Dectin-2-dependent NKT cell activation and serotype-specific antibody production in mice immunized with pneumococcal polysaccharide vaccine.
ABSTRACT: Although thymus-independent type 2 antigens generally do not undergo Ig class switching from IgM to IgG, pneumococcal polysaccharide vaccine (PPV) induces the production of serotype-specific IgG. How this happens remains unclear, however. In the present study, PPV immunization induced production of IgG as well as IgM specific for a serotype 3-pneumococcal polysaccharide in the sera of wild-type (WT) mice, but this phenomenon was significantly reduced in Dectin-2 knockout (KO) mice. Immunization with PPV caused IL-12p40 production in WT mice, but this response was significantly reduced in Dectin-2KO mice. Likewise, immunization with PPV activated natural killer T (NKT) cells in WT mice but not in Dectin-2KO mice. Furthermore, administration of ?-galactosylceramide, recombinant (r)IL-12 or rIFN-? improved the reduced IgG levels in Dectin-2KO mice, and treatment with neutralizing anti-IFN-? mAb resulted in the reduction of IgG synthesis in PPV-immunized WT mice. Transfer of spleen cells from PPV-immunized WT mice conferred protection against pneumococcal infection on recipient mice, whereas this effect was cancelled when the transferred spleen cells were harvested from PPV-immunized Dectin-2KO mice. These results suggest that the detection of PPV antigens via Dectin-2 triggers IL-12 production, which induces IFN-? synthesis by NKT cells and subsequently the production of serotype-specific IgG.
Project description:Dectin-2 is a C-type lectin receptor that recognizes high mannose polysaccharides. Cryptococcus neoformans, a yeast-form fungal pathogen, is rich in polysaccharides in its cell wall and capsule. In the present study, we analyzed the role of Dectin-2 in the host defense against C. neoformans infection. In Dectin-2 gene-disrupted (knockout) (Dectin-2KO) mice, the clearance of this fungus and the inflammatory response, as shown by histological analysis and accumulation of leukocytes in infected lungs, were comparable to those in wild-type (WT) mice. The production of type 2 helper T (Th2) cytokines in lungs was higher in Dectin-2KO mice than in WT mice after infection, whereas there was no difference in the levels of production of Th1, Th17, and proinflammatory cytokines between these mice. Mucin production was significantly increased in Dectin-2KO mice, and this increase was reversed by administration of anti-interleukin 4 (IL-4) monoclonal antibody (MAb). The levels of expression of ?1-defensin, cathelicidin, surfactant protein A (Sp-A), and Sp-D in infected lungs were comparable between these mice. In in vitro experiments, IL-12p40 and tumor necrosis factor alpha (TNF-?) production and expression of CD86 and major histocompatibility complex (MHC) class II by bone marrow-derived dendritic cells and alveolar macrophages were completely abrogated in Dectin-2KO mice. Finally, the disrupted lysates of C. neoformans, but not of whole yeast cells, activated Dectin-2-triggered signaling in an assay with nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing this receptor. These results suggest that Dectin-2 may oppose the Th2 response and IL-4-dependent mucin production in the lungs after infection with C. neoformans, and it may not be required for the production of Th1, Th17, and proinflammatory cytokines or for clearance of this fungal pathogen.
Project description:BACKGROUND:Both HIV positivity and African American (AA) ethnicity are associated with increased incidence of invasive pneumococcal disease (IPD). Poor immune response to pneumococcal polysaccharide-based vaccines may contribute to the race related increased frequency of IPD in African American HIV positive individuals. METHODS:Caucasian and AA HIV-infected (HIV+) individuals 40-65?years old with CD4+ T cells/µl (CD4) >200 on antiretroviral therapy (ART) received either the 13-valent pneumococcal conjugate vaccine (PCV) followed by the 23-valent pneumococcal polysaccharide vaccine (PPV) or PPV only. Serum IgG, IgM and opsonophagocytic antibody responses to serotypes 14 and 23F as well as serum IgG and opsonophagocytic antibody responses to serotype 19A were measured pre- and post-vaccination. We measured serum markers of inflammation in all participants and performed single cell gene expression profiling at the baseline by HD Biomark in Caucasians and African Americans. RESULTS:There were no significant differences in pre-immunization inflammatory markers or post-vaccination IgG and IgM concentrations between Caucasian and African American participants. However, we found significantly lower opsonophagocytic activity in response to serotypes 14 and 19A in the AA group compared to the Caucasian group. There was no association between inflammatory markers and immune response to vaccination, however we found extensive biomodal variation in gene expression levels in single IgM+ memory B cells. Differentially expressed genes may be related to differences in the immune response between ethnic groups. CONCLUSIONS:Distinct racial differences were found in the functional immune response following either PPV and/or PCV/PPV immunization in HIV-positive adults, although these differences were serotype dependent. Decreased ability to respond to vaccination may in part explain racial disparities in pneumococcal disease epidemiology. ClinicalTrials.gov ID: NCT03039491.
