Effects of LT‐K63 and CpG2006 on Phenotype and Function of Murine Neonatal Lymphoid Cells

Abstract

The immature state of the immune system of neonates makes them vulnerable to infectious agents, including Streptococcus pneumoniae. The aim of our study was to analyse and compare the effects of Escherichia coli heat‐labile enterototoxin (LT)‐K63 and CpG2006 on cells and key molecules of the neonatal immune system, using a previously established immunization model with pneumococcal polysaccharide of serotype 1 conjugated to tetanus toxoid (TT) (Pnc1‐TT). The cellular response was evaluated by measuring cytokine secretion and proliferation upon in vitro stimulation with TT, the protein moiety of Pnc1‐TT, and antibody (Ab) to both the polysaccharide (PS) and protein parts of the vaccine were measured by enzyme‐linked immunosorbent assay (ELISA). Antigen (Ag)‐presenting and co‐stimulatory capacity of neonatal B‐cells was evaluated by staining for major histocompatibility complex (MHC)II, CD80, CD86 and CD40. The results showed that both LT‐K63 and CpG2006 significantly enhanced the neonatal Ab response to Pnc1‐TT. Spleen cells from mice receiving LT‐K63 showed enhanced proliferation and interferon (IFN)‐γ, interleukin (IL)‐4, IL‐5 and IL‐10 secretion upon TT stimulation, whereas cells from mice receiving CpG2006 could only enhance IL‐10 secretion. LT‐K63 and to a lesser extent CpG2006 enhanced the capacity of B‐cells to up‐regulate the expression of co‐stimulatory and activation markers compared with those of mice receiving Pnc1‐TT alone. Thus, we conclude that LT‐K63 markedly improves T‐cell activation whereas the direct adjuvant effect of CpG2006 on neonatal B‐cells may partly compensate for lower T‐cell help resulting in enhanced neonatal Ab responses to both the TT and PS parts of the vaccine by both adjuvants.

IC31®, a Two‐Component Novel Adjuvant Mixed with a Conjugate Vaccine Enhances Protective Immunity against Pneumococcal Disease in Neonatal Mice

“…We have previously shown that the TLR9 agonist, CpG2006, enhances Pnc1-TT-induced antibody responses in neonatal mice, and activates B cells and dendritic cells [32,33]. IC31 Ò signals through TLR9 [20] similar to CpG-ODN, but one dose of Pnc1-TT with HD or LD IC31 Ò elicited higher Ab response than when given with CpG2006.…”

“…In the present study the IgG subclass pattern of mice receiving IC31 Ò , revealed strong IgG1 and IgG3 responses with much lower IgG2a and IgG2b responses that nevertheless were slightly higher in the groups receiving IC31 Ò than those receiving no adjuvant. The effects of IC31 Ò on the cellular responses in neonates remain to be studied in order to directly demonstrate if IC31 Ò can overcome the Th2 bias of the neonatal immune system as we have previously shown for LT-K63 [32].…”

The Adjuvant LT-K63 Can Restore Delayed Maturation of Follicular Dendritic Cells and Poor Persistence of Both Protein- and Polysaccharide-Specific Antibody-Secreting Cells in Neonatal Mice

“…Three weeks after neonatal priming with Pnc1-TT with or without LT-K63, 10 7 spleen cells/ml in complete RPMI 1640 were incubated with or without TT (10 mg/ml) in 24-well plates (Nunc) at 37˚C, 5% CO 2 for 48 h (20). Supernatants (kept at 270˚C) were incubated for 2 h in plates (MaxiSorp; Nunc), coated with anti-mouse TNF-a (R&D Systems), and blocked with PBS 1% BSA (Sigma).…”

“…MultiScreen High protein binding immobilon-P membrane plates (Millipore Corporation, Bedford, MA) were coated with 20 mg/ml PPS-1 or 10 mg/ml TT overnight at 37˚C, blocked with complete RPMI 1640, serial dilutions of spleen cell (10 8 cells/ml) in complete RPMI 1640 (Life Technologies BRL, Life Technologies, Paisley, U.K.) (20) were incubated for 5 h at 37˚C, washed and incubated with ALP-goat anti-mouse IgG (Southern Biotechnology Associates) overnight at 4˚C, and developed by 5-bromo-4-chloro-3-indolylphosphate and NBT in AP development buffer (Bio-Rad Labs, Hercules, CA). Spots were counted using a microscope (Zeiss, Oberkochen, Germany) and analyzed with KS ELISPOT (Zeiss).…”

“…Using an early-life murine model of pneumococcal vaccination and challenge that reproduces the main features of human infant response to pneumococcal conjugate vaccines (16), we have reported that coadministration of the nontoxic adjuvant LT-K63 (nontoxic mutant of Escherichia coli heat-labile enterotoxin) (17,18) with pneumococcal polysaccharide of serotype 1 conjugated to tetanus toxoid (Pnc1-TT) enhances protection against pneumococcal infection, correlating with enhanced PPS-specific Ab levels (16), avidity (19), and activation of B cells, T cells (20), and dendritic cells in spleen (21).…”