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.     

Biological activity of immunostimulatory CpG DNA motifs in domestic animals

Bacterial DNA contains a much higher frequency of CpG dinucleotides than are present in mammalian DNA. Furthermore, bacterial CpG dinucleotides are often not methylated. It is thought that these two features in combination with specific flanking bases constitute a CpG motif that is recognized as a "danger" signal by the innate immune system of mammals and therefore an immune response is induced when these motifs are encountered. These immunostimulatory activities of bacterial CpG DNA can also be achieved with synthetic CpG oligodeoxynucleotides (ODN). Recognition of CpG motifs by the innate immune system requires engagement of Toll-like receptor 9 (TLR-9), which induces cell signaling and subsequently triggers a pro-inflammatory cytokine response and a predominantly Th1-type immune response. CpG ODN-induced innate and adaptive immune responses can result in protection in various mouse models of disease. Based on these observations, clinical trials are currently underway in humans to evaluate CpG ODN therapies for cancer, allergy and infectious disease. However, potential applications for immunostimulatory CpG ODN in species of veterinary importance are just being explored. In this review, we will highlight what is presently known about the immunostimulatory effects of CpG ODN in 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.     

CpG oligodeoxynucleotides augment the immune responses of piglets to swine Pasteurella multocida living vaccine in vivo

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) prevent development of T-helper type 2 (Th2) immune response and reverse established allergic responses in mouse models. However, little work on immune responses in piglets has been conducted in vivo. In this report, the ability of a porcine-specific CpG ODN to act as an immunostimulant and enhance immune responses of piglets to swine Pasteurella multocida living vaccine (SPML vaccine) was determined. The titre of IgG and IgG1/IgG2 isotype to SPML vaccine in serum, the proliferation of lymphocytes, SPML-specific interferon-gamma (IFN-gamma) and IL-6, TNF-alpha, IL-4 production of PBMCs in vitro and IFN-gamma, IL-6, TNF-alpha, IL-4, IL-10 in piglets serum were examined to identify the immune responses of the piglets. Immune responses of the piglets vaccinated with SPML and CpG ODN were significantly stronger than responses of piglets vaccinated with SPML alone. All these data summarized that immunostimulatory CpG ODN could modulate the immune response towards a Th1-like response when co-administered to piglets during SPML vaccination, which suggested that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.     

Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs within certain flanking base pairs are recognized as a danger signal by the innate immune system of vertebrates. Using lymphocyte proliferative response (LPR) and IFN-gamma secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on bovine peripheral blood mononuclear cells. ODN composed of a nuclease resistant phosphorothioate backbone and a leading 5'-TCGTCGTT-3' motif with two 5'-GTCGTT-3' motifs were highly stimulatory in both assays. Flow cytometric analysis and cell-specific surface marker labeling determined that B-cells (surface IgM(+)) were the primary cell population responding in the LPR assay. Depletion of T cells (CD3(+)) from the PBMC population did not affect IFN-gamma secretion or B-cell proliferation when cultured with CpG-ODN. However, depletion of monocytes (DH59B(+)) completely abrogated the ability of CpG-ODN to stimulate IFN-gamma secretion, and significantly reduced the B-cell proliferative response. These data establish the identity of an optimal immunostimulatory CpG motif for cattle and demonstrate that monocytes play a pivotal role in the ability of cell populations to respond to CpG-ODN. These data provide insight for future studies investigating the mechanism of CpG-ODN bioactivity and its application in novel vaccine formulations and immunotherapy.     

疫苗佐剂胞嘧啶-鸟嘌呤寡脱氧核苷酸的研究进展

胞嘧啶-鸟嘌呤寡脱氧核苷酸(cytosine phosphate guanidine oligodeoxynucleotide,CpG ODN)是指含有非甲基化的胞嘧啶和鸟嘌呤二核苷酸为核心序列的核苷酸序列,近年来,CpG ODN作为一种新型免疫佐剂的研究越来越多,可诱发机体产生多种免疫学效应,提高系统免疫和黏膜免疫水平,具有安全性高,耐受性强等特点。     

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。     

Vaccination with Newcastle disease vaccine and CpG oligodeoxynucleotides induces specific immunity and protection against Newcastle disease virus in SPF chicken

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) have been proven to be immunoprotective in mouse models. However, little work has been conducted on in vivo immune responses in chicken with CpG ODN. The objective of this study was to investigate the immunoadjuvant effects of CpG ODN to Newcastle disease (ND) vaccine and its protective effects against ND virus in SPF chicken. In this report, the titre of serum IgG to ND vaccine and the proliferation of lymphocytes were monitored in SPF chickens. The results demonstrated that the above-mentioned immune responses were significantly stronger in chickens that received CpG ODN than in the birds that received only ND vaccine. Furthermore, ND vaccine plus CpG ODN protected SPF chicken from challenge with an otherwise lethal dose of ND virus. These data suggest that CpG ODN holds considerable promise as an adjuvant for future vaccines against ND virus.     

CpG-oligodeoxynucleotides enhance porcine immunity to Toxoplasma gondii

Protection against a challenge infection with Toxoplasma gondii VEG strain oocysts was examined in pigs after vaccination with T. gondii RH strain tachyzoites with or without a porcine specific synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. Six groups of pigs were immunized with incomplete Freund's adjuvant (IFA) and either vehicle, tachyzoites alone or in combination with three different doses of CpG ODN or with CpG ODN alone. Protection from challenge was significantly (P < 0.05) improved in pigs vaccinated using CpG ODN as an adjuvant with tachyzoites compared to all other groups. The CpG ODN tachyzoite-immunized pigs also had higher serum parasite specific IgG antibody, no clinical signs of disease, and 52% had no demonstrable tissue cysts after the challenge infection. These data indicate that CpG ODN is a potential safe and effective adjuvant for the T. gondii RH strain vaccine in pigs.     

Subcutaneous, but not intratracheal administration of the TLR9 agonist, CpG DNA transiently reduces parainfluenza-3 virus shedding in newborn lambs

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs signal through TLR9 and activate innate immunity resulting in protection against a variety of parasitic, bacterial and viral pathogens in mouse models. However, few studies have demonstrated protection in humans and large animals. In the present investigations, we evaluated protection by CpG ODN in a parainfluenza-3 (PI-3) virus infection in neonatal lambs. Subcutaneous (SC) injection of CpG ODN induced high levels of 2′5′-A synthetase and significantly reduced PI-3 virus shedding in newborn lambs. Furthermore, pre-treatment of newborn lambs with SC CpG ODN 2 days, but not 6 days prior to the virus challenge was protective. In contrast, intratracheal (IT) administration of CpG ODN induced 2′5′-A synthetase but had no significant impact on PI-3 virus shedding in nasal secretions. We conclude that a systemic administration of CpG ODN and the timing of the treatment are critical for the protection of neonatal lambs against a respiratory viral infection.     

Immunostimulatory CpG oligodeoxynucleotides enhance the induction of bovine CD4+ cytotoxic T-lymphocyte responses against the polymorphic immunodominant molecule of the protozoan parasite Theileria parva

Enhancement of the induction of cytotoxic T-cell responses by immunostimulatory CpG oligodeoxynucleotides has been described in humans and mouse models. The present study attempted to address whether CpG has a similar effect in cattle. Immunisation of cattle with a recombinant form of the polymorphic immunodominant molecule from Theileria parva emulsified with immunostimulatory CpG oligodeoxynucleotides in adjuvant had no effect on the induction of antibody responses including the isotype profile, but significantly enhanced the induction of cytolytic responses that were mediated by CD4+CD3+ T cells utilizing the perforin-granzyme pathway.     

Vaccination of cattle with Mycobacterium bovis culture filtrate proteins and CpG oligodeoxynucleotides induces protection against bovine tuberculosis

Culture filtrate protein (CFP) vaccines have been shown to be effective in small animal models for protecting against tuberculosis while immunisation with these types of vaccines in cattle has been less successful. A study was conducted in cattle to evaluate the ability of selected adjuvants and immunomodulators to stimulate protective immune responses to tuberculosis in animals vaccinated with Mycobacterium bovis CFP. Seven groups of cattle (n=5) were vaccinated with M. bovis CFP formulated with either Emulsigen or Polygen adjuvant alone or in combination with a specific oligodeoxynucleotides (ODN), polyinosinic acid: polycytidylic acid (poly I:C) or poly I:C and recombinant granulocyte-macrophage colony stimulating factor. Two additional groups were vaccinated subcutaneously with BCG or non-vaccinated. In contrast to the strong interferon-gamma (IFN-gamma) responses induced by BCG, the CFP vaccines induced strong antibody responses but weak IFN-gamma responses. The addition of CpG ODN to CFP significantly enhanced cell-mediated responses and elevated antibody responses to mycobacterial antigens. Of the CFP vaccinated groups, the strongest IFN-gamma responses to CFP vaccines were measured in animals vaccinated with CFP/Emulsigen+CpG or CFP/Polygen+CpG. The animals in these two groups, together with those in the BCG and non-vaccinated groups were challenged intratracheally with virulent M. bovis at 13 weeks after the first vaccination and protection was assessed, by examination for presence of tuberculous lesions in the lungs and lymph nodes, 13 weeks later at postmortem. While BCG gave the best overall protection against tuberculosis, significant protection was also seen in animals vaccinated with CFP/Emulsigen+CpG. These results establish an important role for CpG ODN in stimulating protective Th1 responses to tuberculosis in cattle and indicate that a sub-unit protein vaccine can protect these animals against tuberculosis.     

