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This article treats of the history of the Marx Engels Gesamtausgabe, or MEGA, edition of Marx and Engels’ collected works. The edition began under the editorship of David Ryazonov in 1924, and the article traces the evolution of the edition from this beginning until 1990. During this time, the edition came into being as the product of a particular tension: a tension between its claim to scientific status, on the one hand, and the dogmas of Marxism Leninism, on the other. The article explores the political pressures on the MEGA that occurred in the 1930’s, the 1950’s, and the 1970’s, and also the transitional issues that occurred in the wake of 1989. The varying scope of the edition’s contents during these historical periods, and the political and editorial reasons for this variation, are explored. Special attention is given to the relationship between Berlin and Moscow in the 1950’s, 1960’s, and 1970’s, with respect to the edition. Finally, the article notes the greatly reduced support for the MEGA staff in the post 1989 period, and, in conclusion, explores the international reception of the MEGA volumes.

Synthetic oligodeoxynucleotides (ODN) containing cytosine-phosphodiester-guanine (CpG) motifs (CpG-ODN) have been shown to be effective immunoprotective agents and vaccine adjuvants in a variety of bacterial, viral, and protozoan diseases in different animal species. The objective of this study was to compare the immune response of chickens to a killed Escherichia coli vaccine combined with oil in water emulsion or with CpG-ODN. Birds were vaccinated with killed E. coli antigens with either 10 or 50 microg of CpG-ODN on days 10 and 20 of age. At day 30, a virulent isolate of homologous E. coli was applied on a scratch site on the caudal abdominal region. Birds were examined for 10 days post-E. coli challenge, and pathologic and bacteriologic assessments were conducted on all birds that were either found dead or euthanized. The E. coli vaccine group that received no CpG-ODN had a survival rate of 65%. In contrast, groups that received the vaccine with CpG-ODN adjuvant had significantly higher survival rate of 92% (P0.01) with isolation of low numbers of E. coli from internal organs. Total IgG against E. coli antigens was highest in groups that received CpG-ODN as an adjuvant. Birds that received vaccine containing CpG-ODN had minimal inflammatory reaction without tissue necrosis at the injection site. Severe tissue necrosis was present in birds that received vaccine containing oil in water emulsion adjuvant. This study demonstrated that CpG-ODN is an effective vaccine adjuvant in chickens and results in minimal tissue destruction. This study is the first study in which CpG-ODN has been demonstrated to produce an adaptive immune response, at a significant level, against an extracellular bacterial infection in chickens.

The ineffectiveness of simple delivery of soluble antigens to mucosal membranes for immunization has stimulated extensive studies of strategies for appropriate delivery systems and adjuvants. Biphasic lipid vesicles are formulations suitable for the delivery of proteins, peptides, and oligo/polynucleotides. The purpose of these studies was to investigate the ability of biphasic lipid vesicles (as vaccine-targeting adjuvants) containing a bacterial antigen and unmethylated oligonucleotides containing CGdinucleotides - CpG motifs (CpG ODNs) to induce systemic and mucosal immune responses in pigs. Results showed that while the protein, either alone or with CpG ODNs, did not induce mucosal immune responses, administration of antigen and CpG ODNs in biphasic lipid vesicles resulted in induction of both systemic and local antibody responses after immunization using a combined mucosal/systemic approach.

The development of a subunit protein vaccine for bovine tuberculosis which could be used either in combination withMycobacterium bovisBCG (to improve the efficacy of that vaccine) or alone would offer significant advantages over currently available strategies. A study was conducted with cattle to determine the protective efficacy of a strategy based on concurrent immunization with anM. bovisculture filtrate (CFP) vaccine and BCG compared to vaccination with either vaccine alone. One group of calves (10 animals per group) was vaccinated subcutaneously with CFP formulated with Emulsigen and combined with a CpG oligodeoxynucleotide (ODN). A second group was vaccinated with both the CFP vaccine and BCG injected at adjacent sites (CFP-BCG). One further group was vaccinated subcutaneously with BCG, while another group served as nonvaccinated control animals. Vaccination with CFP-BCG induced levels of antigen-specific gamma interferon (IFN-γ) and interleukin-2 (IL-2) in whole-blood cultures that were higher than those induced by vaccination with BCG alone. The combination of CFP and BCG did not enhance the production of antibodies toM. bovisCFP compared to vaccination with CFP alone. Vaccination with CFP alone led to delayed antigen-specific IFN-γ and IL-2 responses. Vaccination with CFP-BCG induced a high level of protection against an intratracheal challenge with virulentM. bovis, based on a significant enhancement of six pathological and microbiological parameters of protection compared with the nonvaccinated group. In contrast, vaccination with BCG alone induced a significant enhancement of protection in only one parameter, while CFP alone induced no protection. These results suggest that a combination of a CpG ODN-formulated protein vaccine and BCG offers better protection against bovine tuberculosis than does BCG alone.

