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Enhanced Protective Immunity Against Filariasis

Final Report Summary - E PIAF (Enhanced Protective Immunity Against Filariasis)

Executive Summary:
Enhanced Protective immunity Against Filarial Infections
HEALTH-2009-4.3.1-1 Contract 242131

Summary description of project

The prime objective of E PIAF (http://filaria.eu/) was identification and testing of candidates for inclusion in a vaccine against onchocerciasis, also known as river blindness. The working hypothesis is that neutralization of excreted-secreted parasite-derived immuno-modulators will lead to expression of a Th2-driven protective immunity

To achieve this goal the following investigations were undertaken.
1 Human studies: Gene expression profiling of patients with different clinical and parasitological presentations of onchocerciasis and other filarial infections has been used to define pathways associated with protective immunity. This information will help determine appropriate formulation of experimental vaccines to promote protection while avoiding unwanted induction of pathology.

2 Animal studies: Parallel studies in the Litomosoides sigmodontis-mouse model to defined protective immune responses evoked by vaccination with irradiated L3 larvae.

3 Parasite studies: Detailed genome and proteome mapping of the developmental (life cycle) stages of Onchocerca spp and other filarial species to identify and characterized the excreted/secreted products that modulate [suppress] potential lethal host [Th2] response. E PIAF has completed the genomes of Onchocerca ochengi, O gutterosa, Litomosdoes sigmodontis, Acanthocheilonema viteae, Dirofilaria immitis and Setaria labiatopapillosa (http://badger.bio.ed.ac.uk/filarial/) and with the completion of the genomes of O volvulus, Wuchereria bancrofti, and Brugia spp by the Sanger Centre and The Broad Institute of MIT and Harvard, this means that the genomes of all the major filariae species have now been determined.

4 Vaccine testing: Use of the L sigmodontis-mouse model to test various formulations of vaccines incorporating selected excreted/secreted parasite antigens an aimed at driving the immune response down protective pathways identified by the microarray analyses. Efficacy was assessed by reduction of blood microfilariae and adult worms. Three vaccine candidate have been identified on the basis of their ability to reduce microfilarial loads by a >90%. These vaccine candidates are now ready to take to Phase I safety trials with the prospect of starting Phase 2 trials by 2020 (http://riverblindnessvaccinetova.org)

5 Preliminary modelling analyses, based on an initial vaccine efficacy of 50% against incoming worms, a 90% reduction of microfilarial load, and an 80% coverage of 1-5 yr olds initially with subsequent annual vaccination of 1 yr olds, suggest that after 15 years of vaccination in areas not previously treated with ivermectin, a vaccine would have a substantial impact, markedly reducing microfilarial load in the young (under 20 years of age) in a range of endemicity scenarios. This highlighted the risk of acquiring heavy infections early in life with the prospect of developing onchocerciasis-related morbidity. This suggests that a vaccine would have a beneficial impact in terms of reducing Onchocerca disease burden in these populations. Moreover, a vaccine could markedly decrease the chance of onchocerciasis infection re-spreading to areas where it is deemed that mass drug administration with ivermectin can be stopped. Therefore, a vaccine would protect the substantial investments made by present and past onchocerciasis control programmes (together, the Onchocerciasis Control Programme in West Africa (OCP) and APOC have cost over US$1 billion–excluding the value of ivermectin), decreasing the chance of disease recrudescence and the potential spread of ivermectin resistance.

It is envisaged that vaccination would be used together with ivermectin in any control or elimination programme but it would also have the benefit of protecting pre-school children from severe disease. Furthermore, the therapeutic application of any vaccine is also a real possibility.

Project Context and Objectives:
The prime objective of E PIAF (http://filaria.eu/) was identification and testing of candidates for inclusion in a vaccine against onchocerciasis, also known as river blindness.

This study represented the sixth successive EU Framework contract aimed at improving control, treatment and prevention of onchocerciasis. From the start of this programme in 1984, the Consortium has taken a dual approach to address these issues. First, refinement of regimes for treatment using existing drugs; and second, vaccine development. Again, at the start of the programme, investigations were primarily limited to human studies because of the absence of suitable animal models for experimental work. Onchocerca volvulus, the causative agent of onchocerciasis does not develop in laboratory mice or other animals, except chimpanzee’s However, support from the EU has facilitated development of two animal models. The first is Onchocerca ochengi in cattle. This parasite is the closest know relative of O volvulus with which it is sympatric across central and west Africa. Like O volvulus, O ochengi lives in skin nodules facilitating relative easy and accurate quantification of infection and because experiment can be performed under conditions of natural exposure, this model provides a convenient and appropriate secondary screen for vaccination studies.

The second model is Litomosoides sigmodontis which can complete its full developmental cycle and produce patent infections in mice. Different mouse strains variable resistance (C57Bl6) and susceptibility (Balb/c) that mimic the parasitological presentations of human filarial infections including patent and latent presentations with regard to presence or absence of microfilariae in the blood.

Using both models, early studies by the Consortium demonstrated the capacity of irradiated L3 larvae to evoke a strong protective immunity against a secondary challenge. Detailed immunological studies have shown that protective immunity is driven by Th2 responses but these responses can be modulated by T regulatory cells. Such responses, and protection, have also been seen following immunisation with excreted/secreted proteins of filariae and other nematode parasites.

Moreover, the Th2 and T-regulatory responses associated with protective immunity in mice are also found in humans who have been exposed to infection for extended periods or their entire lives but have never presented with the parasitological or clinical manifestations of onchocerciasis; the so-called putative immune or endemic normal individuals.

Thus the two models and identification of immune humans provide a framework for vaccine development which has been boosted by the introduction of high-through-put technologies for assessment of immune responses and the new genome and protein sequencing techniques that now permit identification of vaccine candidates from very small quantities of parasite material.

The E PIAF contract has used these new technologies with information from human and animal studies to test the hypothesis is that neutralization of excreted-secreted parasite-derived immuno-modulators will lead to expression of a Th2-driven protective immunity.

The human studies were based in Cameroon, Ghana and Togo, where the Consortium has been working with endemic communities for over 25 years. The work centred on gene expression profiling of patients with different clinical and parasitological presentations of onchocerciasis and other filarial infections to define pathways associated with protective immunity. This information helps determine the appropriate formulation of experimental vaccines to promote protection while avoiding unwanted induction of pathology.

While many individuals present with O volvulus infections alone, in many areas in central Africa, co-infections with Loa loa. Patients with these co-infections are at risk of severe adverse reaction if treated with ivermectin and at a community level, these co-infections prevent implementation of mass drug treatment programmes using ivermectin. It is estimated that 14 million people are excluded from mass ivermectin treatment campaigns and this will not only leave individuals at risk of onchocerciasis disease but will also prevent eradication of the disease from Africa.

Co-infections may also influence the out-come of treatment and interfere with the efficacy of any vaccine. For these reason E PIAF is comparing immune gene expression profiles of patients infected with only O volvulus, Wuchereria bancrofti, L loa or Mansonella perstans; with individuals with co-infection of two (eg O volvulus + L loa) or more (O volvulus + L loa + M perstans) filariae. Cohorts of endemic normals (putative immune) provide [negative] controls.

A data base has been constructed that contains clinical and parasitological together with the gene expression profiles. This unique resource is generating important information about immune responses to filarial infections that not only helps with formulation of experimental vaccines but also help identify biomarkers for diagnosis and prediction of morbidity.

Gene expression studies has also been performed with Litomosoides sigmodontis-mouse model to defined protective immune responses evoked by vaccination with irradiated L3 larvae and recombinant protein and DNA vaccine candidates. This work has confirmed the Th2 basis of protective immunity, and validated the L sigmodontis-mouse model as a primary vaccine screen.

The genome and proteome mapping studies began with collection of the various developmental stages of O ochengi and L sigmodontis. This is arduous work best demonstrated by the fact that to collect 50,000 L3 of O ochengi, colleagues working in Cameroon were required to dissect over 200,000 black flies, which measure just 2 mm in length.

The sequencing work itself was also complicated by the presence of very large quantities of host material. Nevertheless, E PIAF has completed the genomes of Onchocerca ochengi and Litomosoides sigmodontis, and in collaboration with the Gene Pool, Edinburgh, the genomes of O gutterosa, Acanthocheilonema viteae, Dirofilaria immitis and Setaria labiatopapillosa (http://badger.bio.ed.ac.uk/filarial/). E PIAF has therefore made a major contribution to an international effot to establish the genome maps of all the important filarial species that include of the genomes of O volvulus, Wuchereria bancrofti, and Brugia spp by the Sanger Centre, Cambridge and Loa loa at the Broad Institute of MIT and Harvard. E PIAF has also determined the oomplete genome of Wolbachia.

Proteomic studies, performed in Liverpool, were also complicated by the present of large quantities of host material but E PIAF has been successful in completing the transcriptomes of infective L3, adults and microfilariae of L sigmodontis together with the transcriptomes of adults and microfilariae of Onchocerca ochengi. Furthermore, detailed maps of the proteins excreted/secreted by the target filarial species. This work has confirmed the compartmentalisation of “established” vaccine candidates and as important it has led to identification of a series of new vaccine candidates.

Vaccine testing and immunological characterisation of protective responses in the L sigmodontis-mouse model was divided between Bonn, Edinburgh, Paris and Tuebingen. Vaccine efficacy was assessed by reduction of the number of microfilariae in blood and adult worms in the body cavities following challenge with L3 larvae except in Tuebingen were protection was measure by the survival of microfiliae inoculated intravenous after immunisation.

This work has led to ranking of three vaccine candidate that are capable of reducing microfilariae loads >90%. This represents almost a doubling of the efficacy of one of the “established” vaccine candidates and result that can be explained by the formulation of the vaccine that includes an adjuvant that drives Th2 responses and a construct which contains a third protein which targets the filarial antigen to dendritic cells.

In an alternate approach, Tuebingen have identified a series of peptides eluted from MHC class II molecules on cells collected from onchocerciasis patients living in Togo. Over 80 peptides were identified including some derived from the first rank vaccine candidate identified by the “conventional” screening programme. Synthetic peptides corresponding to the eluted molecules are being tested in vaccination experiments.

Preliminary modelling analyses, based on an initial vaccine efficacy of 50% against incoming worms, a 90% reduction of microfilarial load, and an 80% coverage of 1-5 yr olds initially with subsequent annual vaccination of 1 yr olds, suggest that after 15 years of vaccination in areas not previously treated with ivermectin, a vaccine would have a substantial impact, markedly reducing microfilarial load in the young (under 20 years of age) in a range of endemicity scenarios. This highlights the risk of acquiring heavy infections early in life with the prospect of developing onchocerciasis-related morbidity. It also suggests that a vaccine would have a beneficial impact in terms of reducing disease burden in these populations. Moreover, a vaccine could markedly decrease the chance of onchocerciasis infection re-spreading to areas where it is deemed that mass drug administration with ivermectin can be stopped. Therefore, a vaccine would protect the substantial investments made by present and past onchocerciasis control programmes (together, the Onchocerciasis Control Programme in West Africa (OCP) and the African Programme for Onchocerciasis Control (APOC) have cost over US$1 billion–excluding the value of ivermectin), decreasing the chance of disease recrudescence and the potential spread of ivermectin resistance.

The vaccine candidates are now ready to take to Phase I first-in-human safety trials. For this purpose, the E PIAF consortium has joined with three laboratories in the USA that have been working on filarial vaccines for over 20 years and the Sabin Vaccine Institute in a new initiative, “The Onchocerciasis Vaccine for Africa (TOVA)”, [http://riverblindnessvaccinetova.org].

The next step towards human trials will require Good Manufacturing Practice to produce the vaccines to the appropriate standard, however this is a major financial hurdle. The European and Developing Countries Clinical Trials Programme (EDCTP) offers the prospect of support for Phase 2 studies however, there is an absence of programmes/schemes to finance GMP production and phase 1 trials. It is estimated that production (including regulatory processes) of sufficient quantities of a single vaccine for a safety trial will be €2 million while a phase 1 trial may cost up to €16 million.

It is envisaged that vaccination would be used together with ivermectin in any control or elimination programme but it would also have the benefit of protecting pre-school children from severe disease. Ivermectin currently is not given to children under 5 years old who remain at risk of infection and act as a reservoir for transmission. It is also envisaged that a vaccine could find therapeutic application in older individuals.

Added value
E PIAF studies have highlighted the capacity of immune-modulators secreted by filariae to suppress Th2 responses. These Th2 immuno-modulators offer the prospect of new therapeutic agents for treatment of Th2 driven pathologies and diseases such as Multiple Sclerosis (MS), Irritable Bowl Disease (IBD) and psoriasis.

Project public website address:
http://www.riverblindnessvaccinetova.org/
http://filaria.eu/
http://badger.bio.ed.ac.uk/filarial/
http://nematodes.org/genomes/litomosoides_sigmodontis/
http://www.nematodes.org/genomes/onchocerca_ochengi/

Project Results:
E PIAF Final Report

Enhanced Protective immunity Against Filarial Infections
The 2020 vision of a vaccine against river blindness

Objective
The primary objective of the E PIAF project was to identify antigen candidates for inclusion in a vaccine against onchocerciasis, also known as river blindness.

To achieve this goal the following investigations have been undertaken.
1 Gene expression profiling of patients with different clinical and parasitological presentations of onchocerciasis and other filarial infections has been used to define pathways associated with protective immunity. This information will help determine appropriate formulation of experimental vaccines to promote protection while avoiding unwanted induction of pathology.
2 Parallel studies in the Litomosoides sigmodontis-mouse model to defined protective immune responses evoked by vaccination with irradiated L3 larvae.
3 Detailed proteome and genomic analyses of the developmental (life cycle) stages of Onchocerca volvulus and other filarial species to characterized the excreted/secreted products that are implicated in the development of protective immunity and modulation of host immune system.

The working hypothesis is that neutralization of excreted-secreted parasite-derived immune-modulators will lead to expression of protective immunity and thus the basis of a vaccine.

