anti-PspA Response Research Articles

A preliminary study on the application of PspA as a carrier for group A meningococcal polysaccharide.

This study aimed to explore the feasibility of pneumococcal surface protein A (PspA) as a carrier protein. Three recombinant pneumococcal surface proteins from three different clades were expressed by the prokaryotic expression system and conjugated to group A meningococcal polysaccharide (GAMP) to generate three polysaccharide-protein conjugates. The conjugates, unconjugated proteins, GAMP, and GAMP-TT vaccine bulk (used as positive control) were immunized into mice, and their immune effects were assessed by the methods of enzyme-linked immunosorbent assay (ELISA), flow cytometry (FCM), and serum bactericidal assay (SBA). The results showed that the polysaccharide-protein conjugates could produce higher levels of anti-GAMP IgG titers (P < 0.05), higher ratios of Th1/Th2 (P < 0.05), and higher levels of serum bactericidal activity (P < 0.05), compared with the unconjugated GAMP. The conjugation of PspAs to GAMP also enhanced the anti-PspA responses compared with unconjugated PspAs except for PspA3. In conclusion, the results indicated that the three PspAs were appropriate carrier proteins, as demonstrated by the characteristics of T-cell dependent responses to the GAMP, and might protect against group A of epidemic cerebrospinal meningitis.

A bivalent conjugate vaccine containing PspA families 1 and 2 has the potential to protect against a wide range of Streptococcus pneumoniae strains and Salmonella Typhi

Previously we showed that conjugation of pneumococcal surface protein A (PspA) to Vi capsular polysaccharide from Salmonella Typhi enhanced the anti-PspA response without the need to add adjuvant. In the current study conjugates consisting of the α helical regions of PspA families 1 or 2 bound to Vi were used to vaccinate mice to test their ability to protect against a lethal intravenous challenge of a range of various strains of Streptococcus pneumoniae. Conjugate vaccine containing PspA family 1 provided good protection from PspA family 1 challenge strains but offered very little protection against PspA family 2 challenge strains. Similarly, PspA family 2 conjugates provided good protection from PspA family 2 challenge strains and poor protection against PspA family 1 challenge strains. This observation was supported by the low levels of cross-reactivity of PspA antibodies seen in ELISA plates coated with the heterologous PspA family. Cytokine profiles showed a mixed Th1/Th2 response to Vi and the Vi-PspA conjugates. IgG subclass analysis of the anti-Vi response showed a shift from predominantly IgG2a/3 to IgG1 after conjugation to PspA was consistent with other polysaccharide conjugate vaccines. The results demonstrate that conjugation of the α helical region of PspA to Vi enhances its capacity to induce a protective immune response and that a vaccine based on the α helical region of PspA should contain PspA from both families 1 and 2 to achieve broad cross-protection.

Preparation and testing of a Vi conjugate vaccine using pneumococcal surface protein A (PspA) from Streptococcus pneumoniae as the carrier protein

In the current study pneumococcal surface protein A (PspA) was conjugated to Vi capsular polysaccharide from Salmonella Typhi to make available a vaccine against typhoid fever that has the potential to also provide broad protection from Streptococcus pneumoniae. High yielding production processes were developed for the purification of PspAs from families 1 and 2. The purified PspAs were conjugated to Vi with high recovery of both Vi and PspA. The processes developed especially for PspA family 2 could readily be adapted for large scale production under cGMP conditions. Previously we have shown that conjugation of diphtheria toxoid (DT) to Vi polysaccharide improves the immune response to Vi but can also enhance the response to DT. In this study it was shown that conjugation of PspA to Vi enhanced the anti-PspA response and that PspA was a suitable carrier protein as demonstrated by the characteristics of a T-cell dependent response to the Vi. We propose that a bivalent vaccine consisting of PspA from families 1 and 2 bound to Vi polysaccharide would protect against typhoid fever and has the potential to also protect against pneumococcal disease and should be considered for use in developing countries.

Macrophages Pulsed withStreptococcus pneumoniaeElicit a T Cell-Dependent Antibody Response upon Transfer into Naive Mice

Macrophages are less effective than DC at priming naive CD4(+) T cells, suggesting that DC are unique in initiating T cell-dependent Ab responses. We compared the ability of DC and macrophages, pulsed in vitro with Streptococcus pneumoniae, to elicit protein- and polysaccharide-specific Ig isotype production upon adoptive transfer into naive mice. S. pneumoniae-activated DC secreted more proinflammatory and anti-inflammatory cytokines, expressed higher levels of surface MHC class II and CD40, and presented S. pneumoniae or recombinant pneumococcal surface protein A (PspA) to a PspA-specific T hybridoma more efficiently than macrophages. However, upon adoptive transfer into naive mice, S. pneumoniae-pulsed macrophages elicited an IgM or IgG anti-PspA and anti-polysaccharide response comparable in serum titers and IgG isotype distribution to that induced by DC. The IgG anti-PspA response, in contrast to the IgG anti-polysaccharide, to S. pneumoniae-pulsed macrophages was T cell-dependent. S. pneumoniae-pulsed macrophages that were paraformaldehyde-fixed before transfer or lacking expression of MHC class II or CD40 were highly defective in eliciting an anti-PspA response, although the anti-polysaccharide response was largely unaffected. To our knowledge, these data are the first to indicate that macrophages can play an active role in the induction of a T cell-dependent humoral immune response in a naive host.