Plasmid DNA continues to attract interest as a potential vaccine-delivery vehicle. However, the mechanisms whereby immune responses are elicited by plasmids are not fully understood. Although there have been suggestions regarding the importance of CpG motifs in plasmid immunogenicity, the molecular mechanisms by which CpG motifs enhance immune responses to DNA vaccines are not well understood. As Toll-like receptor 9-deficient (TLR9-/-) mice fail to respond to the adjuvant effects of CpG oligonucleotides, we used these mice to determine the effect of CpG motifs in plasmids used for DNA immunization. In the study described below, we report that DNA immunization was as effective in eliciting antigen-specific antibody and at stimulating antigen-specific interferon-gamma (IFN-gamma)-secreting cells in TLR9-/- mice as in TLR9+/+ mice. This study illustrates that DNA vaccines elicit immune responses by multiple mechanisms and demonstrates that TLR9 is not essential for the induction of immune responses following DNA immunization.

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.

CpG oligonucleotides (ODNs) are potent mucosal and systemic adjuvants. For practical applications however, improvements in delivery need to be developed. A mouse model was used to determine if the biological activity of CpG ODNs could be enhanced using a novel delivery system of biphasic lipid vesicles (Biphasix Vaccine-Targeting Adjuvant; VTA). Immunization studies were performed to evaluate the potential of VTA formulations to enhance the immunoadjuvant activity of CpG ODNs following systemic or mucosal administration with gD. Immune responses following immunization were assessed by protection from HSV-1 viral challenge and characterization of serum gD-specific antibody responses using ELISA. VTA formulations in combination with CpG and glycoprotein D (gD) were able to increase gD-specific IgG in serum compared to gD alone, and protect from a lethal HSV-1 challenge following subcutaneous immunization. Following mucosal immunization, VTA formulations in combination with CpG and antigen enhanced mucosal IgA responses compared to CpG and antigen administered in PBS.

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.

The Arabidopsis thalianachlorophyll a/b binding protein underexpressed (cue1) mutant, which has been isolated in a screen for chlorophyll a/b binding protein (CAB) underexpressors, exhibits a reticulate leaf phenotype combined with delayed chloroplast development and aberrant shape of the palisade parenchyma cells. The affected gene in cue1 is a phosphoenolpyruvate (PEP)/phosphate translocator (PPT) of the plastid inner envelope membrane. The proposed function of the PPT in C3-plants is the import of PEP into the stroma as one of the substrates for the shikimate pathway, from which aromatic amino acids and a variety of secondary plant products derive. The mutant phenotype could be: (i) complemented by constitutive overexpression of a heterologous PPT from cauliflower; and (ii) rescued by overexpression of a C4-type pyruvate,orthophosphate dikinase (PPDK). The latter approach indicates that PEP deficiency within plastids triggers developmental constraints in cue1. The impact of the mutation on aspects of primary and secondary metabolism was assessed in cue1 as well as in the individual transformant lines. The majority of the data obtained in this and an accompanying paper suggest that the mutant phenotype is not simply caused by a general restriction of the shikimate pathway because of a defect in a PPT.

The efficacy of oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) as an adjuvant for rabbits was assessed alone and in combination with aluminum hydroxide (CpG/alum). The CpG/alum combination elicited a greater immune response to several antigens compared to Freund's adjuvant. A non-CpG/alum combination did not have the same effects as CpG/alum suggesting that the adjuvanticity was related to the CpG motifs. In addition, we formulated one of the antigens with combinations of CpG ODN and 30 or 10% Emulsigen (Em) [CpG/Em (30%) and CpG/Em (10%)]. Both CpG/Em (30%) and CpG/Em (10%) were more effective than Em, and equivalent to CpG/alum. The CpG/Em (10%) combination caused minimal tissue damage. Our results demonstrate that the addition of CpG ODN to aluminum hydroxide or to 10% Em significantly improves the efficiency of these adjuvants, without enhancing tissue reaction.

