Protection against nasal colonization with Streptococcus pneumoniae by parenteral immunization with a DNA vaccine encoding PspA (Pneumococcal surface protein A)

Pneumococcal surface protein A (PspA) is an important candidate for a cost-effective vaccine with broad coverage against Streptococcus pneumoniae. We have previously shown that intramuscular immunization with PspA as a DNA vaccine induces an immune response characterized by the induction of a balanced IgG1/IgG2a antibody response in BALB/c mice, which was able to efficiently mediate complement deposition onto intact bacteria and to induce protection against an intraperitoneal challenge. We now confirm the results in C57BL/6 mice and further show that the response induced by the DNA vaccine expressing PspA is able to mediate protection against colonization of the nasopharyngeal mucosa even though immunization was given parenterally. Moreover, a positive correlation was observed between IgG1 and the numbers of CFU recovered, whereas an inverse correlation was observed between nasal CFU levels and IgG2a. A positive correlation was also found for IgG1/IgG2a antibody ratios with CFU recovered from the nasopharynx. Therefore, reduction of nasal colonization was strongly associated with increased levels of serum IgG2a complement fixing antibody and low levels of IgG1 antibody which has much less complement fixing activity. Passive transfer of serum from animals immunized with the DNA vaccine expressing PspA was also able to reduce the fraction of mice with high density of colonization of the nasopharynx. Secretion of IFN-γ, but not IL-17, was observed in splenocytes from mice immunized with the DNA vaccine.     

Pneumococcal surface protein A (PspA) is effective at eliciting T cell-mediated responses during invasive pneumococcal disease in adults

Humoral immune response is essential for protection against invasive pneumococcal disease and this property is the basis of the polysaccharide-based anti-pneumococcal vaccines. Pneumococcal surface protein A (PspA), a cell-wall-associated surface protein, is a promising component for the next generation of pneumococcal vaccines. This PspA antigen has been shown to stimulate an antibody-based immunity. In the present study, we evaluated the capacity of PspA to stimulate CD4+ T cells which are needed for the correct development of a B cell based immune response in humans. Cellular immunity to PspA was evaluated by whole-blood culture with different pneumococcal antigens, followed by flow cytometric detection of activated CD4+CD25+ T cells. T cell-mediated immune responses to recombinant PspA proteins were assessed in acute-phase and convalescent blood from adults with invasive pneumococcal disease and in blood from healthy subjects. All cases had detectable antibodies against PspA on admission. We found that invasive pneumococcal disease induced transient T cell depletion but adaptive immune responses strengthened markedly during convalescence. The increased production of both interleukin (IL)-10 and interferon (IFN)-gamma during convalescence suggests that these cytokines may be involved in modulating antibody-based immunity to pneumococcal disease. We demonstrated that PspA is efficient at eliciting T cell immune responses and antibodies to PspA. This study broadens the applicability of recombinant PspA as potent pneumococcal antigen for vaccination against S. pneumoniae.     

E-cadherin is a receptor for the common protein pneumococcal surface adhesin A (PsaA) of Streptococcus pneumoniae

Streptococcus pneumoniae (Pnc) binds to nasopharyngeal (NP) epithelial cells in the first steps of nasopharyngeal carriage and colonization through bacterial adhesins. The pneumococcal surface adhesin A (PsaA) has previously been reported to play a significant role in pneumococcal adherence and colonization. Identification of a receptor for PsaA on human epithelium will aid in understanding the pathogenesis of this bacterium. Using recombinant PsaA covalently bound to fluorescent spheres (fluospheres), we show PsaA binds to NP cells through interaction with the human cellular receptor, E-cadherin. SDS-PAGE silver stain analysis demonstrates binding of PsaA to E-cadherin. Recombinant human E-cadherin binds to and blocks PsaA-coated fluospheres and whole transparent bacteria from adhering to NP cells, but does not block a Pnc PsaA(-) mutant. Recombinant E-selectin and human alpha(5)beta(1) integrin did not bind to or block PsaA-coated fluosphere adherence to NP cells. Likewise, if NP cells were preincubated with anti-E-cadherin antibody, there was a significant decrease (46%, P=0.05) in PsaA-coated fluosphere adherence to the cells. Additionally, when using E-cadherin transfected cells, we observed PsaA-coated fluospheres bind more efficiently to cells which express E-cadherin. This work identifies E-cadherin as a receptor on human epithelial cells for the pneumococcal surface adhesin, PsaA.     

