Bergquist 2010 Chapter 10 – Control of Important Helminthic Infections

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vaccine candidates would mainly be to influence transmission

through targeting the intermediate or reservoir host, when the

infection is zoonotic. Apart from this general scheme, there are

also vaccine candidates targeting the parasites in the definitive

host, in particular the early developmental stages, which shouldreduce the risk of drug failure. Since the biological targets in most

cases are different, vaccination would be synergistic with drug

therapy. This review covers diseases caused by helminthes in

both humans and animals and includes examples of diseases caused

by cestodes, nematodes and trematodes. The focus is on infections

for which vaccine development has been undertaken for a long

time, resulting in products that could realistically become

integrated into control strategies in the near future.

10.1. INTRODUCTION

Initially, based on the early success of anti-viral and anti-bacterial vac-cines, it was believed that development of vaccines against parasiteswould be likewise. The first vaccine candidates of this kind consisted of attenuated or killed whole organisms, but the results were not entirely

successful and work therefore turned towards native parasite antigensand recombinant subunit vaccines. At this point, it was already clear thatthe way forward would be far from simple. Even after the first stumbling block, large-scale antigen production, had been removed by the advent of recombinant DNA technology in the 1980s, work on anti-parasite vac-cines did not immediately take off. The situation has improved since then but chemotherapy still completely dominates the control measures forparasitic diseases of humans and animals alike. However, long-term drugtreatment represents a continuous expense and drug resistance is a per-manent threat; in the veterinary field often a reality. The goal of vaccinedevelopment in this area is not to produce a vaccine capable of inducingsterilizing immunity but to create an adjunct to chemotherapy that wouldreduce the likelihood of vaccinated individuals developing severe infec-tions and thus reduce the burden of disease throughout the world. Anintegrated approach, that is the follow-up of initial drug treatment withvaccination to achieve long-term protection (Bergquist et al., 2008), hasmuch to offer but a repository of specific vaccines useful against thevariety of infectious agents that make up the neglected tropical diseases(NTD) must first be established.

Commercially provided antiparasitic drugs with broad-spectrumaction have successfully been used to control parasitic diseases in live-stock and other domestic animals. However, frequent emergence of drugresistance in the target parasites has become a challenge. In addition,

298 Robert Bergquist and Sara Lustigman



issues regarding drug residues in the environment and food chain havecome to the forefront, boosting interest in alternative control methods andrenewed the appeal of vaccines.

Approaches based on molecular biology technology have resulted inthe elucidation of entire parasite genomes, as well as the identification of individual genes (Abubucker et al., 2008; Berriman et al., 2009; Brindleyet al., 2009; Ghedin et al., 2007; Schistosoma japonicum Genome Sequencingand Functional Analysis Consortium et al., 2009). Without doubt, furtherunderstanding of the role of gene products in parasite biology will lead tothe identification of novel parasite vaccine target antigens. However, forthis aim to be fully realized, strong investment in basic research on thecomplex interplay between parasite and host is necessary. Despite long-

term work on vaccine development, notably in the fields of hookworminfection, leishmaniasis, malaria, onchocerciasis and schistosomiasis, wehave yet to see an effective vaccine being implemented against a humanparasitic disease. On the other hand, for some of these infections, experi-ments using animal models have shown strong promise and there has been clear progress on vaccines against veterinary helminthic infections(Rickard et al., 1995). This may reflect fewer problems to be solved at the biological level with regard to animal vaccine development, but the moreprobable reasons are the much stricter regulatory demands governing

products for human use. The marketability of veterinary products isanother factor, as people who require parasite vaccines are generally notin a position to pay for them.

With regard to veterinary vaccines, the immunological control of Fasciolain sheep and cattle is within reach (Tendler and Simpson, 2008), and highlyeffective recombinant vaccines have already been developed for the preven-tion of Taenia ovis in sheep, T. saginata in cattle, T. solium in pigs andEchinococcus granulosus in livestock (Lightowlers, 2006a,b). While vaccinesagainst T. ovis and T. saginata are purely intended for assistance forfarming, vaccines against T. solium and E. granulosus vould primarily be beneficial to humans. It should, in this connection, be mentioned that trans-mission-blocking Schistosoma japonicum vaccines, which also belong tothis category, are currently in field trials (Da’dara et al., 2008; McManuset al., 2009).

Parasites survive in the host by avoiding or confusing immuneresponses against them, for example through stimulation of factorsdown-regulating the cellular response or through non-specific activationof B-cells. Parasite antigens are complex and difficult to characterise andthe host commonly responds with a range of various defence mechan-isms. Western blotting (Burnette, 1981) is the traditional approach toidentifying potential antigens for vaccine development but few of theantibodies identified by this technique are protective, as they are mostoften raised against non-related intracellular proteins which are released

Vaccine Development and Control of Helminthic Infections 299



when the parasite disintegrates. Protein polymorphism representsanother problem (Rosenzvit et al., 2006) that could make a the vaccineineffective against serotypes not included in the original design. Finally,

laboratory strains of parasites may become atypical, lose their naturalrange of polymorphism or even develop new polymorphisms notseen in nature by being maintained through long-term laboratorypropagation.

10.2. TYPE OF VACCINES

Vaccine production involves a spectrum of designs that can be based onanything between attenuated organisms and complex compounds pro-duced by molecular biology. Although the simplest approaches do notgenerally result in useful products, it does not follow that the mostcomplicated approaches are necessarily superior. To facilitate orientation,a brief guide to the different vaccine types is presented:

1. Vaccines consisting of whole parasites killed in various ways (heating,irradiation, etc.) that conserve their structures.

