体外筛选对犬具有免疫刺激活性的CpG寡脱氧核苷酸

建立并优化犬特异性寡脱氧核苷酸(CpGODN)的体外筛选条件,筛选自行设计的对犬具有免疫刺激活性的CpGODN。用CpGODN2006对犬的免疫细胞进行刺激,测定细胞增殖能力。对免疫细胞的来源、细胞铺板浓度、CpGODN的刺激时间和浓度及增值能力的测定方法等条件进行优化。并对自行设计的CpGODN进行了初步筛选。结果显示,犬脾细胞对CpGODN的反应性比外周血单个核细胞好。在犬脾细胞为6×105个/孔,37℃5%CO2条件下,经10mg/LCpGODN2006刺激48h,使脾细胞明显增生。3H-TdR掺入法测得的结果比MTT法更敏感。自行设计的CpGODNR008能显著地刺激犬脾细胞增生,且优于CpGODN2006(P0.05)。结果表明,优化了犬特异性CpGODN的体外筛选条件,并筛选出了对犬具有免疫刺激活性的CpGODN。     

Development of a simple IgE‐independent anaphylactic model using buckwheat antigen and B‐type CpG oligodeoxynucleotide from Streptococcus thermophilus

We developed a severe anaphylactic model in mice using buckwheat antigen and B‐type CpG‐oligodeoxynucleotides (CpG‐ODNs) from Streptococcus thermophilus genome. In typical systemic anaphylaxis models, animals are challenged with large quantity of antigens via an intravenous (i.v.) route. Here, we showed a simple anaphylactic shock after challenge via intraperitoneal (i.p.) route. The i.p. method is simpler than i.v. administration and has a lower risk for failure. To generate this anaphylactic model, 5‐week‐old female BALB/c mice were first i.p. sensitized with buckwheat antigen mixed with B‐type CpG‐ODN. After 2 weeks, mice were challenged with antigen to induce anaphylactic shock, which was evaluated by scoring the severity symptoms and measuring serum levels of various proteins and splenic cell producing cytokines. Immunoglobulin (Ig)G_2a production and interferon‐γ positive cells were markedly increased in mice immunized with antigen mixed with B‐type CpG‐ODN, whereas serum IgE levels were decreased by B‐type CpG‐ODN. We also examined the effects of various ODNs (A, B and C‐type CpG‐ODNs) and antigens (buckwheat, α‐casein, β‐lactoglobulin and ovalbumin) on anaphylactic severity, and found that the combination of buckwheat and B‐type CpG‐ODN induced the most intense anaphylactic shock. This model is expected to contribute to the study of the prevention of anaphylactic shock.     

Attenuated cytokine responses in porcine lymph node cells stimulated with CpG DNA are associated with low frequency of IFNalpha-producing cells and TLR9 mRNA expression

The immune stimulatory effects of synthetic CpG DNA, on porcine peripheral blood mononuclear cells (PBMC) have been reported, but little is known about CpG-induced responses in other lymphoid tissues of pigs. We investigated innate immune responses induced by CpG DNA in cells from blood, lymph nodes (LN) and spleens of pigs. Porcine PBMC and lymph node cells (LNC) were stimulated in vitro with three classes (A-, B- and C-class) of CpG oligodeoxynucleotides (ODNs), and a non-CpG control ODN. All three classes of CpG ODNs induced significant production of IFNalpha, TNFalpha, IL-1, IL-6 and IL-12 in PBMC. In contrast, in LNC, only IL-12 was stimulated by all three classes of CpG ODNs, while IFNalpha, and IL-6 were induced by A- and C-class ODNs. No TNFalpha was induced in LNC by any of the ODNs. Significant lymphocyte proliferation was induced in PBMC by all three classes of CpG ODNs and non-CpG control. However, in LNC, B- and C-class ODNs induced significant proliferation, while no proliferation was seen with A-class and non-CpG control ODN. All three classes of ODNs induced NK-like cytotoxicity in PBMC and spleen cells, but were less effective in inducing NK cytotoxicity in LNC. We then investigated the reasons for the relatively poor CpG-induced responses in LNC. Our investigations revealed that LNC had a lower frequency of IFNalpha-secreting cells and expressed low levels of TLR9 mRNA compared to PBMC. We conclude that the lower number of IFNalpha-secreting cells and receptor expression may contribute to the attenuated responses in LNC following stimulation with CpG ODN.     