Analysis of a surface protein of Streptococcus pneumoniae recognised by protective monoclonal antibodies

Using two monoclonal antibodies which protect mice from a fatal challenge with S. pneumoniae, we have identified a surface protein antigen on the pneumococcus. These antibodies recognised components of 84 and 76 kD in a cell wall extract of the nonencapsulated strain, R36A, against which they were made. Absorption experiments indicated that both of the antibodies recognised the same two proteins. The proteins detected by the antibodies in the encapsulated type 2 strain D39 and type 3 strain WU2, exhibited different molecular weights than those proteins detected from R36A. Using a colony blot procedure and a quantitative ELISA, we have shown that these antibodies react with 6 of the 21 pneumococcal strains tested. There was no association between reactivity with these anti-protein antibodies and the capsular serotype of the pneumococcal isolates tested.     

A functional epitope of the pneumococcal surface adhesin A activates nasopharyngeal cells and increases bacterial internalization

Pneumococcal surface adhesin A (PsaA) is a putative pneumococcal (Pnc) adhesin known to bind to nasopharyngeal (NP) epithelial cells. This study evaluated the effect of peptides within a functional domain of PsaA on NP cells. Detroit 562 NP cells were treated with synthetic peptides derived from PsaA (P4, P6, and P7; 28, 12, and 16 amino acids, respectively). The P4 peptide also binds to NP cells. Analysis of P4-treated NP cells by transmission electron microscopy revealed major cytological changes. Of 9 cytokines analyzed, a 6-fold increase in FGFb secretion at 3 and 6h (11-fold at 12h) was found post-P4 treatment of NP cells. There was a simultaneous reduction in the secreted levels of IL-6, IL-8, and VEGF. We observed enhancement in the adherence of Pnc strains to P4-treated NP cells (2-38-fold increase). Enhancement in adherence (2-fold increase) to P4-treated NP cells was also recorded with other streptococcal species (Streptococcus mitis and Streptococcus pyogenes). Internalization experiments demonstrated that 45% of the adherent bacteria were actually internalized after pretreatment with P4 peptide as compared to controls. Peptide fragments of P4, P6 and P7 did not activate NP cells to the extent of P4 peptide. The P4-mediated enhancement of Pnc adherence was blocked (100%) by anti-P4 antibodies, confirming the specificity of the P4 sequence for NP cell activation. Our data suggests that this functional domain of PsaA contained within the P4 sequence binds and activates NP cells to facilitate Pnc invasion.     

Anti-pneumococcal deficits of monocyte-derived macrophages from the advanced-age,frail elderly and related impairments in PI3K-AKT signaling

The advanced-age, frail elderly are especially vulnerable to developing pneumococcal infection and disease. Macrophages are critical mediators in the defence against Streptococcus pneumoniae at the upper respiratory tract, however, little is known of their anti-pneumococcal capacity in the elderly. Herein we demonstrate that monocyte-derived macrophages (MDMs) from the advanced-age, frail elderly produce less TNF, IL-6, IL-1β and IL-8 in response to heat-killed S. pneumoniae, which does not appear to be related to mRNA stability or decay. Furthermore, despite maintaining the ability to bind and phagocytose bacteria, MDMs from these individuals have a reduced capacity to kill S. pneumoniae. These defects parallel reduced PI3K-AKT signaling, which can significantly abrogate bacterial killing, but does not affect cytokine responses. Since macrophages are critical in the defence against S. pneumoniae, this study adds valuable insight into the susceptibility of the elderly to pneumococcal disease and highlights the PI3K-AKT signaling pathway as a potential therapeutic target.     

