In vitro cytokine induction by TLR-activating vaccine adjuvants in human blood varies by age and adjuvant.
ABSTRACT: Most infections occur in early life, prompting development of novel adjuvanted vaccines to protect newborns and infants. Several Toll-like receptor (TLR) agonists (TLRAs) are components of licensed vaccine formulations or are in development as candidate adjuvants. However, the type and magnitude of immune responses to TLRAs may vary with the TLR activated as well as age and geographic location. Most notably, in newborns, as compared to adults, the immune response to TLRAs is polarized with lower Th1 cytokine production and robust Th2 and anti-inflammatory cytokine production. The ontogeny of TLR-mediated cytokine responses in international cohorts has been reported, but no study has compared cytokine responses to TLRAs between U.S. neonates and infants at the age of 6months. Both are critical age groups for the currently pediatric vaccine schedule. In this study, we report quantitative differences in the production of a panel of 14 cytokines and chemokines after in vitro stimulation of newborn cord blood and infant and adult peripheral blood with agonists of TLR4, including monophosphoryl lipid A (MPLA) and glucopyranosyl lipid Adjuvant aqueous formulation (GLA-AF), as well as agonists of TLR7/8 (R848) and TLR9 (CpG). Both TLR4 agonists, MPLA and GLA-AF, induced greater concentrations of Th1 cytokines CXCL10, TNF and Interleukin (IL)-12p70 in infant and adult blood compared to newborn blood. All the tested TLRAs induced greater infant IFN-?2 production compared to newborn and adult blood. In contrast, CpG induced greater IFN-?, IL-1?, IL-4, IL-12p40, IL-10 and CXCL8 in newborn than in infant and adult blood. Overall, to the extent that these in vitro studies mirror responses in vivo, our study demonstrates distinct age-specific effects of TLRAs that may inform their development as candidate adjuvants for early life vaccines.
Project description:Newborns display distinct immune responses that contribute to susceptibility to infection and reduced vaccine responses. Toll-like receptor (TLR) agonists may serve as vaccine adjuvants, when given individually or in combination, but responses of neonatal leukocytes to many TLR agonists are diminished. TLR8 agonists are more effective than other TLR agonists in activating human neonatal leukocytes in vitro, but little is known about whether different TLR8 agonists may distinctly activate neonatal leukocytes. We characterized the in vitro immuno-stimulatory activities of a novel benzazepine TLR8 agonist, VTX-294, in comparison to imidazoquinolines that activate TLR8 (R-848; (TLR7/8) CL075; (TLR8/7)), with respect to activation of human newborn and adult leukocytes. Effects of VTX-294 and R-848 in combination with monophosphoryl lipid A (MPLA; TLR4) were also assessed.TLR agonist specificity was assessed using TLR-transfected HEK293 cells expressing a NF-?B reporter gene. TLR agonist-induced cytokine production was measured in human newborn cord and adult peripheral blood using ELISA and multiplex assays. Newborn and adult monocytes were differentiated into monocyte-derived dendritic cells (MoDCs) and TLR agonist-induced activation assessed by cytokine production (ELISA) and co-stimulatory molecule expression (flow cytometry).VTX-294 was ? 100x more active on TLR8- than TLR7-transfected HEK cells (EC50, ? 50 nM vs. ? 5700 nM). VTX-294-induced TNF and IL-1? production were comparable in newborn cord and adult peripheral blood, while VTX-294 was 1 log more potent in inducing TNF and IL-1? production than MPLA, R848 or CL075. Combination of VTX-294 and MPLA induced greater blood TNF and IL-1? responses than combination of R-848 and MPLA. VTX-294 also potently induced expression of cytokines and co-stimulatory molecules HLA-DR and CD86 in human newborn MoDCs.VTX-294 is a novel ultra-potent TLR8 agonist that activates newborn and adult leukocytes and is a candidate vaccine adjuvant in both early life and adulthood.
