The Lymphatic Immune Response Induced by the Adjuvant AS01: A Comparison of Intramuscular and Subcutaneous Immunization Routes.

The liposome-based adjuvant AS01 incorporates two immune stimulants, 3-O-desacyl-4'-monophosphoryl lipid A and the saponin QS-21. AS01 is under investigation for use in several vaccines in clinical development. i.m. injection of AS01 enhances immune cell activation and dendritic cell (DC) Ag presentation in the local muscle-draining lymph node. However, cellular and Ag trafficking in the lymphatic vessels that connect an i.m. injection site with the local lymph node has not been investigated. The objectives of this study were: 1) to quantify the in vivo cellular immune response induced by AS01 in an outbred ovine model, 2) to develop a lymphatic cannulation model that directly collects lymphatic fluid draining the muscle, and 3) to investigate the function of immune cells entering and exiting the lymphatic compartments after s.c. or i.m. vaccination with AS01 administered with hepatitis B surface Ag (HBsAg). We show that HBsAg-AS01 induces a distinct immunogenic cellular signature within the blood and draining lymphatics following both immunization routes. We reveal that MHCII(high) migratory DCs, neutrophils, and monocytes can acquire Ag within muscle and s.c. afferent lymph, and that HBsAg-AS01 uniquely induces the selective migration of Ag-positive neutrophils, monocytes, and an MHCII(high) DC-like cell type out of the lymph node via the efferent lymphatics that may enhance Ag-specific immunity. We report the characterization of the immune response in the lymphatic network after i.m. and s.c. injection of a clinically relevant vaccine, all in real time using a dose and volume comparable with that administered in humans.

Local Antiglycan Antibody Responses to Skin Stage and Migratory Schistosomula of Schistosoma japonicum.

Schistosomiasis is a tropical disease affecting over 230 million people worldwide. Although effective drug treatment is available, reinfections are common, and development of immunity is slow. Most antibodies raised during schistosome infection are directed against glycans, some of which are thought to be protective. Developing schistosomula are considered most vulnerable to immune attack, and better understanding of local antibody responses raised against glycans expressed by this life stage might reveal possible glycan vaccine candidates for future vaccine research. We used antibody-secreting cell (ASC) probes to characterize local antiglycan antibody responses against migrating Schistosoma japonicum schistosomula in different tissues of rats. Analysis by shotgun Schistosoma glycan microarray resulted in the identification of antiglycan antibody response patterns that reflected the migratory pathway of schistosomula. Antibodies raised by skin lymph node (LN) ASC probes mainly targeted N-glycans with terminal mannose residues, Galß1-4GlcNAc (LacNAc) and Galß1-4(Fucα1-3)GlcNAc (LeX). Also, responses to antigenic and schistosome-specific glycosphingolipid (GSL) glycans containing highly fucosylated GalNAcß1-4(GlcNAcß1)n stretches that are believed to be present at the parasite's surface constitutively upon transformation were found. Antibody targets recognized by lung LN ASC probes were mainly N-glycans presenting GalNAcß1-4GlcNAc (LDN) and GlcNAc motifs. Surprisingly, antibodies against highly antigenic multifucosylated motifs of GSL glycans were not observed in lung LN ASC probes, indicating that these antigens are not expressed in lung stage schistosomula or are not appropriately exposed to induce immune responses locally. The local antiglycan responses observed in this study highlight the stage- and tissue-specific expression of antigenic parasite glycans and provide insights into glycan targets possibly involved in resistance to S. japonicum infection.

Incorporation of CpG into a liposomal vaccine formulation increases the maturation of antigen-loaded dendritic cells and monocytes to improve local and systemic immunity.