Molecular mechanisms of lipopolysaccharide-induced cyclooxygenase-2 expression in human neutrophils: involvement of the mitogen-activated protein kinase pathway and regulation by anti-inflammatory cytokines

Neutrophils are an important cellular source of proinflammatory mediators, whose regulation may be of potential benefit for the treatment of a number of inflammatory diseases. However, the mechanisms of lipopolysaccharide (LPS)-induced neutrophil activation and its regulation by anti-inflammatory cytokines have not yet been fully elucidated. Recent studies have revealed that mitogen-activated protein kinases (MAPK) play a crucial role in the generation of proinflammatory mediators in some cell types. Therefore, we conducted this study to determine whether MAPK activation could be involved in prostaglandin E(2) (PGE(2)) production and cyclooxygenase (COX)-2 expression in LPS-stimulated human neutrophils. PD98059 (MEK1 inhibitor) and SB203580 (p38(MAPK) inhibitor) reduced PGE(2) production as well as COX-2 expression in LPS-stimulated neutrophils. In addition, both extracellular signal-regulated protein kinase (ERK) and p38(MAPK) were phosphorylated and activated in time- and dose-dependent manners. Since we previously showed that IL-10 and IL-4 similarly inhibited COX-2 expression in LPS-stimulated neutrophils, we next tested the effects of IL-10 and IL-4 on the phosphorylation and activation of both kinases. IL-10 inhibited the phosphorylation and activation of p38(MAPK), but not ERK. In addition, IL-4 caused a marginal inhibition in the activation of p38(MAPK). Taken together, these results suggest that both ERK and p38(MAPK) pathways are involved in LPS-induced COX-2 expression and PGE(2) production in neutrophils, and IL-10 and IL-4 inhibit neutrophil prostanoid synthesis by down-regulating the activation of p38(MAPK).     

The role of Janus kinase 2 (JAK2) activation in pneumococcal EstA protein-induced inflammatory response in RAW 264.7 macrophages

In a previous study, we demonstrated pneumococcal EstA-induced inflammatory response through NF-κB and MAPK-dependent pathways. Herein, we tested the hypothesis that the Janus kinase 2 (JAK2) activation and associated signaling cascades may also be involved in EstA-induced inflammatory process in RAW 264.7 macrophages. Our immunoblot analysis indicated EstA-induced activation of JAK2, with the phosphorylated protein detected from 1 to 24 h post-stimulation. As type I interferon (IFN) signaling requires the JAK/STAT pathway, we investigated EstA-induced expression of INF-α4 and INF-β by semi-quantitative and quantitative RT PCR. Our results indicated both concentration- and time-dependent increases in both IFN-α4 and IFN-β mRNA expression after EstA challenge, with the highest fold-increases observed at 4 h and 6 h post-stimulation for IFN-α4 and IFN-β mRNA, respectively. Furthermore, we applied a pharmacological approach to demonstrate the effect of JAK2 inhibition on EstA-induced nitric oxide (NO) and pro-inflammatory cytokine production. The JAK2 inhibitor AG-490 reduced significantly (P < 0.05) EstA-induced NO production and the expression of iNOS mRNA in a concentration-dependent manner. Similarly, EstA-induced IL-1β and IL-6 production and their respective mRNA expression were markedly suppressed by AG-490. However, AG-490 had no inhibitory effect on both mRNA and protein levels of TNF-α. Taken together, we demonstrate that JAK2 activation and IFN I signaling are integral parts of EstA-induced inflammatory process. Further studies will elucidate the interaction of the different signaling pathways, the specific downstream targets of JAK2, the kinetics of cytokine release, and if EstA could induce the pro-inflammatory mediators to the same extent in alveolar macrophages.     

