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.

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.

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.

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.

Stimulation of innate immune responses by CpG oligodeoxynucleotide in newborn lambs can reduce bovine herpesvirus-1 shedding.

Stimulation of the innate immune system is potentially very important in neonates who have an immature adaptive immune system and vaccination cannot be used to reduce the risk of infection. CpG oligodeoxynucleotide (ODN) can stimulate innate immune responses in newborn chickens and mice, but similar studies are lacking in other mammalian species. We have shown previously that CpG ODN can both stimulate an acute-phase immune response and induce the antiviral effector molecule, 2'5'-A synthetase, in adult sheep. Therefore, the immunostimulatory activity of A class and B class CpG ODN was evaluated in newborn lambs, and the capacity of CpG ODN-induced responses to reduce viral shedding was evaluated following aerosol challenge with the respiratory pathogen, bovine herpesvirus-1 (BHV-1). In vitro CpG ODN stimulation of peripheral blood mononuclear cells (PBMC) isolated from newborn lambs (3-5 days old) and adult sheep induced equivalent CpG-specific proliferative responses and interferon-alpha (IFN-alpha) secretion. CpG ODN-induced IFN-alpha secretion by neonatal PBMCs was, however, significantly (p < 0.01) enhanced 6 days after subcutaneous (s.c.) injection of 100 microg/kg CpG ODN 2007. Newborn lambs injected s.c. with B class CpG ODN 2007 or the inverted GpC control ODN formulated in 30% Emulsigen (MVP Laboratories, Ralston, NE) displayed CpG ODN-specific increases in body temperature (p < 0.0001), serum 2'5'-A synthetase activity (p = 0.0015), and serum haptoglobin (p = 0.07). CpG ODN-treated lambs also displayed a transient reduction in viral shedding on day 2 postinfection (p < 0.05), which correlated (p < 0.03) with serum 2'5'-A synthetase levels on the day of viral challenge. These observations confirmed that CpG ODNs effectively activate innate immune responses in newborn lambs and CpG ODN-induced antiviral responses correlated with a reduction in viral shedding.

Strategies for enhancing the immunostimulatory effects of CpG oligodeoxynucleotides.

Synthetic oligodeoxynucleotides (ODN) containing CpG sequences are recognized as a "danger" signal by the immune system of mammals. As a consequence, CpG ODN stimulate innate and adaptive immune responses in humans and a variety of animal species. Indeed, the potential of CpG ODN as therapeutic agents and vaccine adjuvants has been demonstrated in animal models of infectious diseases, allergy and cancer and are currently undergoing clinical trials in humans. While CpG ODN are potent activators of the immune system, their biologic activity is often transient, subsequently limiting their therapeutic application. Modifications in the CpG ODN backbone chemistry, various delivery methods including mixing or cross-linking of ODN to other carrier compounds have been shown to significantly enhance the biologic activity of ODN. However, the exact mechanisms that mediate this enhancement of activity are not well understood and may include local cell recruitment and activation, cytokine production, upregulation of receptor expression and increasing the half-life of ODN through creation of a depot. We will review the various approaches that have been used in enhancing the immunostimulatory effects of CpG ODN in vivo and also discuss the possible mechanisms that may be involved in this enhancement.

Quantitative analysis of pro-inflammatory cytokine mRNA expression in Theileria annulata-infected cell lines derived from resistant and susceptible cattle.

The pathogenic mechanisms involved in tropical theileriosis, caused by the tick-borne protozoan parasite Theileria annulata, are unclear. Pathology is associated with the schizont stage of the parasite, which resides within bovine macrophages. Breed-specific differences in pathology have been observed in cattle, several Bos indicus breeds are relatively resistant to tropical theileriosis whilst Bos taurus cattle are highly susceptible. Infected cells express pro-inflammatory cytokines and it has been hypothesized that these cytokines play a major role in the pathology of the disease. Therefore, using quantitative RT-PCR we investigated the expression of the key candidates, interleukin 1 beta (IL-1beta), IL-6 and tumour necrosis factor alpha (TNF-alpha), in T. annulata low passage infected cell lines derived ex vivo from experimental infection of resistant and susceptible cattle. mRNA for each cytokine was detected in all cell lines investigated at levels higher than those observed in resting monocytes. However, the analyses did not identify any breed-specific differences. Therefore, these results are not consistent with the hypothesis that differential regulation of infected cell derived pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) accounts for the breed-related differences in resistance and susceptibility to T. annulata infection. Other, currently unknown mechanisms may be of greater importance.

CpG oligodeoxynucleotide induction of antiviral effector molecules in sheep.

Immunostimulatory CpG oligodeoxynucleotide (ODN) can protect mice against infection by many pathogens but the mechanisms mediating disease protection are not well defined. Furthermore, the mechanisms of CpG ODN induced disease protection in vivo have not been investigated in other species. We investigated the induction of antiviral effector molecules in sheep treated with a class B CpG ODN (2007). Subcutaneous injection of ODN 2007 induced a dose-dependent increase in serum levels of the antiviral effector molecule, 2'5'-A synthetase. Peak levels of enzyme were observed 4 days following ODN injection and enzyme levels remained elevated for the following 3-5 days. Repeated ODN injections induced a more sustained elevation of serum 2'5'-A synthetase activity. Finally, formulation of ODN 2007 in emulsigen increased the level of serum 2'5'-A synthetase activity and this response was CpG-specific. Elevated serum 2'5'-A synthetase activity suggested that CpG ODN acted through the induction of either interferon (IFN)-alpha or IFN-gamma. ODN 2007 did not induce detectable levels of IFN-alpha or IFN-gamma when incubated with peripheral blood mononuclear cells, but both IFN-alpha and IFN-gamma were detected following stimulation of lymph node cells with ODN 2007. CpG ODN induction of 2'5'-A synthetase in vitro correlated with the secretion of both IFN-alpha and IFN-gamma. Furthermore, immunohistochemical staining of skin revealed a marked cellular infiltration at the site of ODN 2007 injection. This cellular infiltration was CpG-specific and consisted of primarily CD172(+) myeloid cells. Many of the cells recruited to the site of ODN 2007 injection expressed IFN-alpha and some IFN-gamma. These observations support the conclusion that localized cell recruitment and activation contribute to CpG ODN induction of antiviral effector molecules, such as interferon and 2'5'-A synthetase.