Liposomal vaccine formulations incorporating stimulants that target innate immune receptors have been shown to significantly increase vaccine immunity. Following vaccination, innate cell populations respond to immune stimuli, phagocytose and process Ag, and migrate from the injection site, via the afferent lymphatic vessels, into the local lymph node. In this study, the signals received in the periphery promote and sculpt the adaptive immune response. Effector lymphocytes then leave the lymph node via the efferent lymphatic vessel to perform their systemic function. We have directly cannulated the ovine lymphatic vessels to detail the in vivo innate and adaptive immune responses occurring in the local draining lymphatic network following vaccination with a liposome-based delivery system incorporating CpG. We show that CpG induces the rapid recruitment of neutrophils, enhances dendritic cell-associated Ag transport, and influences the maturation of innate cells entering the afferent lymph. This translated into an extended period of lymph node shutdown, the induction of IFN-γ-positive T cells, and enhanced production of Ag-specific Abs. Taken together, the results of this study quantify the real-time in vivo kinetics of the immune response in a large animal model after vaccination of a dose comparable to that administered to humans. This study details enhancement of numerous immune mechanisms that provide an explanation for the immunogenic function of CpG when employed as an adjuvant within vaccines.

Transcriptional profile in afferent lymph cells following vaccination with liposomes incorporating CpG.

Vaccine formulations incorporating innate immune stimulants are highly immunogenic; however, the biological signals that originate in the peripheral tissues at the site of injection and are transmitted to the local lymph node to induce immunity remain unclear. By directly cannulating the ovine afferent lymphatic vessels, we have previously shown that it takes 72 hr for mature antigen-loaded dendritic cells and monocytes to appear within afferent lymph following injection of a liposomal formulation containing the Toll-like receptor ligand CpG. In this present study, we characterize the global transcriptional signatures at this time-point in ovine afferent lymph cells as they migrate from the injection site into the lymphatics following vaccination with a liposome antigen formulation incorporating CpG. We show that at 72 hr post vaccination, liposomes alone induce no changes in gene expression and inflammatory profiles within afferent lymph; however, the incorporation of CpG drives interferon, antiviral and cytotoxic gene programmes. This study also measures the expression of key genes within individual cell types in afferent lymph. Antiviral gene signatures are most prominent in lymphocytes, which may play a significant and unexpected role in sustaining the immune response to vaccination at the site of injection. These findings provide a comprehensive analysis of the in vivo immunological pathways that connect the injection site with the local draining lymph node following vaccination.

The developing schistosome worms elicit distinct immune responses in different tissue regions.

Schistosome parasites follow a complex migration path through various tissues, changing their antigenic profile as they develop. A thorough understanding of the antibody response in each tissue region could help unravel the complex immunology of these developing parasites and aid vaccine design. Here we used a novel strategy for analysing the local antibody responses induced by Schistosoma japonicum infection at each site of infection. Cells from rat lymph nodes draining the sites of larval migration (the skin and lungs), the liver-lymph nodes where adults reside and the spleens were cultured to allow the in vivo-induced antibody-secreting cells to release antibody into the media. The amount and isotype of antibodies secreted in the supernatants differed significantly in the different lymph nodes and spleen, corresponding with the migration path of the schistosome worms. In addition, there were significant differences in binding specificity, as determined by surface labelling, western blots and by screening a glycan array. Through capturing the local antibody response, this study has revealed dramatic differences in the quality and specificity of the immune response at different tissue sites, and highlighted the existence of stage-specific protein and carbohydrate antigens. This will provide a valuable tool for the isolation of novel vaccine targets against the larval stages of schistosomes.

Modulation of soluble and particulate antigen transport in afferent lymph by monophosphoryl lipid A.

