Oral antigen exposure in newborn piglets circumvents induction of oral tolerance in response to intraperitoneal vaccination in later life.

BACKGROUND: We previously determined that newborn piglets orally gavaged with Ovalbumin (OVA) responded to systemic OVA re-exposure with tolerance; if adjuvants were included in oral vaccine, piglets responded with antibody-mediated immunity (Vet Immunol Immunopathol 161(3-4):211-21, 2014). Here, we will investigate whether newborn piglets gavaged with a vaccine comprised of OVA plus unmethylated CpG oligodeoxynucleotides (CpG; soluble component; OVA/CpG) combined with OVA plus CpG encapsulated within polyphosphazene microparticles (MP; particulate component) responded with systemic and mucosal immunity. To monitor the response to systemic antigen re-exposure, piglets were i.p.-immunized with OVA plus Incomplete Freund's Adjuvant (IFA) one month later. RESULTS: Newborn piglets (n = 5/group) were gavaged with a combined soluble and particulate vaccine consisting of OVA (0.5-0.05 mg) plus 50 µg CpG and 0.5 mg OVA plus 50 µg CpG encapsulated within a polyphosphazene MP (0.5 mg) referred to as OVA/CpG + MP. Control piglets were gavaged with saline alone. Piglets were i.p. immunized with 10 mg OVA (or saline) in IFA at four weeks of age and then euthanized at eight weeks of age. We observed significantly higher titres of serum anti-OVA immunoglobulin (Ig) IgM, IgA, IgG, IgG1, IgG2 and IgG in piglets immunized with 0.05 mg OVA/CpG + MP relative to saline control animals. Thus, a single oral exposure at birth to a combined soluble and particulate OVA vaccine including adjuvants can circumvent induction of oral tolerance which impacts response to i.p. vaccination in later life. Further, piglets gavaged with 0.05 mg OVA/CpG + MP generated significant anti-OVA IgG and IgG1 titres in lung compared to saline control piglets but results were comparable to titres measured in parenteral control piglets. Peripheral blood mononuclear cells (PBMCs) ex vivo-stimulated with OVA showed markedly decreased production of IL-10 cytokine after 72 hours relative to animal-matched cells incubated with media alone. No production of IFN-γ was observed from any groups. CONCLUSION: Newborn piglets gavaged with low dose soluble and particulate OVA plus CpG ODN and polyphosphazene adjuvants produced antigen-specific antibodies in serum and lung after systemic re-exposure in later life. These data indicate circumvention of oral tolerance but not induction of oral immunity.

B-cell activating factor (BAFF) promotes CpG ODN-induced B cell activation and proliferation.

It is controversial whether naïve B cells are directly activated in response to TLR9 ligand, CpG ODN. Although bovine blood-derived CD21(+) B cells express TLR9 and proliferate in response to CpG in mixed-cell populations, purified bovine B cells do not proliferate significantly in response to CpG ODN, even when the B cell receptor is engaged. When co-cultured with CD14(+) myeloid cells and/or B-cell activating factor (BAFF), a cytokine produced by activated myeloid cells, there was a significant increase in CpG-specific B cell proliferation, and the number of large B cells in general or positive for CD25, all of which are markers for B cell activation. These data suggest that activated myeloid cells and BAFF prime B cells for significant CpG-specific activation. Understanding the signals required to mediate efficient CpG-induced, antigen-independent and T-cell independent activation of B cells has implications for polyclonal B cell activation and the development of autoimmune diseases.

Vaccine Formulation for Infectious Diseases and Adjuvant Mechanisms of Action.

The ultimate goal for vaccination is the generation of a safe and effective immune response that protects against diseases [...].

Polyphosphazenes as Adjuvants for Animal Vaccines and Other Medical Applications.

Polyphosphazenes are a class of experimental adjuvants that have shown great versatility as vaccine adjuvants in many animal species ranging from laboratory rodents to large animal species. Their adjuvant activity has shown promising results with numerous viral and bacterial antigens, as well as with crude and purified antigens. Vaccines adjuvanted with polyphosphazenes can be delivered via systemic and mucosal administration including respiratory, oral, rectal, and intravaginal routes. Polyphosphazenes can be used in combination with other adjuvants, further enhancing immune responses to antigens. The mechanisms of action of polyphosphazenes have not fully been defined, but several systematic studies have suggested that they act primarily by activating innate immunity. In the present review, we will highlight progress in the development of polyphosphazenes as adjuvants in animals and their other medical applications.

The Adjuvants Polyphosphazene (PCEP) and a Combination of Curdlan Plus Leptin Promote a Th17-Type Immune Response to an Intramuscular Vaccine in Mice.