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.

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in particular sequence contexts (CpG ODN) are recognized as a danger signal by the innate immune system of vertebrates. For this reason, CpG ODNs have a potential application as both an adjuvant and nonspecific immune modulator and are currently being evaluated in a number of human and veterinary clinical trials. Given their potent immunostimulatory activity, CpG ODNs could possibly induce adverse reactions. As all adjuvants and immune modulators must be nontoxic to meet safety requirements, it was essential to address the safety aspects of CpG ODNs. The current review summarizes experiments carried out to date to establish the safety of CpG ODNs in animals.

Recent advances in molecular biology, genomics, and immunology are revolutionizing our approach to managing infectious diseases of humans, livestock, and poultry. One of the most interesting additions to the armamentarium of research focusing on controlling infectious diseases has been a better understanding of how the host's innate immune system recognizes "danger" signals. Additionally, there has been recognition of the relationship between the innate and the specific arms of the immune system. For example, the recent discovery that CpG motifs can modulate immune responses has been used both as an adjuvant to enhance the responses to vaccines, as well as a direct immunostimulant to prevent infections. Using an Escherichia coli chicken model, we have been able to prevent cellulitis in chickens with CpG alone. Thus, CpG can be used immunoprophylactically to reduce infectious diseases. In addition, we will describe how CpG formulations with various antigens; recombinant proteins, peptides, and conventional vaccines can enhance immune responses to each of these different vaccine combinations. What is even more interesting is that CpG incorporation in vaccines can shift the immune response from a predominant T helper 2 (Th2)-like immune response generally induced by killed or subunit proteins to a much more balanced Th1-Th2 response. These immunomodulatory effects have significant implications for management of infectious diseases of all vertebrates.

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to be effective immunoprotective agents in murine models for a variety of viral, intracellular bacterial, and protozoan infections. Until now, the use of CpG-ODN to protect against extracellular bacterial infections has not been reported. The objective of this study was to investigate the effect of CpG-ODN against cellulitis and colibacillosis in broiler chickens, using a well-established model. At 22 days of age, birds received CpG-ODN by either the subcutaneous or intramuscular route. Three days later, a virulent isolate of Escherichia coli was applied to a scratch site on the caudal abdominal skin. Birds were examined for 10 days after the E. coli challenge, and pathological and bacteriological assessments were conducted on all birds. The control group of birds receiving no CpG-ODN (2007) had a survival rate of 15%. In contrast, groups that received CpG-ODN (2007) , by either subcutaneous or intramuscular injection, had significantly higher survival rates ( P < 0.0001). Furthermore, the size of the cellulitis lesion was significantly smaller in groups that received CpG-ODN (2007) by the subcutaneous route ( P < 0.01). A dose of as little as 3.16 μg of CpG-ODN (2007) , delivered 3 days prior to challenge by either the subcutaneous or intramuscular route, significantly protected birds against E. coli infection ( P < 0.01). This study demonstrates that CpG-ODN (2007) has both local and systemic protective effects in broiler chickens. This is the first time that CpG-ODN (2007) has been demonstrated to have an immunoprotective effect against an extracellular bacterial infection in any food animal species.

The potential of CpG-enhanced plasmid DNA vectors encoding a truncated secreted form of bovine herpesvirus-1 (BHV-1) glycoprotein D (tgD) to induce enhanced immune responses in cattle was investigated. We created tgD expression plasmids containing 0, 40 or 88 copies of the hexamer 5′ GTCGTT 3′, a known pan-activating CpG motif in several species. The total tgD-specific IgG titre of calves immunized with these plasmids did not correlate with the CpG content of the plasmid backbone. However, the pBISIA88-tgD-vaccinated group showed a significantly lower IgG1:IgG2 ratio than calves immunized with pBISIA40-tgD or pMASIA-tgD, which has no CpG motifs inserted. Antigen-specific lymphocyte proliferation and IFN-γ secretion by peripheral blood mononuclear cells correlated positively with the CpG content of the vectors. In contrast, calves that received a killed BHV-1 vaccine had an IgG1-predominant isotype and low lymphocyte proliferation and IFN-γ levels. Following challenge, the pBISIA88-tgD-immunized group developed the greatest anamnestic response, the highest BHV-1 neutralization titres in serum and a significantly lower level of virus shedding than the saline control group. However, there were no significant differences in clinical symptoms of infection between the DNA-immunized groups and the saline control group. These data indicate that CpG-enhanced plasmids induce augmented immune responses and could be used to vaccinate against pathogens requiring a strong cellular response for protection.