Vaccine adjuvants stimulate the innate immune system and determine the outcome of the immune response induced. A better understanding of their action is therefore crucial to the development of new and safer vaccines. Monophosphoryl lipid A (MPL), a 'detoxified' version of lipolysaccharide, is a promising new adjuvant component in human vaccines. The present study uses an ovine lymphatic cannulation model to study cell recruitment and antigen transport from the injection site into the afferent lymph, and how this is modulated by co-injection with MPL. Compared with saline, MPL injections caused only minor variations in lymph flow and no difference in cell number migrating into the lymph. MPL did, however, cause a significantly increased recruitment of neutrophils and monocytes, but not dendritic cells (DC) into the lymph for the first 12 h. Soluble ovalbumin (OVA) antigen flowed freely into the lymph over a 24-h period and was slightly reduced at 6-9 h in the MPL-injected sites. OVA-coated fluorescent 1-µ beads were initially transported predominantly by neutrophils and, from 24 to 72 h, by DC. MPL induced an increased and more sustained transport of beads by neutrophils and monocytes although it did not increase the phagocytic capacity of these cells. In contrast to aluminium adjuvant, MPL did not increase bead transport by DC at the later time point. These studies provide important new insights in the in vivo action of different adjuvants and the initial events that set up an immune response after vaccination.

Comparative experimental Haemonchus contortus infection of two sheep breeds native to the Canary Islands.

This study compares the susceptibility to Haemonchus contortus infection in two breeds of sheep endemic to the Canary Islands, the Canaria Hair Breed sheep and the Canaria sheep. Sheep were experimentally infected with 20,000 larvae of H. contortus and animals killed on days 7 and 28 post-infection. No difference between sheep breeds were detected in immature worm counts at days 7 or 28 post-infection. However, in comparison to the Canaria sheep breed, the Canaria Hair Breed sheep showed lower mean faecal egg counts, lower adult worm counts, lower number of eggs in utero and female worm stunting. Overall, these data suggest that the Canaria Hair Breed sheep has a greater resistance to H. contortus infection than Canaria sheep, and that this resistance may act at the level of the adult parasite.

Production of monoclonal antibodies reactive with ovine eosinophils.

BACKGROUND: There is strong evidence implicating eosinophils in host defence against parasites as well as allergic disease pathologies. However, a lack of reagents such as monoclonal antibodies (mAbs) specific for eosinophils has made it difficult to confirm the functional role of eosinophils in such disease conditions. Using an established mammary model of allergic inflammation in sheep, large numbers of inflammatory cells enriched for eosinophils were collected from parasite-stimulated mammary glands and used for the generation of mAbs against ovine eosinophils. RESULTS: A panel of mAbs was raised against ovine eosinophils of which two were shown to be highly specific for eosinophils. The reactivity of mAbs 3.252 and 1.2 identified eosinophils from various cell and tissue preparations with no detectable reactivity on cells of myeloid or lymphoid lineage, tissue mast cells, dendritic cells, epithelial cells or other connective tissues. Two other mAbs generated in this study (mAbs 4.4 and 4.10) were found to have reactivity for both eosinophils and neutrophils. CONCLUSION: This study describes the production of new reagents to identify eosinophils (as well as granulocytes) in sheep that will be useful in studying the role of eosinophils in disease pathologies in parasite and allergy models.

Cells, cytokines and other molecules associated with rejection of gastrointestinal nematode parasites.

Infections with gastrointestinal nematodes represent a major problem for human health and animal health and production. The physical size of the parasite, its changing life-cycle stages and the relative inaccessibility to host cells pose unique challenges to the immune system, which has evolved specialized strategies for parasite control. This paper reviews the work performed in the authors' laboratories to identify components that are involved in the natural rejection response against ruminant gastrointestinal nematode parasites, in particular Haemonchus contortus in sheep. The results of these studies indicate that stage-specific antibodies act in concert with effector cells, in particular globular leukocytes (intraepithelial mast cells) and eosinophils, appropriately activated/primed by type 2 (T2) cytokines, to initiate different mechanisms of parasite expulsion and killing. In addition, other molecules, in particular carbohydrate binding galectins, may be involved in strengthening the final effector phase of the rejection response.

The effect of immunosuppression with cyclosporin A on the development of sheep scab.