4-1BB (CD137) differentially regulates murine in vivo protein- and polysaccharide-specific immunoglobulin isotype responses to Streptococcus pneumoniae.

4-1BB (CD137) is induced on activated CD4(+) and CD8(+) T cells and delivers a costimulatory signal upon binding the 4-1BB ligand (4-1BBL) expressed on antigen-presenting cells. Induction of 4-1BB is dependent on activation via the T-cell receptor (TCR) and possibly CD28. It was previously demonstrated that both an in vivo protein (pneumococcal surface protein A [PspA])- and polysaccharide (phosphorylcholine [PC] determinant of teichoic acid)-specific immunoglobulin (Ig) isotype response to Streptococcus pneumoniae was dependent on CD4(+) TCRalphabeta(+) T cells and B7-dependent costimulation through CD28. We thus postulated that 4-1BB costimulation would also play a role in regulating the in vivo anti-PspA and anti-PC response to S. pneumoniae. We demonstrate that mice genetically deficient in 4-1BBL elicit a markedly reduced IgM and IgG anti-PC but normal primary and secondary IgG anti-PspA responses to S. pneumoniae relative to those for wild-type mice. However, injection of an agonistic anti-4-1BB monoclonal antibody (MAb), while having no significant effect on the anti-PC response, strongly inhibits the primary anti-PspA response, the generation of PspA-specific memory, and germinal center formation but does not induce a lasting state of tolerance. In contrast, anti-4-1BB MAb has no effect on the anti-PspA response when injected only at the time of secondary immunization. Delay of the addition of anti-4-1BB leads to progressively less inhibition of the primary response up to day 8. This inhibition is independent of CD8(+) T cells and is associated with the expansion of CD4(+) T cells with an activated phenotype, which is partly dependent on B7-dependent costimulation. These data are the first to suggest a stimulatory role for endogenous 4-1BB-4-1BBL interactions during a humoral immune response to a pathogen and further underscore significant differences in costimulation requirements for an in vivo protein- versus polysaccharide-specific Ig isotype response to an extracellular bacterium.

The mechanism underlying T cell help for induction of an antigen-specific in vivo humoral immune response to intact Streptococcus pneumoniae is dependent on the type of antigen.

Little is known concerning the role of T cells in regulating an anti-polysaccharide Ig response to an intact pathogen. We previously reported that the in vivo Ig responses to Streptococcus pneumoniae (strain R36A), specific for pneumococcal surface protein A (PspA) and for the phosphorylcholine (PC) determinant of C-polysaccharide, were both dependent on TCR-alphabeta(+) T cells and B7-dependent costimulation, although only PspA-specific memory was generated. In this report, we show that the T cell help underlying these two Ag-specific Ig responses is distinct. Using H-Y-specific T cell transgenic mice made "nonleaky" by crossing with mice genetically deficient for TCR-alpha, we demonstrate that the T cell help for the anti-PC, in contrast to the anti-PspA, response is TCR-nonspecific and occurs normally in the absence of germinal center formation, although it is still dependent on B7-dependent costimulation. Consistent with these data, we demonstrate, using cathepsin S(-/-) mice, that although the anti-PC response is largely dependent on CD4(+) T cells, there is a reduced (or lack of) dependence, relative to the anti-PspA response, on the generation of new peptide-MHC class II complexes. In this regard, the T cell help for an optimal anti-PC response is delivered more rapidly than that required for an optimal anti-PspA response. Collectively, these data demonstrate a novel accelerated TCR-nonspecific B7-dependent form of T cell help for augmenting a polysaccharide-specific Ig response to an intact bacterium without the generation of memory.

Endogenous pro- and anti-inflammatory cytokines differentially regulate an in vivo humoral response to Streptococcus pneumoniae.

