[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to characterize and identify the mode of action of IC31®, a two-component vaccine adjuvant. We found that IC31® was accumulated in human peripheral blood monocytes, MHC class II positive cells and monocyte-derived DCs (moDCs) but not in plasmacytoid DCs (pDCs). In the presence of IC31® the differentiation of inflammatory CD1a(+) moDCs and the secretion of chemokines, TNF-α and IL-6 cytokines was inhibited but the production of IFNβ was increased. Sustained addition of IC31® to differentiating moDCs interfered with IκBα phosphorylation, while the level of phospho-IRF3 increased. We also showed that both IC31® and its KLK component exhibited a booster effect on type I IFN responses induced by the specific ligands of TLR3 or TLR7/8, whereas TLR9 ligand induces type I IFN production only in the presence of IC31® or ODN1. Furthermore, long term incubation of moDCs with IC31® caused significantly higher expression of IRF and IFN genes than a single 24 hr treatment. The adjuvant activity of IC31® on the IFN response was shown to be exerted through TLRs residing in the vesicular compartment of moDCs. Based on these results IC31® was identified as a moDC modulatory adjuvant that sets the balance of the NF-κB and IRF3 mediated signaling pathways to the production of IFNβ. Thus IC31® is emerging as a potent adjuvant to increase immune responses against intracellular pathogens and cancer in future vaccination strategies.
PLoS ONE 10/2013; 8(2):e55264. DOI:10.1371/journal.pone.0055264 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The HIV vaccine strategy that, to date, generated immune protection consisted of a prime-boost regimen using a canarypox vector and an HIV envelope protein with alum, as shown in the RV144 trial. Since the efficacy was weak, and previous HIV vaccine trials designed to generate antibody responses failed, we hypothesized that generation of T cell responses would result in improved protection. Thus, we tested the immunogenicity of a similar envelope-based vaccine using a mouse model, with two modifications: a clade C CN54gp140 HIV envelope protein was adjuvanted by the TLR9 agonist IC31®, and the viral vector was the vaccinia strain NYVAC-CN54 expressing HIV envelope gp120. The use of IC31® facilitated immunoglobulin isotype switching, leading to the production of Env-specific IgG2a, as compared to protein with alum alone. Boosting with NYVAC-CN54 resulted in the generation of more robust Th1 T cell responses. Moreover, gp140 prime with IC31® and alum followed by NYVAC-CN54 boost resulted in the formation and persistence of central and effector memory populations in the spleen and an effector memory population in the gut. Our data suggest that this regimen is promising and could improve the protection rate by eliciting strong and long-lasting humoral and cellular immune responses.
PLoS ONE 07/2012; 7(7):e42163. DOI:10.1371/journal.pone.0042163 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Streptococcus pneumoniae is responsible for many vaccine-preventable deaths, annually causing around 1 million deaths in children younger than 5 years of age. A new generation of pneumococcal vaccines based on conserved proteins is being developed. We evaluated the immunogenicities and protective efficacies of four pneumococcal protein vaccine candidates, PcsB, StkP, PsaA, and PspA, in a neonatal mouse model. Mice were immunized three times and challenged intranasally with virulent pneumococci. All four proteins were immunogenic in neonatal mice, and antibody (Ab) responses were significantly enhanced by the novel adjuvant IC31, which consists of an antibacterial peptide (KLKL5KLK) and a synthetic oligodeoxynucleotide, ODN1a, that signals through Toll-like receptor 9 (TLR9). Two single proteins, StkP and PspA, combined with IC31 significantly reduced pneumococcal bacteremia but had no effects on lung infection. Three proteins, PcsB, StkP, and PsaA, were evaluated with alum or IC31. IC31 enhanced Ab responses and avidity to all three proteins, whereas alum enhanced Ab responses and avidity to StkP and PsaA only. Mice receiving the trivalent protein formulation with IC31 had significantly reduced bacteremia and lung infection compared to unvaccinated mice, but the level of protection was dependent on the dose of IC31. When PspA was added to the trivalent protein formulation, the dose of IC31 needed to obtain protective immunity could be reduced. These results demonstrate that a novel pneumococcal protein-based vaccine is immunogenic at an early age of mice and emphasize the benefits of using a combination of conserved proteins and an effective adjuvant to elicit potent protective immunity against invasive pneumococcal disease.
