Final Report Summary - HOOKVAC (Developing and Testing a novel, low-cost, effective HOOKworm VACcine to Control Human Hookworm Infection in endemic countries)
Executive Summary:
The HOOVAC project was implemented by a consortium of scientists from 9 academic institutions and companies in the EU, the USA and Africa. This consortium was the first and only in the world developing a vaccine for human hookworm infection. The HOOKVAC project was intended to contribute to the development of a candidate vaccine against hookworm infection, which ranks number one in terms of years lost from disability from a neglected infectious disease. Within HOOKVAC, the consortium worked on developing a bivalent, low-cost vaccine candidate was clinically tested for the first time in an African disease endemic population.
Previously, from more than 20 potential target proteins involved in the hookworm blood feeding process, two lead proteins have emerged as promising candidate antigens for further clinical development, based on preclinical efficacy testing and favourable human immunoepidemiology: Necator americanus-glutathione-S-transferase-1 (Na-GST-1) and Necator americanus- Aspartic- Protease-1 (Na-APR-1). These antigens from adult hookworms have proven to be safe and non-allergenic as opposed to antigens targeting larval stages which have resulted in significant adverse events. Therefore, both antigens are excellent candidates for vaccine development. Since neither recombinant Na-GST-1 or Na-APR-1 alone have produced complete sterilizing immunity in laboratory animals, it is hypothesized that including both antigens in a vaccine will have a greater chance of success at producing an effective vaccine for humans than either alone. By targeting different steps in the blood digestion pathway of the adult hookworm, the effect of combining them in a bivalent vaccine is expected to be at least additive, if not synergistic.
The HOOKVAC project addressed four main objectives while testing Na-GST-1 and Na-APR-1 as vaccine candidates.
1. Establish safety and immunogenicity of the vaccine candidate in an endemic population;
2. Improve the manufacturing process;
3. Provide clinical proof of concept; and
4. Improve accessibility of the vaccine in endemic areas (sub-Saharan Africa, Southeast Asia and Latin America).
The HOOKVAC program consisted of a series of technical work packages, of which WP2 included a series of clinical trials in a high burden country (Gabon), where both adults as well as children were included in clinical trials to test the safety and efficacy of the vaccine.
The HOOKVAC project delivered the results of the first phase 1 clinical trial conducted in sub-Saharan Africa of two novel Na-APR-1 and Na-GST-1 vaccine candidates against human hookworm infection, It also delivered for the first time, safety and immunogenicity data of Na-APR-1 administered to individuals that are resident in a Necator endemic area.
Co-administration of these candidate hookworm vaccines were observed to be safe and well tolerated when tested in healthy adults and children from this hookworm-endemic region of Gabon. Antigen-specific IgG antibodies were induced to both vaccine antigens in the first study with adult patients. Results on antigen-specific IgG antibodies to children and using a different adjuvant are expected by the end of the summer of 2019.
In WP3 and WP4 scaling of production capacity was successfully investigated. In WP5, the immune responses were studies in-depth in order to get a better understanding on how the parasite interacts with the host immune system. In WP6, we build upon Involving knowledge of end-user communities and health professionals as well as active engagement with advocacy and policy leaders.
The HOOKVAC project has contributed to the development of a human hookworm vaccine, which is a crucial tool on the path to eliminating the disease, as well as a means to reduce poverty, particularly in developing economies. Hookworm infection has been identified as a major cause of poverty in low- and middle-income countries and because of hookworm's potential for additive or synergistic effects on other co-infections of overlapping geographic distributions, thus improving the overall public health economic development in endemic economies. The antigens that have tested in the HOOKVAC product can thus ultimately result in a true anti-poverty vaccine.
Project Context and Objectives:
Human hookworm infection is a neglected tropical disease caused predominantly by the nematode parasite Necator americanus. Recent estimates indicate that approximately 439 million people are infected with hookworm worldwide, with the majority of cases found in the developing regions of South Asia (140 million cases), Sub-Saharan Africa (118 million), Southeast Asia (77 million), East Asia (64.5 million), and the Latin American and Caribbean region (30 million). In these areas, hookworm disease is a major cause of iron-deficiency anemia, a consequence of the adult hookworm's ability to extract blood from the intestinal mucosa and submucosa. The Global Burden of Disease Study 2010 (GBD 2010) estimated that hookworm is responsible for a loss of 3.2 million disability adjusted life years (DALYs), making it one of the leading neglected tropical diseases (along with schistosomiasis and leishmaniasis) in terms of disease burden and a leading cause of anemia in large parts of Africa. Hookworm infection occurs when the larval stages penetrate the skin of a human host. The primary adverse effect of infection, anemia, disproportionately occurs in children and pregnant women with lower iron reserves compared to other populations. Hookworm is hyperendemic among some pediatric populations in sub-Saharan Africa where in countries such as Sierra Leone or Togo one-third of the population under the age of 20 is infected. Children with chronic hookworm infection experience anemia and cognitive and developmental delays with resultant reductions in school performance, attendance and future wage earnings. Approximately 7 million pregnant women in sub-Saharan Africa – almost one third of annual pregnancies in Africa – are also infected, making hookworm disease one of the most common complications of pregnancy in that part of the world. Moreover, many of these individuals – both children and pregnant women – are co-infected with malaria, thereby exacerbating anemia and its sequelae.
The primary approach to hookworm control is mass drug administration with the anthelminthics albendazole or mebendazole. However, a single dose of mebendazole, administered once per year, has yielded low cure rates, particularly with repeated use. One comprehensive meta-analysis showed no impact of mebendazole treatment on improving anemia in hookworm-affected communities. Similarly, for albendazole, given as a single dose, once per year, drug failure has been reported, though less often. Moreover, children can re-acquire hookworm several months after treatment, particularly in geographic locations with significant infectivity or attack rates. These observations may explain a recent finding from GBD 2013 study that overall global hookworm prevalence has remained essentially unchanged over the last 20 years, while the prevalence of other neglected tropical diseases such as lymphatic filariasis, ascariasis, and trachoma has decreased by 25–33% over the same time period.
There is, therefore, a need for new technologies to achieve better control of hookworm infection. A safe and effective anti-hookworm vaccine, as a complement to conventional chemotherapy, may provide a cost-effective means of reaching this goal.
From more than 20 potential target proteins involved in the hookworm blood feeding process, two lead proteins have emerged as promising candidate antigens for further clinical development, based on preclinical efficacy testing and favorable human immunoepidemiology: Necator americanus-glutathione-S-transferase-1 (Na-GST-1) and Necator americanus- Aspartic- Protease-1 (Na-APR-1). These antigens from adult hookworms have proven to be safe and non-allergenic as opposed to antigens targeting larval stages which have resulted in significant adverse events. Therefore, both antigens are excellent candidates for vaccine development, as they (i) target adult N. americanus worms and (ii) have been shown to partially protect laboratory animals against challenge infection with hookworm.
In the HOOKVAC project, these 2 selected antigens were further tested, with the following four main objectives:
1. Establish safety and immunogenicity of the vaccine candidates Necator americanus-glutathione-S-transferase-1 (Na-GST-1) and Necator americanus- Aspartic- Protease-1 (Na-APR-1) in an endemic population;
2. Improve the manufacturing process of Na-GST-1 and Na-APR-1;
3. Provide clinical proof of concept; and
4. Improve accessibility of the vaccine in endemic areas (sub-Saharan Africa, Southeast Asia and Latin America).
Project Results:
The HOOKVAC program was designed through a series of 6 work packages, of which the 1st work package was coordination the project. The S&T objectives were divided over a series of 5 work packages (WP2-WP6)
Clinical Studies (WP2)
Since neither recombinant Na-GST-1 or Na-APR-1 alone have produced complete sterilizing immunity in laboratory animals, it is hypothesized that including both antigens in a vaccine will have a greater chance of success at producing an effective vaccine for humans than either alone. By targeting different steps in the blood digestion pathway of the adult hookworm, the effect of combining them in a bivalent vaccine is expected to be at least additive, if not synergistic. As a single formulation vaccine of these 2 antigens was not available at the start of the project, HOOKVAC initiated Phase I clinical testing in which the separately-formulated antigens were co- administered first in adults (HV-01) and then in school-aged children (HV-02) to validate the safety and immunogenicity of these antigens and explore the optimal doses and vaccination schedule in an endemic African population. Finally, a HV-03 study was designed to assess the safety and immunogenicity of co-administered Na-GST-1 and CPG 10104 (a human TLR-9 stimulator) in an adult African population resident in a hookworm-endemic region, in order to establish the possibility of scaling back to one antigen for further clinical studies.
