CpG-containing Oligodeoxynucleotides Research Articles

Expression of Concern: A Palindromic CpG-Containing Phosphodiester Oligodeoxynucleotide as a Mucosal Adjuvant Stimulates Plasmacytoid Dendritic Cell-Mediated TH1 Immunity.

Expression of Concern: A Palindromic CpG-Containing Phosphodiester Oligodeoxynucleotide as a Mucosal Adjuvant Stimulates Plasmacytoid Dendritic Cell-Mediated TH1 Immunity.

CpG-Containing Oligodeoxynucleotides and Freund Adjuvant in Combination with Alum Augment the Production of Monoclonal Antibodies Against Recombinant HBsAg.

Background:Adjuvants are essential to potentiate the immune response to inoculated antigens and play a central role in vaccine development. Alum is generally used as a classic adjuvant, although it does not stimulate proper immunity, and some of the immunized subjects have low or no antibody response. Efforts have been continued to find more efficient adjuvants for better antibody responses. In the present study, the efficacy of three formulations of adjuvants, i.e. Cysteine p Guanine Oligodeoxynucleotide (CpG ODN), alum, and Freund, in the production of monoclonal anti Hepatitis B Surface Antigen (HBsAg) antibodies was investigated.Methods:To immunize mice, regular hepatitis B vaccine containing recombinant HBsAg and alum was used with CpG ODN or Freund adjuvants, and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by Enzyme-Linked Immunosorbent Assay (ELISA) using HBsAg as coating antigen followed by a limited dilution process.Results:The results showed that by using all three formulations of adjuvants, monoclonal antibody (mAb) specific to HBsAg was successfully generated. It was also found that the mice immunized with (HBsAg + Alum) + CpG had the highest concentration of antibody production in serum and hybridoma supernatants as well as positive clones. Based on these findings, the addition of CpG ODN also induced a higher antibody response compared with Complete Freund’s Adjuvant (CFA).Conclusion:Results of this study showed that CpG and Freund adjuvants could be efficient partners for alum in the immunization period of the process of monoclonal antibody production.

Abstract P4-06-02: Chemoimmunotherapy with cyclophosphamide plus a toll-like receptor 9 (TLR9) agonist eradicates triple negative breast cancer in a murine model

Abstract Background. Toll-like receptors (TLR) recognize conserved molecular patterns expressed by microbes and together with other molecular sensors serve as a first line of defense, inducing soluble and cellular mediators of innate immunity and initiating key steps of an adaptive immune response. The use of TLR agonists for therapeutic purposes relies on the ability of these compounds to induce, at least partially, some of the immune events that occur during natural infections. For example, synthetic CpG-containing oligodeoxynucleotides (ODN) act as TLR9 agonists, mimicking stimulation of the immune system by bacterial or viral DNA. CpG ODNs are being developed for cancer immunotherapy based on their capacity to stimulate innate and adaptive anti-tumor responses. In this study, we examined the efficacy of intratumorally administered CpG-ODN 1826 alone or in combination with immunomodulatory antibodies (anti-PD-1, anti-OX40, anti-CTLA4) or chemotherapy (cyclophosphamide, paclitaxel) in the syngeneic mouse 4T1 breast tumor model. Methods. Tumors were implanted orthotopically in the mammary fat pads on the left and right flanks while only one tumor was injected with CpG-ODN 1826. Therapy began 12-14 days post tumor injection when tumors were 8-10 mm in diameter. CpG-ODN 1826 (100 ug) was administered intratumorally, while immunomodulatory antibodies (anti-PD-1 [200 ug], anti-CTLA4 [100 ug], anti-OX40 [400 ug]) and chemotherapies (cyclophosphamide [150 mg/kg], paclitaxel [10 mg/kg]) were administered intraperitoneally. Tumor volume was monitored on both flanks to assess direct and abscopal/systemic anti-tumor activity. Tumor tissue obtained during the treatment regimen was used to evaluate therapy-induced changes in the immune microenvironment. Results. CpG-ODN 1826, administered intratumorally over 5 consecutive days induced complete regressions in ˜50% of the treated tumors, but only delayed growth in the distant lesions. The immunomodulatory antibodies had little effect on their own and did not add to the therapeutic efficacy of CpG-ODN 1826. The best therapeutic efficacy was obtained with a combination of weekly cyclophosphamide + CpG-ODN 1826 resulting in complete regression of both the CpG injected tumors and the contralateral tumors in 100% of the mice. Conclusions. These data support the clinical investigation of the combination of a TLR9 agonist (CpG-ODN) with cyclophosphamide in women with breast cancer. Citation Format: Campbell MJ, Tandon V, Senman B, Li J, Esserman L. Chemoimmunotherapy with cyclophosphamide plus a toll-like receptor 9 (TLR9) agonist eradicates triple negative breast cancer in a murine model [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-02.