2. Attenuated organisms—live parasites treated so they cannot persist or

cause injury when injected.3. Recombinant vaccines—well-characterised gene products (antigens)

that can be produced in large quantities, consistently and cheaply.4. Peptide vaccines—short synthetic protein sequences including the

desired immuno-dominant epitopes.5. Recombinant-vector vaccines—attenuated virus or bacteria whose

genome has been supplied with extraneous DNA encoding parasiteantigenic determinants to be replicated and expressed by the vector.

6. DNA vaccines—plasmids containing the immune-dominant sequence

(s) capable of producing the desired parasite antigen intracellularly(in host cells) in an approach similar to that of recombinant-vectorvaccines.

Synthetic peptide vaccines are excellent from the point of view thatthey do not carry any danger of contamination with other proteins.In addition, epitopes from different proteins can be incorporated into asingle construct. However, peptides are not immunogenic and are there-fore utterly dependent on adjuvants. Although in theory this very fact

permits the option to select predominantly cell-mediated immunity orantibody production (Al-Sherbiny et al., 2003), we do not yet have accessto the adjuvants needed to achieve this modulation precisely. However,once the field has advanced further and products have been cleared for




assistance in antigen discovery comes from the possibility of probingpreparations by Western blotting (Burnette, 1981) against protective serato identify the most antigenic components. The proteins in question can

be chromatographically analysed and the preferred band(s) identified,extracted and subjected to liquid chromatography followed by massspectrometry for identification. The subsets of proteins, selected on the basis of their involvement in the host–parasite relationship and parasite biology, can then be produced in large amounts by means of recombinantDNA techniques for the routinely required efficacy-testing in animalmodels.

10.3.1. Correlate studiesOnce a panel of protective antigens has been established, the next step isto find out how animals and humans in endemic areas react immunologi-cally to natural infection. The investigation of individual immuneresponses to specific antigens, focusing interest on the underlyingmechanisms in resistance at the molecular level, is one of the keyapproaches to vaccine design. Well-researched antigen panels have beenset up for various parasites with the aim of identifying antibody andcytokine correlates of apparent resistance and apparent susceptibility.The approach was pioneered for S. mansoni antigens (Acosta et al., 2002;Al-Sherbiny et al., 2003; Ribeiro de Jesus et al., 2000). In a comprehensivestudy of this subject, carried out by the Egyptian Reference DiagnosticCenter (ERDC) in Cairo, immune responses against a panel of 10 priorityS. mansoni vaccine candidate molecules were determined in cohorts of humans living in areas where they were exposed to infection daily, andthe results compared with the corresponding data emanating from para-sitological diagnosis (Al-Sherbiny et al., 2003). Responses significantlycorrelating either with resistance or with susceptibility to re-infectionwere demonstrated and it was also possible to group the protectiveresponses to some of the antigens as belonging either to the Th1 or tothe Th2 sphere. This type of study is instructive not only for identifyingthe few antigens that directly and exclusively correlate with resistance, but also by indicating which of them can be modulated to produce thesought-after immune response. In a similar approach from the field of filariasis, Vedi et al. (2008) have identified a 2.0 kb cDNA clone coding forBrugia malayi heavy chain myosin which exhibited strong immunoreac-tivity with bancroftian sera from endemic putatively immune humansubjects. Similarly, sera from individuals immune against Onchocercavolvolus have been used successfully to clone vaccine candidates subse-quently shown to be protective against O. volvulus infective third-stagelarvae in a mouse model (Lustigman et al., 2003).




worse than those produced by potassium aluminium sulphate (alum), theonly widely used adjuvant in human vaccines today. However, sincealum probably acts more as a depot than anything else (Lindblad, 2004),

the need for specific adjuvants is growing. Already, an adjuvant fromGlaxoSmithKline (London, UK) based on MPL and Quil (ASO4) has beenlicensed for use in human vaccines in more than 100 countries(Pichichero, 2008).

10.4. PROGRESS IN VACCINE DEVELOPMENT

Overall, the development of vaccines against parasitic diseases in humans

has not been taken seriously by donor agencies. The main reason for thisis the easy access to cheap, effective drugs which, apart from malariachemotherapy, have not been prone to produce resistance in their targetparasites. In the veterinary field, there is a stronger need for vaccines asdrug resistance is commonplace. This is probably due to the particularlyhigh drug pressure realized when herds and flocks are exposed to regularrecurrent blanket treatments. Also humans are subjected to repeatedchemotherapy against various helminth infections but the risk for drugresistance is relatively low as the treatment cycles promoted by control

programmes rely on lower doses spaced by relatively long intermissions.Still, development of resistance against drugs used for control of humaninfections can definitely not be ruled out, but it may take longer than the20 years commonly mentioned by veterinarians. Notably, the emergenceof resistant strains of the O. volvulus parasite is suggested by reports of onchocerciasis patients failing to respond to ivermectin treatment(Churcher et al., 2009). In addition, an increased frequency of a resistantallele has been identified in Wuchereria bancrofti microfilariae in patients inareas subjected to mass drug administration (MDA) with benzimidazole(Schwab et al., 2005). Both drugs are used for MDA of onchocerciasisand/or lymphatic filariasis (LF).

A typical characteristic of parasitic diseases is that the life cycles of theinfectious agents require that their life forms oscillate between differenthosts which often involve vertebrates both as intermediate and as defini-tive host. Although the intermediate host can also be a gastropod or aninsect (as in schistosomiasis and malaria, respectively), vertebrate hostsother than humans can also play the role of disease reservoir. For exam-ple, in contrast to other schistosome species, S. japonicum infects a broadrange of animals which are sometimes more important for the transmis-sion of the disease than humans (Gray et al., 2007; Wang et al., 2005).Against this background, one might think that vaccination of key vertebrate species involved in the propagation of diseases eventually implicat-ing humans should be part of control activities, yet it is not. In the absence




of any human vaccine against parasites, veterinary vaccines should be triedfor the control of human endemic diseases. It is a positive sign that this issubject to research (Da’dara et al., 2008; Gray et al., 2007; Guo et al., 2006;

Lightowlers, 2004). The current situation with respect to vaccine develop-ment is reviewed below with respect to major zoonotic helminthiases suchas cysticercosis, echinococcosis and schistosomiasis japonica as well as forhuman helminth hookworm infection, LF and onchocerciasis.