The present study confirms that following infection with the ectoparasitic sheep scab mite, Psoroptes ovis, there is a rapid (within 24 h) inflammatory influx of eosinophils and apoptosis of the keratinocytes at the site of infection. In order to investigate whether these inflammatory reactions are important in the maintenance of mite infection, a group of animals were treated daily after the establishment of infection with the potent anti-inflammatory drug, Cyclosporin A. The course of infection was monitored by determining the lesion area and mite numbers, systemic antibody and blood eosinophils, as well as the inflammatory cells and T cell sub-populations within the lesion throughout the 6-week duration of the experiment. These parameters were compared with those in a similar infected control (non-treated) group. In control infected animals, the lesion area and mite numbers increased steadily throughout the 6-week period. In contrast, lesion area and mite numbers were severely depressed in the group which received Cyclosporin A. Local and systemic eosinophils, and systemic antibody were also significantly reduced in the drug treated animals, compared to controls. Surprisingly however, the number of lesional pan T cells, T helper cells, gammadeltaT cells and dendritic cells in Cyclosporin A treated animals were either the same, or significantly (P < 0.05) enhanced when compared to the control infected animals at the termination of the experiment. The results will be discussed in terms of the role of the dermal inflammatory response in the establishment and maintenance of the sheep scab mite.

Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development.

The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11-12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test.

Using the local immune response from the natural buffalo host to generate an antibody fragment library that binds the early larval stages of Schistosoma japonicum.

Antibodies isolated from the local draining inguinal lymph node of field exposed-water buffaloes following challenge with Schistosoma japonicum cercariae showed high reactivity towards S. japonicum antigen preparations and bound specifically to formaldehyde-fixed S. japonicum schistosomules. Using this specific local immune response we produced a series of single-chain antibody Fv domain libraries from the same lymph nodes. Removal of phage that cross reacted with epitopes on adult parasites yielded a single-chain antibody Fv domain-phage library that specifically bound to whole formaldehyde-fixed and live S. japonicum schistosomules. DNA sequencing indicated clear enrichment of the single-chain antibody Fv domain library for buffalo B-cell complementarity determining regions post-selection for schistosomule binding. This study also revealed that long heavy chain complementarity determining regions appear to be an important factor when selecting for antibody binding fragments against schistosomule proteins. The selected single-chain antibody Fv domain-phage were used to probe a schistosome-specific protein microarray, which resulted in the recognition of many proteins expressed across all schistosome life-cycle stages. Following absorption to adult worms, the single-chain antibody Fv domain-phage library showed significantly reduced binding to most proteins, whilst two proteins (NCBI GenBank accession numbers AY915878 and AY815196) showed increased binding. We have thus developed a unique set of host derived single-chain antibody Fv domains comprising buffalo B-cell variable regions that specifically bind to early S. japonicum life-stages.

Exploiting natural immunity to helminth parasites for the development of veterinary vaccines.

The development of subunit vaccines against most parasitic helminth infections will require a better understanding of the different components of a natural rejection process including (1) recognition of parasite antigens; (2) induction of protective immune response phenotypes; and (3) activation of appropriate immune effector mechanisms. While novel technologies have allowed significant progress to be made in the identification of candidate vaccine antigens, the large scale production of these antigens and their presentation to the host with appropriate adjuvant systems remains a major problem in vaccine research. Identification of the molecular interactions involved in the innate immune response to helminth infections and the application of new genomic and proteomic technologies are likely to lead to major advances in these research fields. Gastrointestinal nematode parasites and liver fluke are the most important helminth parasites of production animals. In recent years, a lot of new knowledge has been gathered on the immunobiology of the host-parasite interactions in these two infection systems, which has allowed new vaccination strategies to be considered. Functional genomic technologies such as gene expression analysis by microarrays, promise to further advance our understanding of the molecular pathways leading to protection against parasite infections. This will not only have implications for vaccine research, but also provide novel targets for drug development and genetic selection.

Effects of implantation and early pregnancy on the expression of cytokines and vascular surface molecules in the sheep endometrium.