Novel strategy to enhance antigen presentation and CD8 T cell immunity for curative efficacy of intranasal therapeutic HPV peptide vaccination in preclinical vaginal HPV tumor model

Abstract Immune checkpoint modulation to enhance antitumor immunity while minimizing immune suppressive responses within tumors has proven to be a highly successful cancer treatment strategy, but, often with toxic side effects and financial constraints. Using preclinical models of human papillomavirus (HPV) cancers, we tested vaccination employing adjuvants with diverse modes of action to promote antigen presentation and T cell immunity. We employed the combination of α-galactosylceramide (α-GalCer), a potent inducer of NKT cells and CpG-containing oligo-deoxynucleotide (CpG-ODN) that function through the TLR9 pathway. Since most HPV tumors occur at mucosal tissues, we used the mucosal intranasal route for vaccination with E6 and E7 peptides representing tumor-specific antigens to treat vaginal HPV+ TC-1 tumors in mice. Previously, we observed that vaccination using α-GalCer adjuvant alone reduced tumor growth but, complete and durable tumor regression required supplementing the vaccine with anti-4-1BB checkpoint modulating antibody that has clinical safety concerns. We now observed that intranasal HPV peptide vaccine incorporating α-GalCer and CpG-ODN adjuvants, rendered tumor-free survival advantage. Correlates of tumor protection included doubling the number of CD8 T cells exhibiting enhanced cytotoxic potential within the tumor and 84% increase in the ratio of CD8 T cells to regulatory T cells relative to vaccination with either adjuvant alone. Intranasal vaccination delivering α-GalCer plus CpG-ODN allows for the activation of dendritic cells through both classical and alternative licensing pathways and permits the secretion of a diverse set of chemokines to attract enhanced populations of naïve CD8+ T cells.

Phosphodiester backbone of the CpG motif within immunostimulatory oligodeoxynucleotides augments activation of Toll-like receptor 9

Toll-like receptor 9 (TLR9) stimulatory CpG-containing oligodeoxynucleotides (ODNs) with phosphorothioate backbones have successfully replaced the naturally occurring agonists of TLR9 in drug development due to their increased stability. Replacing the nonbridging oxygen with a sulfur atom in the phosphate linkage of ODNs has been accepted as having a minor impact on the chemical and physical properties of the agonists. Here, we report that the TLR9 binding site exhibits a strong bias in favor of a phosphodiester backbone over the phosphorothioate backbone of the CpG motif. Furthermore, we show that while single point mutations of W47, W96 and K690 within the TLR9 binding site retains full TLR9 activation by phosphodiester-based ODNs, activation by phosphorothioate-based ODNs is strongly impaired. The substitution of a phosphorothioate linkage for a phosphodiester linkage of just the CpG motif considerably improves the activation potency of a phosphorothioate-based oligonucleotide for human B-cells and plasmacytoid dendritic cells, as well as for mouse bone marrow-derived dendritic cells and macrophages. Our results highlight the functional significance of the phosphodiester linkage of a CpG dinucleotide for binding, which is important in designing improved immunostimulatory TLR9 agonists.

Multiple Immunosuppressive Effects of CpG-c41 on Intracellular TLR-Mediated Inflammation.

A growing body of literature suggests that most chronic autoimmune diseases are associated with inappropriate inflammation mediated by Toll-like receptor (TLR) 3, TLR7/8, or TLR9. Therefore, research into blocking TLR activation to treat these disorders has become a hot topic. Here, we report the immunomodulatory properties of a nonstimulatory CpG-containing oligodeoxynucleotide (CpG-ODN), CpG-c41, which had previously only been known as a TLR9 antagonist. In this study, we found that both in vitro and in vivo CpG-c41 decreased levels of various proinflammatory factors that were induced by single activation or coactivation of intracellular TLRs, but not membrane-bound TLRs, no matter what downstream signal pathways the TLRs depend on. Moreover, CpG-c41 attenuated excessive inflammation in the imiquimod-induced psoriasis-like mouse model of skin inflammation by suppressing immune cell infiltration and release of inflammatory factors. We also found evidence that the immunosuppressive effects of CpG-c41 on other intracellular TLRs are mediated by a TLR9-independent mechanism. These results suggest that CpG-c41 acts as an upstream of signaling cascades, perhaps on the processes of ligand internalization and transfer. Taken together, these results suggest that CpG-c41 disrupts various aspects of intracellular TLR activation and provides a deeper insight into the regulation of innate immunity.

