A comparative study of adjuvants effects on neonatal plasma cell survival niche in bone marrow and persistence of humoral immune responses.

The neonatal immune system is distinct from the immune system of older individuals rendering neonates vulnerable to infections and poor responders to vaccination. Adjuvants can be used as tools to enhance immune responses to co-administered antigens. Antibody (Ab) persistence is mediated by long-lived plasma cells that reside in specialized survival niches in the bone marrow, and transient Ab responses in early life have been associated with decreased survival of plasma cells, possibly due to lack of survival factors. Various cells can secrete these factors and which cells are the main producers is still up for debate, especially in early life where this has not been fully addressed. The receptor BCMA and its ligand APRIL have been shown to be important in the maintenance of plasma cells and Abs. Herein, we assessed age-dependent maturation of a broad range of bone marrow accessory cells and their expression of the survival factors APRIL and IL-6. Furthermore, we performed a comparative analysis of the potential of 5 different adjuvants; LT-K63, mmCT, MF59, IC31 and alum, to enhance expression of survival factors and BCMA following immunization of neonatal mice with tetanus toxoid (TT) vaccine. We found that APRIL expression was reduced in the bone marrow of young mice whereas IL-6 expression was higher. Eosinophils, macrophages, megakaryocytes, monocytes and lymphocytes were important secretors of survival factors in early life but undefined cells also constituted a large fraction of secretors. Immunization and adjuvants enhanced APRIL expression but decreased IL-6 expression in bone marrow cells early after immunization. Furthermore, neonatal immunization with adjuvants enhanced the proportion of plasmablasts and plasma cells that expressed BCMA both in spleen and bone marrow. Enhanced BCMA expression correlated with enhanced vaccine-specific humoral responses, even though the effect of alum on BCMA was less pronounced than those of the other adjuvants at later time points. We propose that low APRIL expression in bone marrow as well as low BCMA expression of plasmablasts/plasma cells in early life together cause transient Ab responses and could represent targets to be triggered by vaccine adjuvants to induce persistent humoral immune responses in this age group.

LT-K63 Enhances B Cell Activation and Survival Factors in Neonatal Mice That Translates Into Long-Lived Humoral Immunity.

Adjuvants enhance magnitude and duration of immune responses induced by vaccines. In this study we assessed in neonatal mice if and how the adjuvant LT-K63 given with a pneumococcal conjugate vaccine, Pnc1-TT, could affect the expression of tumor necrosis factor receptor (TNF-R) superfamily members, known to be involved in the initiation and maintenance of antibody responses; B cell activating factor receptor (BAFF-R) and B cell maturation antigen (BCMA) and their ligands, BAFF, and a proliferation inducing ligand (APRIL). Initially we assessed the maturation status of different B cell populations and their expression of BAFF-R and BCMA. Neonatal mice had dramatically fewer B cells than adult mice and the composition of different subsets within the B cell pool differed greatly. Proportionally newly formed B cells were most abundant, but they had diminished BAFF-R expression which could explain low proportions of marginal zone and follicular B cells observed. Limited BCMA expression was also detected in neonatal pre-plasmablasts/plasmablasts. LT-K63 enhanced vaccine-induced BAFF-R expression in splenic marginal zone, follicular and newly formed B cells, leading to increased plasmablast/plasma cells, and their enhanced expression of BCMA in spleen and bone marrow. Additionally, the induction of BAFF and APRIL expression occurred early in neonatal mice immunized with Pnc1-TT either with or without LT-K63. However, BAFF+ and APRIL+ cells in spleens were maintained at a higher level in mice that received the adjuvant. Furthermore, the early increase of APRIL+ cells in bone marrow was more profound in mice immunized with vaccine and adjuvant. Finally, we assessed, for the first time in neonatal mice, accessory cells of the plasma cell niche in bone marrow and their secretion of APRIL. We found that LT-K63 enhanced the frequency and APRIL expression of eosinophils, macrophages, and megakaryocytes, which likely contributed to plasma cell survival, even though APRIL+ cells showed a fast decline. All this was associated with enhanced, sustained vaccine-specific antibody-secreting cells in bone marrow and persisting vaccine-specific serum antibodies. Our study sheds light on the mechanisms behind the adjuvanticity of LT-K63 and identifies molecular pathways that should be triggered by vaccine adjuvants to induce sustained humoral immunity in early life.

Adjuvants Enhance the Induction of Germinal Center and Antibody Secreting Cells in Spleen and Their Persistence in Bone Marrow of Neonatal Mice.

Immaturity of the immune system contributes to poor vaccine responses in early life. Germinal center (GC) activation is limited due to poorly developed follicular dendritic cells (FDC), causing generation of few antibody-secreting cells (ASCs) with limited survival and transient antibody responses. Herein, we compared the potential of five adjuvants, namely LT-K63, mmCT, MF59, IC31, and alum to overcome limitations of the neonatal immune system and to enhance and prolong responses of neonatal mice to a pneumococcal conjugate vaccine Pnc1-TT. The adjuvants LT-K63, mmCT, MF59, and IC31 significantly enhanced GC formation and FDC maturation in neonatal mice when co-administered with Pnc1-TT. This enhanced GC induction correlated with significantly enhanced vaccine-specific ASCs by LT-K63, mmCT, and MF59 in spleen 14 days after immunization. Furthermore, mmCT, MF59, and IC31 prolonged the induction of vaccine-specific ASCs in spleen and increased their persistence in bone marrow up to 9 weeks after immunization, as previously shown for LT-K63. Accordingly, serum Abs persisted above protective levels against pneumococcal bacteremia and pneumonia. In contrast, alum only enhanced the primary induction of vaccine-specific IgG Abs, which was transient. Our comparative study demonstrated that, in contrast to alum, LT-K63, mmCT, MF59, and IC31 can overcome limitations of the neonatal immune system and enhance both induction and persistence of protective immune response when administered with Pnc1-TT. These adjuvants are promising candidates for early life vaccination.

