Still hope for schistosomiasis vaccine

Abstract

Schistosomiasis is a parasitic disease caused by helminths belonging to the Schistosoma genus. Approximately 700 million people are at risk of infection and 200 million people are currently infected. Schistosomiasis is the most important helminth infection, and treatment relies solely on the drug praziquantel. Worries of praziquantel resistance as well as high disease burden are only some of the justifications which support the development of a vaccine against schistosomiasis. To date, only 2 schistosome vaccines have made it into clinical trials: Sh28GST (Bilhvax) and Sm14. However, there are several vaccine candidates, such as TSP-2, sm-p8, and Sm-Cathepsin B, which are generating promising results in pre-clinical studies. Schistosomiasis vaccine development has been an uphill battle, and there are still several hurdles to overcome in the future. Fortunately, the research groups involved in the research for vaccine development have not abandoned their work. Furthermore, in the last few years, schistosomiasis has garnered some additional attention on a global scale due to its significant impact on public health.

Keywords:

• clinical trial
• neglected tropical disease
• schistosomiasis
• public health
• vaccine

Introduction

Schistosomiasis is a fresh-water-borne parasitic disease caused by trematode worms of the genus Schistosoma. More than 200 million people are infected worldwide and approximately 700 million people are at risk of infection.^1,2 The World Health Organization (WHO) considers it the most important human helminth infection not only due to its high morbidity, but also due to its important impact on public health. The high disability rate is influenced by the various schistosomiasis associated conditions seen in patients; these include anemia, chronic pain, malnutrition, and developmental effects in children.^3,4 The infection is most prevalent in tropical and sub-tropical regions with the geographic distribution varying according to the species in questions. The pathology of the disease is due to parasite egg deposition in the tissues which will trigger an immune reaction and consequently cause progressive damage to the organs.

Treatment of schistosomiasis relies on the single drug praziquantel. Praziquantel kills the adult worms by increasing the calcium permeability of the tegument membrane; thereby, disrupting calcium homeostasis and causing paralysis of the worms.⁵ Exclusively relying on praziquantel raises concerns of potential drug resistance; there has already been some observed decreased praziquantel sensitivity in some strains in both Egypt and Senegal.⁶ The outcomes of schistosomiasis control programs have been overall disappointing.⁷ Decades of mass praziquantel administration has had negligible effects on the global prevalence of schistosomiasis. In fact, in certain regions, the disease has expanded to new foci.⁸ Low program coverage represents another issue threatening disease control. In 2011, of the 112 million children in need of praziquantel, only 16 million children received treatment.⁹ Less than 5% of the schistosomiasis affected population is treated with praziquantel. With such this low coverage, it will be difficult for any program to make significant contributions to schistosomiasis control. Since praziquantel does not prevent re-infection, drug administration needs to be constant in order to maintain disease control. This would require continuous support from donors. Vaccines or at least chemotherapy combined with vaccination, present the best strategy for long-term sustained control of schistosomiasis. Since the parasites do not replicate within the human host, even a partially effective anti-schistosome vaccine could significantly contribute to morbidity reduction and disease elimination.^10,11 A multi-component, integrated control program is likely the best option, and could include approaches such as vaccination, drug administration, environmental modifications, improved sanitation, and intermediate host control by molluscicides.

Schistosomiasis Vaccine Development

New antigen discovery has been aided by the major advances in schistosome molecular biology; the genome, the transcriptome, and the tegument proteome. The genomes of S. mansoni and S. japonicum were sequenced in 2009 followed by the genome of S. haematobium in 2012.^12-14 The accessibility of this information in combination with the improving postgenomic technologies has the potential to identify plenty of new vaccine candidates.. The most relevant targets are the excretory/secretory products as well as molecules on the surface of the worm. These are the molecules that constantly and directly interact with the host immune system.