Successful implantation is a highly coordinated process involving changes in cytokines, adhesion molecules, hormones, enzymes and growth factors. This study examines the expression of key cytokines and vascular surface molecules in the pregnant uterus of sheep around the time of implantation. Uterine tissues and uterine washings were collected from non-pregnant and pregnant sheep at 17-19 days post-coitus (dpc), 26-27 and 34-36 dpc. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of caruncular/placentomal tissues revealed that cytokines IL-2, IL-4 and IL-8, which were not detected in non-pregnant uterus, were induced more strongly at 26-27 dpc than at other stages of pregnancy tested. Cytokines LIF, IL-6, IL-10, TNF-alpha were also most highly expressed at 26-27 dpc, expression of them being lower at other time-points during early pregnancy and non-pregnancy. The cytokines IL-1beta, IFN-gamma and TGF-beta were expressed in all non-pregnant and pregnant tissues examined. Enzyme-linked immunosorbent assay (ELISA) performed on uterine washings clearly detected the presence of IL-1alpha protein at 26-27 and 34-36 dpc. Immunohistochemistry revealed that expression of vascular adhesion molecule VCAM-1 in endometrial endothelium was strongly induced at 26-27 dpc in the pregnant endometrium. Expression of CD5 on vascular endothelium was not induced in placentomal tissues until 26-27 dpc and was further increased by 34-36 dpc. These results demonstrate a dynamic change in a wide range of cytokines during early stages of pregnancy, with a critical period around 26-27 dpc. In addition, at 26-27 dpc, expression of the surface/adhesion molecules, CD5 and VCAM-1, is induced on vascular endothelium of the sheep endometrium, possibly as a direct consequence of the changed cytokine environment, and may be involved in directing the changes in leucocyte migration observed during pregnancy.

Cellular and molecular characterisation of the ovine rectal mucosal environment.

Immunohistological characterisation of ovine rectal tissue has revealed the presence of lymphoid follicles, predominantly in the submucosa, that closely resemble those found in intestinal Peyer's patches (PPs). Distinct T (CD4+, CD8+, gammadelta-TCR+) and B (CD21+, CD45R+) lymphocyte staining patterns were observed within and around follicles of the rectal mucosa. In addition, IgA+ and IgE+ cells were also found at this tissue site, with both phenotypes commonly residing in the lamina propria. RT-PCR examination of the cytokines expressed in the rectal mucosal tissue revealed consistently high levels of TGFbeta and IL-8 mRNA, low levels of IL-2 mRNA and no detectable IL-4 mRNA. The presence of lymphoid follicles, IgA+ plasma cells and IgA-inducing cytokines in rectal tissue of sheep indicate that this may be a suitable route for delivering mucosal vaccines.

Antibody isotype profiles in serum and circulating antibody-secreting cells following mucosal and peripheral immunisations of sheep.

The isotype-specific antibody responses of sheep immunised with keyhole limpet hemocyanin by a peripheral route (intramuscular (i.m.) injection) were compared to those induced by immunisation via different mucosal routes: (1) intra-nasal spray; (2) rectal deposition with cholera toxin; (3) injection into the mucosa of the small intestine or rectum. Antigen-specific IgG1 antibodies were induced in the i.m., intra-intestinal and intra-rectal injection groups and in a proportion of the cholera toxin immunised sheep, but not in the intra-nasal immunisation group. IgA was the only antibody isotype detected in serum collected from the intra-nasal immunisation group. No significant differences in serum IgA levels were detected in any of the mucosal immunisation groups as compared to the i.m. injection group. In contrast, analysis of the in vitro antibody profiles secreted by circulating antibody-secreting cells (ASC) revealed significantly higher IgA responses in the supernatants from all mucosal immunisation groups. This suggests that the measurement of antibodies secreted by circulating ASCs may be a better correlate of local mucosal responses in ruminants, as has been previously demonstrated in human studies. In addition to IgG1 and IgA responses, immunisation by direct injection of antigen formulations into the intestinal and rectal mucosa were the only groups to induce consistently high IgG2 antibodies in serum and ASC cultures.

Advances in mucosal vaccination.