10.4.1. Cestode infections

T. solium (see life cycle in the back of volume 72), the cestode whose larvalencystation gives rise to neurocysticercosis in humans, belongs to a zoonotic

disease complex that is endemic in a large number of countries where pigsare kept under unhygienic conditions and without adequate sanitary dis-posal of human faeces. Since the pig is the obligate intermediate host forT. solium, and effective drugs are available, it should be possible to curb thetransmission of this major parasitic disease that frequently affects humanhealth and economy in the developing countries. Since a veterinary vaccinehas also been developed against this disease (Lightowlers, 2004), it is almostsurprising that neurocysticercosis has not already been eliminated. Theexplanation for this lack of progress is that the pig readily establishes new

tapeworm infections in humans, leading to renewed transmission after eachround of treatment. A detailed review of the disease and its present situa-tion is given by Willingham et al. (2010).

A well-designed vaccine strategy should have a good chance to preventinfection in the pig and thus interrupt the parasite life cycle. Indeed,vaccine research has been moving well ahead during the last decade asthe work to decipher this parasite genetically has provided novel antigens.For example, Almeida et al. (2009) generated more than 1500 high-qualityExpressed Sequence Tags (ESTs) from 20 cDNA T. solium mini-libraries.Identification of protective antigens and their production by recombinantDNA technology has been researched by Lightowlers (2003 and 2004).Tenyears ago, Johnson et al. (1989) published the cloning of a recombinantT. ovis antigen which stimulated high levels of protective immunity insheep. Relatively rapidly, this was followed by the development of effec-tive vaccines not only against T. ovis in sheep but also against T. saginata incattle, T. solium in pigs and E. granulosus in livestock, indicating thatreliable, high-level protection against a complex metazoan parasite can be achieved using defined recombinant antigens (Lightowlers, 2006a,b;Lightowlers et al., 2003).

Several approaches based on recombinant antigens have been madetowards development of vaccines against T. solium. For example, twohighly immunogenic oncosphere antigens, TSOL18 and TSOL45, have been shown to induce near-complete protection against experimental




challenge infection in four separate vaccine trials in pigs (Kyngdon et al.,2006; Lightowlers, 2004) and another oncospheral stage-specific 45Wprotein has shown similar results (Luo et al., 2009). In addition, a review

on vaccines against cysticercosis by Sciutto et al. (2008) highlights work onS3Pvac, the only synthetic peptide vaccine that has been tested andproved effective in the field against naturally acquired disease. Thus,from the technical point of view, we have access to vaccines with thecapability to break the parasite life cycle in the pig intermediate host.If correctly deployed and monitored, any of these vaccines would withoutdoubt rapidly lead to the eradication of human cysticercosis.

E. granulosus (for life cycle see back of volume 72 and McManus, 2010)is another cestode stimulating strong immune responses which open

possibilities for the development of vaccines directed against this infec-tion, both in the intermediate and in the definitive host. An effectivestrategy would be to stop the development of adult gravid tapeworms indogs with a vaccine, so preventing the oncosphere from producing hyda-tid cysts in animals, for example sheep. This is a straightforward strategywhich relies on the fact that echinococcosis induces cell-mediated cellularresponses as well as significant antibody production in their human andintermediate hosts. However, for the approach to be successful, a thor-ough understanding of the immune mechanisms involved is required.

The many recent reviews of immunity mechanisms at work in echino-coccosis are evidence of the large body of knowledge amassed so far, forexample Amri et al. (2009), Gottstein and Hemphill (2008), Lightowlers(2006a), Lightowlers and Heath (2004), Torgerson (2009) and Zhang et al.(2008). Clearly, a very high coverage of flocks would need to be achievedfor vaccination alone to be effective, but a combination of vaccination of the sheep and drug treatment of surrounding dogs could achieve a goodlevel of transmission control, even with a less-than-perfect vaccine aspointed out by Torgerson (2006).

EG95, the recombinant E. granulosus vaccine based on an oncosphereprotein containing a glycosylphosphatydilinositol (GPI) anchor and afibronectin domain, is strongly immunogenic and induces effective pro-tection against challenge infection (Gauci et al., 2005). However, its immu-nological coverage may not be as broad as desired, since the E. granulosusgenome also contains several other EG95-related genes which may affectthe efficacy of this vaccine (Chow et al., 2008). Further sequencing hasuncovered a large amount of variability in this organism (Haag et al.,2009) confirming this suspicion. Nevertheless, as the sheep-dog strain of the parasite is responsible for most cases of human disease, EG95 remainspromising.

Although it would be of huge benefit in reducing the effective periodrequired to stop transmission of E. granulosus, no effective vaccine existsagainst canine echinococcosis. Apart from that, however, a long list of




deliverables needs to be consulted to efficiently approach this goal, forexample improved diagnostics, characterization of infection dynamicsand determination of the longevity of protection induced, to mention

just a few. In addition, further down the road, mathematical models willneed to be developed to facilitate impact evaluation. Even incorporationof only a few of these measures should increase the efficiency of controland reduce the time required to achieve prevention of disease transmis-sion. Above all, the scarcity of vaccine candidates for immune protectionagainst adult tapeworms reflects the lack of immune correlates and theambiguity of natural immunity in dogs (Craig et al., 2007; Zhang andMcManus, 2008).