The immunogenicity and protective efficacy of a bovine herpesvirus 1 (BHV-1) subunit vaccine formulated with Emulsigen (Em) and a synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) was determined in cattle. A truncated, secreted version of BHV-1 glycoprotein D (tgD) formulated with Em and CpG ODN at concentrations of 25, 2.5, or 0.25 mg/dose produced a more balanced immune response, higher levels of virus neutralizing antibodies, and greater protection after BHV-1 challenge compared to tgD adjuvanted with either Em or CpG ODN alone. In contrast, tgD formulated with Em and either 25 mg of a non-CpG ODN or another immunostimulatory compound, dimethyl dioctadecyl ammonium bromide, induced similar immunity and protection compared to tgD formulated with Em alone, a finding which confirms the immunostimulatory effect of ODN to be CpG motif mediated. Our results demonstrate the ability of CpG ODN to induce a strong and balanced immune response in a target species.

The adjuvanticity of a synthetic oligodeoxynucleotide containing unmethylated CpG motifs (CpG ODN) was determined in cattle. Calves were immunized with a truncated secreted version of glycoprotein D (tgD) of bovine herpes virus-1 (BHV-1) formulated with alum, CpG ODN, or a combination of both. BHV-1 tgD formulated with CpG ODN or with alum and CpG ODN induced a stronger and more balanced immune response than tgD in alum. This level of immunity was of sufficient magnitude to minimize weight loss and significantly reduce the duration of virus shedding after intranasal viral challenge. Local tissue reactions generated by CpG ODN were very mild and transient, whereas reactions induced by alum or a combination of CpG ODN and alum were moderate in severity and duration. These data demonstrate that CpG ODN causes minimal injection site reactions and yet acts as an effective adjuvant in cattle.

Oligodinucleotides containing CpG motifs stimulate vertebrate immune cells in vitro, have proven efficacy in murine disease models and are currently being tested in human clinical trials as therapies for cancer, allergy, and infectious disease. As there are no known immunostimulatory motifs for veterinary species, the potential of CpG DNA as a veterinary pharmaceutical has not been investigated. Here, optimal CpG motifs for seven veterinary and three laboratory species are described. The preferential recognition of a GTCGTT motif was strongly conserved across two vertebrate phyla, although a GACGTT motif was optimal for inbred strains of mice and rabbits. In a subsequent adjuvanticity trial, the in vitro screening methodology was validated in sheep, representing the first demonstration of CpG DNA efficacy in a veterinary species. These results should provide candidate immunostimulant and therapeutic drugs for veterinary use and enable the testing of CpG DNA in large animal models of human disease.

(2001). The mega project: An edition between a scientific claim and the dogmas of marxism‐leninism. Critique: Vol. 29, No. 1, pp. 188-195.

Proteins strongly synthesized in Corynebacterium glutamicum during nitrogen restriction were examined by two-dimensional gel electrophoresis and microsequencing. Two main groups of enzymes were identified beside miscellaneous proteins, enzymes involved (i) in protein synthesis, and (ii) in carbon metabolism. Biochemical measurements revealed an increase of oxygen consumption during nitrogen starvation, indicating an enhanced energy demand of the cells. By Northern hybridizations, an increased transcription for the gap and fda genes upon nitrogen deprivation was shown.

The only identified component of the scrapie prion is PrPSc, a glycosylinositol phospholipid (GPI)-linked protein that is derived from the cellular isoform (PrPC) by an as yet unknown posttranslational event. Analysis of the PrPSc GPI has revealed six different glycoforms, three of which are unprecedented. Two of the glycoforms contain N-acetylneuraminic acid, which has not been previously reported as a component of any GPI. The largest form of the GPI is proposed to have a glycan core consisting of Man alpha-Man alpha-Man-(NeuAc-Gal-GalNAc-)Man-GlcN-Ino. Identical PrPSc GPI structures were found for two distinct isolates or "strains" of prions which specify different incubation times, neuropathology, and PrPSc distribution in brains of Syrian hamsters. Limited analysis of the PrPC GPI reveals that it also has sialylated glycoforms, arguing that the presence of this monosaccharide does not distinguish PrPC from PrPSc.