Pathogens that enter the body via mucosal surfaces face unique defense mechanisms that combine the innate barrier provided by the mucus layer with an adaptive response typified by the production and transepithelial secretion of pathogen-specific IgA. Both the measurement and induction of mucosal responses pose significant challenges for experimental and practical application and may need to be adapted to the species under study. In particular, for livestock, immunization procedures developed in small rodent models are not always effective in large animals or compatible with management practices. This paper reviews the latest advances in our understanding of the processes that lead to secretory IgA responses and how this relates to the development of mucosal immunization procedures and adjuvants for veterinary vaccines. In addition, it highlights the complex interactions that can take place between the pathogen and the host's immune response, with specific reference to Chlamydia/Chlamydophila infections in sheep.

Afferent lymphatic cannulation as a model system to study innate immune responses to infection and vaccination.

The afferent lymphatics consist of the cells and immunomodulatory signals that are involved in the early response to peripheral stimuli. Examination of this compartment in both homeostatic and stimulatory conditions permits the analysis of the innate biological pathways responsible for the generation of an adaptive immune response in the lymph node. Afferent lymphatic cannulation is therefore an ideal model system to study cellular migration and antigen dispersal kinetics during infection and vaccination. Utilisation of these lymphatic cannulation models has demonstrated the ability to both increase current understanding of infectious diseases, vaccine delivery systems and has the potential to target effector cells and molecules that may be used as novel therapeutic or vaccine targets.

Discovery of novel Schistosoma japonicum antigens using a targeted protein microarray approach.

BACKGROUND: Novel vaccine candidates against Schistosoma japonicum are required, and antigens present in the vulnerable larval developmental stage are attractive targets. Post-genomic technologies are now available which can contribute to such antigen discovery. METHODS: A schistosome-specific protein microarray was probed using the local antibody response against migrating larvae. Antigens were assessed for their novelty and predicted larval expression and host-exposed features. One antigen was further characterised and its sequence and structure were analysed in silico. Real-time polymerase chain reaction was used to analyse transcript expression throughout development, and immunoblotting and enzyme-linked immunosorbent assays employed to determine antigen recognition by antibody samples. RESULTS: Several known and novel antigens were discovered, two of which showed up-regulated transcription in schistosomula. One novel antigen, termed S. japonicum Ly-6-like protein 1 (Sj-L6L-1), was further characterised and shown to share structural and sequence features with the Ly-6 protein family. It was found to be present in the worm tegument and expressed in both the larval and adult worms, but was found to be antigenic only in the lungs that the larvae migrate to and traverse. CONCLUSIONS: This study represents a novel approach to vaccine antigen discovery and may contribute to schistosome vaccine development against this important group of human and veterinary pathogens.

Vaccination with liposomal poly(I:C) induces discordant maturation of migratory dendritic cell subsets and anti-viral gene signatures in afferent lymph cells.

Vaccine formulations administered in the periphery must activate naive immune cells within the lymph node. In this study, we have directly cannulated the ovine lymphatic vessels to investigate the cellular and molecular mechanisms that transfer information from the periphery into the local draining lymph node via the afferent lymph. Inclusion of poly(I:C) into a liposomal vaccine formulation enhances the neutrophil-associated inflammatory immune response in afferent lymph and increases antigen uptake by migratory dendritic cells (DCs). Interestingly, antigen positive migratory DCs undergo discordant maturation, with peak expression of CD86 at 4 h and CD80 at 48-72 h post vaccination. Afferent lymph monocytes up-regulate expression of genes related to inflammatory and anti-viral immune phenotypes following vaccination however show no differentiation into APCs prior to their migration to the local lymph node as measured by surface MHC II expression. Finally, this study reveals the addition of poly(I:C) increases systemic antigen-specific humoral immunity. These findings provide a detailed understanding of the real time in vivo immune response induced by liposomes incorporating the innate immune agonist poly(I:C) utilising a vaccination setting comparable to that administered in humans.