Before leaving the topic of vaccines against echinococcosis, E. multi-

locularis (see life cycle on page tbc) should briefly be mentioned. UnlikeE. granulosus, this species produces multiple, small cysts that spreadthroughout the body in many mammals, including rodents and humans.Although this infection is mainly distributed in the northern hemisphereand is much less common than that caused by E. granulosus, it is referredto here since there are prospects that a vaccine can be developed against it.A tetraspanin candidate (E24) has been cloned from a full-length cDNAlibrary emanating from the E. multilocularis metacestode. Antibodiesagainst this antigen specifically recognize a 25 kDa cyst antigen from

the germinal layer of the E. multilocularis metacestode, highlighting itspotential both for diagnostics and vaccine development (Dang et al.,2009a,b)

10.4.2. Nematodes

The Phylum Nematoda (roundworms) includes tens of thousands of oftenvery diverse species, a large number of which are parasitic. Nematodescausing disease in humans include filarids, hookworms, pinworms andwhipworms, as well as individual species such as Ascaris lumbricoides andTrichinella spiralis. While drug treatment is an adequate approach for mostof these, it is realized that the long-term control of some of them, that ishuman hookworm infection (due to Ancylostoma duodenale or Necatoramericanus), LF (due to W. bancrofti or B. malayi,) and river blindness(caused by O. volvulus) will not be possible with drugs alone. Whileregular, annual or semi-annual chemotherapy is an important part of any public health interventions, high rates of re-infection and the spectreof diminished efficacy of drugs used often and repeatedly, conspire tochip away at the sustainability of this approach. Indeed, macrofilarialdrug cure, such as treatment with the adulticide melarsomine, can evenreduce natural (or induced) protective immunity as shown by a longitu-dinal study in a bovine model (Tchakoute et al., 2006). However, currentlythe treatment is with ivermectin, which has potent efficacy against the




microfilariae only. Duerr et al. (2008), on the other hand, have suggestedthat the resistance against filarial parasites includes a time-dependentcomponent caused by an early immune response with short-term mem-

ory. While vaccine studies are moving forward, clinical studies to investi-gate this conjecture are warranted.Hookworm (see life cycles on back of volume 72) is a leading cause of

maternal and child morbidity in the developing countries of the tropics andsubtropics. Together, the two species of hookworms that infect humans,N. americanus and A. duodenale, infect more than 500 million people world-wide (Hotez and Kamath, 2009; Hotez et al., 2008). The former species iscommon in the Americas, sub-Saharan Africa and Southeast Asia, with A. duodenale mainly found in the Middle East, North Africa and India.

The excretory/secretory (ES) component, a mixture of proteins, carbo-hydrates and lipids emanating from the parasite, represents the host–parasite interface and is probably involved in modulation of the hostimmune responses to promote the survival of the parasite. The doghookworm A. caninum is the common model for the study of hookworminfection and information from its genome coupled with functional geno-mics and proteomics is accelerating the move towards human hookwormcontrol. This work has resulted in the identification of a suite of ESproteins which are important for the parasitic lifestyle and which provide

insights into the biology of hookworm infection. For example, Abubuckeret al. (2008) generated 104,000 genome survey sequences (GSSs) andassembled them into 57.6 Mb of unique sequence, while Mulvenna et al.(2009) identified 105 different proteins and characterised much of the ESproteome.

Vaccine research targets both the larval and adult stages of the worm but vaccine candidates based on the larval forms are in the lead. Thecurrently most promising vaccine candidate is the N. americanus ASP-2(Na-ASP-2) antigen, first shown in secretions from A. duodenale but lateralso isolated from N. americanus (Diemert et al., 2008). A well-controlledstudy has shown this candidate to be safe in animals and capable of inducing protective responses, consisting of both specific IgG antibodiesand cellular immune responses. (Bethony et al., 2008). A Phase I safetytrial has been completed in the United States, while corresponding Phase Itrials in endemic areas are underway (Bethony et al., 2008).

With regard to potential adult worm antigens, vaccine-orientedresearch has focused on how the worm feeds, specifically investigatinghow to interfere with the action of the enzymes involved in the breakingdown of haemoglobin. Indeed, several of the proteins involved in theproteolytic cascade utilized by the adult worm to degrade haemoglobulinfrom host erythrocytes, and thus essential for its nutrition and survival,have been shown to induce protective immune responses. Among these,cysteine protease-haemoglobinase (CP-2) (Loukas et al., 2004), aspartic




protease-haemoglobinase (APR-1) (Loukas et al., 2005) and glutathioneS-transferase (GST) (Zhan et al., 2005) have been selected as possibletargets as they are essential for the digestive pathway. In fact, work

already carried out has shown that vaccination with the two formerantigens has been shown to reduce blood loss and faecal egg counts indogs (Loukas et al., 2004, 2005). Although an attack against the larvalstages by the Na-ASP-2 antigen, combined with interference of the adultworm’s digestive pathway, is theoretically attractive, we are not yet there.Nevertheless, great strides have been made and a partially effectivevaccine should soon be within reach.

LF, also known as elephantiasis, directly affects more than 120 millionpeople with about 10 times more at risk in the 80 countries where the

infection is now endemic (WHO fact sheet No. 102 on lymphatic filariasis,2010). One-third of those infected live in India, one-third in Africa andmost of the remainder in Southeast Asia. The overall prevalence of LF isincreasing due to the rapid, unplanned growth of cities producingexpanded breeding sites for the mosquitoes that transmit the disease.The causative filarial worms W. bancrofti and B. malayi (see life cycle on back of volume 72) lodge in the lymphatic system of humans, the defini-tive host of these parasites. Despite the hostile environment surroundingthem, these nematodes can survive up to 6 years, which they accomplish

by adopting various immunomodulatory strategies. During its lifespan,each worm produces millions of microfilariae that eventually reach andcirculate in the blood, assuring transmission to the mosquito vector.Application of drugs targeting the first-stage-larvae (L1) can block trans-mission but there are also drawbacks such as inadequate drug coverage,reappearance of infection through migration of infected people into con-trolled areas and partial success leading to reduced compliance. This hasled to a call for complementary approaches that include both improvedchemotherapy and vaccine development. Mouse and gerbil models of Brugia infection have provided information on the immune responseelicited by the different stages of these nematodes. Studies by Lawrenceand Devaney (2001) reinforce the concept that the different developmen-tal stages each have their own mechanism of modulating responses lead-ing to the down-regulation of potentially host beneficial immuneresponses. In an approach similar to that used by Almeida et al. (2009)for identifying potential vaccine antigens from T. solium, Nagaraj et al.(2008) have carried out a large-scale analysis of excreted or secretedproteins inferred from EST data. Although this inventory of known andnovel excreted or secreted proteins covers an enormous range of nema-todes, it can be used as a source of new vaccine candidates against filarialworms infecting humans.