The HV-01 study, was a randomized, controlled, double-blind Phase 1 trial was conducted in 32 healthy adults in N. americanus endemic areas of Gabon. Participants were randomised to receive three doses of either co-administered Na-GST-1 plus Na-APR-1 (M74) (30µg or 100µg of each), adjuvanted with Alhydrogel® (aluminium hydroxide gel suspension) together with an aqueous formulation of glucopyranosyl lipid A (GLA-AF) or the hepatitis B vaccine as a comparator. Vaccinations were administered on days 0, 28, and 180. Co-administration of Na-GST-1/Alhydrogel® and Na-APR-1 (M74)/Alhydrogel® was well tolerated at both doses tested. The most common adverse events were mild-to-moderate injection site pain, headache, myalgia, and nausea. No severe or serious adverse events related to the vaccines were recorded. IgG antibodies were induced to each of the vaccine antigens, with increasing IgG levels observed after each vaccination. Vaccination with 100µg of each vaccine antigen with GLA-AF consistently induced IgG sero-conversion. The peak of the IgG response was observed two weeks after the third vaccine administration for both vaccine antigens. Study HV-01 therefore showed that vaccination with recombinant Na-GST-1 and Na-APR-1 (M74) in healthyadults resident in Necator endemic areas of Gabon was safe and induced increasing levels of IgG to each antigen after each vaccine administration. This manuscript presents the first description of Na-APR-1 (M74)/Alhydrogel® in participants from a Necator endemic area. Further clinical development of these vaccines should be advanced with efficacy studies.
The HV-002 study was designed as a double blind, randomized, controlled, dose-escalation Phase 1 clinical trial in hookworm-exposed children aged 6 to 10 years living in the area of Lambaréné, Gabon. Children received three doses of the assigned vaccine(s) delivered intramuscularly (deltoid) on approximately Days 0, 56, and 112 or 180.
In total, 137 children were screened. Out of them, 77 screened failed for a range of reasons including Informed consent withdrawn (05), Hepatitis B vaccine received before (16), Participation in other clinical trials (02), planning to move out from a study area (12), unhealthy (37), positive for Hepatitis B or C virus (03), assent form not provided (02). During the follow-up, the study team reported one unrelated SAE (right inguinal hernia) in participant belonging to the pediatric trial. No vaccine related serious adverse event were reported during the trial.
The team concluded that the co-administered Na-GST-1 and Na-APR-1 vaccine candidate in children living in hookworms endemic area in Gabon were found to be safe and well tolerated. Further serum analysis will assess their immunogenicity, these results will become available at the end of the summer period of 2019, and will be reported separately to the Commission.
In the original design of WP2, we had envisioned that the vaccine product in trial HV-03 would be a bivalent vaccine containing a mix of the 2 antigens that were tested as co-administered products in studies HV01 (adults) and HV02 (children).
Unfortunately, the work conducted under WP3 and WP4 demonstrated that the co-formulation of Na-APR-1 and Na-GST-1 using the backbone of the currently used buffer formulations was not feasible. The alternative of conducting the study with the individual antigens would NOT yield data that are sufficiently different from what we had learned from HV-01, which is described above.
Meanwhile, the Na-GST-1/Alhydrogel hookworm vaccine has been tested in combination with CPG 10104 in one Phase 1 clinical trial in healthy adults in the United States (n=24). In study SVI-GST-03 in healthy, hookworm-naïve American adults initiated in 2014 in Washington, DC, 24 volunteers received three vaccinations (on study days 0, 56 and 112) with up to 100 µg Na-GST-1/Alhydrogel administered with or without up to 500 µg CPG10104 (NCT02143518). Anti-Na-GST-1 IgG antibody responses have been assayed up to one month following the third vaccination, and indicate that the addition of CPG 10104 to Na-GST-1 results in significantly improved humoral immune responses to the vaccine antigen.
Based on these findings, the design of study HV-03 was modified such that it studied whether the same improved vaccine response can be generated in an adult population that lives in a hookworm endemic country. If this would the case, possibly a single antigen based vaccine could be developed, which would be a crucial alternative clinical development strategy in case we do not solve the current technical hurdles in producing a bivalent vaccine
The HV-003 study was therefore set-up in twenty-four eligible adults that were progressively enrolled into 1 of 2 groups over a projected 2-month enrolment period, with each participant followed for 9 months after the final injection. Group enrolment was done in an open sequential fashion, whereas within each Group, investigational product (IP) assignment and vaccination was done in a randomized double-blind fashion. The first 12 subjects was recruited and enrolled into Group 1: (A) 8 subjects received 30 µg Na-GST-1 plus 500 µg CPG 10104 delivered by IM injection in the deltoid muscle according to a 0,2,4-month schedule, and (B) 4 subjects received 100 µg Na-GST-1 delivered by IM injection in the deltoid muscle according to a 0,2,4-month schedule. In Group 2 (A) 8 subjects received 100 µg Na-GST-1 plus 500 µg CPG 10104 delivered by IM injection in the deltoid muscle according to a 0,2,4-month schedule, and (B) 4 subjects received 100 µg Na-GST-1 delivered by IM injection in the deltoid muscle according to a 0,2,4-month schedule. During the follow-up, 2 cases of pregnancy have been reported in each cohort and one resulting in miscarriage in the cohort 1 and 2 suspensions of the vaccine administration after both participants have received their first immunization. No vaccine related serious adverse event were reported in the trial. The conclusion from the study is thus that co-administered Na-GST-1 with CPG 10104 vaccines in adults living in hookworms endemic area in Gabon were found to be safe, well tolerated. Further serum analysis will assess their immunogenicity.
WP3 scaling the production platforms to the level needed for subsequent studies
WP3 was targeted toward scaling the production platforms for both antigens. We purified Na-GST and Na-APR antigens which showed biochemical and immunological comparability to the reference materials that were used in earlier studies.
With regards to Na-GST-1, this scaled production process resulted in over 95% pure intact Na-GST. A similar purity of Na-GST was thus obtained for the batches of development and engineering run, confirming the robustness of the developed purification process. The Na-GST produced under WP3 is similar to the reference material and meets the specifications determined in the monograph with respect to the identity, purity, pH, protein concentration, endotoxin content, total contaminating host cell proteins and residual total DNA. The fermentation is now at a scale of 10-15 litres. A Pichia pastoris working cell bank has been produced starting from a vial of the Sabin master cell bank. Na-GST has been purified in cGMP from the harvested micro-filtered medium according the scalable downstream process and fulfilled specifications as described in the Monograph. A stability study showed that the over 90% pure Na-GST was stable for at least 12 months at below minus 60°C. A comparable in vivo potency was observed for the alhydrogel adsorbed Na-GST obtained by the new scalable and reference process. WP3 has thus delivered a scalable downstream process of P. pastoris Na-GST-1. CMC-reports of the cGMP–manufactured Na-GST batch and the report of the one- year Na-GST stability study have been prepared.
Production of NA-APR-1 has proved a lot more challenging. Before the start of the HOOKVAC program, Na-APR-1 has been produced as a recombinant His-tagged protein with ER retention signal (KDEL) in tobacco plants, the according expression levels are very low. Only milligram quantities have been obtained out of kilograms of plant extracts. One of the major problems is the tendency of the protein to form aggregates. Moreover, for further use as an injectable, it is recommended to produce the Na-APR-1 protein without His-tag.
To this end, an E.coli strain was therefore constructed expressing APR-1 as a cytosolic protein (GI724/M-APR-1). Biochemical analysis of the purified E. coli M-APR pool showed that the current purification method consisting of 2 consecutive anion exchange chromatography steps, had resulted in an at least 92% pure monomeric full size M-APR and that the residual HCP are below 3% (based on anti-E. coli WB). QC analysis showed that the product meets specifications. The overall yield of M-APR is over 30 mg per liter fermentation equivalent, while formation is now at the same level as for Na-GST-1. Stability studies at 3, 6, 9 and 12months showed that the over 90% pure M-APR-1 was stable for at least 12 months at 2-8°C and at below minus 60°C.. Unfortunately, initial formulation experiments showed that Empigen concentrations which is needed for the stability of APR-1 even at 0.01% (w/v) causes structural changes in the alhydrogel-bound Na-GST-1. The M-APR and empigen concentration were respectively 200µg/ml and 0.25% (w/v) in the first generation Drug Substance. These combined data suggest that co-formulation of M-APR and Na-GST1 antigens would only be possible after adaptation of the M-APR purification process. This prompted a change of the scope of WP4.