Project description:Neonatal inflammation, mediated in part through Toll-like receptor (TLR) and inflammasome signaling, contributes to adverse outcomes including organ injury. Pentoxifylline (PTX), a phosphodiesterase inhibitor which potently suppresses cytokine production in newborn cord blood, is a candidate neonatal anti-inflammatory agent. We hypothesized that combinations of PTX with other anti-inflammatory agents, the steroid dexamethasone (DEX) or the macrolide azithromycin (AZI), may exert broader, more profound and/or synergistic anti-inflammatory activity towards neonatal TLR- and inflammasome-mediated cytokine production.Whole newborn and adult blood was treated with PTX (50-200 ?M), DEX (10-10-10-7 M), or AZI (2.5-20 ?M), alone or combined, and cultured with lipopolysaccharide (LPS) (TLR4 agonist), R848 (TLR7/8 agonist) or LPS/adenosine triphosphate (ATP) (inflammasome induction). Supernatant and intracellular cytokines, signaling molecules and mRNA were measured by multiplex assay, flow cytometry and real-time PCR. Drug interactions were assessed based on Loewe's additivity.PTX, DEX and AZI inhibited TLR- and/or inflammasome-mediated cytokine production in newborn and adult blood, whether added before, simultaneously or after TLR stimulation. PTX preferentially inhibited pro-inflammatory cytokines especially TNF. DEX inhibited IL-10 in newborn, and TNF, IL-1?, IL-6 and interferon-? in newborn and adult blood. AZI inhibited R848-induced TNF, IL-1?, IL-6 and IL-10, and LPS-induced IL-1? and IL-10. (PTX+DEX) synergistically decreased LPS- and LPS/ATP-induced TNF, IL-1?, and IL-6, and R848-induced IL-1? and interferon-?, while (PTX+AZI) synergistically decreased induction of TNF, IL-1?, and IL-6. Synergistic inhibition of TNF production by (PTX+DEX) was especially pronounced in newborn vs. adult blood and was accompanied by reduction of TNF mRNA and enhancement of IL10 mRNA.Age, agent, and specific drug-drug combinations exert distinct anti-inflammatory effects towards TLR- and/or inflammasome-mediated cytokine production in human newborn blood in vitro. Synergistic combinations of PTX, DEX and AZI may offer benefit for prevention and/or treatment of neonatal inflammatory conditions while potentially limiting drug exposure and toxicity.
Project description:Newborns have frequent infections and manifest impaired vaccine responses, motivating a search for neonatal vaccine adjuvants. Alum is a neonatal adjuvant but might confer a T(H)2 bias. Toll-like receptor (TLR) agonists are candidate adjuvants, but human neonatal cord blood monocytes demonstrate impaired T(H)1-polarizing responses to many TLR agonists caused by plasma adenosine acting through cyclic AMP. TLR8 agonists, including imidazoquinolines (IMQs), such as the small synthetic 3M-002, induce adult-level TNF from neonatal monocytes, but the scope and mechanisms of IMQ-induced activation of neonatal monocytes and monocyte-derived dendritic cells (MoDCs) have not been reported.We sought to characterize IMQ-induced activation of neonatal monocytes and MoDCs.Neonatal cord and adult peripheral blood monocytes and MoDCs were cultured in autologous plasma; levels of alum- and TLR agonist-induced cytokines and costimulatory molecules were measured. TLR8 and inflammasome function were assayed by using small interfering RNA and Western blotting/caspase-1 inhibitory peptide, respectively. The ontogeny of TLR8 agonist-induced cytokine responses was defined in rhesus macaque whole blood ex vivo.IMQs were more potent and effective than alum at inducing TNF and IL-1? from monocytes. 3M-002 induced robust TLR pathway transcriptome activation and T(H)1-polarizing cytokine production in neonatal and adult monocytes and MoDCs, signaling through TLR8 in an adenosine/cyclic AMP-refractory manner. Newborn MoDCs displayed impaired LPS/ATP-induced caspase-1-mediated IL-1? production but robust 3M-002-induced caspase-1-mediated inflammasome activation independent of exogenous ATP. TLR8 IMQs induced robust TNF and IL-1? in whole blood of rhesus macaques at birth and infancy.IMQ TLR8 agonists engage adenosine-refractory TLR8 and inflammasome pathways to induce robust monocyte and MoDC activation and represent promising neonatal adjuvants.
Project description:Toll like receptor (TLR) ligands are important adjuvant candidates, causing antigen presenting cells to release inflammatory mediators, leading to the recruitment and activation of other leukocytes. The aim of this study was to define the response of human blood derived dendritic cells and macrophages to three TLR ligands acting singly or in combination, Poly I:C (TLR3), GLA (TLR4) and R848 (TLR7/8). Combinations of TLR agonists have been shown to have a synergistic effect on individual cytokines, here we look at the global inflammatory response measuring both cytokines and chemokines. Using a custom Luminex assay we saw dose responses in several mediators including CCL3 (MIP1?), IL-1?, IL-1?, IL-12, CXCL10 (IP-10) and IL-6, all of which were significantly increased by the combination of R848 and GLA, even when low dose GLA was added. The synergistic effect was inhibited by specific MAP kinase inhibitors blocking the kinases p38 and JNK but not MEK1. Combining TLR adjuvants also had a synergistic effect on cytokine responses in human mucosal tissue explants. From this we conclude that the combination of R848 and GLA potentiates the inflammatory profile of antigen presenting cells. Since the pattern of inflammatory mediators released can alter the quality and quantity of the adaptive immune response to vaccination, this study informs vaccine adjuvant design.