Vaccination of stage III/IV melanoma patients with long NY-ESO-1 peptide and CpG-B elicits robust CD8+ and CD4+ T-cell responses with multiple specificities including a novel DR7-restricted epitope

ABSTRACTLong synthetic peptides and CpG-containing oligodeoxynucleotides are promising components for cancer vaccines. In this phase I trial, 19 patients received a mean of 8 (range 1–12) monthly vaccines s.c. composed of the long synthetic NY-ESO-179–108 peptide and CpG-B (PF-3512676), emulsified in Montanide ISA-51. In 18/18 evaluable patients, vaccination induced antigen-specific CD8+ and CD4+ T-cell and antibody responses, starting early after initiation of immunotherapy and lasting at least one year. The T-cells responded antigen-specifically, with strong secretion of IFNγ and TNFα, irrespective of patients' HLAs. The most immunogenic regions of the vaccine peptide were NY-ESO-189–102 for CD8+ and NY-ESO-183–99 for CD4+ T-cells. We discovered a novel and highly immunogenic epitope (HLA-DR7/NY-ESO-187–99); 7/7 HLA-DR7+ patients generated strong CD4+ T-cell responses, as detected directly ex vivo with fluorescent multimers. Thus, vaccination with the long synthetic NY-ESO-179–108 peptide combined with the strong immune adjuvant CpG-B induced integrated, robust and functional CD8+ and CD4+ T-cell responses in melanoma patients, supporting the further development of this immunotherapeutic approach.

Interferon gamma (IFNg) production is associated to disease parameters in TLR9-induced secondary hemophagocytic lymphohistiocytosis (sHLH) in mice

IFNg is the pivotal mediator in the murine model of primary HLH. Mice given repeated injection with the TLR9 agonist, CpG-containing oligodeoxynucleotides, develop pathology resembling the human disease, sHLH. Coadministration of an anti-IL-10 Receptor (R) monoclonal antibody (mAb) with CpG induces more severe disease characterized also by hemophagocytosis (fulminant sHLH).

CpG-mediated modulation of MDSC contributes to the efficacy of Ad5-TRAIL therapy against renal cell carcinoma.

Tumor progression occurs through the modulation of a number of physiological parameters, including the development of immunosuppressive mechanisms to prevent immune detection and response. Among these immune evasion mechanisms, the mobilization of myeloid-derived suppressor cells (MDSC) is a major contributor to the suppression of antitumor T-cell immunity. Patients with renal cell carcinoma (RCC) show increased MDSC, and methods are being explored clinically to reduce the prevalence of MDSC and/or inhibit their function. In the present study, we investigated the relationship between MDSC and the therapeutic potential of a TRAIL-encoding recombinant adenovirus (Ad5-TRAIL) in combination with CpG-containing oligodeoxynucleotides (Ad5-TRAIL/CpG) in an orthotopic mouse model of RCC. This immunotherapy effectively clears renal (Renca) tumors and enhances survival, despite the presence of a high frequency of MDSC in the spleens and primary tumor-bearing kidneys at the time of treatment. Subsequent analyses revealed that the CpG component of the immunotherapy was responsible for decreasing the frequency of MDSC in Renca-bearing mice; further, treatment with CpG modulated the phenotype and function of MDSC that remained after immunotherapy and correlated with an increased T-cell response. Interestingly, the CpG-dependent alterations in MDSC frequency and function did not occur in tumor-bearing mice complicated with diet-induced obesity. Collectively, these data suggest that in addition to its adjuvant properties, CpG also enhances antitumor responses by altering the number and function of MDSC.