WP4 towards the production of a stable bivalent hookworm vaccine
WP4 was originally targeted towards the production of a stable bivalent hookworm vaccine product. It was originally envisioned that we would engage in the development of a formulation process was described allowing co-formulation of the two antigens on Alhydrogel. Such a co-formulated bivalent hookworm vaccine would be more convenient to administer (single injection). Thus would improve subject compliance, significantly reduce vaccine manufacturing costs and simplify logistics related to distribution and storage.
While in WP3 we have shown that the purified Na-GST and Na-APR antigens that were produced showed biochemical and immunological comparability to the reference materials that were used in earlier studies, the initial co-formulation of Na-APR-1 and Na-GST-1 using the backbone of the currently used buffer formulations is not feasible. Specifically, the presence of Empigen BB in the buffer – which is vital to the stability of the Na-APR-1 monomer - impairs the ability of incorporating other molecules such as GST to the mix. When presenting these data to the scientific and ethical advisory board of HOOKVAC, in which representatives with pharmaceutical expertise are engaged, it was advised that such early formulation hurdles are not uncommon, and should definitely not be regarded as a showstopper. Typically, once a (vaccine) product has been optimized in terms of generated the desired immunogenicity, enhanced formulation activities can still lead to an optimized vaccine in terms desired formulation. The required expertise is typically found in the pharmaceutical industry. The required investments from the pharmaceutical industry are more likely to be recruited if the prototype antigens have successfully completed the planned clinical studies. Meanwhile, it was felt that additional pre-formulation and formulation studies could enhance the likelihood of finding a suitable formulation technology. To this end, alternative excipients were identified and screened. Alternative ‘in-solution’-stabilizing excipients and buffer conditions (type, pH) were tested for the alhydrogel- adsorbed antigens. These excipients minimized the destabilizing effect of Empigen, up to 0.016%. GSH had again the strongest stabilizing effect (i.e. strong increase in melting point) and the data favored the formulation at a pH below 7.0. The replacement of Empigen by Tween 20 as detergent in the elution of the polishing step and the increased concentration in the eluate indeed now opens perspectives for the coformulation and development of a prototype Hookworm vaccine based on GST-1 and M-APR-1.
WP5 Towards an understanding of immunological responses to the vaccine
In this workpackage, we characterized the T cell responses in the vaccinated volunteers of study HV-01. This was measured by stimulating PBMC with recombinant Na-GST and Na-APR-M74, at pre (day 0) and after first (day 28) and third (day 180) vaccination and assessed intracellular cytokine (IL2, IL4/IL5/IL13, IL10, IFNγ and TNF) responses. Such data can provide a better characterizing the target population (exposure, pre-existing immunity and coinfections) in order to determine the optimal dose needed to stimulate an effective immune response to a vaccine.
In the HV-01 study, a significant increase in Na-GST specific CD4+ IL2+ and CD4+TNF+ cells as well as cells co-expressing IL2 and TNF was seen after the third vaccination with the high dose vaccine. The vaccination with Na-GST and Na-APR in HV-01, resulted in antigen specific induction of IL2 and TNF expressing T cells, that was seen in the group that received the high dose vaccine. Over the vaccination course an increase in IL2 and TNF was observed with the highest cytokine secretion seen at day 194. However, there was no correlation between the antibodies and the cytokines responses over the vaccination course. Indeed, some participants with high Abs titers had no detectable cells in peripheral blood producing cytokines in response to in vitro stimulation with vaccine candidates. This could be due to the sensitivity of the assay or the residence of the antigen specific T cells in peripheral tissues such as in lymph nodes.
The increased IL2 response following the vaccination schedule, could promote the expansion of effector T cells that can help B cells antibody production. The enhanced TNF response, which is generally related to an early type 1 response, is also known to help the humoral immune response as TNF blockade has been shown to be associated with antibody titer reduction and therefore reduced responses to vaccination. Earlier studies have indicated that multifunctional T cells might play an important role in the immune response to vaccines in general. In our study the magnitude of double cytokine (TNF+IL2+) producing CD4+ T cells was significantly increased from day 0 to day 194, which might suggest that these cells play a role in Na-GST immunogenicity.
In contrast, the vaccination did not result in Na-APR specific CD4+ cytokine producing cells even though detectable antibodies to this candidate was seen following vaccination albeit to a lesser extent than what was seen for Na-GST antibodies. This is in line with the lower sensitivity of detecting immunogenicity based on intracellular cytokine producing CD4+ cells in peripheral blood compared to detection of antibodies. It is possible that higher doses of Na APR are needed for improved antibody responses.
A low cytokines response to APR antigen that was seen at baseline as well as in the group vaccinated with HBV but this was not boosted over time by the vaccines given. Participants included in this trial were hookworm- exposed subjects and a pre-existing response to Na-APR might prevent a response to be mounted to Na-APR. It is also possible that other hematophagous parasites such as malaria parasites express APR. Interestingly, we noticed that the participant with the higher Na-APR antibody titers were positive for malaria before inclusion into the study. We therefore hypothesize that the potential pre-existing immunity to Na-APR due to a pre-exposure or cross reaction with malaria APR, is responsible for the low cytokines response observed at baseline.
WP6 Global Access Strategy
During the HOOKVAC program, a wide portfolio of external activities was initiated, including the press/media engagement, organized meetings and position statements. Several consortium members have led numerous speaking engagements and presentations to a wide array of audiences highlighting the HOOKVAC consortium and increasing awareness of the need for hookworm vaccine development. Further details are provided in the dissemination section of this report.
Potential Impact:
More than 400 million people worldwide are infected with hookworm (1), with the greatest number of cases occurring in sub-Saharan Africa, Southeast Asia, China, and tropical regions of the Americas. Of the two species that infect humans, Necator americanus is the most prevalent, causing 85% of infections. The clinical hallmark of hookworm is iron deficiency anemia (IDA) that results from intestinal blood loss caused by adult parasites feeding at their site of attachment in the small intestine. Currently, the major approach to the control of pediatric hookworm is the annual mass drug administration of a benzimidazole anthelminthic drug such as mebendazole or albendazole. However, widespread mebendazole drug failure for the treatment of hookworm and high rates of post-treatment re-infection with both drugs justify the need to develop a vaccine.
The Human Hookworm Vaccine (HHV) is being developed as a bivalent product containing the Na-GST-1 and Na-APR-1 recombinant proteins, with the indication being the prevention of moderate and heavy hookworm infections caused by N. americanus. The candidate antigens were selected for clinical development based on their protective efficacy in animal trials and immunoepidemiological studies in individuals resident in hookworm-endemic areas (2). Children living in N. americanus endemic regions of Africa, Asia, and the Americas will be the principle targets of vaccination with the HHV because they are at greatest risk of the severe growth, developmental and cognitive impairments that result from hookworm-associated IDA. Vaccine efficacy of at least 80% against moderate and heavy hookworm infections for at least five years after immunization is the goal. The development of the HHV is led by the product development partnership based at Texas Children’s Hospital Center for Vaccine Development at Baylor College of Medicine in Houston, Texas and with key academic and industrial partners including those within the HOOKVAC consortium.
Both Na-GST-1 and Na-APR-1 are components of the blood-feeding pathway of N. americanus (3). Each antigen is intended to induce antibodies that will interfere with the function of the native proteins in the hookworm gut and thus impair worm survival and fecundity. Na-GST-1 can bind heme and other toxic byproducts of the blood degradation process, thereby protecting the adult worm from these harmful molecules (4,5), whereas the aspartic protease Na-APR-1 performs the first step in the enzymatic cleavage of host hemoglobin by the adult hookworm residing in the intestinal tract (6). The HHV is not expected to induce sterilizing immunity. Instead, the aim is to reduce the pathology of hookworm by limiting intestinal blood loss due to moderate and heavy hookworm infections. More than one molecule will be needed to disrupt this complex system of hemoglobin digestion by targeting several points along the blood feeding pathway to increase the effect of each on parasite nutrition and reduce parasite fecundity and survival.
This HOOKVAC project has there achieved an impact in shaping (1) the target product profile of the HHV, (2) provide the basis for the design of tthe clinical trials that will be required before an application for licensure/registration of the HHV can be submitted, (3) shape our preliminary thoughts on the development strategy of the HHV.
Main dissemination activities
Amongst the most notable engagements at a political level is the appointment of Dr. Peter Hotez as a U.S. Science Envoy under 2nd administration of US president Barack Obama. In this role, Dr. Hotez engaged internationally at the citizen and government levels to develop partnerships, improve collaboration, and forge mutually beneficial relationships between other nations and the United States to stimulate increased scientific cooperation and foster economic prosperity. This appointment has led to extensive vaccine diplomacy, including the discussions of the hookworm vaccine, in the Middle East region, particularly in Saudi Arabia, Morocco, and Tunisia. Furthermore, he was instrumental in drafting and sign-on of the first ever NTD community letter to the G7 leaders in advance of the 2015 Summit in Bavaria. Over 100 NTD leaders and organizations signed on to the letter, delivered to the G7 heads of state, calling for greater investment in NTD treatment programs as well as R&D for new technologies.