Interaction of pneumococcal phase variation,host and pressure/gas composition: Virulence expression of NanA,HylA, PspA and CbpA in simulated otitis media

Streptococcus pneumoniae, a leading cause of otitis media (OM), adapts to the host environment and undergoes spontaneous intra-strain phase variations in colony morphology. Transparent (T) phase variants are more efficient in colonizing the nasopharynx while the opaque (O) phase variants exhibit greater virulence during systemic infections. We recently demonstrated that T phase variants exhibited a higher growth rate and greater epithelial adherence and destruction than did O phase variants during interactions with human middle ear (ME) epithelial cells. This study was to delineate the underlying molecular mechanisms. Human ME epithelial cells were preconditioned for 24 h under one of the three simulated ME gas/pressure conditions designed to reflect those for 1) normal ME, 2) ME with Eustachian tube obstruction (ETO) and 3) ME with tympanostomy tube (TT) placement; subsequently exposed to a dose (˜10⁷ CFU/ml) of either T or O phase variants of S. pneumoniae (6A), and then incubated for 1 h and 3 h. Gene expressions coding for pneumococcal NanA, HylA, PspA, and CbpA virulence factors in inoculum, epithelium-attached and free-floating bacteria were assayed using real-time PCR. Result showed significantly higher basal expression levels for NanA and HylA in T inoculums than in O inoculum. Furthermore, striking differences between the two phase variants were observed in the forms of the inocula, significantly higher expression levels for PspA in T inoculum, but for CbpA in O inoculum. The TT condition enhanced the molecular activities of NanA, HylA, and PspA virulence factors in epithelium-attached T phase variants and fluid-floating O variants, followed by the ETO condition. Our study suggests that the pneumococcal virulence of the phase variations are modulated by the pathological ME environment and host–pathogen interaction during the pathogenesis of OM.     

The lack of Pneumococcal surface protein C (PspC) increases the susceptibility of Streptococcus pneumoniae to the killing by microglia

Microglial cells, the resident phagocytes in the brain, share many phenotypical and functional characteristics with peripheral macrophages, suggesting that they may participate in an innate immune response against microorganisms invading the central nervous system (CNS). In this study, we demonstrate that the microglial cells constitutively exhibit antibacterial activity in vitro against Streptococcus pneumoniae. By using a Pneumococcal surface protein C (PspC)-deleted strain and its wild-type counterpart, we found that the extent of such an activity is significantly influenced by the presence of a PspC molecule on the bacterial surface. The PspC– mutant FP20 is indeed more susceptible than the PspC+ strain HB565 to microglial killing. Interestingly, this phenomenon is observed when using a medium supplemented with heat-inactivated foetal bovine serum (FBS). Electron microscopy studies indicate that the microglial cells interact more efficiently with PspC– than with PspC+ pneumococci. Moreover, upon infection with the PspC– mutant, microglial cells produce levels of TNF-, MIP-2, IL-10 and nitric oxide, significantly higher than those observed with PspC+ bacteria. These findings indicate that the lack of PspC significantly enhances the susceptibility of S. pneumoniae to both bactericidal activity and secretory response by the microglial cells, suggesting that this molecule may play an important role in the invasion of CNS by pneumococcus.     

Nasopharyngeal pneumococcal carriage of children attending day care centers in Korea: comparison between children immunized with 7-valent pneumococcal conjugate vaccine and non-immunized