Our aim was to determine whether polyphosphazene (PCEP), Curdlan (ß-glucan, a dectin-1 agonist), and Leptin could act as adjuvants to promote a Th17-type adaptive immune response in mice. Mice were vaccinated via the intramuscular route then boosted three weeks later with Ovalbumin plus: PCEP, Leptin, Curdlan, PCEP+Curdlan, Curdlan+Leptin, or saline. Mice vaccinated with OVA+PCEP and OVA+Curdlan+Leptin showed significantly higher frequency of antigen-specific CD4+ T cells secreting IL-17 relative to OVA-vaccinated mice. No formulation increased the frequency of CD4+ T cells secreting IL-4 or IFNγ. Since activation of innate immunity precedes the development of adaptive immunity, we wished to establish whether induction of Th17-type immunity could be predicted from in vitro experiments and/or from the local cytokine environment after immunization with adjuvants alone. Elevated IL-6 and TGFß with reduced secretion of IL-12 is a cytokine milieu known to promote differentiation of Th17-type immunity. We injected the immunostimulants or saline buffer into murine thigh muscles and measured acute local cytokine production. PCEP induced significant production of IL-6 and reduced IL-12 production in muscle but it did not lead to elevated TGFß production. Curdlan+Leptin injected into muscle induced significant production of TGFß and IL-17 but not IL-6 or IL-12. We also stimulated splenocytes with media or PCEP, Leptin, Curdlan, PCEP+Curdlan, Curdlan+Leptin, PCEP+Leptin, and PCEP+Curdlan+Leptin and measured cytokine production. PCEP stimulation of splenocytes failed to induce significant production of IL-6, IL-12, TGFß, or IL-17 and therefore ex vivo splenocyte stimulation failed to predict the increased frequency of Th17-type T cells in response to the vaccine. Curdlan-stimulated splenocytes produced Th1-type, inducing cytokine, IL-12. Curdlan+/-PCEP stimulated TGF-ß production and Curdlan+Leptin+/- PCEP induced secretion of IL-17. We conclude that PCEP as well as Curdlan+Leptin are Th17-type vaccine adjuvants in mice but that cytokines produced in response to these adjuvants in muscle after injection or in ex vivo cultured splenocytes did not predict their role as a Th17-type adjuvant. Together, these data suggest that the cytokine environments induced by these immunostimulants did not predict induction of an antigen-specific Th17-type adaptive immune response. This is the first report of these adjuvants inducing a Th17-type adaptive immune response.

Elastase-dependent live attenuated swine influenza A viruses are immunogenic and confer protection against swine influenza A virus infection in pigs.

Influenza A viruses cause significant morbidity in swine, resulting in a substantial economic burden. Swine influenza virus (SIV) infection also poses important human public health concerns. Vaccination is the primary method for the prevention of influenza virus infection. Previously, we generated two elastase-dependent mutant SIVs derived from A/Sw/Saskatchewan/18789/02(H1N1): A/Sw/Sk-R345V (R345V) and A/Sw/Sk-R345A (R345A). These two viruses are highly attenuated in pigs, making them good candidates for a live-virus vaccine. In this study, the immunogenicity and the ability of these candidates to protect against SIV infection were evaluated in pigs. We report that intratracheally administrated R345V and R345A induced antigen-specific humoral and cell-mediated immunity characterized by increased production of immunoglobulin G (IgG) and IgA antibodies in the serum and in bronchoalveolar lavage fluid, high hemagglutination inhibition titers in serum, an enhanced level of lymphocyte proliferation, and higher numbers of gamma interferon-secreting cells at the site of infection. Based on the immunogenicity results, the R345V virus was further tested in a protection trial in which pigs were vaccinated twice with R345V and then challenged with homologous A/Sw/Saskatchewan/18789/02, H1N1 antigenic variant A/Sw/Indiana/1726/88 or heterologous subtypic H3N2 A/Sw/Texas/4199-2/9/98. Our data showed that two vaccinations with R345V provided pigs with complete protection from homologous H1N1 SIV infection and partial protection from heterologous subtypic H3N2 SIV infection. This protection was characterized by significantly reduced macroscopic and microscopic lung lesions, lower virus titers from the respiratory tract, and lower levels of proinflammatory cytokines. Thus, elastase-dependent SIV mutants can be used as live-virus vaccines against swine influenza in pigs.

PCEP enhances IgA mucosal immune responses in mice following different immunization routes with influenza virus antigens.

