Goutam Sen

In Vivo Humoral Immune Responses to Isolated Pneumococcal Polysaccharides Are Dependent on the Presence of Associated TLR Ligands

We determined whether T cell-independent Ig isotype responses to isolated pneumococcal polysaccharides (PPS) required TLR signaling in vivo. IgG anti-PPS responses to PPS3, PPS14, and C-polysaccharide (C-PS) were virtually undetectable in TLR2−/− mice, whereas specific IgM induction was variably reduced compared with wild-type mice. All PPS-containing preparations induced IL-6 and TNF-α from wild-type, but not TLR2−/−, macrophages. TLR2 activity was distinct from that of PPS, in that it was phenol extractable. Immunization of wild-type mice with phenol-extracted PPS14 also resulted in a marked reduction in the IgG, although not the IgM-anti-PPS14, response compared with untreated PPS14. The commercial 23-valent PPS vaccine, Pneumovax-23 also contained TLR ligands (TLR2 and TLR4), which were absolutely critical for the IgG-inducing activity of the vaccine in mice. Finally, the commercial pneumococcal conjugate vaccine, Prevnar, contained a TLR2 ligand(s) that substantially enhanced both the primary and secondary anti-PPS responses in mice, especially the type 1 IgG isotypes. These data strongly suggest the absolute need for a distinct, TLR-dependent second signal for inducing in vivo IgG T cell-independent humoral immune responses to isolated pneumococcal polysaccharide Ags and highlight the potential importance of previously unappreciated copurified and/or contaminating TLR ligands in PPS vaccine preparations.

Immunization of aged mice with a pneumococcal conjugate vaccine combined with an unmethylated CpG-containing oligodeoxynucleotide restores defective immunoglobulin G antipolysaccharide responses and specific CD4+-T-cell priming to young adult levels

ABSTRACT Polysaccharide (PS)-protein conjugate vaccines, in contrast to purified PS vaccines, recruit CD4+-T-cell help and restore defective PS-specific humoral immunity in the immature host. Surprisingly, in the immunocompromised, aged host, anti-PS responses to conjugate vaccines are typically no better than those elicited by purified PS vaccines. Although aging leads to defects in multiple immune cell types, diminished CD4+-T-cell helper function has recently been shown to play a dominant role. We show that in response to immunization with purified pneumococcal capsular PS serotype 14 (PPS14) in saline, the T-cell-independent immunoglobulin G (IgG) anti-PPS14 response in aged mice was comparable to that in young mice. In contrast, the T-cell-dependent IgG anti-PPS14 response to a soluble conjugate of PPS14 and pneumococcal surface protein A (PspA) (PPS14-PspA) in saline was markedly defective. This was associated with defective priming of PspA-specific CD4+ T cells. In contrast, immunization of aged mice with PPS14-PspA combined with an unmethylated CpG-containing oligodeoxynucleotide (CpG-ODN) restored IgG anti-PPS14 responses to young adult levels, which were substantially higher than those observed using purified PPS14. This was associated with enhanced PspA-specific CD4+-T-cell priming. Similarly, intact Streptococcus pneumoniae capsular type 14, which contains Toll-like receptor (TLR) ligands, also induced substantial, though modestly reduced, T-cell-dependent (TD) IgG ant-PPS14 responses in aged mice. Spleen and peritoneal cells from aged and young adult mice made comparable levels of proinflammatory cytokines in response to CpG-ODN, although cells from aged mice secreted higher levels of interleukin-10. Collectively, these data suggest that inclusion of a TLR ligand, as an adjuvant, with a conjugate vaccine can correct defective TD IgG anti-PS responses in elderly patients by augmenting CD4+-T-cell help.

Parameters underlying distinct T cell-dependent polysaccharide-specific IgG responses to an intact gram-positive bacterium versus a soluble conjugate vaccine.