Innate immune responses induced by CpG oligodeoxyribonucleotide stimulation of ovine blood mononuclear cells.

Examples exist in the literature that demonstrate that treatment with immunostimulatory cytosine-phosphate-guanosine (CpG)-DNA can protect mice against infection by intracellular pathogens. There are, however, few studies reporting that CpG-DNA offers similar disease protection in other species. In this study, we assessed the potential of a class A and class B CpG oligodeoxynucleotide (ODN) to induce innate immune responses in sheep, an outbred species. Using peripheral blood mononuclear cells, we have for the first time demonstrated CpG-ODN-induced innate immune responses, including natural-killer-like activity [non-major histocompatibility complex (MHC)-restricted cytotoxicity], interferon-alpha secretion and 2'-5'A oligoadenylate synthetase activity, that could contribute to immune protection in sheep. The type and magnitude of these responses were dependent on ODN class and non-MHC-restricted killing was not associated with interferon-gamma production. The latter observation is in contrast with observations reported for mice and humans. These observations support the conclusion that differences in CpG-ODN-induced responses exist among species and that specific ODN sequences can significantly influence innate immune responses.

Standardisation and comparison of serial dilution and single dilution enzyme linked immunosorbent assay (ELISA) using different antigenic preparations of the Babesia (Theileria) equi parasite.

Serial dilution and single dilution enzyme linked immunosorbent assays (ELISA) were standardised and their sensitivity and specificity were compared for serodiagnosis of Babesia equi infection. The antibody titres of 24 donkey sera of known identity were determined separately by serial dilution ELISA using three different B. equi antigens namely whole merozoite (WM), cell membrane (CM) and high speed supernatant (HSS). The ratios of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as the positive/negative (P/N) ratio. The coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of sera and the log10 antibody titres of the same sera were ascertained by serial dilution ELISA. The highest value of 'r' was obtained at a serum dilution of 1:200. From log10 antibody titre of sera (y) and their P/N ratio at a dilution of 1:200 (x), regression equations (y = a + bx) were calculated separately for the three antigens. Test sera were diluted to 1:200, their OD were read in duplicate wells and were converted to the P/N ratio. Antibody titres were predicted from the P/N ratio using a regression equation separately for the three antigens. Titres obtained by both ELISAs were not significantly different from each other, thus confirming that single dilution ELISA could be successfully used to replace conventional serial dilution ELISA. The sensitivity, specificity and predictive value of single dilution ELISA was validated statistically using 42 B. equi disease-positive sera and 106 B. equi disease-negative sera. The WM antigen was found to be the most sensitive with a higher predictive value for negative test sera as compared to the CM or HSS antigens. Sera positive for other equine infections including Babesia caballi showed no cross-reaction with the three B. equi antigens in ELISA, thus the test was immunologically specific. Antibody titres of 109 unknown field donkey/horse sera obtained by serial and single dilution ELISA using the WM antigen did not show any significant difference. Since the single dilution ELISA was found to be more economical, convenient, sensitive, specific than the serial dilution ELISA and has a high predictive value, it is suitable for use in sero-epidemiological studies on B. equi infections in the field.

Bovine and ovine blood mononuclear leukocytes differ markedly in innate immune responses induced by Class A and Class B CpG-oligodeoxynucleotide.

Cytosine-phosphate-guanosine (CpG)-DNA can induce an impressive array of innate immune responses that may directly or indirectly contribute to the clearance of infectious agents. Assays, such as lymphocyte proliferative responses, have been used to demonstrate that the immunostimulatory activity of CpG-DNA is conserved among a broad range of vertebrate species, but no studies have been completed to determine if qualitative differences exist among species for CpG-oligodeoxynucleotide (ODN)-induced innate immune responses. In this study, we assessed the capacity of a Class A (ODN 2216) and a Class B (ODN 2007) CpG-ODN to induce innate immune responses in two closely related species, ovine (n = 28) and bovine (n = 29). The secretion of interferon (IFN)-alpha and IFN-gamma and non-major histocompatability complex (MHC)-restricted cytotoxic activity were assayed with CpG-ODN-stimulated peripheral blood mononuclear cells (PBMC). These investigations revealed significant interspecies and intraspecies variation in the responses. As expected, ODN 2216 was a potent inducer of IFN-alpha secretion by both bovine and ovine PBMC, but ODN 2007 also induced dose-dependent, CpG-specific IFN-alpha secretion by ovine PBMC. In contrast, a significant dose-dependent, CpG-specific IFN-gamma secretion response was only observed following ODN 2216 stimulation of bovine PBMC. Furthermore, both ODN 2216 and ODN 2007 induced CpG-specific non-MHC-restricted cytotoxicity with ovine but not bovine PBMC. Finally, there was not a single assay in which PBMC from all sheep or cattle responded at a detectable level. A striking aspect of these results is that such marked differences in CpG-ODN induced innate responses existed both between and within two closely related species.