Testing this hypothesis has led to identification of three antigens that have proven efficacious in vaccination experiments using the Litomosoides sigmodontis-mouse model.

These vaccine candidates are ready to take to Phase I safety trials with the prospect of starting Phase 2 trials by 2020 [Annex1, http://www.riverblindnessvaccinetova.org/]

Introduction
Disease associate with filarial infections remain a major public health problem in sub-Saharan Africa. Three filarial species are involved: Onchocerca volvulus (causing onchocerciasis or river blindness); Wuchereria bancrofti (lymphatic filariasis); and Loa loa (the eye worm). Onchocerciasis and lymphatic filariasis are the primary causes of morbidity and socio-economic exclusion. The prevalence of each infection varies considerably across the continent and while many patients present with a single infection, many individuals harbour two or more parasite species. Furthermore, a fourth species, Mansonella perstans, which is generally considered to be non-pathogenic, is also highly prevalent across central Africa and sympatric with the pathogenic filariae. This is significant because all filariae excrete/secrete molecules that modulate their host’s immune system to promote their own survival and establish chronic infections, which persist for many years. The presence of a co-infection may influence the efficacy of any vaccination regime.

The primary focus of E PIAF contract has been river blindness but because of the possibility that immune-modulators secreted by one species may influence immune responses to a second filariae in a co-infection, E PIAF investigations have included all human filariae species.

It is estimated that about 37 million people are infected with O volvulus of which over 95% live in sub-Saharan Africa. Additional foci are found in central and south America and the Yemen. Up to 70% infected individuals will present with skin disease of variable severity while eye disease afflicts 10% and 1% are blind.

It is envisaged that vaccination would be used together with ivermectin in any control or elimination programme but it would also have the benefit of protecting pre-school children from severe disease. Furthermore, the therapeutic application of any vaccine is a real possibility.
Control and elimination
Onchocerciasis has been the target of major control programmes for over 40 years. Initially, the Onchocerciasis Control Programme (OCP, 1974-2002) used insecticides to kill larvae of the Simulium spp backfly that transmit the infection. However, since 1985, control has relied on mass treatment with ivermectin, lately through the African Programme for Onchocerciasis Control (APOC). A single annual treatment can kill the larvae of the parasite that live in the skin and which contribute, through inflammatory responses, to the pathology associated with infection. With clearance of microfilariae from the skin, morbidity is greatly reduced and this has been a very significant benefit to the individual and has raised the prospect of elimination and possible eradication of the disease.

However, ivermectin does not kill adult worms, and while prolonged use can reduce female fecundity, sterilization may not be permanent, and once treatment stops, microfilariae can repopulate the skin and return to levels measured before treatment within 1 year. This increases the risk of new disease episodes for the individual as well as resumption of transmission in the community. It can take over 12 years of annual treatment before the female worms appear to be permanently sterilized but in some areas, such as the forest regions of Cameroon, even such a prolonged treatment schedule has failed to prevent transmission.

The feasibility of eliminating river blindness also depends on initial levels of endemicity, patterns of transmission (seasonal versus continuous), the level of residue transmission between treatment periods, therapeutic coverage and compliance with treatment [Annex 2].

Lack of sustainability of treatment (eg funding, staffing, access, conflict etc) and failure to attain adequate therapeutic coverage (number of individuals in a community receiving treatment) are major constraints on any control programme. Indeed, the recent outbreak of Ebola demonstrates all too well the fragility of health services in poor counties and where even routine provision of health care can be severely interrupted. Reliance of a single drug also carries a risk associated with development of resistance and indeed, there is evidence of resistance to ivermectin by O volvulus, which will inevitably spread and become a major problem, as it is already in veterinary use.

Ivermectin is generally well tolerated and safe for use however, in a large area of equatorial west and central Africa where O volvulus is co-endemic with Loa loa, there is a risk of serious adverse reactions to treatment, including coma and death associated with rapid and massive death of L loa microfilariae. This is the most immediate and serious constraint on wider use of ivermectin both with respect to clinical and ethical issues and because of this, individuals and communities often no longer trust its safety. An estimated that 14 million people live in areas at high risk loiasis and thus may be excluded from ivermectin mass treatment programmes.

The problem of contra-indication for ivermectin treatment in areas where O volvulus and L loa are co-endemic could be overcome through use of doxycycline (a tetracycline) which shows selective toxicity for O volvulus due to the presence of Wolbachia, an endosymbiont bacterium. L loa is unusual among filariae in not acting as a host for Wolbachia, thus while the antibiotic can kill O volvulus, it does not kill L loa. Previous work by the E PIAF consortium ( EU FP6 contract Sustainable Control of Onchocerciasis Today and Tomorrow, SCOOTT, INCO-CT-2006-032321) demonstrated that patients co-infected with O volvulus and L loa can be safely treated with doxycycline and administration of this drug using a community-directed process can achieve the required therapeutic coverage for control. However, doxycycline cannot be used in children under 9 years, or in pregnant women and lactating mothers, and these important groups remain at risk of disease and constitute a reservoir of infection. Similarly, children under 5 years, pregnant women and lactating mothers are also excluded from treatment with ivermectin.

These different circumstances argue very strongly for development of a vaccine to ensure elimination of onchocerciasis from Africa and remove the risk of reintroduction of the infection to areas where elimination may have been achieved. Regional elimination of onchocerciasis could be more quickly achieved with a combination of drug treatment and vaccination.

Furthermore, a vaccine would afford protection for children under 5 who are currently excluded from all possible treatment regimes. The long term health and economic benefits of prevention of helminth infections in very young children are well recognized.

Vaccination [Ref 5, 6]
The feasibility of vaccination against filarial infections is founded on three pillars. 1, identification of individuals who have lived for many years with constant exposure to infection yet never present with either clinical or parasitological signs of infection; 2,epidemiological and experimental evidence of cross-protection between O volvulus and O ochengi; and 3, successful immunization of animal models against experimental and natural challenge through vaccination with irradiated infective L3 larvae.

Studies of onchocerciasis and lymphatic filariasis patients, and animal models have identified Th2 responses as playing a major role in protective immunity directed against invading L3 and the microfilariae larvae. However, there is a fine balance between protective immunity on one hand and responses that either fail to kill the parasite or cause disease on the other. Evidence points to a controlling role for T regulatory cells, including some that are antigen-specific, in expression of protective immunity. This discovery arguably holds the key to successful development of a vaccine as it provides both a means to identify possible vaccine candidate antigens and defines the immunological presentational and effector pathways that must be followed to express a protective response.

Hypothesis and plan of work
E PIAF’s working hypothesis is that protective immunity is associated with Th2 responses, however, expression of protective immunity is controlled, in part, by the activity of T regulatory cells.

Vaccination will require: 1, a homing pathogen-specific component[s] (antigen); and 2, a driver[s] (adjuvant) that guides the immune system down the protective pathway.

The E PIAF investigation was divided into 6 [experimental] Work Packages (WPs):
1 Human studies that will define immune responses in different categories of filarial patients (WP1)
2 Animal studies that will determine the nature of vaccine induced protective immunity (WP2)
3 Parasite studies, genomic and post genomic analyses of L3. adult worms and microfilariae (WP3)
4 Determine transcriptome and proteome profiles of the different developmental stages of selected filarial parasites and Wolbachia (WP4)
5 Pathway analyses (in silico) that will identify specific molecular interactions between parasites and the various categories of immune and infected human and animal hosts (WP5)
6 Testing of vaccine candidates in animal models (WP6).

Feed-back loops within this framework helped refine analyses and inform decisions concerning progress (direction and stop-go) of individual experiments and work packages.

WP1, Human studies that define immune responses in different categories of filarial patients [Ref 3, 2, 8, 13, 19, 22, 23, 25, 30, 31, 33, 34].
Protective immunity against filarial nematodes in humans is demonstrated by the fact that a proportion of the population living in hyper-endemic areas show no clinical or parasitological signs of infection despite having life-long exposure. These individuals in onchocerciasis endemic areas are described as putative immune while the term endemic normals is used to describe the corresponding individuals living in areas endemic for lymphatic filariasis. For primary analyses endemic normals/putative immune individuals are regarded as immune to challenge infection. Lymphatic filariasis patients present with patent infections with microfilariae in the blood, and latent infections in which adults are detected by the presence of circulating filarial antigen (CFA), but microfilariae are absent from peripheral blood. This last group, the existence of which only became apparent recently after the introduction of the CFA test, demonstrates that elements of acquired immunity against filarial parasites can be stage-specific. Patent and latent infections are also characteristic of loiasis and again recognition of these different parasitologcal presentations provide the opportunity to investigate stage-specific immunity.

In Ghana (Partner 4, WP 1a,1b) and Cameroon (Partner 2, WP1c) the focus of the work was on detailed clinical examinations and laboratory diagnoses to identify cohorts of microfilaraemic and amicrofilaraemic lymphatic filariasis (LF), onchocerciasis (Ov) and loiasis (Loa) patients, and immune individuals ie people who had lived their entire lives in filarial endemic areas but show no clinical or parasitological signs of infection (also known as endemic normals). In Togo (Partner 5, WP1b) responses of onchocerciasis patients treated with ivermectin have been investigated.

Standard Operating Procedures (SOPs) were established for all immunological assays, collection and recoding, processing and transport of samples for microarray analysis, which are being carried out by a commercial company (Partner 10, Fios Genomics, Edinburgh). These SOPS were trialed in Ghana before being adopted by Cameroon and Togo.

A common scoring system was used for all clinical and parasitological assessments and a minimum of 49 individuals was required for each cohort to ensure sufficient statistical analysis of microarray data. Because of the necessarily strict criteria applied to the clinical examinations, particularly with respect to identification of immune individuals (endemic normals), over 6,000 individuals had to be examined to identify sufficient numbers of eligible persons.

This work took longer than originally anticipated and required extending the catchment areas from which individuals where recruited. Furthermore, in Cameroon, an unexpected high prevalence of co-infection with Mansonella perstans was revealed by the parasitological examinations. This required examination of more individuals than originally expected with associated increase in consumables costs and extension of the scheduled collection period. This discovery also raised to 20 the number of cohorts submitted for microarray analyses with proportional increase in costs for this aspect of the study.

The first blood samples from Ghanaian lymphatic filariasis patients were sent to Edinburgh in June 2011 for QA of mRNA and microarray analysis. Further blood samples from Ghanaian onchocerciasis patients where shipped to Edinburgh in January 2012. The first onchocerciasis, loiasis and M perstans patient samples arrived in Edinburgh in January 2012 and the final samples arrived in September 2012. Primary statistical analyses of the microarray data from these samples have been completed by Fios (Partner 10 Edinburgh). The final batch of samples from Togo arrived in March 2013. All samples have been processed using the Illumina microarray platform and all primary statistical analyses have been performed. The immunological, clinical and parasitological analyses are progressing and the first publication appeared in February 2014 (doi: 10.1371/journal.pntd.0002679). At least 5 more publications are expected over the next 2 years.

Sample tracking: The importance of managing and recording transactions committed on the highly valuable resource of human blood-derived RNA samples cannot be overstated. Partner 1 Edinburgh has invested considerable time in design and implementation of an Excel-based spreadsheet to capture the required clinical information from the Case Report Form, completed by field investigators, to facilitate direct import into the sample tracking database. Data validation techniques, conditional formatting and pre-defined lists to constrain entered data were employed throughout the design in order to minimize transcription errors from the paper-based forms. The spreadsheet, along with the Case Report Form, was standardized for use by the African Partners to ensure consistency of the collected data.

To ensure appropriate and adequate recording a formal laboratory information management system (LIMS) has been established in Edinburgh (Partner 1) by Nigel Binns at the Division of Pathway Medicine. This pool of barcode-tracked material has been collected over a period of many months, involving a considerable effort on the part of the African teams involved. For many of the subjects, it would be impossible to obtain replacement samples, due to the difficulty of re-locating those individuals. It is therefore imperative that this resource is well managed by an enterprise-class sample tracking database in order to maximize the scientific information derived from this resource.

Sample storage: Samples from Cameroon, Ghana and Togo are now permanently store in a specialized cold storage facility at the Royal Infirmary, Edinburgh. These samples may be accessed by any Partner for re-analysis and any future investigation.

Clinical data-base: The biological interpretation and management of high-throughput, multi-dimensional data generated by a multi-disciplinary team located in several different countries requires a centralised repository for storing, interrogating and analyzing a large number of different datasets. For this purpose Partner 1 Edinburgh (Nigel Binns) has developed a dedicated data based to provide a robust and secure depository with the required access controls to meet the needs of E PIAF to mine the vast amounts of generated (clinical, microarray etc) data to extract the maximum informational content that will expedite interpretation of the data in its relevant biological context.

WP1a, Pt 1 Anti-microfilarial immunity in lymphatic filariasis (Partners 4 Ghana; Partner 7 Bonn; Partner 10 Fios Edinburgh) [Ref 2, 18, 19].
The objective of this WP was to use gene expression profiling to compare and contrast immune responses associated with latent and patent infections with W bancrofti with those of individuals who showed no clinical or parasitological signs of infection, ie endemic normals (EN) (Tables 3,4).

A total of 184 human whole blood samples (filarial infected plus endemic normals) were collected from Ghanaians using PaXgene™ tubes. Isolated RNA was analysed using Illumina microarrays (Fios Genomics, Partner 10). Comparisons (group and individual) were made using a cut-off Fold-Change [in expression] at FC ≥1.3 adj. p<0.05.

A total of 418 genes were found to be regulated whereas FC≥1.3 among 6398 genes encoded on the arrays. The apparent relative low detection has been attributed to the presence of globin mRNA in total RNA that may reduce noise-signal ratio. In pair-wise group comparisons (Latent vs. EN; Patent vs EN and Latent +Patent vs. EN) the strongest response was found in the Latent group compared to EN. A very similar profile was found when all infected individuals (Latent + Patent) were combined and compared against EN, although the extent of regulation was lower than when only latent infected is considered. This strong regulation pattern is apparently mostly dependent on the presence of adult worms.