We also carried out jointly carried out meetings with international stakeholders, including key World Health Organization (WHO) representatives in Geneva to discuss pathways to vaccine manufacture, WHO prequalification and broader global uptake. Furthermore, we have had discussions with pharmaceutical companies GSK, Sanofi Pasteur, Merck KGaA and Johnson & Johnson regarding potential collaboration on the vaccine.
Because the consortium was lacking the require proprietary / business expertise in low-cost manufacturing development for such a vaccine, we also encaged with companies in the Developing Countries Vaccine Manufacturers Network DCVMN. With funding from EUROPEAID, we engaged with Indian manufacturers in the hope/expectation that they could be bringing such expertise on low-cost manufacturing vaccines, because of specific skills and knowhow that is lacking with Western manufactures. It was also believed that such collaborative partnerships might also produce vaccines designed from the onset to meet specific implementation characteristics of resource-poor regions, such as heat-stable formulations and single-dose or combination vaccines. Finally, it was believed that Indian manufactures would be interested to take a hookworm vaccine onboard, as the disease is highly prevalent in India. The inspiration for this project was based on a Phase-III clinical trial of low-cost Indian-made rotavirus vaccine, ROTAVAC, which had demonstrated strong efficacy and excellent safety profile.
Exploitation Results
While the HOOKVAC project has been publicly funded via the framework program of the European Commision, it is unlikely that the later phase Ii and II studies (post HOOKVAC project) can be funded via such public funds only. Also, there will be a significant investment needed to further scale industrial production, provide evidence for the funders to back decisions on procurement of the HHV and set-up the logistics for the vaccination campaigns with HHV. We therefore believe that risk baring funding needs to be attracted to guarantee the conduct of phase II and III clinical trials and conduct the other market introduction preparations in parallel.
Attracting such risk baring funding means that we really have to go a different path and pursue a different approach: and this process all starts with making a plan on how the returns of invested capital can be created.
In order to quantify our investment needs and financial liabilities within such a re-payment funding model, the HOOKVAC consortium has done initial explorations an economic model of the vaccine development costs. We call this economic model a business case – although it is intended to be more than a simple profit and loss balance sheet. We target both the investments and returns from our potential investors, but we would also like to point the social economic benefit of our vaccine. In this sense the economic model is a hybrid: economic and social returns are both quantified and presented. At this stage, the model is not intended to perfectly represent the highly complex drug discovery process, as obviously our vaccines are early stage and many aspects are still uncertain. However, it was designed to help determine the current investment needs, to model when the vaccines will start to generate revenue and a positive cash flow, when all investors can be fully reimbursed, and what socio-economic impact can be achieved. This effort has not been done before, at least not in precise quantitative terms – whereas such a quantitative investment case /economic model is essential when talking to investors.
HOOKVAC consortium partners, along with PwC and the Clinton Health Access Initiative at the Clinton Foundation have been developing such an economic model for investment in 2 demonstration projects for neglected infectious diseases vaccine development. One of them is a business case for hookworm vaccine development.
For the first time we have shown how much investment is needed to bring hookworm and schistosomiasis vaccines to the market. We have calculated that the total funding need accumulates to EUR 150 million per vaccine.
In the business cases we demonstrate a positive investment case based on the market introduction in 2 large markets (India and Brazil) for hookworm (and 4 African markets for schistosomiasis).
HHV starts to generate revenue and a positive cash flow as of 2027. After 7 years of commercial operation (in 2032) the cumulative return on sold vaccines equals the full investment requirement (EUR 150 million). The sensitivity analysis show that the internal rate of return will become higher when we will be targeting more countries at a later stage (currently only India and Brazil were modelled);
The business cases also show the business terms for hookworm vaccine are favorable compared to schistosomiasis vaccine. This is a reflection of the fact that the hookworm epidemic is more pronounced in major pharmaceutical markets such as India and Brazil.
With these business cases we would like approach potential investors to invest in these cases. Our ‘series A investment need’ for HHV is 30M-40M EUR, depending on the final selection of phase II clinical studies. This will cover all clinical trial costs and all operating cost up and to the phase II clinical trial. The business cases show that after completion of the phase II studies, it will become more attractive for governments of countries where the disease is endemic to start with financing of the hookworm vaccine. After around 5 years of development when the vaccine candidates move to late stage development we therefore intent to ask developers and governments from India and Brazil (and possibly other funders) to cover the cost of the phase III development. We consider this the ‘series B financing need’.
With this series B investment an (150M), we can pay back series A investors in 2023 (estimated debt by than 50M EUR at an estimated interest of 10%). With series B funding we can subsequently develop the vaccine through the phase III study and do the market introduction of the vaccine. After 7 years of commercial operation the cumulative return on sold vaccines equals the full investment requirement ($150 million). Meaning that by 2033 all investors will have fully been reimbursed.
During the HOOKVAC project, we have engaged with Indian manufacturers in the hope/expectation that they could be bringing such expertise on low-cost manufacturing vaccines, because of specific skills and knowhow that is lacking with Western manufactures. It was also believed that such collaborative partnerships might also produce vaccines designed from the onset to meet specific implementation characteristics of resource-poor regions, such as heat-stable formulations and single-dose or combination vaccines. Finally, it was believed that Indian manufactures would be interested to take a hookworm vaccine onboard, as the disease is highly prevalent in India. The inspiration for this project was based on a Phase-III clinical trial of low-cost Indian-made rotavirus vaccine, ROTAVAC, which had demonstrated strong efficacy and excellent safety profile.
A good example of how a collaboration on hookworm vaccine could take shape, can be derived from a collaboration of Biological-E with Novartis on the development of thyphoid vaccine. In conversations with Biological-E, we have learned that this could serve as a model for hookworm vaccine development. Biological-E is responsible for manufacturing – Novartis is responsible for phase I and II studies, all activities are financed with closed wallets. Funding for the phase III is anticipated to primarily come from public sources. Biological-E and Novartis have already agreed on different territories once the vaccine comes on the market. Of course the hookworm vaccine proposition is a tougher cookie in absence of a travelers market, but they are aware of that and nonetheless believe that elements of the Biological-E Novartis deal could work for us as well, in case an Indian Manufacturer would adopt the manufacturing, and the series A financing would raised from public and private sources in a joint venture between and Indian Manufacturer and selected members of the HOOKVAC consortium.
To engage in meaningful and sustainable relationship with Indian vaccine manufactures, we designed a strategy where the action was initially targeted towards the establishment of long-term partnerships for low-cost manufacture of a vaccine for hookworm as an example case.
From a long list of 5 potential manufacturing partners, we have selected a preferred candidate, with which we have intensified our outreach activities. In the interactions with the vaccine manufactures, we have learned that since currently there are no vaccines for helminth infections developed, a business case would lack a benchmark with already marketed vaccines, as there are no real parasitic vaccine development programs that we could use as model for a sound and credible business case. This presents an extra complication for manufactures to become stakeholders and invest in early stages of the vaccine development processes. With our global partners, we are now defining the profile of the ultimate vaccine product. At the start of the HOOKVAC program, this package was primarily defined in scientific terms. The contacts with various manufacturers, both in the West as well as in the Global South, provides an opportunity for a better translation into a business-oriented value proposition for a hookworm vaccine.
We are now looking at the design of efficacy studies for a preventative vaccine propose treating hookworm-infected volunteers in endemic areas prior to vaccination and determining efficacy by comparing incidence of reinfection or mean intensity of reinfection. Details on the design of such studies are provided in the post-project development plan. In short, a traditional phase II trial to establish this would require a sample size of approximately 1200 volunteers and last for several years. An alternative far more efficient strategy using a hookworm vaccination-challenge model (HVCM) to test the efficacy of candidate vaccines. Critical to CHHI is the manufacture of Necator americanus infective larvae (NaL3) according to current Good Manufacturing Practice (cGMP) and the determination of an inoculum of NaL3 that is safe and reliably induces patent infection. HOOKVAC partner GWU developed such a model successfully in parallel to the studies conducted in the HOOKVAC program. An HVCM trial would last only 3 months following completion of vaccinations and require far fewer volunteers (eg, 12 adults per group, with 1 group serving as unvaccinated infectivity controls), such that multiple vaccine candidates or adjuvant formulations could be assessed simultaneously to facilitate rapid down-selection of the most promising product for phase 3 efficacy testing.
