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.

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.

Liposome-based dry powder vaccine immunization targeting the lungs induces broad protection against pneumococcus

Streptococcus pneumoniae is an important human pathogen. Currently used conjugate vaccines are effective against invasive disease, but protection is restricted to serotypes included in the formulation, leading to serotype replacement. Furthermore, protection against non-invasive disease is reported to be considerably lower. The development of a serotype-independent vaccine is thus important and Pneumococcal surface protein A (PspA) is a promising vaccine candidate. PspA shows some diversity and can be classified in 6 clades and 3 families, with families 1 and 2 being the most frequent in clinical isolates. The ideal vaccine should thus induce protection against the two most common families of PspA. The aim of this work was to develop a liposome-based vaccine containing PspAs from family 1 and 2 and to characterize its immune response. Liposomes (LP) composed of dipalmitoylphosphatidylcholine (DPPC) and 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) with or without α-galactosylceramide (α-GalCer) were produced by microfluidics, encapsulating PspA from clade 1 (PspA1, family 1) and/or clade 4 (PspA4Pro, family 2) followed by spray-drying with trehalose to form nanocomposite microparticles carriers (NCMP). LP/NCMPs showed good stability and preservation of protein activity. LP/NCMPs containing PspA1 and/or PspA4Pro were used for immunization of mice targeting the lungs. High serum IgG antibody titers against both PspA1 and PspA4Pro were detected in animals immunized with LP/NCMPs containing α-GalCer, with a balance of IgG1 and IgG2a titers. IgG in sera from immunized mice bound to pneumococcal strains from different serotypes and expressing different PspA clades, indicating broad recognition. Mucosal IgG and IgA were also detected. Importantly, immunization with LP/NCMPs induced full protection against strains expressing PspAs from family 1 and 2. Furthermore, CD4+ resident memory T cells were detected in the lungs of the immunized animals that survived the challenge.

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.

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.

Pneumococcal Surface Protein A-Hybrid Nanoparticles Protect Mice from Lethal Challenge after Mucosal Immunization Targeting the Lungs.

Pneumococcal disease remains a global burden, with current conjugated vaccines offering protection against the common serotype strains. However, there are over 100 serotype strains, and serotype replacement is now being observed, which reduces the effectiveness of the current vaccines. Pneumococcal surface protein A (PspA) has been investigated as a candidate for new serotype-independent pneumococcal vaccines, but requires adjuvants and/or delivery systems to improve protection. Polymeric nanoparticles (NPs) are biocompatible and, besides the antigen, can incorporate mucoadhesive and adjuvant substances such as chitosans, which improve antigen presentation at mucosal surfaces. This work aimed to define the optimal NP formulation to deliver PspA into the lungs and protect mice against lethal challenge. We prepared poly(glycerol-adipate-co-ω-pentadecalactone) (PGA-co-PDL) and poly(lactic-co-glycolic acid) (PLGA) NPs using an emulsion/solvent evaporation method, incorporating chitosan hydrochloride (HCl-CS) or carboxymethyl chitosan (CM-CS) as hybrid NPs with encapsulated or adsorbed PspA. We investigated the physicochemical properties of NPs, together with the PspA integrity and biological activity. Furthermore, their ability to activate dendritic cells in vitro was evaluated, followed by mucosal immunization targeting mouse lungs. PGA-co-PDL/HCl-CS (291 nm) or CM-CS (281 nm) NPs produced smaller sizes compared to PLGA/HCl-CS (310 nm) or CM-CS (299 nm) NPs. Moreover, NPs formulated with HCl-CS possessed a positive charge (PGA-co-PDL +17 mV, PLGA + 13 mV) compared to those formulated with CM-CS (PGA-co-PDL -20 mV, PLGA -40 mV). PspA released from NPs formulated with HCl-CS preserved the integrity and biological activity, but CM-CS affected PspA binding to lactoferrin and antibody recognition. PspA adsorbed in PGA-co-PDL/HCl-CS NPs stimulated CD80+ and CD86+ cells, but this was lower compared to when PspA was encapsulated in PLGA/HCl-CS NPs, which also stimulated CD40+ and MHC II (I-A/I-E)+ cells. Despite no differences in IgG being observed between immunized animals, PGA-co-PDL/HCl-CS/adsorbed-PspA protected 83% of mice after lethal pneumococcal challenge, while 100% of mice immunized with PLGA/HCl-CS/encapsulated-PspA were protected. Therefore, this formulation is a promising vaccine strategy, which has beneficial properties for mucosal immunization and could potentially provide serotype-independent protection.