In contrast, few and different genes were found more weakly regulated when the patent group was compared to EN. Apparently, the presence of microfilariae is associated with a weaker transcriptional regulation/host response. This might either be part of an immunosuppressive effect trigged by the microfilariae or, alternatively, the weaker response to infection in these individuals allows the development of microfilariae.

Infection with adult worms (Infected vs EN and Latent vs EN) resulted in the up-regulation of several eosinophil-associated genes with CLC and RNASE2 being the most prominent. Both genes were down-regulated in patent infection compared to latent infection indicating their function may compromise microfilariae survival. Moreover, similar results were observed in the onchocerciasis cohort showing high expression of CLC and RNASE2 in infected subjects compared with endemic normals.
Relative to processes measured in endemic normals, the main biological processes influenced by filarial infection include: cell-to-cell signalling, cell death and survival, protein trafficking, cell assembly and morphology as well as cell growth and proliferation. Furthermore, cell cycle, carbohydrate and lipid metabolism were found to be strongly regulated in individuals with latent infections compared with those presenting with a patent infection, whereas cellular assembly and organisation was more activated in the patent infections relative to other groups.

To determine whether the described transcriptomics profiles were biased by secondary factors, such as ivermectin treatment or the study area (geographical location), multiple regression analysis was performed on protein and gene transcription profiles.

Increased transcription levels were found in the Nzema East samples compared to those from Ahanta West. Most of the up-regulated genes in the Nzema East samples were related to the presence of infection and therefore this association may be explained by higher infection intensity in these communities. However, the possibility that environmental and/or genetic difference may also explain differences. For example, individuals in the Nzema East district had more rounds of ivermectin treatment (2X) compared to their Ahanta West counterparts. This increased frequency in the rounds of ivermectin intake significantly influenced the transcriptomic pattern in the Nzema East subjects relative to Ahanta West and could apparently help to explain the observed difference between the two study areas. Therefore caution is required when interpreting human gene expression and protein data since previous treatment history, among others, is likely to impact the immunological signature.

WP1a, Pt2 Gene expression profiling: T cell subsets of W bancrofti infected patients (Bonn Partner 7Bonn; Partner 4 Ghana; Partner 10 Fios Edinburgh) [Ref 2].
In order to obtain more in-depth information about differentially expressed genes in W bancrofti infected individuals, T cell subpopulations from EN, MF+ and MF- individuals were sorted and subsequently analyzed with an Illumina microarray (HT12V4 Array, containing 47,231 probes). FACS sorting was performed with a flow cytometry cell sorter.

Signal intensities of all tested samples were within the normal range. To identify differentially regulated genes within the three cell populations, a combination of filtering for absolute (max-min 100) and relative (max/min 1.5) changes in average expression signals across all conditions, and statistical testing (t-test) was applied. The amounts of differentially expressed genes within each cell population are depicted in Table 3. Out of these up- and down-regulated genes some interesting candidate genes have emerged these include distinct cytokines (IL-10 receptor, TGF-receptor, IL-15), apoptosis (B-cell lymphoma 6 protein (BCL-6), serum/glucocorticoid regulated kinase (SGK), tumour necrosis factor (ligand) superfamily member 10 (TRAIL)), immune regulation (CD83), effector function (CXCR4) and cell signaling (STAT1).

WP1b, Pt1 Gene expression profiling of O volvulus infected patients (Partner 7 Bonn; Partner 4 Ghana; Partner 10 Fios Edinburgh) [Ref 3].
The parasitology, pathology and immune profiles in infection-free volunteers and infected individuals that were Mf+ or Mf- have been compared. The infection-free volunteers came from villages in either Central or Ashanti regions of Ghana, which had received up to 8 or only 1 round of ivermectin treatment respectively. Mf- patients had fewer nodules and decreased IL-10 responses to all stimuli tested. On the other hand, this patient group displayed contrary IL-5 profiles following in vitro stimulation and in plasma and the dampened response in the latter correlated to reduced eosinophils and associated factors but elevated neutrophils. Furthermore, multivariable regression analysis with covariates Mf+/-, ivermectin treatment or region (Central vs Ashanti) revealed that immune responses were associated with different covariates: whereas O volvulus-specific IL-5 responses were primarily associated with presence of microfilariae, IL-10 secretion had a negative correlation with times of individual ivermectin therapy. All plasma parameters (eosinophil cationic protein, IL-5, eosinophils and neutrophils) were highly associated with the presence of microfilariae. With regard to IL-17 secretion, although no differences were observed between groups to filarial-specific or bystander stimuli, these responses were associated with the region. These data indicate that immune responses are affected by both, individual ivermectin therapy, and the number of rounds of ivermectin treatment within the community. Consequently, it appears that a lowered infection pressure due to ivermectin treatment may affect the immune profile of community members even if they have not regularly participated in the programmes.

WP1b, Pt2 T regulatory cells and Ig4 in O volvulus infected individuals (Partner 7 Bonn;, Partner 4 Ghana) [Ref 12, 25].
A further objective was to determine the role of regulatory T cells (Tregs) and IgG4 expression of protective immunity against Onchocerca volvulus. Patients infected with onchocerciasis are classified into three groups: 1, generalized onchocerciasis (GEO) or hypo-responsive individuals who have palpable nodules under their skin but no overt pathology; 2, patients with severe pathology (termed hyper-reactive or Sowda) who have few worms and usually no microfilariae; and 3, putatively immune individuals (PI or endemic normal, EN) who never develop clinical or parasitological signs of infection despite their lifelong exposure to the parasite. Several studies have identified mechanisms and cell types associated with modulation of immune responses in hypo-responsive individuals. Amongst them are regulatory T cells (Tregs), IL-10 producing Tr1 cells, and alternatively activated macrophages (AAMs). Immunologically, GEO patients are considered to have a regulated immune system and are characterized by high levels of IL-10, regulatory T cell populations (Treg) and IgG4. E PIAF supported work carried out by Tomabu Adjobimey showed that Treg clones induce B cells to make IgG4 and that this process required cell-cell contact, IL-10 and TGF-and GITR-GITRL interaction.

IgG4 binds the same receptor as IgE and the ratio of IgG4:IgE is known to regulate type I hypersensitivity reactions and consequential pathology. Treg-mediated modulation can lead to a significant increase in the ratios of IgG4/IgG and IgG4/IgE. Where this regulation is limited, pathology can be severe as seen in the case of Sowda patients that present with strong Th2 responses, high levels of IgE and eosinophilia and very few worms. This critical balance is also reflected in nodule pathology where GEO patients have more IgG4+ plasma cells and Foxp3+ Tregs than those recovered from Sowda patients,

Freshly isolated Treg dampened the production of all IgG subclasses and IgE with the exception of IgG4. Treg also influenced plasma cell formation, B cell proliferation and the levels of activation markers on B cells. This was not observed in the presence of conventional T cells (Tconv). Prior activation of B cells with TLR4-stimuli (LPS) or TLR9-stimuli (CpG) prevented Treg suppression of Ig production and in some situations (eg IgG2a) even enhanced Ig secretion.

This outcome remained when TLR stimuli were present throughout the co-cultures. Although it has been reported that B cells respond weakly to LPS, they can be activated through a combination of CD40L/IL-4 and TLR-triggering.

Triggering TLR on B cells skewed both immunoglobulin and cytokine secretion patterns and surprisingly, Treg within TLR4 and TLR9 but not TLR2-triggered B cell co-cultures up-regulated RORC and produced IL-17. These data suggest that in the presence of bacterial or viral infections, B cells can escape Treg control and thus provide an explanation as to why individuals infected with the same helminth (or exposed to the same allergen) can present with polar (and a spectrum of) immune-pathological symptoms.

WP1c, Effect of co-infection with Loa loa on the progression of immune responses against O volvulus (Partner 2 REFOTDE Cameroon; Partner 1 Edinburgh). [Manuscripts in preparation].
In vast regions of Africa, several species of filariae live in sympatric and co-infect individuals. But little is known on their interactions on the immune response of the host. In onchocerciasis and loiasis, individuals living in areas of co-endemicity can be divided into three groups based on their parasitological status. (i) Individuals exposed but completely free of infection, (ii) individuals infected, but with no microfilariae in the skin or in the blood. (iii) Individuals infected with microfilariae in the blood or in the skin. Different clinical pictures of the infection are also observed in areas of co-endemicity. Some individuals develop clinical manifestations, including blindness, leopard skin, lizard skin, sowda, nodules, oedema, Calabar swelling, whereas others develop mild pathology or are asymptomatic. This work package was designed to (i) identify in areas of co-endemicity L loa, O volvulus in Cameroon, the different parasitological groups (single infection, co-infection, no infection), (ii) to characterize the individuals of those groups clinically and immunologically and establish possible correlations between the immunological, pathological and parasitological status. During the initial stages of this work it was determined that Mansonella perstans was highly prevalent across the study region and it was necessary to include single and co-infections with is parasite as additional study groups because of the potential influence on immune responses.

In total, 32 villages situated in the rain forest area of the South West, Littoral and South regions of Cameroon. In the South West region, the study took place in Mamfe health district (6 villages), Eyumojock health district (5 villages) and Konye health district (4 villages). In the Littoral region, the study took place in 13 villages located in Melong health district. In the South region, the study took place in 4 villages in the Lolodorf health district.

Individuals recruited for the study were those who have been in the study community for at least 5 years, were 18 years and above, and have not taken any microfilaricides for the past two years, who felt in one of the defined parasitological categories of the study (Table 2).

In total 264 samples were collected and processed. Primary statistical analysis has been completed and the immunological, clinical and parasitological analyses are in progress.

WP1d, Pt1 Cytokine and chemokine response profiles of Togolese patient and control cohorts with distinct states of filarial infection (Partner 4 Togo; Partner 8 Tuebingen) [Ref 8, 22, 30, 31].
The effect of residual adult filariae on chemokines and cytokines production was investigated in: 1, in onchocerciasis patients who developed a latent (occult) O volvulus infection (Mf-negative) due to repeated ivermectin treatment; 2 patients who became Mf-negative without ivermectin treatment, presumably through aging and death of female worms; and 3, endemic and non-endemic O volvulus Mf-negative controls.

With latent (occult) O volvulus infection, serum levels of pro-inflammatory chemokines (MCP-1/CCL2, MIP-1a/CCL3, MIP-1b/CCL4, MPIF-1/CCL23 and CXCL8/IL-8) were enhanced and approached the higher concentrations seen in infection-free controls, whilst levels of regulatory and Th2-type cytokines and chemokines (MCP-4/CCL13, MIP-1d/CCL15, TARC/CCL17 and IL-13) were reduced. Levels of Eotaxin-2/CCL24, MCP-3/CCL7 and BCA-1/CXCL13 remained unchanged.

Following initial ivermectin treatment, MCP-1/CCL2, MCP-4/CCL13, MPIF-1/CCL23 and eotaxin-2/CCL24 levels were elevated, perhaps suggesting that monocytes and eosinophils may be associated with microfilariae clearance.

In summary, in latent (occult) and long-standing (expiring) O volvulus infections the serum levels of inflammatory chemokines were enhanced over time while regulatory and Th2-type-promoting cytokines and chemokines were reduced. These changes may reflect a decreasing effector cell activity against microfilariae and an enhanced inflammatory response. Similar results were also obtained in the L sigmodontis-mouse model (WP 2c, Pt 1) again demonstrating the congruence of the human and animal immune responses to filarial infections.




WP1d, Pt2 Enhancement of anti-filarial immunity with selected T cell epitopes (Partner 8 Tuebingen; Partner 5 Togo) [Manuscripts in preparation]
This section of the WP was to identify Onchocerca volvulus specific peptides eluted from MHC class II complexes on antigen presenting cells of onchocerciasis patients as an alternate route to selection of potential vaccine candidates.

The objectives were:
1. To identify filaria antigen-specific T cell epitopes relevant for immunity against filarial infection which are presented by MHC class II haplotypes in the endemic populations.
2. To identify endosymbiont Wolbachia-specific peptides bound by human MHC class ll molecules on dendritic cell; and confirm their immunogenicity in T cell assays.
3. To characterize the immune response to the newly identified T cell epitopes to determine their potential for use in a vaccine.
To initiate this work, HLA typing was performed by PCR amplification with sequence-specific primers (PCR-SSP) on 30 blood samples originating from onchocerciasis patients and endemic controls from the central region of Togo. The HLA-DRB1*01 – HLA-DRB1*16 haplotypes were analysed simultaneously and the most frequent haplotypes found were: DRB1*13 (frequency 0,27), DRB1*11 (0,17), DRB1*08 (0,14), DRB1*03 (0,11), DRB1*15 (0,10), DRB1*07 (0,07) and DRB1*01, DRB1*16, DRB1*04, DRB1*09, DRB1*14 frequencies were below 5%.

Subsequently, the SYFPEITHI algorithm was used to predict epitopes of Onchocerca volvulus- and Wolbachia- specific antigens that should bind MHC-II-DRB1 alleles. More than 40 MHC peptides were identified and synthesized and their antigenicity tested in vitro with peripheral blood cells and sera from filariasis patients and controls. A protein identified included onchocystatin [Ov-Cys] cysteine proteinase inhibitor, CPI which the project has shown in model systems to be protective and is a prime vaccine candidate.

More than 40 O volvulus-specific peptides predicted to bind MHC-II-DRB1 were synthesised and their antigenicity tested in vitro with peripheral blood cells and sera from filariasis patients and controls. In parallel, Wolbachia-specific peptide ligands for MHC-II-DRB1 haplotypes have been identified, selected for synthesis and for validation of their immune recognition.

The immunogenicity of the synthesized peptides was tested in appropriate T cell assays; these experiments performed with PBMC from: onchocerciasis patients, African endemic controls, European blood donors, onchocerciasis patients with advanced ocular disease, those from controlled and randomized ivermectin treatment trials, both of which assessed the association of T cell responses to O volvulus antigens post treatment. The potential of peptide antigens to stimulate distinct cytokines and regulatory chemokine release was evaluated, and furthermore peptides were tested for their antibody binding capacity. cytokine, chemokine and antibody profiles of several hundred human subjects from defined cohorts (onchocerciasis patients at distinct states of infection, endemic controls) were performed.