List of Websites: https://web.archive.org/web/20190618142427/http://www.hookvac.eu/
The HOOVAC project was implemented by a consortium of scientists from 9 academic institutions and companies in the EU, the USA and Africa. This consortium was the first and only in the world developing a vaccine for human hookworm infection. The HOOKVAC project was intended to contribute to the development of a candidate vaccine against hookworm infection, which ranks number one in terms of years lost from disability from a neglected infectious disease. Within HOOKVAC, the consortium worked on developing a bivalent, low-cost vaccine candidate was clinically tested for the first time in an African disease endemic population.
Previously, from more than 20 potential target proteins involved in the hookworm blood feeding process, two lead proteins have emerged as promising candidate antigens for further clinical development, based on preclinical efficacy testing and favourable human immunoepidemiology: Necator americanus-glutathione-S-transferase-1 (Na-GST-1) and Necator americanus- Aspartic- Protease-1 (Na-APR-1). These antigens from adult hookworms have proven to be safe and non-allergenic as opposed to antigens targeting larval stages which have resulted in significant adverse events. Therefore, both antigens are excellent candidates for vaccine development. Since neither recombinant Na-GST-1 or Na-APR-1 alone have produced complete sterilizing immunity in laboratory animals, it is hypothesized that including both antigens in a vaccine will have a greater chance of success at producing an effective vaccine for humans than either alone. By targeting different steps in the blood digestion pathway of the adult hookworm, the effect of combining them in a bivalent vaccine is expected to be at least additive, if not synergistic.
The HOOKVAC project addressed four main objectives while testing Na-GST-1 and Na-APR-1 as vaccine candidates.
1. Establish safety and immunogenicity of the vaccine candidate in an endemic population;
2. Improve the manufacturing process;
3. Provide clinical proof of concept; and
4. Improve accessibility of the vaccine in endemic areas (sub-Saharan Africa, Southeast Asia and Latin America).
The HOOKVAC program consisted of a series of technical work packages, of which WP2 included a series of clinical trials in a high burden country (Gabon), where both adults as well as children were included in clinical trials to test the safety and efficacy of the vaccine.
The HOOKVAC project delivered the results of the first phase 1 clinical trial conducted in sub-Saharan Africa of two novel Na-APR-1 and Na-GST-1 vaccine candidates against human hookworm infection, It also delivered for the first time, safety and immunogenicity data of Na-APR-1 administered to individuals that are resident in a Necator endemic area.
Co-administration of these candidate hookworm vaccines were observed to be safe and well tolerated when tested in healthy adults and children from this hookworm-endemic region of Gabon. Antigen-specific IgG antibodies were induced to both vaccine antigens in the first study with adult patients. Results on antigen-specific IgG antibodies to children and using a different adjuvant are expected by the end of the summer of 2019.
In WP3 and WP4 scaling of production capacity was successfully investigated. In WP5, the immune responses were studies in-depth in order to get a better understanding on how the parasite interacts with the host immune system. In WP6, we build upon Involving knowledge of end-user communities and health professionals as well as active engagement with advocacy and policy leaders.
The HOOKVAC project has contributed to the development of a human hookworm vaccine, which is a crucial tool on the path to eliminating the disease, as well as a means to reduce poverty, particularly in developing economies. Hookworm infection has been identified as a major cause of poverty in low- and middle-income countries and because of hookworm's potential for additive or synergistic effects on other co-infections of overlapping geographic distributions, thus improving the overall public health economic development in endemic economies. The antigens that have tested in the HOOKVAC product can thus ultimately result in a true anti-poverty vaccine.
Project Context and Objectives:
Human hookworm infection is a neglected tropical disease caused predominantly by the nematode parasite Necator americanus. Recent estimates indicate that approximately 439 million people are infected with hookworm worldwide, with the majority of cases found in the developing regions of South Asia (140 million cases), Sub-Saharan Africa (118 million), Southeast Asia (77 million), East Asia (64.5 million), and the Latin American and Caribbean region (30 million). In these areas, hookworm disease is a major cause of iron-deficiency anemia, a consequence of the adult hookworm's ability to extract blood from the intestinal mucosa and submucosa. The Global Burden of Disease Study 2010 (GBD 2010) estimated that hookworm is responsible for a loss of 3.2 million disability adjusted life years (DALYs), making it one of the leading neglected tropical diseases (along with schistosomiasis and leishmaniasis) in terms of disease burden and a leading cause of anemia in large parts of Africa. Hookworm infection occurs when the larval stages penetrate the skin of a human host. The primary adverse effect of infection, anemia, disproportionately occurs in children and pregnant women with lower iron reserves compared to other populations. Hookworm is hyperendemic among some pediatric populations in sub-Saharan Africa where in countries such as Sierra Leone or Togo one-third of the population under the age of 20 is infected. Children with chronic hookworm infection experience anemia and cognitive and developmental delays with resultant reductions in school performance, attendance and future wage earnings. Approximately 7 million pregnant women in sub-Saharan Africa – almost one third of annual pregnancies in Africa – are also infected, making hookworm disease one of the most common complications of pregnancy in that part of the world. Moreover, many of these individuals – both children and pregnant women – are co-infected with malaria, thereby exacerbating anemia and its sequelae.
The primary approach to hookworm control is mass drug administration with the anthelminthics albendazole or mebendazole. However, a single dose of mebendazole, administered once per year, has yielded low cure rates, particularly with repeated use. One comprehensive meta-analysis showed no impact of mebendazole treatment on improving anemia in hookworm-affected communities. Similarly, for albendazole, given as a single dose, once per year, drug failure has been reported, though less often. Moreover, children can re-acquire hookworm several months after treatment, particularly in geographic locations with significant infectivity or attack rates. These observations may explain a recent finding from GBD 2013 study that overall global hookworm prevalence has remained essentially unchanged over the last 20 years, while the prevalence of other neglected tropical diseases such as lymphatic filariasis, ascariasis, and trachoma has decreased by 25–33% over the same time period.
There is, therefore, a need for new technologies to achieve better control of hookworm infection. A safe and effective anti-hookworm vaccine, as a complement to conventional chemotherapy, may provide a cost-effective means of reaching this goal.
From more than 20 potential target proteins involved in the hookworm blood feeding process, two lead proteins have emerged as promising candidate antigens for further clinical development, based on preclinical efficacy testing and favorable human immunoepidemiology: Necator americanus-glutathione-S-transferase-1 (Na-GST-1) and Necator americanus- Aspartic- Protease-1 (Na-APR-1). These antigens from adult hookworms have proven to be safe and non-allergenic as opposed to antigens targeting larval stages which have resulted in significant adverse events. Therefore, both antigens are excellent candidates for vaccine development, as they (i) target adult N. americanus worms and (ii) have been shown to partially protect laboratory animals against challenge infection with hookworm.
In the HOOKVAC project, these 2 selected antigens were further tested, with the following four main objectives:
1. Establish safety and immunogenicity of the vaccine candidates Necator americanus-glutathione-S-transferase-1 (Na-GST-1) and Necator americanus- Aspartic- Protease-1 (Na-APR-1) in an endemic population;
2. Improve the manufacturing process of Na-GST-1 and Na-APR-1;
3. Provide clinical proof of concept; and
4. Improve accessibility of the vaccine in endemic areas (sub-Saharan Africa, Southeast Asia and Latin America).
Project Results:
The HOOKVAC program was designed through a series of 6 work packages, of which the 1st work package was coordination the project. The S&T objectives were divided over a series of 5 work packages (WP2-WP6)
Clinical Studies (WP2)
Since neither recombinant Na-GST-1 or Na-APR-1 alone have produced complete sterilizing immunity in laboratory animals, it is hypothesized that including both antigens in a vaccine will have a greater chance of success at producing an effective vaccine for humans than either alone. By targeting different steps in the blood digestion pathway of the adult hookworm, the effect of combining them in a bivalent vaccine is expected to be at least additive, if not synergistic. As a single formulation vaccine of these 2 antigens was not available at the start of the project, HOOKVAC initiated Phase I clinical testing in which the separately-formulated antigens were co- administered first in adults (HV-01) and then in school-aged children (HV-02) to validate the safety and immunogenicity of these antigens and explore the optimal doses and vaccination schedule in an endemic African population. Finally, a HV-03 study was designed to assess the safety and immunogenicity of co-administered Na-GST-1 and CPG 10104 (a human TLR-9 stimulator) in an adult African population resident in a hookworm-endemic region, in order to establish the possibility of scaling back to one antigen for further clinical studies.
