Cationic liposomes as adjuvants for subunit protein vaccines:their role in the depot effect and antigen processing by APCs

Introduction: The requirement of adjuvants in subunit protein vaccination is well known yet their mechanisms of action remain elusive. Of the numerous mechanisms suggested, cationic liposomes appear to fulfil at least three: the antigen depot effect, the delivery of antigen to antigen presenting cells (APCs) and finally the danger signal. We have investigated the role of antigen depot effect with the use of dual radiolabelling whereby adjuvant and antigen presence in tissues can be quantified. In our studies a range of cationic liposomes and different antigens were studied to determine the importance of physical properties such as liposome surface charge, antigen association and inherent lipid immunogenicity. More recently we have investigated the role of liposome size with the cationic liposome formulation DDA:TDB, composed of the cationic lipid dimethyldioctadecylammonium (DDA) and the synthetic mycobacterial glycolipid trehalose 6,6’-dibehenate (TDB). Vesicle size is a frequently investigated parameter which is known to result in different routes of endocytosis. It has been postulated that targeting different routes leads to different intracellular signaling pathway activation and it is certainly true that numerous studies have shown vesicle size to have an effect on the resulting immune responses (e.g. Th1 vs. Th2). Aim: To determine the effect of cationic liposome size on the biodistribution of adjuvant and antigen, the ensuing humoral and cell-mediated immune responses and the uptake and activation of antigen by APCs including macrophages and dendritic cells. Methods: DDA:TDB liposomes were made to three different sizes (~ 0.2, 0.5 and 2 µm) followed by the addition of tuberculosis antigen Ag85B-ESAT-6 therefore resulting in surface adsorption. Liposome formulations were injected into Balb/c or C57Bl/6 mice via the intramuscular route. The biodistribution of the liposome formulations was followed using dual radiolabelling. Tissues including muscle from the site of injection and local draining lymph nodes were removed and liposome and antigen presence quantified. Mice were also immunized with the different vaccine formulations and cytokine production (from Ag85B-ESAT-6 restimulated splenocytes) and antibody presence in blood assayed. Furthermore, splenocyte proliferation after restimulating with Ag85B-ESAT-6 was measured. Finally, APCs were compared for their ability to endocytose vaccine formulations and the effect this had on the maturation status of the cell populations was compared. Flow cytometry and fluorescence labelling was used to investigate maturation marker up-regulation and efficacy of phagocytosis. Results: Our results show that for an efficient Ag85B-ESAT-6 antigen depot at the injection site, liposomes composed of DDA and TDB are required. There is no significant change in the presence of liposome or antigen at 6hrs or 24hrs p.i, nor does liposome size have an effect. Approximately 0.05% of the injected liposome dose is detected in the local draining lymph node 24hrs p.i however protein presence is low (<0.005% dose). Preliminary in vitro data shows liposome and antigen endocytosis by macrophages; further studies on this will be presented in addition to the results of the immunisation study.

Formulation and characterisation of an effective particulate delivery vehicle for the novel sub-unit vaccine antigen, Ag85B-ESAT-6

This research focused on the formation of particulate delivery systems for the sub-unit fusion protein, Ag85B-ESAT-6, a promising tuberculosis (TB) vaccine candidate. Initial work concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyl dioctadecyl ammonium (DDA). These studies demonstrated that addition of the immunomodulatory trehalose dibehenate (TDB) enhanced the physical stability of the system whilst also adding further adjuvanticity. Indeed, this formulation was effective in stimulating both a cell mediated and humoural immune response. In order to investigate an alternative to the DDA-TDB system, microspheres based on poly(DL-lactide-co-glycolide) (PLGA) incorporating the adjuvants DDA and TDB, either alone or in combination, were first optimised in terms of physico-chemical characteristics, followed by immunological analysis. The formulation incorporating PLGA and DDA emerged as the lead candidate, with promising protection data against TB. Subsequent optimisation of the lead microsphere formulation investigated the effect of several variables involved in the formulation process on physico-chemical and immunological characteristics of the particles produced. Further, freeze-drying studies were carried out with both sugar-based and amino acid-based cryoprotectants, in order to formulate a stable freexe-dried product. Finally, environmental scanning electron microscopy (ESEM) was investigated as a potential alternative to conventional SEM for the morphological investigation of microsphere formulations. Results revealed that the DDA-TDB liposome system proved to be the most immunologically efficient delivery vehicle studied, with high levels of antibody and cytokine production, particularly gamma-interferon (IFN-ϒ), considered the key cytokine marker for anti-mycobacterial immunity. Of the microsphere systems investigated, PLGA in combination with DDA showed the most promise, with an ability to initiate a broad spectrum of cytokine production, as well as antigen specific spleen cell proliferation comparable to that of the DDA-TDB formulation.