The antibody, cytokine and chemokine responsiveness to Onchocerca volvulus-specific and Wolbachia-specific peptides were analyzed in onchocerciasis patients, in infection-free African controls (afriCTRL) and non-exposed European controls (euroCTRL). Peptides were eluted from MHC-complexes on antigen presenting cells (macrophages), their amino acid sequence determined and peptides synthesized in silico for analyses. This study aimed to identify parasite-specific molecules suitable for inclusion in a preventive or therapeutic vaccine against filarial infections and disease.

All onchocerciasis patients presented with strong IgG1 antibody reactivity against the selected peptides. The onchocerciasis negative African samples (afriCTRLs) presented with a similar profile but with lower reactivity values. European sera (euroCTRLs) were negative. The highest antibody reactivity was observed with Wolbachia specific peptides which were detected in 50% of onchocerciasis patients. IgG1 responses to peptide pools were positive in 66% of onchocerciasis patients, 12% afriCTRLs and 0% euroCTRLs.

Positive IgG4 antibody responses to the MHC-eluted (and singly tested) O volvulus peptides were present in 50% of onchocerciasis patients and a similar proportion of afriCTRLs but no euroCTRLs showed reactivity. Thus, onchocerciasis patients will develop strong IgG1 and IgG4 responses against Wolbachia-specific peptides, and a similarly strong IgE reactivity against Wolbachia-specific P1 and O volvulus-specific cystatin P2 (CPI2).

WP1d, Cytokine responses of cells recovered from onchocerciasis patients and stimulated by parasite-specific peptides (Partner 8 Tuebingen; Partner 5 Togo) [Manuscripts in preparation]
Reactivity of MHC-ligand peptide antigens was also evaluated with in vitro peripheral blood cell (PBMC) from patients and controls for their capacity to induce cytokines and chemokines. The peptide-induced cellular production of pro-inflammatory (IL-17b, IL-5) and regulatory (IL-27, IL-10) cytokines, as well as inflammatory and Th1-type and effecter cell-attracting (CXCL-8/IL-8; MIP-3a/LARC/CCL20, IP-10/CXCL10) and Th2-type (MDC/CCL22,TARC/CCL17) chemokines was investigated. The O volvulus-specific peptides (ie PGlyco, PGlyco+Cys, Pool-Wolb, Pool-Ov33) activated the cellular release of the regulatory cytokine IL-27 and pro-inflammatory chemokines CCL20, and both were induced by peptides in higher amounts that by the crude antigen extract of O volvulus.

The onchocystatins (CPIs), the Ov33 peptide pools of O volvulus and the Wolbachia-specific peptides induced cellular IL-27 production in onchocerciasis patients, afriCTRLs but not in euroCTRLs, which reveals previous exposure to filarial parasites are also of the endemic afriCTRLs. Furthermore, the induction of IL-27 was observed in all patient groups, irrespective whether onchocerciasis patients were singly, co-infected or poly-parasitised with Mansonella perstans, Necator americanus, Entamoeba histolytica or Schistosoma mansoni. Repeated exposure and persistence of intestinal helminth or protozoa infections, appeared to lessen production of pro-inflammatory and regulatory cytokines required for parasite control as well as for preventing host tissue and organ damage.

Pro-inflammatory and effector cell (eosinophils and neutrophil granulocyte) attracting IL-5 and CXCL8/IL-8 were only activated by the onchocystatin-derived peptide. In contrast, peptides PGlyco, PGlyco+Cys, Pool-Wolb, Pool-Ov33 suppressed the cellular production of Th2-type chemokines TARC/CCL17 and MDC/CCL22 chemokines, and similarly the release of IP-10 (chemokine induced by interferon-gamma) was suppressed when peripheral blood mononuclear cells were co-cultured with the selected peptide antigens. Furthermore, peptides PGlyco, PGlyco+Cys, Pool-Wolb, Pool-Ov33 induced production of inflammation and cell activating chemokines MIP-3α/LARC/CCL20, and the amounts of MIP-3 secreted were higher than those induced by the crude antigen extract of O volvulus.

In conclusion, parasite-specific peptides presented by MHC-molecules to specific T cells will generate a distinct immune response profile. These MHC-ligands can be isolated and tested for their potential to activate and modulate the cytokine and chemokines response profiles in humans infected and exposed to O volvulus. Several of the identified peptides stimulated pro-inflammatory responses while others including onchocystatin (CPI) of O volvulus suppressed production of Th2-type chemokines. Onchocystatin is one of vaccine candidates identified by conventional methodologies.

WP2 Animal studies [Refs 1, 5, 6, 11-14, 24, 29, 34, 3, 40].
E PIAF is building on earlier work of the consortium supported by earlier EU contracts (FP 5 VARBO, FP6 SCOOTT) that resulted in establishment of both the Litomosoides sigmodontis-mouse and the O ochengi-cattle models of filariases and onchocerciasis.

L sigmodontis is the only filariae to produce patent infections in laboratory mice and, indeed, all the parasitological presentations of human filarial infections can be reproduced in mice. C57Bl are resistant to infection while BALB/c mice are susceptible and present with latent and patent infections. Thus, this model provides both a system for detailed investigation of immune responses to filarial infections and a primary screen for vaccine candidates.

O ochengi is the closest known relative to the human O volvulus with which it is cross-protective (there is considerable overlap in their geographical distribution in Africa). This model provides the opportunity to test vaccine candidates under conditions of natural challenge.

In both mouse and cattle models, immunization with irradiated L3 induces protective immunity against homologous secondary challenge.

Experiments in these models complement the human studies and together the results will help inform the pathway and network analysis that dominated E PIAF work during the final period of the contract.The overall objects of WP2 were:
1, To define the characteristics of immune response of animals demonstrably protected against filarial infection by vaccination;
2, To identify specific parasite host interactions associated with specific immunological, clinical and parasitological status.

WP2a, Vaccination of cattle with irradiated O ochengi L3 (Partner 3 IRAD Cameroon; Partner 6 Liverpool).
Although there is a general knowledge of mechanisms that lead to killing of invading parasites following vaccination with irradiated L3, it remains unclear as to how irradiated L3 stimulate protective immunity. This WP was designed to characterize immune response of animals vaccinated with irradiated L3 larvae. The original intention was to repeat earlier mouse experiments in the O ochengi-cattle model. For this purpose, work in Cameroon has collected and cryopreserved over 50,000 L3, which will be shipped to Partner 6 Liverpool. However, it was not possible to carry out the vaccination experiment within the E PIAF contract period because of insufficient funds. This situation has developed because of increased cost of purchase and maintenance of cattle and reagents combined with the steady devaluation of the Euro against Sterling.

This is extremely disappointing but the cryopreservation of the L3 does mean that once new funding can be secured, the experiments can proceed. The key outputs from this work would be characterization of local responses against L3 and identification of host gene clusters differentially expressed in responses to irradiated L3. These prospective results would contribute to design and formulation of any vaccine.

WP2b, Immunochemotherapy of onchocerciasis (Partner 3 IRAD, Cameroon, Partner 6 Liverpool) [Ref 7].
The aim of this WP was to determine whether a combination of vaccination and chemotherapy (immunochemotherapy) could effectively kill adult worms of O ochengi in cattle, while reducing the length of antibiotic treatment normally required (several weeks). The vaccine antigen used in this experiment was a mutated form of onchocystatin, which induced significant protection in the L sigmodontis mouse model when administered in an immune-prophylactic strategy (data from WP 6 Partner 1 Edinburgh). Immuno-chemotherapy using a combination of inoculation with mutated onchocystatin and a short, continuous regimen of oxytetracycline failed to have any adulticidal activity. In contrast, prolonged intermittent oxytetracycline therapy was ~60% adulticidal.

The rationale underpinning the experiment was derived from previously published data demonstrating that after prolonged oxytetracycline treatment, the neutrophils normally surrounding the adult worms are replaced by eosinophils, which degranulate on the worm cuticle and ultimately kill the parasites. The local neutrophil population is known to be recruited by Wolbachia endosymbionts, and clearance of the bacteria leads to ablation of neutrophilia and a potent, helminthotoxic eosinophil response. Immunization against a immune-modulatory protein associated with the adult female worm, prior to a short antibiotic treatment, was hypothesised to generate an increase in anti-filarial antibodies, which could facilitate antibody-mediated cellular cytotoxicity against the worm surface. If sufficiently strong, the anti-onchocystatin immune response might be expected to facilitate eosinophil-mediated killing of the adult worms while the surrounding neutrophil population was still only partially depleted.

A single inoculation with mutated onchocystatin in alum was found to induce a modest increase in anti-onchocystatin antibodies, although the immune response was highly variable in the treatment group that also received a short oxytetracycline regimen. Nodular eosinophil numbers in the immunochemotherapy group were elevated at 12 weeks post-treatment, but not significantly so; whereas in the prolonged oxytetracycline treatment group, local eosinophilia increased markedly by 36 weeks post-treatment as observed previously. Taken together, these findings indicate that the immunochemotherapy strategy may have enhanced eosinophilic activity in the nodule, but this was not of sufficient intensity or duration to achieve adult worm killing.

The mechanism of adult worm killing was further investigated by extensive, label-free quantitative proteomic analysis of adult female worms following short (ineffective) or prolonged (adulticidal) oxytetracycline regimens in the absence of vaccination. Surprisingly, of 1,480 proteins that were detected in quantifiable amounts from the isolated female worms, ~70% were of bovine origin, with most of the remainder derived from the filarial worm, and <1% from Wolbachia. No proteins were found to be differentially expressed following ineffective treatment, whereas adulticidal therapy resulted in 114 significant changes (2-fold) in protein abundance, mostly at 36 weeks post-treatment, and 101 of these proteins were host-derived.

Ninety-five bovine proteins were down-regulated, of which the vast majority appeared to be of neutrophil origin. The largest single category was comprised of granule proteins, such as antimicrobial proteins (cathelicidins, peptidoglycan recognition protein, azurocidin,  defensins, cathepsin G, neutrophil elastase, and bactericidal/permeability-increasing protein), haptoglobin, alkaline phosphatase and lactotransferrin. However, the most strongly down-regulated protein (~15-fold),  defensin C7, was of unknown cellular origin. Anti-peptide polyclonal antibodies raised against this molecule specifically stained cells of macrophage-like morphology in nodule sections by immunohistochemistry. A major proportion of at least one cathelicidin, dodecapeptide, was found to be associated with extracellular crystalline deposits on the worm surface. Surprisingly, several down-regulated bovine proteins appeared to derive from blood, including haemoglobin subunits, serotransferrin, C3 preprotein, and transferrin receptor 1. This may indicate interference with ingestion of blood by the parasites following prolonged antibiotic treatment. Although only seven proteins of Wolbachia origin were present in quantifiable amounts in the worms, all of these were down-regulated (but not eliminated) after adulticidal therapy.

Very few proteins were found to be significantly increased in response to adulticidal treatment. There was some evidence for tissue re-modelling, probably involving macrophages and fibroblasts, with significant increases in the expression of extracellular matrix proteins (fibulin 2 and fibrillin 1) and macrophage-derived enzymes (dihydropyrimidinase-related protein 3 and macrophage metalloelastase). This may reflect resorption of the nodule as worm viability is compromised. Importantly, abundance of an eosinophil granule component related to major basic protein, proteoglycan 3, was increased by >5-fold. Thus, this helminthotoxic product is a key contender for the primary effector mechanism against adult O ochengi. The only filarial proteins found to be differentially expressed in this study were two enzymes of carbohydrate metabolism and two isoforms of cuticular collagen. Up-regulation of the latter suggests an attempt to repair the worm surface following eosinophil-mediated damage.

In conclusion, these data strongly support a model in which neutrophils, attracted to Wolbachia-derived products, surround adult O. ochengi worms and prevent induction of local eosinophilia by an unknown mechanism. Following prolonged antibiotic therapy, Wolbachia is depleted, the neutrophil population in the nodule abates, and an influx of eosinophils degranulates on the worm surface, ultimately leading to death of most of the parasites.

WP 2c, Pt 1 Immunity against microfilariae (Partner 7 Bonn; Partner 9 Paris) [11, 12, 40]
This work demonstrated that immunization with microfilariae together with the adjuvant alum prevents mice from developing high microfilaraemia after challenge infection. Immunization achieved 70% to 100% protection in the peripheral blood and in the pleural space and furthermore strongly reduced the microfilarial load in mice that remained microfilaraemic. Protection was associated with the impairment of intrauterine filarial embryogenesis and with local and systemic microfilarial-specific host IgG, as well as IFN-γ secretion by host cells from the site of infection. Furthermore immunization significantly reduced adult worm burden.

Partner 9 Paris has also described a role for CXCL12/CXCR4 in clearance of microfilariae from blood of susceptible BALB/c and resistant C57Bl/6 mice infected with L sigmodontis. A comparative analyses has shown that rapid parasite clearance was associated with a L sigmodontis-specific CXCL12-dependent cell response in C57BL/6 mice. CXCL12 was produced mainly by pleural mesothelial cells during infection. Conversely, the delayed parasite clearance in BALB/c mice was neither associated with an increase in CXCL12 levels nor with cell influx into the pleural cavity. Remarkably, interfering with the CXCL12/CXCR4 axis in both strains of mice delayed filarial development, as evidenced by the postponement of the fourth moulting process. Furthermore, the in vitro growth of stage 4 filariae was favoured by the addition of low amounts of CXCL12. The CXCL12/CXCR4 axis thus appears to have a dual effect on the L sigmodontis life cycle: by acting as a host-cell restriction factor for infection, and as a growth factor for worms.