BACKGROUND: We previously demonstrated that polyphosphazenes, particularly PCEP, enhance immune responses in mice immunized subcutaneously and intranasally. The objective of the present study was to investigate the efficacy of polyphosphazenes as adjuvants when delivered through different routes of vaccine administration. METHODS: BALB/c mice were immunized through intranasal, subcutaneous, oral and intrarectal delivery with vaccine formulations containing either influenza X:31 antigen alone or formulated in PCEP. Serum and mucosal washes were collected and assayed for antigen-specific antibody responses by ELISA, while splenocytes were assayed for antigen-specific cytokine production by ELISPOT. RESULTS: Intranasal immunization with PCEP+X:31 induced significantly higher IgA titers in all mucosal secretions (lung, nasal, and vaginal) compared to the other routes. Serum analysis showed that all mice given the PCEP+X:31 combination showed evidence of enhanced IgG2a titers in all administered routes, indicating that PCEP can be effective as an adjuvant in enhancing systemic immune responses when delivered via different routes of administration. CONCLUSIONS: We conclude that PCEP is a potent and versatile mucosal adjuvant that can be administered in a variety of routes and effectively enhances systemic and local immune responses. Furthermore, intranasal immunization was found to be the best administration route for enhancing IgA titers, providing further evidence for the potential of PCEP as a mucosal adjuvant.

Characterization of bovine Toll-like receptor 8: ligand specificity, signaling essential sites and dimerization.

Toll-like receptors (TLRs) are a family of highly conserved germline-encoded pattern-recognition receptors (PRRs) and are essential for host immune response. Little is known regarding the activation mechanism of TLRs especially of the TLR7/8/9 subfamily. Here we cloned and characterized bovine TLR8 (bTLR8) and found that it is highly responsive to two TLR7 ligands, imiquimod and gardiquimod, in transfected cell lines. Using the transfected cell lines as model systems, we analyzed by mutagenesis the roles of potentially important regions of bTLR8 in receptor signaling: 5 insertions in leucine rich repeats (LRRs) of the ectodomain (ECD), 9 N-glycosylation sites, all the cysteines, an aspartate conserved between TLRs, the transmembrane (TM) domain and different cytoplasmic regions. All 5 insertions, 2 N-glycosylation sites, most of the cysteines, the conserved aspartate, the TM and each of the cytoplasmic regions are essential for TLR8 signaling. We also showed that bTLR8 undergoes dimerization/self-association which was not affected by imidazoquinoline stimulation. This observation together with kinetics of activation suggested that a ligand-induced dimer conformational switch is mainly responsible for TLR8 activation. All the TLR8 signaling essential sites were examined for their requirement in dimerization; no single mutation or group of mutations affected the dimerization. However, among the impaired TLR8 mutants, all those containing mutations in the transmembrane or cytoplasmic regions and only two within the ECD (N515D and D536A) showed dominant negative inhibition to wild type receptor, whereas the others, all within the ECD, did not compete with wild type TLR8. A model for activation of bTLR8 was described based on these data.

Multiple molecular regions confer intracellular localization of bovine Toll-like receptor 8.

Toll-like receptors (TLRs), a family of highly conserved germline-encoded pattern-recognition receptors (PRRs), are essential for the host immune response. The cellular localization of TLR proteins determines the access to certain sources of ligands and thus the triggering of downstream cellular signaling. The TLR7/8/9 subfamily proteins are localized intracellularly but the molecular elements determining the cellular localization of these proteins are not fully understood. Here we demonstrated that the bovine TLR8 (bTLR8) protein is localized in the ER cellular compartment of transfected cells before and after cell activation. Using chimeric constructs, we showed that the bTLR8 transmembrane (TM) and cytoplasmic (CP) regions could direct the bovine herpesvirus 1 (BHV-1) glycoprotein D (gD) extracellular domain (ECD) to an intracellular localization. Furthermore, the bTLR8 TM, the linker region between the TM and TIR domains, and the TIR-tail region all partially contributed to the intracellular localization. However, truncation of the bTLR8 with the TM and CP regions removed did not alter its intracellular localization, suggesting that ectodomain (ECD) itself contains intracellular information. Indeed, the bTLR8 ECD also targeted the gD ECD to the intracellular localization. Our results suggest that multiple regions, including ECD, TM, linker and TIR-tail regions of bTLR8, are involved in determining the localization of cellular ER compartment.

Porcine TLR8 and TLR7 are both activated by a selective TLR7 ligand, imiquimod.

Toll-like receptors (TLRs) are a family of highly conserved germline-encoded pattern-recognition receptors (PRR), which are utilized by the innate immune system to recognize microbial components, known as pathogen-associated molecular patterns (PAMP). We cloned and characterized porcine TLR7 and TLR8 genes from pig lymph node tissue. Sequence analysis showed that the aa sequence identities of porcine TLR7 with human, mouse and bovine TLR7 are 85, 78 and 90%, respectively, whereas porcine TLR8 aa sequence identities with human, mouse and bovine TLR8 are 73, 69 and 79%, respectively. Both porcine TLR7 and TLR8 proteins were expressed in cell lines and were N-glycosylated. The stimulatory activity of TLR7 and TLR8 ligands to porcine and human TLR7 and TLR8 in transiently transfected Cos-7 and 293T cells were analyzed using a NF-kappaB reporter assay. Two imidazoquinoline molecules, imiquimod and gardiquimod, markedly activated both porcine TLR7 and TLR8 whereas only human TLR7, but not TLR8, was activated by the ligands. Therefore, receptor specificity for porcine TLR8 is clearly species specific. We further showed that porcine TLR7 and TLR8 are located intracellularly and are mainly within the endoplasmic reticulum. Moreover, activation of transfected cells and porcine PBMC by TLR7 ligands was inhibited by bafilomycin A(1) indicating the requirement of endosomal/lysosomal acidification for activation of the receptors.