Infection and immunity 01/2012; 80(1):461-8. DOI:10.1128/IAI.05801-11 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Streptococcus pneumoniae is a major human pathogen, causing high morbidity and mortality in children, and also in the elderly, who are particularly susceptible to S. pneumoniae infections due to the dysregulated function of the aged immune system. As the current generation of polysaccharide vaccines do not provide sufficient protection for elderly, new vaccination strategies are urgently needed. To learn whether pneumococcal proteins are able to induce adaptive immune responses in adults in different age groups, we determined serum IgG antibody titers and T cell immunity (IFN-γ, IL-17A and IL-5 production) to three pneumococcal antigens, PcsB, StkP and PsaA, that are components of an investigational protein-based pneumococcal vaccine, IC47. Therefore, sera and PBMCs of 108 healthy adults in three different age groups (young, middle-aged and elderly) were analyzed by ELISA and ELISpot, respectively. We found naturally acquired antibodies to all three proteins in all age groups against all three antigens. However, elderly individuals had significantly lower IgG levels to PcsB and PsaA compared to those of younger donors. There was no significant age-related difference in the overall rate of T cell immunity for the three pneumococcal proteins. We found that the Th17 response was dominant in all age groups and was frequently combined with a Th1 or Th2 response in young and middle-aged subjects. However, in elderly persons there was a lower percentage of PBMC samples producing more than one cytokine upon antigenic stimulation. The narrow cytokine secretion pattern was the most striking difference between elderly and younger adult age groups. Our results demonstrate that in the majority of adults there is a naturally acquired humoral and cellular immune response to the three pneumococcal proteins tested. The dominance of the Th17 response is especially interesting in the light of new insights regarding the role of Th17 cells in mucosal protection against this pathogen.
[Show abstract][Hide abstract] ABSTRACT: New TB vaccines are urgently needed because of the apparent lack of effect of the BCG vaccine on rates of adult contagious pulmonary tuberculosis and the risk of disseminated BCG disease in immunocompromised individuals. Since BCG appears to protect children, the primary target for vaccine development is a booster vaccine for adults but such vaccines ideally need to be able to efficiently prime mycobacterially naïve individuals as well as boost individuals previously vaccinated with BCG and those latently infected with TB. Protective immunity against Mycobacterium tuberculosis depends mainly on the generation of a Th1-type cellular immune response characterized by interferon-gamma (IFN-γ) production. In the present study, we monitored safety and IFN-γ responses in healthy BCG-vaccinated and prior or latently TB-infected individuals receiving a novel vaccine composed of the fusion protein Ag85B-ESAT-6 combined with the adjuvant IC31(®), administered at 0 and 2 months. Vaccination caused few local or systemic adverse effects besides transient soreness at the injection site, but it elicited strong antigen-specific T cell responses against Ag85B-ESAT-6 and both the Ag85B and ESAT-6 components, that could be augmented by second vaccination. The strong responses persisted through 32 weeks of follow-up, indicating the induction of a persistent memory response in the vaccine recipients.
[Show abstract][Hide abstract] ABSTRACT: Tuberculosis (TB) remains a major killer worldwide. The only available TB-vaccine, the nearly century-old Mycobacterium bovis BCG, has had only a limited effect on TB incidence. Therefore, developing new TB vaccines is a key priority, and the first new generation TB vaccines are now being tested in clinical trials. Here we describe the development and first testing in humans of a novel, wholly synthetic TB subunit vaccine. This vaccine has proven safe and highly immunogenic in all species in which it was tested, including mice, guinea pigs, non-human primates and humans. Most encouragingly, following vaccination in humans, strong IFN-γ responses persisted through at least 2½ years of follow-up, indicating induction of a substantial memory response by this new TB vaccine. These findings encourage further preclinical and clinical studies with TB subunit vaccines and cellular immunity-stimulating new adjuvants.
Human vaccines 12/2010; 6(12):1007-15. DOI:10.4161/hv.6.12.13143 · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon-regulatory factor 4 (IRF4) is essential for the development of T helper 2 (Th2) and Th17 cells. Herein, we report that IRF4 is also crucial for the development and function of an interleukin-9 (IL-9)-producing CD4(+) T cell subset designated Th9. IRF4-deficient CD4(+) T cells failed to develop into IL-9-producing Th9 cells, and IRF4-specific siRNA inhibited IL-9 production in wild-type CD4(+) T cells. Chromatin-immunoprecipitation (ChIP) analyses revealed direct IRF4 binding to the Il9 promoter in Th9 cells. In a Th9-dependent asthma model, neutralization of IL-9 substantially ameliorated asthma symptoms. The relevance of these findings is emphasized by the fact that the induction of IL-9 production also occurs in human CD4(+) T cells accompanied by the upregulation of IRF4. Our data clearly demonstrate the central function of IRF4 in the development of Th9 cells and underline the contribution of this T helper cell subset to the pathogenesis of asthma.
