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.

Characterization of Pili Protein 67 kDa Streptococcus pneumoniae: New Candidate for Virulence Factor-Based Pneumococcal Antigen Vaccine.

INTRODUCTION: Streptococcus pneumoniae is a Gram-positive diplococci bacteria that causes infectious diseases such as otitis, meningitis, and pneumonia. Streptococcus pneumoniae has various virulence factors, one of which is pilus. In addition to being immunogenic, pilus S. pneumoniae also plays a role in bacterial adhesion to host cells and biofilm formation. The S. pneumoniae pilus found in this study consisted of several proteins with various molecular weights, one of which was a 67 kDa protein. OBJECTIVE: This study aimed to determine the characteristics of the 67 kDa pilus protein, including its capacity as hemagglutinin and adhesin and its amino acid sequence (AA). METHODS: The LCMS/MS method is used to determine the AA sequence of the 67 kDa pilus protein. The AA structure was analyzed through BLASTP by matching it with the sequence of the protein data bank of S. pneumoniae (taxid: 1313). The ProtParam tool from ExPASY was used to calculate various physical and chemical parameters of the protein, while for evaluating its immunogenicity, the VaxiJen V2.0 online server was used. RESULTS: The results of this study indicate that the 67 kD a pilus protein, is an anti-hemagglutinin protein and has a role as an adhesin protein. Adhesion tests show the action between protein concentration and the number of bacteria attached to enterocyte cells. LCMS/MS test results obtained by BLASTP showed that the 67 kDa pilus protein had three AA sequences (ITYMSPDFAAPTLAGLDDATK, AEFVEVTK, and LVVSTQTALA), which had similarities with the A backbone chain of S. pneumoniae pilus. The physicochemical test showed that the protein is hydrophilic and nonpolar, while the antigenicity test showed that the protein is antigenic. CONCLUSION: Based on these characteristics, it can be concluded that the 67 kDa S. pneumoniae pilus protein can be used as a vaccine candidate for pneumococcus.

Development of a novel flow cytometry method for detecting pneumococcal-specific B cells.

Antigen-specific B cell identification by flow cytometry is crucial for investigating their immunophenotype, subset distribution, and kinetics post-infection or immunization. Methods using biotinylated polysaccharide antigens have been described, but there is still room for improvement regarding sensitivity and applicability. The aim of this study was the development and validation of a multimer bead-based method for detecting pneumococcal polysaccharide serotypes (PS)-specific B cells following pneumococcal immunization. PS was chemically biotinylated and mounted on anti-biotin beads, and labeled with phycoerythrin (PE)-conjugated anti-biotin antibody to form a PS-multimer used for cell staining. Labeled beads were washed to remove excess fluorochrome and diminish non-specific labeling and background noise. Optimal ratios of PS-bead conjugate to PE and PS-multimer to cells were determined with titration assays. Comparison between the PS-multimer and a PS-PE monomer revealed enhanced detection of PS-specific cells and considerable signal amplification, attributed to the multimeric form of the detection probe and increased availability of antigen epitopes. To validate the specificity of the method, a competition assay using unbound PS was performed. Following pre-incubation with increasing PS concentrations, detection of PS-specific B cells with the PS-multimer was inhibited in a stepwise manner. Pre-incubation with excess PS completely blocked the fluorescent signal. This novel bead-based flow cytometry approach is a sensitive method demonstrating high specificity. It generated enhanced signals, provided clear-cut results, and was easily applicable, not requiring B cell pre-enrichment. It could be modified to adapt other antigens of interest, especially polysaccharides and proteins that could be used to probe antigen-specific B cell responses. The study of such responses may elucidate the underlying mechanisms involved in the establishment of long-term protection, provide evidence-based rationale for improving currently available vaccines and vaccination strategies, and pave the way for future vaccine development.

Microparticles entrapping pneumococcal protein SP0845 show improved immunogenicity and temperature stability.

Protein based vaccines are the most safe and affordable strategy to combat pneumococcal disease circumventing the limitations of conventional polysaccharide-based vaccines like serotype dependence, high cost and inability to be administered to immunocompromised. SP0845 is a highly conserved vaccine candidate shown to provide protection against heterologous strains of Streptococcus pneumoniae, the primal cause of pneumonia. However, the associated poor immunogenicity warrants the need for adjuvants and multiple doses to mount desired responses. The present study relates to improve the immunogenicity of pneumococcal protein SP0845 by use of poly lactic acid biodegradable polymer microparticles. The immunization studies showed that microparticles elicited higher antibody response compared to alum adjuvanted protein and this immunopotentiation was achieved without the use of any additional adjuvant. They were also capable of eliciting secondary antibody response upon boosting after four months. Further, the particles upon storage at 25 and 37 °C for one month were still capable of mounting an immune response equivalent to those stored in cold chain. Thus, using microparticles entrapping SP0845 for immunization not only improve the immunogenicity but also offer better temperature stability. This can greatly reduce the cost and increase access of protein-based vaccine to resource limited settings.