A large-scale, proteomic analysis to identify the ES products of the L3,L3 to L4 moulting, adult male, adult female and microfilarial stages of the




filarial parasite B. malayi has recently been published (Bennuru et al.,2009). This analysis provides extended insight into the host–parasiteinteraction, while the reported abundance of a number of previously

characterised immunomodulatory proteins in the ES of microfilariaeincreases the chances of identifying novel vaccine candidates.Various vaccine candidates have already been put forward. For exam-

ple, Veerapathran et al. (2009), focusing on the key role of GST for thesurvival of the parasite in the host, achieved 61% protection againstB. malayi challenge infection in subsequent vaccination studies in the jird model. This work built on the effect of human and mice anti-GSTantibodies in an antibody-dependent cellular cytotoxicity (ADCC) assay(Veerapathran et al., 2009). In another development, Vedi et al. (2008)

showed the potential of B. malayi recombinant myosin as a vaccine in arodent model. The authors reported a 76% reduction in microfilarial burden and a 54–58% lower adult worm establishment that was conferredthrough the induction of both humoral and cellular immunities (Vediet al., 2008). There are also other antigens worth mentioning, for exampleALT-2 (Ramachandran et al., 2004), a microfilarial soluble 38 kDa proteaseisolated from B. malayi (Krithika et al., 2005) and a zinc-containing175 kDa collagenase (Pokharel et al., 2006) that have shown significantprotection against B. malayi in animal models. Any of these antigens

have the potential to be developed into a useful vaccine but there is stilla long way before a vaccine against LF will reach the stage of clinicaltrials.

Contrary to LF, in which the adult worms cause the pathology, themicrofilariae constitute the culprit in onchocerciasis, a disease caused bythe filarial worm O. volvulus. The infection is transmitted by the Simulium blackfly vector and is almost exclusively found in Africa. However,isolated foci also exist in Yemen and six countries in central and SouthAmerica (WHO, 2009a). Onchocerciasis affects an estimated 40 millionpeople, causing visual impairment in half a million; the disease alsocauses depigmentation and a severe, unrelenting itching. For years, con-trol activities were based on insecticides sprayed by aircraft over the blackfly breading sites but with the donation of MectizanÒ (ivermectin), by the U.S. Company Merck & Co. in 1987 (Colatrella, 2008), controloperations changed towards chemotherapy. Regrettably, resistance tothis drug appeared in the veterinary field early on (Coles et al., 2005;Egerton et al., 1988; Xu et al., 1998). The risk for a parallel situation vis-a-vis O. volvulus is therefore a worry and it seems already to be emerging(Boussinesq, 2008; Lustigman and McCarter, 2007; Osei-Atweneboanaet al., 2007). Above all, this development is a prompt for immediate andincreased activities in the vaccine field.

Like so many other parasitic diseases, there is a lack of good animalmodels as O. volvulus can develop fully only in humans. In a recent review




(Allen et al., 2008), however, Litomosoides sigmodontis in mice andO. ochengi in cattle are placed in the context of how these models can better our ability to control the human disease. The case for vaccine

development with regard to O. volvulus has recently been reviewed byLustigman and Abraham (2009) and several other authors have alsostressed the need for a vaccine (Boussinesq, 2008; Cook et al., 2001).Importantly, protective immunity against O. volvulus larvae has now been definitively demonstrated in humans, cattle and mice, thereby prov-ing the conceptual underpinnings that a vaccine can be produced againstthis infection (Lustigman and Abraham, 2009). As noted by Nutman(2002), an additional modality, complementing chemotherapy and vectorcontrol, is conditional to eliminate onchocerciasis. This makes it all the

more important to develop a vaccine before resistance has spread widelyrendering an integrated approach impossible. Vaccine studies supported by the Edna McConnell Clark Foundation USA resulted in the identifica-tion of 15 protective antigens out of 44 screened that induced significant but partial protection, using the diffusion chamber model in mice(Lustigman et al., 2002). Additional numbers of antigens with protectiveproperties have been reported in the last decade, for example Ov-FBA-1(McCarthy et al., 2002), Ov-ASP-1 (MacDonald et al., 2004), Ov-ALT-1(Wu et al., 2004), Ov-GAPDH (Erttmann et al., 2005), Ov-AST-1 (Borchert

et al., 2007) and paramyosin (Erttmann and Bu¨

ttner, 2009). However,as with vaccines against LF, much more work is needed before a vaccinecan be put to use. Recent reports using the cow model and O. ochengihave clearly proved the possibility of developing vaccines againstO. volvulus as well (Achukwi et al., 2007; Makepeace et al., 2009;Tchakoute et al., 2006).