Effective TRAIL-based immunotherapy requires both plasmacytoid and CD8α dendritic cells

It is now appreciated that there are distinct subsets of dendritic cells (DC) with specialized functions. Plasmacytoid DC (pDC) and CD8α DC can contribute to the priming, activation and function of antitumor CD8 T cells; however, their specific roles and necessity in stimulating antitumor immunity are not clearly understood. We examined the importance of pDC and CD8α DC during immunotherapy of an orthotopic model of metastatic renal cell carcinoma. Immunotherapy that utilizes a recombinant adenovirus encoding tumor necrosis factor-related apoptosis-inducing ligand (Ad5-TRAIL) in combination with an immunostimulatory CpG-containing oligodeoxynucleotide (CpG) resulted in the clearance of primary and metastatic tumors in wild-type (WT) replete BALB/c mice and prolonged survival. In comparison, mice deficient in either pDC (accomplished using a depleting mAb specific for PDCA1) or CD8α DC (through utilization of CD8α DC-deficient Batf3(-/-) BALB/c mice) had uncontrolled tumor growth and high mortality after Ad5-TRAIL/CpG administration. The ineffectiveness of Ad5-TRAIL/CpG therapy in the anti-PDCA1-treated and Batf3(-/-) BALB/c mice was marked by an altered activation phenotype of the DC, as well as significantly reduced expression of type I IFN-stimulated genes and IL-15/IL-15R complex production. In addition, pDC-depleted and Batf3(-/-) BALB/c mice had significantly decreased effector CD8 T cell infiltration in the primary tumor site compared with WT mice after therapy. These data collectively suggest that pDC and CD8α DC carry out independent, but complementary, roles that are necessary to initiate an efficacious antitumor immune response after Ad5-TRAIL/CpG therapy.

A palindromic CpG-containing phosphodiester oligodeoxynucleotide as a mucosal adjuvant stimulates plasmacytoid dendritic cell-mediated T(H)1 immunity.

BackgroundCpG oligodeoxynucleotides (ODNs), resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC)-mediated TH1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA.MethodsTH1 and TH2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT) through nasal vaccination.ResultsG9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of TH1, but not TH2, -type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs.ConclusionsG9.1 is a promising mucosal adjuvant for induction of pDC-mediated TH1 immunity.

A limited CpG-containing oligodeoxynucleotide therapy regimen induces sustained suppression of allergic airway inflammation in mice

BackgroundCpG-containing oligodeoxynucleotides (CpG-ODNs) are potent inhibitors of T helper 2 mediated allergic airway disease in sensitised mice challenged with allergen. A single treatment has transient effects but a limited series...

Systemic Toll-Like Receptor Ligation and Selective Killing of Dendritic Cell Subsets Fail To Dissect Priming Pathways for Anti-Vaccinia Virus CD8+T Cells

CD8⁺ T cell responses can be generated by direct or cross-priming mechanisms, and several mouse models have been used to reveal which of these is the most important pathway for various viruses. Among these models is systemic treatment of mice with a CpG-containing oligodeoxynucleotide (CpG) to mature all dendritic cells (DCs), rendering them incapable of cross-presentation. A second is the use of cytochrome c (cytc) as a selective poison of the subsets of DCs able to cross-present antigen. In this study, using two vaccinia virus (VACV) strains, namely, WR and MVA, we found that the CpG and cytc methods gave conflicting data. Moreover, we show for both strains of VACV that treatment of mice with CpG and cytc inhibited CD8⁺ T cell responses to antigens designed to prime exclusively by direct presentation. Further investigation of the CpG method found that the extent to which priming is inhibited depends on the antigen examined, immunization route, replication ability of the virus, and, crucially, immunization dose. We suggest that greater caution is required when interpreting data using these methods and that priming pathways for antiviral CD8⁺ T cells are not simply separated according to DC subsets or their maturation state.

Induction of HCA587-Specific Antitumor Immunity with HCA587 Protein Formulated with CpG and ISCOM in Mice

HCA587 (also known as MAGE-C2) is a “cancer-testis” antigen highly expressed in a number of malignancies with unique immunological properties, making it a promising target for tumor immunotherapy. In this report, we demonstrated that HCA587 protein, when formulated with adjuvants CpG–containing oligodeoxynucleotides (CpG ODN) and ISCOM, was capable of inducing a potent cellular and humoral immune response as indicated by the presence of a large number of HCA587-specific, IFN-γ-producing CD4+ T cells and high levels of HCA587-specific antibodies. More importantly, vaccination with HCA587 conferred protection against challenge with HCA587-expressing B16 melanoma in prophylactic and therapeutic settings. In analysis of the mechanisms underlying the protective effect, we showed that the vaccination was followed by enhanced accumulation of tumor-infiltrating lymphocytes (TILs) with enrichment of conventional CD4+ T cells but reduced representation of Treg cells. Further, the antitumor effect was largely abrogated in mice either depleted of CD4+ T cells or deficient for IFN-γ. These results indicate that HCA587 protein vaccine possesses evident antitumor activity in a mouse model and holds promise for treatment of human cancers.