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.

CD14+ cells are required for IL-12 response in bovine blood mononuclear cells activated with Toll-like receptor (TLR) 7 and TLR8 ligands.

Single-stranded viral RNA (ssRNA) was recently identified as the natural ligand for TLR7 and TLR8. ssRNA sequences from viruses, as well as their synthetic analogues stimulate innate immune responses in immune cells from humans and mice, but their immunostimulatory activity has not been investigated in ruminants. In the present investigations, we tested whether synthetic RNA oligoribonucleotides (ORN) can activate immune cells from cattle. In vitro incubation of bovine peripheral blood mononuclear cells (PBMCs) with ORN-induced production of IL-12, IFN-gamma and TNF-alpha. No significant induction of IFN-alpha was observed. Depletion of CD14+ cells from PBMC abrogated the IL-12 response and consequently the IFN-gamma response, suggesting that CD14+ cells are required for PBMC immune activation with ORN. Consistent with these findings, the putative receptors for ORN (TLR7 and TLR8) were expressed at higher levels in the CD14+ fraction than the CD14- PBMC fraction. Pre-treatment of PBMC with bafilomycin (an inhibitor of phagosomal acidification) prior to stimulation with ORN abolished the cytokine responses, confirming that the receptor(s) which mediate the ORN-induced responses are intracellular. These results demonstrate for the first time that the TLR7/8 agonist ORN's have strong immune stimulatory effects in cattle, and suggest that further investigation on the potential of TLR7/8 ligands to activate innate and adaptive immune responses in domestic animals are warranted.

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.

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.

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.

Immune mechanisms and therapeutic potential of CpG oligodeoxynucleotides.

Unmethylated CpG motifs in bacterial DNA and synthetic oligodeoxynucleotides activate immune cells that express Toll-like Receptor 9. Activation through this receptor triggers cellular signaling that leads to production of a proinflammatory and a Th1-type, antigen-specific immune response. The immunostimulatory effects of CpG oligodeoxynucleotides confer protection against infectious disease, allergy and cancer in animal models, and clinical trials have been initiated. However, CpG oligodeoxynucleotides may exacerbate disease in some situations. We will review current concepts in the mechanisms of activating Toll-like Receptor 9 with CpG oligodeoxynucleotides and highlight opportunities for using large animal models to better determine the mechanisms of action.

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.

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.

Adoptive transfer of BALb/c mouse splenocytes reduces lesion severity and induces intestinal pathophysiologic changes in the Mycobacterium avium Subspecies paratuberculosis beige/scid mouse model.

Successful immune reconstitution would enhance resistance of beige/scid mice to chronic infection with Mycobacterium avium subspecies paratuberculosis, but may cause damage to intestinal tissue. Therefore, we investigated the effect of adoptive transfer of BALB/c mouse splenocytes on lesion severity and intestinal physiology in beige/scid mice infected with M. paratuberculosis. Mice were inoculated intraperitoneally (i.p.) with M. paratuberculosis, and two weeks later were inoculated i.p. with viable spleen cells from immune-competent BALB/c mice. Mice were necropsied 12 weeks after infection when engraftment of lymphocytes, clinical disease, pathologic lesions, and intestinal electrophysiologic parameters were evaluated. Lymphocytes were rare in control beige/scid mice not inoculated with spleen cells. In contrast, high numbers of CD4+, CD8+, and B220+ lymphocytes were detected in the spleen of all beige/scid mice (n = 24) inoculated with spleen cells, indicating that adoptive transfer resulted in successful engraftment of donor lymphocytes (immune reconstitution). Immune reconstitution of M. paratuberculosis-infected beige/ scid mice significantly reduced the severity of clinical disease and pathologic lesions, and numbers of bacteria in the liver. However, intestinal electrophysiologic parameters studied in vitro indicated that intestinal tissues from reconstituted beige/scid mice had reduced short-circuit current responses (due to reduced ion secretion) following electrical, glucose, and forskolin stimulation. These abnormal responses suggested that neural or epithelial cells in the intestine were damaged. We conclude that successful immune reconstitution of beige/scid mice enhance their resistance to M. paratuberculosis infection, but may cause pathophysiologic changes associated with intestinal inflammation.

The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles.

Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-γ) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks.