Protection of neonatal chicks against a lethal challenge of Escherichia coli using DNA containing cytosine-phosphodiester-guanine motifs

Oligodeoxynucleotides (ODN) containing cytosine-phosphodiester-guanine (CpG) motifs have been shown to be effective immunoprotective agents in murine models for a variety of viral, intracellular bacterial, and protozoan infections. We recently have shown that CpG ODN protects against extracellular bacterial infections in mature chickens. The objective of this study was to investigate the effect of CpG ODN on Escherichia coli septicemia in neonatal broiler chicks. Two-day-old chicks, or embryonated eggs that had been incubated for 18 or 19 days, received 50 microg CpG ODN. Three days after exposure to CpG ODN, a virulent isolate of E. coli was inoculated subcutaneously in the neck of each bird. Birds were examined for 7 days post-E. coli challenge and dinical, pathologic, and bacteriologic assessments were conducted. The control group of birds that received no CpG ODN had a survival rate of 0% to 20%. In contrast, groups that received CpG ODN, either by intramuscular or in ovo routes, had significantly higher survival rates (P < 0.0001). Bacterial counts in air sacs were significantly lower when birds or embryos were treated with CpG ODN as compared with controls. A dose as low as 10 microg of CpG ODN, administered intramuscularly, was able to protect birds significantly against E. coli challenge. Formulation of CpG ODN with 30% Emulsigen did not enhance the protection. This study demonstrates that CpG ODN has systemic protective effects in broiler chicks against E. coli infections. This is the first time that CpG ODN has been demonstrated to have an immunoprotective effect against a bacterial infection in chicks following in ovo delivery.     

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 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.     

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。     

团头鲂对3种致病菌外膜蛋白(OMP)的免疫应答

利用从柱状黄杆菌(Flavobacterium columnare)、嗜水气单胞菌(Aeromonas hydrophila)和温和气单胞菌(Aeromonas sobria)中提取的外膜蛋白(outer membrane protein,OMP)作为免疫原,注射接种团头鲂(Megalobrama amblycephala)后,通过测定受免鱼的交叉凝集抗体效价、肾脏和血液中吞噬细胞的吞噬活性和采用A.hydrophila活菌攻毒方法,比较了3种致病菌OMP对团头鲂的免疫原性。试验结果表明,从3种致病菌中提取的OMP对团头鲂均具有较强的免疫原性,受免鱼的血清中存在对3种致病菌的交叉凝集抗体,与对照鱼相比,肾脏和血液中吞噬细胞对吞噬原的吞噬活性和对A.hy-drophila活菌攻毒的相对免疫保护率(relative percent survival,RPS)明显上升。说明在3种致病菌的OMP上不同程度地存在共同保护性抗原。     

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.     

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。     

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.     

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-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.     

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.     

In vivo immunostimulatory effects of CpG oligodeoxynucleotide in cattle and sheep

Oligodeoxynucleotides (ODN) containing cytosine-phosphate-guanosine (CpG) motifs have been shown to activate the innate immune system and protect mice and chicken from bacterial and viral infections. Unfortunately, similar studies in other veterinary species are lacking. In this study we assessed the in vivo immunostimulatory effects of CpG ODN 2007, an ODN with previously demonstrated in vitro biological activity. The in vivo effects of ODN 2007 were compared in two closely related outbred species, sheep and cattle, to determine if there were common biological responses. We demonstrated that subcutaneous (s.c.) injection of the CpG ODN induces an acute phase response in the form of a transient fever, a mild transient increase in circulating neutrophils and elevated serum haptoglobin in both sheep and cattle. Sheep injected with CpG ODN also exhibited increased serum 2'5'-oligoadenylate (2'5'-A) synthetase activity, but no increase in serum 2'5'-A synthetase was detected in cattle. The ODN-induced responses were stronger in animals injected with CpG ODN formulated in 30% emulsigen than phosphate buffer saline (PBS) alone. These in vivo data demonstrate for the first time that a CpG ODN induces acute phase immunostimulatory responses in sheep and cattle. However, CpG ODN-induced antiviral effector molecule 2'5'-A synthetase was detected only in sheep but not in cattle.     

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.     