Influence of microbial antigen formulation and delivery route on the immune response

Recent technological advances have resulted in the production of safe subunit and synthetic small peptide vaccines. Unfortunately, these vaccines are weakly or non-immunogenic in the absence of an immunological adjuvant (agents that can induce strong immunity to antigens). In addition, in order to prevent and/or control infection at the mucosal surface, stimulation of the mucosal immune system is essential. This may be achieved via the common mucosal immune system by exposure to antigen at a mucosal surface remote from the area of infection. Initial studies investigated the potential of multiple emulsions in effecting oral absorption and the subsequent immune responses to a lipopolysaccharide vaccine (LPS) after immunisation. Nasal delivery of LPS was carried out in parallel work using either aqueous solution or gel formulations. Tetanus toxoid vaccine in simple solution was delivered to guinea pigs as free antigen or entrapped in DSPC liposomes. In addition, adsorbed tetanus toxoid vaccine was delivered nasally free or in an aerosil gel formulation. This work was extended to investigate guinea pigs immunised by various mucosal routes with a herpes simplex virus subunit vaccine prepared from virus infected cells and delivered in gels, multiple emulsions and liposomes. Comparable serum antibody responses resulted but failed to produce enhanced protection against vaginal challenge when compared to subcutaneous immunisation with alhydrogel adjuvanted vaccine. Thus, immunisation of the mucosal surface by these methods may have been inadequate. These studies were extended in an attempt to protect against HSV genital challenge by construction of an attenuated Salmonella typhimurium HWSH aroA mutant expressing a cloned glycoprotein D-l gene fused to the Es-cherichia coli lac z promoter. Preliminary work on the colonisation of guinea pigs with S. typhimurium HWSH aroA mutants were carried out, with the aim of using the guinea pig HSV vaginal model to investigate protection.

The histogenesis of lymphoid organs in Tilapia mossambica and antibody responses in adults and fry

The thymic anlagen appears in Tilapia mossambica at 2 days post hatching and becomes lymphoid at 5 days. Lymphoid cells were first seen in the pronephros at 14 days and in the spleen at approximately five weeks of age. Differentiation into red and white pulp regions was seen by 10 weeks of age. Light and electron microscopic studies of adult lymphoid organ revealed increases in size and lymphoid cell numbers. Adult thymus develops a clearer corticomedullary differentiation of thymic corpuscles in the medulla and in the splenic red and white pulp became more distinct. Melanomacrophage centres were seen in spleen and pronephros. Adult fish gave primary and secondary antibody responses following challenge with sheep red bloods cells (SRBC), Escherichia coli (E. coli) and human gamma globulin (HGG). Plaque forming cell and immunocytoadherence assays revealed that head kidney and spleen were major sites for antibody production and development of antigen reactive cells. Proliferative activity in these organs was revealed using autoradiography and scintillation counting. Increased levels of pyroninophilia were also seen following antigenic challenge. Pilot studies on adults revealed that they were capable of rejecting first and second set allografts and leucocytes from spleen and head kidney proliferated in mixed leucocyte cultures. Antibody responses to SRBC, E. coli and HGG develop at about 10-12 weeks of age. Fry given either a single injection of SRBC at 10 weeks or two injections of the same antigen at 10 weeks and 12 days later, failed to respond to a further challenge with SRBC 56 days after the first injection (A time when animals would normally respond positively to this antigen). Injection of E. coli at the same times resulted in a prolonged antibody response.