Der Artikel beschreibt die Edition des ersten »Kapital«-Bandes in der MEGA und gibt eine Übersicht über die in den verschiedenen Auflagen stattfindende Theorieentwicklung und deren Ursachen.

Background Cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) has been used successfully to induce immune responses against viral and intracellular organisms in mammals. The main objective of this study was to test the effect of CpG-ODN on antigen presenting cells of young foals. Methods Peripheral blood monocytes of foals (n = 7) were isolated in the first day of life and monthly thereafter up to 3 months of life. Adult horse (n = 7) monocytes were isolated and tested once for comparison. Isolated monocytes were stimulated with IL-4 and GM-CSF (to obtain dendritic cells, DC) or not stimulated (to obtain macrophages). Macrophages and DCs were stimulated for 14–16 hours with either CpG-ODN, LPS or not stimulated. The stimulated and non-stimulated cells were tested for cell surface markers (CD86 and MHC class II) using flow cytometry, mRNA expression of cytokines (IL-12, IFNα, IL-10) and TLR-9 using real time quantitative RT-PCR, and for the activation of the transcription factor NF-κB p65 using a chemiluminescence assay. Results The median fluorescence of the MHC class II molecule in non-stimulated foal macrophages and DCs at birth were 12.5 times and 11.2 times inferior, respectively, than adult horse cells (p = 0.009). That difference subsided at 3 months of life (p = 0.3). The expression of the CD86 co-stimulatory molecule was comparable in adult horse and foal macrophages and DCs, independent of treatment. CpG-ODN stimulation induced IL-12p40 (53 times) and IFNα (23 times) mRNA expression in CpG-ODN-treated adult horse DCs (p = 0.078), but not macrophages, in comparison to non-stimulated cells. In contrast, foal APCs did not respond to CpG-ODN stimulation with increased cytokine mRNA expression up to 3 months of age. TLR-9 mRNA expression and NF-kB activation (NF-kB p65) in foal DCs and macrophages were comparable (p > 0.05) to adult horse cells. Conclusion CpG-ODN treatment did not induce specific maturation and cytokine expression in foal macrophages and DCs. Nevertheless, adult horse DCs, but not macrophages, increased their expression of IL-12 and IFNα cytokines upon CpG-ODN stimulation. Importantly, foals presented an age-dependent limitation in the expression of MHC class II in macrophages and DCs, independent of treatment.

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.

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 (p0.01) 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 (p0.0001), 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 (p0.05), which correlated (p0.03) 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.

Previous studies have shown that protection against equine influenza virus (EIV) is partially mediated by virus-specific IgGa and IgGb. In this study we tested whether addition of a CpG ODN formulation to a commercial killed virus vaccine would enhance EIV-specific IgGa and IgGb antibody responses, and improve protection against an experimental EIV challenge. Thirty naive horses were assigned to one of three groups and vaccinated as follows: 10 were given vaccine (Encevac TC4, Intervet Inc.) alone, 10 were given vaccine plus 0.25 mg CpG ODN 2007 formulated with 30% Emulsigen (CpG/Em), and 10 controls were given saline. All horses were challenged with live virus 12 weeks after the final vaccination. Antibody responses were tested by single radial hemolysis (SRH) and ELISA, and protection was evaluated by determination of temperature, coughing, and clinical scores. Killed virus vaccine combined with CpG/Em induced significantly greater serologic responses than did the vaccine alone. All antibody isotypes tested increased after the addition of CpG/Em, although no shift in relative antibody isotypes concentrations was detected. Vaccination significantly improved protection against challenge but the differences between the two vaccine groups were not statistically significant. This study is the first demonstration that CpG/Em enhances antigen-specific antibody responses in horses and supports its potential to be used as an adjuvant for vaccines against equine infections.

CpG oligonucleotides (ODNs) are potent mucosal and systemic adjuvants. For practical applications however, improvements in delivery need to be developed. A mouse model was used to determine if the biological activity of CpG ODNs could be enhanced using a novel delivery system of biphasic lipid vesicles (Biphasix Vaccine-Targeting Adjuvant; VTA). Immunization studies were performed to evaluate the potential of VTA formulations to enhance the immunoadjuvant activity of CpG ODNs following systemic or mucosal administration with gD. Immune responses following immunization were assessed by protection from HSV-1 viral challenge and characterization of serum gD-specific antibody responses using ELISA. VTA formulations in combination with CpG and glycoprotein D (gD) were able to increase gD-specific IgG in serum compared to gD alone, and protect from a lethal HSV-1 challenge following subcutaneous immunization. Following mucosal immunization, VTA formulations in combination with CpG and antigen enhanced mucosal IgA responses compared to CpG and antigen administered in PBS.

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-α) 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.

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 (P0.0001). 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.

Fully synthetic peptide vaccines possess attractive cost and safety attributes. However, peptide vaccines that induce cell-mediated immunity require both selection of appropriate peptides and the development of adjuvant formulations supporting the induction of cellular immunity. An adjuvant formulation composed of emulsigen and the synthetic CpG motif containing ODN2007 was tested in cattle for its ability to induce cellular immunity after peptide vaccination, and compared to Rv3019c DNA vaccination. Peptides from the protective Mycobacterium bovis antigen Rv3019c were included into the vaccine on the basis of their frequent and strong recognition by T cells from M. bovis infected or BCG vaccinated cattle. Following peptide vaccination, strong IFN-gamma and proliferative T cell responses were observed. Proliferative, but no significant IFN-gamma responses were induced by DNA vaccination. Peptide vaccination boosted responses primed by DNA vaccination. In conclusion, emulsigen and CpG motif containing ODN constitute a promising adjuvant formulation to deliver peptides to veterinary species.

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-γ (IFN-γ) responses induced by BCG, the CFP vaccines induced strong antibody responses but weak IFN-γ 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-γ 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.

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.

A large number of studies demonstrated the immunostimulatory effects of CpG oligonucleotides (ODN), particularly in mice. In the present study, we evaluated the ability of lipid-based delivery systems to enhance the adjuvant effect of CpG-ODN and protect against infection in a porcine pleuropneumonia model. Increased levels of OmlA-specific antibody were detected in animals immunised with OmlA and CpG-ODN formulated in the delivery system Biphasix-vaccine targeting adjuvant (VTA), compared to pigs immunised with VTA without CpG-ODN or CpG-ODN alone. In addition, the responses induced by VTA/CpG formulation were similar to those induced by the commercial adjuvant VSA; however, VTA formulations caused significantly less tissue damage than VSA.

Subunit vaccines against tuberculosis show promise but require administration with adjuvants to stimulate relevant immune responses for protection. Guinea pigs are the model of choice for evaluating protective immunity to aerogenic challenge with virulent mycobacteria, but few studies have been undertaken to identify suitable adjuvants for vaccine screening in this species. Here, we compare the efficacy of several adjuvants to induce T cell responses to culture filtrate protein in guinea pigs. We report that of several adjuvants tested, the most promising was CpG ODN formulated in an aqueous emulsion. This adjuvant induced type 1 T cell responses equivalent to that of FIA, as measured by delayed-type hypersensitivity reactions (DTH), antigen-specific T cell proliferation and antigen-specific IgG1 and IgG2 responses. These data demonstrate the potential for CpG motif based adjuvants for use in TB vaccine screening in guinea pigs, and other diseases where a type 1 T cell response is required.

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.

Vaccine adjuvants must have the capacity to increase protective immune responses with minimal side effects. Conventional adjuvants not only cause undesirable tissue site reactions, but often induce T-helper type 2 (Th2)-biased responses which may be undesirable in certain disease scenarios. Oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) are novel adjuvants known to promote Th1-type immune responses. In this study, we compared various mineral oil, metabolizable oil and non-oil adjuvants alone and in combination with CpG ODN for their ability to augment immune responses to a truncated secreted form of bovine herpesvirus (BHV) glycoprotein D (tgD). All adjuvants tested induced Th2-biased immune responses characterized by a predominance of serum IgG1 as well as interleukin-4 (IL-4) production by in vitro stimulated splenocytes. The inclusion of CpG ODN in these formulations not only increased immune responses, but more importantly enhanced serum IgG2a levels and production of interferon-γ (IFN-γ) by splenocytes, indicating a more balanced or Th1-type response. The use of a mineral oil-based adjuvant at reduced doses in combination with CpG ODN attenuated the tissue damage while not compromising the magnitude of the immune response in both mice and sheep. In addition, reduced amounts of mineral oil combined with CpG ODN induced a more balanced Th1/Th2 immune response than the mineral oil used alone. Our results clearly demonstrate that CpG ODN can be used to enhance magnitude and balance of an immune response while reducing the amount of mineral oil and hence undesirable side effects of vaccine adjuvants.