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。     

Enhancement of mucosal immune responses by intranasal co-delivery of Newcastle disease vaccine plus CpG oligonucleotide in SPF chickens in vivo

Immunostimulatory CpG oligodeoxynucleotides (ODN) have been tested as immunoadjuvants for various vaccines in mice and human. Findings from previous reports suggest that CpG ODN can be used to enhance magnitude and balance of an immune response while reducing undesirable side effects of commercial vaccine, when delivered by parenteral route. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant in mice, but data on mucosal immune responses induced by CpG ODN in other animals, especially in chickens, are scarce. Herein, we evaluated intranasal (IN) delivery of CpG ODN with newcastle disease (ND) vaccine (NDV) to determine its potential as a mucosal adjuvant to a commercial vaccine. CpG ODN augmented systemic (IgG in serum, T cell proliferation) and mucosal (IgA in intestinal washings and feces) immune responses against antigen. CpG ODN stimulated effectively both systemic and mucosal immune responses when delivered intranasally. Results from this study indicate that stimulatory CpG ODN is a potential effective mucosal adjuvant for the NDV in SPF chickens and may be applicable to husbandry animals.     

Enhancement of mucosal immune responses by intranasal co-delivery of Newcastle disease vaccine plus CpG oligonucleotide in SPF chickens in vivo

Immunostimulatory CpG oligodeoxynucleotides (ODN) have been tested as immunoadjuvants for various vaccines in mice and human. Findings from previous reports suggest that CpG ODN can be used to enhance magnitude and balance of an immune response while reducing undesirable side effects of commercial vaccine, when delivered by parenteral route. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant in mice, but data on mucosal immune responses induced by CpG ODN in other animals, especially in chickens, are scarce. Herein, we evaluated intranasal (IN) delivery of CpG ODN with newcastle disease (ND) vaccine (NDV) to determine its potential as a mucosal adjuvant to a commercial vaccine. CpG ODN augmented systemic (IgG in serum, T cell proliferation) and mucosal (IgA in intestinal washings and feces) immune responses against antigen. CpG ODN stimulated effectively both systemic and mucosal immune responses when delivered intranasally. Results from this study indicate that stimulatory CpG ODN is a potential effective mucosal adjuvant for the NDV in SPF chickens and may be applicable to husbandry animals.     

A review of CpGs and their relevance to aquaculture

CpG oligodeoxynucleotides (ODN) have been described as functioning as natural adjuvants because they promote professional antigen presenting cell (APC) function and co-stimulate lymphocytes. The majority of studies into the immune effects of CpG ODN to date have been carried out on mammals where they are proving very successful at stimulating innate and adaptive immune responses in a variety of species as well as protecting them from bacterial, viral and protozoan pathogens. Fish also possess the ability to raise both innate and adaptive immune responses to invading pathogens and interest in the effect of CpG ODN on the piscine immune system is growing. Various studies have now been carried out to elicit the effects of CpG ODN on diverse fish species showing that 31 different B-class CpG ODN exert various immune responses both in vivo and in vitro in salmonids, cyprinids and pleuronectiformes. These responses include activation of macrophages, proliferation of leucocytes and stimulation of cytokine expression. CpG ODN have also been shown to be protective against bacterial and viral challenge as well as against pathogenic amoebae. As would be expected these effects are all dependent on not only the ODN sequence and length but on the concentration and the species in which it is being used. This review provides the first comprehensive overview of all CpG ODN tested in fish to date and brings together all the work carried out in this field.     

Immune Enhancement Activity of Different Types of CpG Motifs on the Recombinant Eg95 Antigen of Echinococus granulosus

To study the immune enhancement activity of CpG motifs on the recombinant Eg95 antigen of Echinococcus granulosus,Escherichia coli BL21(DE3) containing pET-32a-3Eg95 was grown in LB medium,and IPTG was then added for induction.The recombinant Eg95 protein was expressed and later harvested by affinity chromatography.The obtained antigen was mixed either with the conventional adjuvant Quli-A or with different types of CpG motifs (pUC18-CpG or CpG ODN),to prepare vaccine candidates for mice immunization.The average levels of serum IgG antibody were tested by indirect ELISA and the quantification of cytokine expression were determined by Real-time quantitative PCR after in vitro stimulation.The results identified that the size of the recombinant protein was 56 ku as predicted,and the recombinant protein could be recognized by serum from Echinococcus granulosus-infected sheep.The average levels of serum IgG antibody (D_{450 nm}) of the pUC18-CpG and CpG ODN groups on 14,28 and 42 d were 3.10,3.03,3.22 and 2.98,3.12, 3.27,respectively.Antibody levels from both CpG groups were significantly higher compared to the conventional adjuvant Quli-A group (P<0.05) The average level of serum IgG antibody in CpG ODN group was a little higher than that in pUC18-CpG group(P>0.05).After stimulation by recombinant protein 3Eg95,the relative expression levels of IFN-β,IFN-γ and TNF-α of pUC18-CpG and CpG ODN groups were 6.88,2.35,6.28 and 5.03,2.85,7.07,respectively,and both were higher than that of the Quli-A group (P<0.05).The results indicated that pUC18-CpG and CpG ODN had significant immune enhancement property on Eg95 antigen in both humoral and cellular immune response,and the immune enhancement activity of CpG ODN was slightly better than that of the pUC18-CpG.Hence,pUC18-CpG and CpG ODN could be used as efficient immune adjuvants enhancer in future vaccines against Echinococcus granulosus infection.     