[Show abstract][Hide abstract] ABSTRACT: Though widely used, the BCG vaccine has had little apparent effect on rates of adult pulmonary tuberculosis. Moreover, the risk of disseminated BCG disease in immunocompromised individuals means that improved TB vaccines ideally need to be able to efficiently prime mycobacterially-naïve individuals as well as boost individuals previously vaccinated with BCG. Protective immunity against Mycobacterium tuberculosis is thought to depend on the generation of a Th1-type cellular immune response characterized by interferon-gamma (IFN-gamma) production. In the present study, we monitored safety and IFN-gamma responses in healthy TB-naïve humans receiving an entirely novel vaccine, composed of the fusion protein Ag85B-ESAT-6, administered at 0 and 2 months either as recombinant protein alone or combined with two concentrations of the novel adjuvant IC31. Vaccination did not cause local or systemic adverse effects besides transient soreness at the injection site, but it elicited strong antigen-specific T cell responses against H1 and both the Ag85B and the ESAT-6 components. These strong responses persisted through 2.5 years of follow-up, indicating the induction of a substantial memory response in the vaccine recipients.
[Show abstract][Hide abstract] ABSTRACT: Originally identified as antiviral substances produced by infected cells, type I interferons (IFN-I) are now known to have a wide range of additional activities within both the innate and adaptive immune response. Here we review properties of IFN-I contributing to their 'natural immune adjuvant' character, and their important role for the function of complete Freund's adjuvant (CFA) and the TLR9-dependent immune adjuvant IC31. We show data to demonstrate that treatment with IFN-I boosts the ability of vaccine/adjuvant combinations to induce peptide-specific CTL in both young and old mice. We view these findings in the perspective of previous clinical applications of IFN-I for vaccination.
[Show abstract][Hide abstract] ABSTRACT: Phosphorylation of transcription factor STAT-1 on Y701 regulates subcellular localization whereas phosphorylation of the transactivating domain at S727 enhances transcriptional activity. In this study, we investigate the impact of STAT-1 and the importance of transactivating domain phosphorylation on the induction of peptide-specific CTL in presence of the TLR9-dependent immune adjuvant IC31. STAT-1 deficiency completely abolished CTL induction upon immunization, which was strongly reduced in animals carrying the mutation of the S727 phospho-acceptor site. A comparable reduction of CTL was found in mice lacking the type I IFN (IFN-I) receptor, whereas IFN-gamma-deficient mice behaved like wild-type controls. This finding suggests that S727-phosphorylated STAT-1 supports IFN-I-dependent induction of CTL. In adoptive transfer experiments, IFN-I- and S727-phosphorylated STAT-1 were critical for the activation and function of dendritic cells. Mice with a T cell-specific IFN-I receptor ablation did not show impaired CTL responses. Unlike the situation observed for CTL development S727-phosphorylated STAT-1 restrained proliferation of naive CD8(+) T cells both in vitro and following transfer into Rag-deficient mice. In summary, our data reveal a dual role of S727-phosphorylated STAT-1 for dendritic cell maturation as a prerequisite for the induction of CTL activity and for T cell autonomous control of activation-induced or homeostatic proliferation.
The Journal of Immunology 09/2009; 183(4):2286-93. DOI:10.4049/jimmunol.0901383 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IC31 is a novel adjuvant which combines the immunostimulatory effects of an 11-mer antibacterial peptide (KLKL(5)KLK) and a synthetic oligodeoxynucleotide (ODN1a) which is a Toll-like receptor 9 agonist without containing cytosine phosphate guanine (CpG) motifs. The effects of IC31 on neonatal immune response to vaccination have not been reported. Neonatal mice were immunized once or twice with a Streptococcus pneumoniae serotype 1 polysaccharide conjugate containing Tetanus Toxoid (Pnc1-TT) carrier protein, with or without IC31 or CpG-ODN. IC31 significantly enhanced IgG1, IgG2a and IgG2b antibodies (Ab) to the serotype 1 polysaccharide. One dose of Pnc1-TT and low dose IC31 elicited high Ab levels that protected the neonatal mice completely from bacteraemia and significantly reduced lung infection following i.n. challenge with serotype 1 pneumococcal strain. One-sixth of an adult murine dose of IC31 was sufficient and optimal for induction of protective immunity in neonatal mice. Two doses of Pnc1-TT with or without adjuvants protected the neonatal mice completely, but more rapid Ab response was observed when IC31 was given with the Pnc1-TT. IC31 is a promising new adjuvant for neonatal vaccinations, rapidly enhancing protective humoral responses when combined with Pnc1-TT.