WP2c, Pt2 Immunity to Litomosoides sigmodontis microfilariae, characterization of models of patency and non-patency in different strains of mice (Partner 8 Tuebingen) [1, 12, 40]
Different strains of mice differ widely in their capability to eliminate circulating microfilariae (mf). Cytokine profiles following intravenous injection of microfilariae (mf) in mf-resistant CBA/Ca and mf-permissive BALB/c-mice were [temporally] measured at different anatomical compartments revealing site-specific differences in immune responses against microfilariae. IFN- and IL-5 play an important role for the fast clearance of blood microfilariae in CBA/Ca mice. The cytokine profile following intra-pleural inoculation did not show a distinct pattern of IFN- and IL-5 production, but indicated a crucial role for IL-12. Interestingly, after non-specific stimulation of naïve BALB/c mice with LPS plasma of those animals contained more IFN- than plasma of CBA/Ca animals. The relative lack of early IFN- secretion (6h post injection) in mf-resistant CBA/Ca mice indicates that capability to react faster against microfilariae is not necessarily associated with a stronger innate immunity per se. The same result was found in mf-resistant congenic C.D1 and permissive BALB/c mice. Using bio-imaging technology with radiolabeled microfilaria and qPCR with filaria-specific primer results it was determined that elimination of microfilariae occurred in the same organs irrespective of inoculation site (intra-pleural or intravenous). The lungs and the spleen play a crucial role in the destruction of microfilariae.

The development of filarial parasites inside their vertebrate hosts is controlled by different host genetic factors. While BALB/c is the only inbred mouse strain known to produce patent infections others such as CBA/Ca and DBA/1 allow development of fertile adult worms but not of circulating microfilariae. BALB/c, DBA/1, DBA/2, C57BL/6, B10.D2 and CBA/Ca mice were used to develop a panel of congenic lines that differ in their tolerability to circulating microfilariae. In previous studies using serial backcross strategies and microsatellite analyses we identified a single gene locus (microfilaremia resistance, mfr) that regulates the innate resistance to microfilaraemia in all of these mouse strains. Back-cross of susceptible mfr BALB-allele onto resistant mouse strains permits assessment of the impact of MHC-haplotype on parasitaemia irrespective of other background genes. Interestingly, congenic CB.C mfr BALB (H-2k) and D1.C mfr balb (H-2q) strains originating from resistant CBA/Ca and DBA/1 mice respectively, tolerate circulating microfilariae even longer than the original BALB/c strain (H-2d).

These findings suggest that innate resistance against microfilariae is regulated by the mfr-locus alone; and second, that MHC haplotype is decisive for the development of a mf-targeting adaptive immunity.

As C57BL/6 is the most important strain in terms of existing knockout mutants, susceptible congenic B6.C strain mfr BALB can now be used in future studies to evaluate the impact of a variety of host immune factors on a mf-tolerating genetic background. In addition, this approach may allow the identification of peptide-based vaccines against microfilariae using different genetic backgrounds with varying MHC-haplotypes therefore mimicking the human situation much better (Unger et al. 2014). Interestingly, the same disparate outcome following infection with L sigmodontis in congenic mice could also be observed after injection of microfilariae of Brugia malayi but not with those of Acanthocheilonema viteae. These similarities in the innate immunity to microfilariae of L sigmodontis and Brugia spp probably reflect the antigen and biochemical similarities of their surface structures. No difference between the congenic strains could be observed after infection with the gastrointestinal nematode Angiostrongylus costaricensis, with the trematode Schistosoma mansoni, with the protozoan Plasmodium berghei ANKA or with the gram-negative Enterobacterium Yersinia enterocolitica.

WP3, Parasite studies (Partner 6 Liverpool, Partner 1 Edinburgh) [Ref 1, 4, 9, 10, 13-17, 21, 26-29, 32, 35-37, 39].
Maintenance of the Litomosoides sigmodontis was a major activity of the European partners. Although the Consortium has great deal of experience with this parasite, unpredictable dip in production of L3 larvae occur and for this reason, and for the purpose of validating vaccination experiments, the life cycle is maintained in the 4 laboratories.

Over 50,000 Onchocerca ochengi L3 were collected though dissection of blackflies (Simulium spp). This work was undertaken by Partner 2 REFOTDE, Cameroon. To put this into perspective, it should be noted that each blackfly carries an average of just 2.5 L3. The L£ were frozen in liquid nitrogen for storage and shipment to Partner 6 Liverpool. The cost of air freight between West Africa and Europe rose over 3 fold over the period of the contract due to increased regulation and safety procedures.

WP3, Pt 1 Parasite Genome analyses (Partner 1 Edinburgh, M Blaxter and The Gene Pool Edinburgh; Partner 6 Liverpool, B Makepeace) [Ref 9, 10, 14, 21, 26-28]
Underpinning science for drug target definition and vaccine candidate discovery, genomics has the ability to deliver the complete catalogue of genes in a target species. Using transcriptomics, the expression patterns of those genes can be investigated, and genes associated with particular life cycle stages, or biological processes (such as responses to drugs) identified. The genome sequence is also a key substrate for the coordination and contextualization of proteomic data and population variation data.

E PIAF has sponsored or co-ordinated the sequencing of the genomes of six filarial nematodes, as part of a global effort to sequence 14 species of filariae. E PIAF has focused on the bovine Onchocerca ochengi; and rodent filariae Litomosoides sigmodontis and Acanthocheilonema viteae. In addition, the dog heartworm Dirofilaria immitis has been completed in collaboration with the Swiss Tropical and Public Health Institute.

The O volvulus genome has been completed by the Sanger Centre and Sara Lustigman, New York Blood Centre and the Broad Institute has sequenced Loa loa.

Using these new data Partner 1 Edinburgh has built interactive genome exploration databases for the community, and provided bespoke analyses to many members of the consortium. The genomics data have in particular allowed the teams to exhaustively survey filarial nematodes to identify homologues of key vaccine targets, and thus defining the levels of variability to be expected in each protein and identifying the key, conserved functional residues. For example, the IP generated around the six-SXC vaccine candidate (UK registration no 1402352.7 see below) was significantly enhanced by our being able to supply the orthologous sequences from all our target species. The presence of the endosymbiont Wolbachia was traced through the nematode phylogeny, and complete assemblies of three Wolbachia genomes released. Analyses of these genomes refined the understanding of the roles of Wolbachia in filarial biology, and confirmed pan-Wolbachia drug targets.

A spin-off project generated genome sequence for one of the major non-filarial parasites of cattle, the lungworm Dictyocaulus viviparus.

The data generated is freely available from the project webservers at:
http://badger.bio.ed.ac.uk/filarial/
http://nematodes.org/genomes/litomosoides_sigmodontis/ for L sigmodontis and http://www.nematodes.org/genomes/onchocerca_ochengi for O ochengi.

These can be analyzed offline or within the project web portal. The raw data have been submitted to the public ENA database, and the assemblies and annotations are being submitted.

WP3, Pt2 Post-genomics of filarial parasites (Partner 6 Liverpool; Partner 1 Edinburgh) [Ref 4, 16, 17].
E PIAF work focused on transcriptome and proteome mapping of accessible developmental stages of L sigmodontis (L3, adult male and female worms, and microfilariae), O ochengi, (L3, adult male and female worms, and microfilariae) and adult O volvulus. The transcriptomes of infective L3, adults and microfilariae of L sigmodontis have been mapped together with those of adults and microfilariae of Onchocerca ochengi.

Both transcriptome and proteome analyses of the filariae are complicated by the presence of the endosymbiont Wolbachia. However, because Wolbachia proteins are also implicated in the development of immune responses and the expression of protective immunity and pathology of filariae, Partner 6 Liverpool (Ben Makepeace) has also determined the complete genome of this bacterium. Partner 6 Liverpool with Partner 1 Edinburgh used novel genome data obtained for Litomosoides sigmodontis and Onchocerca ochengi to enable high-throughput, stage-specific proteomic analyses of each parasite. This work also received substantial and essential support from Partner 2 REFOTDE Cameroon, who provided ~10 million O ochengi microfilariae, several thousand adult male worms, ethanol-fixed nodules for collagenase digestion of adult female worms, and nodule fluid pooled from several hundred nodules.

For L sigmodontis, the focus was on excretory-secretory proteins (ESP, the “secretome”) released during in vitro culture of infective larvae, pre-gravid adult females, gravid adult females, adult males, immature microfilariae (Mf) and blood-derived microfilariae. We identified 302 unique proteins, of which ~65% were only found in gravid adult female ESP. The excretory-secretory products of parasitic worms represent the “frontline” in its interaction with the host. These products are known to have immune-modulatory roles in parasite invasion and long-term persistence of infection. The filarial nematode Litomosoides sigmodontis is a tractable experimental model for filariasis, as it can produce transmissible offspring in BALB/c mice.

Label-free quantitative proteomic analyses of the adult and infective L3 life stages of L. sigmodontis somatic and excretory/ secretory products was performed using high resolution shotgun LC-MS/MS, to identify >400 proteins against a draft L sigmodontis genome assembly. Each ES protein profile was different, with only 14 proteins shared between the three life stages. The protein abundance profile of the ES differed greatly to that of the corresponding somatic protein extract, suggesting that the ES products were not the result of leakage from moribund or dead worms. The predominant ES protein families were protease inhibitors, proteases, lipid-binding proteins and antioxidants. The cysteine protease inhibitor Ls-cystatin, a key vaccine candidate, was one of the most abundant species present in the ES of both adults and the infective L3. Several members of the transthyretin-like protein family were also well represented in adults, consistent with earlier studies on the ES of Ostertagia ostertagi and Brugia malayi. Several previously characterised filarial antigens, including FAR1, leucyl aminopeptidase, galectin and RAL2 were also highly enriched in the ES material. A putative immunosupressive protein containing ShTK toxin domains was abundant in adult stages. In addition, a novel protein product highly expressed in the ES exhibited homology to an apolipophorin from Ascaris suum (a lipid-binding protein). Venom allergen antigen-like proteins were abundant in the L3 ES and unique to this stage. However, only three proteins from the Wolbachia endosymbiont of L. sigmodontis were detected and at low abundance, suggesting a limited involvement in any immune-modulatory action on the host via ES protein material

Although the complexity of the adult female secretome had previously been indicated from some studies on Brugia malayi, this is the first time that gravid and pre-gravid females had been directly compared and the ESP accurately quantified. This supports the hypothesis that the gravid female actively secretes proteins with immune-modulatory functions that protect her microfilariae offspring.

Four novel proteins were determined to be especially abundant in the ESP of gravid females and immature microfilariae: a predicted 28.5 kDa filarial-specific protein, a zonadhesin/SCO-spondin-like protein, a vitellogenin, and a protein containing six ShK toxin domains. The ShK protein contained features that are conserved with canonical metridin peptides from sea anemones, which can block mammalian Kv1.3 potassium channels. As Kv1.3 channels are highly expressed in memory T-cells, this strongly suggests that the L sigmodontis ShK protein has an immune-modulatory role. Orthologous proteins with an equivalent six-ShK domain structure were identified in all other sequenced filarial genomes, including those from human pathogens such as O volvulus. Moreover, mice have been successfully protected from challenge infection with L sigmodontis (Edinburgh Partner1, see below). These novel six-ShK proteins are the subject of a joint patent (UK registration no 1402352.7 submitted 11th February 2014) between Partner 6 Liverpool and Partner 1 Edinburgh, which seeks to protect embodiments in the area of vaccines for human and veterinary filarial infections.

Partner 6 Liverpool in collaboration with Partner 2 REFOTDE, Cameroon and Partner 3 IRAD, Cameroon) also carried out an in-depth, stage-specific proteomic analysis of O ochengi, which encompassed the structural proteomes of infective larvae, adult male and female worms, and intrauterine microfilariae. In addition, we obtained ex vivo nodule fluid from abattoir-derived material and identified excretory-secretory host and parasite-derived products. A total of 4,638 unique filarial proteins and 165 unique endobacterial (that is, Wolbachia-derived) proteins were detected in whole worm extracts, while 159 and 790 filarial and bovine proteins, respectively, in nodule fluid. Protein domains enriched in individual stages included low-density lipoprotein receptor repeat class B (in adult females), ubiquitin (in adult males), and galectins (in infective larvae). In addition, several domains were over-represented across several stages, such as intermediate filament proteins (in all stages except microfilariae), annexins (in adults only), and peptidases of the M16 family (in adult males and infective larvae). The latter family includes mitochondrial processing peptidases that cleave signal peptides from nuclear-encoded proteins which are imported into the mitochondrion. Their enrichment in infective larvae and adult males may reflect a greater importance of aerobic metabolism in these stages, which undergo extensive migration in the definitive host. The peptidase M16 family has previously been identified as a potential drug target in filarial worms.

The nodular secretome of O ochengi displayed remarkable similarities with that of gravid adult female L sigmodontis, with a predominance of transthyretin-like proteins, cysteine proteinase inhibitors and a large von Willebrand type-D domain protein. A class of antioxidant proteins, peroxiredoxins, was also enriched in nodule fluid; alongside immunoglobulin domain proteins such as a massive molecule related to DIG-1 from Caenorhabditis elegans. The significance of the presence of the DIG 1 orthologue in filarial secretions is not known, but ESP from other filarial species contain related proteins. Importantly, an O ochengi orthologue of the six-ShK domain protein from L sigmodontis was also abundant in nodule fluid, and is 100% identical at the amino-acid level to a protein encoded in the O volvulus genome. Bovine host proteins identified in nodular secretions were dominated by antimicrobial proteins, such as cathelicidins and peptidoglycan-recognition protein, as well as other proteins known to be highly expressed in neutrophils (haptoglobin, calgranulins). These data strongly corroborate the proteomic findings of Partner 3 IRAD, Cameroon with respect to host proteins associated with the adult female worm surface and indicated that the nodular immune response is characterized by a neutrophilia induced by Wolbachia.