To confirm the effect of 7-valent pneumococcal conjugate vaccine (PCV7), pneumococcal nasopharyngeal (NP) carriage was compared between vaccinated (3 + 1 doses PCV7) and non-vaccinated children. Vaccinated subjects were recruited from highly vaccinated regions (≥ 60%), Seoul and Incheon whereas control subjects were recruited from Jeju Island where vaccination rates are low (< 15%). NP swabs were obtained from 400 children aged 18-59 months. Serotype and antibiotic susceptibility was analyzed. Pneumococcal carriage rate was 18.0% (36/200) and 31.5% (63/200) for the vaccinated and control group, respectively. Among those vaccinated, 41.7% (15/36) of the serotypes were vaccine-related type (VRT: 6A, 6C, 19A) with the most common serotype 6C. The next common type was non-typable/non-capsule 30.6% (11/36) followed by non-vaccine type 16.7% (6/36) and vaccine type (VT) serotypes were found in only 11.1% (4/36). In contrast, 52.4% (33/63) of the isolates in the control group were VT. Resistance rates for penicillin and erythromycin were lower in the vaccine group (vaccine vs control; penicillin 45.2% vs 71.4%, erythromycin 74.2% vs 90.5%, P < 0.05). Multi-drug resistance was also lower in vaccinated subjects (vaccine vs control; 45.2% vs 69.8%, P < 0.05). PCV7 reduces carriage in VT which leads to replacement of pneumococci by antibiotic susceptible VRT or non-vaccine type strains.     

Expression and secretion of chemotactic cytokines IL-8 and MCP-1 by human endothelial cells after Rickettsia rickettsii infection: regulation by nuclear transcription factor NF-kappaB

Infection of endothelial cells (EC) with Rickettsia rickettsii results in Rocky Mountain spotted fever, an acute illness characterized by systemic inflammation. Interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are important chemokines for activating neutrophils and monocytes, respectively, and recruiting these circulating immune cells to the sites of inflammation. In this study, we have measured the expression and secretion of these chemokines during R. rickettsii infection of cultured human EC. In comparison to uninfected controls, increased mRNA expression of IL-8 and MCP-1 in R. rickettsii-infected EC was evident as early as 3 h and was sustained up to 21 h. Subsequent analysis of culture supernatants revealed significantly enhanced secretion of both chemokines at 3, 8, and 18 h post-infection (5-28-fold increase in IL-8 and 4-16-fold increase in MCP-1). The presence of peptide-aldehyde compound MG132 to inhibit proteasome-mediated degradation of the inhibitory protein IkappaBalpha and synthetic peptide SN-50 to inhibit the nuclear translocation of nuclear factor-kappa B (NF-kappaB) resulted in significant inhibition of the chemokine response. Also, T24 cells expressing a super-repressor mutant of IkappaBalpha (to render NF-kappaB inactivatable) secreted significantly lower quantities of IL-8 than mock-transfected cells. A neutralizing antibody against IL-1alpha or an IL-1 specific receptor antagonist had no effect on the early phase of R. rickettsii-induced NF-kappaB activation and IL-8/ MCP-1 secretion at 3 h. Both of these treatments, however, diminished late-phase NF-kappaB activation by about 33% and only partially suppressed the infection-induced chemokine release at 21 h. Thus, while chemokine response early during the infection likely depends on the direct activation of NF-kappaB, subtle autocrine effects of newly synthesized IL-1alpha may contribute, in part, to the control of NF-kappaB activation and chemokine production at later times. These findings implicate a prominent role for host EC in recruiting immune cells to the site of inflammation during Rickettsia infection and provide important insights to further our understanding of the pathogenesis of spotted fever group rickettsioses.     

Retention of structure,antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles

Pneumococcal surface protein A (PspA) is a choline-binding protein which is a virulence factor found on the surface of all Streptococcus pneumoniae strains. Vaccination with PspA has been shown to be protective against a lethal challenge with S. pneumoniae, making it a promising immunogen for use in vaccines. Herein the design of a PspA-based subunit vaccine using polyanhydride nanoparticles as a delivery platform is described. Nanoparticles based on sebacic acid (SA), 1,6-bis-(p-carboxyphenoxy)hexane (CPH) and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), specifically 50:50 CPTEG:CPH and 20:80 CPH:SA, were used to encapsulate and release PspA. The protein released from the nanoparticle formulations retained its primary and secondary structure as well as its antigenicity. The released PspA was also biologically functional based on its ability to bind to apolactoferrin and prevent its bactericidal activity against Escherichia coli. When the PspA nanoparticle formulations were administered subcutaneously to mice they elicited a high titer and high avidity anti-PspA antibody response. Together these studies provide a framework for the rational design of a vaccine against S. pneumoniae based on polyanhydride nanoparticles.     