The HV-01 study, was a randomized, controlled, double-blind Phase 1 trial was conducted in 32 healthy adults in N. americanus endemic areas of Gabon. Participants were randomised to receive three doses of either co-administered Na-GST-1 plus Na-APR-1 (M74) (30µg or 100µg of each), adjuvanted with Alhydrogel® (aluminium hydroxide gel suspension) together with an aqueous formulation of glucopyranosyl lipid A (GLA-AF) or the hepatitis B vaccine as a comparator. Vaccinations were administered on days 0, 28, and 180. Co-administration of Na-GST-1/Alhydrogel® and Na-APR-1 (M74)/Alhydrogel® was well tolerated at both doses tested. The most common adverse events were mild-to-moderate injection site pain, headache, myalgia, and nausea. No severe or serious adverse events related to the vaccines were recorded. IgG antibodies were induced to each of the vaccine antigens, with increasing IgG levels observed after each vaccination. Vaccination with 100µg of each vaccine antigen with GLA-AF consistently induced IgG sero-conversion. The peak of the IgG response was observed two weeks after the third vaccine administration for both vaccine antigens. Study HV-01 therefore showed that vaccination with recombinant Na-GST-1 and Na-APR-1 (M74) in healthyadults resident in Necator endemic areas of Gabon was safe and induced increasing levels of IgG to each antigen after each vaccine administration. This manuscript presents the first description of Na-APR-1 (M74)/Alhydrogel® in participants from a Necator endemic area. Further clinical development of these vaccines should be advanced with efficacy studies.
The HV-002 study was designed as a double blind, randomized, controlled, dose-escalation Phase 1 clinical trial in hookworm-exposed children aged 6 to 10 years living in the area of Lambaréné, Gabon. Children received three doses of the assigned vaccine(s) delivered intramuscularly (deltoid) on approximately Days 0, 56, and 112 or 180.
In total, 137 children were screened. Out of them, 77 screened failed for a range of reasons including Informed consent withdrawn (05), Hepatitis B vaccine received before (16), Participation in other clinical trials (02), planning to move out from a study area (12), unhealthy (37), positive for Hepatitis B or C virus (03), assent form not provided (02). During the follow-up, the study team reported one unrelated SAE (right inguinal hernia) in participant belonging to the pediatric trial. No vaccine related serious adverse event were reported during the trial.
The team concluded that the co-administered Na-GST-1 and Na-APR-1 vaccine candidate in children living in hookworms endemic area in Gabon were found to be safe and well tolerated. Further serum analysis will assess their immunogenicity, these results will become available at the end of the summer period of 2019, and will be reported separately to the Commission.
In the original design of WP2, we had envisioned that the vaccine product in trial HV-03 would be a bivalent vaccine containing a mix of the 2 antigens that were tested as co-administered products in studies HV01 (adults) and HV02 (children).
Unfortunately, the work conducted under WP3 and WP4 demonstrated that the co-formulation of Na-APR-1 and Na-GST-1 using the backbone of the currently used buffer formulations was not feasible. The alternative of conducting the study with the individual antigens would NOT yield data that are sufficiently different from what we had learned from HV-01, which is described above.
Meanwhile, the Na-GST-1/Alhydrogel hookworm vaccine has been tested in combination with CPG 10104 in one Phase 1 clinical trial in healthy adults in the United States (n=24). In study SVI-GST-03 in healthy, hookworm-naïve American adults initiated in 2014 in Washington, DC, 24 volunteers received three vaccinations (on study days 0, 56 and 112) with up to 100 µg Na-GST-1/Alhydrogel administered with or without up to 500 µg CPG10104 (NCT02143518). Anti-Na-GST-1 IgG antibody responses have been assayed up to one month following the third vaccination, and indicate that the addition of CPG 10104 to Na-GST-1 results in significantly improved humoral immune responses to the vaccine antigen.
Based on these findings, the design of study HV-03 was modified such that it studied whether the same improved vaccine response can be generated in an adult population that lives in a hookworm endemic country. If this would the case, possibly a single antigen based vaccine could be developed, which would be a crucial alternative clinical development strategy in case we do not solve the current technical hurdles in producing a bivalent vaccine
The HV-003 study was therefore set-up in twenty-four eligible adults that were progressively enrolled into 1 of 2 groups over a projected 2-month enrolment period, with each participant followed for 9 months after the final injection. Group enrolment was done in an open sequential fashion, whereas within each Group, investigational product (IP) assignment and vaccination was done in a randomized double-blind fashion. The first 12 subjects was recruited and enrolled into Group 1: (A) 8 subjects received 30 µg Na-GST-1 plus 500 µg CPG 10104 delivered by IM injection in the deltoid muscle according to a 0,2,4-month schedule, and (B) 4 subjects received 100 µg Na-GST-1 delivered by IM injection in the deltoid muscle according to a 0,2,4-month schedule. In Group 2 (A) 8 subjects received 100 µg Na-GST-1 plus 500 µg CPG 10104 delivered by IM injection in the deltoid muscle according to a 0,2,4-month schedule, and (B) 4 subjects received 100 µg Na-GST-1 delivered by IM injection in the deltoid muscle according to a 0,2,4-month schedule. During the follow-up, 2 cases of pregnancy have been reported in each cohort and one resulting in miscarriage in the cohort 1 and 2 suspensions of the vaccine administration after both participants have received their first immunization. No vaccine related serious adverse event were reported in the trial. The conclusion from the study is thus that co-administered Na-GST-1 with CPG 10104 vaccines in adults living in hookworms endemic area in Gabon were found to be safe, well tolerated. Further serum analysis will assess their immunogenicity.
WP3 scaling the production platforms to the level needed for subsequent studies
WP3 was targeted toward scaling the production platforms for both antigens. We purified Na-GST and Na-APR antigens which showed biochemical and immunological comparability to the reference materials that were used in earlier studies.
With regards to Na-GST-1, this scaled production process resulted in over 95% pure intact Na-GST. A similar purity of Na-GST was thus obtained for the batches of development and engineering run, confirming the robustness of the developed purification process. The Na-GST produced under WP3 is similar to the reference material and meets the specifications determined in the monograph with respect to the identity, purity, pH, protein concentration, endotoxin content, total contaminating host cell proteins and residual total DNA. The fermentation is now at a scale of 10-15 litres. A Pichia pastoris working cell bank has been produced starting from a vial of the Sabin master cell bank. Na-GST has been purified in cGMP from the harvested micro-filtered medium according the scalable downstream process and fulfilled specifications as described in the Monograph. A stability study showed that the over 90% pure Na-GST was stable for at least 12 months at below minus 60°C. A comparable in vivo potency was observed for the alhydrogel adsorbed Na-GST obtained by the new scalable and reference process. WP3 has thus delivered a scalable downstream process of P. pastoris Na-GST-1. CMC-reports of the cGMP–manufactured Na-GST batch and the report of the one- year Na-GST stability study have been prepared.
Production of NA-APR-1 has proved a lot more challenging. Before the start of the HOOKVAC program, Na-APR-1 has been produced as a recombinant His-tagged protein with ER retention signal (KDEL) in tobacco plants, the according expression levels are very low. Only milligram quantities have been obtained out of kilograms of plant extracts. One of the major problems is the tendency of the protein to form aggregates. Moreover, for further use as an injectable, it is recommended to produce the Na-APR-1 protein without His-tag.
To this end, an E.coli strain was therefore constructed expressing APR-1 as a cytosolic protein (GI724/M-APR-1). Biochemical analysis of the purified E. coli M-APR pool showed that the current purification method consisting of 2 consecutive anion exchange chromatography steps, had resulted in an at least 92% pure monomeric full size M-APR and that the residual HCP are below 3% (based on anti-E. coli WB). QC analysis showed that the product meets specifications. The overall yield of M-APR is over 30 mg per liter fermentation equivalent, while formation is now at the same level as for Na-GST-1. Stability studies at 3, 6, 9 and 12months showed that the over 90% pure M-APR-1 was stable for at least 12 months at 2-8°C and at below minus 60°C.. Unfortunately, initial formulation experiments showed that Empigen concentrations which is needed for the stability of APR-1 even at 0.01% (w/v) causes structural changes in the alhydrogel-bound Na-GST-1. The M-APR and empigen concentration were respectively 200µg/ml and 0.25% (w/v) in the first generation Drug Substance. These combined data suggest that co-formulation of M-APR and Na-GST1 antigens would only be possible after adaptation of the M-APR purification process. This prompted a change of the scope of WP4.
WP4 towards the production of a stable bivalent hookworm vaccine
WP4 was originally targeted towards the production of a stable bivalent hookworm vaccine product. It was originally envisioned that we would engage in the development of a formulation process was described allowing co-formulation of the two antigens on Alhydrogel. Such a co-formulated bivalent hookworm vaccine would be more convenient to administer (single injection). Thus would improve subject compliance, significantly reduce vaccine manufacturing costs and simplify logistics related to distribution and storage.