Project description:<label>BACKGROUND</label>Streptococcus pneumoniae is a respiratory pathogen causing severe lung infection that may lead to complications such as bacteremia. Current polysaccharide vaccines have limited serotype coverage and therefore cannot provide maximal and long-term protection. Global efforts are being made to develop a conserved protein vaccine candidate. PrtA, a pneumococcal surface protein, was identified by screening a pneumococcal genomic expression library using convalescent patient serum. The prtA gene is prevalent and conserved among S. pneumoniae strains. Its protective efficacy, however, has not been described. Mucosal immunization could sensitize both local and systemic immunity, which would be an ideal scenario for preventing S. pneumoniae infection.<label>METHODS</label>We immunized BALB/c mice intranasally with a combination of a PrtA fragment (amino acids 144-1041) and Th17 potentiated adjuvant, curdlan. We then measured the T-cell and antibody responses. The protective efficacy conferred to the immunized mice was further evaluated using a murine model of acute pneumococcal pneumonia and pneumococcal bacteremia.<label>RESULTS</label>There was a profound antigen-specific IL-17A and IFN-? response in PrtA-immunized mice compared with that of adjuvant control group. Even though PrtA-specific IgG and IgA titer in sera was elevated in immunized mice, only a moderate IgA response was observed in the bronchoalveolar lavage fluid. The PrtA-immunized antisera facilitated the activated murine macrophage, RAW264.7, to opsonophagocytose S. pneumoniae D39 strain; however, PrtA-specific immunoglobulins bound to pneumococcal surfaces with a limited potency. Finally, PrtA-induced immune reactions failed to protect mice against S. pneumoniae-induced acute pneumonia and bacterial propagation through the blood.<label>CONCLUSIONS</label>Immunization with recombinant PrtA combined with curdlan produced antigen-specific antibodies and elicited IL-17A response. However, it failed to protect the mice against S. pneumoniae-induced infection.
Project description:BACKGROUND:While the 23-valent pneumococcal polysaccharide vaccine (PPV23) is routinely used in Canada and some other countries to prevent pneumococcal infection in adults with chronic kidney disease (CKD), patients develop a suboptimal antibody response to PPV23 due to their immune dysfunction. The 13-valent pneumococcal conjugate vaccine (PCV13) has superior immunogenicity in some categories of immunocompromised adults; however, its effect on the immune response in CKD patients has only been addressed by two recent studies with conflicting results. The effect of PPV23 or PCV13 on B cells in these patients has not been previously studied. We studied the absolute numbers and proportions of B cells and subpopulations in two groups of adult patients with severe CKD pre- and 7?days post-immunization with PCV13: pneumococcal vaccine naïve and previously immunized with PPV23 (over one year ago). RESULTS:PPV23 immunized patients had significantly lower proportions and absolute numbers of class switched memory (CD19?+?CD27?+?IgM-), as well as lower absolute numbers of IgM memory (CD19?+?CD27?+?IgM+) and class switched B cells (CD19?+?CD27-IgM-) compared to PPV23 naïve patients. Following PCV13 immunization, the differences in absolute numbers of B-cell subpopulations between groups remained significant. The PPV23 immunized group had higher proportions of CD5- B cells along with lower proportions and absolute numbers of CD5+ B cells compared to PPV23 naïve patients both pre- and post-immunization with PCV13. However, previous PPV23 immunization did not have a noticeable effect on the numbers of total IgG or serotype 6B and 14 specific antibody-secreting cells detected 7?days post-immunization with PCV13. Nevertheless, fold increase in anti-serotype 14 IgG concentrations 28?days post-PCV13 was greater in PPV23 naïve than in previously immunized patients. CONCLUSIONS:The results suggest that immunization with PPV23 may result in long-term changes in B-cell subpopulations such as increased prevalence of CD5- B cells and decreased prevalence of class switched memory B cells in the peripheral blood. Because previous immunization with PPV23 in patients with CKD is associated with a significant decrease in the total class switched memory B cells in response to subsequent immunization with PCV13, this may reduce PCV13 immunogenicity in the setting of PPV23 followed by PCV13. TRIAL REGISTRATION:Registered February 24, 2015 at ClinicalTrials.gov (NCT02370069).