10.4.3. Trematodes

Several species of flatworm threaten human health. Some exist only inSoutheast Asia, that is Clonorchis sinensis, Paragonimus spp. andOpisthorchis spp. (Keiser and Utzinger, 2005), while others are globallydistributed, for example Fasciola and Schistosoma. The diseases caused byClonorchis, Paragonimus, Opisthorchis and also Fasciola may be groupedtogether as foodborne treamatodiases due to the way they are transmit-ted. Apart from F. hepatica (McManus and Dalton, 2006; Vilar et al., 2003),the few antigens reported for this group mainly regard diagnostic use butreports on protective antigens have started to appear (Zhou et al., 2008).Interestingly, in this connection, the schistosomiasis Sm14-FABP vaccinecandidate (Tendler and Simpson, 2008; Vilar et al., 2003) cross-reacts withFasciola antigens (see below).

With close to 800 million people in 74 countries at risk, and directlyaffecting more than 200 million (Steinmann et al., 2006; WHO, 2009b),




schistosomiasis is the second-most, socio-economically devastating para-sitic disease after malaria. Five species cause human schistosomiasis butonly two exist in Southeast Asia (for life cycle see back of volume 72).

S. japonicum is the only species in the People’s Republic of China(P.R. China) and the Philippines, while small pockets of S. mekongi infectionconstitute serious, local problems in Cambodia and Lao People’s Demo-cratic Republic (Attwood et al., 2008). In contrast to all other schistosomiasisspecies, S. japonicum is a zoonotic infection affecting a wide range of animals,including wild and domestic ungulates as well as rodents, which all act asreservoirs. The only available drug is praziquantel, which is also one of thefew not fully subsidized drugs. With an estimated 423 million tabletsneeded globally every year (The Carter Center Schistosomiasis Control

Program, Atlanta, USA, 2010), the total expenditure needed for control isstaggering even though the average cost per dose is less than 20 cents.Although modern schistosomiasis control has clearly shown that che-

motherapy alone is capable of reducing morbidity in the human host(WHO, 2002a,b), rapid re-infection is a reminder that the impact of drugtreatment on transmission is marginal. The case for schistosomiasisvaccine development is based on the understanding that vaccination,even if not 100% effective, would contribute to long-term reduction of egg-excretion from the host. An effective vaccine would also contribute to

a positive trade-off regarding the aggressive inflammatory response thathas been observed following interrupted chemotherapy in children livingin high-transmission areas (Olveda et al., 1996; Reimert et al., 2008). Theunderlying reason for this ‘rebound morbidity’ is unclear but is probablydue to interruption of the Th1 response reducing the modulation thatnormally takes place during the course of natural infection.

Theargumentssupporting theutility of a vaccine againstschistosomiasis, based on more than 50 years of laboratory and field research, are strong.For example, it has long been known that humans living in schistosome-endemic areas develop some degree of protection naturally (Butterworthet al., 1985) and the injection of mice with irradiated schistosome cercariaeconsistently induce 60–85% protection (Dean, 1983). Vaccine developmentwas originally focused on S. mansoni but a panel of well-characterisedS. japonicum antigens have now also shown protective efficacy in animals justifying support for further consideration (McManus and Loukas, 2008).Due to its wide spectrum of final hosts, a ‘transmission-blocking’ veterinaryvaccine is the priority in areas in which S. japonicum is endemic. The possibility that this approach could pay off is supported by studies in P.R. Chinashowing that the animal–snail–human transmission cycle is more prominentthan the human–snail–human cycle in sustaining the infection in the field(Gray et al., 2007). An additional advantage in S. japonicum experimentationis that the access to full-size animal models escapes the limitations of themouse model ( Johansen et al., 2000; Zhu et al., 2006).




More than a hundred schistosome antigens have been identified andcharacterised but few have shown sufficient promise to be selected forfurther development. Despite the fact that Sh28-GST, a S. haematobium

GST molecule, is the current lead candidate, the great majority of Schisto-soma candidate vaccine antigens, reviewed by Bergquist and Colley(1998), derive from S. mansoni. The most well-researched are Sm28-GSTand Sh28-GST (Capron, 1998; Capron et al., 2005), paramyosin (Pearceet al., 1988), triose phosphate isomerase (Sm28-TPI) (Harn et al., 1992),Sm37-GADPH (Goudot-Crozel et al., 1989), Sm14-FABP (Tendler andSimpson, 2008; Vilar et al., 2003) and Sm-p80 calpain (Ahmad et al.,2009; Siddiqui et al., 2005). There are also multiple antigenic peptide(MAP) constructs made from various integrated membrane antigens

such as Sm10, Sm23, Sm28-TPI and Sm28-GST (Argiro et al., 2000; Ferruet al., 1997; Ribeiro de Jesus et al., 2000). The average protection of theseantigens, which have been tested either as native, full-length antigens,recombinant antigens, MAP constructs or as DNA vaccines, is around50% (in some cases higher) in the various animal models used.

The vaccine candidates mentioned above have been developed duringthe last two decades. Meanwhile, technology has become progressivelymore sophisticated resulting in an unprecedented expansion of parasitesequence databases. This accumulation of molecular data allows rational

vaccine discovery such as the two novel S. mansoni vaccine candidatesdetected at the parasite surface by proteomics which was recentlyreported by DeMarco and Verjovski-Almeida (2009).

Although fund raising for vaccine development has become anincreasingly uphill exercise, nationally available funds have beeninvested in Brazil and France, supporting the road toward clinical trialsfor Sm14-FASB and Sh28-GST, respectively. Industrial scale-up was firstachieved for Sh28-GST, now in clinical trials under the name of Bilhvax(Capron et al., 2005). This vaccine candidate has successfully passedPhase I/II clinical trials and been shown to be safe, producing Th2cytokines (IL-5 and IL-13) followed by high titres of neutralizing antibo-dies after three injections. Chemotherapy followed by immunization wasfelt to be the most appropriate modality and the best time to give thevaccine seems to be about three months after treatment, when patientshave switched to the Th2 type of response which takes time to occur andis generally not seen until after drug treatment. Therefore, the Phase IItrials of Bilhvax were based on this model, including both primary clinicaland secondary parasitological endpoints in measuring efficacy.