All three classes of CpG ODNs up-regulate IP-10 gene in pigs

The analysis of CpG ODN induced innate immune responses in different animal species has shown substantial similarities and differences in levels and types of induced cytokines profile. The objectives of these studies were to identify innate immune biomarkers activated by three classes of CpG ODNs in pigs. For this purpose, we investigated the kinetics of innate immune responses in immune cells from pigs following in vitro and in vivo stimulation with CpG ODNs. The mRNA expression of cytokine and chemokine genes were assayed by SYBR^@ green based quantitative real time PCR. A-class CpG ODN induced significant but transient levels of IFN-γ, IL-12 (P40), IL-6, IL-4 and TNF-α mRNA, C-class CpG ODN induced significant level of IFN-γ, IFN-α and IL-12 mRNA and the lowest level of IL-4 (Th-2 type) mRNA. A very low level of some cytokines stimulation was observed by GC ODNs. It is noteworthy, that IL-12 (P35) mRNA was significantly stimulated by B-class GpC ODN 7909. Interestingly, all classes of CpG ODNs induced significant level of IP-10 at 12 h post stimulation. These in vitro and in vivo observations suggest that interferon-γ inducible protein 10 (IP-10) may be a reliable biomarker for immune activity induced by CpG ODNs in pigs.     

CpG oligodeoxynucleotides stimulate canine and feline immune cell proliferation.

Oligodeoxynucleotides (ODNs) with unmethylated CpG dinucleotide motifs may be useful as non-specific immune system stimulants and adjuvants for protein or nucleic acid vaccines in humans and other primates. They may also be useful in cancer immunotherapy and in the modulation of allergic responses or mucosal immunity. To begin to determine the potential utility of CpG ODN technology in small animal veterinary medicine, we developed procedures to analyze the effects of CpG ODN on canine and feline blood, spleen and lymph node (LN) cells. We find that certain CpG ODN cause good lymphocyte proliferation (as monitored by [(3)H]-thymidine incorporation) in both canine and feline spleen and LN cells, but not in blood. This overall stimulatory effect of CpG ODN on spleen and LN cells is CpG dependent. The reverse sequences, GpC ODNs, do not cause significant lymphocyte proliferation in the cat; however, dogs are more sensitive to stimulation by the non-specific immune effects of the phosphorothioate backbone. We conclude that unmethylated CpG ODNs may also have potential uses as immune stimulants for vaccines and other antimicrobial agents in veterinary medicine for companion animals.     

CpG寡核苷酸增强鸡新城疫疫苗免疫效力的研究

为了探讨CpG寡核苷酸（CpG oligonucleotide,CpG ODN）对鸡新城疫疫苗免疫效力的影响,将CpG2007与鸡淋巴细胞共孵育,测定淋巴细胞增殖率,结果发现CpG2007对鸡淋巴细胞具有显著的刺激活性。将CpG2007与不同浓度的新城疫抗原混合,制备灭活疫苗,免疫健康雏鸡。分别于免疫后不同时间采血,测定抗体效价和细胞因子表达量,并进行攻毒保护试验。结果发现,添加CpG ODN佐剂的试验组均比对应相同抗原剂量的免疫对照组的抗体水平高,产生抗体速度快;抗原剂量降低10倍的佐剂试验组与高抗原剂量免疫对照组抗体水平和攻毒保护率均相当,表明CpG ODN能显著增强新城疫疫苗的免疫效力,能促进机体产生更强烈的免疫应答,是有效的疫苗佐剂候选物质。     

Novel B regs down-regulate TLR9-induced cytokine responses in sheep Peyer's patches

Regulation of TLR responses is required in the intestine to prevent unnecessary responses to commensal microorganisms and maintain tissue homeostasis. Several mechanisms have been proposed for the regulation of TLR responses in intestinal epithelial and lamina propria cells. However, whether this regulation occurs in Peyer's patches (PP) is not known. While investigating cellular responses to the TLR9 agonist CpG ODN, we observed that PP cells responded poorly to CpG ODN stimulation despite expressing TLR9. We hypothesized that PP cells produced the immunoregulatory cytokine IL-10 which suppressed TLR-induced cytokine responses. In vitro neutralization of IL-10 or depletion of CD21+ B cells from PP resulted in significant increases in IL-12, IFNγ and IFNα responses in PP cells stimulated with CpG ODN. Essentially, our investigation have identified a novel population of IL-10-secreting B cells in PP with regulatory functions (B(regs)). These B(regs) may play an important role in the maintenance of intestinal homeostasis.     