Proinflammatory cytokines play a critical role in innate host defense against extracellular bacteria. However, little is known regarding the effects of these cytokines on the adaptive humoral response. Mice injected with a neutralizing anti-tumor necrosis factor alpha (TNF-alpha) monoclonal antibody (MAb) at the time of primary immunization with intact Streptococcus pneumoniae (strain R36A) showed a substantial reduction in both the primary immunoglobulin G (IgG) response specific for the cell wall protein, pneumococcal surface protein A (PspA), as well as in the development of PspA-specific memory. In contrast, anti-TNF-alpha MAb injected only at the time of secondary immunization with R36A failed to alter the boosted anti-PspA response. TNF-alpha was required only within the first 48 to 72 h after primary immunization with R36A and was induced both by non-B and non-T cells and by lymphoid cells, within 2 to 6 h after immunization, with levels returning to normal by 24 h. Thus, the early innate release of TNF-alpha was critical for optimal stimulation of the subsequent adaptive humoral response to R36A. Additional proinflammatory (interleukin 1 [IL-1], IL-6, IL-12, and gamma interferon [IFN-gamma]) as well as anti-inflammatory (IL-4 and IL-10) cytokines were also transiently induced. Mice genetically deficient in IL-6, IFN-gamma, or IL-12 also showed a reduced IgG anti-PspA response of all IgG isotypes. In contrast, IL-4(-/-) and IL-10(-/-) mice immunized with R36A showed a significant elevation in the IgG anti-PspA response, except that there was decreased IgG1 in IL-4(-/-) mice. In this regard, a marked enhancement in the induction of proinflammatory cytokines was observed in the absence of IL-10, relative to controls. Ig isotype titers specific for the phosphorycholine determinant of C-polysaccharide were similarly regulated, but to a much more modest degree. These data suggest that proinflammatory and anti-inflammatory cytokines differentially regulate an in vivo protein- and polysaccharide-specific Ig response to an extracellular bacteria.

Enhanced protective antibody responses to PspA after intranasal or subcutaneous injections of PspA genetically fused to granulocyte-macrophage colony-stimulating factor or interleukin-2.

Antibody to pneumococcal surface protein A (PspA) has been shown to be protective for Streptococcus pneumoniae infections in mice. In an attempt to define a model for inducing protective antibody to PspA in the absence of adjuvant, we designed two genetic fusions, PspA-interleukin-2 [IL-2]) and PspA-granulocyte-macrophage colony-stimulating factor (GM-CSF). These constructs maintained high cytokine function in vitro, as tested by their activity on IL-2 or GM-CSF-dependent cell lines. While intranasal immunization with PspA induced no detectable anti-PspA response, both PspA-IL-2 and PspA-GM-CSF stimulated high immunoglobulin G1 (IgG1) antibody responses. Interestingly, only the PspA-IL-2, not the PspA-GM-CSF, construct stimulated IgG2a antibody responses, suggesting that this construct directed the response along a TH1-dependent pathway. Comparable enhancement of the anti-PspA response with similar isotype profiles was observed after subcutaneous immunization as well. The enhancement observed with PspA-IL-2 was dependent on IL-2 activity in that it was not seen in IL-2 receptor knockout mice, while PspA in alum induced high-titer antibody in these mice. The antibody was tested for its protective activity in a mouse lethality model using S. pneumoniae WU-R2. Passive transfer of 1:90 dilutions of sera from mice immunized with PspA-IL-2 and PspA-GM-CSF elicited protection of CBA/N mice against intravenous challenge with over 170 50% lethal doses of capsular type 3 strain WU2. Only 0.17 microg or less of IgG antibody to PspA was able to provide passive protection against otherwise fatal challenge with S. pneumoniae. The data demonstrate that designing protein-cytokine fusions may be a useful approach for mucosal immunization and can induce high-titer systemic protective antibody responses.

Oral immunization with PspA elicits protective humoral immunity against Streptococcus pneumoniae infection

Streptococcus pneumoniae is a major respiratory mucosal pathogen affecting infants and children. Although a polysaccharide-based vaccine has been useful in adult populations, it does not elicit protective immunity in infants and young children. Pneumococcal surface protein A (PspA) is a highly immunogenic surface protein produced by all strains of Streptococcus pneumoniae. Previous studies have shown that systemic immunization of mice with PspA can elicit protective immunity against fatal pneumococcal infection. In this study, we demonstrated that oral immunization with PspA could elicit protective immune responses against pneumococcal infection. When mice were orally immunized with PspA alone, low levels of PspA-specific immunoglobulin G (IgG) responses were induced in serum; none was induced in secretion. On the other hand, when PspA was given orally with the mucosal adjuvant cholera toxin (CT), significant levels of IgG and IgA anti-PspA responses were induced in serum. The major IgG subclass was IgG1, followed by IgG2b, a profile of antibody response supported by Th2-type cells. In addition, all mice orally immunized with PspA and CT were protected from the lethal challenge with capsular serotype 3 S. pneumoniae A66. These results suggested that an oral PspA vaccine may be a useful means of preventing pneumococcal disease.