While in WP3 we have shown that the purified Na-GST and Na-APR antigens that were produced showed biochemical and immunological comparability to the reference materials that were used in earlier studies, the initial co-formulation of Na-APR-1 and Na-GST-1 using the backbone of the currently used buffer formulations is not feasible. Specifically, the presence of Empigen BB in the buffer – which is vital to the stability of the Na-APR-1 monomer - impairs the ability of incorporating other molecules such as GST to the mix. When presenting these data to the scientific and ethical advisory board of HOOKVAC, in which representatives with pharmaceutical expertise are engaged, it was advised that such early formulation hurdles are not uncommon, and should definitely not be regarded as a showstopper. Typically, once a (vaccine) product has been optimized in terms of generated the desired immunogenicity, enhanced formulation activities can still lead to an optimized vaccine in terms desired formulation. The required expertise is typically found in the pharmaceutical industry. The required investments from the pharmaceutical industry are more likely to be recruited if the prototype antigens have successfully completed the planned clinical studies. Meanwhile, it was felt that additional pre-formulation and formulation studies could enhance the likelihood of finding a suitable formulation technology. To this end, alternative excipients were identified and screened. Alternative ‘in-solution’-stabilizing excipients and buffer conditions (type, pH) were tested for the alhydrogel- adsorbed antigens. These excipients minimized the destabilizing effect of Empigen, up to 0.016%. GSH had again the strongest stabilizing effect (i.e. strong increase in melting point) and the data favored the formulation at a pH below 7.0. The replacement of Empigen by Tween 20 as detergent in the elution of the polishing step and the increased concentration in the eluate indeed now opens perspectives for the coformulation and development of a prototype Hookworm vaccine based on GST-1 and M-APR-1.
WP5 Towards an understanding of immunological responses to the vaccine
In this workpackage, we characterized the T cell responses in the vaccinated volunteers of study HV-01. This was measured by stimulating PBMC with recombinant Na-GST and Na-APR-M74, at pre (day 0) and after first (day 28) and third (day 180) vaccination and assessed intracellular cytokine (IL2, IL4/IL5/IL13, IL10, IFNγ and TNF) responses. Such data can provide a better characterizing the target population (exposure, pre-existing immunity and coinfections) in order to determine the optimal dose needed to stimulate an effective immune response to a vaccine.
In the HV-01 study, a significant increase in Na-GST specific CD4+ IL2+ and CD4+TNF+ cells as well as cells co-expressing IL2 and TNF was seen after the third vaccination with the high dose vaccine. The vaccination with Na-GST and Na-APR in HV-01, resulted in antigen specific induction of IL2 and TNF expressing T cells, that was seen in the group that received the high dose vaccine. Over the vaccination course an increase in IL2 and TNF was observed with the highest cytokine secretion seen at day 194. However, there was no correlation between the antibodies and the cytokines responses over the vaccination course. Indeed, some participants with high Abs titers had no detectable cells in peripheral blood producing cytokines in response to in vitro stimulation with vaccine candidates. This could be due to the sensitivity of the assay or the residence of the antigen specific T cells in peripheral tissues such as in lymph nodes.
The increased IL2 response following the vaccination schedule, could promote the expansion of effector T cells that can help B cells antibody production. The enhanced TNF response, which is generally related to an early type 1 response, is also known to help the humoral immune response as TNF blockade has been shown to be associated with antibody titer reduction and therefore reduced responses to vaccination. Earlier studies have indicated that multifunctional T cells might play an important role in the immune response to vaccines in general. In our study the magnitude of double cytokine (TNF+IL2+) producing CD4+ T cells was significantly increased from day 0 to day 194, which might suggest that these cells play a role in Na-GST immunogenicity.
In contrast, the vaccination did not result in Na-APR specific CD4+ cytokine producing cells even though detectable antibodies to this candidate was seen following vaccination albeit to a lesser extent than what was seen for Na-GST antibodies. This is in line with the lower sensitivity of detecting immunogenicity based on intracellular cytokine producing CD4+ cells in peripheral blood compared to detection of antibodies. It is possible that higher doses of Na APR are needed for improved antibody responses.
A low cytokines response to APR antigen that was seen at baseline as well as in the group vaccinated with HBV but this was not boosted over time by the vaccines given. Participants included in this trial were hookworm- exposed subjects and a pre-existing response to Na-APR might prevent a response to be mounted to Na-APR. It is also possible that other hematophagous parasites such as malaria parasites express APR. Interestingly, we noticed that the participant with the higher Na-APR antibody titers were positive for malaria before inclusion into the study. We therefore hypothesize that the potential pre-existing immunity to Na-APR due to a pre-exposure or cross reaction with malaria APR, is responsible for the low cytokines response observed at baseline.
WP6 Global Access Strategy
During the HOOKVAC program, a wide portfolio of external activities was initiated, including the press/media engagement, organized meetings and position statements. Several consortium members have led numerous speaking engagements and presentations to a wide array of audiences highlighting the HOOKVAC consortium and increasing awareness of the need for hookworm vaccine development. Further details are provided in the dissemination section of this report.
Potential Impact:
More than 400 million people worldwide are infected with hookworm (1), with the greatest number of cases occurring in sub-Saharan Africa, Southeast Asia, China, and tropical regions of the Americas. Of the two species that infect humans, Necator americanus is the most prevalent, causing 85% of infections. The clinical hallmark of hookworm is iron deficiency anemia (IDA) that results from intestinal blood loss caused by adult parasites feeding at their site of attachment in the small intestine. Currently, the major approach to the control of pediatric hookworm is the annual mass drug administration of a benzimidazole anthelminthic drug such as mebendazole or albendazole. However, widespread mebendazole drug failure for the treatment of hookworm and high rates of post-treatment re-infection with both drugs justify the need to develop a vaccine.
The Human Hookworm Vaccine (HHV) is being developed as a bivalent product containing the Na-GST-1 and Na-APR-1 recombinant proteins, with the indication being the prevention of moderate and heavy hookworm infections caused by N. americanus. The candidate antigens were selected for clinical development based on their protective efficacy in animal trials and immunoepidemiological studies in individuals resident in hookworm-endemic areas (2). Children living in N. americanus endemic regions of Africa, Asia, and the Americas will be the principle targets of vaccination with the HHV because they are at greatest risk of the severe growth, developmental and cognitive impairments that result from hookworm-associated IDA. Vaccine efficacy of at least 80% against moderate and heavy hookworm infections for at least five years after immunization is the goal. The development of the HHV is led by the product development partnership based at Texas Children’s Hospital Center for Vaccine Development at Baylor College of Medicine in Houston, Texas and with key academic and industrial partners including those within the HOOKVAC consortium.
Both Na-GST-1 and Na-APR-1 are components of the blood-feeding pathway of N. americanus (3). Each antigen is intended to induce antibodies that will interfere with the function of the native proteins in the hookworm gut and thus impair worm survival and fecundity. Na-GST-1 can bind heme and other toxic byproducts of the blood degradation process, thereby protecting the adult worm from these harmful molecules (4,5), whereas the aspartic protease Na-APR-1 performs the first step in the enzymatic cleavage of host hemoglobin by the adult hookworm residing in the intestinal tract (6). The HHV is not expected to induce sterilizing immunity. Instead, the aim is to reduce the pathology of hookworm by limiting intestinal blood loss due to moderate and heavy hookworm infections. More than one molecule will be needed to disrupt this complex system of hemoglobin digestion by targeting several points along the blood feeding pathway to increase the effect of each on parasite nutrition and reduce parasite fecundity and survival.
This HOOKVAC project has there achieved an impact in shaping (1) the target product profile of the HHV, (2) provide the basis for the design of tthe clinical trials that will be required before an application for licensure/registration of the HHV can be submitted, (3) shape our preliminary thoughts on the development strategy of the HHV.
Main dissemination activities
Amongst the most notable engagements at a political level is the appointment of Dr. Peter Hotez as a U.S. Science Envoy under 2nd administration of US president Barack Obama. In this role, Dr. Hotez engaged internationally at the citizen and government levels to develop partnerships, improve collaboration, and forge mutually beneficial relationships between other nations and the United States to stimulate increased scientific cooperation and foster economic prosperity. This appointment has led to extensive vaccine diplomacy, including the discussions of the hookworm vaccine, in the Middle East region, particularly in Saudi Arabia, Morocco, and Tunisia. Furthermore, he was instrumental in drafting and sign-on of the first ever NTD community letter to the G7 leaders in advance of the 2015 Summit in Bavaria. Over 100 NTD leaders and organizations signed on to the letter, delivered to the G7 heads of state, calling for greater investment in NTD treatment programs as well as R&D for new technologies.