Discriminating antigen and non-antigen using proteome dissimilarity III:tumour and parasite antigens

Computational genome analysis enables systematic identification of potential immunogenic proteins within a pathogen. Immunogenicity is a system property that arises through the interaction of host and pathogen as mediated through the medium of a immunogenic protein. The overt dissimilarity of pathogenic proteins when compared to the host proteome is conjectured by some to be the determining principal of immunogenicity. Previously, we explored this idea in the context of Bacterial, Viral, and Fungal antigen. In this paper, we broaden and extend our analysis to include complex antigens of eukaryotic origin, arising from tumours and from parasite pathogens. For both types of antigen, known antigenic and non-antigenic protein sequences were compared to human and mouse proteomes. In contrast to our previous results, both visual inspection and statistical evaluation indicate a much wider range of homologues and a significant level of discrimination; but, as before, we could not determine a viable threshold capable of properly separating non-antigen from antigen. In concert with our previous work, we conclude that global proteome dissimilarity is not a useful metric for immunogenicity for presently available antigens arising from Bacteria, viruses, fungi, parasites, and tumours. While we see some signal for certain antigen types, using dissimilarity is not a useful approach to identifying antigenic molecules within pathogen genomes.

Quantitative online prediction of peptide binding to the major histocompatibility complex

With its implications for vaccine discovery, the accurate prediction of T cell epitopes is one of the key aspirations of computational vaccinology. We have developed a robust multivariate statistical method, based on partial least squares, for the quantitative prediction of peptide binding to major histocompatibility complexes (MHC), the principal checkpoint on the antigen presentation pathway. As a service to the immunobiology community, we have made a Perl implementation of the method available via a World Wide Web server. We call this server MHCPred. Access to the server is freely available from the URL: http://www.jenner.ac.uk/MHCPred. We have exemplified our method with a model for peptides binding to the common human MHC molecule HLA-B*3501.

A Therapeutic Antibody for Cancer, Derived from Single Human B Cells.

Some patients with cancer never develop metastasis, and their host response might provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early-stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high-affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation and release of anaphylatoxins, promotes CDC of tumor cell lines, and inhibits tumor growth in vivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery.

Antigen Drives Regulatory B10 Cell Development and Function

B cells mediate immune responses via the secretion of antibody and interactions with other immune cell populations through antigen presentation, costimulation, and cytokine secretion. Although B cells are primarily believed to promote immune responses using the mechanisms described above, some unique regulatory B cell populations that negatively influence inflammation have also been described. Among these is a rare interleukin (IL)-10-producing B lymphocyte subset termed “B10 cells.” B cell-derived IL-10 can inhibit various arms of the immune system, including polarization of Th1/Th2 cell subsets, antigen presentation and cytokine production by monocytes and macrophages, and activation of regulatory T cells. Further studies in numerous autoimmune and inflammatory models of disease have confirmed the ability of B10 cells to negatively regulate inflammation in an IL-10-dependent manner. Although IL-10 is indispensable to the effector functions of B10 cells, how this specialized B cell population is selected in vivo to produce IL-10 is unknown. Some studies have demonstrated a link between B cell receptor (BCR)-derived signals and the acquisition of IL-10 competence. Additionally, whether antigen-BCR interactions are required for B cell IL-10 production during homeostasis as well as active immune responses is a matter of debate. Therefore, the goal of this thesis is to determine the importance of antigen-driven signals during B10 cell development in vivo and during B10 cell-mediated immunosuppression.

Chapter 3 of the dissertation explored the BCR repertoire of spleen and peritoneal cavity B10 cells using single-cell sequencing to lay the foundation for studies to understand the full range of antigens that may be involved in B10 cell selection. In both the spleen and peritoneal cavity B10 cells studied, BCR gene utilization was diverse, and the expressed BCR transcripts were largely unmutated. Thus, B10 cells are likely capable of responding to a wide range of foreign and self-antigens in vivo.

Studies in Chapter 4 determined the predominant antigens that drive B cell IL-10 secretion during homeostasis. A novel in vitro B cell expansion system was used to isolate B cells actively expressing IL-10 in vivo and probe the reactivities of their secreted monoclonal antibodies. B10 cells were found to produce polyreactive antibodies that bound multiple self-antigens. Therefore, in the absence of overarching active immune responses, B cell IL-10 is secreted following interactions with self-antigens.