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。     

Enhancement of phagocytic and chemokinetic activities of rainbow trout head kidney cells by C-reactive protein

OBJECTIVE: To investigate immunostimulating activity of purified rainbow trout (Oncorhynchus mykiss) C-reactive protein (CRP) on trout phagocytic cells. ANIMALS: 20 rainbow trout and 2 rabbits. PROCEDURE: The effect of CRP on phagocytic activity of head kidney (HK) cells was examined by use of a phagocytosis assay with plastic particles. The enhancing effect of CRP on migration activity of HK cells was examined by use of the blind well assay. RESULTS: Glass-adherent cells from clinically normal trout had increased dose-dependent phagocytic activity against plastic particles when cells were incubated in the presence of CRP. Pretreatment of particles with CRP also enhanced phagocytic activity of the cells, indicating an opsonic effect of CRP. Rabbit anti-trout CRP serum suppressed the enhancing activity of CRP. The HK cells had significant dose-dependent chemokinetic activity against CRP that was not inhibited by anti-CRP serum, indicating that a CRP-antibody complex also could be chemokinetic. CONCLUSIONS: Rainbow trout CRP has immunostimulating activity for HK cells, resulting in enhanced phagocytic and chemokinetic activities.     

Supernatants from leucocytes treated with melanin-concentrating hormone (MCH) and alpha-melanocyte stimulating hormone (alpha-MSH) have a stimulatory effect on rainbow trout (Oncorhynchus mykiss) phagocytes in vitro

Melanin-concentrating hormone (MCH) and alpha-melanocyte stimulating hormone (alpha-MSH) are widespread vertebrate neuropeptides. In teleost fish the peptides are involved in the hormonal control of skin pigmentation, but they have also been shown to modulate corticosteroid secretion in both fish and mammals. alpha-MSH has additional potent anti-inflammatory actions in mammals and both peptides stimulate leucocyte phagocytosis in rainbow trout in vitro. The effects of these peptides on phagocytosis and the release of immunomodulatory factors by rainbow trout head kidney leucocytes were investigated in vitro. Neither MCH nor alpha-MSH had any effect on the adherence of phagocytes to glass slides or the activity of isolated phagocytes. When added to mixed leucocyte suspensions, however, MCH (50 and 100nM) and alpha-MSH (1 and 10nM) significantly increased the percentage of cells undergoing phagocytosis and MCH (50nM), but not alpha-MSH, stimulated the phagocytic index. In subsequent experiments, isolated phagocytes were exposed to supernatants derived from mixed leucocyte suspensions exposed to MCH (50 and 100nM) and alpha-MSH (1 and 10nM). Supernatants from leucocytes exposed to all doses of the peptides significantly increased the percentage phagocytosis and those from cells stimulated with MCH (100nM) and alpha-MSH (1 and 10nM) increased the phagocytic index of the phagocytes. The results suggest that cells other than phagocytes are required for MCH and alpha-MSH to exert their stimulatory effects on trout phagocytic cells through the release of one or more macrophage-activating factors.     

Innate immune responses induced by classes of CpG oligodeoxynucleotides in ovine lymph node and blood mononuclear cells

CpG ODN signal through Toll-like receptor 9 (TLR9) and trigger a cascade of events that lead to activation of innate and adaptive immune responses. Our current understanding of the immunobiology of host responses to CpG is based largely on studies on peripheral blood mononuclear cells (PBMC) and splenocytes. Little is known regarding CpG-induced responses in other lymphoid tissues. In the present study, we investigated responses induced by CpG in both PBMC and lymph nodes. Cells were isolated from the superficial cervical lymph node (LNC) and blood and then stimulated with CpG ODN (either A-, or B- or C-class ODN). Cytokine production was assayed by ELISA, and lymphocyte proliferation was determined by (3)H-thymidine incorporation. NK-like cytotoxicity was analyzed by lysis of (51)Cr-labelled target cells. All three classes of CpG induced IFNalpha and IFNgamma in LNC. In contrast, only A and C-class ODN induced IFNalpha and IFNgamma in PBMC. Moreover, the IFN levels in LNC were 20-40-fold higher than in PBMC. Furthermore, all classes of ODN induced higher IL-12 levels in LNC (five- to six-fold) than in PBMC. Both B and C-class ODN induced good proliferative responses in PBMC and LNC, but the A-class ODN did not induce proliferation of PBMC and only induced moderate proliferation of LNC. A-class ODN induced significant NK-like activity in LNC. Thus, all three classes of CpG ODN induced similar responses in LNC, and these responses were consistently higher than in PBMC. These observations indicate that CpG ODN-induced responses differ between blood and lymph nodes, and suggest that the functional classification of CpG ODN based on PBMC responses may not be directly applicable to cells from other immune tissues.