Pneumococcal surface protein a-hybrid nanoparticles protect mice from lethal challenge after mucosal immunization targeting the lungs

Pneumococcal disease remains a global burden, with current conjugated vaccines offering protection against the common serotype strains. However, there are over 100 serotype strains, and serotype replacement is now being observed, which reduces the effectiveness of the current vaccines. Pneumococcal surface protein A (PspA) has been investigated as a candidate for new serotype-independent pneumococcal vaccines, but requires adjuvants and/or delivery systems to improve protection. Polymeric nanoparticles (NPs) are biocompatible and, besides the antigen, can incorporate mucoadhesive and adjuvant substances such as chitosans, which improve antigen presentation at mucosal surfaces. This work aimed to define the optimal NP formulation to deliver PspA into the lungs and protect mice against lethal challenge. We prepared poly(glycerol-adipate-co-ω-pentadecalactone) (PGA-co-PDL) and poly(lactic-co-glycolic acid) (PLGA) NPs using an emulsion/solvent evaporation method, incorporating chitosan hydrochloride (HCl-CS) or carboxymethyl chitosan (CM-CS) as hybrid NPs with encapsulated or adsorbed PspA. We investigated the physicochemical properties of NPs, together with the PspA integrity and biological activity. Furthermore, their ability to activate dendritic cells in vitro was evaluated, followed by mucosal immunization targeting mouse lungs. PGA-co-PDL/HCl-CS (291 nm) or CM-CS (281 nm) NPs produced smaller sizes compared to PLGA/HCl-CS (310 nm) or CM-CS (299 nm) NPs. Moreover, NPs formulated with HCl-CS possessed a positive charge (PGA-co-PDL +17 mV, PLGA + 13 mV) compared to those formulated with CM-CS (PGA-co-PDL −20 mV, PLGA −40 mV). PspA released from NPs formulated with HCl-CS preserved the integrity and biological activity, but CM-CS affected PspA binding to lactoferrin and antibody recognition. PspA adsorbed in PGA-co-PDL/HCl-CS NPs stimulated CD80+ and CD86+ cells, but this was lower compared to when PspA was encapsulated in PLGA/HCl-CS NPs, which also stimulated CD40+ and MHC II (I-A/I-E)+ cells. Despite no differences in IgG being observed between immunized animals, PGA-co-PDL/HCl-CS/adsorbed-PspA protected 83% of mice after lethal pneumococcal challenge, while 100% of mice immunized with PLGA/HCl-CS/encapsulated-PspA were protected. Therefore, this formulation is a promising vaccine strategy, which has beneficial properties for mucosal immunization and could potentially provide serotype-independent protection.

Pneumococcal vaccines: past findings, present work, and future strategies

The importance of Streptococcus pneumoniae has been well established. These bacteria can colonize infants and adults without symptoms, but in some cases can spread, invade other tissues and cause disease with high morbidity and mortality. The development of pneumococcal conjugate vaccines (PCV) caused an enormous impact in invasive pneumococcal disease and protected unvaccinated people by herd effect. However, serotype replacement is a well-known phenomenon that has occurred after the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) and has also been reported for other PCVs. Therefore, it is possible that serotype replacement will continue to occur even with higher valence formulations, but the development of serotype-independent vaccines might overcome this problem. Alternative vaccines are under development in order to improve cost effectiveness, either using proteins or the pneumococcal whole cell. These approaches can be used as a stand-alone strategy or together with polysaccharide vaccines. Looking ahead, the next generation of pneumococcal vaccines can be impacted by the new technologies recently approved for human use, such as mRNA vaccines and viral vectors. In this paper, we will review the advantages and disadvantages of the addition of new polysaccharides in the current PCVs, mainly for low- and middle-income countries, and we will also address future perspectives.