One of the most important contributions in antischistosomal vaccine studies was the radiation attenuated cercariae vaccine (RA vaccine). Studies demonstrated that a single immunization with radiation attenuated cercariae can attain protection levels of 60–70% in animal models, and these levels can be increased further with repeated immunizations.^15-18 The findings from RA vaccine studies provided crucial knowledge concerning the induction of protective immunity. The RA vaccine is a proof of concept that has paved the way for the development of molecular vaccines against schistosomiasis. These encouraging results along with major advances in molecular technologies prompted the WHO, approximately 20 y ago, to commence the testing of the most promising vaccine candidates. In order to be considered significant, protection levels needed to reach the 40% threshold established by the WHO.¹⁹

Recombinant glutathione S-transferase cloned from S. haematobium (Sh28GST) was the first of only 2 schistosome antigens to enter into clinical trials. The vaccine named Bilhvax is formulated with an alum hydroxide adjuvant. In the pre-clinical studies, the vaccine significantly inhibited female worm fecundity and egg viability.²⁰ The Institut Pasteur and the French Institute National de la Santé et de la Recherche Médicale took Bilhvax through both Phase 1 and 2 clinical testing September 1998 marked the start of the Phase I clinical trials in Lille. The volunteers did not experience any adverse effects and no cross-reactivity was observed with the human GST(Pi).²⁰ The Phase Ib trials were carried out in St. Louis, Senegal known to be endemic. The trial consisted of 2 groups of 12 healthy African children, between the ages of 6 and 10 y old. The children received 2 injections and they tolerated the vaccine with no sign of adverse effects.²⁰ Bilhvax was being marketed as a pediatric vaccine. Unfortunately, no new information has made available about the status of the vaccine; thereby, rendering specialists in the field skeptical of its future.

The S. mansoni fatty acid binding protein Sm14 is the second candidate to enter clinical trials. In mice, the recombinant Sm14 protein generated protection levels of 67% against S. mansoni challenge, and complete protection against Fasciola hepatica challenge.²¹ A vaccine formulated with Sm14 would have the potential to provide protection against 2 parasitic diseases, but also prevent economical losses caused by veterinary fascioliasis which causes annual losses of over USD 3 billion to the livestock industry.⁸ The Sm14 clinical trials are the result of an initiative between Oswaldo Cruz Foundation (FIOCRUZ), the Brazilian governmental financial agency (FINEP), and Alvos Biotecnologia.⁸ The Phase I clinical trial commenced in March of 2011, in Brazil. This initial phase is meant to determine the safety and tolerability of the vaccine composed of Sm14 with glucopyranosyl lipid adjuvant (GLA) in healthy adult volunteers.²² The next step will be to perform Phase II trials in Brazil and Egypt.

Although Sh28GST and Sm14 are the only schistosome antigens to have entered into clinical trials. However, there are currently several other vaccine candidates which are generating promising results in pre-clinical studies and have the potential to enter into clinical testing in the near future. The S. mansoni tetraspanin extracellular loop 2 (_EC-2) of Sm-TSPS-2 has been shown to protect mice from a schistosome challenge. Mice immunized with the recombinant _EC-2 of Sm TSP-2 plus Freund complete (prime) or Freund incomplete (2 boosts) adjuvant have a 57% reduction in worm burden and a 64% reduction in hepatic egg burden compared to control mice.²³ The promising results seen with _EC-2 of Sm-TSP-2 prompted the Human Hookworm Vaccine Initiative (HHVI) to select _EC-2 of Sm-TSP-2 for development as a human vaccine candidate.¹⁹ HHVI is a group dedicated to the development of vaccines against helminth infections. Tetraspanins have already been used to generate veterinary vaccines; thereby, providing an encouraging precedent for this type of antigen.²⁴ The promising pre-clinical data in combination with the successful scale-up expression of the antigen suggest that Sm-TSP-2 is ready for Phase I trials.²⁵

The S. mansoni calpain large subunit, Sm-p80, another vaccine candidate is generating promising results in pre-clinical studies. Worm burden and hepatic egg burden were reduced by 70% and 75%, respectively, in mice immunized with a formulation of recombinant Sm-p80 and CpG dinucleotides compared to controls.²⁶ The promising results observed in the mouse model, prompted the group to continue their experiments in a nonhuman primate model; the baboon. Experimentally immunized baboons had a 58% decrease in worm burden compared to the controls.²⁷ These levels of protection have previously never been seen with a schistosome antigen in large animal models. Furthermore, in addition to prophylactic efficiency, the Sm-p80 immunizations could also induce killing of established adult worms and protect against urinary schistosomiasis caused by S. haematobium.²⁸