Leucine-rich Repeat 11 of Toll-like Receptor 9 Can Tightly Bind to CpG-containing Oligodeoxynucleotides, and the Positively Charged Residues Are Critical for the High Affinity

TLR9 is a receptor for sensing bacterial DNA/CpG-containing oligonucleotides (CpG ODN). The extracellular domain (ECD) of human TLR9 (hTLR9) is composed of 25 leucine-rich repeats (LRR) contributing to the binding of CpG ODN. Herein, we showed that among LRR2, -5, -8, and -11, LRR11 of hTLR9 had the highest affinity for CpG ODN followed by LRR2 and -5, whereas LRR8 had almost no affinity. In vitro, preincubation with LRR11 more significantly decreased CpG ODN internalization, subsequent NF-κB activation, and cytokine release than with LRR2 and -5 in mouse peritoneal macrophages treated with CpG ODN. The LRR11 deletion mutant of hTLR9 conferred decreased cellular responses to CpG ODN. Single- or multiple-site mutants at five positively charged residues of LRR11 (LRR11m1-9), especially Arg-337 and Lys-367, were shown to contribute to hTLR9 binding of CpG ODN. LRR11m1-9 showed reduced inhibition of CpG ODN internalization and CpG ODN/TLR9 signaling, supporting the above findings. Prediction of whole hTLR9 ECD-CpG ODN interactions revealed that Arg-337 and Lys-338 directly contact CpG ODN through hydrogen bonding, whereas Lys-347, Arg-348, and His-353 contribute to stabilizing the shape of the ligand binding region. These findings suggested that although all five positively charged residues within LRR11 contributed to its high affinity, only Arg-337 and Lys-338 directly interacted with CpG ODN. In conclusion, the results suggested that LRR11 could strongly bind to CpG ODN, whereas mutations at the five positively charge residues reduced this high affinity. LRR11 may be further investigated as an antagonist of hTLR9.

CpG-containing oligodeoxynucleotides increases resistance of Anopheles mosquitoes to Plasmodium infection

Unmethylated CpG dinucleotide motifs in bacterial DNA or in synthetic oligodeoxynucleotides (ODN) are potent stimulators of the vertebrate innate immune system. However, the potential of these DNA species to modulate mosquito immunity have not been explored. In the present study, we investigated the effects of CpG-ODN on the outcome of Plasmodium infection in insects and on the modulation of mosquito immunity to Plasmodium. Anopheles stephensi and Anopheles gambiae mosquitoes inoculated with CpG-ODN showed significant reductions in the prevalence of Plasmodium infection, intensity of Plasmodium infection, and number of eggs produced. Microarrays were used to elucidate the transcriptional profiles of the fat bodies of CpG-ODN-treated mosquitoes. In total, 172 genes were differentially expressed, of which 136 were up-regulated and 36 were down-regulated. The major functional class of CpG-ODN-regulated genes encoded immune response-related proteins (31%). Within this group, genes associated with coagulation/wound healing were the most frequently represented (23%). Knockdown of a transglutaminase gene that was up-regulated by the CpG-ODN and chemical inhibition of the enzyme resulted in a significant increase in Plasmodium infection. Mosquitoes that were treated with CpG-ODNs were found to be less susceptible to Plasmodium infection. Transcriptional profiling of the fat body suggests that protection is associated with coagulation/wound healing. We show for the first time that transglutaminase activity plays a role in the control of Plasmodium infection.

Dendritic Cell-Mediated Vaccination Relies on Interleukin-4 Receptor Signaling to Avoid Tissue Damage after Leishmania major Infection of BALB/c Mice

Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor alpha (IL-4Rα)-deficient (CD11ccreIL-4Rα−/lox) BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2×105 stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11ccreIL-4Rα−/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4Rα-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4Rα signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.