Project description:The efficacy of different vaccines in protecting elderly individuals against Streptococcus pneumoniae infections is not clear. In the current study, aged mice (22-25 months old) exhibited significantly increased susceptibility to respiratory infection with serotype 3 S. pneumoniae relative to younger adult mice, regardless of whether mice were naive or immunized with native pneumococcal polysaccharide (PPS; Pneumovax23) or protein-PPS conjugate (Prevnar-13) vaccines. Nonetheless, Pneumovax-immunized aged mice developed limited bacteremia following respiratory challenge and exhibited significantly increased survival following systemic challenge relative to Prevnar-immune aged mice and young mice that had received either vaccine. This was explained by >10-fold increases in PPS-specific immunoglobulin G (IgG) levels in Pneumovax-immunized aged mice relative to other groups. Remarkably, PPS3-specific B-cell expansion, IgG switching, plasmablast differentiation, and spleen and bone marrow antibody-secreting cell frequencies were 10-fold higher in aged mice following Pneumovax immunization relative to young mice, due to significantly increased B-1b cell participation. In summary, this study highlights (1) the need to devise strategies to enhance respiratory immunity in aged populations, (2) the diverse responses young and aged populations generate to Pneumovax vs Prevnar vaccines, and (3) the potential value of exploiting B-1b cell responses in aged individuals for increased vaccine efficacy.
Project description:A promising alternative vaccine candidate to reduce the burden of pneumococcal diseases is the protein antigen PspA (Pneumococcal surface protein A). Since concomitant colonization with two or more pneumococcal strains is very common in children, we aimed to determine if immunization with PspA would be able to control co-colonization. We evaluated nasal immunization with recombinant PspA (rPspA) in a model of co-colonization with two strains expressing different PspAs. Mice were immunized intranasally with rPspAs from clades 1 to 4 (rPspA1, rPspA2, rPspA3 or rPspA4) using whole-cell pertussis vaccine (wP) as adjuvant. Mice were then challenged with a mixture of two serotype 6B isolates St491/00 (PspA1) and St472/96 (PspA4). Immunization with rPspA1+wP and rPspA4+wP reduced colonization with both strains and the mixture of rPspA1+rPspA4+wP induced greater reduction than a single antigen. Immunization rPspA1+rPspA4+wP also reduced colonization when challenge experiments were performed with a mixture of isolates of serotypes 6B (PspA3) and 23F (PspA2). Furthermore, none of the tested formulations led to a pronounced increase in colonization of one isolate over the other, showing that the vaccine strategy would not favor replacement. Interestingly, the adjuvant wP by itself already led to some reduction in pneumococcal colonization, indicating the induction of non-specific immune responses. Anti-rPspA IgG was observed in serum, nasal wash (NW) and bronchoalveolar lavage fluid (BALF) samples, whereas animals inoculated with formulations containing the adjuvant wP (with or without rPspA) showed higher levels of IL-6 and KC in NW and increase in tissue macrophages, B cells and CD4+T cells in BALF.