Evaluation of polymer choice on immunogenicity of chitosan coated PLGA NPs with surface-adsorbed pneumococcal protein antigen PspA4Pro

Polymeric nanoparticles (NPs) are recognized as potential delivery vehicles for vaccines. PLGA is a biocompatible polymer synonymous with polymeric NPs, which can be coated with other polymers such as chitosan that has intrinsic adjuvant properties as well as mucoadhesive properties. Numerous modifications and variations exist for PLGA and chitosan, which can influence the NP characteristics and the resulting immunogenicity. The current study investigated variations for making chitosan coated PLGA NPs incorporating recombinant pneumococcal surface protein A from family 2, clade 4 (PspA4Pro) antigen as a vaccine targeting the vast majority of pneumococcal strains and determine the effect of the polymers on particle size, surface charge, and surface marker upregulation on a dendritic cell (DC) line in vitro. PLGA variations tested with the ester-terminal group had the greatest detriment for prospective vaccine use, due to the lowest PspA4Pro adsorption and induction of CD40 and CD86 cell surface markers on DCs. The negatively charged chitosans exhibited the lowest surface marker expressions, similar to the uncoated NP, supporting the commonly accepted notion that positive surface charge augments immunogenic effects of the NPs. However, the study indicated that NPs made from PLGA with an acid terminated group, and chitosan HCl salt, exhibit particle characteristics, antigen adsorption efficiency and immunogenicity, which could be most suitable as a vaccine formulation.

Serum levels of anti-PspA and anti-PspC IgG decrease with age and do not correlate with susceptibility to experimental human pneumococcal colonization

Older adults are at increased risk of pneumococcal disease. This work aims to evaluate whether there is any decrease in serum IgG against variants of the antigens Pneumococcal surface protein A (PspA) and Pneumococcal surface protein C (PspC) in healthy adults with increasing age. Levels of IgG against PspA and PspC variants were determined by ELISA in serum samples comparing volunteers 18–30 years of age with volunteers who were 50–70+ before and after an experimental pneumococcal colonization challenge. The serotype 6B strain used in the challenge belongs to a minor group of pneumococcal isolates expressing two PspC variants. There was a decrease in levels of IgG with increasing age for the most common PspA variants and for all PspC variants analyzed. No correlation was found between basal levels of IgG against these antigens and protection against colonization. There was an increase in levels of IgG against PspA variants that are more cross-reactive with the variant expressed by the challenge strain post challenge in younger individuals who became colonized. Since the challenge strain used in our study expresses two different PspC variants, an increase in serum IgG against all PspC variants tested was observed in younger individuals who became colonized. For some of the antigen variants tested, a decrease in serum IgG was observed in young volunteers who were challenged but did not become colonized. Serum IgG antibodies against PspA and PspC variants thus decrease with age in healthy adults, but there is no correlation between levels of IgG against these antigens and protection against human experimental colonization. Though no correlation between naturally induced serum IgG antibodies against PspA and PspC and protection against colonization was observed, these results do not rule out the protective potential of these antigens as vaccines against pneumococcal infections.

Efficacy of a protein vaccine and a conjugate vaccine against co-colonization with vaccine-type and non-vaccine type pneumococci in mice