Project description:A therapeutic vaccine for human Chagas disease (American trypanosomiasis caused by Trypanosoma cruzi) is under development based on the success of vaccinating mice with DNA constructs expressing the antigens Tc24 and Tc-TSA-1. However, because DNA and nucleic acid vaccines produce less than optimal responses in humans, our strategy relies on administering a recombinant protein-based vaccine, together with adjuvants that promote Th1-type immunity. Here we describe a process for the purification and refolding of recombinant TSA-1 expressed in Escherichia coli. The overall yield (20-25%) and endotoxin level of the purified recombinant TSA-1 (rTSA-1) is suitable for pilot scale production of the antigen for use in phase 1 clinical trials. Mice infected with T. cruzi were treated with rTSA-1, either alone or with Toll-like receptor 4 (TLR-4) agonist adjuvants including monophosphoryl lipid A (MPLA), glucopyranosyl lipid A (GLA, IDRI), and E6020 (EISEI, Inc). TSA-1 with the TLR-4 agonists was effective at reducing parasitemia relative to rTSA-1 alone, although it was difficult to discern a therapeutic effect compared to treatment with TLR-4 agonists alone. However, rTSA-1 with a 10 ug dose of MPLA optimized reductions in cardiac tissue inflammation, which were significantly reduced compared to MPLA alone. It also elicited the lowest parasite burden and the highest levels of TSA-1-specific IFN-gamma levels and IFN-gamma/IL-4 ratios. These results warrant the further evaluation of rTSA-1 in combination with rTc24 in order to maximize the therapeutic effect of vaccine-linked chemotherapy in both mice and non-human primates before advancing to clinical development.
Project description:Each year millions of neonates die due to vaccine preventable infectious diseases. Our study seeks to develop novel neonatal vaccines and improve immunogenicity of early childhood vaccines by incorporating TLR agonist-adjuvant combinations that overcome the inherent neonatal Th2 bias and stimulate Th1 polarizing response from neonatal APCs. We systematically stimulated cord blood mononuclear cells with single and multiple combinations of TLR agonists and measured levels of IL-12p70, IFN-?, IFN-?, IL-10, IL-13, TNF-?, IL-6 and IL-1? from cell culture supernatants. APC-specific surface expression levels of costimulatory markers CD40, CD83 and PD-L1 were assessed by flow cytometry. Whole blood assays were included to account for the effect of plasma inhibitory factors and APC intracellular TNF-? and IL-12p40 secretions were measured. We found robust Th1 polarizing IL-12p70, IFN-? and IFN-? responses when cord blood APCs were stimulated with TLR agonist combinations that contained Poly I:C, Monophosphoryl Lipid A (MPLA) or R848. Addition of class A CpG oligonucleotide (ODN) to Th1 polarizing TLR agonist combinations significantly reduced cord blood IL-12p70 and IFN-? levels and addition of a TLR2 agonist induced significantly high Th2 polarizing IL-13. Multi-TLR agonist combinations that included R848 induced lower inhibitory PD-L1 expression on cord blood classical dendritic cells than CpG ODN-containing combinations. Incorporation of combination adjuvants containing TLR3, TLR4 and TLR7/8 agonists to neonatal vaccines may be an effective strategy to overcome neonatal Th2 bias.
Project description:Newborns and young infants are at higher risk for infections than adults, and manifest suboptimal vaccine responses, motivating a search for novel immunomodulators and/or vaccine adjuvants effective in early life. In contrast to most TLR agonists (TLRA), TLR8 agonists such as imidazoquinolines (IMQs) induce adult-level Th1-polarizing cytokine production from human neonatal cord blood monocytes and are candidate early life adjuvants. We assessed whether TLR8-activating IMQ congeners may differ in potency and efficacy in inducing neonatal cytokine production in vitro, comparing the novel TLR7/8-activating IMQ analogues Hybrid-2, Meta-amine, and Para-amine to the benchmark IMQ resiquimod (R848).TLRA-induced NF-?B activation was measured in TLR-transfected HEK cells. Cytokine production in human newborn cord and adult peripheral blood and in monocyte-derived dendritic cell cultures were measured by ELISA and multiplex assays. X-ray crystallography characterized the interaction of human TLR8 with Hybrid-2.Hybrid-2 selectively activated both TLR7 and 8 and was more potent than R848 in inducing adult-like levels of TNF-?, and IL-1?. Consistent with its relatively high in vitro activity, crystallographic studies suggest that absence in Hybrid-2 of an ether oxygen of the C2-ethoxymethyl substituent, which can engage in unfavorable electrostatic and/or dipolar interactions with the carbonyl oxygen of Gly572 in human TLR8, may confer greater efficacy and potency compared to R848.Hybrid-2 is a selective and potent TLR7/8 agonist that is a candidate adjuvant for early life immunization.