Systemic Treatment with CpG-B after Sublethal Rickettsial Infection Induces Mouse Death through Indoleamine 2,3-Dioxygenase (IDO)

Due to its strong immune stimulatory effects through TLR9, CpG-containing oligodeoxynucleotides (CpG ODN) have been tested in multiple clinical trials as vaccine adjuvant for infectious diseases and cancer. However, immune suppression induced by systemic administration of CpGs has been reported recently. In this study, we evaluated the impact of CpGs in an acute rickettsiosis model. We found that systemic treatment with type B CpG (CpG-B), but not type A CpG (CpG-A), at 2 days after sublethal R. australis infection induced mouse death. Although wild-type (WT) B6 and IDO−/− mice showed similar survival rates with three different doses of R. australis infection, treatment with CpG-B after sublethal infection consistently induced higher mortality with greater tissue bacterial loads in WT but not IDO−/− mice. Also, CpG-B treatment promoted the development of higher serum concentrations of proinflammatory cytokines/chemokines through IDO. Furthermore, while T cell-mediated immune responses enhanced by CpG-B were independent of IDO, treatment with CpG-B promoted T cell activation, PD-1 expression and cell apoptosis partially through IDO. A depletion study using anti-mPDCA-1 mAb indicated that plasmacytoid dendritic cells (pDC) were not required for CpG-B-induced death of R. australis-infected mice. Additionally, the results in iNOS−/− mice suggested that nitric oxide (NO) was partially involved in CpG-B-induced death of R. australis-infected mice. Surprisingly, pre-treatment with CpG-B before administration of a lethal dose of R. australis provided effective immunity in WT, IDO−/− and iNOS−/− mice. Taken together, our study provides evidence that CpGs exert complex immunological effects by both IDO-dependent and -independent mechanisms, and that systemic treatment with CpGs before or after infection has a significant and distinct impact on disease outcomes.

Contribution of human neutrophils in the development of protective immune response during in vitro Leishmania major infection

Stimulation of neutrophils may potentiate immunity to Leishmania major. CpG-containing oligodeoxynucleotide (ODN) has immune stimulatory effects and has been suggested as adjuvants and therapeutics to potentiate efficacy of vaccines and treatments against leishmaniasis. Here, we examined the stimulatory effect of synthetic ODN containing CpG motifs class A and B on cytokine production by neutrophils. Neutrophils from healthy donors responded to CpG-ODN type A, but not to class B, with secretion of IL-8 and following GM-CSF pretreatment with TNF-α production. To test whether neutrophil responses were altered in cutaneous leishmaniasis (CL) and to better understand the role of neutrophils in susceptibility and resistance to disease, we evaluated cytokine responses in GM-CSF preconditioned neutrophils from asymptomatic (Leishmanin skin test positive, LST+) and nonhealing CL individuals to CpG-ODN class A and assessed the expression levels of toll-like receptors (TLR2), 4 and 9. LST+ and healthy donor, but not nonhealing CL neutrophils, responded with TNF-α secretion. Neutrophils from nonhealing CL displayed increased mRNA expression levels of TLR2, 4 and 9 compared to neutrophils from LST+ or healthy donors. Therefore, failure to cure CL is associated with reduced ability of neutrophils to secrete TNF-α and correlates with high TLR 2, 4 and 9 expressions.

Immunization with Live and DeadChlamydia muridarumInduces Different Levels of Protective Immunity in a Murine Genital Tract Model: Correlation with MHC Class II Peptide Presentation and Multifunctional Th1 Cells

Mice that were intranasally vaccinated with live or dead Chlamydia muridarum with or without CpG-containing oligodeoxynucleotide 1862 elicited widely disparate levels of protective immunity to genital tract challenge. We found that the frequency of multifunctional T cells coexpressing IFN-γ and TNF-α with or without IL-2 induced by live C. muridarum most accurately correlated with the pattern of protection against C. muridarum genital tract infection, suggesting that IFN-γ(+)-producing CD4(+) T cells that highly coexpress TNF-α may be the optimal effector cells for protective immunity. We also used an immunoproteomic approach to analyze MHC class II-bound peptides eluted from dendritic cells (DCs) that were pulsed with live or dead C. muridarum elementary bodies (EBs). We found that DCs pulsed with live EBs presented 45 MHC class II C. muridarum peptides mapping to 13 proteins. In contrast, DCs pulsed with dead EBs presented only six MHC class II C. muridarum peptides mapping to three proteins. Only two epitopes were shared in common between the live and dead EB-pulsed groups. This study provides insights into the role of Ag presentation and cytokine secretion patterns of CD4(+) T effector cells that correlate with protective immunity elicited by live and dead C. muridarum. These insights should prove useful for improving vaccine design for Chlamydia trachomatis.