Following analyses of the transcriptome and proteome of Wolbachia from O ochengi attention was paid the basis of antibiotic tolerance in Wolbachia, which is reflected by the prolonged and currently impracticable tetracycline regimens required to kill adult filarial worms (4-6 weeks of daily therapy). Due to the major challenges inherent in obtaining deep transcriptome and proteome coverage of Wolbachia in filarial worms, we performed short-term doxycycline treatment experiments in vitro on a Wolbachia strain from Drosophila that has been stably transfected into a mosquito cell line. Wolbachia not only survived three days of doxycycline exposure at physiological concentrations, but showed extensive adaptive responses at the level of nucleotide synthesis, energy metabolism and protein secretion via the twin-arginine translocase pathway (all up-regulated); whereas outer membrane proteins were generally down-regulated. Furthermore, Wolbachia upregulated a phosphate ABC transporter ATPase associated with antimicrobial resistance in free-living bacteria, which may act as an efflux pump.

These findings provide a mechanistic explanation for the observed tolerance of Wolbachia to tetracycline antibiotics in the absence of classical genetic resistance mechanisms. They also will be of utility in evaluating the relative efficacy of other antibiotic classes (eg rifamycins, fluoroquinolones) against Wolbachia, and the ability of combination regimens to overcome tolerance and thus shorten regimens.

WP4, Microarray analyses and gene profiling of human and murine samples (Partner 10 Fios Genomic, Edinburgh) [Ref 3, 25].
This utilised the expression data generated in WP1 and WP2 to evaluate the molecular mechanisms of infection and immunity using computational methods. Partner 10, Fios Genomics Edinburgh have performed data analysis, including quality control, statistical, network and pathway analyses, of both human and mouse data to better understand the host response to infection.

Whole blood samples were collected from the following human study cohorts:
WP1a: Anti-microfilarial immunity in lymphatic filariasis (Ghana). Extraction and whole transcriptome microarray profiling of 187 subjects utilizing the Illumina HT12v4 array chip

WP1b: Protective immunity against Onchocerca volvulus (Ghana). Extraction & whole transcriptome microarray profiling of 167 subjects utilising the Illumina HT12v4 array chip

WP1c: Onchocerca-Loa co-infections (Cameroon). Extraction and whole transcriptome microarray profiling of 336 subjects utilising the Illumina HT12v4 array chip

WP1d: Onchocerciasis MHC restriction (Togo). Extraction & whole transcriptome microarray profiling of 42 subjects (both globin depleted & not) utilising the Illumina HT12v4 array chip

The work has involved profiling of approximately 700 human subjects. Fios Genomics have performed whole-genome gene expression profiling of all samples from whole blood collected into PAX tubes. Partner 1 Edinburgh performed RNA extractions and quality control of mRNA and carried out all primary statistical analysis. Results were then sent to the respective Partners in Ghana and Bonn (WP1a,1b), Cameroon (WP1c) and Togo and Tuebingen (WP1d) for the biological analyses. The first publication concerning gene expression profiling of onchocerciasis patients from Ghana (WP1a) has been published. It is expected that at least 5 other papers will be published over the next 2 years.

The same arrangements were made for Work Packages 2a and 2b involving murine vaccination experiments (Partner 7 Bonn and Partner 9 Paris).

WP2a: Basis of immunity to irradiated L3 (mouse). Extraction & whole transcriptome microarray profiling of 71 samples utilising the Illumina WG6 v2 array chip

WP2c: Immunity to microfilaria (mouse). Extraction & whole transcriptome microarray profiling of 132 samples utilising the Illumina WG6 v2 array chip

Approximately 200 individual expression profiles have been established for four different groups of mice (naïve, mock or sham vaccinated control, irradiated larvae, normal [non-irradiated] larvae) before and after immunisation and challenge. Inoculation with irradiated larvae compared with un-irradiated larvae produced a stronger Th1 response, in the form of activation of the interferon-stimulated genes S100A8 and Glrx and repression of Cd74 while inoculation or challenge with larvae induced the Th2 marker, Cxcr4. All mice challenged with normal larvae showed up-regulation of genes involved in eosinophil-based responses (Ear2 and Ear3)

The general conclusion from the microarray analyses performed so far suggest that human latent infection and mice that mimic latent infection (ie mice inoculated with either microfilaria plus alum or irradiated larvae) produce stronger responses in a subset of Th1 markers and pathways eg natural killer cell-mediated cytotoxicity. Further analyses will be required to determine the general mechanisms of protective immunity in humans and mice but the balance of data collected to date supports the conclusion that protective immunity is a Th2 driven process. It is expected that these studies will continue for at least a year after the completion of the E PIAF contract.

WP5, Pathway analysis (Partner 1 Edinburgh; Partner 10 Fios Genomics Edinburg)
A pathway-biology approach based on computation modelling to identify pathogen-host protein-protein interactions and correlates of protective immunity. Microarray and clinical data-bases have been completed (Partner 1 Edinburgh). Network analysis integrated with the statistical analysis will be used to identify clusters of coordinately expressed genes and subsequently mapped to functional groupings. Pathways responsible for initiation and propagation of inflammatory and immune responses will be identified distinguish between different clinical, parasitological and pathological presentations. The protective immunological pathways and effectors identified through the microarray analyses of both human and murine responses, will inform in silico logic-based systems-level bioinformatics and computation modeling to identify pathogen-host protein-protein interactions with parasite antigens (including those from predictions of MHC class II via the SYFPEITHI algorithm and/or directly identified by mass-spectrometry, WP2d Partner 8 Tuebingen) and correlates of protective immunity to assist with selection of potential vaccine candidates. Furthermore, this work is also identifying interactions and pathways that may lead to development of pathology and which must not be activated by vaccination. This work, which primarily uses proprietary software (Ingenuity Pathway Analysis) will continue well beyond the end of the E PIAF contract. The focus is on defining profiles leading to expression of protective immunity for the purpose of improving formulation for experimental vaccines (WP6). It is expected that these studies will continue for at least a year after the completion of the E PIAF contract.

WP6, Testing of experimental vaccines.
WP6a, Host-parasite protein-protein interactions
It is anticipated that the bioinformatics and pathway analyses will identify clusters of molecular interactions between parasite and hosts for which sequence data will be available for the proteins involved. This data facilitates application of yeast-2-hydrid systems to confirm specific protein-protein interactions. This approach offers the prospect for a high through-put screen of interactions that have the potential to drive protective immune responses. However, this WP has been cancelled because of insufficient funds brought about by a combination of general inflation, increased cost of animal experiments, salaries and the fall in the value of the Euro against Sterling.

WP6b, Pt1 Primary vaccination against Litomosoides sigmodontis using purified MHC ligands isolated ex vivo from dendritic cells of microfilariae-infected mice (Partner 8 Tuebingen) [Manuscripts in preparation]
The objective of this work package which was led by Partner 8 Tuebingen was to identify vaccine candidates against microfilarial stage of L sigmodontis from putative MHC II ligands. The method employed was a combination of in silico prediction and wet-lab assays. In silico prediction is based on available genome and transcriptomics data of related parasite species, Blast algorithm, and the T-cell epitope prediction program SYFPEITHI. The SYFPEITHI is based on position-specific scoring matrices (PSSM) to calculate the affinity score of peptides from a given protein with MHC molecules. The matrices are manually generated based on expert knowledge and the occurrence of amino acids in naturally processed MHC ligands from the SYFPEITHI database.

Crude extracts of microfilariae were co-cultured with mouse bone marrow-derived immature dendritic cells to induce maturation, antigen processing and antigen presentation. The putative MHC II-binding peptides were purified and their sequences have been determined by LC-MS/MS and database searching using Mascot program. Those originated from L sigmodontis or its bacterial endosymbiont Wolbachia were selected by matching the sequences with the transcriptomics databases of L sigmodontis (determined by GenePool, Partner 1 Edinburgh) and Wolbachia. From these peptides, those homologues in mouse were excluded. The remaining peptides showing high affinity with MHC II molecules, and predicted by SYFPEITHI were synthesized. Their immunogenicity was assessed by T cell proliferation assay ex vivo. Vaccine candidate peptides were formulated with adjuvants and evaluated for maximum immune protection in vivo. Six microfilariae-derived peptides were isolated from in vitro- stimulated murine dendritic cells. A Blast search in protein database identified the putative protein origin of these peptides.

WP6b, Pt2 Protective immunity induced by synthetic peptides derived from cystatin (CPI) with microfilariae vaccine (Partner 8 Tuebingen) [Manuscripts in preparation]
Following successful DNA vaccinations performed by Partner 1 Edinburgh, modified versions of the two filarial proteins Abundant Larval Transcript (ALT) and cystatin (CPI, cysteine protease Inhibitor), namely Acidic Domain Deleted ALT (Ls-ADDALT) and mutated CPI (Ls-muCPI) were used for the screening of immunogenic peptides.

Eleven peptides, each of around 30 amino acid length with overlapping sequences covering both L sigmodontis proteins, were synthesized and covalently linked to keyhole limpet haemocyanin (KLH), and the same peptides were biotinylated for quantification of peptide-specific antibodies in ELISA. Seven out of the eleven peptides were identified as immunogenic epitopes contained within LsALT-1 and LsCPI-2 proteins, and subsequently synthesized and conjugated with keyhole limpet haemocyanin (KLH). In addition single peptides were conjugated to a second adjuvant, N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3Cys). BALB/C mice were vaccinated three times with one-week intervals with each of the selected peptides from ALT1 and CPI and two weeks after the final inoculation, animals were challenged by intra-venous injection of microfilariae.

Plasma samples were collected at intervals during vaccination and post infection for assessment of antibody responses. Blood-circulating microfilariae were monitored until they were cleared from the blood.

Vaccination with peptides derived from Ls-CPI using KLH as carrier protein and Pam3Cys as adjuvant induces strong humoral and cellular responses in BALB/c mice, and immune protection was indicated in five groups by shorter half-life of microfilariae in blood circulation, compared to non-vaccinated controls. These peptides are promising candidates for Mf stage-specific vaccine formulation.

Protective immunity, as judged by reduction in the half-life of microfilariae in the blood, was demonstrable in mice vaccinated with four synthetic peptides. Strong antibody responses were detected in plasma collected from these groups together with secretion of MIP-1α and mixed Th1- and Th2- type cellular activity.

WP6b, Pt 3 Vaccination experiments (Partner 1 Edinburgh) [Ref 6].
The work of Partner 1 Edinburgh (Simon Babayan) tested new vaccine candidates derived from proteome and pathway analyses in the Litomosoides sigmodontis mouse model and to optimize vaccine design in continuation of previous work supported by EU funding (SCOOTT and VARBO).

Proof-of-principle has been obtained for development of protective responses using DNA vaccines targeting parasite secreted proteins which modulate host immune responses can be protective. The vaccine formulations that achieved best protection contained plasmids expressing functionally- inactivated parasite proteins coupled to a single-chain Fv antibody that specifically recognizes the dendritic cell surface protein, DEC-205. This targeting to antigen presenting cells was further enhanced by the co-administration of plasmids expressing host cytokines MIP-1a (activation and recruitment of dendritic cells), Flt3L and IL-4.

The optimal formulations established in that basis included a genetically modified filariae antigen, cysteine protease inhibitor 1 (CPIm), and at least one structural antigen, of which thioredoxin peroxidase (TPX) performed most reliably. A novel antigen was identified in collaboration with Partner 6 Liverpool, LsSxC, has also shown substantial protective capacity. Mechanisms that underlie the protective effects of CPIm involve increased dendritic cell activation during the initial immunisation phase, leading to increased T cell expansion. Immunisation with the CPIm also leads to enhance production of IL12 and IL6 and decreased production of IL10.

The protective responses evoked by these vaccine regimes are associated with a Th2
response in which IgG1 antibodies predominate although the most protective vaccine formulation did not produce the strongest humoral nor cellular response. Thus, adult worm burden and microfilariae numbers remain the best indicators of vaccine efficacy.

Sufficient data exists to take the modified cystatin to Phase 1 first-in-trials

E PIAF concluding comments
The most important output from the E PAF contract has been identification of 3 filarial proteins for which there is sufficient evidence to take to Phase I first-in-human safety trials.

The lead candidate is mutated cysteine protease inhibitor (CPIm). The effect of the mutation is to remove the immune-modulatory function of the native protein. Immunization with CPIm induces an antibody response that neutralizes the immune-modulatory function of native CPI produced by the filarial worms.

It is hypothesized that parasite-derived immune-modulators are responsible for long survival of filarial and chronic nature of the pathology they may cause. Neutralization of the immune-modulators restores the full competence of the host’s immune system enabling vaccinated hosts to express an effective protective response that will also target other [critical] parasite antigens.

E PIAF work has also shown that the efficacy of experimental vaccines can be enhanced by a formulation that targets delivery of the filarial antigen to dendritic cells. The efficacy of a vaccine is best measured by a reduction in the numbers of circulating microfilariae. Appropriate adjuvant formulation of the CPIm antigen resulted in a >95% reduction in the number of circulating microfilariae in mice challenged with L sigmodontis. This compared with a <40% reduction following immunization with antigen without the corresponding adjuvant formulation.

The relevance of data collected from the L sigmodontis-mouse model to human immune responses has been validated through microarray analyses that reveal prominent Th2 responses in animals and humans associated with expression of protective immunity. Furthermore the parasitological presentations of human filarial infections are mimicked in the L sigmodontis-mouse model.

Results of the various E PIAF vaccination studies have been shared with colleagues in the US who have also been working in this area but with different model systems. There was remarkable congruence of data obtained on both sides of the Atlantic. This led to the formation of a larger consortium for the purpose of developing a vaccine against onchocerciasis (river blindness) and the launch of The Onchocerciasis Vaccine for Africa (TOVA) initiative (http://www.riverblindnessvaccinetova.org/).

Current control of onchocerciasis relies on mass administration of ivermectin. This has been a great success in reducing morbidity in many regions but the claim that this drug alone will eradicate onchocerciasis from Africa is not supported by recent modeling exercises. A major problem is encountered in central Africa where ivermectin is contra-indicated because of the risk of severe adverse reaction in patients co-infected with Loa loa. Consequently, many areas situated in the central belt of Sub-Saharan Africa remain without satisfactory onchocerciasis control because of low levels of therapeutic coverage and/or compliance with ivermectin. An estimated 14 million people are excluded from mass drug treatment programmes because of this problem. Vaccination would help control and ultimately eliminate onchocerciasis in these problematic areas.