CpG oligodeoxynucleotides stimulate immune cell proliferation but not specific antibody production in rainbow trout (Oncorhynchus mykiss)

Bacterial DNA and CpG ODN have both been shown to have immunostimulatory effects in mammals, activating APCs and inducing a potent Th1 type immune response. They have also been shown to have a strong adjuvant effect and up-regulate MHC class 2 expression in murine cells, augment human and murine NK cell lytic activity, activate human B cells and induce murine B cell proliferation. However, little work has been carried out with regard to their effects on the piscine immune system. Here it is shown that various CpG ODN induce proliferation of peripheral blood leucocytes, spleen and head kidney cells from rainbow trout although, at the range of concentrations tested CpG ODN 2133 lacked the ability to induce specific antibody production to a protein antigen.     

Characteristics of oligodeoxyribonucleotides that induce interferon (IFN)-alpha in the pig and the phenotype of the IFN-alpha producing cells

The immunostimulatory effects of oligodeoxyribonucleotides (ODN) containing unmethylated CpG dinucleotides (CpG-ODN) in certain base contexts have been extensively studied in man and mice. One major action is their ability to trigger production of massive amounts of interferon-alpha (IFN-alpha) by plasmacytoid dendritic cells (PDC), also referred to as natural IFN-alpha/beta producing cells (NIPC). The present study using porcine PBMC activated by CpG-ODN or plasmid DNA revealed a considerable variation in the IFN-alpha production in response to various CpG-ODN constructs. Several phosphodiester ODNs, such as 5' TTTTCAATTCGAAGATGAAT 3' (ODN H), and the plasmid pcDNA3 all required pre-incubation with lipofectin in order to induce IFN-alpha. Intact unmethylated CpGs were also important, because methylation or substitution of the cytosines and CpG-inversion strongly reduced the IFN-alpha induction by single- or double-stranded forms of ODN H. Certain CpG-ODNs that contained flanking phosphorothioate or phosphodiester poly-G sequences were potent inducers of IFN-alpha without pre-incubation with lipofectin, for instance the ODN 2216 (5' GGGGGACGATCGTCGGGGGG 3'). While poly-G sequences have been suggested to increase uptake of ODNs by cells, they did not obviate the need for lipofectin when added to the ODN H. However, they resulted in up to five-fold increases of the IFN-alpha levels caused by ODN H upon lipofection, indicating other enhancing effects of poly-G sequences on the induction of IFN-alpha. The identity of the IFN-alpha producing cells (IPC) stimulated by CpG-ODN or plasmid DNA was studied by means of flow cytometry using combined staining for intracellular IFN-alpha and surface markers. Approximately 1-3 IPC/10(3) PBMC were detected, compared to only 3 IPC/10(4) PBMC stimulated by Aujeszky's disease virus. The IPC frequencies were confirmed by detection of IFN-alpha mRNA positive cells by in situ hybridisation. The IPC induced by CpG-ODN or plasmid DNA had a similar phenotype, expressing CD2 and CD4 and intermediate levels of MHC class II and the myeloid marker SWC3, but not the markers of T and B cells or monocytes (CD3, CD21 and CD14). Consequently, porcine IPC that respond to CpG-DNA seem to correspond to the PDC/NIPC.     