Widespread use of pneumococcal conjugate vaccines (PCVs) has led to substitution of vaccine-type (VT) strains by non-vaccine type (NVT) strains in nasopharyngeal carriage. We compared the efficacy of PCV13 and a nasal protein formulation containing pneumococcal surface protein A (PspA) adjuvanted with the whole-cell pertussis vaccine (wP) in the protection against co-colonization challenge models in mice with VT and NVT strains expressing different PspAs. Immunized mice were challenged with two different mixtures: i. VT4 (PspA3) + NVT33 (PspA1) and ii. VT23F (PspA2) + NVT15B/C (PspA4). Results from the first mixture showed a reduction in loads of VT4 strain in the nasopharynx of mice immunized with PCV13. A statistical difference between the loads of the VT and NVT strains was observed, indicating a competitive advantage for the NVT strain in PCV13-immunized animals. In the second mixture, no reduction was observed for the VT23F strain, probably due to low levels of anti-23F polysaccharide IgG induced by PCV13. Interestingly, a combination of the PspA formulation containing wP with PCV13 led to a reduction in colonization with both strains of the two mixtures tested, similar to the groups immunized nasally with wP or PspA plus wP. These results indicate that a combination of vaccines may be a useful strategy to overcome pneumococcal serotype replacement

Evaluation of inactivated Bordetella pertussis as a delivery system for the immunization of mice with Pneumococcal Surface Antigen A

Pneumococcal Surface Protein A (PspA) has been successfully tested as vaccine candidate against Streptococcus pneumoniae infections. Vaccines able to induce PspA-specific antibodies and Th1 cytokines usually provide protection in mice. We have shown that the whole cell pertussis vaccine (wP) or components from acellular pertussis vaccines, such as Pertussis Toxin or Filamentous Hemagglutinin (FHA), are good adjuvants to PspA, suggesting that combined pertussis-PspA vaccines would be interesting strategies against the two infections. Here, we evaluated the potential of wP as a delivery vector to PspA. Bordetella pertussis strains producing a PspA from clade 4 (PspA4Pro) fused to the N-terminal region of FHA (Fha44) were constructed and inactivated with formaldehyde for the production of wPPspA4Pro. Subcutaneous immunization of mice with wPPspA4Pro induced low levels of anti-PspA4 IgG, even after 3 doses, and did not protect against a lethal pneumococcal challenge. Prime-boost strategies using wPPspA4Pro and PspA4Pro showed that there was no advantage in using the wPPspA4Pro vaccine. Immunization of mice with purified PspA4Pro induced higher levels of antibodies and protection against pneumococcal infection than the prime-boost strategies. Finally, purified Fha44:PspA4Pro induced high levels of anti-PspA4Pro IgG, but no protection, suggesting that the antibodies induced by the fusion protein were not directed to protective epitopes.

Streptococcus pneumoniae augments inflammatory process in asthma model without changing Th2 profile

Studies suggested that some aspects of asthma exacerbation by Spn infection remain unclear. Objective: to evaluate possible mechanism that worsen inflammation caused by Spn in an experimental model of chronic allergic inflammation. Methods: 30 BALB/c mice were divided in 4 groups: SAL (non-sensitized group), STREP (animals challenged with Spn), OVA (ovalbumin sensitized group), OVAST (OVA sensitized and challenged with Spn). OVA and OVAST groups received intraperitoneal injections of ovalbumin (OVA) solution (days 1 and 14). OVA challenges were performed on days 22, 24, 26, and 28. Afterwards, animals were challenged with pneumococcal strains M10 (11A)(50ul/bacteria in saline). After 12h, lung mechanics and bronchoalveolar lavage (BAL) were performed. Animals were euthanized, lungs removed for immunohistochemistry and morphometric analysis. Results: Challenge with Spn in OVA sensitized group induces an increase in of total cells (46.33±13.22x104cells/mL), neutrophils (23.70±14.39x104cells/mL), macrophages (7.70±2.03x104cells/mL) and eosinophils (14.52±13.88x104cells/mL) in BALF as well as increasing in polymorphonuclear cells (0.152±0.06mm2) and expression of IL-17 (12.12±2.67mm2) in peribronchovascular area in lung compared to OVA group (p<0.05). There were an increase in tissue damping (27.01±7.25cmH2O/mL/s(1-a)); expression of IL-5 (10.15±3.39mm2) and IL-13 (8.85±3.56mm2) in peribronchovascular area were observed in OVA groups compared to other groups (p<0.05). Conclusion: Challenge with Spn, in this model induces an increasing in lung inflammation by increasing IL-17 without changes in Th2 profile.