Our group has chosen to target S. mansoni Cathepsin B (Sm-CB) as a vaccine candidate. Immunization of mice with Sm-CB adjuvanted with CpG oligodeoxynucleotides conferred a 59% decrease in worm burden. Hepatic and intestinal egg burdens were decreased by 56% and 54% respectively compared to control groups.²⁹ Sm-CB formulated with Montanide ISA 720 VG decreased worm, hepatic egg, and intestinal egg burdens by 60%, 62%, and 56% respectively compared to control groups. Antibody production was significantly augmented in the vaccinated mice; both formulations elicited Sm-CB specific total immunoglobulin G endpoint titers that were greater than 120,000. Furthermore, analysis of cytokine secretion levels revealed that immunization with Sm-CB plus CpG resulted in a T-helper (Th) 1 biased response whereas immunization with Sm-CB plus Montanide led to a mixed Th1/Th2 response. Our results highlight the potential of Sm-CB as a strong vaccine candidate against schistosomiasis.

There are also several S. japonicum antigens which are undergoing pre-clinical studies for vaccine development; both DNA vaccines and recombinant protein vaccines are being tested. There is an added level to consider when researching vaccine development for S. japonicum; zoonotic transmission from reservoir hosts. For this schistosome species, there is the possibility of developing a transmission-blocking veterinary vaccine. Most of the S. japonicum antigens being tested are membrane proteins, muscle components, or enzymes. Examples of some vaccine candidates include Sj26GST, Paramyosin (Sj97), S. japonicum very low-density lipoprotein binding protein (SVLBP), Serpins, and S. japonicum triose-phosphate isomerase.³⁰

Hurdles To Overcome

The introduction of praziquantel had a significant impact on schistosomiasis control. Mass administration of praziquantel resulted in never before seen reductions in morbidity, and thereby creating an illusion that schistosomiasis was a disease of the past. In consequence, research for a schistosomiasis vaccine suffered from a lack of funding due to the misconception that this disease was not an urgent public health issue. This lack of interest from the scientific community presented a major hurdle in the development for a vaccine against schistosomiasis as such research is costly. The funding situation has slightly improved over the last years greatly due to the reassessment of its effect on the global burden of disease and the inability of praziquantel to disrupt disease transmission.³⁰ Furthermore, the schistosomiasis vaccine field has recently garnered attention as a result of a Gates Foundation grant of USD 2.85 million. The grant was awarded to Dr. Afzal A. Siddiqui of Texas Tech University Health Sciences Center to support a proof of concept trial of Sm-p80/GLA-SE schistosomiasis vaccine.³¹

Human correlate studies have represented a major step forward in the development of an anti-schistosome vaccine. These studies revealed that a balance between positive and negative responses rather than biased responses correlate with resistance to infection. This lack of a straight-forward response has rendered the testing of potential vaccine antigens difficult to standardize. Independent testing has revealed that certain schistosome antigens require Th2 responses for protection whereas others require Th1 responses.^33–36 Once the antigen has been selected, one must consider formulation. The adjuvant used is important for the stimulation of the desired immune response. Several adjuvants have been tested for schistosomiasis vaccine formulation: CpG dinucleotides, alum, Freund's complete/incomplete adjuvant are just a few examples. The adjuvant selection is one of many factors that influence the results of pre-clinical studies. Other factors include the animal model, the vaccination schedule, the vaccine delivery route, and the dose for parasite challenge. A standardized method to test upcoming antigens is necessary in order to obtain consistently reproducible data. Next, we must ask whether our method of testing one antigen at a time is correct. Developing an anti-parasitic vaccine is not a simple task. These pathogens are complex and, they have developed an arsenal of mechanisms to evade our defenses and establish an infection. Studies developing vaccines against other parasitic infections, such as malaria and leishmania, suggest that an optimal anti-parasitic formulation might be composed of more than a single antigen and a defined adjuvant.^37,38 Perhaps testing multi-antigen vaccine formulations earlier on in pre-clinical trials may accelerate the discovery of the most effective composition.