E PIAF has supported Hugo Turner in use of an onchocerciasis transmission dynamics framework (EpiOncho) to assess the potential long-term impact of a hypothetical vaccine against O volvulus (Annex 2). Preliminary modelling analyses, based on an initial vaccine efficacy of 50% against incoming worms, a 90% reduction of microfilarial load, and an 80% coverage of 1-5 yr olds initially with subsequent annual vaccination of 1yr olds, suggest that after 15 years of vaccination in areas not previously treated with ivermectin, a vaccine would have a substantial impact, markedly reducing microfilarial load in the young (under 20 years of age) in a range of endemicity scenarios.

Another major benefit of a vaccine will be protection of children. Ivermectin is currently not approved for paediatrics use and children under 5 years are excluded for mass treatment programmes. These children are exposed to and become infected with O volvulus. It is envisage that a vaccine against onchocerciasis would be a component of nation Expanded Programmes for Immunization (EPI) of infants.

The EPIAF project is the six successive framework contract supported by the European Commission and awarded to our Consortium. These contracts have targeted a onchocerciasis vaccine from a point at which almost nothing was known about protective immune response in human and there were no animal models for experimentation. The European Commission has supported development two animal models, the molecular and genomic analyses of the parasite and, through E PIAF, development of experimental vaccines that are efficacious in animal models.

The E PIAF project has brought the prospect of vaccination within sight. As indicated in the TOVA Prospectus (http://www.riverblindnessvaccinetova.org/) subject to funding, 3 onchocerciasis vaccines could be taken to Phase 2 efficacy trials by 2020.

The European and Developing Countries Clinical Trials Programme (EDCTP) offers the prospect of support for Phase 2 studies however, there is an absence of programmes/schemes to fund for production of the vaccines to Good Manufacturing Practice and first in human safety trials. Without financial support for this work, the investment made by the Commission and E PIAF Consortium partners may not be taken to fruition and benefit the people of sub-Saharan Africa afflicted with onchocerciasis.

Added Value
In addition to the identification of onchocerciasis vaccine candidates, E PIAF has also contributed to the mapping of the genomes of various filarial nematodes and Wolbachia. These data are freely available to all investigators and provide a fundamental resource for the discovery of new drugs and explanations for mechanisms of resistance against existing therapies. For example, E PIAF studies have provided a mechanistic explanation for the observed tolerance of Wolbachia to tetracycline antibiotics.

The microarray analyses performed on mRNA collected from cohorts presenting with different parasitological and clinical manifestations of onchocerciasis and from patients co-infected with Loa loa and/or Mansonella perstans are unique. This work is helping determine the role of T regulatory processes in, on one hand, expression of protective immunity and, on the other, development of pathology. This work has direct implications for the formulation of the onchocerciasis vaccine and our understanding of Th2 driven immunological pathways.

E PIAF studies have highlighted the critical role played by immune-modulators secreted by filariae is the survival of the parasites as a consequence of suppression of protective Th2 responses. However, while filariae immuno-modulators may be bad news for onchocerciasis and lymphatic filariasis patients, these groups of molecules may be good news for people (in industrialize as well as developing countries) suffering from Th2 driven pathologies and diseases such as Multiple Sclerosis (MS), Irritable Bowl Disease (IBD) and psoriasis. Work by others has shown the clinical benefit of infection with [live] nematodes on the progression of IBD. The proteomic and genomic work of E PIAF in identification of the spectrum of immune-modulators secreted by filariae now offers the opportunity to test the therapeutic potential of a range of new [recombinant] anti-Th2 agents in the treatment of MS, IBD and psoriasis.

To end
The E PIAF project has brought the prospect of vaccination within sight. As indicated in the TOVA Prospectus (http://www.riverblindnessvaccinetova.org/) subject to funding, 3 onchocerciasis vaccines could be taken to Phase 2 efficacy trials by 2020.

Web sites
http://badger.bio.ed.ac.uk/filarial/
http://nematodes.org/genomes/litomosoides_sigmodontis/ http://www.nematodes.org/genomes/onchocerca_ochengi
http://filaria.eu/
http://www.riverblindnessvaccinetova.org/


Publications to date
1 Ajendra J, Specht S, Neumann AL, Gondorf F, Schmidt D, Gentil K, Hoffmann WH, Taylor MJ, Hoerauf A, Hübner MP (2014). ST2 deficiency does not impair Type 2 immune responses during chronic filarial infection but leads to an increased microfilaremia due to an impaired splenic microfilarial clearance. PLoS ONE 9(3): e93072. doi: 10.1371/journal.pone.0093072
2 Arndts K, Deininger S, Specht S, Klarmann U, Mand S, Adjobimey T, Debrah A, Batsa L, Kwarteng A, Epp C, Taylor M, Adjei O, Layland L, Hoerauf A (2012). Elevated adaptive immune responses are associated with latent infections of Wuchereria bancrofti. PLoS Negl Trop Dis 6(4):e1611. doi: 10.1371/journal.pntd.0001611
3 Arndts K, Specht S, Debrah AY, Tamarozzi F, Klarmann Schulz U, Mand S, Batsa L, Kwarteng A, Taylor M, Adjei O, Martin C, Layland LE, Hoerauf A (2014). Immunoepidemiological profiling of onchocerciasis patients reveals associations with microfilaria loads and ivermectin intake on both individual and community levels. PLoS Negl Trop Dis 20, 8:e2679. doi: 10.1371/journal.pntd.0002679
4 Armstrong SD, Babayan SA, Lhermitte-Vallarino N, Gray N, Xia D, Martin C, Kumar S, Taylor DW, Blaxter ML, Wastling JM, Makepeace BL (2014). Comparative analysis of the secretome from a model filarial nematode (Litomosoides sigmodontis) Reveals Maximal Diversity in Gravid Female Parasites. Mol Cell Proteomics 13, 2527-44. doi: 10.1074/mcp.M114.038539. Epub 2014 Jun 23
5 Babayan SA, Allen JE, Taylor DW (2012). Future prospects and challenges of vaccines against filariasis. Parasite Immunol 34: 243-53. doi: 10.1111/j.1365-3024.2011.01350.x.
6 Babayan SA, Luo H, Gray N, Taylor DW, Allen JE (2012). Deletion of parasite immune modulatory sequences combined with immune activating signals enhances vaccine mediated protection against filarial nematodes. PLoS Negl Trop Dis 6(12): e1968. doi: 10.1371/journal.pntd.0001968. Epub 2012 Dec 27
7 Bah GS, Ward EL, Srivastava A, Trees AJ, Tanya VN, Makepeace BL, 2014. Efficacy of three-week oxytetracycline or rifampicin monotherapy compared with a combination regimen against the filarial nematode Onchocerca ochengi. Antimicrob Agents Chemother 58: 801-810. doi: 10.1128/AAC.01995-13. Epub 2013 Nov 18.
8 Banla M, Tchalim S, Karabou PK, Gantin RG, Agba AI, Kére-Banla A, Helling-Giese G, Heuschkel C, Schulz-Key H, Soboslay PT. Sustainable control of onchocerciasis: ocular pathology in onchocerciasis patients treated annually with ivermectin for 23 years: a cohort study. PLoS One. 2014 Jun 2;9(6):e98411. doi: 10.1371/journal.pone.0098411
9 Blaxter M and Koutsovoulos G (2014). The evolution of parasitism in Nematoda. Parasitology, 1-14. doi: 10.1017/S0031182014000791
10 Blaxter M, Kumar S, Kaur G, Koutsovoulos G, Elsworth B. (2012). Genomics and transcriptomics across the diversity of the Nematoda. Parasite immunology 34, 108-120. doi: 10.1111/j.1365-3024.2011.01342.x
11 Bouchery T, Dénécé G, Attout T, Ehrhardt K, Lhermitte-Vallarino N, Hachet-Haas M, Galzi JL, Brotin E, Bachelerie F, Gavotte L, Moulia C, Bain O, Martin C. (2012) The chemokine CXCL12 is essential for the clearance of the filaria Litomosoides sigmodontis in resistant mice. PLoS One 7(4):e34971. doi: 10.1371/journal.pone.0034971. Epub 2012 Apr 12
12 Bouchery T, Ehrhardt K, Lefoulon E, Hoffmann W, Bain O, Martin C. (2012) Differential tissular distribution of Litomosoides sigmodontis microfilariae between microfilaremic and amicrofilaremic mice following experimental infection. Parasite 19(4):351-8. doi: 10.1051/parasite/2012194351
13 Bouchery T, Lefoulon E, Karadjian G, Nieguitsila A, Martin C (2013) The symbiotic role of Wolbachia in Onchocercidae and its impact on filariasis. Clin Microbiol Infect 19(2):131-40. doi: 10.1111/1469-0691.12069
14 Buck AH and Blaxter M (2013). Functional diversification of argonautes in nematodes: an expanding universe. Biochemical Society transactions 41, 881-886. doi: 10.1042/BST20130086
15 Comandatore F, Sassera D, Montagna M, Kumar S, Koutsovoulos G, Thomas G, Repton C, Babayan SA, Gray N, Cordaux R, Darby A, Makepeace B, Blaxter M (2013). Phylogenomics and analysis of shared genes suggest a single transition to mutualism in Wolbachia of nematodes. Genome biology and evolution. doi: 10.1093/gbe/evt125
16 Darby AC, Armstrong SD, Bah GS, Kaur G, Hughes MA, Kay SM, Koldkjær P, Rainbow L, Radford AD, Blaxter ML, Tanya VN, Trees AJ, Cordaux R, Wastling JM, Makepeace BL 2012. Analysis of gene expression from the Wolbachia genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis. Genome Res 22: 2467-2477. doi: 10.1101/gr.138420.112. Epub 2012 Aug 23
17 Darby AC, Gill AC, Armstrong SD, Hartley CS, Xia D, Wastling JM, Makepeace BL ( 2014). Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress. ISME J 8: 925–937. doi: 10.1038/ismej.2013.192. Epub 2013 Oct 24
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19 Debrah AY, Mand S, Marfo-Debrekyei Y, Batsa L, Albers A, Specht S, Klarmann U, Pfarr K Adjei O and Hoerauf A (2011). Macrofilaricidal Activity in Wuchereria bancrofti after 2 Weeks Treatment with a Combination of Rifampicin plus Doxycycline. J Parasitol Res DOI: 10.1155/2011/201617
20 Gentil K, Lentz CS, Mushin RRM, Kamath AD, Mutluer Ö, Specht S, Hübner MP, Hoerauf A (2014). Eotaxin-1 is involved in parasite clearance during chronic filarial infection. Parasite Immunology;36, 60-77. doi: 10.1111/pim.12079
21 Godel C, Kumar S, Koutsovoulos G, Ludin P, Nilsson D, Comandatore F, Wrobel N, Thompson M, Schmid CD, Goto S, Bringaud F, Wolstenholme A, Bandi C, Epe C, Kaminsky R, Blaxter M, Maser P (2012). The genome of the heartworm, Dirofilaria immitis, reveals drug and vaccine targets. FASEB journal 26, 4650-4661. doi: 10.1096/fj.12-205096
22 Hegewald J, Gantin RG, Lechner CJ, Huang X, Agosssou A, Agbeko YF, Soboslay PT, Köhler C. (2015) Cellular cytokine and chemokine responses to parasite antigens and fungus and mite allergens in children co-infected with helminthes and protozoa parasites. J Inflamm (Lond). 2015 Jan 20;12:5. doi: 10.1186/s12950-015-0050-y
23 Hoerauf A, Pfarr K, Mand S, Debrah AY, Specht S (2011). Filariasis in Africa-treatment challenges and prospects. Clin Microbiol Infect 17, 977-985. doi: 10.1111/j.1469-0691.2011.03586.x
24 Karadjian G, Berrebi D, Dogna N, Vallarino-Lhermitte N, Bain O, Landau I, Martin C (2014) Co-infection restrains Litomosoides sigmodontis filarial load and plasmodial P yoelii but not P chabaudi parasitaemia in mice. Parasite 21, 16. doi: 10.1051/parasite/2014017
25 Katawa G, Layland LE, Debrah AY, von Horn C, Batsa L, Kwarteng A, Arriens S, Taylor DW, Specht S, Hoerauf A, Adjobimey T (2015). Hyperreactive Onchocerciasis is Characterized by a Combination of Th17-Th2 Immune Responses and Reduced Regulatory T Cells. PLoS Negl Trop Dis. 2015 Jan 8;9(1):e3414. doi: 10.1371/journal.pntd.0003414
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27 Kumar S and Blaxter ML (2011). Simultaneous genome sequencing of symbionts and their hosts. Symbiosis, 55, 119-126. doi: 10.1007/s13199-012-0154-6
28 Kumar S, Jones M, Koutsovoulos G, Clarke M, Blaxter M (2013). Blobology: exploring raw genome data for contaminants, symbionts and parasites using taxon-annotated GC-coverage plots. Frontiers in Genetics 4, 237. doi 10.3389/fgene.2013.00237
29 Landmann F, Bain O, Martin C, Uni S, Taylor MJ, Sullivan W (2012) Both asymmetric mitotic segregation and cell-to-cell invasion are required for stable germline transmission of Wolbachia in filarial nematodes. Biol Open. 1(6):536-47. doi: 10.1242/bio.2012737
30 Lechner CJ, Gantin RG, Seeger T, Sarnecka A, Portillo J, Schulz-Key H, Karabou PK, Helling-Giese G, Heuschkel C, Banla M, Soboslay PT (2012). Chemokines and cytokines in patients with an occult Onchocerca volvulus infection. Microbes Infect. 2012 May;14(5):438-46. doi: 10.1016/j.micinf.2011.12.002.
31 Lechner CJ, Komander K, Hegewald J, Huang X, Gantin RG, Soboslay PT, Agossou A, Banla M, Köhler C. Cytokine and chemokine responses to helminth and protozoan parasites and to fungus and mite allergens in neonates, children, adults, and the elderly. Immun Ageing. 2013 Jul 15;10(1):29. doi: 10.1186/1742-4933-10-29
32 Lefoulon E, Gavotte L, Junker K, Barbuto M, Uni S, Landmann F, Laaksonen S, Saari S, Nikander S, de Souza Lima S, Casiraghi M, Bain O, Martin C (2012) A new type F Wolbachia from Splendidofilariinae (Onchocercidae) supports the recent emergence of this supergroup. Int J Parasitol 42,1025-36. doi: 10.1016/j.ijpara.2012.09.004
33 Mand S, Debrah A, Klarmann U, Batsa L, Marfo-Debrekyei, Kwarteng A, Specht S, Belda-Domene A, Fimmers R, Taylor M, Adjei O, Hoerauf A (2012). Doxycycline improves filarial lymphedema independent of active filarial infection: a randomized controlled trial. CID, doi: 10.1093/cid/cis486
34 Makepeace BL, Martin C, Turner JD and Specht S (2012). Granulocytes in helminth infection - who is calling the shots? Curr Med Chem 19: 1567-1586. doi: 10.2174/092986712799828337
35 Nieguitsila A, Frutos R, Moulia C, Lhermitte-Vallarino N, Bain O, Gavotte L, Martin C (2013). Fitness cost of Litomosoides sigmodontis filarial infection in mite vectors; implications of infected haematophagous arthropod excretory products in host-vector interactions. Biomed Res Int 2013:584105. doi: 10.1155/2013/584105
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Potential Impact:
Enhanced Protective Immunity Against Filariasis (E PIAF)
HEALTH-2009-4.3.1-1 Contract 242131

E PIAF impact on onchcerciasis control
The E PIAF project is the fulmination of 6 successive EU Framework contracts that have targeted control of onchocerciasis and demonstrates the long-term commitment of the Consortium to help control and eliminate this disease. As one of the recognised Neglected Tropical Diseases, onchocerciasis was included in the London Declaration on Neglected Tropical Diseases of January 2012 which called for sustained efforts to expand and extend drug access programmes to ensure the necessary supply of drugs and other interventions to help control human onchocerciasis (river blindness).