Project description:Due to functionally distinct cell-mediated immunity, newborns and infants are highly susceptible to infection with intracellular pathogens. Indeed, neonatal Ag-presenting dendritic cells (DCs) demonstrate impaired Th1 responses to many candidate adjuvants, including most TLR agonists (TLRAs). Combination adjuvantation systems may provide enhanced immune activation but have typically been developed without regard to the age of the target population. We posited that distinct combinations of TLRAs and C-type lectin receptor agonists may enhance Th1 responses of newborn DCs. TLRA/C-type lectin receptor agonist combinations were screened for enhancement of TNF production by human newborn and adult monocyte-derived DCs cultured in 10% autologous plasma or in newborn cord, infant, adult, and elderly whole blood. Monocyte-derived DC activation was characterized by targeted gene expression analysis, caspase-1 and NF-?B studies, cytokine multiplex and naive autologous CD4+ T cell activation. Dual activation of newborn DCs via the C-type lectin receptor, macrophage-inducible C-type lectin (trehalose-6,6-dibehenate), and TLR7/8 (R848) greatly enhanced caspase-1 and NF-?B activation, Th1 polarizing cytokine production and autologous Th1 polarization. Combined activation via TLR4 (glycopyranosyl lipid adjuvant aqueous formulation) and Dectin-1 (?-glucan peptide) acted synergistically in newborns and adults, but to a lesser extent. The degree of synergy varied dramatically with age, and was the greatest in newborns and infants with less synergy in adults and elders. Overall, combination adjuvant systems demonstrate markedly different immune activation with age, with combined DC activation via Macrophage-inducible C-type lectin and TLR7/8 representing a novel approach to enhance the efficacy of early-life vaccines.
Project description:Newborns are highly susceptible to infectious diseases, which may be due to impaired immune responses. This study aims to characterize the ontogeny of neonatal TLR-based innate immunity during the first month of life. Cellularity and Toll-like receptor (TLR) agonist-induced cytokine production were compared between cord blood obtained from healthy neonates born after uncomplicated gestation and delivery (n=18), neonatal venous blood obtained at the age of one month (n=96), and adult venous blood (n=17). Cord blood TLR agonist-induced production of the Th1-polarizing cytokines IL-12p70 and IFN-alpha was generally impaired, but for TLR3, 7 and 9 agonists, rapidly increased to adult levels during the first month of life. In contrast, TLR4 demonstrated a slower maturation, with low LPS-induced IL-12p70 production and high IL-10 production up until the age of one month. Polarization in neonatal cytokine responses to LPS could contribute to neonatal susceptibility to severe bacterial infection.
Project description:In this study we have investigated the potential of mycobacterial proteins as candidate subunit vaccines for bovine tuberculosis. In addition, we have explored the use of TLR-ligands as potential adjuvants in cattle. In vitro screening assays with whole blood from Mycobacterium bovis-infected and BCG-vaccinated cattle demonstrated that fusion protein constructs were most commonly recognised, and the ID83 fusion protein was selected for further immunisation studies. Furthermore, glucopyranosyl lipid A (GLA) and resiquimod (R848), agonists for TLR4 and TLR7/8 respectively, stimulated cytokine production (IL-12, TNF-?, MIP-1? and IL-10) in bovine dendritic cell cultures, and these were formulated as novel oil-in-water emulsions (GLA-SE and R848-SE) for immunisation studies. Immunisation with ID83 in a water-in-oil emulsion adjuvant (ISA70) induced both cell mediated and humoral immune responses, as characterised by antigen-specific IFN-? production, cell proliferation, IgG1 and IgG2 antibody production. In comparison, ID83 immunisation with the novel adjuvants induced weaker (ID83/R848-SE) or no (ID83/GLA-SE) antigen-specific IFN-? production and cell proliferation. However, both did induce ID83-specific antibody production, which was restricted to IgG1 antibody isotype. Overall, these results provide encouraging preliminary data for the further development of ID83 in vaccine strategies for bovine TB.