Once the vaccine candidate has been selected and shown to elicit high levels of protection in animal models, it must be produced with good manufacturing practices (GMP) and in large scale; if it cannot, it is of little value. Some very promising antigens, such as paramyosin and MAP4, have been abandoned because they could not pass this rate limiting step.¹⁰ This implies that we might have to compromise antigen efficacy for the ability to be scaled-up with GMP. It is important not to overlook the importance of the production process as it can be as challenging to navigate through as the initial antigen discovery and pre-clinical stages. Furthermore, a schistosomiasis vaccine would be delivered to some of the poorest populations; therefore, it would need to be produced cheaply. A schistosomiasis vaccine would require costing below USD 1–2 per dose, which restricts the use of certain techniques and equipment.¹⁹ Therefore, the focus should be on expressing the proteins of interest in low cost yeast and bacterial expression vectors such as Pichia pastoris and Escherichia coli.

Schistosomiasis vaccine development is tedious and costly. Maintaining the life cycle of the parasite requires the appropriate housing of the intermediate snail host. The pre-clinical phase usually involves immunological and protection studies carried out in the mouse model. There are worries that the mouse model is too dissimilar from human schistosomiasis, and that results from vaccine pre-clinical studies cannot translate to real life applicability. One of the major concerns stems from the fact that laboratory animals living in a controlled environment are being used test a vaccine that will be administered to populations living in schistosomiasis endemic regions. These models do not take into account co-infections. The majority of individuals infected with a schistosome species also suffer from malaria, HIV, or soil-transmitted helminth infections.^39-41 The laboratory mice are pathogen free; therefore, these studies do not take into account an underlying infection's effect on vaccine efficacy. Furthermore, individuals living in schistosomiasis endemic regions have likely already been infected and treated with praziquantel at least once in their lives. Usually, pre-clinical studies do not analyze vaccine success in previously infected mice that were cured with praziquantel. These different real life scenarios highlight the complexity of introducing a new vaccine in endemic settings. Furthermore, it is important to learn lessons from the ASP-2 based hookworm vaccine trial. The vaccine was safe and well tolerated in US volunteers. However, once introduced in a hookworm endemic region of Brazil, the vaccine elicited immunoglobulin-E (IgE) mediated allergic responses in the volunteers; thereby, halting the clinical trial.^42,43 These individuals had parasite specific IgE antibodies which had developed from past infections. The unfortunate outcome of this study needs to be kept in mind when developing a schistosomiasis vaccine. It is essential to perform human correlate studies in order to test a candidate antigen's reactivity with IgE in serum samples from high-risk endemic populations. In terms of better models for vaccine efficacy, baboons would be a good choice because they develop a disease which is similar to that seen in humans.^44,45 However, they are significantly more expensive than mice and they require more extensive maintenance. Furthermore, there are more ethical hurdles from institutional review boards and regulatory agencies to overcome when conducting studies on baboons.

Summary

Schistosomiasis affects a large portion of the World's population and many more are at risk of infection. Due to its mortality and impact on public health, Schistosomiasis is the most important helminth infection; yet, it is a neglected tropical disease. The development of a vaccine against schistosomiasis has the potential to contribute a long-lasting decrease in disease spectrum and transmission. A vaccine would lessen the morbidity of the disease by decreasing worm burden and egg production. Furthermore, used in combination with praziquantel, it could prolong the intervals between required drug treatments. The feasibility of developing a vaccine against schistosomiasis is supported by convincing arguments. For instance, immunization of mice with a single dose of cercarial RA vaccine induces high levels of protection. Further supporting evidence includes the effective development and application of veterinary recombinant vaccines against parasitic worms.²⁹ Bilhvax and Sm14 are the only antischistosome vaccines to have entered clinical trials. Unfortunately, little information has been made available concerning the results generated from the different phases. There are currently several antigens that are in the research pipeline and are advancing in pre-clinical trials. This growth in new antigen discovery is the result of advances in schistosome molecular biology. Based on their pre-clinical data, TSP-2, Sm-p80, and Sm-CB appear to be promising vaccine candidates. The vaccine formulations based on these antigens are being refined, and hopefully, they will enter into clinical trials in the near future. Once the technical and economical hurdles of vaccine development are surpassed, the next step would be to determine the best way to make the vaccine globally accessible.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

The National Reference Center for Parasitology is supported by Public Health Agency of Canada/National Microbiology Laboratory grant MOA 4500299739, the Foundation of the Montreal General Hospital and the Research Institute of the McGill University Health Center.