Chapter 5 of this dissertation investigated whether foreign antigens are capable of driving B10 cell expansion and effector activity during an active immune response. In a model of contact-induced hypersensitivity, in vitro B cell expansion was again used to isolate antigen-specific B10 clones, which were required for optimal immunosuppression.

The studies described in this dissertation shed light on the relative contributions of BCR-derived signals during B10 cell development and effector function. Furthermore, these investigations demonstrate that B10 cells respond to both foreign and self-antigens, which has important implications for the potential manipulation of B10 cells for human therapy. Therefore, B10 cells represent a polyreactive B cell population that provides antigen-specific regulation of immune responses via the production of IL-10.

Immunotherapy for the treatment of poor prognosis cancers

Cancer is amongst the leading causes of death worldwide and the number one cause in the developed world. Every year there are close to 10 million cancer related deaths and this corresponds to hundreds of millions of euro in health care costs and lost productivity, placing a substantial drain on the economy. The efficacy of traditional treatment modalities for cancer therapy, such as surgery, radiotherapy and chemotherapy has plateaued, and while they are undoubtedly effective at prolonging patient lifespan, there is a high rate of adverse side effects and fatal reoccurrence. Currently, there is a huge amount of interest in the areas of cancer immunosurveillance and cancer immuno-editing, which explain some of the complex interactions between the host immune system and cancer. If left unchecked, cancerous malignancies have the ability to generate an immunosuppressive microenvironment, effectively shielding themselves from elimination and promoting tumour growth and progression. To overcome this, the potential of the immune system must be harnessed and the work undertaken in this thesis sought to contribute to this goal. Focus was placed on using novel therapies, combining tumour ablation with immune-modulating antibodies to maximise tumour elimination in an immune dependent manner, to overcome immunosuppression and promote immune activation. Chapter 2 focuses on the use of ECT as a method of tumour ablation and its effects on the immune system. ECT proved to be effective at inhibiting the tumour growth both in vitro and in vivo, and conferred significant survival advantages in both small and large animal models. More importantly, ECT proved to cause tumour death in an immune dependent manner, displaying the hallmarks of Immunogenic Cell Death, increases in immune cell infiltration and generating tumour-specific immune responses. Chapter 3 focuses on combining ECT with immune checkpoint blockade inhibitors; anti- CTLA-4 and anti-PD-1. Both combinations proved to be effective at inhibiting both primary and distal tumour growth, indicating the generation of tumour specific immune responses and prolonged animal survival. In addition, the treatments caused increases in the levels of certain intra-tumoural immune cell subsets and modulated the cytokine profile of treated animals in a way that was favourable overall. Chapter 4 focuses on the combining ECT with an anti-iCOS agonist antibody, capable of causing immune co-stimulation. This novel combinational therapy proved to be the most effective by far, with a high cure rate achieved across a number of different in vivo tumour models. Total regression was seen in both primary and distal tumours, as well as spontaneous metastases, with the tumour specific immune response generated conferring total protection to animals on tumour rechallenge. Overall the data presented here adds further insight into the area of cancer immunotherapy with some of the novel combinational therapies demonstrating substantial clinic potential.

The Burkholderia Cenocepacia OmpA-Like Protein BCAL2958: Identification, Characterization, and Detection of Anti-BCAL2958 Antibodies in Serum from B. Cepacia Complex-Infected Cystic Fibrosis Patients

Respiratory infections by bacteria of the Burkholderia cepacia complex (Bcc) remain an important cause of morbidity and mortality among cystic fibrosis patients, highlighting the need for novel therapeutic strategies. In the present work we have studied the B. cenocepacia protein BCAL2958, a member of the OmpA-like family of proteins, demonstrated as highly immunogenic in other pathogens and capable of eliciting strong host immune responses. The encoding gene was cloned and the protein, produced as a 6× His-tagged derivative, was used to produce polyclonal antibodies. Bioinformatics analyses led to the identification of sequences encoding proteins with a similarity higher than 96 % to BCAL2958 in all the publicly available Bcc genomes. Furthermore, using the antibody it was experimentally demonstrated that this protein is produced by all the 12 analyzed strains from 7 Bcc species. In addition, results are also presented showing the presence of anti-BCAL2958 antibodies in sera from cystic fibrosis patients with a clinical record of respiratory infection by Bcc, and the ability of the purified protein to in vitro stimulate neutrophils. The widespread production of the protein by Bcc members, together with its ability to stimulate the immune system and the detection of circulating antibodies in patients with a documented record of Bcc infection strongly suggest that the protein is a potential candidate for usage in preventive therapies of infections by Bcc.