IgG anti-polysaccharide (PS) responses to both intact Streptococcus pneumoniae (Pn) and PS conjugate vaccines are dependent on CD4+ T cells, B7-dependent costimulation, and CD40-CD40-ligand interactions. Nevertheless, the former response, in contrast to the latter, is mediated by an ICOS-independent, apoptosis-prone, extrafollicular pathway that fails to generate PS-specific memory. We show that pre-existing PS-specific Igs, the bacterial surface or particulation, selective recruitment of B cell subsets, or activation and recruitment of Pn protein-specific CD4+ T cells do not account for the failure of Pn to generate PS-specific IgG memory. Rather, the data suggest that the critical factor may be the lack of covalent attachment of PS to protein in intact Pn, highlighting the potential importance of the physicochemical relationship of PS capsule with the underlying bacterial structure for in vivo induction of PS-specific Igs.

Transgenic expression of Bcl-xL or Bcl-2 by murine B cells enhances the in vivo antipolysaccharide, but not antiprotein, response to intact Streptococcus pneumoniae.

IgG antipolysaccharide (PS) and antiprotein responses to Streptococcus pneumoniae (Pn) are both CD4+ T cell dependent. However, the primary IgG anti-PS response terminates more quickly, uses a shorter period of T cell help, fails to generate memory, and is more dependent on membrane Ig (mIg) signaling. We thus determined whether this limited anti-PS response to Pn reflected a greater propensity of PS-specific B cells to undergo apoptosis. We used mice that constitutively expressed the antiapoptotic protein Bcl-xL or Bcl-2 as a B cell-specific transgene. Both transgenic (Tg) mice exhibited increased absolute numbers of splenic B-1 and peritoneal B-1b and B-2 cells, subsets implicated in anti-PS responses, but not in marginal zone B (MZB) cells. Both Tg mouse strains elicited, in an apparently Fas-independent manner, a more prolonged and higher peak primary IgM and IgG anti-PS, but not antiprotein, response to Pn, but without PS-specific memory. A similar effect was not observed using purified PS or pneumococcal conjugate vaccine. In vitro, both splenic MZB and follicular Tg B cells synthesized DNA at markedly higher levels than their wild-type counterparts, following mIg cross-linking. This was associated with increased clonal expansion and decreased apoptosis. Using Lsc−/− mice, the Pn-induced IgG response specific for the capsular PS was found to be almost entirely dependent on MZB cells. Collectively, these data suggest that apoptosis may limit mIg-dependent clonal expansion of PS-specific B cells during a primary immune response to an intact bacterium, as well as decrease the pool of PS-responding B cell subsets.

A novel tetrameric gp3501-470 as a potential Epstein-Barr virus vaccine

Infectious mononucleosis and B-cell transformation in response to infection with Epstein-Barr virus (EBV) is dependent upon binding of the EBV envelope glycoprotein gp350 to CD21 on B-cells. Gp350-specific antibody comprises most of the EBV neutralizing activity in the serum of infected patients, making this protein a promising target antigen for a prophylactic EBV vaccine. We describe a novel, tetrameric gp350-based vaccine that exhibits markedly enhanced immunogenicity relative to its monomeric counterpart. Plasmid DNA was constructed for synthesis, within transfected CHO cells, of a tetrameric, truncated (a.a. 1-470) gp350 protein (gp350(1-470)). Tetrameric gp350(1-470) induced ≈ 20-fold higher serum titers of gp350(1-470)-specific IgG and >19-fold enhancements in neutralizing titers at the highest dose, and was >25-fold more immunogenic on a per-weight basis than monomeric gp350(1-470). Further, epidermal immunization with plasmid DNA encoding gp350(1-470) tetramer induced 8-fold higher serum titers of gp350(1-470)-specific IgG relative to monomer. Tetrameric gp350(1-470) binding to human CD21 was >24-fold more efficient on a per-weight basis than monomer, but neither tetramer nor monomer mediated polyclonal human B-cell activation. Finally, the introduction of strong, universal tetanus toxoid (TT)-specific CD4+ T-cell epitopes into the tetrameric gp350(1-470) had no effect on the gp350(1-470)-specific IgG response in naïve mice, and resulted in suppressed gp350(1-470)-specific IgG responses in TT-primed mice. Collectively, these data suggest that tetrameric gp350(1-470) is a potentially promising candidate for testing as a prophylactic EBV vaccine, and that protein multimerization, using the approach described herein, is likely to be clinically relevant for enhancing the immunogenicity of other proteins of vaccine interest.