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.

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.

Blue light controls the development of sporangiophores in the zygomycete Phycomyces blakesleeanus Burgeff. Light represses the production of microsporangiophores and enhances the development of macrosporangiophores. Inhibition of the biosynthesis of tetrahydrobiopterin, a cofactor of NO synthase, inhibits this photomorphogenesis. Light induces production of citrulline from arginine in the mycelium and in sporangiophores. The citrulline-forming activity is dependent on NADPH, independent of calcium, and inhibited by NO synthase inhibitors. It is reduced in tetrahydrobiopterin-depleted mycelium. Light induces emission of NO from the developing fungus in the same order of magnitude as citrulline formation from arginine. The NO donor sodium nitroprusside can replace the light effect on sporangiophore development, and inhibitors of NO synthase repress it. We suggest that a fungal NO synthase is involved in sporangiophore development and propose its participation in light signaling.

Publisher Summary This chapter describes matrix-assisted laser desorption/ionization mass spectrometry of the scrapie prion protein (PrPSc). PrPSc is a GPI-anchored membrane sialoglycoprotein encoded by a chromosomal gene and is implicated in the development of a unique group of fatal neurodegenerative diseases, including several human diseases, scrapie of sheep, and bovine spongiform encephalopathy of cattle. PrpSc is derived from a normal cellular isoform (PrPC) by an unidentified posttranslational event that causes physical changes and alters the location of PrP in the cell. PrPSc is more resistant to proteolysis, and after limited digestion by proteinase-K, an N-terminally truncated form accumulates. In a study described in the chapter, extensive peptide mapping of PrPSc was carried out using enzyme digestion. The study also included HPLC separation of the resulting peptides followed by amino acid analysis, Edman sequencing, and mass spectrometry. Mass spectra were obtained on a laser desorption time-of-flight mass spectrometer constructed at the Rockefeller University. The molecular mass of deglycosylated but not delipidated PrPSc was measured as 25,540 Da, approximately 150–200 Da higher than anticipated.

Scrapie prions are composed largely, if not entirely, of PrPSc molecules. The prion isolates Sc237 and 139H exhibit markedly different incubation times in Syrian, Armenian, and Chinese hamsters, as well as in transgenic (Tg) 81 mice expressing Syrian hamster PrP (SHaPrP). Repassage of prions from transgenic mice or Chinese hamsters into Syrian hamsters revealed that the original properties of the prion isolates are retained. When Syrian hamsters were first inoculated with 139H prions and subsequently challenged with Sc237 prions, the incubation period was determined by the faster Sc237 isolate. Regional mapping studies demonstrated different kinetics and patterns of PrPSc accumulation for Sc237 and 139H prions in the brains of Syrian hamsters as well as Tg(SHaPrP)7 mice. That distinct prion isolates induce different region-specific accumulations of PrPSc in brain suggests a novel mechanism for propagation of isolates whereby they replicate in particular sets of neurons. The prion isolates could be targeted to specific CNS cells by differing conformations of PrPSc, post-translational modifications of PrPSc such as Asn-linked glycosylation, or an as yet undetected macromolecule complexed with PrPSc in the prion.

Viroids are plant pathogens distinguished from viruses by the absence of a protein coat and by their small size. They are circular single-stranded RNA molecules consisting of a few hundred nucleotides, the smallest having about 240 and the largest about 600 nucleotides. Since viroids can have only a very limited coding capacity and as there is no experimental evidence for a viroid-coded translation product, one has to assume that viroid replication and pathogenesis depend completely on the enzyme systems of the host (reviews 1–5). Thus, their genetic information is the RNA structure, the ability to undergo structural transitions and the capability to interact with host cell factors. Most results reported here were obtained from studies on the potato spindle tuber viroid (PSTVd)