Inflammatory response to Streptococcus pneumoniae was attenuated by moderate aerobic exercise training

Moderate aerobic exercise training may alter immune system. Studies suggested that S. pneumoniae remained an important cause of mortality. Objective: To study mechanisms for attenuating inflammatory process involving pneumococcal infection by moderate aerobic exercise. Methods: 38Balb/C mice were divided into 4 groups: Control (C), Moderate Aerobic Exercise Training (MAT), S. pneumonia infection (IF), MAT+IF groups. Moderate intensity treadmill training was performed over 4weeks, 5x/week, 60min/session in MAT groups. After 72h of last exercise training, IF groups were challenged with pneumococcal strains M10 (11A; 50ul/bacteria in saline) and 12h after, lung function and bronchoalveolar lavage (BAL) were performed. Afterwards, animals were euthanized, lungs removed to proceed immunohistochemistry and morphometric analysis in lung parenchyma. Results: Bacterial inoculation resulted in increase in: total cells (77.66±54.02x104cells/mL), neutrophils (73.78±50.88x104cells/mL), resistance (0.77±0.08cmH2O.mL-1.s;) and elastance (31.86±8.16cmH2O.mL-1.s) of respiratory system and expression of IL-17 (817.88±217.59mm2)(p<0.05). MAT in animals submitted to bacterial challenge presented a decrease of these parameters (p<0.05). MAT+IF group showed an increase in expression of IL-1ra (886.04±274.07mm2), TLR2 (708.28±161.48mm2) and TLR4 (1444.11±723.92mm2) compared to IF group (p<0.05). Conclusion: These results suggest that MAT attenuated inflammatory process in an animal model of bacterial infection by increasing anti-inflammatory mediators, increasing Toll-like receptors that anticipated host defense, helping in resolution of inflammation.

High intensity interval training control inflammation in mice exposed to Streptococus pneumoniae

S.pneumoniae is an important cause of pneumonia. Exercise training is a stimulator of immune system. High Intensity Interval Training (HIIT) have been gain adepts, although their benefits remain unclear. Objective: Evaluate if HIIT prior to S.pneumoniae infection in mice alters lung inflammation. Methods: 38Balb/C mice were divided into 4 groups: Sedentary (SED),HIIT (HIIT), Infection (IF),HIIT+infection (HIIT/IF). HIIT was performed in a treadmill altering 26 session: 1min of 75%¨maximum capacity training and 30s of 50% maximum capacity training, over 4w, 5x/w. 72h after last training, IF groups were challenged with pneumococcal strains M10 (11A)(50 ul/bacteria in saline).Lung function and bronchoalveolar lavage (BAL) were performed 12h after challenge. Afterwards, animals were euthanized, lungs removed to immunohistochemistry and morphometric analysis in lung parenchyma. Results: Pneumococcal inoculation induces an increase in lung resistance (0.74±0.07cmH2O.mL-1.s) and elastance (31.86±8.16 cmH2O.mL-1.s) of respiratory system, in total cells (77.66±54.02x104cells/mL) and neutrophils (73.47±50.88x104cells/mL) in BALF, expression of IL-17 (817.88±217.59mm2) and collagen fibers content in lung parenchyma (17.24±4.54%) (p<0.05). HIIT in inoculated animals resulted in a reduction of all parameters (p<0.05), except for lung resistance. HIIT groups presented increasing expression of IL-1ra (698.64±432.42mm2), CuZnSOD (576.42±138.18mm2), IL-33 (990.07±212.47mm2)(p<0.001). Conclusion: HIIT attenuated inflammatory process induced by S.pneumoniae, increasing antiinflammatory mediators and antioxidant enzymes, reducing proinflammatory mediators.