Effects of CpG-B ODN on the protein expression profile of swine PBMC

The CpG motif within bacterial DNA is a potent immuno-stimulatory moiety. Here, using a 2-D electrophoretic approach, we investigated the effect of synthetic oligodeoxynucleotide containing a B type CpG motif (CpG-B ODN) on the protein expression profile of swine peripheral blood mononuclear cells (PBMC). We found that several proteins including spondin 1, N-acetolactate alpha linked acidic dipeptidase; V kappa light chain, T cell receptor variable alpha chain, heat shock protein (Hsp) 60, Hsp70, KIAA0857 protein, and PNAS-146 were up-regulated in PBMC by CpG-B ODN stimulation. Further studies showed that CpG-B ODN-mediated Hsp60, Hsp70 and Hsp90 expressions were closely associated with the TLR9 signalling pathway. Pretreatment with inhibitors of Hsp70, Hsp90 and TLR9 all blocked the CpG-B ODN-mediated anti-apoptotic effect in swine PBMC. These results suggest that CpG-B ODN treatment of swine PBMC may enhance the expression of biologically active proteins, notably spondin 1, V kappa light chain, T cell receptor variable alpha chain and Hsps, which may play an important role in CpG-B ODN-mediated activation of immune responses and enhancement of swine PBMC survival.     

In vivo effects of CpG oligodeoxynucleotide on Eimeria infection in chickens

Poultry coccidiosis is the major parasitic disease of poultry and, until now, no recombinant vaccine has been developed. Short oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (CpG ODNs) have been shown to be effective immunoprotective agents and vaccine adjuvants in mammalian systems. Their use in poultry to protect against intracellular parasites has not been reported to date. The present work investigated the effects of CpG ODN treatment on host susceptibility to Eimeria infection in two chicken strains with different genetic background, SC and TK. The data show that CpG ODN enhanced the birds' resistance to coccidiosis in a normally susceptible chicken strain (TK), as shown by reduced oocyst shedding and improved weight gain. CpG treatment had a differential effect on body weight gains and serum antibody responses, depending on the chicken strain and ODN dose, delivery route, and backbone. This study shows for the first time that CpG ODNs could be used as immunoprotective agents in Eimeria-infected chickens to enhance resistance to the pathogen and improve performance. Future research is needed to optimize their use alone and as vaccine adjuvants that may lead to better and more efficient vaccine applications.     

CpG ODN对水貂免疫增强效果的研究

为获得对水貂具有免疫刺激活性的CpG ODN序列,设计合成了45种含不同CpG基序的DNA序列,应用MTS比色法测定合成CpG ODNs刺激水貂PBMC增殖的能力,结果有11条CpG ODN对水貂PBMC有刺激活性（SI＞2）;应用SI值大于5的6种CpG ODN分别与水貂伪狂犬灭活疫苗联合免疫水貂,免疫后经水貂血清抗伪狂犬中和抗体效价测定和水貂PBMC非特异性增殖效应检测,结果有3个CpG ODN序列对水貂具有较好免疫增强作用,分别是CpG-21(ATCGATTTGTCGTTATCGAT)、CpG-23(ATCGATGAACGTTAACGTTTC)和CpG-24(AACGTTCATCGATATCGATGT)。本研究获得3条对水貂具有较好免疫刺激活性的CpG ODN序列,可为水貂新型CpG佐剂的研究提供参考。     

Interferon-gamma and B-cell Activating Factor (BAFF) promote bovine B cell activation independent of TLR9 and T-cell signaling

We previously reported that CD21(+) B cells purified from bovine blood do not respond to CpG-ODN stimulation unless either CD14(+) monocytes or B-cell Activating Factor (BAFF), a cytokine produced by activated monocytes, are present. In this report, we present evidence that CD14(+) monocytes are critical for CpG-specific lymphocyte proliferation within the peripheral blood mononuclear cell (PBMC) population but that this response is not mediated by soluble factors produced by CpG-activated monocytes. We further determine that bovine monocytes stimulated with IFN-γ induce expression of the BAFF gene and that recombinant IFN-γ and BAFF induced robust B cell activation when cultured in the absence of CpG ODN. These data suggest that CpG-stimulated monocytes may indirectly promote B cell activation by promoting release of cytokines and/or other soluble factors from accessory cells which in turn act on CpG-stimulated B cells to promote antigen-independent and T cell independent B cell activation. Understanding the T cell independent signals that induce B cell activation has important implications for understanding B cell development in locations where T cells are limited and in understanding polyclonal B cell activation that may contribute to autoimmune diseases.     