Characterization of cancer/testis antigen MAGE-A11 for immunotherapy of prostate cancer

Les antigènes testiculaires du cancer sont des cibles idéales pour l’immunothérapie du cancer car ce sont des protéines immunogéniques dont l’expression est restreinte aux cellules germinales et au cancer. Le but de cette étude est d’évaluer le potentiel de MAGE-A11, un antigène testiculaire du cancer, comme cible pour développer un vaccin contre le cancer de la prostate. Pour ce faire, l’anticorps monoclonal 5C4 qui a la capacité de reconnaître la présence de MAGE-A11 dans les tissus fixés et inclus en paraffine a été produit. De plus, l’expression de MAGE-A11 a été analysée sur plusieurs lignées de cellules cancéreuses. Il a été démontré que MAGE-A11 est exprimé dans plusieurs types de cancers notamment dans le cancer du côlon et du cerveau. Finalement, nous avons identifié trois épitopes du CMH classe II HLA-DR1 dans la protéine MAGE-A11 confirmant ainsi l’immunogénicité de cet antigène et son potentiel comme cible pour l’immunothérapie du cancer.

Molecular and serological detection of occult hepatitis B virus among healthy hepatitis B surface antigen-negative blood donors in Malaysia.

Background: Occult hepatitis B infections are becoming a major global threat, but the available data on its prevalence in various parts of the world are often divergent. Objective: This study aimed to detect occult hepatitis B virus in hepatitis B surface antigen-negative serum using anti-HBc as a marker of previous infection. Patient and Methods: A total of 1000 randomly selected hepatitis B surface antigen-negative sera from blood donors were tested for hepatitis B core antibody and hepatitis B surface antibody using an ELISA and nested polymerase chain reaction was done using primers specific to the surface gene (S-gene). Results: Of the 1000 samples 55 (5.5%) were found to be reactive, of which 87.3% (48/55) were positive for hepatitis B surface antibody, indicating immunity as a result of previous infection however, that does not exclude active infection with escaped mutant HBV. Nested PCR results showed the presence of hepatitis B viral DNA in all the 55 samples that were positive for core protein, which is in agreement with the hepatitis B surface antibody result. Conclusion: This study reveals the 5.5% prevalence of occult hepatitis B among Malaysian blood donors as well as the reliability of using hepatitis B core antibody in screening for occult hepatitis B infection in low endemic, low socioeconomic settings.

Structural determination and gynecological tumor diagnosis using antibody chip captured proteins

Purpose: To identify markers for gynecological tumor diagnosis using antibody chip capture. Methods: Marker proteins, including cancer antigen 153 (CA153), CA125, and carcinoembryonic antigen (CEA), were analyzed using antibody chip capture of serum samples. Fifteen agglutinin types that specifically recognized five common glycans (fucose, sialic acid, mannose, N - acetylgalactosamine, and N-acetylglucosamine) were used to detect marker protein glycan levels. The levels of CA153, CA125, and CEA from 49 healthy control samples, 31 breast cancer samples, 24 cervical cancer samples, and 19 ovarian cancer samples were used to measure the glycan levels of these marker proteins. Results: In breast cancer samples, CA153 and CA125 were down-regulated (p < 0.01), while differences in ovarian cancer samples were not statistically significant (p > 0.01). The total accuracy was 85.1 %, with 96.8 % accuracy for breast cancer, 75 % in cervical cancer, and 78.9 % in ovarian cancer. Cross-validation analyses showed that breast cancer had 93.5 % accuracy, cervical cancer was 66.7 %, and ovarian cancer was 68.4 %, leading to 78.4 % total accuracy (58/74). Conclusions: The results indicate that better clinical diagnosis of gynecological tumors can be obtained by monitoring changes in glycan levels of serum proteins and types of proteoglycan changes.