[Show abstract][Hide abstract] ABSTRACT: We showed previously that Tyk2(-/-) natural killer cells lack the ability to lyse leukemic cells. As a consequence, the animals are leukemia prone. Here, we show that the impaired tumor surveillance extends to T cells. Challenging Tyk2(-/-) mice with EL4 thymoma significantly decreased disease latency. The crucial role of Tyk2 for CTL function was further characterized using the ovalbumin-expressing EG7 cells. Tyk2(-/-) OT-1 mice developed EG7-induced tumors significantly faster compared with wild-type (wt) controls. In vivo assays confirmed the defect in CD8(+) cytotoxicity on Tyk2 deficiency and clearly linked it to type I IFN signaling. An impaired CTL activity was only observed in IFNAR1(-/-) animals but not on IFNgamma or IL12p35 deficiency. Accordingly, EG7-induced tumors grew faster in IFNAR1(-/-) and Tyk2(-/-) but not in IFNgamma(-/-) or IL12p35(-/-) mice. Adoptive transfer experiments defined a key role of Tyk2 in CTL-mediated tumor surveillance. In contrast to wt OT-1 cells, Tyk2(-/-) OT-1 T cells were incapable of controlling EG7-induced tumor growth.
Cancer Research 02/2009; 69(1):203-11. DOI:10.1158/0008-5472.CAN-08-1705 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have monitored the effects of KLKL(5)KLK (KLK), a derivative of a natural cationic antimicrobial peptide (CAP) on isolated membrane vesicles, and investigated the partition of the peptide within these structures. KLK readily interacted with fluorescent dyes entrapped in the vesicles without apparent pore formation. Fractionation of vesicles revealed KLK predominantly in the membrane. Peptide-treated vesicles appeared with generally disorganized bilayers. While KLK showed no effect on osmotic resistance of human erythrocytes, dramatic decrease in core and surface membrane fluidity was observed in peptide-treated erythrocyte ghosts as measured by fluorescence anisotropy. Finally, CD spectroscopy revealed lipid-induced random coil to beta-sheet and beta-sheet to alpha-helix conformational transitions of KLK. Together with the oligonucleotide oligo-d(IC)(13) [ODN1a], KLK functions as a novel adjuvant, termed IC31. Among other immunological effects, KLK appears to facilitate the uptake and delivery of ODN1a into cellular compartments, but the nature of KLK's interaction with the cell surface and other membrane-bordered compartments remains unknown. Our results suggest a profound membrane interacting property of KLK that might contribute to the immunostimulatory activities of IC31.
Cell Biology International 09/2008; 32(11):1449-58. DOI:10.1016/j.cellbi.2008.08.024 · 1.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The compromised immune responses in the elderly as well as the threat of pandemic influenza necessitate the development of improved influenza vaccines. This study provides evidence that IC31, a two-component synthetic adjuvant signalling through TLR-9, augments humoral and cellular immune responses to seasonal influenza vaccines. Experiments performed in young adult mice showed increased HI titres and higher levels of IgG2a antibodies that were accompanied by the induction of IFN-gamma producing CD4(+) T cells after single vaccination with reduced doses of vaccine antigens, even 200 days after single immunisation. Importantly, similar effects were seen in aged mice, although most pronounced upon booster immunisation. Thus, IC31 fulfils important criteria of novel influenza vaccine adjuvants.
[Show abstract][Hide abstract] ABSTRACT: Vaccine efficacy largely depends upon DC targeting and activation. The most potent TLR soluble ligands induce diffuse DC activation, which may be associated with marked pro-inflammatory responses and possibly adverse effects. This raises the concern that effective vaccine adjuvants may similarly rely on widespread DC activation. Using a promising candidate vaccine against tuberculosis (fusion protein of Ag85B and 6-kDa early secretory antigenic target (ESAT-6)) formulated in the potent IC31 adjuvant, DC targeting and activation was studied in vivo, following the fate of antigen and adjuvant in the draining lymph nodes, to define the magnitude of DC targeting/activation required in vivo to induce protective vaccine responses. Unexpectedly, protective IFN-gamma-mediated Ag85B-ESAT-6/IC31 responses were associated to the activation of a minute population (less than 0.3%) of CD11c(+) lymph node DC, without detectable systemic pro-inflammatory responses. This activated peripheral tissue-derived DC population, characterized by enhanced CD80, CD86, CD40 and IL-12p40 expression, was only identified when focusing on adjuvant- or antigen-labeled CD11c(+) DC, which were found to support T cell proliferation. Immunization with aluminum hydroxide adjuvant (Alum) resulted in a similar proportion of antigen-associated DC but without detectable enhancement of CD80, CD86, CD40 or IL-12p40 expression. Thus, potent protective IFN-gamma-producing responses may be elicited by the exquisite activation of a minute number of in vivo targeted DC.