Efficacy of a protein vaccine and a conjugate vaccine against co-colonization with vaccine-type and non-vaccine type pneumococci in mice

Widespread use of pneumococcal conjugate vaccines (PCVs) has led to substitution of vaccine-type (VT) strains by non-vaccine type (NVT) strains in nasopharyngeal carriage. We compared the efficacy of PCV13 and a nasal protein formulation containing pneumococcal surface protein A (PspA) adjuvanted with the whole-cell pertussis vaccine (wP) in the protection against co-colonization challenge models in mice with VT and NVT strains expressing different PspAs. Immunized mice were challenged with two different mixtures: i. VT4 (PspA3) + NVT33 (PspA1) and ii. VT23F (PspA2) + NVT15B/C (PspA4). Results from the first mixture showed a reduction in loads of VT4 strain in the nasopharynx of mice immunized with PCV13. A statistical difference between the loads of the VT and NVT strains was observed, indicating a competitive advantage for the NVT strain in PCV13-immunized animals. In the second mixture, no reduction was observed for the VT23F strain, probably due to low levels of anti-23F polysaccharide IgG induced by PCV13. Interestingly, a combination of the PspA formulation containing wP with PCV13 led to a reduction in colonization with both strains of the two mixtures tested, similar to the groups immunized nasally with wP or PspA plus wP. These results indicate that a combination of vaccines may be a useful strategy to overcome pneumococcal serotype replacement

Evaluation of inactivated Bordetella pertussis as a delivery system for the immunization of mice with Pneumococcal Surface Antigen A

Pneumococcal Surface Protein A (PspA) has been successfully tested as vaccine candidate against Streptococcus pneumoniae infections. Vaccines able to induce PspA-specific antibodies and Th1 cytokines usually provide protection in mice. We have shown that the whole cell pertussis vaccine (wP) or components from acellular pertussis vaccines, such as Pertussis Toxin or Filamentous Hemagglutinin (FHA), are good adjuvants to PspA, suggesting that combined pertussis-PspA vaccines would be interesting strategies against the two infections. Here, we evaluated the potential of wP as a delivery vector to PspA. Bordetella pertussis strains producing a PspA from clade 4 (PspA4Pro) fused to the N-terminal region of FHA (Fha44) were constructed and inactivated with formaldehyde for the production of wPPspA4Pro. Subcutaneous immunization of mice with wPPspA4Pro induced low levels of anti-PspA4 IgG, even after 3 doses, and did not protect against a lethal pneumococcal challenge. Prime-boost strategies using wPPspA4Pro and PspA4Pro showed that there was no advantage in using the wPPspA4Pro vaccine. Immunization of mice with purified PspA4Pro induced higher levels of antibodies and protection against pneumococcal infection than the prime-boost strategies. Finally, purified Fha44:PspA4Pro induced high levels of anti-PspA4Pro IgG, but no protection, suggesting that the antibodies induced by the fusion protein were not directed to protective epitopes.

Progress in mucosal immunization for protection against pneumococcal pneumonia.

Introduction: Lower respiratory tract infections are the fourth cause of death worldwide and pneumococcus is the leading cause of pneumonia. Nonetheless, existing pneumococcal vaccines are less effective against pneumonia than invasive diseases and serotype replacement is a major concern. Protein antigens could induce serotype-independent protection, and mucosal immunization could offer local and systemic immune responses and induce protection against pneumococcal colonization and lung infection. Areas covered: Immunity induced in the experimental human pneumococcal carriage model, approaches to address the physiological barriers to mucosal immunization and improve delivery of the vaccine antigens, different strategies already tested for pneumococcal mucosal vaccination, including live recombinant bacteria, nanoparticles, bacterium-like particles, and nanogels as well as, nasal, pulmonary, sublingual and oral routes of vaccination. Expert opinion: The most promising delivery systems are based on nanoparticles, bacterial-like particles or nanogels, which possess greater immunogenicity than the antigen alone and are considered safer than approaches based on living cells or toxoids. These particles can protect the antigen from degradation, eliminating the refrigeration need during storage and allowing the manufacture of dry powder formulations. They can also increase antigen uptake, control release of antigen and trigger innate immune responses.