23-valent polysaccharide vaccine (PPSV23)-targeted serotype-specific identification of Streptococcus pneumoniae using the loop-mediated isothermal amplification (LAMP) method.

Reports of invasive disease due to Streptococcus pneumoniae have declined since the introduction of pneumococcal conjugate vaccines (PCV7 and PCV13). The incidence of invasive diseases due to S. pneumoniae that are not addressed by the vaccines, however, has increased in children and adults, creating a global public health problem. Previously, we established the loop-mediated isothermal amplification (LAMP) method for a PCV13 serotype-specific assay. In the current study, we developed a rapid, simple, and cost-effective assay to detect serotypes in the 23-valent pneumococcal polysaccharide vaccine (PPSV23) using the LAMP method. In this study, LAMP primer sets for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F of S. pneumoniae were developed. The reactivity, specificity, and sensitivity of LAMP assays were determined and compared to those of conventional PCR. The feasibility of LAMP assays in clinical application in patients with invasive pneumococcal diseases was validated by defining the detection limit of the LAMP assay with bacterial genomic DNA-spiked blood specimens. The specificity of each LAMP assay was determined using 44 serotypes of pneumococcal strains. Their sensitivity was 100 copies per reaction versus 103 to 106 copies per reaction for PCR assays. Using DNA-spiked blood specimens, excluding the LAMP assay that targeted serotype 22F (103 copies per reaction), the limit of detection of the LAMP assay was similar to that with purified DNA as the template (102 copies per reaction), compared with 103 to >106 copies per reaction for PCR assays. In conclusion, a rapid and simple LAMP-based PPSV23-targeted serotype detection assay was developed for use in many countries. This study is the first report of a LAMP-based assay for identification of PPSV23 serotypes. Further evaluation of this assay is needed through surveillance and vaccine efficacy studies.

Characterization of protective immune responses induced by pneumococcal surface protein A in fusion with pneumolysin derivatives.

Pneumococcal surface protein A (PspA) and Pneumolysin derivatives (Pds) are important vaccine candidates, which can confer protection in different models of pneumococcal infection. Furthermore, the combination of these two proteins was able to increase protection against pneumococcal sepsis in mice. The present study investigated the potential of hybrid proteins generated by genetic fusion of PspA fragments to Pds to increase cross-protection against fatal pneumococcal infection. Pneumolisoids were fused to the N-terminus of clade 1 or clade 2 pspA gene fragments. Mouse immunization with the fusion proteins induced high levels of antibodies against PspA and Pds, able to bind to intact pneumococci expressing a homologous PspA with the same intensity as antibodies to rPspA alone or the co-administered proteins. However, when antibody binding to pneumococci with heterologous PspAs was examined, antisera to the PspA-Pds fusion molecules showed stronger antibody binding and C3 deposition than antisera to co-administered proteins. In agreement with these results, antisera against the hybrid proteins were more effective in promoting the phagocytosis of bacteria bearing heterologous PspAs in vitro, leading to a significant reduction in the number of bacteria when compared to co-administered proteins. The respective antisera were also capable of neutralizing the lytic activity of Pneumolysin on sheep red blood cells. Finally, mice immunized with fusion proteins were protected against fatal challenge with pneumococcal strains expressing heterologous PspAs. Taken together, the results suggest that PspA-Pd fusion proteins comprise a promising vaccine strategy, able to increase the immune response mediated by cross-reactive antibodies and complement deposition to heterologous strains, and to confer protection against fatal challenge.

Identification of a novel pneumococcal vaccine antigen preferentially expressed during meningitis in mice.