Project description:Infections with Streptococcus pneumoniae cause substantial morbidity and mortality, particularly in children in developing nations. Polysaccharide-conjugate vaccines provide protection against both invasive disease and colonization, but their use in developing countries is limited by restricted serotype coverage and expense of manufacture. Using proteomic screens, we recently identified several antigens that protected mice from pneumococcal colonization in a CD4(+) T cell- and interleukin-17A (IL-17A)-dependent manner. Since several of these proteins are lipidated, we hypothesized that their immunogenicity and impact on colonization are in part due to activation of Toll-like receptor 2 (TLR2), a receptor for lipoproteins. Here we show that lipidated versions of the antigens elicited significantly higher activation of both human embryonic kidney cells engineered to express TLR2 (HEK-TLR2) and wild-type (WT) murine macrophages than nonlipidated mutant antigens. Lipoprotein-stimulated secretion of proinflammatory cytokines was ?10× to ?100× lower in murine TLR2-deficient macrophages than in WT macrophages. Subcutaneous immunization of C57BL/6 mice with protein subunit vaccines containing one or two of these lipoproteins or protein fusion constructs bearing N-terminal lipid adducts elicited a robust IL-17A response and a significant reduction in colonization compared with immunization with alum alone. In contrast, immunization of Tlr2(-/-) mice elicited no detectable IL-17A response and no protection against pneumococcal colonization. These experiments suggest that the lipid moieties enhance the immunogenicity and protective efficacy of pneumococcal TH17 antigens through activation of TLR2. Thus, triggering TLR2 with an antigen-specific protein subunit formulation is a possible strategy for the development of a serotype-independent pneumococcal vaccine that would reduce pneumococcal carriage.
Project description: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.
Project description:Background and Aim: Recently, the 23-valent IgG-assay was suggested as screening assay to identify poor responders to pneumococcal polysaccharide (PnPS)-vaccination with the serotype-specific assay as a second-line test. However, in a low pre-test probability general hospital setting predicting good responders could be more valuable to reduce the number of samples needing serotyping. Methods: Serotype-specific PnPS antibody-assays were performed for suspected immunodeficiency in two Dutch general hospitals (Jeroen Bosch Hospital, 's-Hertogenbosch; Elisabeth Tweesteden Hospital, Tilburg). 23-Valent PnPS antibody-assays were subsequently performed in archived material. Data were analyzed using receiver operating characteristic curves (AUC) and agreement indices (ICC). Results: Sera of 284 patients (348 samples) were included; 23-valent IgG-titres and the corresponding sum of PnPS-serotype specific antibodies showed moderate correlation (ICC = 0.63). In 232 conjugated-pneumococcal-vaccine-naïve patients (270 samples), a random 23-valent IgG-titer could discriminate between samples with and without ?7/11, ?7/13, or ?6/9 pneumococcal serotypes when both cut-off values 0.35 and 1.0 ?g/ml were used (AUC 0.86 and 0.92, respectively). All patients with a pre-immunization-titer ?38.2 ?g/ml and/or post-immunization-titer ?96.1 ?g/ml and none with a post-immunization-titer ?38.5 ?g/ml exhibited a good response to PnPS vaccination. Using these breakpoints as screening test to predict good responders, only 24% of patients would require further serotyping, as opposed to 68% if breakpoints to predict poor responders would have been used. Conclusion: In a low pre-test probability setting, the 23-valent IgG-assay proved to be a reliable screening test for good responders in conjugated-pneumococcal-vaccine-naïve patients, reducing the overall number of patient samples needing further serotyping, thus reducing overall costs of pneumococcal vaccination response assessment.
Project description:Pneumococcal surface protein A (PspA) is a candidate antigen for the composition of protein-based vaccines against Streptococcus pneumoniae. While searching for efficient adjuvants for PspA-based vaccines, our group has described the potential of combining PspA with the whole-cell pertussis vaccine (wP). When given to mice through the nasal route, a formulation composed of PspA from clade 5 (PspA5) and wP (PspA5-wP) induced high levels of antibodies and protection against challenges with different pneumococcal strains. PspA5-wP also induced the secretion of interleukin 17 (IL-17) by splenocytes and the infiltration of leukocytes in the lungs after challenge. Here, we show that protection against a pneumococcal invasive challenge was completely abrogated in ?MT(-/-) mice, which are deficient in the maturation of B cells, illustrating the importance of antibodies in the survival elicited by the PspA5-wP vaccine. Moreover, passive immunization showed that IgG purified from the sera of mice immunized with PspA5-wP conferred significant protection to naive mice, whereas the respective F(ab')2 did not. Additionally, in vivo depletion of complement abolished protection against the pneumococcal challenge. The combination of PspA5 with wild-type or mutant Bordetella pertussis strains or with purified components showed that the pertussis toxin (PT)-containing formulations induced the highest levels of antibodies and protection. This suggests that the adjuvant activity of wP in the PspA5 model is mediated at least in part by PT. The sera from mice immunized with such formulations displayed high IgG binding and induction of complement deposition on the pneumococcal surface in vitro, which is consistent with the in vivo results.