CpG寡脱氧核苷酸的免疫刺激特性及应用研究进展

CpG基序是指含有非甲基化的胞嘧啶（C）和鸟嘌呤（G）二核苷酸为核心序列的基序为5′-R-D-CpG-Y-Y-3′的DNA或寡脱氧核苷酸序列,是DNA具有免疫刺激活性的结构基础。随着对CpG特性研究的不断深入,CpG寡脱氧核苷酸在激活先天性免疫、作为疫苗佐剂、对变态反应进行免疫治疗、抗肿瘤应用及基因治疗等方面均显示出较广阔的应用前景。现就CpG寡脱氧核苷酸的免疫激活作用、细胞识别及结构特征、治疗疾病方面的应用及毒理作用加以探讨。     

不同类型CpG基序对细粒棘球蚴Eg95抗原的免疫增强作用研究

为探究不同类型CpG基序对细粒棘球蚴（Echinococus granulosus）Eg95抗原的免疫增强作用,将pET-32a-3Eg95重组大肠杆菌,经IPTG诱导表达,亲和层析纯化重组蛋白pET-32a-3Eg95。将重组蛋白分别与常规佐剂Quli-A、pUC18-CpG和CpG ODN混合制备基因工程亚单位疫苗样品,免疫小鼠。通过间接ELISA方法检测抗体水平,通过实时荧光定量PCR方法测定体外刺激试验后细胞因子的相对表达量,检测不同佐剂在体液免疫和细胞免疫方面对Eg95抗原免疫效果的增强作用。结果显示,诱导表达重组蛋白的大小约为56 ku,与理论值相符,用羊细粒棘球蚴阳性血清检测有特异性条带;pUC18-CpG组和CpG ODN组小鼠免疫后14、28、42 d的抗体平均水平（D_{450 nm}）分别为3.10、3.03、3.22和2.98、3.12、3.27,与Quli-A组抗体平均水平相比均差异显著（P<0.05）;CpG ODN组血清中抗体平均水平略高于pUC18-CpG组,但差异不显著（P>0.05）。重组蛋白刺激后pUC18-CpG组与CpG ODN组IFN-β、IFN-γ和TNF-α的相对表达量分别为6.88、2.35、6.28和5.03、2.85、7.07,与Quli-A组相比差异均显著（P<0.05）。因此相对于常规佐剂Quli-A,pUC18-CpG和CpG ODN在体液免疫和细胞免疫方面对Eg95抗原都有显著的免疫增强作用,CpG ODN的免疫增强作用略优于pUC18-CpG,二者均可作为免疫佐剂,增强现有包虫病基因工程疫苗抗原的免疫效果。     

Identification of a potent immunostimulatory oligodeoxynucleotide from Streptococcus thermophilus lacZ

Immunostimulatory sequences of oligodeoxynucleotides (ODNs), such as CpG ODNs, are potent stimulators of innate immunity. Here, we identified a strong immunostimulatory CpG ODN, which we named MsST, from the lac Z gene of Streptococcus (S.) thermophilus ATCC19258, and we evaluated its immune functions. In in vitro studies, MsST had a similar ability as the murine prototype CpG ODN 1555 to induce inflammatory cytokine production and cell proliferation. In mouse splenocytes, MsST increased the number of CD80+CD11c+and CD86+CD11c+ dendritic cells and CD4+CD25+ regulatory T cells. We also analyzed the effects of MsST on the expression of regulatory cytokines by real-time quantitative PCR. MsST was more potent at inducing interleukin-10 expression than the ODN control 1612, indicating that MsST can augment the regulatory T cell response via Toll-like receptor 9, which plays an important role in suppressing T helper type 2 responses. These results suggest that S. thermophilus , whose genes include a strong Immunostimulatory sequence-ODN, is a good candidate for a starter culture to develop new physiologically functional foods and feeds.