Streptococcus pneumoniae is the most common cause of severe bacterial meningitis in children, the elderly, and immunocompromised individuals. To identify virulence factors preferentially expressed during meningitis, we conducted niche-specific genome-wide in vivo transcriptomic analysis after intranasal infection of mice with serotype 4 or 6A pneumococci. The expression of 34 bacterial genes was substantially altered in brain tissue of mice infected with either of the 2 strains. Ten upregulated genes were common to both strains, 7 of which were evaluated for their role in the development of meningitis. One previously uncharacterized protein, α-glycerophosphate oxidase (GlpO), was cytotoxic for human brain microvascular endothelial cells (HBMECs) via generation of H(2)O(2). A glpO deletion mutant was defective in adherence to HBMECs in vitro as well as in progression from the blood to the brain in vivo. Mutant bacteria also induced markedly reduced meningeal inflammation and brain pathology compared with wild type, despite similar levels of bacteremia. Immunization of mice with GlpO protected against invasive pneumococcal disease and provided additive protection when formulated with pneumolysin toxoid. Our results provide the basis of a strategy that can be adapted to identify genes that contribute to the development of meningitis caused by other pathogens.

Phenotypic analysis of pneumococcal polysaccharide-specific B cells.

The phenotype of B cells responsible for the production of anti-pneumococcal polysaccharide Ab has been unclear. Although individuals that respond poorly to the 23-valent pneumococcal polysaccharide (PPS) vaccine, Pneumovax, such as children <2 y, the asplenic, and a subset of common variable immunodeficiency patients, are profoundly deficient or lack IgM memory cells (CD27(+)IgM(+)), they are also deficient in the switched memory (CD27(+)IgM(-)) compartment. Direct characterization of PPS-specific B cells has not been performed. In this study, we labeled PPS14 and PPS23F with fluorescent markers. Fluorescently labeled PPS were used in FACSAria flow cytometry to characterize the phenotype of PPS-specific B cells obtained from 18 young adults pre- and postimmunization with Pneumovax. The labeled PPS were capable of inhibiting binding of Ab to the native PPS. Similarly, the native PPS were able to inhibit binding of PPS-specific B cells in a flow cytometric assay demonstrating specificity and functionality. Phenotypic analysis of unselected B cells, pre- and postimmunization, demonstrated a predominance of naive CD27(-)IgM(+) cells accounting for 61.5% of B cells. Likewise, the PPS-specific B cells obtained preimmunization consisted primarily of naive, CD27(-) B cells, 55.4-63.8%. In contrast, the PPS-specific B cells obtained postimmunization were predominantly IgM memory cells displaying the CD27(+)IgM(+), 54.2% for PPS14 and 66% for PPS23F, significantly higher than both unselected B cells and PPS-specific B cells. There was no significant difference in switched memory B cell populations (CD27(+)IgM(-)) between groups. These results suggest a dominant role of IgM memory cells in the immune response to pneumococcal polysaccharides.

Combat pneumococcal infections: adhesins as candidates for protein-based vaccine development.

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the upper respiratory tract in humans. However, these apparently harmless bacteria have also a high virulence potential and are known as the etiologic agent of respiratory and life-threatening invasive diseases. Dissemination of pneumococci from the nasopharynx into the lungs or bloodstream leads to community-acquired pneumonia, septicaemia and meningitis. Traditionally, pneumococcal diseases are treated with antibiotics and prevented with polysaccharide-based vaccines. However, due to the dramatic increase in antibiotic resistance and limitations of the current available vaccines, the burden of diseases remains high. Thus, combating pneumococcal transmission and infections has emphasized the need for a new generation of protein-based vaccines. Interactions of pneumococci with soluble host proteins or cellular receptors are crucial for adherence, colonization, transmigration of host barriers and immune evasion. Therefore, surface-exposed proteins involved in these pathogenic processes and virtually expressed by all pneumococcal strains and serotypes are the prime potential targets for an immunogenic and highly protective pneumococcal-derived carrier protein of a vaccine. In this review, we will address the state of the art in deciphering, i). the conservation, distribution and pathogenic role of recently discovered pneumococcal adhesins in colonization and invasive diseases, ii). the interactions of these virulence factors with host-proteins and receptors, iii). the subversion of the host immune and cellular responses, and iv). the potential of pneumococcal adhesins as vaccine candidates.

Pneumococcal whole-cell vaccine: optimization of cell growth of unencapsulated Streptococcus pneumoniae in bioreactor using animal-free medium.

The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al- eryR was originally cultured in Todd-Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al- eryR and kanR, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al- kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al- kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.

Memory B cells in common variable immunodeficiency: clinical associations and sex differences.

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by impaired antibody responses, recurrent infections, inflammatory, autoimmune and malignancy-related conditions. We evaluated the relationship between memory B cell phenotype, sex, age at diagnosis, immunologic and clinical conditions in 105 CVID subjects from one medical center. Reduced numbers of switched memory B cells (cutoff

Evaluation of humoral immune function in patients with bronchiectasis.