European Journal of Immunology 06/2008; 38(5):1247-56. DOI:10.1002/eji.200737889 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: With the exception of some live vaccines, e.g. BCG, subunit vaccines formulated with "classical" adjuvants do not induce similar responses in neonates as in adults. The usual neonatal profile is characterized by lower levels of TH1-associated biomarkers. This has hampered the development of new neonatal vaccines for diseases that require early protection. Tuberculosis is one of the major targets for neonatal immunization. In this study, we assessed the immunogenicity of a novel candidate vaccine comprising a mycobacterial fusion protein, Ag85B-ESAT-6, in a neonatal murine immunization model.
The Ag85B-ESAT-6 fusion protein was formulated either with a classical alum based adjuvant or with the novel IC31 adjuvant. Following neonatal or adult immunization, 3 parameters were studied in vivo: (1) CD4(+) T cell responses, (2) vaccine targeting/activation of dendritic cells (DC) and (3) protection in a surrogate mycobacterial challenge model. Conversely to Alum, IC31 induced in both age groups strong Th1 and Th17 responses, characterized by multifunctional T cells expressing IL-2 and TNF-alpha with or without IFN-gamma. In the draining lymph nodes, a similarly small number of DC contained the adjuvant and/or the antigen following neonatal or adult immunization. Expression of CD40, CD80, CD86 and IL-12p40 production was focused on the minute adjuvant-bearing DC population. Again, DC targeting/activation was similar in adults and neonates. These DC/T cell responses resulted in an equivalent reduction of bacterial growth following infection with M. bovis BCG, whereas no protection was observed when Alum was used as adjuvant.
Neonatal immunization with the IC31-adjuvanted Ag85B-ESAT-6 subunit vaccine elicited adult-like multifunctional protective anti-mycobacterial T cell responses through the induction of an adult pattern of in vivo DC activation.
PLoS ONE 02/2008; 3(11):e3683. DOI:10.1371/journal.pone.0003683 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptor (TLR) agonists have a proven potential to become the adjuvants of the next generation when admixed and formulated with all kinds of vaccine compositions. The quality and magnitude of a vaccine-induced immune response is often strongly facilitated by TLR agonists, with the result that protection is increased and expanded toward type 1-driven immunity. DNA oligodeoxynucleotides bind to TLR9 and have been tested in a variety of vaccine settings with encouraging results. Combining oligodeoxynucleotides with poly-L-arginine (IC30) or certain artificial antimicrobial peptides dramatically improves and synergizes with the adjuvant action of TLR9 agonists, a notion that has prompted the development of IC31, an adjuvant with a promising profile in both preclinical and clinical trials.
[Show abstract][Hide abstract] ABSTRACT: IC31, the combination of a novel immunostimulatory oligodeoxynucleotide containing deoxy-Inosine/deoxy-Cytosine (ODN1a) and the antimicrobial peptide KLKL(5)KLK, represents a promising novel adjuvant signaling via the TLR9/MyD88-dependent pathway of the innate immune system. In mice, IC31 induces potent peptide-specific type 1 cellular immune responses, as well as mainly type 1 dominated protein-specific cellular and humoral immune responses. In addition, cytotoxic T lymphocytes were induced, able to kill efficiently target cells in vivo. Activation of murine dendritic cells by IC31 induced efficiently proliferation of naïve CD4(+) TCR transgenic T cells (DO.11.10) as well as their differentiation into IFN-gamma- and IL-4-producing T cells in vitro.
[Show abstract][Hide abstract] ABSTRACT: In this study, we evaluated the potential of a novel synthetic adjuvant designated IC31 for the ability to augment the immune response and protective efficacy of the well-known mycobacterial vaccine antigen, Ag85B-ESAT-6. The IC31 adjuvant, consisting of a vehicle based on the cationic peptide KLKL(5)KLK and the immunostimulatory oligodeoxynucleotide ODN1a signalling through the TLR9 receptor, was found to promote highly efficient Th1 responses. The combination of Ag85B-ESAT-6 and IC31 exhibited significant levels of protection in the mouse aerosol challenge model of tuberculosis and a detailed analysis of the immune response generated revealed the induction of CD4 T cells giving rise to high levels of IFN-gamma secretion. Furthermore, the combination of Ag85B-ESAT-6/IC31 was found to confer efficient protection in the guinea pig aerosol model of tuberculosis infection and is at present moving towards clinical testing.