. Our results contrast with data from the literature using other pneumococcal strains, showing that the role of PspA and PspC in colonization can vary depending on the background of the knockout strain studied. BHN418 has been selected for its capacity to colonize humans in experimental challenge studies and may have redundant factors that compensate for the lack of PspA and PspC during nasopharyngeal colonization of mice.

Immune responses and protection against Streptococcus pneumoniae elicited by recombinant Bordetella pertussis adenylate cyclase (CyaA) carrying fragments of pneumococcal surface protein A, PspA

Streptococcus pneumoniae is a common agent of important human diseases such as otitis media, pneumonia, meningitis and sepsis. Current available vaccines that target capsular polysaccharides induce protection against invasive disease and nasopharyngeal colonization in children, yet their efficacy is limited to the serotypes included in the formulations. The virulence factor Pneumococcal Surface Protein A (PspA) interacts with host immune system and helps the bacteria to evade phagocytosis. Due to its essential role in virulence, PspA is an important vaccine candidate. Here we have tested a delivery system based on the adenylate cyclase toxin of Bordetella pertussis (CyaA) to induce immune responses against PspA in mice. CyaA was engineered to express fragments of the N-terminal region of PspAs from clades 2 and 4 (A2 and A4) and the resulting proteins were used in immunization experiments in mice. The recombinant CyaA-A2 and CyaA-A4 proteins were able to induce high levels of anti-PspA antibodies that reacted with pneumococcal strains expressing either PspA2 or PspA4. Moreover, reactivity of the antibodies against pneumococcal strains that express PspAs from clades 3 and 5 (PspA3 and PspA5) was also observed. A formulation containing CyaA-A2 and CyaA-A4 was able to protect mice against invasive pneumococcal challenges with isolates that express PspA2, PspA4 or PspA5. Moreover, a CyaA-A2-A4 fusion protein induced antibodies at similar levels and with similar reactivity as the formulation containing both proteins, and protected mice against the invasive challenge. Our results indicate that CyaA-PspA proteins are good candidates to induce broad protection against pneumococcal isolates.

Peptide linker increased the stability of pneumococcal fusion protein vaccine candidate

Streptococcus pneumoniae is a bacterial pathogen exclusive to humans, responsible for respiratory and systemic diseases. Pneumococcal protein vaccines have been proposed as serotype-independent alternatives to currently used conjugated polysaccharide vaccines, which have presented limitations regarding their coverage. Previously in our group, pneumococcal surface protein A (PspA) and detoxified pneumolysin (PdT) were genetically fused and the hybrid protein protected mice against pneumococcal challenge, offered higher cross-protection against different strains and showed greater opsonophagocytosis rate than co-administered proteins. As juxtaposed fusion was unstable to upscale production of the protein, flexible (PspA-FL-PdT) and rigid (PspA-RL-PdT) molecular linkers were inserted between the antigens to increase stability. This work aimed to produce recombinant fusion proteins, evaluate their stability after linker insertion, both in silico and experimentally, and enable the production of two antigens in a single process. The two constructs with linkers were cloned into Escherichia coli and hybrid proteins were purified using chromatography; purity was evaluated by SDS-PAGE and stability by Western blot and high performance size exclusion chromatography. PspA-FL-PdT showed higher stability at −20°C and 4°C, without additional preservatives. In silico analyses also showed differences regarding stability of the fusion proteins, with molecule without linker presenting disallowed amino acid positions in Ramachandran plot and PspA-FL-PdT showing the best scores, in agreement with experimental results. Mice were immunized with three doses and different amounts of each protein. Both fusion proteins protected all groups of mice against intranasal lethal challenge. The results show the importance of hybrid protein structure on the stability of the products, which is essential for a successful bioprocess development.

Pneumococcal Surface Proteins as Virulence Factors, Immunogens, and Conserved Vaccine Targets.

Streptococcus pneumoniae is an opportunistic pathogen that causes over 1 million deaths annually despite the availability of several multivalent pneumococcal conjugate vaccines (PCVs). Due to the limitations surrounding PCVs along with an evolutionary rise in antibiotic-resistant and unencapsulated strains, conserved immunogenic proteins as vaccine targets continue to be an important field of study for pneumococcal disease prevention. In this review, we provide an overview of multiple classes of conserved surface proteins that have been studied for their contribution to pneumococcal virulence. Furthermore, we discuss the immune responses observed in response to these proteins and their promise as vaccine targets.