Bronchiectasis is a chronic debilitating condition characterized by abnormal dilated thick-walled bronchi. To investigate humoral immune function in bronchiectatic patients, this study was performed. Forty patients with established diagnosis of bronchiectasis, who were referred from two tertiary care pulmonology centers in Tehran, were investigated in this study. Immunoglobulin isotypes concentrations and IgG-subclasses were measured by nephelometry and enzyme-linked immunosorbent assay (ELISA) methods, respectively. All patients received unconjugated pneumococcal vaccine, and blood samples were taken before and 21 days after vaccination. Specific antibodies against whole pneumococcal antigens were measured using the ELISA method. Fifteen (37.5%) out of 40 patients were diagnosed to have defects in antibody mediated immunity including 5 (12.5%) patients with immunoglobulin class deficiency (2 with common variable immunodeficiency and 3 with IgA deficiency), 3 (7.5%) with IgG subclass deficiency and 7 (17.5%) patients had Specific antibody deficiency (SAD) against polysaccharide antigen despite normal levels of serum immunoglobulins and IgG subclasses. Our study along with several other studies confirmed that all patients with bronchiectasis should undergo thorough immunological evaluation in order to identify the presence of the underlying immunologic defect. This evaluation should include serum immunoglobulins, IgG subclasses concentrations and also determination of serum antibodies against pneumococcal antigens. Early diagnosis and appropriate treatment will prevent the subsequent complications and improve quality of life of affected individuals.

Analysis of type II secretion of recombinant pneumococcal PspA and PspC in a Salmonella enterica serovar Typhimurium vaccine with regulated delayed antigen synthesis.

Recombinant attenuated Salmonella vaccines (RASVs) have been used extensively to express and deliver heterologous antigens to host mucosal tissues. Immune responses can be enhanced greatly when the antigen is secreted to the periplasm or extracellular compartment. The most common method for accomplishing this is by fusion of the antigen to a secretion signal sequence. Finding an optimal signal sequence is typically done empirically. To facilitate this process, we constructed a series of plasmid expression vectors, each containing a different type II signal sequence. We evaluated the utilities of these vectors by fusing two different antigens, the alpha-helix domains of pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC), to the signal sequences of beta-lactamase (bla SS), ompA, and phoA and the signal sequence and C-terminal peptide of beta-lactamase (bla SS+CT) on Asd(+) plasmids under the control of the P(trc) promoter. Strains were characterized for level of expression, subcellular antigen location, and the capacity to elicit antigen-specific immune responses and protection against challenge with Streptococcus pneumoniae in mice. The immune responses to each protein differed depending on the signal sequence used. Strains carrying the bla SS-pspA and bla SS+CT-pspC fusions yielded the largest amounts of secreted PspA and PspC, respectively, and induced the highest serum IgG titers, although all fusion proteins tested induced some level of antigen-specific IgG response. Consistent with the serum antibody responses, RASVs expressing the bla SS-pspA and bla SS+CT-pspC fusions induced the greatest protection against S. pneumoniae challenge.

Differential V gene expression detected in the immune response to Streptococcus pneumoniae capsular polysaccharide between elderly and young adults.

Streptococcus pneumoniae is one of the major causative agents of respiratory infections in the elderly population. The 23-valent pneumococcal polysaccharide vaccine is recommended for use in this age group. However, research has indicated that the protective efficacy of the vaccine declines with age. Although similar levels of antibody induction are seen in both young and elderly adults, following immunization with this vaccine, recent studies have indicated that the elderly possess antibodies with lower opsonophagocytic activity and avidity than young adults. We investigated whether a shift in V(H) gene usage may be responsible for this observation. To this end we utilized anti-idiotypic determinants to detect V(H)1 and V(H)3 gene usage by antibodies to pneumococcal capsular polysaccharides in both young and elderly subjects by enzyme-linked immunosorbent assay (ELISA). We found no significant difference in V(H)3 idiotypic expression in antibody responses to capsular polysaccharide from serotype 14 (PPS14). In response to PPS14 a significant higher level of V(H)1 idiotypic expressing antibodies was detected in the elderly as compared with young adults. V(H)1 idiotypic expression in response to capsular polysaccharide from serotype 4 (PPS4) was identical in young and elderly individuals. V(H)3 idiotypic expression in the elderly response to PPS4 was significantly lower than that seen in young individuals. These patterns of idiotypic expression are discussed in relation to recent studies of functional activity of pneumococcal reactive antibodies from young and aged humans.