Industrial scale-up has also been achieved for Sm14-FABP (Tendlerand Simpson, 2008). Interestingly, thanks to a shared antigen betweenFasciola and Schistosoma, both natural infection and experimental animalresearch show cross-protection (Vilar et al., 2003). The former parasitecauses great losses in sheep and cattle breeding and can also infect




humans. Commercial interest in a vaccine for veterinary applications hascontributed to move this vaccine candidate into advanced veterinary fieldtrials which made it possible to piggyback the adoption of the same

molecule for developing a human vaccine against schistosomiasis(Tendler and Simpson, 2008). A collaborative initiative in Brazil for thescale-up of Sm14-FABP according to Good Manufacturing Practice (GMP)was established between the government-funded research centreOswaldo Cruz Foundation (FIOCRUZ) and Butantan, a producer of vac-cines for the Brazilian Ministry of Health. The previously used laboratory-scale expression system was substituted for systems based on vectorsappropriate for GMP production. Studies of the gene structure of Sm14were undertaken and provided the basis for functional and structural

analysis to access the preparation of a more stable form of the antigen based on site-directed mutagenesis. Stability and functionality (fatty acid binding) quality control assays were designed and developed. The newconstruct provided a highly purified protein in large yields with pre-served protective activity for both parasites opening the way to Phase Isafety trials (Ramos et al., 2003, 2009).

As S. japonicum is a zoonotic infection, the possibility of creating atransmission-blocking vaccine for livestock offers a shortcut in the devel-opment of vaccines for P.R. China and the Philippines. This and the recent

publication of the S. japonicum genome (Schistosoma japonicum GenomeSequencing and Functional Analysis Consortium et al., 2009) have nodoubt contributed to the increase in activities focused on this species inthe last few years (Da’dara et al., 2008; McManus and Dalton, 2006;McManus and Loukas 2008; Zhu et al., 2004, 2006). Based on the notionthat reduced schistosome infection in water buffaloes would also reducedisease transmission to humans, randomised double blind trials in water buffaloes using DNA vaccines encoding well-researched S. japonicumantigens (Sj28-TPI, Sj23) have taken place in P.R. China (Da’dara et al.,2008). The results were all close to 50% protection which exceeds thehypothetical level predicted by mathematical modelling to be needed toachieve a significant reduction of schistosome transmission.

10.5. INDUSTRIAL VACCINE PRODUCTION

It has been a long road in the development of vaccines against parasiticdiseases, and as scientific hurdles are overcome and we are reaching theindustrial level, the identification of partners and financial support growsin importance. This undertaking is as critical as dealing with the science, but the problems may be unfamiliar as they are played out in the realworld of commerce and politics. The commercial-scale production of GMP-grade material, required at the clinical-trials level, is not only




more expensive than the previous developmental steps but can also bemore difficult. Indeed, the transfer from the research laboratory to indus-try amounts to a real bottleneck, capable of making or breaking a vaccine

candidate. In fact, two of the most promising schistosomiasis vaccinecandidates had to be shelved for this reason (Bergquist and Colley,1998). In one case, sustained industrial production was not possible evenafter large amounts had been made in the laboratory and commercial-grade production had originally achieved production at the gram-level by the commercial partner. Another predicament, stressed by Lightowlers(2006a), is that the uncertain market potential of the product generallydashes the hope to attract the interest of bio-pharmaceutical companiesin the industrialized countries. However, there are real possibilities in

the developing, endemic countries as they have a vested interest inproducing vaccines for their own needs. For example, the vaccine produc-tion facility at the Research Institute of Tropical Medicine (RITM) in thePhilippines, which was established using a modular, turnkey approach forthe production of certified GMP-grade biological materials according toStandard Operating Procedures (SOP). At present, RITM is producingBCG to cover national needs. However, with this facility in place, addinga few more modules would not be an insurmountable barrier. Thus, multi-purpose industrial plants can be established in endemic countries, not

only for use as vaccine research/development laboratories but also for batch scale-up for clinical trials and, eventually, for full-scale vaccineproduction.

Vaccine development requires long-term commitment as well as sus-tained, high-level funding (Todd and Colley, 2002) and, as shown inFig. 10.1, the process is one of increasing risks. Once promising antigenshave been identified and tested in pilot studies, the researchers must learnto master laboratory large-scale production and focus on implementationof the vaccine in the field. At this point, the workload multiplies asactivities become more multifaceted requiring a different infrastructure,and when this has been put in place, the demanding phase of applied fieldstudies begins. Like the move from the bench to the field, the change fromexperimental approaches to industrial GMP-grade production of antigenmaterial is one of increasing complexity. In fact, there are steps involvedin the process (ringed in the figure) which are critical to the developmen-tal chain: for example without convincing, independent protection stud-ies the project must revert back to the bench and, if large amounts of standard material cannot be produced in a sustainable manner, the devel-opmental chain breaks and no further work is possible even with vaccinecandidates shown to be protective and overall strongly experimentallysupported. Finally, after safety and immunogenicity have been shown(Phase I), there is still no guarantee that the vaccine will prove effective inthe field (Phase II/III). However, when all is said and done, the fact that




we are entering this phase is testimony to a series of successes in thelaboratory and field which augur well for the future.

Clinical-trials are time consuming and constitute the most labour-intensive part of the whole process. A Phase I trial can be carried outwith limited staff and funding but the steps to follow require multifacetedactivities. Already at the Phase II level, the need for staff multiplies andresource consumption increases logarithmically, as this step entails vac-cination and follow-up testing of large numbers of people in an endemicarea. The even longer observation periods needed to follow the immuno-logical responses in the subjects participating in trials to show proof of efficacy (Phase III) may well hamper securing the funds needed. The finallevel (Phase IV) represents the follow-up of an already licensed productfor hidden side effects, an activity that requires even larger populationdata and goes on for a long time indeed. After that, regulatory authorities,involving many administrative levels, will have their say and the hurdlesto be overcome at this stage often require considerable additional input.The consequence is that the time from discovery to ready product takesdecades rather than years.