Induction of In Vivo Antipolysaccharide Immunoglobulin Responses to Intact Streptococcus pneumoniae Is More Heavily Dependent on Btk-Mediated B-Cell Receptor Signaling than Antiprotein Responses

ABSTRACT The relative role of Btk-dependent B-cell receptor (BCR) signaling in the induction of antipolysaccharide (anti-PS) and antiprotein immunoglobulin (Ig) responses to an intact extracellular bacterium in vivo is unknown. Btklow mice exhibit reduced BCR signaling but largely restore B-cell development. Btklow mice immunized with intact Streptococcus pneumoniae elicit reduced anti-PS but normal antiprotein Ig responses. Immunization of Btklow mice with PS-protein conjugate in saline results in an even more profound defect in the anti-PS but not antiprotein response, which is largely restored by use of a CpG-containing oligodeoxynucleotide as an adjuvant. These data demonstrate a greater dependence on Btk-mediated BCR signaling for physiologic anti-PS relative to antiprotein responses, as well as the existence of a compensatory Toll-like-receptor-mediated signaling pathway naturally triggered in response to intact bacterial pathogens.

Endogenous CD4 CD25 Regulatory T Cells Play No Apparent Role in the Acute Humoral Response to Intact Streptococcus pneumoniae

ABSTRACT Immunoglobulin G (IgG) antiprotein and antipolysaccharide responses to intact Streptococcus pneumoniae are CD4+-T-cell dependent and therefore might be under the negative control of CD4+ CD25+ regulatory T cells. Injection of anti-interleukin 2 receptor α (anti-IL-2Rα) MAb to deplete regulatory T cells, injection of agonistic MAb against glucocorticoid-induced tumor necrosis factor receptor family-related protein to inhibit regulatory-T-cell function, and adoptive transfer of regulatory-T-cell-depleted CD4+ T cells into athymic nude mice each had no effect on either the primary or secondary protein- or polysaccharide-specific IgG response to intact S. pneumoniae. Surprisingly, anti-IL-2Rα MAb also had no effect on the IgG response to intact S. pneumoniae in MyD88−/− mice or to a soluble protein-polysaccharide conjugate injected into wild-type mice in the absence of adjuvant. Collectively, these data are the first to suggest that, in contrast to their role in limiting chronic cell-mediated immunity, regulatory T cells may play no significant role in an acute humoral immune response to an intact extracellular bacterial pathogen.

B and CD4 + T-cell expression of TLR2 is critical for optimal induction of a T-cell-dependent humoral immune response to intact Streptococcus pneumoniae

TLR2−/− mice immunized with Streptococcus pneumoniae (Pn) elicit normal IgM, but defective CD4+ T‐cell‐dependent type 1 IgG isotype production, associated with a largely intact innate immune response. We studied the T‐cell‐dependent phosphorylcholine (PC)‐specific IgG3 versus the T‐cell‐independent IgM response to Pn to determine whether TLR2 signals directly via the adaptive immune system. Pn‐activated TLR2−/− BMDC have only a modest defect in cytokine secretion, undergo normal maturation, and when transferred into naïve WT mice elicit a normal IgM and IgG3 anti‐PC response, relative to WT BMDC. Pn synergizes with BCR and TCR signaling for DNA synthesis in purified WT B and CD4+T cells, respectively, but is defective in cells lacking TLR2. Pn primes TLR2−/− mice for a normal CD4+ T‐cell IFN‐γ recall response. Notably, TLR2−/− B cells transferred into RAG‐2−/− mice with WT CD4+T cells, or TLR2−/− CD4+T cells transferred into athymic nude mice, each elicit a defective IgG3, in contrast to normal IgM, anti‐PC response relative to WT cells. These data are the first to demonstrate a major role for B‐cell and CD4+ T‐cell expression of TLR2 for eliciting an anti‐bacterial humoral immune response.