Interaction of pneumococcal phase variation and middle ear pressure/gas composition: an in vitro model of simulated otitis media

Streptococcus pneumoniae, a leading cause of otitis media (OM), undergoes spontaneous intra-strain variations in colony morphology. Transparent (T) variant is more efficient in colonizing the nasopharynx while the opaque (O) variant exhibits greater virulence during systemic infections. We hypothesized that changes in middle ear (ME) gas pressure/composition during Eustachian tube (ET) dysfunction and the treatment of that dysfunction, e.g., tympanostomy tube (TT) insertion, play a role in selecting the S. pneumoniae variant that can efficiently colonize/infect the ME mucosa. Human ME epithelial cells were preconditioned for 24h under one of three conditions that simulated (1) normal ME, (2) ME with ET obstruction (ETO) and (3) ME with TT; subsequently exposed to a dose (approximately 10(7)CFU/ml) of either T or O variant of S. pneumoniae, and then incubated for 1h and 3h. Under the simulated ETO and TT conditions, T variant exhibited a higher growth rate and greater epithelial adherence and killing than did O variants. Attachment of T variant to epithelial cells was documented by scanning electron microscopy. These results suggest that the T variant is more highly adapted to various ME environments than the O variants.     

Changes in serotype distribution and antibiotic resistance of nasopharyngeal isolates of Streptococcus pneumoniae from children in Korea, after optional use of the 7-valent conjugate vaccine

We investigated serotype distribution and antimicrobial resistance of pneumococcal carriage isolates from children after optional immunization with the 7-valent pneumococcal conjugate vaccine (PCV7) in Korea. From June 2009 to June 2010, 205 (16.5%) pneumococcal isolates were obtained from 1,243 nasopharyngeal aspirates of infants and children at Seoul National University Children's Hospital, Korea. Serotype was determined by Quellung reaction and antibiotic susceptibility was tested by E-test. The results were compared to previous studies done in the pre-PCV7 period. In this study, the most common serotypes were 6A (15.3%), 19A (14.7%), 19F (10.2%), 35B (7.3%), and 6D (5.6%). The proportion of PCV7 serotypes decreased from 61.9% to 23.8% (P < 0.001). The overall penicillin nonsusceptibility rate increased from 83.5% to 95.4% (P = 0.001). This study demonstrates the impact of optional PCV7 vaccination in Korea; the proportion of all PCV7 serotypes except 19F decreased while antimicrobial resistant serotypes 6A and 19A further increased.     

Role of tau kinases (CDK5R1 and GSK3B) in Parkinson's disease: a study from India

Glycogen synthase kinase-3β (GSK3B) and cyclin-dependent kinase 5 (CDK5) are the 2 major protein kinases involved in abnormal phosphorylation of tau. To determine their potential role in the pathogenesis of Parkinson's disease (PD) we analyzed 2 functional single nucleotide polymorphisms (SNPs) of GSK3B (rs334558 and rs6438552) and rs735555 of CDK5 regulatory subunit 1 (CDK5R1) in 373 PD cases and 346 healthy controls of eastern India. The C,C and T,C haplotypes of GSK3B were respectively moderately associated with increased risk and protection for late onset PD (LOPD) (odds ratio [OR], 1.399; 95% confidence interval [CI], 1.069-1.829; p = 0.015, and OR, 0.436; 95% CI, 0.222-0.853; p = 0.016, respectively). Moreover, moderate to significant interaction between different loci were observed for the entire PD cohort or late onset PD only. However, among these interactions, individuals carrying the (C/C) genotype at both loci (rs6438552 and rs735555) had almost twice the risk of developing PD than those without this genotypic combination (OR, 1.871; 95% CI, 1.181-2.964; p = 0.009). Thus, synergistic effect between the 2 major tau kinases, through these SNPs, appears to determine the risk profile for PD.