The situation regarding vaccines for the veterinary market are some-what less constrained than that of vaccines aimed to be used in humans.

Antigens andhost responses

Identification

Phase I

Field studies

Antigenproduction

Industrial-scale

Clinical trials

Vaccine development:

discovery to implementation

Main objective

Study areaSubjectsNumberTime

Endpoints

Safety

Nonendemic

Healthy adults> 20

About 3 months

Adverse effects

Laboratory-scale

Selection

Phase IV

Expenditure

Protection studies

Efficacy General impact

Endemic Endemic

Infected/healthy Infected/healthy>1000 Many thousands

Up to 5 years > 5 years

Immunogenicity

Endemic

Infected/noninf.>100

About 3 years

Immune responses Protection Long-term effects

Phase II Phase III

FIGURE 10.1 Many steps are involved in the process of vaccine development, most of

them more technically demanding (symbolized by larger script) than the previous one.

The three encircled stages in this process are critical, that is convincing protection in

animal models, ability to scale-up antigen production and showing impact in the field

(evidence of human protection).




This may explain the rapid progress of vaccines against E. granulosus,T. solium and F. hepatica. Rather than intended for prevention of infectionsin livestock per se, some vaccines against zoonotic parasites, notably that

against T. solium, have been developed to assist with the control of transmission of the human disease. Although animals rather thanhumans are the recipients of these vaccines, the main aim is not protectionof livestock but the control of human disease resulting from ingestion of infected meat. However, as these diseases occur primarily in the develop-ing world, vaccines against them are of little commercial interest. Forexample, the T. ovis vaccine was registered by the New Zealand AnimalRemedies Board in February 1994 (Rickard et al., 1995). In spite of the highefficacy of this vaccine, which has the capability to wipe out neurocysti-

cercosis if properly applied, the product has yet to be generally applied inthe endemic areas on a large scale.Table 10.1 summarizes the current situation in a way that illuminates

differences and similarities between vaccine-related activities in the vari-ous fields covered. An obvious difference is that progress has been par-ticularly strong in the field of schistosomiasis vaccines. This does notreflect that this is an easier parasite to work with but is rather a reflectionof the relatively large amounts of financial support available for thisparasite in the 1980s and 1990s. At this time, the UNICEF/UNDP/

World Bank/WHO Special Programme for Research and Training inTropical Diseases (TDR), the United States National Institutes of Health(NIH) and the Edna McConnell Clark Foundation (EMCF) (USA) ran largewell-funded research programmes; first on schistosomiasis and later ononchocerciasis. In addition, the United States Agency for InternationalDevelopment (USAID) and the Government of Egypt supported a 10-year general Schistosomiasis Research Project (SRP) continued by SVDP,a specific Schistosomiasis Vaccine Development Project (Bergquist andColley, 1998).

10.6. CONCLUDING REMARKS

Vector control and drug administration have reduced infection and dis-ease rates for many parasitic diseases significantly. However, as we arecurrently learning from the field of onchocerciasis, ivermectin’s selectiveactivity on microfilariae, the need for 10–15 years of annual treatments,logistical obstacles and the emergence of drug-resistant strains demandalternative strategies. This is also the case for schistosomiasis, in whichlarge-scale drug distribution is proving a stopgap solution which must befollowed up by the development of an integrated control process.

Helminthic diseases of both humans and animals tend to occur togethergeographically but have historically been targeted by disease-specific




TABLE 10.1 Overview of candidates for some helminth vaccines

Parasite Antigen discovery Industrial scale-up Phase I trial Phase

T. solium* >10

E. granulosus >20

E. multilocularis <10

Hookworm >10 1**

W. bancrofti >10

B. malayi >10

O. volvulus >10S. mansoni >100*** 3

S. haematobium >10***

S. japonicum <100***

S. mekongi ?

Food-borne infection <5

* Vaccine for pigs intended to benefit the human host by breaking the transmission cycle.** Two vaccine candidates upscaled.

*** Corresponding antigens exist in the various species; only a few are species-specific.



treatment programs which are now being integrated into a strategy of preventive chemotherapy. Although these programmes have made con-siderable progress reducing prevalence and intensity of disease, this

approach runs the risk not only of having to be continued indefinitely but also of inducing drug resistance without the long-term protection avaccine can offer as a complementary intervention.

A particularly interesting observation is that transmission-blockingvaccines developed for intermediate or reservoir hosts have largely beensuccessful, for example the pioneering T. solium vaccine initiative againsthuman cysticercosis is being emulated by a vaccine against S. japonicumtargeting the water buffalo reservoir. The fact that these veterinary vac-cines also contribute to creation of healthier animal herds is worth

highlighting.The successful vaccines against taeniases show that it is indeed possible to create a sterilizing vaccine against a multicellular parasite. Regret-fully though, it seems that, despite the availability of excellent vaccines,they will not be widely applied without a commercial incentive. This canonly be remedied by convincing international donor agencies and privatefoundations to support large-scale vaccination projects and/or by publichealth provision of vaccines through local production in the endemiccountries themselves.

The current accumulation of molecular data and expansion of parasitesequence databases is providing a fresh start by permitting a more ratio-nal approach to vaccine discovery. A range of new vaccines can beexpected during the next few years provided international donor organi-zations and private foundations can agree on a joint, major NTD vaccineinitiative. The task ahead is to assure product delivery by convincingpotential donors that vaccine production in the developing world is arealistic goal worth supporting.

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Bergquist 2010 Chapter 10 – Control of Important Helminthic Infections

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