We also carried out jointly carried out meetings with international stakeholders, including key World Health Organization (WHO) representatives in Geneva to discuss pathways to vaccine manufacture, WHO prequalification and broader global uptake. Furthermore, we have had discussions with pharmaceutical companies GSK, Sanofi Pasteur, Merck KGaA and Johnson & Johnson regarding potential collaboration on the vaccine.
Because the consortium was lacking the require proprietary / business expertise in low-cost manufacturing development for such a vaccine, we also encaged with companies in the Developing Countries Vaccine Manufacturers Network DCVMN. With funding from EUROPEAID, we engaged with Indian manufacturers in the hope/expectation that they could be bringing such expertise on low-cost manufacturing vaccines, because of specific skills and knowhow that is lacking with Western manufactures. It was also believed that such collaborative partnerships might also produce vaccines designed from the onset to meet specific implementation characteristics of resource-poor regions, such as heat-stable formulations and single-dose or combination vaccines. Finally, it was believed that Indian manufactures would be interested to take a hookworm vaccine onboard, as the disease is highly prevalent in India. The inspiration for this project was based on a Phase-III clinical trial of low-cost Indian-made rotavirus vaccine, ROTAVAC, which had demonstrated strong efficacy and excellent safety profile.
Exploitation Results
While the HOOKVAC project has been publicly funded via the framework program of the European Commision, it is unlikely that the later phase Ii and II studies (post HOOKVAC project) can be funded via such public funds only. Also, there will be a significant investment needed to further scale industrial production, provide evidence for the funders to back decisions on procurement of the HHV and set-up the logistics for the vaccination campaigns with HHV. We therefore believe that risk baring funding needs to be attracted to guarantee the conduct of phase II and III clinical trials and conduct the other market introduction preparations in parallel.
Attracting such risk baring funding means that we really have to go a different path and pursue a different approach: and this process all starts with making a plan on how the returns of invested capital can be created.
In order to quantify our investment needs and financial liabilities within such a re-payment funding model, the HOOKVAC consortium has done initial explorations an economic model of the vaccine development costs. We call this economic model a business case – although it is intended to be more than a simple profit and loss balance sheet. We target both the investments and returns from our potential investors, but we would also like to point the social economic benefit of our vaccine. In this sense the economic model is a hybrid: economic and social returns are both quantified and presented. At this stage, the model is not intended to perfectly represent the highly complex drug discovery process, as obviously our vaccines are early stage and many aspects are still uncertain. However, it was designed to help determine the current investment needs, to model when the vaccines will start to generate revenue and a positive cash flow, when all investors can be fully reimbursed, and what socio-economic impact can be achieved. This effort has not been done before, at least not in precise quantitative terms – whereas such a quantitative investment case /economic model is essential when talking to investors.
HOOKVAC consortium partners, along with PwC and the Clinton Health Access Initiative at the Clinton Foundation have been developing such an economic model for investment in 2 demonstration projects for neglected infectious diseases vaccine development. One of them is a business case for hookworm vaccine development.
For the first time we have shown how much investment is needed to bring hookworm and schistosomiasis vaccines to the market. We have calculated that the total funding need accumulates to EUR 150 million per vaccine.
In the business cases we demonstrate a positive investment case based on the market introduction in 2 large markets (India and Brazil) for hookworm (and 4 African markets for schistosomiasis).
HHV starts to generate revenue and a positive cash flow as of 2027. After 7 years of commercial operation (in 2032) the cumulative return on sold vaccines equals the full investment requirement (EUR 150 million). The sensitivity analysis show that the internal rate of return will become higher when we will be targeting more countries at a later stage (currently only India and Brazil were modelled);
The business cases also show the business terms for hookworm vaccine are favorable compared to schistosomiasis vaccine. This is a reflection of the fact that the hookworm epidemic is more pronounced in major pharmaceutical markets such as India and Brazil.
With these business cases we would like approach potential investors to invest in these cases. Our ‘series A investment need’ for HHV is 30M-40M EUR, depending on the final selection of phase II clinical studies. This will cover all clinical trial costs and all operating cost up and to the phase II clinical trial. The business cases show that after completion of the phase II studies, it will become more attractive for governments of countries where the disease is endemic to start with financing of the hookworm vaccine. After around 5 years of development when the vaccine candidates move to late stage development we therefore intent to ask developers and governments from India and Brazil (and possibly other funders) to cover the cost of the phase III development. We consider this the ‘series B financing need’.
With this series B investment an (150M), we can pay back series A investors in 2023 (estimated debt by than 50M EUR at an estimated interest of 10%). With series B funding we can subsequently develop the vaccine through the phase III study and do the market introduction of the vaccine. After 7 years of commercial operation the cumulative return on sold vaccines equals the full investment requirement ($150 million). Meaning that by 2033 all investors will have fully been reimbursed.
During the HOOKVAC project, we have engaged with Indian manufacturers in the hope/expectation that they could be bringing such expertise on low-cost manufacturing vaccines, because of specific skills and knowhow that is lacking with Western manufactures. It was also believed that such collaborative partnerships might also produce vaccines designed from the onset to meet specific implementation characteristics of resource-poor regions, such as heat-stable formulations and single-dose or combination vaccines. Finally, it was believed that Indian manufactures would be interested to take a hookworm vaccine onboard, as the disease is highly prevalent in India. The inspiration for this project was based on a Phase-III clinical trial of low-cost Indian-made rotavirus vaccine, ROTAVAC, which had demonstrated strong efficacy and excellent safety profile.
A good example of how a collaboration on hookworm vaccine could take shape, can be derived from a collaboration of Biological-E with Novartis on the development of thyphoid vaccine. In conversations with Biological-E, we have learned that this could serve as a model for hookworm vaccine development. Biological-E is responsible for manufacturing – Novartis is responsible for phase I and II studies, all activities are financed with closed wallets. Funding for the phase III is anticipated to primarily come from public sources. Biological-E and Novartis have already agreed on different territories once the vaccine comes on the market. Of course the hookworm vaccine proposition is a tougher cookie in absence of a travelers market, but they are aware of that and nonetheless believe that elements of the Biological-E Novartis deal could work for us as well, in case an Indian Manufacturer would adopt the manufacturing, and the series A financing would raised from public and private sources in a joint venture between and Indian Manufacturer and selected members of the HOOKVAC consortium.
To engage in meaningful and sustainable relationship with Indian vaccine manufactures, we designed a strategy where the action was initially targeted towards the establishment of long-term partnerships for low-cost manufacture of a vaccine for hookworm as an example case.
From a long list of 5 potential manufacturing partners, we have selected a preferred candidate, with which we have intensified our outreach activities. In the interactions with the vaccine manufactures, we have learned that since currently there are no vaccines for helminth infections developed, a business case would lack a benchmark with already marketed vaccines, as there are no real parasitic vaccine development programs that we could use as model for a sound and credible business case. This presents an extra complication for manufactures to become stakeholders and invest in early stages of the vaccine development processes. With our global partners, we are now defining the profile of the ultimate vaccine product. At the start of the HOOKVAC program, this package was primarily defined in scientific terms. The contacts with various manufacturers, both in the West as well as in the Global South, provides an opportunity for a better translation into a business-oriented value proposition for a hookworm vaccine.
We are now looking at the design of efficacy studies for a preventative vaccine propose treating hookworm-infected volunteers in endemic areas prior to vaccination and determining efficacy by comparing incidence of reinfection or mean intensity of reinfection. Details on the design of such studies are provided in the post-project development plan. In short, a traditional phase II trial to establish this would require a sample size of approximately 1200 volunteers and last for several years. An alternative far more efficient strategy using a hookworm vaccination-challenge model (HVCM) to test the efficacy of candidate vaccines. Critical to CHHI is the manufacture of Necator americanus infective larvae (NaL3) according to current Good Manufacturing Practice (cGMP) and the determination of an inoculum of NaL3 that is safe and reliably induces patent infection. HOOKVAC partner GWU developed such a model successfully in parallel to the studies conducted in the HOOKVAC program. An HVCM trial would last only 3 months following completion of vaccinations and require far fewer volunteers (eg, 12 adults per group, with 1 group serving as unvaccinated infectivity controls), such that multiple vaccine candidates or adjuvant formulations could be assessed simultaneously to facilitate rapid down-selection of the most promising product for phase 3 efficacy testing.
List of Websites: https://web.archive.org/web/20190618142427/http://www.hookvac.eu/