Endogenous IL-1R1 Signaling Is Critical for Cognate CD4+ T Cell Help for Induction of In Vivo Type 1 and Type 2 Antipolysaccharide and Antiprotein Ig Isotype Responses to Intact Streptococcus pneumoniae, but Not to a Soluble Pneumococcal Conjugate Vaccine

MyD88−/− mice exhibit defective innate, diminished CD4+ T cell-dependent (TD) type 1, but enhanced type 2, humoral immunity in response to intact Streptococcus pneumoniae (Pn). Because type 1 IL-1R (IL-1R1) signaling is MyD88 dependent, a role for endogenous IL-1 was determined. IL-1R1−/−, in contrast to MyD88−/−, mice exhibited relatively intact innate splenic cytokine expression in response to Pn. Nevertheless, IL-1R1−/−, like MyD88−/−, mice were more sensitive to killing with live Pn relative to wild-type controls. Although IL-1R1−/− mice elicited a normal T cell-independent IgM antipolysaccharide (PS) response to heat-killed Pn, the induction of PS- and protein-specific cognate, but not noncognate, TD type 1 and type 2 IgG isotypes were markedly reduced. Additionally, CD4+ T cells from Pn-primed IL-1R1−/− mice failed to elicit IFN-γ, IL-5, or IL-13 secretion upon restimulation with Pn in vitro, whereas MyD88−/− mice secreted normal levels of IFN-γ and enhanced levels of IL-5 and IL-13. In contrast, IgG responses to a soluble, pneumococcal protein-PS conjugate, with or without adjuvant, showed little dependence on IL-1R1 and normal CD4+ T cell priming. These data are the first to demonstrate a nonredundant role for endogenous IL-1 in TD induction of humoral immune responses to an intact pathogen, although not a pathogen-derived soluble conjugate, suggesting that antigenic context is a key determinant for IL-1 dependence. These data further suggest that IL-1 may be critical for preserving CD4+ Th2 function in the presence, but not absence, of MyD88-dependent signaling via TLRs.

Novel Synthetic (Poly)Glycerolphosphate-Based Antistaphylococcal Conjugate Vaccine

ABSTRACT Staphylococcal infections are a major source of global morbidity and mortality. Currently there exists no antistaphylococcal vaccine in clinical use. Previous animal studies suggested a possible role for purified lipoteichoic acid as a vaccine target for eliciting protective IgG to several Gram-positive pathogens. Since the highly conserved (poly)glycerolphosphate backbone of lipoteichoic acid is a major antigenic target of the humoral immune system during staphylococcal infections, we developed a synthetic method for producing glycerol phosphoramidites to create a covalent 10-mer of (poly)glycerolphosphate for potential use in a conjugate vaccine. We initially demonstrated that intact Staphylococcus aureus elicits murine CD4+ T cell-dependent (poly)glycerolphosphate-specific IgM and IgG responses in vivo. Naive mice immunized with a covalent conjugate of (poly)glycerolphosphate and tetanus toxoid in alum plus CpG-oligodeoxynucleotides produced high secondary titers of serum (poly)glycerolphosphate-specific IgG. Sera from immunized mice enhanced opsonophagocytic killing of live Staphylococcus aureus in vitro. Mice actively immunized with the (poly)glycerolphosphate conjugate vaccine showed rapid clearance of staphylococcal bacteremia in vivo relative to mice similarly immunized with an irrelevant conjugate vaccine. In contrast to purified, natural lipoteichoic acid, the (poly)glycerolphosphate conjugate vaccine itself exhibited no detectable inflammatory activity. These data suggest that a synthetic (poly)glycerolphosphate-based conjugate vaccine will contribute to active protection against extracellular Gram-positive pathogens expressing this highly conserved backbone structure in their membrane-associated lipoteichoic acid.