The most well know international effort against onchocerciasis is the [World Bank/WHO] African Programme for Onchocerciasis Control (APOC) which comes to an end in 2015. Discussions are underway to incorporate the objectives of APOC with control of lymphatic filariasis in a new Programme for the Elimination of Neglected Diseases in Africa (PENDA) to begin in 2016 PENDA has the new aim of eliminating Onchocerca volvulus, the causative agent of onchocerciasis by 2025. However, the establishment of PENDA is uncertain any extension of the work conducted by APOC beyond this year is similarly uncertain: it is possible that control of onchocerciasis in Africa will be the sole responsibility of national governments.

APOC’s work has been predicated on sole use of mass drug administration (MDA) of ivermectin (MectizanTM). This drug has been very effective in reducing morbidity because it is very effective at killing the microfilariae larvae, but it does not kill adult worms.

The possibility of eliminating and even eradicating onchocerciasis from Africa using ivermectin has been discussed extensity by various organisations (eg the Mectizan Foundation) in addition to APOC. Work in Mali and Senegal has been cited as demonstrating the feasibility of achieving this goal. However, the ecology and epidemiology of onchocerciasis in these countries is far from the situation found the rain forest regions of West and Central Africa. For example, in Cameroon (where E PIAF partners have been working for over 20 years), after a decade of mass ivermectin treatment, the burden of onchocerciasis disease (morbidity) has been reduced but, most critical, transmission is still going (Annex 3).

A known deficiency of mass drug treatment programmes is the fact that ivermectin cannot be used in areas where onchocerciasis and loiasis are co-endemic due to the risk of severe adverse reactions following drug treatment. It is estimated that 12-14 million people live in such high risk areas in central Africa and are potentially affected by this contraindication. Communities in these areas often do not receive supportive treatment, onchocerciasis transmission rates remain high, and reintroduction of the infection to neighbouring communities from which the disease has been eliminated is an ongoing threat.

In regions where co-infections do not prevent the implementation of mass ivermectin treatment local emergence of resistance (eg in Ghana) is compromising the outcome. Development of resistance to ivermectin should not come as a surprise as it is very well known in veterinary usage. Once established, resistance to ivermectin on the part of O volvulus will inevitably spread.

Even given ideal conditions the possibility of eradicating onchocerciasis from Africa using ivermectin alone is unrealistic.

Development of new tools (including drugs, diagnostics and vaccines) will be required to ensure onchocerciasis elimination and remove the risk of reintroduction of the infection to areas where elimination may have been achieved. Such new tools would potentiate or enhance the efficiency of ivermectin treatments and address the identified deficiencies of the current MDA programmes.

With completion of the E PIAF contract, the possibility of adding vaccination to the onchocerciasis control tool box is a reality.

E PIAF has identified three antigens that have been shown capable of protecting mice against secondary challenge with infective L3 larvae and thus are onchocerciasis vaccine candidates.

The target population for vaccination is pre-school children (as part of the Expanded Programmes of Immunisation) who are currently excluded from treatment with ivermectin. Mathematical modelling based on an initial vaccine efficacy of 50% against incoming worms, a 90% reduction of microfilariae load, and an 80% coverage of 1-5 yr olds initially with subsequent annual vaccination of 1 yr olds, suggest that after 15 years of vaccination in areas not previously treated with ivermectin, a vaccine would have a substantial impact, markedly reducing microfilariae load in the young (under 20 years of age) in a range of endemicity scenarios. This has important implications as studies have highlighted the crucial role of acquiring heavy infections earlier in life regarding the risk of developing onchocerciasis-related morbidity and mortality. This suggests that a vaccine would have a beneficial impact in terms of reducing onchocerciasis disease burden in these populations. Moreover, a vaccine could markedly decrease the chance of onchocerciasis infection re-spreading to areas where it is deemed that mass drug administration with ivermectin can be stopped. Therefore, a vaccine would protect the substantial investments made by present and past onchocerciasis control programmes (together, the Onchocerciasis Control Programme in West Africa (OCP) and APOC have cost over US$1 billion, excluding the value of ivermectin), decreasing the chance of disease recrudescence and the potential spread of ivermectin resistance.

The next step is to take one or more to Phase I human safety trials. This will require producing quantities of the antigen to Good Manufacturing Practice and taking these formulations through mandatory/regulatory toxicity tests in animals. Successful completion of animal toxicity test should enable the antigens to be taken through phase I in human volunteers. A two-stage process is envisaged with the primary tests being performed in Europe with Europeans of West African origin but who have never travelled to Africa. The second phase would be carried out in Ghana.

Successful completion of Phase 1 safety trials will permit efficacy trails to be initiated in a staggered manner in communities where only Onchocerca volvulus is endemic (Ghana) and subsequently in Cameroon where O volvulus and Loa loa are co-endemic ie areas where ivermectin is contraindicated for mass treatment programmes.

If production of the vaccine were to be commissioned by the end of 2015, the animal toxicity and Phase 1 human trials could be completed by 2018. Efficacy trials could follow by 2020.

E PIAF impact on other filarial infections
The genomic and proteomic studies performed as part of E PIAF have demonstrated the presence of homologues of the onchocerciasis vaccine candidates in other human filariae including Wuchereria bancrofti the causative agent of lymphatic filariasis (elephantiasis) and Dirofilaria immitis, the dog heart worm.

E PIAF consortium members are in discussion with a major veterinary company to carry out safety and vaccination trials in dogs.

Development of a vaccine against lymphatic filariasis would require some additional laboratory studies appropriate animal models (eg Brugia malayi in gerbils). However, assuming demonstration of efficacy in such models (and TOVA Partners have some data in this area, see Annex 1), the lead time for Phase 1 trails would be about 5 years. Over 120 million people are currently infected with W bancroft or B malayi) and nearly 1.4 billion people in 73 countries are threatened by lymphatic filariasis. As with onchocerciasis, mass drug administration is used for control. In this case a combination of albendazole and ivermectin or diethylcarbamazine citrate is used. These regimes are very effective but again as seen in onchocerciasis, a vaccine would find application for the treatment of pre-school children.

The gene expression profiles of filarial infected individuals and the genomes and proteomes of the parasites can also provide a starting point for data mining for new drugs for both human and veterinary (Dirofilaria immitis, the dog heart worm) application.

E PIAF advances for the small holder livestock producers
Livestock [ruminant] production is a key foundation for food security and economic viability, especially in developing countries. As a consequence, infectious diseases that depress livestock productivity have a disproportionate impact on the health and livelihood of local populations. In the developing world, even more so than in developed countries, livestock are typically infected by multiple parasite groups throughout their lives. Although microparasitic diseases can be more virulent, the ubiquitous presence of chronic macroparasitic infections is of primary concern due to the systemic immunosuppression that helminths and ticks induce to ensure their own survival and transmission. The direct global cost of parasitic worms of livestock is ~$1,900M per annum, and the cost of tick infection, which are major vectors for other diseases, is estimated in excess of $14 billion. Further, there is extensive evidence that immunosuppression itself can facilitate opportunistic infections by other pathogens, adversely affect responses to standard vaccination, and there is evidence that the immunosuppressed phenotype induced by these parasites can be transmitted to the offspring. Current macroparasiticidal interventions, where effective, rely on repeated and often expensive drug treatment, rarely lead to immunity against reinfection, and are logistically unfeasible in most of the developing world. The most cost-effective alternative may be vaccination, but there are still no publicly available effective vaccines against macroparasites.
E PIAF has demonstrated that neutralisation of parasite-driven immunosuppression is a practical target for intervention in the case of filarial infections. The time is right to extend this approach to
a broader range of common parasites that continually immunosuppress small holder livestock [ruminants] in developing countries. Most livestock are infected by multiple species of ticks and nematodes, and targeting only one of those might be insufficient to allow productivity gains.

Restoration of protective immune function and productivity of livestock by targeting molecular motifs shared by their most abundant immunosupressors provides a practical target for a pan=spefic vaccine. This could be achieved by taking advantage of the phylogenetic proximity of tick and nematodes (both belong to the ecdysozoa group) to safely target motifs of immunosuppressive proteins that (a) are conserved amongst several tick and nematode species, (b) are specific to invertebrates to prevent autoimmune responses, and (c) have, or are likely to have, a role in immunosuppression based on homology with known immune-suppressors and on in vitro screening.

This area would be a natural extension of the E PIAF studies and if successful could provide quantifiable gains to livestock productivity (weight gain and milk production) through a new generation of vaccines that target pathogen immune evasion strategies.

E PIAF and autoimmune diseases
The onchocerciasis vaccine candidates include potent Th2 immuno-modulators. These may have therapeutic application for treatment of Th2 driven autoimmune diseases such as multiple sclerosis and psoriasis, and allergies for which a number of in vitro and in vivo models for the target disease exist. This area requires funding for proof-of-principle studies and E PIAF Partners are exploring both commercial and public funding opportunities for such work.

Project meetings, Dissemination, Scientific meetings
Project meetings
September 2012 - Paris, Mid-term meeting and formal review
January 2013 - Edinburgh. Ingenuity training session.
May 2013 - Edinburgh. Data analysis and Biolayout Express training session.
December 2013 - Buea, Cameroon Data analysis of Cameroon dataset.
January 2014, Edinburgh, Filarial Pathway workshop

E PIAF publications in peer-reviewed scientific journals will be posted on the E PIAF web site as they become available.

In addition to papers in the primary scientific literature, E PIAF studies have been presented at numerous scientific meetings while the Co-ordinator has also presented to broader interest groups (eg Scottish Parliament).

Arguably the most important presentation was to the 38th session of the Technical Consultative Committee of the African Programme for Onchocerciasis Control (APOC) which will be held in Ouagadougou, from 10 to 14 March 2014 at which the Co-ordinator, Dr Ben Makepeace and Dr Martin Walker (Imperial College) presented the Onchocerciasis Vaccine for Africa TOVA initiative (Annex 1, http://riverblindnessvaccinetova.org) and the case for inclusion of a vaccine in onchocerciasis control programmes in order to achieve elimination of the infection from Africa.

Other scientific meetings attended by individual members of the Consortium 2012-2014
1, American Society of Tropical Medicine and Hygiene, Atlanta, USA 11-15 November 2012,
2, Congrès de la Société Française de Parasitologie. Dijon, 14-15 May 2013.
3, Institut Pasteur. "Insights into filarial biology: biodiversity, migration, and control of the infection", 27 June 2013.
4, 15th International Congress of Immunology, Italie, 22-27 August 2013.
5, 11th World Congress on Inflammation, Natal. Brésil. "The chemokine receptor CXCR4 and its ligand CXCL12 limit filarial infection", 21-25 September 2013.
6, American Society of Tropical Medicine and Hygiene, Atlanta, USA, 11-15 November 2012.
7, Gordon Research Conferences Molecular Mechanisms in Lymphatic Function & Disease, Lucca, Italy, 9-14 March 2014.
8, British Society for Parasitology, Cambridge, UK, 6-9 April 2014.
9 Congrès de la Société Française de Parasitologie, 22-23 May 2014.
10, 8th International Wolbachia conference, Igls, Autriche, 6-11 June 2014.
11, International Filariasis Meeting, Paris, France, 26-27 September 2014.


Patent application
A patent has been filed for the use of a unique immuno-modulator identified through the proteomic analysis of excreted/secreted filarial products. This molecule is an addition to the three antigens discussed in the narrative of this report

Details of this patent can be found in Annex 5 (Makepeace, Benjamin (2014) Vaccines, Polypeptides, and Nucleic Acids. 1402352.7)

Dissemination
The most important dissemination activity has been the production of the TOVA prospectus (Annex 1, http://riverblindnessvaccinetova.org) which has been followed by an Editorial in plos Neglected Diseases and submitted as Annex 6.

List of Websites:
http://www.riverblindnessvaccinetova.org/
http://filaria.eu/
http://badger.bio.ed.ac.uk/filarial/
http://nematodes.org/genomes/litomosoides_sigmodontis/
http://www.nematodes.org/genomes/onchocerca_ochengi/