Adjuvanted rush immunotherapy using CpG oligodeoxynucleotides in experimental feline allergic asthma

Allergic asthma is driven by relative overexpression of Th2 cell-derived cytokines in response to aeroallergens. In independent studies, both allergen-specific rush immunotherapy (RIT) and CpG oligodeoxynucleotides (ODN) showed promise in blunting eosinophilic inflammation in a model of feline allergic asthma. We hypothesized that RIT using allergen and CpG ODN would work synergistically to dampen the asthmatic phenotype in experimentally asthmatic cats. Twelve cats with asthma induced using Bermuda grass allergen (BGA) were studied. Of these, six were administered adjuvanted BGA RIT using CpG ODN #2142; six were administered placebo (saline) RIT and later crossed over to adjuvanted RIT. Over 2 days, subcutaneous CpG ODN (0.5ng/kg) with BGA (increasing doses every 2h from 20 to 200microg) was administered. Adverse events were recorded and compared with historical controls. Percentage of eosinophils in bronchoalveolar lavage fluid (BALF), % peripheral CD4+CD25+ T regulatory cells (Tregs), lymphocyte proliferation in response to ConA, and cytokine concentrations in BALF were measured over 2 months. Group mean BALF % eosinophils for the adjuvanted RIT cats were significantly lower at week 1 and month 1 (p=0.03 for both), and marginally significantly lower at month 2 (p=0.09) compared with placebo RIT cats. By the end of the study, 8/12 treated cats had BALF % eosinophils within the reference range for healthy cats. Adjuvanted RIT, but not placebo RIT, cats had significant decreases in the ConA stimulation index over time (p=0.05). BALF IL-4 concentrations were significantly higher at week 1 in adjuvanted RIT cats compared with baseline and month 2, and also with placebo RIT cats at week 1. No significant differences were detected between treatments or over time for IL-10 or IFN-gamma concentrations in BALF or for %Tregs cells in peripheral blood. Adjuvanted RIT using CpG ODN in experimental feline asthma dampens eosinophilic airway inflammation. Adverse effects associated with adjuvanted RIT were less severe compared with a historical, non-adjuvanted RIT protocol. The exact mechanism(s) by which adjuvanted RIT alters the aberrant allergic immune response were not elucidated in this study.     

Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides

Exposure to bacterial DNA generates a "danger signal" that stimulates cellular elements of the mammalian immune system to proliferate and/or secrete cytokines. Stimulation is critically dependent on hexameric motifs that contain an unmethylated CpG dinucleotide: these are commonly found in bacterial but not vertebrate DNA. Different motifs are optimally stimulatory in different species. This work examines whether oligodeoxynucleotides (ODNs) containing CpG motifs stimulate peripheral blood mononuclear cells from pigs. Results show that pigs respond to CpG ODN by proliferating and secreting IL-6, IL-12 and TNF-alpha. By screening a large panel (>100) of ODNs, the palindromic hexamer 'ATCGAT' was identified as being optimally active in all animals examined (N=10). These findings are the first to establish the immunostimulatory activity of CpG ODN in pigs, and suggest that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.     

Enhancing mucosal immunity in mice by recombinant adenovirus expressing major epitopes of porcine circovirus-2 capsid protein delivered with cytosine-phosphate-guanosine oligodeoxynucleotides

A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4⁺ T cells and IFN-γ-producing CD8⁺ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3⁺, CD3⁺CD4⁺CD8^-, and CD3⁺CD4^-CD8⁺ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.     

TLR9 agonists: immune mechanisms and therapeutic potential in domestic animals

Toll like receptors (TLRs) are transmembrane glycoproteins that recognize conserved microbial molecules. Engagement of TLRs activates innate and adaptive immunity. TLR-mediated activation of immune cells results in upregulation of cytokines, chemokines and costimulatory molecules. These early innate responses control pathogen spread and initiates adaptive immune responses. Synthetic CpG oligodeoxynucleotides (ODN), agonists for TLR9, had shown great promise as immunotherapeutic agents and vaccine adjuvants in laboratory animal models of infectious disease, allergy and cancer. However, it has become apparent that CpG ODN are less potent immune activators in domestic animals and humans. The disparity in immune responses between rodents and mammals has been mainly attributed to differences in cellular expression of TLR9 in the various species. In this article, our current understanding of the immune mechanisms, as well as the potential applications of CpG ODN will be reviewed, with particular emphasis on domestic animals.     