Studies of MHC class I antigen presentation & the origins of the immunopeptidome

La présentation d'antigène par les molécules d'histocompatibilité majeure de classe I (CMHI) permet au système immunitaire adaptatif de détecter et éliminer les agents pathogènes intracellulaires et des cellules anormales. La surveillance immunitaire est effectuée par les lymphocytes T CD8 qui interagissent avec le répertoire de peptides associés au CMHI présentés à la surface de toutes cellules nucléées. Les principaux gènes humains de CMHI, HLA-A et HLA-B, sont très polymorphes et par conséquent montrent des différences dans la présentation des antigènes. Nous avons étudié les différences qualitatives et quantitatives dans l'expression et la liaison peptidique de plusieurs allotypes HLA. Utilisant la technique de cytométrie de flux quantitative nous avons établi une hiérarchie d'expression pour les quatre HLA-A, B allotypes enquête. Nos résultats sont compatibles avec une corrélation inverse entre l'expression allotypique et la diversité des peptides bien que d'autres études soient nécessaires pour consolider cette hypothèse. Les origines mondiales du répertoire de peptides associés au CMHI restent une question centrale à la fois fondamentalement et dans la recherche de cibles immunothérapeutiques. Utilisant des techniques protéogénomiques, nous avons identifié et analysé 25,172 peptides CMHI isolées à partir des lymphocytes B de 18 personnes qui exprime collectivement 27 allotypes HLA-A,B. Alors que 58% des gènes ont été la source de 1-64 peptides CMHI par gène, 42% des gènes ne sont pas représentés dans l'immunopeptidome. Dans l'ensemble, l’immunopeptidome présenté par 27 allotypes HLA-A,B ne couvrent que 17% des séquences exomiques exprimées dans les cellules des sujets. Nous avons identifié plusieurs caractéristiques des transcrits et des protéines qui améliorent la production des peptides CMHI. Avec ces données, nous avons construit un modèle de régression logistique qui prédit avec une grande précision si un gène de notre ensemble de données ou à partir d'ensembles de données indépendants génèrerait des peptides CMHI. Nos résultats montrent la sélection préférentielle des peptides CMHI à partir d'un répertoire limité de produits de gènes avec des caractéristiques distinctes. L'idée que le système immunitaire peut surveiller des peptides CMHI couvrant seulement une fraction du génome codant des protéines a des implications profondes dans l'auto-immunité et l'immunologie du cancer.

African swine fever virus transmission cycles in Central Europe: evaluation of wild boar-soft tick contacts through detection of antibodies against Ornithodoros erraticus saliva antigen.

BACKGROUND African swine fever (ASF) is one of the most complex viral diseases affecting both domestic and wild pigs. It is caused by ASF virus (ASFV), the only DNA virus which can be efficiently transmitted by an arthropod vector, soft ticks of the genus Ornithodoros. These ticks can be part of ASFV-transmission cycles, and in Europe, O. erraticus was shown to be responsible for long-term maintenance of ASFV in Spain and Portugal. In 2014, the disease has been reintroduced into the European Union, affecting domestic pigs and, importantly, also the Eurasian wild boar population. In a first attempt to assess the risk of a tick-wild boar transmission cycle in Central Europe that would further complicate eradication of the disease, over 700 pre-existing serum samples from wild boar hunted in four representative German Federal States were investigated for the presence of antibodies directed against salivary antigen of Ornithodoros erraticus ticks using an indirect ELISA format. RESULTS Out of these samples, 16 reacted with moderate to high optical densities that could be indicative of tick bites in sampled wild boar. However, these samples did not show a spatial clustering (they were collected from distant geographical regions) and were of bad quality (hemolysis/impurities). Furthermore, all positive samples came from areas with suboptimal climate for soft ticks. For this reason, false positive reactions are likely. CONCLUSION In conclusion, the study did not provide stringent evidence for soft tick-wild boar contact in the investigated German Federal States and thus, a relevant involvement in the epidemiology of ASF in German wild boar is unlikely. This fact would facilitate the eradication of ASF in the area, although other complex relations (wild boar biology and interactions with domestic pigs) need to be considered.