Pneumococcal polysaccharide abrogates conjugate-induced germinal center reaction and depletes antibody secreting cell pool, causing hyporesponsiveness.

BACKGROUND: Plain pneumococcal polysaccharide (PPS) booster administered during second year of life has been shown to cause hyporesponsiveness. We assessed the effects of PPS booster on splenic memory B cell responses and persistence of PPS-specific long-lived plasma cells in the bone marrow (BM). METHODS: Neonatal mice were primed subcutanously (s.c.) or intranasally (i.n.) with pneumococcal conjugate (Pnc1-TT) and the adjuvant LT-K63, and boosted with PPS+LT-K63 or saline 1, 2 or 3 times with 16 day intervals. Seven days after each booster, spleens were removed, germinal centers (GC), IgM(+), IgG(+) follicles and PPS-specific antibody secreting cells (AbSC) in spleen and BM enumerated. RESULTS: PPS booster s.c., but not i.n., compromised the Pnc1-TT-induced PPS-specific Abs by abrogating the Pnc1-TT-induced GC reaction and depleting PPS-specific AbSCs in spleen and limiting their homing to the BM. There was no difference in the frequency of PPS-specific AbSCs in spleen and BM between mice that received 1, 2 or 3 PPS boosters s.c.. Repeated PPS+LT-K63 booster i.n. reduced the frequency of PPS-specific IgG(+) AbSCs in BM. CONCLUSIONS: PPS booster-induced hyporesponsiveness is caused by abrogation of conjugate-induced GC reaction and depletion of PPS-specific IgG(+) AbSCs resulting in no homing of new PPS-specific long-lived plasma cells to the BM or survival. These results should be taken into account in design of vaccination schedules where polysaccharides are being considered.

Hyporesponsiveness following booster immunization with bacterial polysaccharides is caused by apoptosis of memory B cells.

BACKGROUND: Repeated immunizations with polysaccharide (PS) vaccines cause hyporesponsiveness through undefined mechanisms. We assessed the effects of a PS booster on immune responses, frequency, and survival of PS-specific B-cell subpopulations in spleen and bone marrow. METHODS: Neonatal mice were primed with meningococcus serotype C (MenC) conjugate MenC-CRM(197)+CpG1826, boosted with MenC-CRM(197), MenC-PS, or saline; subsequently, bromodeoxyuridine (BrdU) was injected daily intraperitoneally. MenC-PS-specific cells were labeled with fluorescent MenC-PS and phenotyped by flow cytometry. RESULTS: After MenC-PS booster, proliferating (BrdU(+)) MenC-PS-specific naive B cells (CD138(-)/B220(+); P = .0003) and plasma cells (CD138(+)/B220(-); P = .0002) in spleen were fewer than after saline booster. BrdU(+) MenC-PS-specific plasma cells were also reduced in bone marrow (P = .0308). Compared to saline, MenC-PS booster reduced BrdU(+) IgG(+) MenC-PS-specific B cells in spleen (P = .0002). Twelve hours after the MenC-PS booster, an increased frequency of apoptotic (AnnexinV(+)) MenC-PS-specific B cells in spleen was observed compared with MenC-CRM(197) (P = .0286) or saline (P = .001) boosters. CONCLUSIONS: We demonstrated that the MenC-PS booster significantly reduced the frequency of newly activated MenC-PS-specific B cells-mostly switched IgG(+) memory cells-by driving them into apoptosis. It shows directly that apoptosis of PS-specific memory cells is the cause of PS-induced hyporesponsiveness. These results should be taken into account prior to consideration of the use of PS vaccines.

Competition for FcRn-mediated transport gives rise to short half-life of human IgG3 and offers therapeutic potential.

Human IgG3 displays the strongest effector functions of all IgG subclasses but has a short half-life for unresolved reasons. Here we show that IgG3 binds to IgG-salvage receptor (FcRn), but that FcRn-mediated transport and rescue of IgG3 is inhibited in the presence of IgG1 due to intracellular competition between IgG1 and IgG3. We reveal that this occurs because of a single amino acid difference at position 435, where IgG3 has an arginine instead of the histidine found in all other IgG subclasses. While the presence of R435 in IgG increases binding to FcRn at neutral pH, it decreases binding at acidic pH, affecting the rescue efficiency-but only in the presence of H435-IgG. Importantly, we show that in humans the half-life of the H435-containing IgG3 allotype is comparable to IgG1. H435-IgG3 also gave enhanced protection against a pneumococcal challenge in mice, demonstrating H435-IgG3 to be a candidate for monoclonal antibody therapies.