Bovine toll-like receptor 9: a comparative analysis of molecular structure, function and expression

Non-methylated CpG motifs, present in viral and bacterial DNA, are one of many pathogen-associated molecular patterns (PAMP) recognized by the mammalian innate immune system. Recognition of this PAMP occurs through a specific interaction with toll-like receptor 9 (TLR9) and this interaction can induce cytokine responses that influence both innate and adaptive immune responses. Previous investigations determined that both the flanking sequences in synthetic CpG oligodeoxynucleotides (CpG ODN) and the cellular pattern of TLR9 expression can influence species-specific responses to CpG ODN. Therefore, the structure, function and cellular distribution of bovine TLR9 were compared with what is known for mice and human. Analysis of the bovine TLR9 gene revealed greater sequence homology between cattle and humans than cattle and mice Similar CpG motifs induced optimal activation of both human and bovine leukocytes and these motifs were distinct from those which activated mouse leukocytes. Functional analyses with CpG ODN stimulated bovine blood leukocytes revealed that class A CpG ODN were more potent inducers of interferon-alpha (IFN-alpha) than class B CpG ODN. Furthermore, magnetic activated cell sorting of bovine blood leukocyte subpopulations implicated dendritic cells but not monocytes in the regulation of CpG ODN-induced IFN secretion. Thus, the cellular pattern of CpG ODN-induced responses in cattle shared many similarities with human leukocytes. Collectively, these analyses revealed substantial conservation of TLR9 structure and TLR9 function in blood leukocytes of humans, cattle and other domestic species.     

In vivo administration of ligands for chicken toll-like receptors 4 and 21 induces the expression of immune system genes in the spleen

Toll-like receptors (TLRs) are a group of conserved proteins that play an important role in pathogen recognition in addition to the initiation and regulation of innate and adaptive immune responses. To date, several TLRs have been identified in chickens, each recognizing different ligands. TLR stimulation in chickens has been shown to play a role in host-responses to pathogens. However, the mechanisms through which TLRs modulate the chicken immune system have not been well examined. The present study was conducted to characterize the kinetics of responses to TLR4 and TLR21 stimulation in chickens following intramuscular injections of their corresponding ligands, lipopolysaccharide (LPS) and CpG oligodeoxynucleotides (ODNs), respectively. To this end, relative expression of cytokine genes in the spleen was determined at 2, 6, 12 and 24 h after injection of TLR ligands. The results indicated that LPS strongly induced the up-regulation of some immune system genes early on in the response to treatment, including interferon (IFN)-γ, interleukin (IL)-10, and IL-1β. Furthermore, treatment with CpG ODN promoted the up-regulation of major histocompatibility complex (MHC)-II, IFN-γ and IL-10. The response to CpG ODN appeared to be somewhat delayed compared to the response to LPS. Moreover, we found a significant increase in IFN-α gene expression in response to LPS but not CpG ODNs. Future studies may be aimed to further characterize the molecular mechanisms of TLR activation in chickens or to exploit TLR agonists as vaccine adjuvants.     

Systemic innate immune responses following intrapulmonary delivery of CpG oligodeoxynucleotides in sheep

Mucosal delivery of CpG oligodeoxynucleotide (ODN) in mice has been shown to induce potent innate immunostimulatory responses and protection against infection. We evaluated the efficacy of CpG ODN in stimulating systemic innate immune responses in sheep following delivery to the pulmonary mucosa. Intrapulmonary (IPM) administration of B-Class CpG ODN in saline induced transient systemic responses which included increased rectal temperatures, elevated serum 2'5'-A synthetase and haptoglobin concentrations. The ODN dose required to induce detectable systemic responses following IPM delivery could be reduced by approximately 80% if the CpG ODN was administered in 30% emulsigen instead of saline. Intrapulmonary B-Class CpG ODN formulated in 30% emulsigen produced similar effects when compared to those seen following SC injection. These responses were CpG ODN-specific since control GpC ODN did not induce any detectable response. Intrapulmonary administration of both B-Class and the newly described C-Class CpG ODN produced similar effects indicating that both classes of CpG ODN were comparably effective in stimulating innate immune system following mucosal delivery. Administration of CpG ODN directly into the lungs or delivery of CpG ODN via an intratracheal (IT) infusion also produced similar systemic responses. These observations support the conclusion that mucosal delivery of CpG ODN is an effective route for induction of systemic acute phase responses and antiviral effector molecules in large animals, and may be helpful in controlling systemic infections.